EA048715B1 - ANTIBODY-DRUG CONJUGATES - Google Patents

Description

Field of technology

The present invention relates to new drug conjugates, drugs, drug-linker compounds, methods for their preparation, pharmaceutical compositions containing said drug conjugates, and their use as antitumor agents.

Prior art

Ecteinascidins are extremely potent antineoplastic agents isolated from the marine tunicate Ecteinascidia turbinata. One of these compounds, trabectedin, is used to treat patients with advanced and metastatic soft tissue sarcoma (STS) who have failed or are contraindicated for therapy with anthracyclines and ifosfamide, and to treat relapsed platinum-sensitive ovarian cancer in combination with pegylated liposomal doxorubicin.

U.S. Patent No. 5,149,804 describes Ecteinascidin 722 (ET-722), isolated from the Caribbean tunicate Ecteinascidia turbinata, and its structure. ET-722 protects mice in vivo at extremely low concentrations against P388 lymphoma, B16 melanoma, and Lewis lung sarcoma.

ET-722

WO 03066638 describes certain synthetic analogues of ET-722 and their cytotoxic activity against tumor cells. In particular, WO 03066638 describes compounds 1-3 with their cytotoxic activity against a panel of cancer cell lines.

MeO MeO

2 3

Another compound described in WO 03/014127, lurbinectedine, is currently undergoing clinical trials for the treatment of cancer. Lurbinectedine has the following chemical structure

MeO

lurbinectedin

WO 2018197663 relates to novel ecteinascidin derivatives that show promising antitumor activity. One of the compounds disclosed in such patent application is

- 1,048,715 are currently undergoing phase I clinical trials for the prevention and treatment of solid tumors.

Cancer treatment has advanced significantly in recent years due to the development of pharmaceuticals that more effectively target and kill cancer cells. Researchers have taken advantage of cell surface receptors and antigens selectively expressed by target cells, such as cancer cells, to develop antibody-based pharmaceuticals that bind, for example, tumors, tumor-specific or tumor-associated antigens. To do this, cytotoxic molecules such as chemotherapeutic drugs, bacteria, plant toxins and radionuclides have been chemically linked to monoclonal antibodies that bind tumor-specific or tumor-associated cell surface antigens.

Thus, ADCs represent a challenging area of development given the complex structure of the payload, linker, and antibody, but there remains a need to develop additional ADCs.

Brief summary of the invention

There is a need for new active drug conjugates. The present invention satisfies this need. It also provides new drugs and drug-linker compounds for use in preparing the drug conjugates of the present invention, methods for preparing the new drug conjugates of the present invention, pharmaceutical compositions containing said drug conjugates, and their use as antitumor agents.

In a first aspect of the present invention there is provided a drug conjugate comprising a drug moiety covalently attached to the remainder of the drug conjugate having the formula [D-(X) _b -(AA) _w -(T)g-(L)-] _n -Ab, where:

D is a grouping of a drug of the following formula (IH) or its pharmaceutically acceptable salt or stereoisomer ^R 3

(IH), where:

the wavy line denotes the point of covalent attachment to (X) _b if present, or to (AA) _w if present, or to (T)g if present, or to (L);

Y represents -NH- or -O-;

R1 represents -OH or -CN;

R2 represents the group -C(=O)R _a ;

R3 represents hydrogen or the -OR _b group;

R ₄ is selected from hydrogen, -CH2OH and -CH2NH2;

R _a is unsubstituted C ₁ -C ₁₂ -αalkyl;

R _b is unsubstituted C ₁ -C ₁₂ -alkyl;

X represents an extending group selected from:

-COO-(C ₁ -C ₆ -alkylene)NH-;

-COO-CH ₂ -(unsubstituted phenylene)-NH-;

-COO-(C ₁ -C ₆ -alkylene)NH-COO-CH ₂ -(unsubstituted phenylene)-NH-;

-COCH2NH-COCH2-NH-;

-COCH2NH-;

-COO-(C ₁ -C ₆ -alkylene)S-;

-COO-(C1-C6-alkylene)NHCO(C1-C6-αalkylene)S-;

T is an extending group selected from the group consisting of -CO-( _C1 - _C6alkylene )-NH-, -CO-( _C1 - _C6alkylene )-[O-( _C2 - _C6alkylene )] _j- NH-, -C00-( _C1 - _C6alkylene )-[0-( _C2 - _C6alkylene )] _jNH- ; where j is an integer from 1 to 25;

(AA)w is represented by formula (II):

- 2 048715

where the wavy lines represent the point of covalent attachment to (X) _b if present or to the drug moiety (wavy line on the left) and to (T)g if present or to the linker (wavy line on the right); and

R ₂₁ in each case is selected from the group consisting of hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, -CH2CH2COOH, (CH2)3NHC(=NH)NH2, -(C^^N^ -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, (CH2bNHCONH2, -(CH2)4NHCONH2, phenyl, cyclohexyl,

and w is an integer in the range 0 to 6;

L is a linker group selected from the group consisting of:

ABOUT _

About S

5» ¹¹ / $ C Ri 9—|\j

ABOUT

NH where the wavy lines represent the point of covalent attachment to Ab (wavy line on the right) and to (T)g if present, or (AA)w if present, or (X) _b if present, or D (wavy line on the left);

R ₁₉ is unsubstituted -C ₁ -C ₁₂ -alkylene-;

R ₃₀ represents a -C ₁ - ₆ alkylene- group;

M is unsubstituted -C _1-6 alkylene- _;

b is equal to the integer 0 or 1;

g is equal to the integer 0 or 1;

Ab is an anti-HER2 antibody or antigen-binding fragment thereof; and n is the ratio of the [D-(X) _b -(AA)w-(T) _g -(L)-] group to Ab, and ranges from 1 to 20.

Preferably, in the drug conjugate of the invention, D is a compound of the formula:

or a pharmaceutically acceptable salt thereof.

It is also preferred that in the drug conjugate of the invention D is a compound of the formula:

or a pharmaceutically acceptable salt thereof.

Preferably, in the drug conjugate of the invention, the salt is selected from hydro

- 3 048715 chloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate, paratoluenesulfonate, sodium, potassium, calcium, ammonium, ethylenediamine, ethanolamine, N,N-dialkyleneethanolamine, triethanolamine and essential amino acids.

Preferably, the drug conjugate of the invention is selected from formulas (IV), (V) and (VI):

Where

X, T, AA, w, b, g, D, Ab and n are as defined above;

R ₁₉ is unsubstituted -C ₁ -C ₈ -alkylene-;

R30 represents a -C2- _C4 -alkylene- group; and

M is unsubstituted -C ₁ -C ₃ -alkylene-;

or selected from formulas (IV), (V) and (VI):

(VI) where

X, T, AA, w, b, g, D, Ab and n are as defined above;

R ₁₉ is unsubstituted -C ₁ - ₆ alkylene-;

R ₃₀ represents a -C ₂ -C ₄ alkylene- group; and

M is unsubstituted -C ₁ -C ₃ -alkylene-.

Preferably, in the drug conjugate of the invention (AA) _w is represented by formula (II), wherein:

R21 in each case is selected from the group consisting of hydrogen, methyl, isopropyl, sec-butyl, benzyl, indolylmethyl, -(CH2) ₃ NHCONH2, -(CH2) ₄ NH2, -(CH2) ₃ NHC(=NH)NH2 and -(CH ₂ ) ₄ NHC(=NH)NH ₂ ;

or where w is 0 or 2, and when w is 2, then (AA) _w is represented by formula (III):

where the wavy lines represent the point of covalent attachment to (X) _b if present or to the drug moiety (wavy line on the left) and to (T) _g if present or to the linker (wavy line on the right);

R ₂₂ is selected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl; and

R23 is selected from methyl, -(CH2)4NH2, -(CH2)3NHCONH2 and -(CH2)3NHC(=NH)NH2.

- 4 048715

Preferably, in the drug conjugate of the invention, X is a stretching group selected from the group consisting of the following groups

-COO-(C2-C4alkylene)NH-;

-COO-CH ₂ -phenylene-NH-;

- COO-(C ₂ -C ₄ αalkylene)NH-COO-CH ₂ -(phenylene)-NH-;

- COCH2NH-COCH2-NH-;

- COO-(C ₂ -C ₄ alkylene)S-;

-COO-(C ₂ -C ₄ alkylene)NHCO(C ₁ -C ₃ alkylene)S-; and b is 0 or 1;

or X is an extension group selected from the group consisting of the following groups:

- COO-CH ₂ -phenylene-NH-;

- COO(CH ₂ ) ₃ NHCOOCH ₂ -phenylene-NH-;

- COO(CH2)3NH-;

-COO(CH2)3-S-;

-COO(CH ₂ ) ₃ NHCO(CH ₂ ) ₂ S-; and b is 0 or 1.

More preferably, in the above drug conjugate, b is 1.

Preferably, in the drug conjugate of the invention, T is a stretching group selected from the group consisting of -CO-(C ₁ -C ₄ alkylene)NH-, -CO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-, -COO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-, where j is an integer from 1 to 10; and g is 0 or 1;

or T is an extending group selected from the group consisting of -CO(C ₁ -C ₄ -alkylene)NH-, -CO-(C ₁ -C ₄ -alkylene)-[O-(C ₂ -C ₄ -alkylene)]j-NH-, -COO-(C ₁ -C ₄ -alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-; where j is an integer from 1 to 5; and g is 0 or 1.

Preferably, in the drug conjugate of the invention, D is a drug moiety of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

R1 represents CN or OH;

R2 is C(=O)R _a , where R _a is unsubstituted C ₁ -C ₆ αalkyl;

R3 represents hydrogen or a -OR _b group, where R _b represents an unsubstituted C _1-6 alkyl group,

R4 is selected from hydrogen, -CH2OH and -CH2NH2; and

Y represents -NH- or -O-;

or D is a moiety of a drug of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R ₁ is CN or OH; R ₂ is acetyl; R3 is hydrogen or methoxy; R4 is hydrogen or -CH2OH; and

Y represents -NH- or -O-;

or D is a moiety of a drug of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R1 is CN; R2 is acetyl; R3 is methoxy; _R4 is hydrogen; and

Y represents -NH- or -O-.

More preferably, in the above drug conjugate, R3 is methoxy. Also more preferably, in the above drug conjugate, Y is -NH-.

Preferably, in the drug conjugate of the invention, D is selected from:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the wavy line denotes the point of covalent attachment to (X) _b if present, or to (AA) _w if present, or to (T) _g if present, or to (L).

Preferably, in the drug conjugate of the invention, the Ab moiety is Trastuzumab or an antigen-binding fragment or immunologically active portion thereof.

Preferably, the drug conjugate of the invention is a conjugate

- 5 048715 antibody-drug selected from the group consisting of:

where n is from 2 to 6 and each is an α-HER2 antibody such as Trastuzumab, or an antigen-binding fragment or immunologically active portion thereof.

- 6 048715

More preferably, in the above drug conjugate, each

is Trastuzumab or an antigen-binding fragment or immunologically active portion thereof. Also more preferably, in the above drug conjugate n is 3, 4 or 5.

Preferably, the drug conjugate of the invention is an antibody-drug conjugate in isolated or purified form.

As will be explained in more detail and illustrated by examples below, the drug conjugates of formula [D-(X)b-(AA) _w -(T) _g -(L)-] _n -Ab of the present invention represent a breakthrough in solving the problems described above relating to the need for additional drug conjugates in addition to those based on the three major families of cytotoxic drugs currently used as payloads that exhibit excellent antitumor activity.

In a further aspect the present invention provides a compound of formula D-(X)b-(AA)w-(T)g-L1 or formula D-(X)b-(AA)w-(T)g-H, wherein:

L1 is a linker group:

where the wavy line denotes the point of covalent attachment to (T) _g if present, or to (AA) _w if present, or to (X) _b if present, or to D;

R19 is unsubstituted -C ₁ -C ₁₂ -αalkylene-;

b is equal to the integer 0 or 1;

g is equal to the integer 0 or 1;

w is an integer in the range 0 to 6;

where, for compounds of the formula D-(X-) _b (AA) _w -(T) _g -H, b+w+g^;

each of D, X, T and AA is as defined in the first aspect of the invention.

Preferably, the compound of formula D-(X) _b -(AA) _w -(T) _g -L ₁ or formula D-(X) _b -(AA) _w -(T) _g -H according to the invention is

Preferably, in the compound of formula D-(X) _b -(AA) _w -(T)gH according to the invention b+w+g^0.

Preferably, in the drug conjugate of the invention, b+g+w is not equal to 0; or

- 7 048715 b+w is not equal to 0; or when w is not equal to 0, then b is 1; or when w is 0, then b is 1.

In a further aspect of the present invention there is provided the use of a drug conjugate of the invention as a drug. Preferably, the drug is a drug for the treatment of a cancer selected from HER2 positive lung cancer, including HER2 positive NSCLC, HER2 positive gastric cancer, HER2 positive colorectal cancer, HER2 positive breast cancer, HER2 positive pancreatic carcinoma, HER2 positive endometrial cancer, HER2 positive bladder cancer, HER2 positive cervical cancer, HER2 positive esophageal cancer, HER2 positive gallbladder cancer, HER2 positive uterine cancer, HER2 positive salivary duct cancer and HER2 positive ovarian cancer. More preferably, the drug is a drug for the treatment of a cancer selected from HER2 positive cancer selected from HER2 positive breast cancer, HER2 positive ovarian cancer and HER2 positive gastric cancer. More preferably, the drug is a drug for the treatment of HER2 positive breast cancer.

In a further aspect, the present invention provides a pharmaceutical composition comprising a drug conjugate of the invention and a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a method for preventing or treating cancer, comprising administering to a patient in need thereof an effective amount of a drug conjugate of the invention, wherein the cancer is selected from HER2 positive lung cancer, including HER2 positive NSCLC, HER2 positive gastric cancer, HER2 positive colorectal cancer, HER2 positive breast cancer, HER2 positive pancreatic carcinoma, HER2 positive endometrial cancer, HER2 positive bladder cancer, HER2 positive cervical cancer, HER2 positive esophageal cancer, HER2 positive gallbladder cancer, HER2 positive uterine cancer, HER2 positive salivary duct cancer and HER2 positive ovarian cancer. Preferably, the cancer is a HER2 positive cancer selected from HER2 positive breast cancer, HER2 positive ovarian cancer and HER2 positive gastric cancer. More preferably, the cancer is HER2 positive breast cancer.

Preferably, in the drug conjugate of the invention, n is in the range of 1-12, 1-8, 3-8, 3-6, 3-5 or is equal to 1, 2, 3, 4, 5 or 6, more preferably 3, 4 or 5, or 4.

In a further aspect of the present invention there is provided a method for producing a drug conjugate of the invention comprising conjugating an Ab moiety comprising at least one antigen-binding site and a drug D, wherein Ab and D are as defined herein;

where obtaining an antibody-drug conjugate of formula (G) or (G'):

includes the following stages:

(1) interaction of the drug D-H of formula (Ш)-Н:

- 8 048715

where the substituents in the definitions (IH)-H are as defined herein, with a compound of formula (D') or (E):

(2) partially reducing one or more disulfide bonds in the antibody to be conjugated to produce a reduced antibody Ab-SH having free thiol bonds to produce a compound of formula (F) or (F'), respectively:

groups:

AЬ recovery / \ --------► Ab-SH

S---S disulfide bonds' ^and (3) reacting the partially reduced antibody Ab-SH having free thiol groups with the compound of formula (F) or (F') obtained in step (1) to obtain the desired antibody-drug conjugate of formula (G) or (G'), respectively:

- 9 048715

(θ’)·

Detailed Description of Preferred Embodiments

In a further aspect of the present invention, a drug moiety D is disclosed for use in an antibody-drug conjugate. In a further aspect of the present invention, a drug moiety D is disclosed for use as a payload in an antibody-drug conjugate. In a further aspect of the present invention, the use of a drug moiety D as described herein in the manufacture of an antibody-drug conjugate is disclosed.

In a further aspect the present invention discloses medicinal products of formula (IA) ^R 3

+-0 (ΙΑ), where

Y represents -NH- or -O-;

R1 represents -OH or -CN;

R2 represents the group -C(=O)R _a ;

R3 represents hydrogen or an ORb group;

R ₄ is selected from hydrogen, -CH2OH and -CH2NH2;

R _a is unsubstituted C ₁ -C ₁₂ -αalkyl;

Rb is unsubstituted C ₁ -C ₁₂ -alkyl;

provided that when R4 is hydrogen, then Y is -O-.

In a further aspect of the present invention, there is disclosed the use of a drug conjugate of the present invention in the manufacture of a medicament for the treatment of cancer.

In another aspect of the present invention, a kit is disclosed comprising a therapeutically effective amount of a drug conjugate of the present invention and a pharmaceutically acceptable carrier.

The following applies to all aspects of the present invention:

In the compounds of the present invention, the alkyl groups may be branched or unbranched and preferably have from 1 to about 12 carbon atoms. Another preferred class of alkyl groups has from 1 to about 6 carbon atoms. Even more preferred are alkyl groups having 1, 2, 3 or 4 carbon atoms. Methyl, ethyl, n-propyl, isopropyl and butyl, including n-butyl, isobutyl, sec-butyl and tert-butyl, are particularly preferred alkyl groups in the compounds of the present invention.

In the compounds of the present invention, the alkylene groups in the definition of R19 are straight or branched alkylene groups having from 1 to 12 carbon atoms, and alkylene

- 10 048715 The alkylene groups in the definitions of M, X, T and R ₃₀ are straight or branched alkylene groups having from 1 to 6 carbon atoms. Preferably, the alkylene groups in the definition of R19 are straight or branched alkylene groups having from 1 to 8 carbon atoms, more preferably straight or branched alkylene groups having from 1 to 6 carbon atoms. For M, straight or branched alkylene groups having from 1 to 3 carbon atoms are preferred. In the definition of X, the alkylene groups in the definition of X are preferably straight or branched alkylene groups having from 2 to 4 carbon atoms. For T, straight or branched alkylene groups having from 2 to 4 carbon atoms are preferred. In the definition of R ₃₀ , preferred are straight or branched alkylene groups having from 2 to 4 carbon atoms, most preferably a straight alkylene group having 3 carbon atoms. For the avoidance of doubt, the term alkylene is used to designate alkanediyl groups.

In one embodiment, D may be a moiety of a drug of formula (IH) or a pharmaceutically acceptable salt thereof:

where the wavy line denotes the point of covalent attachment to (X) _b if present, or to (AA) _w if present, or to (T)g if present, or to (L);

Y is selected from -NH- and -O-;

R1 represents -OH or -CN;

R2 represents the group -C(=O)R _a ;

R ₃ represents hydrogen or the -OR _b group;

R ₄ is selected from hydrogen, -CH2OH and -CH2NH2;

R _a is unsubstituted C ₁ -C ₁₂ -alkyl;

R _b is unsubstituted C ₁ -C ₁₂ alkyl.

Preferred compounds of general formula (IH) and drugs of general formula (IA) or b, or their pharmaceutically acceptable salts, are those having the general formula a or stereoisomer:

It should be noted that when the compounds have the general formula a or b, R4 may not be hydrogen.

Preferred drug groupings and drugs include groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -NH-;

and R1; _R2 ; _R3 ; R4; R _a and R _b are as defined above.

Preferred drug groupings and drugs include groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

and R ₁ ; R ₂ ; R ₃ ; R ₄ ; R _a and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

R1 is -OH;

- 11 048715 and Y; R2; _R3 ; R4; R _a and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Ri represents -CN;

and Y; R2; _R3 ; _R4 ; R _a and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

R2 is a group -C(=O)R _a , where R _a is unsubstituted C _1-6 alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl; and Y; R ₁ ; R ₃ ; R4 and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

R ₃ represents hydrogen or the -OR _b group;

where _{R b} is unsubstituted C _{1_6} alkyl.

Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ is hydrogen and methoxy, with methoxy being the most preferred R ₃ group; and Y; R ₁ ; R2; R4 and Ra are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

R4 is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, R4 may be hydrogen or -CH2OH. Most preferably, R4 is hydrogen; and Y; R ₁ ; R2; R ₃ ; R _a and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -NH-;

R ₁ is -OH;

and R2; R3; R4; Ra and Rb are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -NH-;

R2 is -C(=O)R _a ; where R _a is unsubstituted C ₁ -C ₆ alkyl. Particularly preferred Ra is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R ₂ is acetyl; and R ₁ ; R ₃ ; R4 and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -NH-;

R3 is hydrogen or an -ORb group; wherein Rb is unsubstituted _C1-6 -alkyl. Particularly preferred _Rb is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R3 is hydrogen and methoxy, with methoxy being the most preferred _R3 group, and _R1 ; R2; R4 and _Ra are as defined above.

Additional preferred drug groupings include groupings of the general formula (IH), where:

Y represents -NH-;

R ₄ is selected from hydrogen, -CH ₂ OH and -CH ₂ NH ₂ . More preferably, R ₄ may be hydrogen or -CH2OH. Most preferably, R ₄ is hydrogen; and R ₁ ; R 2 ; R ₃ ; R _a and R _b are as defined above.

Additional preferred medicinal products of general formula (IA), where:

Y represents -NH-;

R4 is selected from -CH2OH and -CH2NH2. Most preferred R4 is -CH2OH;

and R1; R2; R3; Ra and Rb are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -NH-; R ₁ represents -OH;

- 12 048715

R2 is a group -C(=O)R _a , where R _a is unsubstituted C _1-6 -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl; and R ₃ ; R ₄ and Rb are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -NH-; R1 represents -OH;

R ₃ is hydrogen or an -ORb group; wherein Rb is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ is hydrogen and methoxy, with methoxy being the most preferred R ₃ group; and R2; R4 and _Ra are as defined above.

Additional preferred drug groupings include groupings of the general formula (IH), where:

Y represents -NH-; R1 represents -OH;

R ₄ is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, R ₄ may be hydrogen or -CH2OH. Most preferably, R ₄ is hydrogen; and R2; R ₃ ; R _a and R _b are as defined above.

Additional preferred medicinal products of general formula (IA), where:

Y represents -NH-; R1 represents -OH;

R ₄ is selected from -CH2OH and -CH2NH2. Most preferred R ₄ is -CH2OH;

and R2; _R3 ; R _a and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -NH-;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R ₃ is hydrogen or a group -OR _b ; wherein R _b is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ is hydrogen and methoxy, with methoxy being the most preferred R3 group;

and R1 and R4 are as defined above.

Additional preferred drug groupings include groupings of the general formula (IH), where:

Y represents -NH-;

R2 is a -C(=O)Ra group, where _Ra is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred Ra is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R4 is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, R4 may be hydrogen or -CH2OH. Most preferably, R4 is hydrogen;

and R1; _R3 and _Rb are as defined above.

Additional preferred medicinal products include medicinal products of the general formula (IA), where:

Y represents -NH-;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R ₄ is selected from -CH2OH and -CH2NH2. Most preferred R ₄ is -CH2OH;

and R1; _R3 and _Rb are as defined above.

Additional preferred drug groupings include groupings of the general formula (IH), where:

Y represents -NH-;

R ₃ is hydrogen or an -OR _b group; wherein R _b is unsubstituted Ci-6-alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl

- 13 048715 la, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ is hydrogen and methoxy, with methoxy being the most preferred R ₃ group;

R ₄ is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, R ₄ may be hydrogen or -CH2OH. Most preferably, R ₄ is hydrogen;

and R1; R2 and R _a are as defined above.

Additional medicinal products include medicinal products of the general formula (IA), where:

Y represents -NH-;

R ₃ is hydrogen or a -ORb group; wherein R _b is unsubstituted Ci-6-alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R3 is hydrogen and methoxy, with methoxy being the most preferred R3 group;

R4 is selected from -CH2OH and -CH2NH2. Most preferred R4 is -CH2OH;

and R1; R2 and R _a are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -NH-; R _i represents -OH;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl.

The most preferred R2 is acetyl;

R ₃ is hydrogen or a group -OR _b ; wherein R _b is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ is hydrogen and methoxy, with methoxy being the most preferred R ₃ group;

and R ₄ is as defined above.

Additional preferred drug groupings include groupings of the general formula (IH), where:

Y represents -NH-; R _i represents -OH;

R2 is a -C(=O)R _a group, where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred Ra is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R4 is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, R4 may be hydrogen or -CH2OH. Most preferably, R4 is hydrogen;

and R ₃ and R _b are as defined above.

Additional preferred medicinal products include medicinal products of the general formula (IA), where:

Y represents -NH-; R _i represents -OH;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R4 is selected from -CH2OH and -CH2NH2. Most preferred R4 is -CH2OH;

and R ₃ and R _b are as defined above.

Additional preferred drug groupings include groupings of the general formula (IH), where:

Y represents -NH-;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R3 is hydrogen or a -ORb group; wherein Rb is unsubstituted _{C1.6 -} alkyl. Particularly preferred _Rb is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R3 is hydrogen and methoxy, with methoxy being the most preferred R3 group;

R4 is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, R4 may be hydrogen or _-CH2OH . Most preferably, _R4 is hydrogen;

and R _i is as defined above.

- 14 048715

Additional preferred medicinal products include medicinal products of the general formula (IA), where:

Y represents -NH-;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R ₃ is hydrogen or an -ORb group; wherein R _b is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ are hydrogen and methoxy, with methoxy being the most preferred R ₃ group;

R4 is selected from -CH2OH and -CH2NH2. Most preferred R4 is -CH2OH;

and R1 is as defined above.

Additional preferred drug groupings include groupings of the general formula (IH), where:

Y represents -NH-; R ₁ represents -OH;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R ₃ is hydrogen or an -OR _b group; wherein R _b is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R 3 are hydrogen and methoxy, with methoxy being the most preferred R ₃ group;

R4 is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, _R4 may be hydrogen, _-CH2OH . Most preferably, _R4 is hydrogen.

Additional preferred medicinal products include medicinal products of the general formula (IA), where:

Y represents -NH-;

R1 is -OH;

R2 is a group -C(=O) _Ra , where _Ra is unsubstituted _{C1.6 -} alkyl. Particularly preferred Ra is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R3 is hydrogen or a -ORb group; wherein Rb is unsubstituted Ci-6-alkyl. Particularly preferred _Rb is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert- _butyl . More preferred _R3 are hydrogen and methoxy, with methoxy being the most preferred _R3 group, _R4 is selected from _-CH2OH and _-CH2NH2 . Most preferred _R4 is _-CH2OH .

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-; Ri represents -OH;

and R2; R3; R4; Ra and Rb are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R ₂ is acetyl;

and R1; _R3 ; R4 and _Rb are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

R3 is hydrogen or an -ORb group; wherein Rb is unsubstituted _{C1.6 -} alkyl. Particularly preferred _Rb is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred _R3 are hydrogen and methoxy, with methoxy being the most preferred _R3 group;

- 15 048715 and R1; R2; R4 and R _a are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

R4 is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, _R4 may be hydrogen or -CH2OH. Most preferably, _R4 is hydrogen;

and R1; R2; R ₃ ; R _a and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-; R1 represents -OH;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

and R ₃ ; R4 and R _b are as defined above.

Additional preferred drug groupings and drugs include groupings of general formula (IH) and drugs of formula (IA), where:

Y represents -O-; R1 represents -OH;

R ₃ is hydrogen or a group -OR _b ; wherein R _b is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R3 are hydrogen and methoxy, with methoxy being the most preferred R3 group;

and R2; R4 and Ra are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-; R _i represents -OH;

R ₄ is selected from hydrogen, -CH ₂ OH and -CH ₂ NH ₂ . More preferably, R ₄ may be hydrogen or -CH ₂ OH. Most preferably, R ₄ is hydrogen;

and R ₂ ; R ₃ ; R _a and R _b are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

R2 is a group -C(=O) _Ra , where _Ra is unsubstituted _{C1.6 -} alkyl. Particularly preferred Ra is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R ₃ is hydrogen or a group -OR _b ; wherein R _b is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ are hydrogen and methoxy, with methoxy being the most preferred R ₃ group;

and Ri and R4 are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred Ra is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R ₂ is acetyl;

R ₄ is selected from hydrogen, -CH ₂ OH and -CH ₂ NH ₂ . More preferably, R ₄ may be hydrogen or -CH ₂ OH. Most preferably, R ₄ is hydrogen;

and R1; _R3 and _Rb are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

R3 is hydrogen or an -ORb group; wherein Rb is unsubstituted _{C1.6 -} alkyl. Particularly preferred _Rb is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R3 are hydrogen and methoxy, with methoxy being the most preferred _R3 group;

R ₄ is selected from hydrogen, -CH ₂ OH and -CH ₂ NH ₂ . More preferably, R ₄ can be

- 16 048715 hydrogen or -CH2OH. Most preferred R ₄ is hydrogen;

and R1; R2 and R _a are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-; R1 represents -OH;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R ₃ is hydrogen or an -ORb group; wherein R _b is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ are hydrogen and methoxy, with methoxy being the most preferred R ₃ group;

and R ₄ is as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-; R1 represents -OH;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R4 is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, R4 may be hydrogen or -CH2OH. Most preferably, R4 is hydrogen;

and R ₃ and Rb are as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R ₃ is hydrogen or a -ORb group; wherein Rb is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ are hydrogen and methoxy, with methoxy being the most preferred R3 group;

Id is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, R ₄ may be hydrogen or -CH2OH. Most preferably, R ₄ is hydrogen;

and R1 is as defined above.

Additional preferred drug groupings and drugs include drug groupings of general formula (IH) and drugs of general formula (IA), where:

Y represents -O-;

R1 is -OH;

R2 is a group -C(=O)R _a , where R _a is unsubstituted _C1.6 -alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl;

R ₃ is hydrogen or an -OR _b group; wherein R _b is unsubstituted C ₁ . ₆ -alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ are hydrogen and methoxy, with methoxy being the most preferred R ₃ group;

R4 is selected from hydrogen, -CH2OH and -CH2NH2. More preferably, _R4 may be hydrogen or -CH2OH. Most preferably, R4 is hydrogen.

The following preferred substituents (when permitted by possible substituent groups) apply to drug groups of formula (IH) and to drug groups of formula (IA).

Particularly preferred R1 is -OH.

Particularly preferred R2 is a group -C(=O)R _a , where R _a is unsubstituted C ₁ . ₆ alkyl. Particularly preferred R _a is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. Most preferred R2 is acetyl.

Particularly preferred R ₃ is hydrogen or an OR _b group, wherein Rb is unsubstituted O^-alkyl. Particularly preferred R _b is selected from unsubstituted methyl, unsubstituted ethyl, unsubstituted n-propyl, unsubstituted isopropyl, unsubstituted n-butyl, unsubstituted isobutyl, unsubstituted sec-butyl and unsubstituted tert-butyl. More preferred R ₃ are hydrogen and methoxy, with methoxy being the most preferred R ₃ group.

Particularly preferred R4 is selected from hydrogen, -CH2OH and -CH2NH2. Even more preferred R4 is hydrogen or -CH2OH, and most preferred R4 is hydrogen.

Particularly preferred drug groupings and drugs according to the present invention include: Drug groupings of formula (IH), wherein

Y represents -NH-; and

R4 is selected from hydrogen, -CH2OH and -CH2NH2. Drugs of formula (IA), where

Y represents -NH-; and

R ₄ is selected from -CH ₂ OH and -CH ₂ NH ₂ .

Groups of medicinal products of formula (IH) or medicinal products of formula (IA), where

Y represents -O-;

R ₄ is selected from hydrogen, -CH ₂ OH and -CH ₂ NH ₂ .

Groups of medicinal products of formula (IH) or medicinal products of formula (IA), where

R2 represents the group -C(=O)R _a ;

R3 represents hydrogen or an -ORb group;

R4 is selected from hydrogen, -CH2OH and -CH2NH2; R _a is unsubstituted C^alkyl; and R _b is unsubstituted C ₁ . ₆ -alkyl.

More preferred drug moieties according to the present invention include drug moieties of formula (IH), wherein

Y represents -NH-;

R2 represents a -C(=O)R _a group; _R3 represents hydrogen or a -OR _b group; R4 represents hydrogen or -CH2OH; R _a represents unsubstituted _{C1_6} alkyl; and R _b represents unsubstituted C^alkyl. Drugs of formula (IA), where

Y represents -NH-;

R2 represents a -C(=O)R _a group; _R3 represents hydrogen or a -OR _b group; R4 represents -CH2OH; R _a represents unsubstituted C ₁ .6 alkyl; and R _b represents unsubstituted C ₁ .6 alkyl. Moieties of formula (IH) or drugs of formula (IA), wherein

Y represents -O-;

R ₂ is a -C(=O)R _a group; R ₃ is hydrogen or a -OR _b group; R ₄ is hydrogen or -CH2OH; R _a is unsubstituted C ₁ . ₆ alkyl; and R _b is unsubstituted C ₁ . ₆ -alkyl.

Moieties of formula (IH) or medicinal products of formula (IA), wherein R ₂ is a -C(=O)R _a group; R ₃ is hydrogen or a -OR _b group; R ₄ is hydrogen or -CH2OH; R _a is unsubstituted C ₁ . ₆ alkyl; and R _b is unsubstituted C _{1 .} 6-alkyl.

Particularly preferred drug groupings according to the present invention include:

Groupings of formula (IH), where

Y represents -NH-;

R ₂ is a -C(=O)R _a group; R ₃ is hydrogen or methoxy; R ₄ is hydrogen or -CH2OH; and R _a is unsubstituted C ₁ . ₆ -alkyl. Drugs of formula (IA), wherein

Y represents -NH-;

R ₂ is a -C(=O)R _a group; R ₃ is hydrogen or methoxy; R ₄ is -CH2OH; and R _a is unsubstituted C ₁ . ₆ -alkyl. Moieties of formula (IH) or drugs of formula (IA), wherein

Y represents -O-;

R2 represents the group -C(=O)Ra;

R3 represents hydrogen or methoxy;

R4 is hydrogen or -CH2OH; and

R _a is unsubstituted C ₁ . ₆ alkyl. Moieties of formula (IH) or drugs of formula (IA), where

R ₂ represents the group -C(=O)R _a ;

- 18 048715

R3 represents hydrogen or methoxy;

R ₄ is hydrogen or -CH2OH; and

Ra is selected from methyl, ethyl, n-propyl, isopropyl and butyl, including n-butyl, sec-butyl, isobutyl and tert-butyl.

Even more preferred drug groupings according to the present invention include: Groupings of formula (IH), wherein

Y is -NH-; R2 is acetyl; R3 is methoxy; and

R4 represents hydrogen. Medicines of formula (IA), where

Y is -NH-; R2 is acetyl; R3 is methoxy; and R4 is -CH2OH.

Groupings of formula (IH) or drugs of formula (IA), where

Y represents -O-;

R2 is acetyl;

R ₃ is methoxy; and

R4 represents hydrogen. Moieties of formula (IH) or drugs of formula (IA), where

R2 is acetyl;

R ₃ is methoxy; and

R4 represents hydrogen. Moieties of formula (IH) or drugs of formula (IA), where

R1 is -OH;

R2 is acetyl;

R ₃ is methoxy; and

R4 is hydrogen.

The grouping according to the present invention has the formula:

or a pharmaceutically acceptable salt thereof; wherein D is covalently attached through an amino group to (X)b if present, or to (AA) _w if present, or to (T)g if present, or to (L).

Particularly preferred are groupings of the formula:

MeO MeO

or a pharmaceutically acceptable salt thereof; wherein D is covalently attached through an amino group to (X) _b if present, or to (AA) _w if present, or to (T) _g if present, or to (L).

- 19 048715

Medicine formula:

In further preferred embodiments, the preferences described above for the different substituents are combined. The present invention also relates to such combinations of preferred substitutions (if this is permissible for possible groups of substituents) in the drug groups of formula (IH) and in the drugs of formula (IA) according to the present invention.

For the avoidance of doubt, the compounds described above may be a drug moiety D and covalently attached through an amino group to (X) _b if present, or (AA) _w if present, or (T) g if present, or (L). Thus, upon conjugation, the covalent bond replaces a proton in the amino group of the compound.

Preferred drug conjugates of the present invention are provided below. The preferred definitions of (X) _b , (AA) _w , (T) g and (L) as set forth below apply to all of the compounds with the drug moiety D described above. Preferred drug conjugates of the present invention include:

a drug conjugate of the formula [D-(X) _b -(AA) _w -(T)g-(L)-]n-Ab according to the present invention, wherein L is a linker group selected from the group consisting of:

where the wavy lines represent the point of covalent attachment to Ab (the wavy line on the right) and to (T) _g if present, or to (AA) _w if present, or to (X) _b if present,

- 20 048715 or to D (wavy line on the left);

R ₁₉ is unsubstituted -C ₁ -C ₁₂ -alkylene-;

R ₃₀ represents a -C _1-6 -alkylene- group;

M is an unsubstituted -C _1-6 -alkylene-. drug conjugate of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention, selected from formulas (IV), (V) and (VI):

Where

X and T are extenders as defined herein; each AA is independently an amino acid unit as defined herein;

w is an integer in the range 0 to 6;

b is equal to the integer 0 or 1;

g is equal to the integer 0 or 1;

where b+g+w is not necessarily equal to 0;

D represents the drug group;

Ab is an anti-HER2 antibody or its antigen-binding fragment;

n is the ratio of the group [D-(X) _b -(AA) _w -(T) _g -(L)-], where L is defined in formula (IV), (V) or (VI), to the moiety containing at least one antigen-binding site, and is in the range from 1 to 20;

R ₁₉ is unsubstituted -C ₁ -C ₈ -alkylene-;

R ₃₀ represents a -C ₂ -C ₄ alkylene- group; and

M is unsubstituted -C ₁ -C ₃ -alkylene-.

Preferably, when in the definition of a drug conjugate of the formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab

L is as defined in the preferred definitions for the said group above, and (AA) _w is represented by formula (II):

^L R ₂ i ^j w (II) where the wavy lines represent the point of covalent attachment to (X) _b if present, or to the drug moiety (wavy line on the left) and to (T) _g if present, or to the linker (wavy line on the right); and

R ₂₁ in each case is selected from the group consisting of hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, -CH2CH2COOH, (CH2^NHC(=NH)NH2, -(CH^N^ -(CH2)4NHC(=NH)NH2,

-(CH2)4NH2, (CH2)3NHCONH2, -(CH2)4NHCONH2, phenyl, cyclohexyl,

and w is an integer in the range 0 to 6;

a drug conjugate of the formula [D-(X) _b -(AA) _w -(T)g-(L)-] _n -Ab according to the first aspect of the present invention, wherein L is as defined in the preferred definitions for said group above, and (AA) _w is represented by formula (II), wherein:

R21 in each case is selected from the group consisting of hydrogen, methyl, isopropyl, sec-butyl, benzyl, indolylmethyl, -(CH2)3NHCONH2, -(CH2)4NH2, -(CH2)3NHC(=NH)NH2 and -(CH2)4NHC(=NH)NH2; and w is an integer in the range from 0 to 6;

a drug conjugate of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the first aspect

- 21 048715 of the present invention, wherein L is as defined in the preferred definitions for said group above, wherein w is 0 or 2 and when w is 2 then (AA) _w is represented by formula (III), wherein:

(III) the wavy lines represent the point of covalent attachment to (X) _b if present, or to the drug moiety (wavy line on the left) and to (T)g if present, or to the linker (wavy line on the right);

R ₂₂ is selected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl; and R ₂₃ is selected from methyl, -(CH2)4NH2, -(CH2)3NHCONH2 and -(CH2)3NHC(=NH)NH2.

In embodiments of the present invention, b+g+w is not equal to 0. In further embodiments, b+w is not equal to 0. In still further embodiments, when w is not equal to 0, then b is equal to 1. Furthermore, it is preferred that in the definition of a drug conjugate of formula

[D-(X)b-(AA)w-(T)g-(L)-] _n -Ab,

L and (AA) _w are as defined in the preferred definitions for the groups indicated above, and X is an extending group selected from:

-COO-(C1-C6alkylene)-NH-;

-COO-CH ₂ -(unsubstituted phenylene)-NH-;

- COO-(C ₁ -C ₆ -alkylene)NH-COO-CH ₂ -(unsubstituted phenylene)-NH-;

- COCH2NH-COCH2-NH-;

- COCH2NH-;

-COO-(C ₁ -C ₆ -alkylene)S-;

-COO-(C ₁ -C ₆ alkylene)NHCO(C ₁ -C ₆ alkylene)S-; and b is 0 or 1, preferably 1.

a drug conjugate of the formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention, wherein L and (AA) _w are as defined in the preferred definitions for the groups indicated above, and X is a stretcher group selected from the group consisting of:

-COO-(C2-C4-alkylene)NH-;

-COO-CH ₂ -(unsubstituted phenylene)-NH-;

- COO-(C ₂ -C ₄ alkylene)NH-COO-CH ₂ -(unsubstituted phenylene)-NH-;

- COCH2NH-COCH2-NH-;

- COO-(C ₂ -C ₄ -alkylene)S-;

-COO-( _C2 - _C4 -alkylene)NHCO( _C1 - _C3 -alkylene)S-; and b is 0 or 1, preferably 1.

a drug conjugate of the formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention, wherein L and (AA) _w are as defined in the preferred definitions for the groups indicated above, and X is a stretcher group selected from the group consisting of:

-COO-CH ₂ -(unsubstituted phenylene)-NH-;

- COO(CH ₂ ) ₃ NHCOOCH ₂ -(unsubstituted phenylene)-NH-;

- COO(CH2)3NH-;

- COO(CH2)3-S-;

- COO(CH2)3NHCO(CH2)2S-; and b is 0 or 1, preferably 1.

a drug conjugate of the formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention, wherein L, (AA) _w and (X) _b are as defined in the preferred definitions for the groups indicated above, and T is a stretcher group selected from the group consisting of:

- CO-(C1-C6alkylene)-NH-;

- CO-(C ₁ -C ₆ -alkylene)-[O-(C ₂ -C ₆ -alkylene)] _j -NH-;

- COO-(C ₁ -C ₆ -alkylene)-[O-(C ₂ -C ₆ -alkylene)] _j -NH-;

where j is an integer from 1 to 25, and g is 0 or 1.

a drug conjugate of the formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention, wherein L, (AA) _w and (X) _b are as defined in the preferred definitions for the groups indicated above, and T is a stretcher group selected from the group consisting of:

- CO-(Cl-C ₄ -alkylene)NH-;

- CO-(Cl-C ₄ -alkylene)-[O-(C ₂ -C ₄ -alkylene)] _j -NH-;

- COO-(C1-C ₄ -alkylene)-[O-(C ₂ -C ₄ -alkylene)]|-KH-;

where j is an integer between 1 and 10; and g is 0 or 1.

- 22 048715 a drug conjugate of the formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention, wherein L, (AA) _w and (X) _b are as defined in the preferred definitions for the groups indicated above, and T is a stretcher group selected from the group consisting of:

-CO-(C ₁ -C ₄ alkylene)NH-;

-CO-(c ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ αalkylene)]j-NH-;

-COO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-;

where j is an integer from 1 to 5; and g is 0 or 1.

A preferred drug conjugate of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention is one wherein L, (AA)w, (X)b and (T)g are as defined above and wherein D is a compound of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 is CN or OH, and more preferably R1 is CN.

Another preferred drug conjugate of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention is one wherein L, (AA) _w , (X) _b and (T) _g are as defined above and wherein D is a compound of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2 is C(=O)R _a , wherein R _a is unsubstituted C ₁ . ₆ alkyl, and more preferably R2 is acetyl.

Another preferred drug conjugate of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention is one wherein L, (AA)w, (X)b and (T)g are as defined above and wherein D is a compound of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ₃ is hydrogen or a group -OR _b , wherein R _b is an unsubstituted C ₁ .6 alkyl group, and more preferably R ₃ is hydrogen or methoxy. Most preferably R ₃ is methoxy.

Another preferred drug conjugate of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention is one wherein L, (AA) _w , (X) _b and (T) _g are as defined above and wherein D is a compound of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R4 is selected from hydrogen, -CH2OH and -CH2NH2, and more preferably R4 is hydrogen or -CH2OH. Most preferably R ₄ is hydrogen.

Another preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA)w, (X)b and (T)g are as defined above and wherein D is a compound of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y is -NH- or -O-. A further preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA)w, (X)b and (T)g are as defined above and wherein D is a compound of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R1 is -CN or -OH; R2 is -C(=O)R _a , where R _a is unsubstituted C ₁ -C ₆ αalkyl;

R ₃ represents hydrogen or an -OR _b group, where R _b represents an unsubstituted C _{1-6 alkyl} group;

R4 is hydrogen, -CH2OH, or -CH2NH2; and

Y represents -NH- or -O.

A further preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA)w, (X)b and (T)g are as defined above and wherein D is a compound of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

R ₁ represents -CN or -OH;

R ₂ is acetyl;

R ₃ represents hydrogen or methoxy, more preferably methoxy;

R ₄ is hydrogen or -CH ₂ OH; and

Y represents -NH- or -O-.

A further preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA)w, (X)b and (T)g are as defined above and wherein D is a compound of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R1 is -CN; R2 is acetyl; R3 is methoxy; R4 is hydrogen; and

Y represents -NH- or -O-.

A further preferred drug conjugate of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention is one wherein L, (AA) _w , (X) _b and (T) _g are as defined above and wherein D is selected from:

- 23 048715

or a pharmaceutically acceptable salt or stereoisomer thereof; wherein the wavy lines represent the point of covalent attachment to (X) _b if present, or to (AA) _w if present, or to (T) _g if present, or to (L).

A further preferred drug conjugate of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention is one wherein L, (AA) _w , (X) _b , (T) _g and D are as defined above and wherein the Ab moiety comprising at least one antigen-binding site is an anti-HER2 antibody or an antigen-binding fragment thereof.

A further preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA)w, (X) _b , (T) _g and D are as defined above and the Ab moiety comprising at least one antigen-binding site is an antibody, a single-domain antibody or an antigen-binding fragment thereof.

A further preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA)w, (X) _b , (T) _g and D are as defined above and the Ab moiety comprising at least one antigen-binding site is a monoclonal antibody, a polyclonal antibody or a bispecific antibody, and wherein the antibody or antigen-binding fragment thereof is of any species, preferably human, mouse or rabbit origin.

A further preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA)w, (X) _b , (T) _g and D are as defined above and the moiety A _b comprising at least one antigen-binding site is an antibody or antigen-binding fragment thereof selected from the group consisting of a human antibody, an antigen-binding fragment of a human antibody, a humanized antibody, an antigen-binding fragment of a humanized antibody, a chimeric antibody, an antigen-binding fragment of a chimeric antibody, a glycosylated antibody and a glycosylated antigen-binding fragment. A further preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA) _w , (X) _b , (T) _g and D are as defined above and the Ab moiety comprising at least one antigen-binding site is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab') ₂ fragment and a Fv fragment. A further preferred drug conjugate of the formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention is one wherein L, (AA) _w , (X) _b , (T) _g and D are as defined above and the Ab moiety comprising at least one antigen-binding site is an antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof is a monoclonal antibody that immunospecifically binds to cancer cell antigens, viral antigens, antigens of cells that produce autoimmune antibodies associated with an autoimmune disease, microbial antigens and, preferably, a monoclonal antibody that immunospecifically binds to cancer cell antigens. A further preferred drug conjugate of formula [D-(X)b-(AA)w-(T)g-(L)-]n-Ab according to the present invention is one wherein L, (AA) _w , (X) _b , (T) _g and D are as defined herein and the Ab moiety comprising at least one antigen-binding site is Trastuzumab or an antigen-binding fragment or immunologically active portion thereof.

Particularly preferred drug conjugates of formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab according to the present invention include the following: (a) a drug conjugate according to the present invention, wherein:

L is selected from the group consisting of:

- 24 048715

Where:

the wavy lines represent the point of covalent attachment to Ab (wavy line on the right) and to (T) _g if present, or to (AA) _w if present, or to (X) _b if present, or to (D) (wavy line on the left);

R19 is unsubstituted -C ₁ -C ₁₂ -alkylene-;

R ₃₀ represents a -C ₁ -C ₆ -alkylene- group;

M is unsubstituted -C ₁ -C ₆ -alkylene-;

(AA) _w is represented by formula (II):

R21 ^j w (II) where the wavy lines represent the point of covalent attachment to (X) _b if present, or to the drug moiety (wavy line on the left) and to (T) _g if present, or to the linker (wavy line on the right);

R21 in each case is selected from the group consisting of hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, -CH2CH2COOH, (ONΛΊΚ)NII)Nll·, -(CH)^ -(CH2)4NHC(=NH)NH2, -(CH ₂ ) ₄ NH ₂ , (CH2) ₃ NHCONH2, -(CH ₂ ) ₄ NHCONH ₂ , phenyl, cyclohexyl,

w is an integer in the range 0 to 6;

where X is an extension group selected from the groups:

-COO-(C ₁ -C ₆ -alkylene)NH-, -COO-CH ₂ -(unsubstituted phenylene) -NH-, -COO-(C ₁ -C ₆ -alkylene)NHСOO-CH ₂ -(unsubstituted phenylene) -NH-, -COCH2NH-COCH2-NH-, -COCH ₂ -NH-,-COO-(C ₁ -C ₆ -alkylene)S-, -COO-(C1-C6-alkylene)NHCO(C1-C6-alkylene)S-;

b is 0 or 1, preferably 1;

where T is an extending group selected from the groups -CO-(C ₁ -C ₆ alkylene)-NH-, -CO-(C ₁ -C ₆ alkylene)-[O-(C ₂ -C ₆ alkylene)]j-NH- and -COO-(C ₁ -C ₆ alkylene)-[O-(C ₂ -C ₆ alkylene)]j-NH-, where j is an integer from 1 to 25;

g is 0 or 1;

D is a grouping of a drug of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, where:

R2 is C(=O) _Ra , where Ra is unsubstituted _C1 - _C6 -alkyl;

R3 represents hydrogen or an -OR _b group, where R _b represents an unsubstituted C _1-6 alkyl group;

R ₄ is selected from hydrogen, -CH ₂ OH and -CH ₂ NH ₂ ;

the Ab moiety comprising at least one antigen-binding site is an antibody or an antigen-binding fragment thereof and is selected from the group consisting of a human antibody, an antigen-binding fragment of a human antibody, a humanized antibody, an antigen-binding fragment of a humanized antibody, a chimeric antibody, an antigen-binding fragment of a chimeric antibody, a glycosylated antibody and a glycosylated antigen-binding fragment; and n is the ratio of the [D-(X) _b -(AA) _w -(T) _g -(L)-] moiety to the Ab moiety comprising at least one antigen-binding site and is in the range from 1 to 12.

(b) a drug conjugate according to the present invention selected from formulae (IV), (V) and (VI):

- 25 048715

Where

R ₁₉ is unsubstituted -C1-C ₈ -alkylene-;

R30 represents a -C2- _C4 alkylene- group;

M is unsubstituted -C ₁ -C ₃ -alkylene-;

(AA) _w is represented by formula (II)

where the wavy lines represent the point of covalent attachment to (X)b if present, or to the drug moiety (wavy line on the left) and to (T)g if present, or to the linker (wavy line on the right);

R21 in each case is selected from the group consisting of hydrogen, methyl, isopropyl, sec-butyl, benzyl, indolylmethyl, -(C^^NHCON^, -(CH ₂ )4NH ₂ , -(CH2) ₃ NHC(=NH)NH2 and -(CH2)4NHC(=NH)NH2;

w is an integer from 0 to 6;

X is an extension group selected from the group consisting of:

-COO-(C ₂ -C ₄ -alkylene)NH-, -COO-CH ₂ -(unsubstituted phenylene)-NH-, -COO-(C ₂ -C ₄ -alkylene)NHСOO-CH ₂ -(unsubstituted phenylene) -NH-, -COCH2NH-COCH2-NH-, -COO-(C ₂ -C ₄ -alkylene)S- and -COO-(C ₂ -C ₄ -alkylene)NHCO(C ₁ -C ₃ -alkylene)S-;

b is 0 or 1, preferably 1;

where T is an extending group selected from the groups -CO-(C ₁ -C ₄ alkylene)-NH-, -CO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH- and -COO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-, where j is an integer from 1 to 10;

g is 0 or 1;

D is a grouping of a drug of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof, where:

R ₂ is acetyl;

R3 is hydrogen or methoxy, preferably R3 is methoxy;

R ₄ is hydrogen or -CH ₂ OH, preferably R ₄ is hydrogen;

the Ab moiety comprising at least one antigen-binding site is an antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody that immunospecifically binds to cancer cell antigens, viral antigens, antigens of cells that produce autoimmune antibodies associated with an autoimmune disease, microbial antigens, and preferably, a monoclonal antibody that immunospecifically binds to cancer cell antigens; and n is the ratio of the group [D-(X) _b -(AA) _w -(T) _g -(L)-], where L is as defined in formulae (IV), (V) or (VI), to the Ab moiety comprising at least one antigen-binding site, and is in the range of 3 to 8.

(c) A drug conjugate according to the present invention of formula (IV):

- 26 048715

Where

R ₁₉ is unsubstituted C ₂ -C ₅ alkylene-;

w is 0 or 2, and when w is 2, then (AA) _w is represented by formula (III):

where R ₂₂ is isopropyl, R ₂₃ is selected from methyl and -(CH ₂ ) ₃ NHCONH ₂ , and the wavy lines represent the point of covalent attachment to (X) _b (wavy line on the left) and to (T)g when present or to the linker (wavy line on the right); and

X represents the group -COOCH ₂ -(unsubstituted phenylene)-NH;

b is equal to 1;

T represents an extending group of the formula -CO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)] ₄ -NH-;

g is 0 or 1;

or formula (VI)

where R ₁₉ is unsubstituted -C2-C ₅ alkylene-;

R ₃₀ is -C ₃ alkylene-;

w is 0 or 2, and when w is 2, then (AA) _w is represented by formula (III):

where R ₂₂ is isopropyl, R ₂₃ is selected from methyl and -(CH ₂ ) ₃ NHCONH ₂ , and the wavy lines represent the point of covalent attachment to (X) _b (wavy line on the left) and to (T) _g when present or to the linker (wavy line on the right); and

X represents the group -COOCH ₂ -(unsubstituted phenylene)-NH;

b is equal to 1;

T represents an extending group of the formula -CO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)] ₄ -NH-;

g is 0 or 1;

D represents a drug grouping selected from

- 27 048715 or a pharmaceutically acceptable salt or stereoisomer thereof; where the wavy line denotes the point of covalent attachment to (X)b;

the Ab moiety comprising at least one antigen-binding site is Trastuzumab or an antigen-binding fragment or immunologically active portion thereof; and n is the ratio of the group [D-(X) _b -(AA) _w -(T) _g -(L)-], where L is as defined in formula (IV), to the Ab moiety comprising at least one antigen-binding site, and is in the range of 3 to 5, and is preferably 4.

(d) an antibody-drug conjugate according to the present invention selected from the group consisting of:

- 28 048715

where n is from 2 to 6, more preferably 3, 4 or 5, and each

is an anti-HER2 antibody such as trastuzumab, or an antigen-binding fragment or immunologically active portion thereof.

More preferably, the antibody-drug conjugate is selected from the group consisting of:

such as trastuzumab, or an antigen-binding fragment or immunologically active portion thereof,

- 29 048715

where n is from 2 to 6, more preferably 3, 4 or 5, and is selected from an anti-HER2 antibody such as trastuzumab, or an antigen-binding fragment or immunologically active portion thereof,

where n is from 2 to 6, more preferably 3, 4 or 5, and is selected from an anti-HER2 antibody such as trastuzumab, or an antigen-binding fragment or immunologically active portion thereof,

wherein n is from 2 to 6, more preferably 3, 4 or 5, and is selected from an anti-HER2 antibody such as trastuzumab, or an antigen-binding fragment or immunologically active portion thereof.

It is particularly preferred that the antibody-drug conjugates of the present invention be in isolated or purified form.

Preferred compounds of formula D-(X) _b -(AA) _w -(T) _g -L1 or formula D-(X) _b -(AA) _w -(T) _g -H according to the present invention include:

a compound of formula D-(X)b-(AA) _w -(T) _g -L1 or of formula D-(X) _b -(AA) _w -(T) _g -H, wherein each of D, X, AA, T, L1, b, g and w is as defined herein;

- 30 048715 but additionally, where, if the compound is a compound of the formula D-(X)b-(AA)w-(T)g-H, then b+w+g^0.

a compound of formula D-(X)b-(AA)w-(T)g-L1 or formula D-(X)b-(AA)w-(T)g-H according to the present invention, wherein

L1 is a linker of the formula

where the wavy line denotes the point of covalent attachment to (T) _g if present, or to (AA)w if present, or to (X)b if present, or to D;

R19 is unsubstituted -C ₁ -C ₁₂ -alkylene-; and each of D, X, AA, T, b, g and w is as defined herein; but wherein if the compound is a compound of formula D-(X) _b -(AA) _w -(T) _g -H, then b+w+g^0.

a compound of formula D-(X)b-(AA)w-(T)g-L1 or formula D-(X)b-(AA)w-(T)gH according to the present invention, wherein: L ₁ is a linker of formula

where the wavy line denotes the point of covalent attachment to (T)g if present, or to (AA) _w if present, or to (X) _b if present, or to D;

R1g is unsubstituted -C1 _- C8-alkylene-;

(AA) _w is represented by formula (II):

where the wavy lines represent the point of covalent attachment to (X) _b , if present, or to D (wavy line on the left) and to (T)g if present, or to L1 or to the hydrogen atom (wavy line on the right);

where R21 in each case is selected from the group consisting of hydrogen, methyl, isopropyl, sec _- butyl, benzyl, indolylmethyl, -( _CH2 ) _3NhCoNH2 , -(CH2) _4NH2 , -( _CH2 ) _3NHC (=NH)NH2 and -( _CH2 ) _4NhC (=NH)NH2, _and w is an integer from 0 to 6;

X is an extension group selected from the group consisting of:

-COO-(C ₂ -C ₄ alkylene)NH-, -COO-CH ₂ -(unsubstituted phenylene)-NH, -COO-(C ₂ -C ₄ alkylene)NH-COOCH ₂ -(unsubstituted phenylene)-NH- , -COCH2NH-COCH2-NH-, -COO-(C ₂ -C ₄ alkylene)S- and -COO-(C ₂ -C ₄ alkylene)-NHCO(C ₁ -C ₃ -alkylene)S-;

T is an extending group selected from the groups -CO-(C ₁ -C ₄ -alkylene)-NH-; -CO(C ₁ -C ₄ -alkylene)-[O-(C ₂ -C ₄ -alkylene)]j-NH- and -COO-(C ₁ -C ₄ -alkylene)-[O-(C ₂ -C ₄ -alkylene)]j-NH-, where j is an integer from 1 to 10;

b is 0 or 1;

g is 0 or 1;

where, if the compound is a compound of formula D-(X) _b -(AA) _w -(T) _g -H, then b+w+g^0; and

D is a grouping of a drug of formula (IH), or a pharmaceutically acceptable salt or stereoisomer thereof, where:

- 31 048715

(IH), where the wavy line in (IH) denotes the point of covalent attachment to (X) _b if present, or to (AA) _w if present, or to (T) _g if present, or to L1;

R1 represents -OH or -CN;

R2 represents a group -C(=O)R _a , where R _a represents unsubstituted C _1-6 -alkyl;

R3 represents hydrogen or an -OR _b group, where R _b represents an unsubstituted C1-6 alkyl group;

R is selected from hydrogen, -CH2OH and -CH2NH2; and Y is -NH- or -O-.

a compound of formula D-(X) _b -(AA) _w -(T) _g -L1 or formula D-(X) _b -(AA) _w -(T) _g -H according to the present invention, wherein: L1 is a group of formula:

where the wavy line denotes the point of covalent attachment to (T) _g if present, or to (AA) _w if present, or to (X) _b if present, or to D;

R ₁₉ is unsubstituted -C _1-6 -alkylene-;

w is 0 or 2, and when w is 2, then (AA) _w is represented by formula (III):

(III) where the wavy lines represent the point of covalent attachment to X (wavy line on the left) and to (T) _g if present, or to L ₁ or to the hydrogen atom (wavy line on the right);

R ₂₂ is selected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl;

R23 is selected from methyl, -(C^^N^, -(CH^NHCONH and -(CH2ENHC(=NH)NH2;

X represents an extending group selected from:

-COO-CH ₂ -(unsubstituted phenylene)-NH-, -COO(CH ₂ ) ₃ NHCOO-CH ₂ -(unsubstituted phenylene)-NH, -COO-(CH2) ₃ NH-, -COO(CH ₂ ) ₃ -S- and -COO-(CH ₂ ) ₃ NHCO-(CH ₂ ) ₂ S-;

where T is an extending group selected from the groups -CO-(C _{1 -C4} alkylene)-NH-, -CO(C ₁ -C4 alkylene)-[O-(C ₂ -C4 alkylene)]j-NH- and -COO-(C ₁ -C4 alkylene)-[O-(C ₂ -C4 alkylene)]j-NH-, where j is an integer from 1 to 5;

b is 0 or 1;

g is 0 or 1;

where, if the compound is a compound of formula D-(X) _b -(AA) _w -(T) _g -H, then b+w+g^0; and

D is a grouping of a medicinal product of formula (IH) or a pharmaceutically acceptable salt or stereoisomer thereof:

- 32 048715

where the wavy line in (IH) denotes the point of covalent attachment;

R1 represents -CN or -OH;

R2 is acetyl;

R ₃ represents hydrogen or methoxy, preferably methoxy;

R4 represents hydrogen or -CH2OH, preferably hydrogen;

Y represents -NH- or -O-.

a compound of formula D-(X) _b -(AA) _w -(T) _g -L1 or formula D-(X) _b -(AA) _w -(T) _g -H according to the present invention, wherein

L1 is a linker of the formula

where the wavy line denotes the point of covalent attachment to (T)g if present, or to (AA) _w if present, or to (X)ь if present, or to D;

R19 is unsubstituted -C ₂ -C ₆ -alkylene-;

w is 0 or 2, and when w is 2, then (AA) _w is represented by formula (III):

R ₂₂ is isopropyl, R2 is selected from methyl and -(CH ₂ ) ₃ NHCONH ₂ , where the wavy lines represent the point of covalent attachment to X (wavy line on the left) and to (T) _g when present, or to L ₁ or to a hydrogen atom (wavy line on the right);

X represents an extending group selected from the groups -COO- _CH2- (unsubstituted phenylene)-NH-, -COO( _CH2 ) _3NHCOO - _CH2- (unαsubstituted phenylene)-NH, -COO-( _CH2 ) ₃ )NH-, -COO( _CH2 ) ₃ -S- and -COO-(CH2)3NHCO-(CH2)2S-;

where T is an extending group selected from the groups -CO-(C ₁ -C ₄ -alkylene)-NH-, -CO-(C ₁ -C ₄ -alkylene)-[O-(C ₂ -C ₄ -alkylene)]j-NH- and -COO-(C ₁ -C ₄ -alkylene)-[O-(C ₂ -C ₄ -alkylene)]j-NH-, where j is an integer from 1 to 5;

b is 0 or 1;

g is 0 or 1;

where, if the compound is a compound of formula D-(X) _b -(AA) _w -(T) _g -H, then bw-g+0; and D is a drug moiety selected from:

- 33 048715 or a pharmaceutically acceptable salt or stereoisomer thereof; where the wavy line denotes the point of covalent attachment.

a compound of formula D-(X)b-(AA) _w -(T) _g -L ₁ or formula D-(X) _b -(AA) _w -(T) _g -H according to the present invention, wherein: L ₁ is a group of formula:

where the wavy line denotes the point of covalent attachment to (T)g if present, or to (AA) _w if present, or to (X)b if present, or to D;

R ₁₉ is unsubstituted -C ₂ -C ₅ -alkylene-;

w is 0 or 2, and when w is 2, then (AA) _w is represented by formula (III):

where R ₂₂ is isopropyl, R23 is selected from methyl and -(CH ₂ )3NHCONH ₂ , where the wavy lines represent the point of covalent attachment to X (wavy line on the left) and to (T) _g if present, or to L1 or to a hydrogen atom (wavy line on the right);

X represents the group -COO-CH ₂ -(unsubstituted phenylene)-NH-;

T represents the group -CO-(CH ₂ ) ₂ -[O-(CH ₂ ) ₂ ] ₄ -NH-;

b is 0 or 1;

g is 0 or 1;

where, if the compound is a compound of formula D-(X) _b -(AA) _w -(T) _g -H, then b+w+g#0; and D is a drug moiety selected from:

or a pharmaceutically acceptable salt or stereoisomer thereof; where the wavy line denotes the point of covalent attachment.

a compound of formula D-X-(AA)w-(T)g-L1 selected from:

- 34 048715

MeO

The term pharmaceutically acceptable salts or stereoisomers in the drug conjugates of the present invention refers to any pharmaceutically acceptable salt or stereoisomeric form or any other compound that, when administered to a patient, is capable of providing a compound as described herein, directly or indirectly. However, it should be appreciated that salts that are not pharmaceutically acceptable are also within the scope of the invention, as they may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs and derivatives can be accomplished by methods known in the art.

For example, pharmaceutically acceptable salts of the compounds provided herein are synthesized from the parent compound, which contains a basic or acidic group, by conventional chemical methods. Typically, such salts are prepared, for example, by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. Non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of alkali metal addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and alkaline organic salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylethanolamine, triethanolamine salts, and salts of basic amino acids.

The drug conjugate of the present invention may be in crystalline form, either as free compounds or as solvates (e.g. hydrates), and both forms are intended to be within the scope of the present invention. Methods of solvation are generally known in the art.

Any compound which is a prodrug of a drug conjugate of the present invention is within the scope and spirit of the invention. The term prodrug is used in its broadest sense and encompasses those derivatives which are converted in vivo into the compounds of the present invention. Such derivatives may be apparent to those skilled in the art and include, for example, compounds wherein the free hydroxy group is converted into an ester derivative. Many suitable prodrugs are well known to those skilled in the art and can be found, for example, in Burger Medicinal Chemistry and Drug Discovery ^6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Applications of Prodrugs (H. Bundgaard ed., 1985,

- 35 048715

Harwood Academic Publishers), the contents of which are incorporated herein by reference.

With respect to the compounds of the present invention, pharmacologically acceptable esters are not particularly limited and can be selected by a person skilled in the art. In the case of said esters, it is preferable that such esters can be cleaved by a biological process such as hydrolysis in vivo. The group constituting said esters (a group shown as R when the esters thereof are expressed as -COOR) may be, for example, a _C1 - _C4 -alkoxy- _C1 - _C4- alkyl group such as methoxyethyl, 1-ethoxyethyl, 1-methyl-1methoxyethyl, 1-(isopropoxy)ethyl, 2-methoxyethyl, 2-ethoxyethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl, isopropoxymethyl, butoxymethyl or tert-butoxymethyl; A C1- _C4 alkoxylated C1- _C4 alkoxy-C1- _C4 alkyl group, such as 2-methoxyethoxymethyl; A C6- _C1o -aryloxy- _C1 - _C4 alkyl group, such as phenoxymethyl; A halogenated C1- _C4 alkoxy-C1- _C4 alkyl group, such as 2,2,2-trichloroethoxymethyl or bis(2-chloroethoxy)methyl; A C1- _C4 alkoxycarbonyl-C1- _C4 alkyl group, such as methoxycarbonylmethyl; A cyano-C1- _C4 alkyl group, such as cyanomethyl or 2-cyanoethyl; A C1- _C4 alkylthiomethyl group, such as phenylthiomethyl or naphthylthiomethyl; a C1- _C4 alkylsulfonylC1 _{-C4 lower} alkyl group which may be optionally substituted by halogen atom(s), such as 2-methanesulfonylethyl or 2-trifluoromethanesulfonylethyl; a _C6 - _C10 arylsulfonylC1- _{C4 alkyl} group, such as 2-benzenesulfonylethyl or 2-toluenesulfonylethyl; A C ₁ -C ₇ α-liphatic acyloxyC ₁ -C ₄ alkyl group such as formyloxymethyl, acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl, valeryloxymethyl, isovaleryloxymethyl, hexanoyloxymethyl, 1-formyloxyethyl, 1-acetoxyethyl, 1-propionyloxyethyl, 1-butyloxyethyl, 1-pivaloyloxyethyl, 1-valeryloxyethyl, 1-isovaleryloxyethyl, 1-hexanoyloxyethyl, 2-formyloxyethyl, 2-acetoxyethyl, 2-propionyloxyethyl, 2-butyryloxyethyl, 2-pivaloyloxyethyl, 2-valeryloxyethyl, 2-isovaleryloxyethyl, 2-hexanoyloxyethyl, 1-formyloxypropyl, 1-acetoxypropyl, 1-propionyloxypropyl, 1-butyryloxypropyl, 1-pivaloyloxypropyl, 1-valeryloxypropyl, 1-isovaleryloxypropyl, 1-hexanoyloxypropyl, 1-acetoxybutyl, 1-propionyloxybutyl, 1-butyryloxybutyl, 1-pivaloyloxybutyl, 1-acetoxypentyl, 1-propionyloxypentyl, 1-butyryloxypentyl, 1-pivaloyloxypentyl or 1-pivaloyloxyhexyl; a _C5 - _C6 -cycloalkylcarbonyloxy- _C1 - _C4 -alkyl group such as cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl, 1-cyclopentylcarbonyloxyethyl, 1-cyclohexylcarbonyloxyethyl, 1-cyclopentylcarbonyloxypropyl, 1-cyclohexylcarbonyloxypropyl, 1-cyclopentylcarbonyloxybutyl or 1-cyclohexylcarbonyloxybutyl; a _C6 - _C10 -arylcarbonyloxy-C1- _C4 -alkyl group such as benzoyloxymethyl; C ₁ -C ₆ -alkoxycarbonyloxy-C ₁ -C ₄ -alkyl group such as methoxycarbonyloxymethyl, 1-(methoxycarbonyloxy)ethyl, 1-(methoxycarbonyloxy)propyl, 1-(methoxycarbonyloxy)butyl, 1-(methoxycarbonyloxy)pentyl, 1-(methoxycarbonyloxy)hexyl, ethoxycarbonyloxymethyl, 1-(ethoxycarbonyloxy)ethyl, 1-(ethoxycarbonyloxy)propyl, 1-(ethoxycarbonyloxy)butyl, 1-(ethoxycarbonyloxy)pentyl, 1-(ethoxycarbonyloxy)hexyl, propoxycarbonyloxymethyl, 1-(propoxycarbonyloxy)ethyl, 1-(propoxycarbonyloxy)propyl, 1-(propoxycarbonyloxy)butyl, isopropoxycarbonyloxymethyl, 1-(isopropoxycarbonyloxy)ethyl, 1-(isopropoxycarbonyloxy)butyl, butoxycarbonyloxymethyl, 1-(butoxycarbonyloxy)ethyl, 1-(butoxycarbonyloxy)propyl, 1-(butoxycarbonyloxy)butyl, isobutoxycarbonyloxymethyl, 1-(isobutoxycarbonyloxy)ethyl, 1-(isobutoxycarbonyloxy)propyl, 1-(isobutoxycarbonyloxy)butyl, tert-butoxycarbonyloxymethyl, 1-(tert-butoxycarbonyloxy)ethyl, pentyloxycarbonyloxymethyl, 1-(pentyloxycarbonyloxy)ethyl, 1-(pentyloxycarbonyloxy)propyl, hexyloxycarbonyloxymethyl, 1-(hexyloxycarbonyloxy)ethyl or 1-(hexyloxycarbonyloxy)propyl; A _C5 - _C6 cycloalkyloxycarbonyloxy-C1-C4 alkyl group such as cyclopentyloxycarbonyloxymethyl, 1-(cyclopentyloxycarbonyloxy)ethyl, 1-(cyclopentyloxycarbonyloxy)propyl, 1-(cyclopentyloxycarbonyloxy)butyl, cyclohexyloxycarbonyloxymethyl, 1-(cyclohexyloxycarbonyloxy)ethyl, 1-(cyclohexyloxycarbonyloxy)propyl or 1-(cyclohexyloxycarbonyloxy)butyl; [5-(C1- _C4 -alkyl)-2-oxo-1,3-dioxolen-4-yl]methyl group, such as (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl, (5-ethyl-2-oxo-1,3-dioxolen-4-yl)methyl, (5-propyl-2-oxo-1,3-dioxolen-4-yl)methyl, (5-isopropyl-2-oxo-1,3-dioxolen-4-yl)methyl or (5-butyl-2-oxo-1,3-dioxolen-4-yl)methyl; [5-(phenyl which may optionally be substituted with _C1 - _C4 -alkyl, _C1 - _C4 -alkoxy or halogen atoms)-2-oxo-1,3-dioxolen-4-yl]methyl group, such as (5-phenyl-2oxo-1,3-dioxolen-4-yl)methyl, [5-(4-methylphenyl)-2-oxo-1,3-dioxolen-4-yl]methyl, [5-(4methoxyphenyl)-2-oxo-1,3-dioxolen-4-yl]methyl, [5-(4-fluorophenyl)-2-oxo-1,3-dioxolen-4-yl]methyl or [5-(4-chlorophenyl)-2-oxo-1,3-dioxolen-4-yl]methyl; or a phthalidyl group which may be optionally substituted with C1- _C4 alkyl or C1- _C4 alkoxy groups, such as phthalidyl, dimethylphthalidyl or dimethoxyphthalidyl, and is preferably a pivaloyloxymethyl group, a phthalidyl group or a (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl group, and more preferably a (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl group.

Any compound mentioned herein is intended to represent such specific compound as well as certain variants or forms. In particular, the compounds mentioned

- 36 048715 mentioned herein may have asymmetric centers and therefore exist in different enantiomeric forms. All optical isomers and stereoisomers of the compounds mentioned herein and mixtures thereof are considered within the scope of the present invention. Thus, any given compound mentioned herein is intended to be any of the racemate, one or more enantiomeric forms, one or more diastereomeric forms, one or more atropisomeric forms and mixtures thereof. In particular, drug conjugates of the formula [D-(X) _b -(AA) _w -(T)g-(L)] _n -Ab and compounds of the formula DX-(AA) _w -(T) _g -L1 or DX-(AA) _w -(T) _g -H may include enantiomers depending on their asymmetry, or diastereoisomers. Stereoisomerism around a double bond is also possible, so in some cases a molecule could exist as an (E)-isomer or a (Z)-isomer. If a molecule contains multiple double bonds, each double bond will have its own stereoisomerism, which may be the same or different from the stereoisomerism of other double bonds in the molecule. Individual isomers and mixtures of isomers are within the scope of the present invention.

In addition, the compounds mentioned herein may exist as geometric isomers (i.e., cis and trans isomers), as tautomers, or as atropisomers. In particular, the term tautomer refers to one of two or more structural isomers of a compound that exist in equilibrium and are readily convertible from one isomeric form to another. Common tautomeric pairs are amine-imine, amide-imide, keto-enol, lactam-lactim, etc. It is further understood that any compound mentioned herein is intended to represent hydrates, solvates, and polymorphs and mixtures thereof when such forms exist in a medium. In addition, the compounds mentioned herein may exist in isotopically labeled forms. All geometric isomers, tautomers, atropisomers, hydrates, solvates, polymorphs and isotopically labeled forms of the compounds mentioned herein, and mixtures thereof, are considered within the scope of the present invention.

Protected forms of the compounds described herein are contemplated as being within the scope of the present invention. Suitable protecting groups are well known to those skilled in the art. A general review of protecting groups in organic chemistry is provided by Wuts, PGM and Greene TW in Protecting Groups in Organic Synthesis, ^4th Ed. Wiley-Interscience and Kocienski PJ in Protecting Groups, ^3rd Ed. Georg Thieme Verlag. These references provide sections on protecting groups for OH, amino, and SH groups. All of these references are incorporated by reference in their entirety.

Within the scope of the present invention, a protecting group for OH is defined as an O-linked group resulting from the protection of OH by forming a suitable protected OH group. Examples of such protected OH groups include ethers, silyl ethers, esters, sulfonates, sulfenates and sulfinates, carbonates and carbamates. In the case of ethers, the OH protecting group may be selected from methyl, methoxymethyl, methylthiomethyl, (phenyldimethylsilyl)methoxymethyl, benzyloxymethyl, p-methoxybenzyloxymethyl, [(3,4-dimethoxybenzyl)oxy]methyl, p-nitrobenzyloxymethyl, ortho-nitrobenzyloxymethyl, [(R)-1-(2-nitrophenyl)ethoxy]methyl, (4-methoxyphenoxy)methyl, guaiacolmethyl, [(p-phenylphenyl)oxy]methyl, tert-butoxymethyl, 4-pentenyloxymethyl, siloxymethyl, 2-methoxyethoxymethyl, 2-cyanoethoxymethyl, bis(2-chloroethoxy)methyl, 2,2,2-trichloroethoxymethyl, 2(trimethylsilyl)ethoxymethyl, methoxymethyl, O-bis(2-acetoxyethoxy)methyl, tetrahydropyranyl, tetrahydropyranyl fluoride, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl, 4-methoxytetrahydrothiopyranyl, S,S-4-methoxytetrahydrothiopyranyl dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl, 1-(4-chlorophenyl)-4-methoxypiperidin-4-yl, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanebenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-hydroxyethyl, 2-bromoethyl, 1-[2(trimethylsilyl)ethoxy]ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 1-methyl-1-phenoxyethyl, 2,2,2-trichloroethyl, 1,1-dianisyl-2,2,2-trichloroethyl, 1,1,1,3,3,3-hexafluoro-2-phenylisopropyl, 1-(2-cyanoethoxy)ethyl, 2-trimethylsilylethyl, 2(benzylthio)ethyl, 2-(phenylselenyl)ethyl, tert-butyl, cyclohexyl, 1-methyl-1'-cyclopropylmethyl, allyl, prenyl, cinnamyl, 2-phenylyl, propargyl, napa-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, 2,4-dinitrophenyl, 2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, ortho-nitrobenzyl, napa-nitrobenzyl, pentadienylnitrobenzyl, pentadienylnitropiperonyl, halobenzyl, 2,6-dichlorobenzyl, 2,4-dichlorobenzyl, 2,6-difluorobenzyl, p-cyanobenzyl, benzyl fluoride, 4-alkoxybenzyl fluoride, trimethylsilylxylyl, p-phenylbenzyl, 2-phenyl-2-propyl, p-acylaminobenzyl, p-azidobenzyl, 4-azido-3-chlorobenzyl, 2-trifluoromethylbenzyl, 4-trifluoromethylbenzyl, p-(methylsulfinyl)benzyl, napa-siletanylbenzyl, 4-acetoxybenzyl, 4-(2-trimethylsilyl)ethoxymethoxybenzyl, 2-naphthylmethyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, 2-quinolinylmethyl, 6-methoxy-2-(4-methylphenyl)-4-quinolinemethyl, 1-pyrenylmethyl, diphenylmethyl, 4-methoxydiphenylmethyl, 4-phenyldiphenylmethyl, p,p'-dinitrobenzhydryl, 5

- 37 048715 dibenzosuberyl, triphenylmethyl, tris(4-tert-butylphenyl)methyl, α-naphthyldiphenylmethyl, paramethoxyphenyldiphenylmethyl, di(para-methoxyphenyl)phenylmethyl, tri(para-methoxyphenyl)methyl, 4(4'-bromophenacyloxy)phenyldiphenylmethyl, 4,4',4-tris(4,5-dichlorophthalimidophenyl)-methyl, 4,4',4tris(levulinoyloxyphenyl)methyl, 4,4',4-tris(benzoyloxyphenyl)-methyl, 4,4'-dimethoxy- 3-[No (imidazolylmethyl)]trityl, 4,4'-dimethoxy-3-[N(imidazolylethyl)carbamoyl]trityl, bis(4methoxyphenyl)-1'-pyrenylmethyl, 4-(17-tetrabenzo[a,c,g,i]fluorenylmethyl)-4,4-dimethoxytrityl, 9anthryl, 9-(9-phenyl)xanthenyl, 9-phenylthioxanthyl, 9 -(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, 4,5-bis(ethoxycarbonyl)-[1,3]-dioxolan-2-yl, S,S-benzisothiazolyl dioxide. In the case of silyl ethers, the OH protecting group may be selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylhexylsilyl, 2-norbornyldimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, tribenzylsilyl, tripara-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-tert- butylmethylsilyl, bis(tert-butyl)1-pyrenylmethoxysilyl, tris(trimethylsilyl)silyl, (2-hydroxystyryl)dimethylsilyl, (2hydroxystyryl)diisopropylsilyl, tert-butylmethoxyphenylsilyl, tert-butoxydiphenylsilyl, 1,1,3,3-tetraisopropyl-3-[2-(triphenylmethoxy)ethoxy]-disiloxan-1-yl and silyl fluoride. In the case of esters, the OH protecting group together with the oxygen atom of the unprotected OH to which it is attached form an ester which may be selected from formate, benzoyl formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trichloroacetamidate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, para-chlorophenoxyacetate, phenylacetate, diphenylacetate, 3-phenylpropionate, bifluorocarbonyl propanoate, 4-pentenoate, 4-oxopentanoate, 4,4-(ethylenedithio)pentanoate, 5[3-bis(4-methoxyphenyl)hydroxymethylphenoxy]levulinate , pivaloate, 1-adamantoate, crotonate, 4-methoxycrotonate, benzoate, para-phenylbenzoate, 2,4,6-trimethylbenzoate, 4-bromobenzoate, 2,5-difluorobenzoate, para-nitrobenzoate, picolinate, nicotinate, 2-(azidomethyl)benzoate, 4-azidobutyrate, (2-azidomethyl)phenylacetate, 2-{[(tritylthio)oxy ]methyl }benzoate, 2-{[(4methoxytritylthio)oxy]methyl} benzoate, 2-{[methyl(tritylthio)amino]methyl} benzoate, 2-{{[(4methoxytrityl)thio]methylamino}methyl}benzoate, 2-(allyloxy)phenylacetate, 2-(prenyloxymethyl)benzoate, 6-(levulinyloxymethyl)-3 -methoxy-2-nitrobenzoate, 6-(levulinyloxymethyl)-3 -methoxy-4-nitrobenzoate, 4-benzyloxybutyrate, 4-trialkylsilyloxybutyrate, 4-acetoxy-2,2-dimethylbutyrate, 2,2-dimethyl-4pentenoate, 2-iodobenzoate, 4-nitro-4-methylpentanoate, ortho-(dibromomethyl)benzoate, 2formylbenzenesulfonate, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2(chloroacetoxymethyl)benzoate, 2-[(2-chloroacetoxy)ethyl]benzoate, 2-[2-(benzyloxy)ethyl]benzoate, 2-[2-( 4methoxybenzyloxy)ethyl]benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate , ortho-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl-N,N,N',N'-tetramethylphosphorodiamidate and 2-chlorobenzoate. In the case of sulfonates, sulfenates and sulfinates, the OH protecting group together with the oxygen atom of the unprotected OH to which it is attached form a sulfonate, sulfenate or sulfinates which may be selected from sulfate, allyl sulfonate, methanesulfonate, benzyl sulfonate, tosylate, 2-[( 4-nitrophenyl)ethyl]sulfonate, 2-trifluoromethylbenzenesulfonate, 4-monomethoxytrityl sulfenate, alkyl 2,4-dinitrophenyl sulfenate, 2,2,5,5-tetramethylpyrrolidin-3-one-1-sulfinate and dimethylphosphinothioyl. In the case of carbonates, the OH protecting group together with the oxygen atom of the unprotected OH to which it is attached form a carbonate which may be selected from methyl carbonate, methoxymethyl carbonate, 9-fluorenylmethyl carbonate, ethyl carbonate, bromoethyl carbonate, 2-(methylthiomethoxy)ethyl carbonate, 2,2,2 -trichloroethyl carbonate, 1,1-dimethyl-2,2,2-trichloroethyl carbonate, 2-(trimethylsilyl)ethyl carbonate, 2-[dimethyl(2-naphthylmethyl)silyl]ethyl carbonate, 2-(phenylsulfonyl)ethyl carbonate, 2(triphenylphosphonio)ethyl carbonate, cis-[4-[[(methoxytrityl)sulfenyl]oxy]-tetrahydrofuran-3yl]oxycarbonate, isobutyl carbonate, tert-butyl carbonate, vinyl carbonate, allyl carbonate, cinnamyl carbonate, propargyl carbonate, para-chlorophenyl carbonate, para-nitrophenyl carbonate, 4-ethoxy-1naphthyl carbonate, 6-bromo-7-hydroxycoumarin-4-ylmethyl carbonate, benzyl carbonate, orthonitrobenzyl carbonate, para-nitrobenzyl carbonate, para-methoxybenzyl carbonate, 3,4dimethoxybenzylcarbonate, anthraquinon-2-ylmethylcarbonate, 2-dansilethylcarbonate, 2-(4nitrophenyl)ethylcarbonate, 2-(2,4-dinitrophenyl)ethylcarbonate, 2-(2-nitrophenyl)propylcarbonate, 2(3,4-methylenedioxy-6 -nitrophenyl)propyl carbonate, 2-cyano-1-phenylethyl carbonate, 2-(2pyridyl)amino-1-phenylethyl carbonate, 2-[N-methyl-N-(2-pyridyl)]amino-1-phenylethyl carbonate, phenacyl carbonate, 3',5'-dimethoxybenzoyl carbonate, methyl dithiocarbonate and S-benzyl thiocarbonate. And in the case of carbamates, the protecting group for OH together with the oxygen atom unprotected OH to which it is attached forms a carbamate which may be selected from dimethylthiocarbamate, Nphenylcarbamate and N-methyl-N-(ortho-nitrophenyl)carbamate.

Within the scope of the present invention, an amino protecting group is defined as an N-linked group resulting from the protection of an amino group by forming a suitable protected amino group. Examples of protected amino groups include carbamates, ureas, amides, heterocyclic

- 38 048715 systems, N-alkylamines, N-alkenylamines, N-alkynylamines, N-arylamines, imines, enamines, N metal derivatives, N-N derivatives, N-P derivatives, N-Si derivatives and N-S derivatives. In the case of carbamates, the amino protecting group together with the amino group to which it is attached form a carbamate which may be selected from methyl carbamate, ethyl carbamate, 9-fluorenyl methyl carbamate, 2,6-di-tert-butyl-9-fluorenyl methyl carbamate, 2,7-bis(trimethylsilyl)fluorenyl methyl carbamate, 9-(2-sulfo)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluorenyl methyl carbamate, 17-tetrabenzo[a,c,g,i]fluorenyl methyl carbamate, 2-chloro-3-indenyl methyl carbamate, benz[1]inden-3-yl methyl carbamate, 1,1-dioxobenzo[b]thiophene-2-ulmethylcarbamate, 2-methylsulfonyl-3-phenyl-1-prop-2enylcarbamate, 2,7-di-tert-butyl-[9,(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate, 2,2,2trichloroethylcarbamate, 2-trimethylsilylethylcarbamate, (2-phenyl-2-trimethylsilyl)ethylcarbamate, 2phenylethylcarbamate, 2-chloroethylcarbamate, 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2dibromoethylcarbamate, 1,1-dimethyl-2,2,2-trichloroethylcarbamate, 2-(2'-pyridyl)ethylcarbamate, 2-(4'pyridyl)ethylcarbamate, 2,2-bis(4'-nitrophenyl)ethylcarbamate, 2-[(2-nitrophenyl)dithio]-1phenylethylcarbamate, 2-(N,N-dicyclohexylcarboxamido)-ethylcarbamate, tert-butylcarbamate, fluoride BOC-carbamate, 1-adamantylcarbamate, 2-adamantylcarbamate, 1-(1-adamantyl)-1-methylethylcarbamate, 1methyl-1-(4-biphenylyl)ethylcarbamate, 1-(3,5-di-tert-butylphenyl)-1-methylethylcarbamate, triisopropylsilyloxycarbamate, vinylcarbamate, allylcarbamate, prenylcarbamate, 1-isopropylallylcarbamate, cinnamylcarbamate, 4-nitrocinnamylcarbamate, 3-(3'-pyridyl)prop-2-enylcarbamate, hexadienylcarbamate, propargylcarbamate, 1,4-but-2-inylbiscarbamate, 8-quinolylcarbamate, N-hydroxypiperidinylcarbamate, alkyldithiocarbamate, benzylcarbamate, 3,5-di-tert-butylbenzylcarbamate, p-methoxybenzylcarbamate, p-nitrobenzylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate, 4-trifluoromethylbenzylcarbamate, benzyl fluoridecarbamate, 2-naphthylmethylcarbamate, 9-anthrylmethylcarbamate, diphenylmethylcarbamate, 4phenylacetoxybenzylcarbamate, 4-azidobenzylcarbamate, 4-azidomethoxybenzylcarbamate, meta-chloro-paraacyloxybenzylcarbamate, para-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2(trifluoromethyl)-6-chromonylmethylcarbamate, 2-methylthioethylcarbamate, 2-methylsulfonylethylcarbamate, 2(p-toluenesulfonyl)ethylcarbamate, 2-(4-nitrophenylsulfonyl)ethylcarbamate, 2-(2,4dinitrophenylsulfonyl)ethylcarbamate, 2-(4-trifluoromethylphenylsulfonyl)ethylcarbamate, [2-(1,3-dithianyl)]methylcarbamate, 2-phosphonioethylcarbamate, 2-[phenyl(methyl)sulfonio]ethylcarbamate, 1-methyl-1(triphenylphosphonio)ethylcarbamate, 1,1-dimethyl-2-cyanoethylcarbamate, 2-dansylethylcarbamate, 2-(4-nitrophenyl)ethylcarbamate, 4-methylthiophenylcarbamate, 2,4-dimethylthiophenylcarbamate, meta-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, αmethylnitropiperolinecarbamate, ortho-nitrobenzylcarbamate, 3,4-dimethoxy-6-nitrobenzylcarbamate, phenyl(ortho-nitrophenyl)methylcarbamate, 2-nitrophenylethylcarbamate, 6-nitroveratrilcarbamate, 4methoxyphenacylcarbamate, 3',5'-dimethoxybenzoincarbamate, 9-xanthenylmethylcarbamate, N-methyl-N-(opmonitrophenyl)carbamate, tert-amylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1methyl-1-cyclopropylmethylcarbamate, cyclobutylcarbamate, cyclopentylcarbamate, cyclohexylcarbamate, isobutylcarbamate, isobornylcarbamate, cyclopropylmethylcarbamate, para-decyloxybenzylcarbamate, diisopropylmethylcarbamate, 2,2-dimethoxycarbonylvinylcarbamate, ortho-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propylcarbamate, butynylcarbamate, 1,1dimethylpropynylcarbamate, 2-iodoethylcarbamate, 1-methyl-1-(4'-pyridyl)ethylcarbamate, 1-methyl-1-(paraphenylazophenyl)ethylcarbamate, n-{n'-methoxyphenylazo)benzylcarbamate, para-(phenylazo)benzylcarbamate, 2,4,6-trimethylbenzylcarbamate, isonicotinylcarbamate, 4-(trimethylammonium)benzylcarbamate, para-cyanobenzylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, phenylcarbamate, 2,4,6-tri-tert-butylphenylcarbamate, 1-methyl-1-phenylethylcarbamate and S-benzylthiocarbamate. In the case of ureas, the protecting groups for the amino group can be selected from phenothiazinyl-(10)-carbonyl, N'-para-toluenesulfonylaminocarbonyl, N'-phenylaminothiocarbonyl, 4-hydroxyphenylaminocarbonyl, 3-hydroxytryptaminocarbonyl and N'-phenylaminothiocarbonyl. In the case of amides, the amino protecting group together with the amino group to which it is attached form an amide which may be selected from formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, pent-4-enamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanylamide, benzamide, p-phenylbenzamide, ortho-nitrophenylacetamide, 2,2-dimethyl-2(ortho-nitrophenyl)acetamide, ortho-nitrophenoxyacetamide, 3-(ortho-nitrophenyl)propanamide, 2-methyl-2(ortho-nitrophenoxy)propanamide, 3-methyl-3-nitrobutanamide, ortho-nitrocinnamide, ortho-nitrobenzamide, 3-(4-tert-butyl-2,6-dinitrophenyl)-2,2-dimethylpropanamide, ortho(benzoyloxymethyl)benzamide, 2-(acetoxymethyl)benzamide, 2-[(tert-butyldiphenylsiloxy)methyl]benzamide, 3-(3',6'-dioxo-2',4',5'-trimethylcyclohexa-1',4'-diene)-3,3-dimethylpropionamide, ortho-hydroxy-trans-cinnamide, 2-methyl-2-(ortho-phenylazophenoxy)propanamide, 4-chlorobutanamide, acetoacetamide, 3-(para-hydroxyphenyl)propanamide, (N-dithiobenzyloxycarbonylamino)acetamide and N-acetylmethylninamide. In the case of heterocyclic systems, the protecting group for the amino group together with the amino group to which it is attached form a heterocyclic system, which can be selected from 4,5-diphenyl-3-oxazolin-2-one, N

- 39 048715 phthalimide, N-dichlorophthalimide, N-tetrachlorophthalimide, N-4-nitrophthalimide, N-thiodiglycoloyl, N-dithiasuccinimide, N-2,3-diphenylmaleimide, N-2,3-dimethylmaleimide, N-2,5-dimethylpyrrole, N-2,5-bis(triisopropylsiloxy)pyrrole, N-1,1,4,4-tetramethyldisilylasecyclopentane adduct, N1,1,3,3-tetramethyl-1,3-disilaisoindoline, N-diphenylsilyldiethylene, N-5-substituted-1,3-dimethyl1,3,5-triazacyclohexan-2-one, N-5-substituted-1,3-benzyl-1,3,5-triazacyclohexan-2-one, 1-substituted-3,5-dinitro-4-pyridone and 1,3,5-dioxazine. In the case of N-alkyl-, N-alkenyl-, N-alkynyl- or N-arylamines, the protecting group for the amino group can be selected from N-methyl, N-tert-butyl, N-allyl, N-prenyl, N-cinnamyl, N-phenylallyl, N-propargyl, N-methoxymethyl, N-[2(trimethylsilyl)ethoxy]methyl, N-3-acetoxypropyl, N-cyanomethyl, N-2-azanorbornenes, N-benzyl, N-4-methoxybenzyl, N-2,4-dimethoxybenzyl, N-2-hydroxybenzyl, N-ferrocenylmethyl, N-2,4-dinitrophenyl, ortho-methoxyphenyl, para-methoxyphenyl, N-9-phenylfluorenyl, N-fluorenyl, N-2-picolylamine Noxide, N-7-methoxycoumar-4-ylmethyl, N-diphenylmethyl, N-6uc(4methoxyphenyl)methyl, N-5-dibenzosuberyl, N-triphenylmethyl, N-(4-methylphenyl)diphenylmethyl and N(4-methoxyphenyl)diphenylmethyl. In the case of imines, the protecting group for the amino group can be selected from N-1,1-dimethylthiomethylene, N-benzylidene, N-p-methoxybenzylidene, N-diphenylmethylene, N-[2-pyridyl)mesityl]methylene, N-(N',N'-dimethylaminomethylene), N-(N',N'-dibenzylaminomethylene), N-(N-tert-butylaminomethylene), N,N-isopropylidene, N-p-nitrobenzylidene, N-salicylidene, N5-chlorosalicylidene, N-(5-chloro-2-hydroxyphenyl)phenylmethylene, N-cyclohexylidene and N-tert-butylidene. In the case of enamines, the protecting group for the amino group can be selected from N-(5,5-dimethyl-3-oxo-1-cyclohexenyl), N-2,7-dichloro-9-fluorenylmethylene, N-1-(4,4-dimethyl-2,6dioxocyclohexylidene)ethyl, N-(1,3-dimethyl-2,4,6-(1H,3H,5H)-trioxopyrimidin-5-ylidene)methyl, N4,4,4-trifluoro-3-oxo-1-butenyl and N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl). In the case of N-metal derivatives, the protecting group for the amino group can be selected from N-borane, N-diphenylborine ether, N-diethylborine ether, N-9-borabicyclononane, N-difluoroborine ether and 3,5-bis(trifluoromethyl)phenylboronic acid; and also including Nphenyl(pentacarbonylchromium)carbenyl, N-phenyl(pentacarbonyltungsten)carbenyl, Nmethyl(pentacarbonylchromium)carbenyl, N-methyl(pentacarbonyltungsten)carbenyl, N-copper chelate, N-zinc chelate and 18-crown-6 derivative. In the case of N-N derivatives, the amino protecting group together with the amino group to which it is attached form an N-N derivative which can be selected from N-nitroamino, N-nitrosoamino, amine N-oxide, azide, triazene derivative and N-trimethylsilylmethyl-N-benzylhydrazine. In the case of N-P derivatives, the amino protecting group together with the amino group to which it is attached form an N-P derivative which can be selected from diphenylphosphonamide, dimethylthiophosphonamide, diphenylthiophosphonamide, dialkyl phosphoroamidate, dibenzyl phosphoroamidate, diphenyl phosphoroamidate and iminotriphenylphosphorane. In the case of N-Si derivatives, the NH2 protecting group can be selected from tert-butyldiphenylsilyl and triphenylsilyl. In the case of N-S derivatives, the protected amino group can be selected from N-sulfenyl or N-sulfonyl derivatives. N-sulfenyl derivatives can be selected from benzenesulfenamide, 2-nitrobenzenesulfenamide, 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 1-(2,2,2-trifluoro-1,1-diphenyl)ethylsulfenamide and N-3-nitro-2-pyridinesulfenamide. The N-sulfonyl derivatives can be selected from methanesulfonamide, trifluoromethanesulfonamide, tert-butylsulfonamide, benzylsulfonamide, 2(trimethylsilyl)ethanesulfonamide, para-toluenesulfonamide, benzenesulfonamide, orthoanisylsulfonamide, 2-nitrobenzenesulfonamide, 4-nitrobenzenesulfonamide,

2,4-dinitrobenzenesulfonamide, 2-naphthalenesulfonamide, 4-(4',8'dimethoxynaphthylmethyl)benzenesulfonamide, 2-(4-methylphenyl)-6-methoxy-4-methylsulfonamide, 9-anthracenesulfonamide, pyridine-2-sulfonamide, benzothiazole-2-sulfonamide, phenacylsulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide, 2,4,6-trimethoxybenzenesulfonamide, 2,6-dimethyl-4-methoxybenzenesulfonamide, pentamethylbenzenesulfonamide, 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide, 4-methoxybenzenesulfonamide, 2,4,6-trimethylbenzenesulfonamide, 2,6-dimethoxy-4-methylbenzenesulfonamide, 3-methoxy-4-tert-butylbenzenesulfonamide and 2,2,5,7,8-pentamethylchroman-6-sulfonamide.

Within the scope of the present invention, a protecting group for SH is defined as an S-linked group resulting from the protection of an SH group by forming a suitable protected SH group. Examples of such protected SH groups include thioethers, disulfides, silyl thioethers, thioesters, thiocarbonates and thiocarbamates. In the case of thioethers, the protecting group for SH can be selected from S-alkyl, S-benzyl, S-para-methoxybenzyl, S-opmo-hydroxybenzyl, S-para-hydroxybenzyl, S-ortho-acetoxybenzyl, S-napa-acetoxybenzyl, S-para-nitrobenzyl, S-orthotitrobenzyl, S-2,4,6-trimethylbenzyl, S-2,4,6-trimethoxybenzyl, S-4-picolyl, S-2-picolyl-Noxide, S-2-quinolinylmethyl, S-9-anthrylmethyl, S-9-fluorenylmethyl, S-xanthenyl, Sferrocenylmethyl, S-diphenylmethyl, S-bis(4-methoxyphenyl)methyl, S-5-dibenzosuberyl, Striphenylmethyl, 4-methoxytrityl, S-diphenyl-4-pyridylmethyl, S-phenyl, S-2,4-dinitrophenyl, S-2quinolyl, S-tert-butyl, S-1-adamantyl, S-methoxymethyl, S-isobutoxymethyl, S-benzyloxymethyl,

- 40 048715

S-1-ethoxyethyl, S-2-tetrahydropyranyl, S-benzylthiomethyl, S-phenylthiomethyl, S-acetamidomethyl (Acm), S-trimethylacetamidomethyl, S-benzamidomethyl, S-allyloxycarbonylaminomethyl, S-N[2,3,5,6-tetrafluoro-4-(N'-piperidino)phenyl-N-allyloxycarbonylaminomethyl, S-phthalimidomethyl, Sphenylacetamidomethyl, S-acetylmethyl, S-carboxymethyl, S-cyanomethyl, S-(2-nitro-1-phenyl)ethyl, S-2-(2,4-dinitrophenyl)ethyl, S-2-(4'-pyridyl)ethyl, S-2-cyanoethyl, S-2-(trimethylsilyl)ethyl, S-2,2bis(carboethoxy)ethyl, S-(1-meta-nitrophenyl-2-benzoyl)ethyl, S-2-phenylsulfonylethyl, S-1-(4methylphenylsulfonyl)-2-methylprop-2-yl and S-napa-hydroxyphenacyl. In the case of disulfides, the protected SH group can be selected from S-ethyl disulfide, S-tert-butyl disulfide, S-2nitrophenyl disulfide, S-2,4-dinitrophenyl disulfide, S-2-phenylazophenyl disulfide, S-2carboxyphenyl disulfide and S-3-nitro-2-pyridyl disulfide. In the case of silyl thioethers, the protecting group for SH can be selected from the list of groups listed above for the protection of OH with silyl ethers. In the case of thioethers, the protecting group for SH can be selected from Sacetyl, S-benzoyl, S-2-methoxyisobutyryl, S-trifluoroacetyl, S-N-[[parabiphenylyl)isopropyloxy]-carbonyl]-N-methyl-γ-aminothiobutyrate and S-N-(tert-butoxycarbonyl)-Nmethyl-γ-aminothiobutyrate. In the case of thiocarbonate, the protecting group for SH can be selected from S2,2,2-trichloroethoxycarbonyl, S-tert-butoxycarbonyl, S-benzyloxycarbonyl, S-paramethoxybenzyloxycarbonyl and S-fluorenylmethylcarbonyl. In the case of thiocarbamate, the protected SH group can be selected from S-(N-ethylcarbamate) and S-(N-methoxymethylcarbamate).

The mention of these groups should not be interpreted as limiting the scope of the invention, since they have been mentioned as merely illustrative of protecting groups for the OH, amino and SH groups, but other groups having the indicated function may be known to those skilled in the art and it should be understood that they are also encompassed by the present invention.

For a more concise description, some quantitative expressions given in this document are not supplemented with the term approximately.

It is understood that, whether or not the term approximately is explicitly used, each quantity stated herein is intended to refer to an actual given value and is also intended to refer to an approximation to such given value that is reasonably derivable based on ordinary skill in the art, including equivalents and approximations implied by experimental and/or measurement conditions for such given value.

Antibody-drug conjugates (ADCs) are a targeted strategy for delivering a cytotoxic molecule to a cancer cell (see, for example, International Patent Applications WO-A2004/010957, WO-A-2006/060533 and WO-A-2007/024536). Such compounds are commonly referred to as drug-toxin-radionuclide conjugates. Tumor cell killing occurs upon binding of the drug conjugate to the tumor cell and release and/or activation of the cytotoxic activity of the drug moiety. The selectivity provided by drug conjugates minimizes toxicity to normal cells, thereby increasing patient tolerability of the drug. Three examples of antibody-drug conjugates of this type that have received market approval include: Gemtuzumab ozogamicin for acute myelogenous leukemia, Brentuximab vedotin for relapsed and treatment-resistant Hodgkin lymphoma or anaplastic large cell lymphoma, and ado-Trastuzumab emtansine for breast cancer, particularly HER2+.

The effectiveness of cancer chemotherapy drugs typically depends on differences in growth rates, biochemical pathways, and physiological characteristics between cancerous and normal tissues. Consequently, most standard chemotherapeutic agents are relatively nonspecific and exhibit dose-limiting toxicity that contributes to suboptimal therapeutic effects. One approach to selectively targeting malignant cells over healthy tissues is to use specific monoclonal antibodies (mAbs) that recognize tumor-associated antigens expressed on the surface of tumor cells [Meyer, D. L. & Senter, P. D. (2003) Recent advances in antibody drug conjugates for cancer therapy. Annu. Rep. Med. Chem., 38, 229–237; Chad, R. V. (2008) Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc. Chem. Res. 41, 98–107]. More than 30 immunoglobulin G (IgG) and related agents have been approved over the past 25 years, primarily for cancer and inflammatory diseases.

An alternative strategy is to find a chemical conjugate of small antineoplastic molecules with mAbs, used both as carriers (extended half-life) and as targeting agents (selectivity). Considerable effort has been devoted to the use of monoclonal antibodies (mAbs) for targeted drug delivery due to their high selectivity for tumor-associated antigens, favorable pharmacokinetics, and relatively low intrinsic toxicity. mAb-drug conjugates (ADCs) are prepared by covalently linking anticancer drugs to mAbs, typically through a conditionally stable linker system. After binding to cell surface antigens, the mAb used

- 41 048715 for most ADCs, they are actively transported to lysosomes or other intracellular compartments where enzymes, low pH, or reducing agents promote drug release. However, there are a limited number of ADCs currently in development.

Antigens must have high selectivity for tumor cells to limit toxicity and off-target effects. A variety of tumor-associated antigens have been investigated in preclinical models and in clinical trials, including antigens overexpressed on B cells (e.g., CD20, CD22, CD40, CD79), T cells (CD25, CD30), carcinoma cells (HER2, EGFR, EpCAM, EphB2, PSMA), endothelial (endoglin), or stromal cells (fibroblast-activated protein), to name a few [Teicher BA. Antibody-drug conjugate targets. Curr Cancer Drug Targets 9(8):982–1004, 2009]. An important property of ADC targets is their ability to be internalized; this may be an intrinsic feature of the antigen itself or may be induced by binding of the antibody to its antigen. Indeed, ADC internalization is critical to reduce the toxicity associated with extracellular delivery of the drug payload.

In the case of conjugated small molecules, and in contrast to the enormous diversity of putative antigen targets, a limited number of families of cytotoxic drugs used as payloads in ADCs are currently under active investigation in clinical trials: calicheamicin (Pfizer), duocarmycins (Synthon), pyrrolobenzodiazepines (Spirogen), irinotecan (Immunomedics), maytansinoids (DM1 and DM4; ImmunoGen+Genentech/Roche, Sanofi-Aventis, Biogen Idec, Centocor/Johnson & Johnson, Millennium/Takeda), and auristatins (MMAE and MMAF; Seattle Genetics+Genentech/Roche, MedImmune/AstraZeneca, Bayer-Schering, Celldex, Progenics, Genmab). Calicheamicin, duocarmycins, and pyrrolobenzodiazepines are DNA minor groove binding proteins, irinotecan is a topoisomerase I inhibitor, and maytansinoids and auristatins are tubulin depolymerizing agents.

Interestingly, a representative of three of these cytotoxic ADCs has reached late-stage clinical trials: Trastuzumab emtansine (T-DM1), trastuzumab linked to a maytansinoid semisynthetic drug by a stable linker (FDA approval on February 22, 2013 for advanced HER2 positive breast cancer); Inotuzumab ozogamicin (CMC-544), a humanized anti-CD22 mAb (G5/44, IgG4) conjugated to calicheamicin by an acid-labile linker (acetylphenoxy-butane) (B-cell non-Hodgkin lymphoma); Brentuximab vedotin, a humanized anti-CD30 mAb linked to monomethyl auristatin E (MMAE) via a maleimidocaproylvalyl-citrullinyl-p-aminobenzylcarbamate linker (FDA approval August 19, 2011 for anaplastic large cell lymphoma and Hodgkin lymphoma).

Linkers represent a key component of ADC structures. Several classes of second-generation linkers have been investigated, including acid-labile hydrazone linkers (lysosomes) (e.g. gemtuzumab and inotuzumab ozogamicin); disulfide-based linkers (reducing intracellular environment); non-cleavable thioether linkers (catabolic degradation in lysosomes) (e.g. trastuzumab emtansine); peptide linkers (e.g. citrulline-valine) (lysosomal protease-like cathepsin-B) (e.g. brentuximab vedotin): see, e.g., WO-A-2004/010957, WOA-2006/060533 and WO-A-2007/024536. Purification of antibody-drug conjugates by size exclusion chromatography (SEC) has also been described [see, e.g., Liu et al., Proc. Natl. Acad. Sci. USA, 93: 8618–8623 (1996) and Chari et al., Cancer Research, 52: 127–131 (1992)].

Trastuzumab (Herceptin) is a monoclonal antibody that interacts with the HER2/neu receptor. Its main use is to treat certain breast cancers. HER receptors are proteins that are embedded in the cell membrane and transmit molecular signals from outside the cell (molecules called EGF) into the cell and turn genes on and off. HER proteins stimulate cell proliferation. In some cancers, especially some types of breast cancer, HER2 is overexpressed and causes cancer cells to multiply uncontrollably.

The HER2 gene is amplified in 20-30% of early stage breast cancers, causing it to overexpress epidermal growth factor (EGF) receptors in the cell membrane. In some cancers, HER2 can signal without growth factors entering and binding to the receptor, making its action in the cell constitutive; however, trastuzumab is not effective in this case.

The HER2 pathway promotes cell growth and division when it is functioning normally; however, when it is overexpressed, cell growth accelerates beyond normal limits. In some types of cancer, this pathway is used to accelerate cell growth and proliferation and, therefore, tumor formation. In cancer cells, HER2 protein can be expressed at levels up to 100 times higher than in normal cells (2 million versus 20,000 per cell). This overexpression results in strong and persistent proliferation signaling and, therefore, tumor formation. HER2 overexpression also causes checkpoint inhibition, which promotes even greater proliferation.

In the compounds of the present invention, Ab is a moiety comprising at least one antigen-binding site. In an alternative embodiment, Ab may be any suitable agent that is capable of binding to a target cell, preferably an animal cell and more preferably a human cell. Examples of such agents include lymphokines, hormones,

- 42 048715 growth factors and nutrient transfer molecules (eg, transferrin). In another example, the Ab may be an aptamer and may include a nucleic acid or peptide aptamer.

If Ab is a moiety comprising at least one antigen-binding site, this moiety is preferably an antigen-binding peptide or polypeptide. In a preferred embodiment, this moiety is an antibody or an antigen-binding fragment thereof.

The term antibody in the drug conjugates of the present invention refers to any immunoglobulin, preferably a full-length immunoglobulin. Preferably, the term encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies such as bispecific antibodies, and fragments thereof, as long as they exhibit the desired biological activity. The antibodies may be of any species of origin, but are preferably of rodent origin, such as a rat or mouse, a human, or a rabbit. Alternatively, the antibodies, preferably monoclonal antibodies, may be humanized, chimeric, or antibody fragments. The term chimeric antibodies may also include primatized antibodies comprising variable domain antigen-binding sequences of non-human primate (e.g., old world monkey, monkey, etc.) origin and human constant region sequences. Immunoglobulins can also be of any type (eg, IgG, IgE, IgM, IgD, and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of the immunoglobulin molecule.

The term monoclonal antibody refers to a substantially homogeneous population of antibody molecules (i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts) produced by a single clone of a B cell line, often a hybridoma. Importantly, each monoclonal antibody has the same antigen specificity - that is, it is directed against a single determinant of the antigen.

The production of monoclonal antibodies can be carried out by methods known in the art. However, by way of example, monoclonal antibodies can be produced by hybridoma technology (Kohler et al. (1975) Nature 256:495), the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), or the EBV hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, the monoclonal antibody can be produced using recombinant DNA techniques (see, US 4,816,567) or isolated from antibody phage libraries using the methods described in Clackson et al. (1991) Nature, 352:624628; Marks et al. (1991) J. Mol. Biol., 222:581-597.

Polyclonal antibodies are antibodies directed against different determinants (epitopes). This heterogeneous population of antibodies can be obtained from the serum of immunized animals using various methods well known in the art.

The term bispecific antibody refers to an artificial antibody consisting of two different monoclonal antibodies. They may be engineered to bind either to two adjacent epitopes on a single antigen, thereby increasing avidity and specificity, or to bind two different antigens for a variety of applications, but especially for recruiting cytotoxic T cells and natural killer (NK) cells or retargeting toxins, radionuclides or cytotoxic drugs for cancer treatment (Holliger & Hudson, Nature Biotechnology, 2005, 23(9), 1126-1136). A bispecific antibody may have a hybrid immunoglobulin heavy chain with the first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing the second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a simple means of separation (WO 94/04690; Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigues et al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992, Bio/Technology 10:163-167; Carter et al., 1995, J. of Hematotherapy 4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681).

Methods for producing hybrid or bispecific antibodies are known in the art. In one method, bispecific antibodies can be produced by fusing two hybridomas into a single quadroma by chemical cross-linking or genetic fusion of two different Fab or scFv modules (Holliger & Hudson, Nature Biotechnology, 2005, 23(9), 1126-1136).

The term chimeric antibody refers to an antibody in which different portions are derived from different animal species. For example, a chimeric antibody may have a variable region from a mouse and a constant region from a human. In contrast, a humanized antibody is predominantly human, even if it contains non-human portions. Specifically, humanized antibodies are human immunoglobulins (the recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (the donor antibody), such as mouse, rat, rabbit, or non-human primate, that have the desired specificity, affinity, and capacity. In some cases, residues from the framework region (FR) of the human immunoglobulin are replaced by the corresponding

- 43 048715 residues that are not human. Moreover, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. In general, a humanized antibody will include substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to loops of a non-human immunoglobulin and all or substantially all of the FRs belong to human immunoglobulin sequences. A humanized antibody will optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin.

Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, can be produced by recombinant DNA techniques known in the art. Fully human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the usual manner with a selected antigen. Monoclonal antibodies directed against the antigen can be produced using conventional hybridoma technology. The human immunoglobulin transgenes contained in the transgenic mice are rearranged during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced using such a method. For a review of this method for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this method for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., CH2A Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which is incorporated herein by reference in its entirety. Other human antibodies can be obtained commercially, e.g., from Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA).

The term antigen-binding fragment in the drug conjugate of the present invention refers to a portion of a full-length antibody, wherein such antigen-binding antibody fragments retain the antigen-binding function of the corresponding full-length antibody. The antigen-binding fragment may comprise a portion of a variable region of an antibody, wherein said portion comprises at least one, two, preferably three CDRs selected from CDR1, CDR2 and CDR3. The antigen-binding fragment may also comprise a portion of an immunoglobulin light and heavy chain. Examples of antibody fragments include Fab, Fab', F(ab')2, scFv, di-scFv, sdAb and BiTE (bispecific T-cell-engaging activators), Fv fragments including nanobodies, diabodies, diabody-Fc fusions, triabodies and tetrabodies; minibodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementarity determining region) and epitope-binding fragments of any of the above that immunospecifically bind to a target antigen such as cancer cell antigens, viral antigens or microbial antigens, single-chain or single-domain antibody molecules including heavy chain-only antibodies such as the camel VHH and shark V-NAR domains; and multispecific antibodies formed from antibody fragments. By comparison, a full-length antibody, referred to as an antibody, is one that contains the VL and VH domains as well as the complete light and heavy chain constant domains.

An antibody may also have one or more effector functions, which refer to a biological activity attributed to an Fc region (a native sequence Fc region or an amino acid sequence variant Fc region engineered by methods known in the art to alter receptor binding) of the antibody. Examples of effector functions of an antibody include CIq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

An antibody may also be a functionally active fragment (also referred to herein as an immunologically active portion), derivative, or analog of an antibody that immunospecifically binds to a target antigen, such as a cancer cell antigen, a viral antigen, or a microbial antigen, or other tumor cell-associated antibodies. In this regard, functionally active means that the fragment, derivative, or analog is capable of eliciting anti-idiotypic antibodies that recognize the same antigen that recognizes the antibody from which the fragment, derivative, or analog is derived. In particular, in a representative embodiment, the antigenicity of the idiotype of an immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind an antigen, synthetic peptides containing the CDR sequences can be used in antigen binding assays by any binding assay known in the art (e.g., BIAcore assay), see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md; Kabat E et al., 1980, J. of Immunology

- 44 048715

125(3):961–969).

The term antibody may also include a fusion protein of an antibody or a functionally active fragment thereof, for example, in which the antibody is fused via a covalent bond (e.g., a peptide bond) either at the N-terminus or at the C-terminus, to an amino acid sequence of another protein (or a portion thereof, such as at least a portion of a 10, 20, or 50 amino acid protein) that is not an antibody. The antibody or fragment thereof may be covalently linked to another protein at the N-terminus of a constant domain.

In addition, the antibody or antigen-binding fragments of the present invention may include analogs and derivatives of antibodies or antigen-binding fragments thereof that are either modified, for example, by covalent attachment of any type of molecule, so long as such covalent attachment allows the antibody to retain immunospecificity for binding to the antigen. Examples of modifications include glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of the numerous chemical modifications may be carried out by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. In addition, the analog or derivative may contain one or more amino acids that do not exist in nature.

The antibodies or antigen-binding fragments of the present invention may also have modifications (e.g., substitutions, deletions, or additions) in the Fc domain of the antibody. In particular, the modifications may be in the Fc hinge region and result in enhanced binding to the FcRn receptor (WO 97/34631).

In one embodiment, the antibody in the drug conjugate of the present invention may be any antibody or antigen-binding fragment thereof, preferably a monoclonal antibody, that is useful in the treatment of a disease, preferably a cancer, and more preferably a cancer selected from lung cancer including NSCLC, gastric cancer, colorectal cancer, breast cancer, pancreatic carcinoma, endometrial cancer, bladder cancer, cervical cancer, esophageal cancer, gallbladder cancer, uterine cancer, salivary duct cancer, ovarian cancer, renal cancer, leukemia, multiple myeloma and lymphoma, wherein the cancer is preferably a HER2 positive cancer, wherein HER2 positive cancers include HER2 positive lung cancer including HER2 positive NSCLC, HER2 positive gastric cancer, HER2 positive colorectal cancer, HER2 positive breast cancer, HER2 positive pancreatic carcinoma, HER2 positive endometrial cancer, HER2 positive bladder cancer, HER2 positive cancer cervical cancer, HER2 positive esophageal cancer, HER2 positive gallbladder cancer, HER2 positive uterine cancer, HER2 positive salivary duct cancer and HER2 positive ovarian cancer, more preferably HER2 positive breast cancer, HER2 positive ovarian cancer and HER2 positive gastric cancer, most preferably HER2 positive breast cancer.

Antibodies that may be useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovary), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas) such as EGF receptor protein 2 (breast cancer), MAGE-I (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MUClKLH (breast cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinoma), P21 (carcinoma), MPG (melanoma), and Neu oncogenic product (carcinoma). Some specific useful antibodies include, but are not limited to, BR96 mAb (Trail, P. A., et al., Science (1993) 261, 212-215), BR64 (Trail, P. A., et al., Cancer Research (1997) 57, 100-105), anti-CD40 antigen mAbs such as S2C6 mAb (Francisco, J. A., et al., Cancer Res. (2000) 60:3225-3231), anti-CD70 antigen mAbs such as 1F6 mAb, and anti-CD30 antigen mAbs such as AClO (Bowen, M. A., et al. (1993) J. Immunol., 151:5896-5906; Wahl et al., 2002 Cancer Res. 62(13):37363742). Many other internalizing antibodies that bind to tumor-associated antigens can be used and have been described (Franke, A. E., et al., Cancer Biother Radiopharm. (2000) 15:459-476; Murray, J. L. (2000) Semin Oncol, 27:64-70; Breitling, F. and Dubel, S., Recombinant Antibodies, John Wiley and Sons, New York, 1998). Other tumor-associated antigens include, but are not limited to,

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BMPR1B, E16,

STEAP1, STEAP2, 0772P. MPF, Napi3b, Sema5b, PSCA hlg, ETBR, MSG783, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Ra, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79A, CXCR5, HLA -DOB, P2X5, CD72, LY64, FCRH1, IRTA2 and TENB2.

In a further embodiment, the antibody or antigen-binding fragment binds to an epitope that is present on a cell, such as a tumor cell.

Preferably, if the cell is a tumor cell, the tumor cell epitope is not present on non-tumor cells or is present at a lower concentration or in a different spatial configuration than on tumor cells.

The antibody or antigen-binding fragment may bind to an epitope present in the context of one of the following antigens: CA125, CA15-3, CA19-9 L6, Lewis Y, Lewis X, alpha fetoprotein, CA 242, placental alkaline phosphatase, prostate-specific antigen, prostatic acid phosphatase, epidermal growth factor such as EGF receptor protein 2, MAGE-I, MAGE-2, MAGE-3, MAGE-4, anti-transferrin receptor, p97, MUCl-KLH, CEA, gp100, MARTI, PSA, IL-2 receptor, CD20, CD52, CD33,CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, Neu oncogenic product, BMPR1B, E16, STEAP1, STEAP2, 0772Р. MPF, Napi3b, Sema5b, PSCA hlg, ETBR, MSG783, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Ra, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79A, CXCR5, HLA-DOB, P2X5, CD72, LY64, FCRH1, IRTA2, TENB2.

In one embodiment, if the antigen is ErBB2 (also known as ERBB2, CD340, or HER2; these terms may be used interchangeably), the antibody or antigen-binding fragment may bind to one or more of the following epitopes: ARHC L (SEQ ID NO: 1), QNGS (SEQ ID NO: 2), and PPFCVARC PSG (SEQ ID NO: 3). These epitopes correspond to positions 557561, 570-573, and 593-603, respectively, of the human HER2 polypeptide sequence (Accession: NM_004448, Version: NM_004448.3).

An antibody that binds a molecular target or antigen of interest, such as an ErBB2 antigen, is one that is capable of binding the antigen with sufficient affinity such that the antibody is useful for targeting a cell expressing the antigen. If the antibody is one that binds ErBB2, it typically binds ErBB2 preferentially over other ErbB receptors and may be one that does not significantly cross-react with other proteins such as EGFR, ErbB 3, or ErBB4. In such embodiments, the extent of binding of the antibody to these non-ErBB2 proteins (e.g., cell surface binding to an endogenous receptor) will be less than 10%, as determined by fluorescence-activated cell sorting (FACS) or radioimmunoprecipitation (RIA) analysis. Occasionally, the anti-ErBB2 antibody will not significantly cross-react with the rat neu protein, such as described in Schecter et al., Nature 312:513-516 (1984) and Drebin et al., Nature 312:545-548 (1984).

The antibody of the drug conjugate or the target may be selected from the antibody or target in the table below. Such antibodies are immunospecific for the target antigen and may be obtained commercially or by any method known in the art, such as, for example, recombinant expression methods.

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Table 1

Therapeutic monoclonal antibodies

Name Trade name Target 3F8 GD2 ganglioside 8N9 B7-NZ Abagovomab SA-125 (imitation) Abituzumab CD51 Adecatumumab Ersam Alemtuzumab Campath, Lemtrada CD52 Altumomab Hybri-ceaker SEA Amatu ximab Mesothelium Andecaliximab gelatinase B Anetumab MSLN Aprutumab FGFR2 Askrinvacumab Activin receptor-like kinase 1 Atezolizumab Tecentriq PD-L1 Atinumab RTN4 Avelumab In avenci o PD-L1 Azintuxizumab CD319 Bavituximab phosphatidylserine BCD-100 PD-1 Belantamab VSMA Bemarituzumab FGFR2

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Bermekimab Xilonix ILIA Bersanlimab ICAM-1 Besilesomab Scintimun CEA-related antigen Bevacizumab Avastin VEGF-A Bivatuzumab CD44 v6 Blontuvetmab Biontress CD20 Brentuximab Adcentris CD30 (TNFRSF8) Brontictuzumab Notch 1 Cabyralizumab CSF1R Kamidanlumab CD25 Camrelizumab Programmed cell death 1 Cantuzumab MUC-1 Capromab Prostascint prostate carcinoma cells Carlu may MSR-1 Carotuximab endoglin Catumaxomab Removab ErSAM, CD3 CBR96 Lewis-Y antigen Cemiplimab PCDC1 Cergutuzumab IL2 Cetrelimab Programmed cell death 1 Cetuximab Erbitux EGFR Cibisatamab SEACAM5 Cirmtuzumab ROR1 Cixutumumab IGF-1 receptor (CD221) Clivatuzumab hPAM4-Cide MUC1 Codrituzumab glypican 3 Cofetuzumab RTK7 Coltuximab CD19 Conatumumab TRAIL-R2

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Cuzatuzumab CD70 Dacetuzumab CD40 Dalotuzumab IGF-1 receptor (CD221) Dapirolizumab pegol CD 154 (CD40L) Daratu mumab Darzalex CD38 Dectrecumab IL-13 Demcizumab DLL4 Denintuzumab CD19 Denosumab Prolia RANKL Depatu xizumab EGFR Derlotuximab Histone complex Detumomab B-cell lymphoma Dinutuximab Uni tuxin GD2 ganglioside Dostarlimab PCDP1 Drositumab DR5 DS-8201 HER2 Duligotuzumab ERBB3 (HER3) Durvalumab Imfinzi PD-L1 Dusigitumab ILGF2 Ecromeximab GD3 ganglioside Edrecolomab Panorex Ersam Elgemtumab ERBB3 (HER3) Elotuzumab Empliciti SLAMF7 Elsilimomab IL-6 Emactuzumab CSF1R Emibetuzumab HHGFR Enapotamab AXL Enavatuzumab TWEAK receptor

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Enfortumab nectin-4 Enlimomab pegol ICAM-1 (CD54) Enoblituzumab CD276 Ensituximab 5AC Epitumomab episialin Epratuzumab CD22 Ertumaxomab Rexomun HER2/neu, CD3 Etaracizumab Abegrin integrin ανβ3 Etigilimab TIGIT Faricimab VEGF-A and Ang-2 Farletuzumab folate receptor 1 FBTA05 Lymphomun CD20 Fibatuzumab Ephrin receptor AZ Ficlatuzumab HGF Figitumumab IGF-1 receptor (CD221) Flanvotumab TYRP1 (glycoprotein 75) Futuximab EGFR Galiximab CD80 G anitumab 1 receptor (CD221) G atipotuzumab MUC1 G edivumab H emagglutinin HA Gemtuzumab My lotarg CD33 G ilvetmab PCDC1 G irentuximab Rencarex carbonic anhydrase 9 (CA-IX) G lembatumumab GPNMB IB 1308 PD1 Ibritumomab Zevalin CD20 Ikrukumab VEGFR-1 Ifabotuzumab ERNAZ

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Iladatuzumab CD97B IMAB362 CLDN18.2 Imalumab MIF Imaprelimab MCAM Imgatuzumab EGFR Indatuximab SDC1 Indusatumab GUCY2C Inebilizumab CD19 Inotuzumab Without a doubt CD22 Intetumumab CD51 Ipilimumab Yervoy CD 152 Iratumumab CD30 (TNFRSF8) Izatuximab CD38 Iscalimab CD40 Istiratumab IGF1R, CD221 Labetuzumab CEA-Cide CEA Lacnotuzumab CSF1,MCSF Ladiratuzumab LIV-1 Laprituximab EGFR Lendalizumab C5 Lenzilumab CSF2 Leronlimab CCR5 Lesofavumab H emagglutinin HA Lexatumumab TRAIL-R2 Lifastuzumab Sodium phosphate cotransporter Lilotomab CD37 Lintuzumab CD33 Lirilumab KIR2D Loncastuximab CD19

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Losatuxizumab EGFR, ERBB1 HER1 Lorvotuzumab CD56 Lucatumumab CD40 Lumiliksimab CD23 (IgE receptor) Lumretuzumab ERBB3 (HER3) Lupartumab LYPD3 Lutikizumab Interleukin 1 alpha Mapatumumab TRAIL-RI Margetuximab HER2 Matuzumab EGFR Milatuzumab CD74 Minretumomab TAG-72 Mirvetuximab Folate receptor alpha Mitumomab GD3 ganglioside Modotuximab EGFR extracellular domain III Mogamulizumab Poteligeo CCR4 Monalizumab NKG2A Morolimumab Rhesus factor Mosunetuzumab CD3E, MS4A1, CD20 Moxetumomab CD22 Namilumab CSF2 Naratuximab CD37 Narnatumab RON Navicixizumab DLL4 Naxitamab C-Met Necitumumab Portrazza EGFR Nerelimomab TNF-α Nesvakumab angiopoietin 2 Nimotuzumab Theracim, EGFR

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Theraloc Nivolumab Opdivo PD-1 Obinutuzumab Gazyva CD20 Ocaratuzumab CD20 Ofatumumab Arzerra CD20 Olaratumab Lartruvo PDGF-R a Oleklumab 5'-nucleotidase Omburtamab CD276 Onartuzumab Human hepatocyte growth factor receptor kinase Ontuxizumab TOPIC1 Onvatilimab VSIR Oregovomab OvaRex SA-125 Otelixizumab CD3 Otlertuzumab CD37 Pamrevlumab CTGF Panitumumab Vectibix EGFR Pancomab Tumor-specific glycosylation MUC1 Parsatuzumab EGFL7 Pazotuxizumab Folate hydrolase Patritumab ERBB3 (HER3) PDR001 PD-1 Pembrolizumab Keytruda PD1 Pemtumomab Theragyn MUC1 Pertuzumab Omnitarg HER2/neu Pidilizumab PD-1 Pinatuzumab CD22 Pintumomab adenocarcinoma antigen

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Pogalizumab Member of 4 TNFR superfamily Polatuzumab CD79B Presalizumab ICOSL Pritu mumab vimentin Racotumomab Vaxira Ganglioside NGNA Radretumab extra domain-B of fibronectin Ramucirumab Cyramza VEGFR2 Relatlimab LAG3 Remtolumab Interleukin 17 alpha, TNF Ril otu mumab HGF Rituximab MabThera, Rituxan CD20 Robatu mumab IGF-1 receptor (CD221) Romilkimab Interleukin 13 Rosmantuzumab Root platelet-specific spondin 3 Rovalpituzumab DLL3 Sacituzumab TROP-2 Samalizumab CD200 Samrotamab LRRC15 Satumomab TAG-72 Selicrelumab CD40 Seribantumab ERBB3 (HER3) Setrusumab SOST Sibrotuzumab FAP SGN-CD19A CD19 Siltuximab Sylvant IL-6 Sintilimab PD-1 Sirtratumab SLITRK6

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Sofituzumab CA-125 Sontuzumb episialin Spartalizumab PDCD1, CD279 Tabalumab BAFF Takatuzumab AFP-Cide alpha-fetoprotein Talacotuzumab CD 123 Tamtuvetmab Tactress CD52 Taplitumomab CD19 Tarextumab Notch receptor Tavolimab CD 134 Telizotuzumab HGFR Tenatumomab tenascin C Tepoditamab Dendritic cell-associated lectin 2 Tesidolumab C5 Tetulomab CD37 Tigatuzumab TRAIL-R2 Timigutuzumab HER2 Timol mab AOSZ Tiragolumab TIGIT Tislelizumab PCDC1, CD279 Tizotumab Coagulation factor III Tomusotuximab EGFR, HER1 Tositumomab Bexxar CD20 Tovetumab CD 140a Trastuzumab Herceptin HER2/neu TRBS07 Ektomab GD2 ganglioside Tregalizumab CD4 Tremelimumab CTLA-4

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Tucotuzumab Ersam Ublituximab MS4A1 Ulocuplumab CXCR4 (CD 184) Urelumab 4-1BB (CD137) Utomilumab 4-1BB (CD137) Vadastuximab CD33 Vanalimab CD40 Vandortuzumab STEAP1 Vantiktumab Burn-associated receptor Vanicusumab angiopoietin 2 Vapaliksimab AOC3 (VAP-1) Varisacumab VEGF-A Varlilumab CD27 Vatelizumab ITGA2 (CD49b) Veltuzumab CD20 Vesencumab NRP1 Volociximab integrin α5βι Vonlerolizumab CD 134 Vopratelimab ICOS Vorsetuzumab CD70 Votumumab HumaSPECT tumor antigen CTAA16.88 Vunakizumab Interleukin 17 alpha Xentuzumab IGF1, IGF2 HMAV-5574 CD19 Zalutumumab HuMax-EGFr EGFR Zanolimumab HuMax-CD4 CD4 Zatuximab HER1 Zenocutuzumab ERBB3,HER3 Ziralimumab CD 147 (basigin) Zolbetuximab CLDN18

In addition to the above, the antibody of the antibody-drug conjugate may be Vitaxin, which is a humanized antibody for the treatment of sarcoma; Smart IDIO, which is a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma; Oncolim, which is a radioactively labeled mouse anti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma; and Allomune, which is a humanized anti-CD2 mAb for the treatment of Hodgkin's disease or non-Hodgkin's lymphoma.

The antibody of the drug conjugate may also be any antibody fragment known for the treatment of any disease, preferably cancer. Again, such antibody fragments are immunospecific for the target antigen and may be obtained commercially or by any method known in the art, such as, for example, recombinant expression methods. Examples of such available antibodies include any of the following table.

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Table 2

Fragments of therapeutic monoclonal antibodies

Fragment type/format Name Trade name Target P(aЬ’)г/ humanized alacizumab pegol VEGFR2 Fab/mice anatumomab TAG-72 Fab/sheep CroFab Snake venom Fab/sheep DigiFab Digoxin Fab/sheep Digibind Digoxin Fab’/mice arcitumomab CEA-scan CEA Fab’/mice bectumomab LymphoScan CD22 BiTE/mice blinatumomab Blincyto CD19 Fab/ humanized Citatuzumab Ersam scFv/chimeric humanized duvortuxizumab CD 19, CD3E

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scFv/human gancotamab unknown P(aЬ’)2/mice igovomab Indimacis-125 CA-125 Fab/mice nacolomab C242 antigen Fab/mice naptumomab 5T4 Fab/mice nofetumomab unknown scFv/ humanized oportuzumab Vicinium Ersam BiTE/mice solitomab Ersam Fab/ humanized Thromboview D-dimer Fab/PEGylated humanized CDP791 VEGF Fab/ bispecific humanized MDX-H210 Her2/Neu & CD64 (yFcRl) (ScFv) ₄ mouse streptavidin fused CC49 antigen TAG-72 pancarcinomas Human ScFv fused to β-lactamase SGN-17 P97 antigen Human ScFv fused to PEG F5 scFv-PEG Immunoliposome Neg2 Diabody (Vh-Vl)2 human C6.5K-A Her2/Neu Diabody (Vh-Vl)2 human L19 LI9-yIFN EDB domain fibronectin Diabody (Vl-Vh)2 human T84.66 SEA Minibody (scFv-Ch3) ₂ , mouse-human chimera (minibody) T84.66 SEA Minibody, a mouse chimera- 10N8 Neg2

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human (minibody) ScFv Fc dimer ( _SC F _V )2-Fc, mouse-human chimera (minibody) T84.66 SEA Bispecific scFv (Vl-Vh-Vh-Vl), mice g28M CD28 and MAP Bispecific (Vl-VhVh-Vl), origin unknown ΒΪΤΕΜΤ103 CD19 and CD3 Bispecific scFv (Vl-Vh-Vh-Vl), origin unknown BiTE Er-CAM and CD3 Bispecific tandem diabody (VHVL-VH-VL) (mice) tandab CD19& CD3 νΐιΗ-β-lactamase, fused, camel nanobody SEA Dab/human anti-TNFa dAb TNFa VhH/camel nanobody TNFa VhH/camel nanobody von Willebrand factor

Fab fragment, antigen binding (one arm) F(ab')2 fragment, antigen binding including the hinge region (both arms) Fab' fragment, antigen binding including the hinge region (one arm) scFv single chain variable fragment di-scFv dimeric single chain variable fragment (Holliger & Hudson, Nature Biotechnology, 2005, 23(9), 1126-1136). In a preferred embodiment, the antibody in the drug conjugates of the present invention targets a cell surface antigen.

In preferred embodiments, the antibody in the drug conjugates of the present invention may bind to a receptor encoded by the ErbB gene. The antibody may specifically bind to an ErbB receptor selected from EGFR, HER2, HER3 and HER4. Preferably, the antibody in the drug conjugate may specifically bind to the extracellular domain of the HER2 receptor and inhibit the growth of tumor cells that overexpress the HER2 receptor. The antibody of the drug conjugate may be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody or a humanized antibody. Preferably, the humanized antibody may be huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (Trastuzumab), particularly preferably Trastuzumab. The antibody may also be an antibody fragment, such as a Fab fragment.

Other antibodies may include:

(1) αanti-CD4 antibodies. The antibody of the drug conjugate may be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody, or a humanized antibody;

(2) anti-CD5 antibodies. The antibody of the drug conjugate may be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody, or a humanized antibody;

(3) anti-CD13 antibodies. The antibody of the drug conjugate may be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody, or a humanized antibody;

(4) anti-CD20 antibodies. The antibody of the drug conjugate may be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody, or a humanized antibody. Preferably, the humanized antibody is Rituximab or an antibody fragment thereof, such as a Fab fragment; and (5) anti-CD30 antibodies. The antibody of the drug conjugate may be a monoclonal antibody, such as a mouse monoclonal antibody, a chimeric antibody, or a humanized antibody.

- 59 048715 titer. Preferably, the humanized antibody is Brentuximab vedotin or an antibody fragment thereof.

In one embodiment, the antibody-drug conjugate may exhibit one or more of the following actions:

(1) increased cytotoxicity (or decreased cell survival), (2) increased cytostatic activity (cytostasis), (3) increased binding affinity to the target antigen or epitope, (4) increased internalization of the conjugate, (5) decreased side effects in the patient, and/or (6) an improved toxicity profile.

Such an increase may be in comparison to a known antibody-drug conjugate in the art that binds the same or a different epitope or antigen.

Methods for producing antibody-drug conjugates.

The antibody-drug conjugates of the present invention can be prepared by techniques well known in the art. Methods for conjugating moieties comprising antibodies with at least one antigen-binding site, for example antibodies to a number of different drugs, using a variety of processes have been previously described and illustrated, for example, in WO-A-2004/010957, WO-A-2006/060533 and WO-A-2007/024536, the contents of which are incorporated herein by reference. These involve the use of a linker group that derivatizes the drug, toxin or radionuclide so that it can then be attached to a moiety such as an antibody. Attachment to a moiety such as an antibody is typically accomplished in one of three ways: via free thiol groups on cysteines following partial reduction of disulfide groups on the antibody; via free amino groups on lysines in the antibody; and via free hydroxyl groups on serines and/or threonines in the antibody. The mode of attachment varies depending on the site of attachment of the moiety, such as an antibody. Purification of antibody-drug conjugate by size exclusion chromatography (SEC) has also been described [see, e.g., Liu et al., Proc. Natl. Acad. Set (USA), 93: 8618-8623 (1996) and Chari et al., Cancer Research, 52: 127-131 (1992)].

As noted above, a method for producing a drug conjugate of the present invention is provided, comprising conjugating a moiety Ab containing at least one antigen-binding site and a drug D of formula (IH), wherein Ab and D are as defined herein.

One example of a method for producing a drug conjugate of the present invention comprises producing antibody-drug conjugates of formula (G) or (G') of the present invention as follows:

(θ’), where said method includes the following steps:

(1) interaction of a drug (D-H) of formula (IH)-H:

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where the substituents in the definitions (IH)-H are as defined above for formula (IH), with a compound of formula (D') or (E):

(2) partially reducing one or more disulfide bonds in the antibody to be conjugated to produce a reduced antibody Ab-SH having free thiol bonds to produce a compound of formula (F) or (F'), respectively:

groups:

,, recovery ., , /A\ -----------► Ab-SH

S---S disulfide ~ ; and (3) reacting the partially reduced antibody Ab-SH having free thiol groups with the compound of formula (F) or (F') obtained in step (1) to obtain the desired antibody-drug conjugate of formula (G) or (G'), respectively:

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In another preferred embodiment of this method, the antibody is selected from an anti-HER2 antibody such as Trastuzumab, or an antigen-binding fragment or immunologically active portion thereof. Moreover, the partial reconstitution of this monoclonal antibody is carried out using tris[2-carboxyethyl]phosphine hydrochloride (TCEP).

Another example of a method for producing a drug conjugate of the present invention includes producing antibody-drug conjugates of formula (W) or (W') of the present invention below:

where the said method includes the following stages:

(1) interaction of the antibody with 2-iminothiolane hydrochloride (Troth's reagent) to obtain a thiol-activated antibody:

Ab- _NH2

Η Ab-N

_NH2Cl

SH

NH ₂ Cl (2) reacting the thiol-activated antibody with a compound of formula (F) or (F'), to obtain the desired antibody-drug conjugate of formula (W) or (W'), respectively.

In another preferred embodiment of this method, the antibody is selected from an anti-HER2 antibody, such as Trastuzumab, or an antigen-binding fragment or immunologically active portion thereof.

- 62 048715

The method for producing a drug conjugate may also include producing antibody-drug conjugates of formula (O) or (P) below:

where the said method includes the following stages:

(1) either:

(a) interaction of a medicinal product of formula (IH)-H:

where the substituents in the definitions (IH)-H are as defined above, with a compound of formula X2-C(O)-X ₁ , where X1 and X2 are leaving groups, to obtain a compound of formula (B):

and the point of attachment of the -(C=O)X1 group is the free -NH2 group of the compound of formula DH, or (b) reacting said drug (D-H) of formula (IH)-H, as defined above, with 4-nitrophenyl chloroformate to obtain a compound of formula (J):

and the point of attachment of the (4-nitrophenyl)-O-CO- group is the same as for the X1(CO) group in (a) above;

(2) either:

(c) reacting the compound of formula (B) obtained in step (1) with a hydroxy compound of formula HO-(CH ₂ ) _1-6 NHProt ^NH and removing the Prot ^NH group from the conjugated compound to obtain a compound of formula (C):

followed by interaction of the obtained compound of formula (C) with a compound of formula

- 63 048715

Me-SS-(CH ₂ )1.3-CO ₂ H to obtain a compound of formula (K): OO

D^O—(CH ₂ ) ₁ . ₆ -N^\(CH ₂ ) ₁ . ₃ ^SMe Η (K), or (d) reacting the compound (J) obtained in step (1) with a compound of formula HO-(CH ₂ )1. ₃ SProt ^SH and removing the Prot ^NH group from the conjugated compound to obtain a compound of formula (L): ⁰ η

ΙΑ1

DO(CH ₂ ) ₁ _ ₃ - _S - _S -(CH ₂ )i.3-O^D (L), (3) reaction of (K) or (L), obtained in step (2), with dithiothreitol under conditions of disulfide reduction to obtain a compound of formula (M) and (N), respectively:

OO9

LAA

DO(CH ₂ )^NH ^A (CH ₂ )i.3-SH ^D °( ^ch 2)i-3-SH (Μ)(Ν) (4) reacting the antibody to be conjugated with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate to derivatize said antibody at one or more lysine groups with a succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carbonyl group:

O _ -X °\

Ab-NH ₂ + SMCC -----Ab—Ν Υ-Λ (5) reacting the derivatized antibody obtained in step (4) with either (M) or (N) obtained in step (3) to obtain the desired antibody-drug conjugate of formula (O) or (P):

The compound of formula _X2- C(O)-X1 is preferably 1,1'-carbonyldiimidazole. Similarly, the hydroxy compound reacting with the compound of formula (B) is preferably HO-( _CH2 ) _2-4 - ^NHProtNH , and more preferably HO-( _CH2 ) ₃ - ^NHProtNH .

The compound reacting with the compound of formula (C) to obtain the compound of formula (K) is preferably 3-(methyldisulfanyl)propanoic acid.

The compound HO-(CH ₂ ) _1-3 SProt ^SH , which reacts with the compound of formula (J) to obtain the compound of formula (L), is preferably HO-(CH ₂ ) ₃ SProt ^SH .

If attachment to a linker-drug moiety is via free thiol groups in cysteines after partial reduction of disulfide groups in a moiety containing at least one antigen-binding site, such as a monoclonal antibody, partial reduction is typically accomplished by first diluting to a suitable concentration and buffering the solution before partially reducing the disulfide bonds by adding a suitable reducing agent such as tris[2-carboxyethyl]phosphine hydrochloride (TCEP) or dithiothreitol

- 64 048715 (DTT). By selecting appropriate ratios of the moiety to be reduced, such as a monoclonal antibody, and the reducing agent, the reaction conditions, and the reduction time, it is possible to obtain a desired ratio of free thiol to group, for example, four free thiol groups per monoclonal antibody.

A partially reduced moiety, such as a partially reduced monoclonal antibody having free thiol groups, prepared as described above, is then reacted with a linker-drug compound of the invention of the formula D-(X) _b -(AA) _w -(T) _g -L1 (wherein the L1 group in such a compound is a maleimide group that can freely react with thiol groups). The resulting antibody-drug conjugates are purified by any suitable means known in the art, for example, size exclusion chromatography (SEC) [see, for example, Liu et al., Proc. Natl. Acad. Sci. USA, 93: 8618-8623 (1996) and Chari et al., Cancer Research, 52: 127-131 (1992)].

In one preferred embodiment of the present invention, the partially reconstituted monoclonal antibody is Trastuzumab or an antigen-binding fragment or immunologically active portion thereof.

Lysines in a moiety containing at least one antigen-binding site, such as a monoclonal antibody, can first be reacted with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. The free amine group on the antibody can be reacted with an N-hydroxysuccinimide ester to produce a maleimide-activated antibody:

SMCC

The maleimide-activated antibody can then react with a compound of the formula D-(X) _b -(AA) _w -(T) _g -H, which has a reactive thiol group.

Alternatively, lysines in a moiety containing at least one antigen-binding site, such as a monoclonal antibody, can first be reacted with 2-iminothiolane hydrochloride (Traut's reagent). The free amine group of the antibody can be reacted with an imide thiolactone to produce a thiol-activated antibody.

^SH

Ab-NH ₂ + Ч/ ---► Ab-N- ₊ nh ₂ ci nh ₂ ⁺ Cl “thiol-activated antibody

One specific example of methods for producing antibody-drug conjugates of the formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab of the present invention by conjugation via free thiol groups in cysteines after partial reduction of disulfide groups in the antibody is shown in Fig. 1.

Another specific example of methods for producing antibody-drug conjugates of the formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab of the present invention by conjugation via free amino groups in lysines after reaction of the antibody with Troth's reagent is shown in Fig. 2.

Compositions containing the antibody-drug conjugate of the invention and their use.

Also provided is a pharmaceutical composition comprising a drug conjugate of the present invention and a pharmaceutically acceptable carrier. Examples of forms of administration of a drug conjugate having the general formula [D-(X) _b -(AA) _w -(T) _g -(L)-] _n -Ab of the present invention include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injections or infusions. Preferably

- 65 048715 compositions are administered parenterally. The pharmaceutical compositions of the invention can be prepared in such a way as to provide the drug conjugate of the present invention with bioavailability when the composition is administered to an animal, preferably a human. The compositions can take the form of one or more dosage units, where, for example, a tablet can represent a single dosage unit, and a container with an antibody-drug conjugate of the present invention in an aerosol form can contain a plurality of dosage units.

The pharmaceutically acceptable carrier or vehicle may be in particulate form, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be a liquid, wherein the compositions are, for example, an oral syrup or an injection liquid. Furthermore, the carrier(s) may be gaseous, to provide an aerosol composition useful, for example, in inhalation administration. The term carrier refers to a diluent, adjuvant or excipient with which the antibody-drug conjugate of the present invention is administered. Such pharmaceutical carriers may be liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers may be saline, acacia, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. In one embodiment, when administered to an animal, the antibody-drug conjugates of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the antibody-drug conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene glycol, water, ethanol and the like. The present compositions, if desired, may also contain minor amounts of wetting or emulsifying agents or pH buffering agents.

When the composition is intended for oral administration, the composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as solid or liquid.

As a solid composition for oral administration, the composition can be made in the form of a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition usually contains one or more inert diluents. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.

When the composition is in the form of a capsule (e.g. a gelatin capsule), it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or fatty oil.

The composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. The liquid may be useful for oral administration or for delivery by injection. When the composition is intended for oral administration, it may contain one or more sweeteners, preservatives, dyes/pigments and flavor enhancers. One or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonic agent may also be included in the composition for injection.

The preferred route of administration is parenteral administration, including, but not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intranasal, intracerebral, intraventricular, intrathecal, intravaginal, or transdermal. The preferred route of administration is left to the discretion of the practitioner and will depend in part on the site of the disease (such as a cancer site). In a more preferred embodiment, the present antibody drug conjugates of the present invention are administered intravenously.

Liquid compositions of the invention, whether solutions, suspensions or other similar form, may also include one or more of the following: sterile diluents such as water for injection, physiological solution, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, polyethylene glycols, glycerol or other solvents; antibacterial agents such as

- 66 048715 as benzyl alcohol or methylparaben; and agents for adjusting tonicity such as sodium chloride or dextrose. The parenteral composition may be enclosed in an ampoule, disposable syringe or multiple-dose vial made of glass, plastic or other material. The preferred adjuvant is saline.

The amount of the drug conjugate of the present invention that is effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical methods. In addition, in vitro or in vivo assays can optionally be used to help determine optimal dosage ranges. The exact dose used in the compositions will also depend on the route of administration and the severity of the disease or disorder, and should be determined in accordance with the judgment of the practitioner and the circumstances of each patient.

The compositions contain an effective amount of the drug conjugate of the present invention so as to obtain a suitable dosage. The correct dosage of the compounds will vary depending on the particular formulation, the route of administration and the particular site of application, the host and the disease being treated, for example cancer and, in this case, the type of tumor. Other factors such as age, body weight, sex, diet, time of administration, rate of elimination, condition of the host, drug combinations, sensitivity of the reaction and severity of the disease should be taken into account. The administration can be carried out continuously or intermittently within the maximum tolerated dose.

The drug conjugate of the present invention or compositions may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous membranes.

In particular embodiments, it may be desirable to administer one or more drug conjugates of the present invention or compositions locally to an area in need of treatment. In one embodiment, administration may be by direct injection into the site (or former site) of a cancer, tumor, or neoplastic or pre-neoplastic tissue. In another embodiment, administration may be by direct injection into the site (or former site) of an autoimmune disease manifestation.

Pulmonary administration may also be used, for example, by inhaler or nebulizer and formulation with an aerosolizing agent, or by perfusion in fluorocarbon or synthetic pulmonary surfactant. In some embodiments, the antibody-drug conjugate of the present invention or compositions may be formulated as suppositories with conventional binding agents and carriers such as triglycerides.

The present compositions may take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin.

Pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended for administration by injection can be prepared by combining a drug conjugate of the present invention with water to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

The inventors have found that the drug conjugates and compositions of the present invention are particularly effective in the treatment of cancer.

Thus, as described above, the present invention provides a method of treating a patient in need thereof, especially a human afflicted with cancer, comprising administering to the affected individual a therapeutically effective amount of a drug conjugate or composition of the present invention. The present invention provides a drug conjugate of the present invention for use in the treatment of cancer, and more preferably a cancer selected from lung cancer, including NSCLC, gastric cancer, colorectal cancer, breast cancer, pancreatic carcinoma, endometrial cancer, bladder cancer, cervical cancer, esophageal cancer, gallbladder cancer, uterine cancer, salivary duct cancer, ovarian cancer, renal cancer, leukemia, multiple myeloma and lymphoma. Most preferably, the cancer is breast cancer. The cancer is preferably a HER2 positive cancer, wherein HER2 positive cancers include HER2 positive lung cancer including HER2 positive NSCLC, HER2 positive gastric cancer, HER2 positive colorectal cancer, HER2 positive breast cancer, HER2 positive pancreatic carcinoma, HER2 positive endometrial cancer, HER2 positive bladder cancer, HER2 positive cervical cancer, HER2 positive esophageal cancer, HER2 positive gallbladder cancer, HER2 positive uterine cancer, HER2 positive salivary duct cancer and HER2 positive ovarian cancer, more preferably HER2 positive breast cancer, HER2 positive ovarian cancer and HER2 positive gastric cancer, most preferably

- 67 048715 positive HER2 breast cancer.

The drug conjugates and compositions of the present invention are useful for inhibiting the proliferation of a tumor cell or a cancer cell or for treating cancer in an animal. The drug conjugate and compositions of the present invention can be used respectively in a variety of conditions for the treatment of cancer in animals. The conjugates of the invention comprising a Drug-Linker-Moiety comprising at least one antigen-binding site can be used to deliver a Drug or a unit of a Drug to a tumor cell or a cancer cell. Without being bound by theory, in one embodiment, the Moiety comprising at least one antigen-binding site of the drug conjugate of the present invention binds or associates with a cancer cell or an antigen associated with a tumor cell, and the drug conjugate of the present invention can be taken up into the tumor cell or cancer cell via receptor-mediated endocytosis. The antigen may be attached to a tumor cell or a cancer cell, or may be an extracellular matrix protein associated with a tumor cell or a cancer cell. Once inside the cell, one or more specific sequences in the Linker unit are hydrolytically cleaved by one or more tumor cell or cancer cell associated proteases or hydrolases, resulting in the release of the Drug or Drug-Linker Compound. The released Drug or Drug-Linker Compound then freely migrates within the cell and induces cytotoxic activity. In an alternative embodiment, the Drug or Drug unit is cleaved from the drug conjugate of the present invention outside the tumor cell or cancer cell, and the Drug or Drug-Linker Compound then enters the cell.

In one embodiment, the Moiety comprising at least one antigen-binding site binds to a tumor cell or a cancer cell. In another embodiment, the Moiety comprising at least one antigen-binding site binds to a tumor cell or cancer cell antigen that is on the surface of the tumor cell or cancer cell. In another embodiment, the Moiety comprising at least one antigen-binding site binds to a tumor cell or cancer cell antigen that is an extracellular matrix protein associated with the tumor cell or cancer cell.

The specificity of the Moiety comprising at least one antigen-binding site for a particular tumor cell or cancer cell may be important for determining those tumors or cancer types that are most effectively treated. For example, the drug conjugates of the present invention having a Trastuzumab unit may be useful for the treatment of antigen-positive carcinomas, including leukemia, lung cancer, colon cancer, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's lymphoma), solid tumors such as sarcoma and carcinomas, multiple myeloma, renal cancer and melanoma. The cancer may preferably be lung cancer, colorectal cancer, breast cancer, pancreatic carcinoma, renal cancer, leukemia, multiple myeloma, lymphoma or ovarian cancer. For example, the drug conjugates of the present invention having a Rituximab unit may be useful for treating tumors that express CD-20, such as hematological cancers, including leukemias and lymphomas. For example, the drug conjugates of the present invention having an anti-CD4 antibody unit may be useful for treating tumors that express CD-4, such as hematological cancers, including lymphomas. For example, the drug conjugates of the present invention having an anti-CD5 antibody unit may be useful for treating tumors that express CD-5, such as hematological cancers, including leukemias and lymphomas. For example, the drug conjugates of the present invention having an anti-CD13 antibody unit may be useful for treating tumors that express CD-13, such as hematological cancers, including leukemias and lymphomas.

Other specific types of cancer that can be treated with the drug conjugates of the present invention include, but are not limited to: cancers of the hematopoietic system, including all forms of leukemia; lymphomas such as Hodgkin's disease, non-Hodgkin's lymphoma and multiple myeloma.

In particular, the drug conjugates and compositions of the present invention exhibit excellent activity in the treatment of breast cancer.

The drug conjugates and compositions of the present invention provide conjugation-specific tumor or cancer targeting, thereby reducing the overall toxicity of these conjugates. The linker units stabilize the antibody-drug conjugate in the blood, but are cleaved by tumor-specific proteases and hydrolases inside the cell, releasing the Drug.

The drug conjugates and compositions of the present invention may be administered to an animal that is also receiving surgery as a cancer treatment. In one embodiment of the present invention, the additional treatment is radiation therapy.

- 68 048715

In a specific embodiment of the present invention, the drug conjugate or composition of the present invention may be administered with radiotherapy. Radiotherapy may be administered simultaneously, before or after treatment with the drug conjugate or composition of the present invention. In one embodiment, the drug conjugate or composition of the present invention is administered simultaneously with radiation therapy. In another specific embodiment, radiation therapy is administered before or after administration of the drug conjugate or composition of the present invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (for example, up to three months), before or after administration of the antibody-drug conjugate or composition of the present invention.

With regard to radiation therapy, any radiation therapy protocol can be used depending on the type of cancer being treated. For example, but not limited to, X-rays can be used; in particular, high-energy megavoltage radiation (radiation with an energy greater than 1 MeV) can be used to treat deep tumors, and electron beam and orthovoltage X-rays can be used for skin cancer. Radioisotopes that emit gamma rays can also be administered, such as radioactive isotopes of radium, cobalt, and other elements.

The present invention discloses a kit comprising a therapeutically effective amount of a drug conjugate of the present invention and a pharmaceutically acceptable carrier. In particular, a kit is disclosed comprising a composition of the present invention and, optionally, instructions for use in the treatment of cancer and, more particularly, a cancer selected from lung cancer, including NSCLC, gastric cancer, colorectal cancer, breast cancer, pancreatic carcinoma, endometrial cancer, bladder cancer, cervical cancer, esophageal cancer, gallbladder cancer, uterine cancer, salivary duct cancer, ovarian cancer, renal cancer, leukemia, multiple myeloma and lymphoma.

In particular, said kit is intended for use in the treatment of cancer, and more preferably, a cancer selected from lung cancer, including NSCLC, gastric cancer, colorectal cancer, breast cancer, pancreatic carcinoma, endometrial cancer, bladder cancer, cervical cancer, esophageal cancer, gallbladder cancer, uterine cancer, salivary duct cancer, ovarian cancer, kidney cancer, leukemia, multiple myeloma and lymphoma. Most preferably, the kit is intended for use in the treatment of breast cancer.

Brief description of graphic materials

The invention is illustrated by diagrams, for example, in the accompanying graphic materials, in which:

Fig. 1 is a schematic illustration of one of the methods of the present invention, wherein the conjugation of the antibody is carried out via free thiol groups;

Fig. 2 is a schematic illustration of one of the methods of the present invention, wherein the conjugation of the antibody is carried out via free amino groups.

- 69 048715

Examples

The present invention is further illustrated by means of the following non-limiting examples. The following abbreviations are used in the examples:

CDI, 1, G -Carbonyldiimidazole

DIPEA, N, L-diisopropylethylamine

No, Hexane

EtOAc, Ethyl acetate

DCM, Dichloromethane

NMP, A-Methyl-2-pyrrolidone

DMF, Dimethylformamide

EDC, A-(3-dimethylaminopropyl)-A'-ethylcarbodiimide hydrochloride

EDTA, Ethylenediaminetetraacetic acid

MeOH, Methanol

DTT, Dithiothreitol

Ru, Pyridine

THF, Tetrahydrofuran

TCEP, Tris[2-carboxyethyl]phosphine hydrochloride

MS, 6-Maleimidocaproyl

Fmoc, 9-Fluorenylmethoxycarbonyl

Cit, Citrulline

Vai, Valin

DMSO, Dimethyl sulfoxide

Trt, Triphenylmethyl

- 70 048715

HOBt, 1-Hydroxybenzotriazole

DIPCDI, -diisopropylcarbodiimide

TFA, Trifluoroacetic acid

PABOH, 4-Aminobenzyl alcohol bis-PNP, bis(4-Nitrophenyl)carbonate

NAC, TV-Acetylcysteine

SEC, Size Exclusion Chromatography

HPLC, High Performance Liquid Chromatography

ADC, Antibody Drug Conjugate

ATCC, American Type Culture Collection

DMEM, Dulbecco's Modified Eagle's Medium

RPMI, Rosmell Park Memorial Institute Medium

ITS, Insulin-transferrin-sodium selenite medium supplement

FCS, Fetal Calf Serum

SRB, Sulforhodamine B

PBS, Phosphate buffered saline

DZ, dose dependent

UV, Ultraviolet

SMCC, Succinimidyl 4-(/U-maleimidomethyl)cyclohexane-1-carboxylate

LAR, Linker to Antibody Ratio

Example 1. Synthesis of drugs.

Compounds 1 and 2 were prepared according to the procedures described in WO 2003066638 (examples 69 and 65, respectively, on pp. 112-116).

Compound 4 was prepared according to the procedure described in WO 2003066638 (Example 12 on pages 61-62).

Compounds 8-S and 8-R were prepared according to the procedure described in WO 2018197663 (Example 8 on pp. 97-98).

Compound 16-S was prepared according to the procedure described in WO 2018197663 (Example 19 on page 117).

Example 1-1.

5-S

To a solution of 4 (35 mg, 0.054 mmol) in acetic acid (0.7 mL, 0.08 M) was added L-tryptophanol (36 mg, 0.189 mmol, Sigma-Aldrich). The reaction mixture was stirred at 50 °C for 3 h and then acetic acid was evaporated. Aqueous saturated NaHCO ₃ solution was added and the mixture was extracted with CH ₂ Cl ₂ . The combined organic layers were dried over Na ₂ SO ₄ . Flash chromatography (Hexane:EtOAc, 1:1) gave purified compound 5-S (28 mg, 63%).

Rf=0.25 (Hexane:EtOAc, 1:1).

1H NMR (400 MHz, CDCl ₃ ): δ 7.72 (s, 1H), 7.35 (d, J=7.9 Hz, 1H), 7.26 (d, J=7.9 Hz, 1H), 7.12 (t, J=7.9 Hz, 1H), 7.02 (t, J=8.0 Hz, 1H), 6.62 (s, 1H), 6.25 (s, 1H), 6.03 (s, 1H), 5.91-5.80 (m, 1H), 5.75 (s, 1H), 5.175.04 (m, 3H), 4.60 (s, 1H), 4.41 (s, 1H), 4.36 (d, J=11.5 Hz, 1H), 4.29 (dd, J=11.7, 2.1 Hz, 1H), 4.22 (d, J=2.7 Hz, 1H), 3.81 (s, 3H), 3.59-3.44 (m, 3H), 3.35 (dd, J= 11.1, 9.0 Hz, 1H), 2.97-2.64 (m, 5H), 2.61 (dd, J=15.3, 4.6 Hz, 1H), 2.43-2.29 (m, 1H), 2.37 (s, 3H), 2.28 (s, 3H), 2.05 (s, 3H).

ESI-MS m/z: Calculated for C ₄₄ H ₄ 5N ₅ O ₉ S: 819.3. Found: 820.3 (M+1)+.

- 71 048715

To a solution of 5-S (26 mg, 0.032 mmol) in

_PdCl2 ( _{PPh3 ) 2} (13 mg, 0.02 mmol), acetic acid ₍ 0.069 mL, 1.2 mmol) and _HSnBu3 (0.17 mL, 0.64 mmol) were added to CH2Cl2. The reaction mixture was stirred at 23°C for 2 h. The crude material was concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 1:9 to 9:1) gave purified compound 6-S (17 mg, 68%).

Rf=0.15 (Hexane: EtOAc, 1:1).

1H NMR (400 MHz, CDCl ₃ ): δ 7.74 (s, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.26 (d, J=7.9 Hz, 1H), 7.12 (t, J=8.0 Hz, 1H), 7.02 (t, J=7.9 Hz, 1H), 6.63 (s, 1H), 6.26 (s 1H), 6.03 (s, 1H), 5.82 (s, 1H), 5.15 (d, J=11.6 Hz, 1H), 4.59 (s, 1H), 4.50 ( d, J=5.1 Hz, 1H), 4.41 (s, 1H), 4.29 (dd, J=11.7, 2.1 Hz, 1H), 4.22 (d, J=2.7 Hz, 1H), 3.903.81 (m, 1H), 3.80 (s, 3H), 3.67-3.49 (m, 1H), 3.49 (d, J=5.2 Hz, 1H), 3.36 (dd, J=11.0, 9.1 Hz, 1H), 3.11-2.85 (m, 3H), 2.60 (dd, J=15.3, 4.5 Hz, 1H), 2.42 (d, J=15.3 Hz, 1H), 2.38-2.22 (m, 2H), 2.35 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H).

ESI-MS m/z: Calculated for C ₄₁ H ₄₁ N ₅ O ₉ S: 779.3. Found: 780.2 (M+1) ⁺ .

To a solution of 6-S (14 mg, 0.018 mmol) in _CH3CN : _H2O (1.39:1, 1.3 mL, 0.015 M) was added _AgNO3 (61 mg, 0.36 mmol). After 17 h at 23 °C, the reaction mixture was quenched with a 1:1 mixture of saturated aqueous solutions of brine and _NaHCO3 , stirred for 15 min, diluted _{with CH2Cl2} , stirred for 5 min and extracted _{with CH2Cl2} . _The combined organic layers were dried over anhydrous _Na2SO4 , filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography ( _CH2Cl2 : _{CH3OH ,} 99:1 to 85:15) to give purified 7-S (3 mg, 22%).

Rf=0.15 (CH2CECH3OH, 9:1). ¹ H NMR (500 MHz, CD3OD): δ 7.70 (s, 1H), 7.35 (d, J=7.9 Hz, 1H), 7.26 (d, J=7.9 Hz, 1H), 7.11 (t, J=8.2 Hz, 1H), 7.02 (t, J=8.2 Hz, 1H), 6.63 (s, 1H), 6.23 (s, 1H), 6.01 (s, 1H), 5.76 (s, 1H), 5.26 (d, J=11.5 Hz, 1H), 4.92 (s, 1H), 4.54 (s, 1H), 4.48 (s, 2H), 4.37 (d, J=5.3 Hz, 1H), 4.21 (d, J=10.2 Hz, 1H), 3.80 (s, 3H), 3.67-3.50 (m, 4H), 3.36 (t, J=10.2 Hz, 1H), 3.04-2.82 (m, 3H), 2.61 (dd, J=15.2, 5.8 Hz, 1H), 2.42-2.28 (m, 2H), 2.36 (s, 3H), 2.27 (s, 3H), 2.02 (s, 3H).

ESI-MS m/z: Calculated for C ₄₀ H ₄₂ N ₄ O ₁₀ S: 770.3. Found: 753.2 (MH ₂ O+1) ⁺ .

Example 1-2.

To a solution of 4 (400 mg, 0.62 mmol) in acetic acid (8 ml, 0.08 M) was added 8-S (468 mg, 2.13 mmol).

- 72 048715

The reaction mixture was stirred at 52 °C for 17 h and then acetic acid was evaporated. Aqueous saturated NaHCO ₃ solution was added and the mixture was extracted with CH ₂ Cl ₂ . The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 1:1) gave purified compound 9-S (325 mg, 62%).

Rf=0.30 (Hexane:EtOAc, 1:1).

1H NMR (400 MHz, CDCl ₃ ): δ 7.65 (s, 1H), 7.16 (d, J=8.6 Hz, 1H), 6.78 (s, 1H), 6.77 (m, 1H), 6.62 (s, 2H), 6.23 (d, J=1.3 Hz, 2H), 6.02 (d, J=1.3 Hz, 2H), 5.85 (dddd, J=17.1, 10.2, 6.8, 5.8 Hz, 1H), 5.75 (s, 1H), 5.15-5.00 ( m, 3H), 4.59 (s, 1H), 4.43-4.22 (m, 4H), 3.80 (s, 3H), 3.78 (s, 3H), 3.53 (d, J=12.9 Hz, 2H), 3.46 (d, J=5.0 Hz, 1H), 3.38 (s, 1H), 2.93 (s, 1H), 2.86 (d, J =4.4 Hz, 1H), 2.85-2.70 (m, 2H), 2.58 (dd, J=15.2, 4.6 Hz, 1H), 2.42-2.30 (m, 2H), 2.37 (s, 3H), 2.26 (s, 3H), 2.04 (s, 3H).

ESI-MS m/z: Calculated for C ₄₅ H ₄₇ N ₅ O ₁₀ S: 849.9. Found: 850.3 (M+1)+.

9-S 10-S

To a solution of 9-S (325 mg, 0.38 mmol) in _{CH2Cl2 were} added _PdCl2 ( _PPh3 ) ₂ (160 mg, 0.23 mmol), acetic acid (0.82 mL, 14.2 mmol) and _HSnBu3 (1.7 mL, 6.27 mmol). The reaction mixture was stirred at 23 °C for 1.5 h. The crude material was concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 1:9 to 9:1) gave purified compound 10-S (180 mg, 59%).

Rf=0.15 (Hexane:EtOAc, 1:1).

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.19 (s, 1H), 6.79 (m, 2H), 6.65 (s, 1H), 6.26 (s, 1H), 6.03 (d, J=1.4 Hz, 2H), 5.77 (d, J=11.5 Hz, 1H), 5.10 (s, 1H), 4.59 (s, 1H), 4.48 (d, J=4.9 Hz, 1H), 4.39-4.29 (m, 3H), 3.79 (s, 3H), 3.79 (s, 3H), 3.64-3.33 (m, 4H), 3.03-2.90 (m, 4H), 2.59 (d, J=14.6 Hz, 2H), 2.44-2.32 (m, 2H), 2.37 (s, 3H), 2.26 (s, 3H), 2.04 (s, 3H).

ESI-MS m/z: Calculated for C ₄₂ H ₄₃ N ₅ O ₁₀ S: 809.3. Found: 810.3 (M+1) ⁺ .

10-S 11-S

To a solution of 10-S (180 mg, 0.22 mmol) in _CH3CN : _H2O (1.39:1, 16 mL, 0.015 M) was added _AgNO3 (756 mg, 4.40 mmol). After 18 h at 23°C, the reaction mixture was quenched with a 1:1 mixture of saturated aqueous solutions of brine and _NaHCO3 , stirred for 15 min, diluted _{with CH2Cl2} , stirred for 5 min and extracted _{with CH2Cl2} . _The combined organic layers were dried over anhydrous _Na2SO4 , filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (CH ₂ Cl ₂ :CH ₃ OH, 99:1 to 85:15) to give purified 11-S (100 mg, 56%).

Rf=0.35 (CH ₂ Cl ₂ :CH ₃ OH, 9:1).

1H NMR (500 MHz, CD3OD): δ 7.15 (dd, J=8.8, 0.6 Hz, 1H), 6.82 (dd, J=2.5, 0.6 Hz, 1H), 6.68 (dd, J=8.9, 2.5 Hz, 1H), 6.56 (s, 1H), 6.27 (d, J=1.3 Hz, 1H), 6.08 (d, J=1.3 Hz, 1H), 5.31 (d, J=11.5 Hz, 1H), 4.62-4.55 (m, 1H), 4.44 (ddtd , J=4.9, 1.5, 1.0, 0.5 Hz, 2H) 4.38-4.27 (m, 1H), 4.25-4.18 (m, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.64 (d, J=4.8 Hz, 1H), 3.61-3.42 (m, 3H), 3.13-2.95 (m, 3H), 2.80 (dd, J=10.4, 5.4 Hz, 2H), 2.68 (dd, J=15.1, 4.2 Hz, 2H), 2.55 (d, J=15.4 Hz, 1H), 2.51-2.36 (m, 3H), 2.34 (s, 3H), 2.29 (s, 3H) , 2.00 (s, 3H).

¹³ C NMR (126 MHz, CD ₃ OD): δ 172.6, 169.2, 155.1, 148.0, 147.2, 144.7, 142.4, 142.1, 133.1, 132.6, 132.2, 131.1, 128.2, 125.5, 122.2, 122.0, 116.3, 112.9, 112.8, 111.4, 109.0, 103.5, 100.9, 91.0, 66.6, 65.0, 61.8, 60.3, 59.2, 57.1, 56.1, 51.7, 47.2, 45.5, 43.8, 39.0, 28.2, 25.4, 20.6, 16.3, 9.5.

ESI-MS m/z: Calculated for C41H44N4O11S: 800.3. Found: 783.4 (MH ₂ O+1) ⁺ .

- 73 048715 (+)-HR-ESI-TOF-MS m/z 800.2796 [M+H]+ (Calculated for C41H44N4O11S: 800.2727).

Example 1-3.

9-R

To a solution of 4 (400 mg, 0.62 mmol) in acetic acid (8 mL, 0.08 M) was added 8-R (468 mg, 2.13 mmol). The reaction mixture was stirred at 52 °C for 17 h and then acetic acid was evaporated. Aqueous saturated NaHCO ₃ solution was added and the mixture was extracted with CH ₂ Cl ₂ . The combined organic layers were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 1:1) gave purified compound 9-R (390 mg, 77%).

Rf=0.30 (Hexane: EtOAc, 1:1).

1H NMR (400 MHz, CDCl ₃ ): δ 7.64 (s, 1H), 7.14 (d, J=8.8 Hz, 1H), 6.81 (d, J=2.6 Hz, 1H), 6.74 (dd, J=8.8, 2.4 Hz, 1H), 6.59 (s, 1H), 6.18 (d, J=1.4 Hz, 1H), 5.97 (d, J=1.4 Hz, 1H), 5.91-5.80 (m, 1H), 5.79 (s, 1H), 5.154.92 (m, 3H ), 4.62 (s, 1H), 4.42-4.23 (m, 2H), 4.23-4.03 (m, 3H), 3.79 (s, 3H), 3.78 (s, 3H), 3.68-3.48 (m, 2H), 3.43 (d, J=5.1 Hz, 2H), 3.01-2.68 (m, 3H), 2.57-2.41 (m, 3H) , 2.39 (s, 3H), 2.25 (s, 3H), 2.22-2.20 (m, 1H), 2.07 (s, 3H).

ESI-MS m/z: Calculated for C ₄₅ H ₄₇ N ₅ O ₁₀ S: 849.9. Found: 850.4 (M+1) ⁺ .

9-R 10-R

To a solution of 9-R (390 mg, 0.46 mmol) in _{CH2Cl2 were} added _PdCl2 ( _PPh3 ) ₂ (193 mg, 0.28 mmol), acetic acid (1.0 mL, 17.2 mmol), and _HSnBu3 (2.04 mL, 7.60 mmol). The reaction mixture was stirred at 23 °C for 1.5 h. The crude material was concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 1:9 to 9:1) gave purified compound 10-R (210 mg, 57%).

Rf=0.15 (Hexane:EtOAc, 1:1).

1H NMR (400 MHz, CDCl ₃ ): δ 7.16 (d, J=8.8 Hz, 1H), 6.82 (d, J=2.4 Hz, 1H), 6.76 (dd, J=8.8, 2 ,5 Hz, 1H), 6.62 (s, 1H), 6.22 (d, J=1.5 Hz, 1H), 6.00 (d, J=1.5 Hz, 1H), 5.03 (d, J=11.5 Hz, 1H), 4.62 (s, 1H), 4.51 (d, J=5.0 Hz, 1H ), 4.36 (s, 1H), 4.23-4.11 (m, 3H), 3.85 (dd, J=8.1, 4.1 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.72-3.57 (m, 1H), 3.45 (d, J=5.2 Hz, 2H), 3.12 (d, J=17.6 Hz, 1H), 2.99 (dd, J=17.9, 9.6 Hz, 1H), 2.54 (s, 2H), 2.46 (d, J=14.7 Hz, 2H), 2.38 (s, 3H), 2.33-2.19 (m, 1H), 2.26 (s, 3H), 2.08 (s, 3H).

ESI-MS m/z: Calculated for C ₄₂ H ₄₃ N ₅ O ₁₀ S: 809.3. Found: 810.5 (M+1) ⁺ .

10-R 11-R

To a solution of 10-R (210 mg, 0.26 mmol) in CH ₃ CN:H ₂ O (1.39:1, 18 mL, 0.015 M) was added AgNO ₃ (883

- 74 048715 mg, 5.20 mmol). After 18 h at 23 °C, the reaction mixture was quenched with a 1:1 mixture of saturated aqueous solutions of brine and NaHCO ₃ , stirred for 15 min, diluted with CH ₂ Cl ₂ , stirred for 5 min and extracted with CH ₂ Cl ₂ . The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (CH ₂ Cl ₂ :CH ₃ OH, 99:1 to 85:15) to give purified 11-R (140 mg, 66%).

Rf=0.30 (CH2C12:CH3OH, 9:1).

¹ H NMR (500 MHz, CD3OD): δ 7.13 (dd, J=8.8, 0.6 Hz, 1H), 6.82 (d, J=8.8 Hz, 1H), 6.67 (dd, J=8 ,8, 2.5 Hz, 1H), 6.61 (s, 1H), 6.25 (d, J=1.3 Hz, 1H), 6.07 (d, J=1.4 Hz, 1H), 5.21 (d, J=11.3 Hz, 1H), 4.80-4.72 (m, 2H), 4.58 (s, 1H), 4.45 (d, J=5.4 Hz, 1H), 4.21 (d, J=2.7 Hz, 1H), 4.16-4.06 (m, 1H), 3.75 (s, 3H), 3.72 (s, 3H), 3.64 (d, J=7.7 Hz, 2H), 3.55-3.48 (m, 3H), 3.16 (d, J=17.6 Hz, 1H) , 3.03 (dd, J=17.7, 9.8 Hz, 1H), 2.76-2.63 (m, 2H), 2.32 (s, 3H), 2.29 (s, 3H), 2.25-2.11 (m, 2H), 2.04 (s, 3H).

¹³ C NMR (126 MHz, CD ₃ OD): δ 171.6, 153.6, 146.6, 145.9, 143.4, 141.3, 140.9, 132.1, 131.1, 130.8, 129.7, 126.4, 121.2, 120.7, 114.8, 112.1, 111.5, 110.0, 108.8, 107.6, 107.6, 102.1, 99.5, 89.6, 65.4, 63.1, 60.1, 59.0, 57.8, 55.9, 54.7, 52.7, 45.9, 26.6, 25.1, 24.2, 19.4, 19.1, 14.8, 13.0, 8.2.

ESI-MS m/z: Calculated for C ₄₁ H ₄₄ N ₄ O11S: 800.3. Found: 783.3 (M-H ₂ O+1)+. (+)-HR-ESI-TOF-MS m/z 800.2781 [M+H]+ (Calculated for C41H44N4O11S: 800.2727). Example 1-4.

13

To a solution of 4 (350 mg, 0.54 mmol) in acetic acid (7 mL, 0.08 M) was added 2-benzofuran-3-yl-ethylamine hydrochloride (12) (1.52 g, 7.70 mmol, Sigma Aldrich). The reaction mixture was stirred at 52 °C for 72 h and then acetic acid was evaporated. Aqueous saturated NaHCO ₃ solution was added and the mixture was extracted with CH ₂ Cl ₂ . The combined organic layers were dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 1:1) gave purified compound 13 (180 mg, 42%).

Rf=0.5 (Hexane:EtOAc, 1:1).

1H NMR (400 MHz, CDCl ₃ ): δ 7.39-7.29 (m, 2H), 7.23-7.07 (m, 2H), 6.64 (s, 1H), 6.19 (d, J=1.3 Hz, 1H), 6.04 (d, J=1.3 Hz, 1H), 5.97-5.80 (m, 1H), 5.78 (s, 1H), 5.19-4.97 (m, 3H), 4.54 (s, 1H), 4.36 (dd, J=4.8, 1.6 Hz, 1H), 4.31 (s, 1H), 4.20 (dd, J=11.4, 1.9 Hz, 2H), 3.80 (s, 3H), 3.59-3.49 (m, 1H), 3.47 (dd, J=7.0, 2.9 Hz, 1H), 3.25 (ddd, J=11.4, 8.1, 5.0 Hz, 1H), 3.04 (d, J=18.0 Hz, 1H), 2.98-2.72 (m, 5H), 2.59-2.49 (m, 2H), 2.37 (s, 3H), 2.27 (s, 3H), 2.23-2.12 (m, 1H), 2.07 (s, 3H).

ESI-MS m/z: Calculated for C ₄₃ H ₄₂ N ₄ O ₉ S: 790.9. Found: 791.5 (M+1) ⁺ .

14

To a solution of 13 (320 mg, 0.40 mmol) in _CH2Cl2 were added _PdCl2 ( _PPh3 ) ₂ (170 mg, 0.24 mmol), acetic acid (0.86 mL, 15 mmol) and _HSnBu3 (1.78 mL, _6.60 mmol). The reaction mixture was stirred at 23°C for 1.5 h. The crude material was concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 1:9 to 9:1) gave purified compound 14 (130 mg, 43%).

Rf=0.15 (Hexane:EtOAc, 1:1).

1H NMR (400 MHz, CDCl3): δ 7.40-7.31 (m, 2H), 7.19-7.10 (m, 2H), 6.65 (s, 1H), 6.19 (d, J=1.5 Hz, 1H), 6.04 (d, J=1.5 Hz, 1H), 5.05 (d, J=11.5 Hz, 1H), 4.43(s, 1H), 4.51-4.47 (m, 1H), 4.30 (s, 1H), 4.21 (d, J=2.3 Hz, 2H), 3.86-3.76 (m, 2H), 3.79 (d, J=1.9 Hz, 2H), 3.46 (d, J=4.7 Hz, 1H), 3.29-3.22 (m, 1H), 3.19 (d, J=17.9 Hz, 1H), 2.99 (dd, J=17.9, 9.4 Hz, 1H), 2.83 (s, 1H), 2.53 ( dt, J=7.9, 4.8 Hz, 2H), 2.35 (s, 3H), 2.33-2.23 (m, 1H), 2.27 (s, 3H), 2.20-2.14 (m, 1H), 2.07 (m, 3H).

- 75 048715

ESI-MS m/z: Calculated for C ₄₀ H ₃₈ N ₄ O ₉ S: 750.8. Found: 751.9 (M+1)+.

15

To a solution of 14 (130 mg, 0.17 mmol) in _CH3CN :H2O (1.39:1, 12 mL, 0.015 M) was added _AgNO3 (578 mg, 3.40 mmol). After 3 h at 23 °C, a 1:1 mixture of saturated aqueous solutions of brine and _NaHCO3 was added, stirred for 15 min, diluted with _CH2Cl2 , stirred for 5 min and extracted _{with CH2Cl2 . The} combined organic layers were dried over anhydrous _Na2SO4 , filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography ( _CH2Cl2 : _CH3OH , 99:1 to 85:15) to afford purified compound 15 ₍ 80 mg, 64% ₎ .

Rf=0.25 (CH2C12:CH3OH, 9:1).

¹ H NMR (500 MHz, CD3OD): δ 7.45-7.34 (m, 2H), 7.26-7.09 (m, 2H), 6.60 (s, 1H), 6.06 (d, J=1.1 Hz, 1H), 6.24 (d, J=1.1 Hz, 1H), 5.24 (d, J=11.5 Hz, 1H), 4.74 (s, 1H), 4.52 (s, 1H), 4.47 (d, J=4.9 Hz, 1H), 4.19-4.09 (m, 2H), 3.74 (s , 3H), 3.64 (d, J=9.2 Hz, 1H), 3.57 (d, J=4.9 Hz, 1H), 3.43-3.37 (m, 1H), 3.20-3.09 (m, 1H), 3.04 (dd, J=17.8, 9.5 Hz, 1H), 2.96-2.90 (m, 1H), 2.83 (d, J=15, 4 Hz, 1H), 2.59-2.56 (m, 2H), 2.34 (s, 3H), 2.30 (s, 3H), 2.10-2.02 (m, 1H), 2.05 (s, 3H).

¹³ C NMR (126 MHz, CD3OD): δ 171.9, 170.7, 156.0, 150.5, 148.7, 147.0, 144.8, 142.4, 142.1, 132.6, 131.2, 128.6, 125.5, 124.7, 123.8, 122.3, 121.2, 120.2, 116.8, 114.9, 114.0, 112.3, 103.5, 91.4, 90.7, 63.7, 62.3, 60.4, 58.7, 57.1, 47.2, 43.5, 40.8, 39.3, 28.2, 21.5, 20.6, 16.2, 9.6.

ESI-MS m/z: Calculated for C ₃₉ H ₃₉ N ₃ O _i0 S: 741.8. Found: 724.9 (M-H ₂ O+1) ⁺ .

(+)-HR-ESI-TOF-MS m/z: 741.2416 [M+H]+ (Calculated for C ₃₉ H ₃₉ N ₃ O ₁₀ S: 741.2356).

Example 1-5.

17-S

To a solution of 4 (150 mg, 0.24 mmol) in CH3CN (15 mL, 0.016 M) were added 16-S (230 mg, 1.20 mmol) and cyanuric chloride (TCC) (45 mg, 30%). The reaction mixture was stirred for 24 h at 85 °C and then aqueous saturated _NaHCO3 solution was added and the mixture was extracted _{with CH2Cl2} . _The combined organic layers were dried over anhydrous _Na2SO4 , filtered and concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 9:1 to 1:9) gave purified 17-S (145 mg, 73% yield).

1H NMR (400 MHz, CDCI3): δ 7.35 (dt, J=8.2, 0.9 Hz, 1H), 7.31 (ddd, J=7.6, 1.5, 0.7 Hz, 1H), 7.20 (ddd, J=8.4, 7.2, 1.5 Hz, 1H), 7.13 (td, J=7.4, 1.1 Hz, 1H), 6.62 (s, 1H), 6.20 (d, J=1.5 Hz, 1H), 6.05 (d, J=1.4 Hz, 1H), 5.85 (m, 1H), 5.74 (s, 1H), 5.16-5.08 (m, 3H), 4.58 (s, 1H), 4.40-4.32 (m, 2H), 4.28 (dd, J=11.5, 2.2 Hz, 1H), 4.19 (d, J=2.9 Hz, 1H), 3.80 (s, 3H), 3.58-3.53 (m, 1H), 3.50 (dd, J=11.3, 4.1 Hz, 2H), 3.42-3.30 (m, 1H), 2.96 (s, 1H), 2.90-2.73 (m, 4H), 2.58 (dd, J=15.7, 4.9 Hz, 1H), 2.52 (d, J=15.0 Hz, 1H), 2.37 (s, 3H), 2.36-2.26 (m, 2Н), 2.28 (s, 3Н), 2.04 (s, 3H).

ESI-MS m/z: 821.3 (M+H) ⁺ .

- 76 048715

17-S 18-S

To a solution of 17-S (140 mg, 0.17 mmol) in CH2C12 were added PdCl2(PPh3)2 (19 mg, 0.027 mmol), acetic acid (0.097 mL, 1.70 mmol) and HSnBu3 (1.65 mL, 6.12 mmol).

The reaction mixture was stirred for 3 h at 23 °C. The crude material was concentrated in vacuo. Flash chromatography (Hexane:EtOAc, 1:9 to 9:1) gave purified compound 18-S (94 mg, 71% yield).

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.35 (dt, J=8.2, 0.9 Hz, 1H), 7.31 (dt, J=7.6, 1.0 Hz, 1H), 7.20 ( ddd, J=8.3, 7.2, 1.5 Hz, 1H), 7.13 (td, J=7.4, 1.1 Hz, 1H), 6.62 (s, 1H), 6.20 (d, J=1.4 Hz, 1H), 6.05 (d, J=1.4 Hz, 1H), 5.09 (dd, J=11.5, 1.1 Hz, 1H), 4.58 (s, 1H), 4.52 (d, J=5.0 Hz, 1H), 4.39-4.35 (m, 1H), 4.27 (dd, J=11.5, 2.1 Hz, 1H), 4.20 (d, J=2.6 Hz, 1H), 3.85 (d, J=18.3 Hz, 1H ), 3.79 (s, 3H), 3.54-3.44 (m, 2H), 3.34 (dd, J=11.2, 9.2 Hz, 1H), 3.04-2.97 (m, 2H), 2.92 (tt, J=8.6, 4.3 Hz, 1H), 2.60-2.47 (m, 2H), 2.35 (s, 3H), 2.34-2.28 (m, 2H), 2.28 (s, 3H), 2.05 (s, 3H).

ESI-MS m/z: 781.3 (M+H)+.

18-S 19-S

To a solution of 18-S (90 mg, 0.11 mmol) in _CH3CN : _H2O (1.39:1, 8 mL, 0.015 M) was added _AgNO3 (580 mg, 3.45 mmol). After 18 h at 23°C, a 1:1 mixture of saturated aqueous solutions of brine and _NaHCO3 was added, stirred for 15 min, diluted _{with CH2Cl2} , stirred for 5 min and extracted _{with CH2Cl2} . The combined organic layers were dried over _{anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash chromatography (CH2Cl2:CH3OH , 99 :} 1 to 85:15) to afford purified compound 19 _- S (60 mg, 68% yield).

1H NMR (400 MHz, CDCl ₃ ): δ 7.35 (d, J=8.1 Hz, 1H), 7.32-7.28 (m, 1H), 7.19 (td, J=8.3, 7.8, 1, 4 Hz, 1H), 7.16-7.09 (m, 1H), 6.58 (s, 1H), 6.18 (d, J=1.5 Hz, 1H), 6.04 (d, J=4.6 Hz, 1H), 5.18 (d, J=11.3 Hz, 1H), 4.91 (s, 1H ), 4.63 (s, 1H), 4.60-4.46 (m, 2H), 4.18 (d, J=10.8 Hz, 2H), 3.83-3.71 (m, 2H), 3.78 (s, 1H), 3.69 (s, 1H), 3.56-3.44 (m, 2H), 3.32 (t, J=10.3 Hz, 1H), 3.08-2.86 ( m, 2H), 2.54 (dd, J=15.6, 5.0 Hz, 2H), 2.37-2.23 (m, 2H), 2.32 (s, 3H), 2.27 (s, 3H), 2.04 (s, 3H).

ESI-MS m/z: 754.3 (MH ₂ O+H) ⁺ .

Example 2. Synthesis of linkers.

Obtaining LIN 1. MC-Val-Cit-PABC-PNP.

Reaction scheme.

(a) Preparation of LIN 1-1.MC-Val-Cit-OH.

- 77 048715

LIN 1-1.

Cl-TrtCl resin (20 g, 1.49 mmol/g) (Iris Biotech, Ref.: BR-1065, 2-Chlorotrityl chloride resin (200400 mesh, 1% DVB, 1.0-1.6 mmol/g), CAS 42074-68-0) was placed on a filter press plate. 100 mL of DCM was added to this resin and the mixture was stirred for 1 h. The solvent was removed by vacuum filtration. A solution of Fmoc-Cit-OH (11.83 g, 29.78 mmol) and DIPEA (17.15 mL, 98.45 mmol) in DCM (80 mL) was added and the mixture was stirred for 10 min. Afterwards, DIPEA (34.82 mmol, 199.98 mmol) was added and the mixture was stirred for 1 h. The reaction was quenched by adding MeOH (30 mL) after stirring for 15 min. The resulting Fmoc-Cit-O-TrtCl resin was subjected to the following washes/treatments: DCM (5x50 mLx0.5 min), DMF (5x50 mLx0.5 min), piperidine:DMF (1:4, 1x1 min, 2x10 min), DMF (5x50 mLx0.5 min), DCM (5x50 mLx0.5 min). The final wash with piperidine gave NH2 _- Cit-O-TrtCl resin. Calculated loading: 1.15 mmol/g.

NH2 _- Cit-O-TrtCl resin obtained above was washed with DMF (5x50 mLx0.5 min) and a solution of Fmoc-ValOH (31.22 g, 91.98 mmol), HOBt (11.23 g, 91.98 mmol) in DMF (100 mL) was added to this _NH2 -Cit-OTrtCl resin, stirred and DIPCDI (14.24 mL, 91.98 mmol) was added and the mixture was stirred for 1.5 h. The reaction was stopped by washing with DMF (5x50 mLχ0.5 min). The Fmoc-Val-Cit-O-TrtCl resin thus obtained was treated with piperidine:DMF (1:4, 1x1 min, 2x10 min) and washed with DMF (5x50 mlχ0.5 min). A final wash with piperidine gave NH ₂ -Val-Cit-O-TrtCl resin.

A solution of 6-maleimidocaproic acid (MC-OH) (9.7 g, 45.92 mmol), HOBt (6.21 g, 45.92 mmol) in DMF (100 mL) was added to the _NH2 -Val-Cit-O-TrtCl-resin obtained above, stirred, and DIPCDI (7.12 mL, 45.92 mmol) was added and the mixture was stirred for 1.5 h. The reaction was stopped by washing with DMF (5x50 mLχ0.5 min) and DCM (5x50 mLχ0.5 min).

The peptide was cleaved from the resin by TFA:DCM treatments (1:99, 5x100 mL). The resin was washed with DCM (7x50 mLχ0.5 min). The combined filtrates were evaporated to dryness under reduced pressure and the resulting solid was triturated with Et2O and filtered to afford LIN 1-1 (7.60 g, 71%) as a white solid.

1H NMR (500 MHz, DMSO-d ₆ ): δ 12.47 (s, 1H), 8.13 (d, J=7.3 Hz, 1H), 7.74 (d, J=9.0 Hz, 1H), 6.99 ( s, 2H), 5.93 (s, 1H), 5.35 (s, 2H), 4.20 (dd, J=9.0, 6.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.36 (t, J=7.0 Hz, 2H), 3.00-2.88 (m , 2H), 2.21-2.12 (m, 1H), 2.11-2.03 (m, 1H), 1.98-1.86 (m, 1H), 1.74-1.62 (m, 1H), 1.61-1.50 (m, 1H), 1.501.31 (m, 6H), 1.21-1.11 (m, 2H), 0.84 (d, J=6.8 Hz, 3H), 0.80 (d, J=6.8 Hz, 3H).

ESI-MS m/z: Calculated for C ₂ iH ₃₃ N ₅ O ₇ : 467.2. Found: 468.3 (M+H) ⁺ .

(b) Obtaining LIN 1-2. MC-Val-Cit-PABOH.

LIN 1-2.

To a solution of LIN 1-1 (1.6 g, 3.42 mmol) and 4-aminobenzyl alcohol (RAVON) (0.84 g, 6.84 mmol) in DCM (60 mL) was added a solution of HOBt (0.92 g, 6.84 mmol) in DMF (5 mL). DIPCDI (1.05 mL, 6.84 mmol) was added, the reaction mixture was stirred for 2 h at 23 °C, Et ₂ O (150 mL) was added and the resulting solid was filtered in a filter press under vacuum to give LIN 1-2 (1.31 g, 67%).

¹ H NMR (500 MHz, DMSO-d6): δ 9.88 (s, 1H), 8.03 (d, J=7.6 Hz, 1H), 7.77 (dd, J=12.2, 8.5 Hz, 1H ), 7.53 (d, J=8.2 Hz, 2H), 7.21 (d, J=8.2 Hz, 2H), 6.99 (s, 3H), 6.01-5.92 (m, 1H), 5.39 (s, 2H), 5.07 (s, 1H), 4.41 (s, 2H), 4.39-4.31 (m, 1H) , 4.23-4.12 (m, 1H), 3.36 (t, J=7.0 Hz, 2H), 3.06-2.97 (m, 1H), 2.96-2.90 (m, 1H), 2.222.03 (m, 2H), 2.01-1.88 (m, 1H), 1.76-1.62 (m, 1H), 1.63-1.28 (m, 6H) , 1.25-1.11 (m, 2H), 0.84 (d, J=6.9 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H).

ESI-MS m/z: Calculated for C ₂₈ H ₄₀ N ₆ O ₇ : 572.3. Found: 573.3 (M+H)+.

(c) Obtaining LIN 1. MC-Val-Cit-PAB-PNP.

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LIN 1.

To a solution of LIN 1-2 (500 mg, 0.87 mmol) and bis(4-nitrophenyl)carbonate (bis-PNP) (2.64 g, 8.72 mmol) in DCM:DMF (8:2, 25 mL) was added DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred for 20 h at 23 °C and poured onto a silica gel column (DCM:CH ₃ OH, 50:1 to 10:1) to afford pure target LIN 1 (364 mg, 57%).

Rf=0.40 (CH ₂ Cl ₂ :CH3OH, 9:1).

1H NMR (400 MHz, CDC13/CD3OD): δ 9.45 (s, 1H), 8.23 (d, J=8.3 Hz, 2H), 7.59 (d, J=8.5 Hz, 2H), 7.35 (d , J=8.3 Hz, 2H), 7.34 (d, J=8.5 Hz, 2H), 6.65 (s, 2H), 5.20 (s, 2H), 4.56 (dt, J=10.5, 5.4 Hz, 1H), 4.15 (d, J=7.2 Hz, 1H), 3.46 (dd, J=8.0, 6.4 Hz, 2H), 3.16-2.89 (m, 2H), 2.21 (dd, J=8.3, 6.6 Hz, 2H), 2.06-1.97 (m, 1H), 1.901.83 (m, 1H), 1.73-1.46 (m, 7H), 1.34-1.20 (m, 2H), 0.91 (d, J=6.7 Hz, 3H), 0.90 (d, J=6.7 Hz, 3H).

¹³ C NMR (125 MHz, CDCI3/CD3OD) δ 174.4, 172.4, 171.1, 170.6, 160.5, 155.5, 152.5, 145.3, 138.7, 134.1, 129.9, 129.5, 125.2, 121.8, 120.0, 70.6, 59.0, 53.2, 37.5, 35.8, 30.6, 29.6, 29.3, 28.1, 26.2, 26.2, 25.1, 19.1, 18.1.

ESI-MS m/z: Calculated for C^H^NyOv 737.3. Found: 738.3 (M+H)+.

Obtaining LIN-2. MC2-PEG4-Val-Cit-PABC-PNP.

Reaction scheme.

.- Fmoc-Cit-OH RAVON .-Fmoc-Val-OH DIPCDI .- Fmoc-PEG4-OH _ipo ,

L MC9 ΓΊΜ nUBt

Cl ινι^ζ-uri g MC ₂ -PEG ₄ -Val-Cit-OH --------------*

DIPCDI, HOBt, DMF CH ₂ CI ₂ , DMF

Chlorotrityl resin LIN 2-1 >100%

Bis-PNP DIPEA

MC2-PEG4-Val-Cit- _ _and MC2-PEG4-Val-Cit-PABC-PNP

PABOH _CH2CI2 , _DMF

LIN 2-2 45% LIN-2

a) Obtaining LIN 2-1. MC2-PEG4-Val-Cit-OH.

LIN 2-1.

Cl-TrtCl resin (5 g, 1.49 mmol/g) was placed on a filter press plate. CH2Cl2 (25 mL) was added to the resin and the mixture was stirred for 1 h at 23 °C. The solvent was removed by vacuum filtration. A solution of Fmoc-Cit-OH (2.95 g, 7.44 mmol) and DIPEA (4.29 mL, 24.61 mmol) _{in CH2Cl2} (20 mL) was added and the mixture was stirred for 10 min at 23 °C. More DIPEA (8.70 mL, 49.99 mmol) was added and the mixture was stirred for 1 h at 23 °C. The reaction was quenched by adding MeOH (10 mL) and stirred for 15 min at 23 °C. Fmoc-Cit-O-TrtCl resin was subjected to the following washes/treatments: CH2C12 (5x15 mLx0.5 min), DMF (5x15 mLx0.5 min), piperidine:DMF (1:4, 15 mL, 1x1 min, 2x10 min), DMF (5x15 mLx0.5 min), CH2C12 (5x15 mLx0.5 min). Calculated loading: 1.17 mmol/g.

NH2 _- Cit-O-TrtCl resin was washed with DMF (5x15 mLx0.5 min) and a solution of Fmoc-Val-OH (7.80 g, 22.99 mmol) and HOBt (2.80 g, 24.5 mmol) was added to BDMF (25 mL) to this NH2 _- Cit-O-TrtCl resin, then DIPCDI (3.56 mL, 24.5 mmol) was added at 23 °C. The reaction mixture was stirred for 1.5 h at 23 °C. The reaction was stopped by washing with DMF (5x15 mLx0.5 min). Fmoc-Val-Cit-O-TrtCl resin was treated with piperidine:DMF (1:4, 15 ml, 1x1 min, 2x10 min) and washed with DMF (5x15 mlx0.5 min).

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A solution of 15-(9-fluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoic acid (Fmoc-NH-PEG4-OH) (4.27 g, 8.75 mmol) and HOBt (1.18 g, 8.72 mmol) in DMF (30 mL) was added to NH2Val-Cit-O-TrtCl-resin, then DIPCDI (1.35 mL, 8.72 mmol) was added at 23 °C. The reaction mixture was stirred for 24 h at 23 °C. The reaction was stopped by washing with DMF (5x15 mLx0.5 min). Fmoc-NH-PEG4-Val-Cit-O-TrtCl resin was treated with piperidine:DMF (1:4, 15 ml, 1x1 min, 2x10 min) and washed with DMF (5x15 mlx0.5 min).

A solution of 3-(maleimido)propionic acid (MC2-OH) (3.95 g, 23.35 mmol) and HOBt (3.16 g, 23.37 mmol) in DMF (30 mL) was added to NH ₂ -PEG4-Val-Cit-O-TrtCl-resin, followed by the addition of DIPCDI (3.62 mL, 23.37 mmol) at 23 °C. The reaction mixture was stirred for 2 h at 23 °C. The reaction was quenched by washing with DMF (5x15 mLx0.5 min) and CH ₂ Cl ₂ (5x15 mLx0.5 min).

The peptide was cleaved from the resin by treatments with TFA: _CH2Cl2 ( _1:99 , 5x50 mL). The resin was washed _{with CH2Cl2} (7x50 mLx0.5 min). The combined filtrates were evaporated to dryness under reduced pressure, the resulting solid was triturated with _Et2O and filtered to give LIN 2-1 (4.59 g, 87% yield) as a white solid.

1H NMR (300 MHz, CDCl ₃ ): δ 7.67-7.57 (m, 1H), 7.44 (d, J=8.3 Hz, 1H), 7.11 (t, J=5.4 Hz, 1H), 6.73 (s, 2H), 4.49 (d, J=7.2 Hz, 1H), 4.35 (t, J=7.7 Hz, 1H), 3.82 (t, J=7.0 Hz, 2H), 3.74 (t, J=6.2 Hz, 2H), 3.68-3.56 (m, 13H), 3.56-3.45 (m, 2H), 3.39 (q, J=5.4 Hz, 2H), 3.17 (s, 2H), 2.55 (q, J=7.0, 6.0 Hz, 4H), 2.16-1.99 (m, 1H), 1.91 (s, 1H), 1.75 (s, 1H), 1.43 (s, 2H), 0.94 (d, = 9.7 Hz, 3H), 0.93 (d, = 9.7 Hz, 3H).

ESI-MS m/z: 673.3 (M+H)+.

(b) Preparation of LIN 2-2. MC2-PEG4-Val-Cit-PABOH.

LIN 2-2.

To a solution of LIN 2-1 (1.5 g, 2.22 mmol) and 4-aminobenzyl alcohol (RAVON) (0.55 g, 4.45 mmol) in CH2Cl2 (60 mL) was added a solution of HOBt (0.60 g, 4.45 mmol) in DMF (5 mL), followed by the addition of DIPCDI (0.69 mL, 4.45 mmol) at 23 °C. The reaction mixture was stirred for 5 h at 23 °C, Et2O (150 mL) was added and the resulting solid was vacuum filtered to afford crude LIN 22 (2.37 g, >100% yield), which was used in the next step without further purification.

1H NMR (500 MHz, DMSO-d6): δ 7.57 (d, J=8.6 Hz, 2H), 7.30 (d, J=8.6 Hz, 2H), 6.81 (s, 2H), 4.58 (s, 1H), 4.56 (s, 2H), 4.50 (dd, J=9.1, 5.1 Hz, 1H), 4.21 (d, J=7.0 Hz, 1H), 3.80-3.68 (m, 4H), 3.65-3.59 (m, 12H), 3.55-3.47 (m, 1H), 3.20 (dd, J=13.6, 6.9 Hz, 1H), 3.12 (dt, J=13.5, 6.7 Hz, 1H), 2.55 (td, J=6.1, 2.1 Hz, 2H), 2.46 (t, J=6.9 Hz, 2H), 2.15-2.07 (m, 1H), 1.95-1.88 (m, 1H), 1.79-1.70 (m, 1H), 1.67-1.50 (m, 2H), 0.99 (d, J=7.0 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H).

ESI-MS m/z: 778.4 (M+H)+.

(c) Obtaining LIN 2. MC2-PEG4-Val-Cit-PABC-PNP.

To a solution of LIN 2-2 (1.73 g, 2.22 mmol) and bis(4-nitrophenyl)carbonate (bis-PNP) (3.38 g, 11.12 mmol) in DCM:DMF (8:2, 75 mL) was added DIPEA (1.16 mL, 6.07 mmol) at 23 °C. The reaction mixture was stirred for 19 h at 23 °C and poured onto a silica gel column (CH ₂ Cl ₂ :CH ₃ OH, 50:1 to 10:1) to afford pure LIN 2 (945 mg, 45% yield).

1H NMR (500 MHz, CD3OD): δ 8.22 (d, J=9.2 Hz, 2H), 7.61 (d, J=8.6 Hz, 2H), 7.34 (d, J=9.2 Hz, 2H), 7.33 (d, J=8.6 Hz, 2H), 6.67 (s, 2H), 4.57-4.47 (m, 1H), 4.23-4.12 (m, 1H), 3.78-3.76 (m, 12H), 3.63-3.50 (m, 16H), 3.49-3.41 (m, 2H), 3.34-3.25 (m, 2H), 3.18-3.03 (m, 2H), 2.51 (t, J=5.9 Hz, 2H), 2.45 (t, J=7.2 Hz, 2H), 2.13-1.99 (m, 1H), 1.92-1.84 (m, 1H), 1.73-1.62 (m, 1H), 1.55-1.45 (m, 2H), 0.92 (d, J=6.8 Hz, 3H), 0.90 (d, J=6.8 Hz, 3H).

¹³ C NMR (75 MHz, CDCl ₃ /CD ₃ OD): δ 174.4, 172.9, 172.4, 172.4, 171.6, 170.9, 170.8, 170.7, 163.7, 155.8, 155.7, 152.5, 145.4, 138.8, 134.1, 131.3, 130.4, 129.2, 128.7, 125.7, 124.9, 121.8, 119.8 (x2), 115.1, 70.2 (x2),

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70.1 (x2), 70.0, 69.9, 69.8, 69.0, 66.9, 59.2, 53.5, 39.0, 36.0, 34.4,34.1,30.4,29.0, 18.5, 17.5.

ESI-MS m/z: 943.4 (M+H)+.

Ri=0.20 (CH ₂ Cl ₂ :CH ₃ OH, 9:1).

Obtaining LIN 3. MC2-PEG4-Val-Ala-PABC-PNP.

Reaction scheme.

.- Fmoc-Ala-OH .- Fmoc-Val-OH PABOH .- Fmoc-PEG4-OH DIPCDI / 4.-MC2-OH HOBt

L Λ--- ^Cl --------------► MC2-PEG4-Val-Ala-OH ------►

DIPCDI, HOBt, DMF CH ₂ CI ₂ , DMF

Chlorotrityl resin 87% ^IN 3-1

Bis-PNP DIPEA MC2-PEG4-Val- --------► MC2-PEG4-Val-Ala-PABC-PNP

Ala- _{PABOH CH2CI2} , DMF

LIN 3-2 59% LIN 3 (a) Preparation of LIN 3-1. MC2-PEG4-Val-Ala-OH.

LIN 3-1.

Cl-TrtCl resin (5 g, 1.49 mmol/g) was placed on a filter press plate. _CH2Cl2 ( ₂₅ mL) was added to the resin and the mixture was stirred for 1 h at 23 °C. The solvent was removed by vacuum filtration. A solution of Fmoc-Ala-OH (2.31 g, 7.41 mmol) and DIPEA (4.28 mL, 24.61 mmol) _{in CH2Cl2} (20 mL) was added and the mixture was stirred for 10 min at 23 °C. More DIPEA (8.60 mL, 49.37 mmol) was added and the reaction mixture was stirred for 1 h at 23 °C. The reaction was quenched by adding MeOH (10 mL) and stirred for 15 min at 23 °C. Fmoc-Ala-O-TrtCl resin was subjected to the following washes/treatments: _CH2Cl2 ( _5x15 mlx0.5 min), DMF (5x15 mlx0.5 min), piperidine:DMF (1:4, 15 ml, 1x1 min, 2x10 min), DMF (5x15 mlx0.5 min), _CH2Cl2 (5x15 mlx0.5 min). Calculated loading: 1.34 mmol _/ g.

NH ₂ -Ala-O-TrtCl resin was washed with DMF (5 x 15 mL x 0.5 min) and a solution of Fmoc-Val-OH (9.09 g, 26.79 mmol) and HOBt (3.62 g, 26.79 mmol) in DMF (25 mL) was added to NH ₂ -Ala-O-TrtCl resin, then DIPCDI (4.14 mL, 26.79 mmol) was added at 23 °C. The mixture was stirred for 1.5 h at 23 °C. The reaction was stopped by washing with DMF (5 x 15 mL x 0.5 min). The Fmoc-Val-Ala-O-TrtCl resin was treated with piperidium:DMF (1:4, 15 ml, 1x1 min, 2x10 min) and washed with DMF (5x15 mlx0.5 min).

A solution of 15-(9-fluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoic acid (Fmoc-NH-PEG4-OH) (4.90 g, 8.75 mmol) and HOBt (1.35 g, 9.98 mmol) in DMF (30 mL) was added to NH2Val-Ala-O-TrtCl-resin, then DIPCDI (1.55 mL, 10.0 mmol) was added at 23 °C. The reaction mixture was stirred for 22 h at 23 °C. The reaction was stopped by washing with DMF (5x15 mLχ0.5 min). Fmoc-NH-PEG4-Val-Ala-O-TrtCl resin was treated with piperidine:DMF (1:4, 15 ml, 1x1 min, 2x10 min) and washed with DMF (5x15 mlx0.5 min).

A solution of 3-(maleimido)propionic acid (MC2-OH) (4.53 g, 26.78 mmol) and HOBt (3.62 g, 26.77 mmol) in DMF (30 mL) was added to NH ₂ -PEG4-Val-Ala-O-TrtCl-resin, followed by the addition of DIPCDI (4.15 mL, 26.80 mmol) at 23 °C. The reaction mixture was stirred for 2 h at 23 °C. The reaction was quenched by washing with DMF (5x15 mLx0.5 min) and CH ₂ Cl ₂ (5x15 mLx0.5 min).

The peptide was cleaved from the resin by treatments with TFA: _CH2Cl2 ( _1:99 , 5x50 mL). The resin was washed _{with CH2Cl2} (7x50 mLx0.5 min). The combined filtrates were evaporated to dryness under reduced pressure, the resulting solid was triturated with _Et2O and filtered to give L 3-1 (4.73 g, 87% yield) as a white solid.

1H NMR (500 MHz, CDCl3): δ 7.67 (bs, 1H), 7.31 (d, J=8.9 Hz, 1H), 7.17 (d, J=7.0 Hz, 1H), 6.85 (t, J =5.6 Hz, 1H), 6.72 (s, 2H), 4.51 (q, J=7.1 Hz, 1H), 4.38 (dd, J=8.9, 6.9 Hz, 1H), 3.84 (t, J=7.1 Hz, 2H), 3.75 (t, J=5, 9 Hz, 2H), 3.69-3.59 (m, 12H), 3.55 (t, J=5.1 Hz, 2H), 3.41 (qd, J=5.0, 1.7 Hz, 2H), 2.62-2.49 (m, 4H), 2.19-2.01 (m, 1H), 1.44 (d, J=7.2 Hz, 3H), 0.95 (d, J=11.9 Hz, 1H), 0.94 (d, J=11.9 Hz, 1H).

(b) Preparation of LIN 3-2. MC2-PEG4-Val-Ala-PABOH.

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LIN 3-2.

To a solution of LIN 3-1 (1.84 g, 3.13 mmol) and 4-aminobenzyl alcohol (RAVON) (0.77 g, 6.27 mmol) in CH ₂ Cl ₂ (70 mL) was added a solution of HOBt (0.84 g, 6.27 mmol) in DMF (5 mL), then DIPCDI (0.97 mL, 6.27 mmol) was added at 23 °C. The reaction mixture was stirred for 5 h at 23 °C, Et ₂ O (150 mL) was added and the resulting solid was vacuum filtered to give crude LIN 32 (1.74 g, 81% yield), which was used in the next step without further purification.

¹ H NMR (500 MHz, DMSO-d ₆ ): δ 7.58 (d, J=8.5 Hz, 2H), 7.30 (d, J=8.5 Hz, 2H), 6.81 (s, 2H), 4.56 (s, 2H), 4.52-4.41 (m, 1H), 4.21 (d, J=6.7 Hz, 1H). 3.91 (p, J=6.5 Hz, 1H), 3.81-3.67 (m, 4H), 3.65-3.54 (m, 12H), 3.49 (t, J=5.5 Hz, 2H), 2.56 (dd, J=6.6, 5.5 Hz, 2H), 2.46 (t, J=6.9 Hz, 2H), 2.12 (h, J=6.8 Hz, 1H), 1.45 (d, J=7.2 Hz, 3H), 1.00 (d, J=12.1 Hz, 3H), 0.98 (d, J=12.1 Hz, ZN).

(c) Obtaining LIN 3. MC2-PEG4-Val-Ala-PABC-PNP.

LIN 3.

To a solution of LIN 3-2 (1.74 g, 2.51 mmol) and bis(4-nitrophenyl)carbonate (bis-PNP) (3.82 g, 12.57 mmol) in CH ₂ Cl ₂ :DMF (8:1, 70 mL) was added DIPEA (1.31 mL, 7.54 mmol) at 23 °C. The reaction mixture was stirred for 20 h at 23 °C and poured onto a silica gel column (CH ₂ Cl ₂ :CH ₃ OH, 50:1 to 10:1) to afford pure LIN 3 (1.26 g, 59% yield).

1H NMR (500 MHz, CDCl3): δ 8.82 (s, 1H), 8.27 (d, J=9.2 Hz, 2H), 7.73 (d, J=8.6 Hz, 2H), 7.38 (d, J=9.1 Hz, 4H), 7.15 (dd, J=21.8, 7.2 Hz, 2H), 6.69 (s, 2H), 6.62 (t, J=5.7 Hz, 1H), 5.24 (s, 2H), 4.67 (p, J=7.2 Hz, 1H), 4.24 (dd, J=6.8, 5.7 Hz, 1H), 3.91-3.76 (m, 2H), 3.71 (ddd, J=10.1, 6.1, 4.3 Hz, 1H), 3.66-3.54 (m, 14H), 3.53 (t, J=5.1 Hz, 1H), 3.46-3.33 (m, 2H), 2.76-2.57 (m, 1H), 2.57-2.42 (m, 2H), 2.33-2.19 (m, 1H), 1.46 (d, J=7.1 Hz, 3H), 1.01 (d, J=12.1 Hz, 3H), 1.00 (d, J=12.1 Hz, 3H).

¹³ C NMR (75 MHz, CD3OD): δ 173.0, 172.1, 171.6 (χ2), 170.7, 163.8, 155.7, 152.5, 145.4, 140.3, 138.9, 134.1, 130.4, 129.1, 125.6, 124.8, 121.9, 119.7, 115.1, 70.2, 70.1 (χ3), 70.0, 69.9, 69.8, 69.0, 66.9, 59.1, 53.4, 49.7, 39.0, 36.0, 34.3, 34.1, 30.4, 18.3, 17.3, 16.6.

ESI-MS m/z: 857.3 (M+H)+.

Rf=0.45 (CH ₂ Cl ₂ :CH ₃ OH, 9:1).

Example 3. Synthesis of compounds of the formula DX-(AA) _w -(T) _g -L1.

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Obtaining the DL-1 compound.

To a solution of 1 (15 mg, 0.019 mmol) and L1 (14 mg, 0.019 mmol) in 1-methyl-2-pyrrolidone (NMP) (1 mL, 0.019 M) was added DIPEA (3 μL, 0.019 mmol) at 23 °C. After 27 h, EtOAc was added and the reaction mixture was washed with water and the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The resulting residue was purified by preparative HPLC to afford pure DL1 (7.5 mg, 29% yield).

1H NMR (500 MHz, CD ₃ OD): δ 7.58 (d, J=8.6 Hz, 1H), 7.50 (d, J=8.6 Hz, 1H), 7.32 (d, J=8.6 Hz , 1H), 7.21 (d, J=8.6 Hz, 1H), 7.11 (dd, J=8.7, 1.8 Hz, 1H), 6.82 (t, J=2.0 Hz, 1H), 6.77 (s, 2H), 6.67 (ddd, J=8.9, 2.5, 1.3 Hz, 1H) , 6.58 (s, 1H), 6.23 (dd, J=3.0, 1.3 Hz, 1H), 6.07 (t, J=1.4 Hz, 1H), 5.64 (ddd, J=12.4, 4.9, 1.8 Hz, 1H), 5.21 (dd, J=22.0, 11.1 Hz, 1H), 5.19-5.11 (m, 1H), 5.14- 5.04 (m, 1H), 5.04-4.96 (m, 1H), 4.75 (s, 1H), 4.70 (s, 1H), 4.58 (s, 1H), 4.50 (ddd, J=8.7, 5.1, 3.3 Hz, 1H), 4.30 (d, J=3.1 Hz, 1H), 4.22-4.11 (m, 3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.58-3.53 (m, 1H), 3.50-3.44 (m, 2H), 3.35 (s, 3H), 3.24-3.17 (m, 2H), 3.11 (ddd, J=13.7, 10.6, 6.6 Hz, 1H), 3.02 (dd , J=17.5, 9.8 Hz, 1H), 2.90-2.84 (m, 2H), 2.76 (dd, J=15.3, 2.4 Hz, 1H), 2.59 (dd, J=7.0, 4.9 Hz, 2H), 2.36-2.24 (m, 6H) , 2.14-2.07 (m, 1H), 2.10-1.97 (m, 4H), 2.04 (s, 3H), 1.93-1.86 (m, 1H), 1.79-1.71 (m, 1H), 1.66-1.60 (m, 2H), 1.59-1.53 (m, 4H), 1.35-1.25 (m, 4H), 0.97 (m, 6H).

ESI-MS m/z: 1352.2 (MH ₂ O+H)+.

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Obtaining the DL-2 compound.

To a solution of 2 (21 mg, 0.027 mmol) in dimethylformamide (DMF) (2 mL, 0.013 M) was added L1 (22 mg, 0.029 mmol), 1-hydroxybenzotriazole (HOBt, 3.9 mg, 0.029 mmol) and DIPEA (26 µL, 0.15 mmol) at 23 °C. After 72 h, EtOAc was added and the reaction mixture was washed with water and the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated in vacuo. The resulting residue was purified by preparative HPLC to afford pure DL2 (3.5 mg, 9% yield).

1H NMR (400 MHz, CDCl ₃ ): δ 7.74 (d, J=7.8 Hz, 1H), 7.47 (dd, J=21.6, 8.1 Hz, 2H), 7.23 (d, J=7 ,8 Hz, 1H), 7.12 (d, J=8.2 Hz, 1H), 7.07 (d, J=8.2 Hz, 1H), 6.77 (s, 2H), 6.64 (s, 2H), 6.54 (s, 1H), 6.16 (s, 1H), 5.97 (s, 1H), 5.63 (d, J =17.2 Hz, 1H), 5.11 (d, J=12.5 Hz, 1H), 5.01 (s, 1H), 4.90 (d, J=12.2 Hz, 1H), 4.66 (s, 1H), 4.50 (s, 1H), 4.29-4.19 (m, 2H), 4.13-4.08 (m, 1H), 3.74 (s, 3H), 3.70 (s, 3H), 3.68 (s, 3H), 3.43 (t, J=7.1 Hz, 2H), 3.34 (t, J=1.9 Hz, 1H), 3.33 (s, 2H), 3.08 (s, 2H), 2.98-2.72 (m, 5H), 2.50 (d , J=16.0 Hz, 1H), 2.33 (s, 3H), 2.26 (s, 3H), 2.22-2.14 (m, 3H), 1.99 (s, 3H), 1.81 (s, 1H), 1.63-1.50 (t, J=7.4 Hz, 4H), 1.48-1.39 (m, 4H), 1.28-1.19 (m, 3H), 0.90- 0.86 (m, 6H). ESI-MS m/z: 1379.5 (M+H)+.

Example 4. Preparation of antibody-drug conjugates (ADC).

This example describes the synthesis of antibody-drug conjugates of the present invention. It should be noted that these syntheses are illustrative and that the processes described may be applicable to all compounds and antibodies described herein.

Example 4a. Production of anti-CD 13 monoclonal antibody.

Ahtu-CD13 monoclonal antibodies were prepared by well-known methods commonly used in the art. Briefly, BALB/c mice were immunized with human umbilical cord endothelial cells. For this purpose, 1.5E7 cells were injected into the mice intraperitoneally on days -45 and -30 and intravenously on day -3. On day 0, the spleens of these animals were removed and the spleen cells were fused with SP2 murine myeloma cells at a ratio of 4:1 according to standard hybridoma preparation techniques and distributed in 96-well tissue culture plates (Costar Corp., Cambridge, MA). After 2 weeks, hybridoma culture supernatants were collected and their reactivity toward the cell line used in the immunization step was tested by flow cytometry. Positive supernatants were analyzed by immunofluorescence staining of the appropriate cells used as antigens. Hybridomas showing specific staining, immunoprecipitation pattern and cell distribution were selected and cloned and subcloned by limiting dilution.

Once clones were selected, cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C for 3-4 days until the medium turned pale yellow. At this point, two-thirds of the medium was removed, centrifuged at 1000 x g for 10 min to pellet the cells, and the supernatant was either centrifuged again for further purification at 3000 x g for 10 min or filtered through 22 μm membranes. The clarified supernatant was subjected to precipitation with

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55% ammonium sulfate saturation and the resulting pellet was resuspended in 100 mM Tris-HCl pH 7.8 (1 ml per 100 ml of the original clarified supernatant) and dialyzed at 4°C for 16-24 h against 5 L of 100 mM Tris-HCl pH 7.8 with 150 mM NaCl, changing the dialysate at least three times. The dialyzed product was finally loaded onto a Protein A-Sepharose column and the corresponding monoclonal antibody was eluted with 100 mM sodium citrate pH 3.0 or, alternatively, with 1 M glycine pH 3.0. Fractions containing the antibody were neutralized with 2 M Tris-HCl pH 9.0 and then dialyzed against PBS and stored at -80°C until use.

Preparation of antibody-drug conjugate ADC1 with Trastuzumab and DL1.

(a) Receiving Trastuzumab.

Trastuzumab (purchased from Roche as a white lyophilized powder for concentrated solution for infusion) was dissolved in 5 ml of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns in phosphate buffer (50 mM, pH 8.0). Trastuzumab concentration (17.0 mg/ml) was determined by measuring absorbance at 280 nm.

(b) Partial reconstitution of Trastuzumab to yield partially reconstituted Trastuzumab.

Trastuzumab solution (0.5 mL, 8.5 mg, 56.6 nmol) was diluted to a concentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partial reduction of disulfide bonds in the antibody was performed by adding 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP) solution (34 μL, 170 nmol, 3 equiv). The reduction reaction mixture was stirred for 90 min at 20°C. Ellman's assay was performed immediately after reduction to yield a free thiol to antibody ratio (FTAR) of 5.0.

(c) Obtaining ADC1.

To a solution of partially reduced Trastuzumab (0.2 mL, 2 mg, 13.3 nmol) was added DMA (39.4 μL) followed by the addition of freshly prepared DL1 solution (10 mM in DMA, 10.6 μL, 106 nmol, 8 equiv). The conjugation reaction was stirred for 30 min at 20 °C and excess drug was quenched by the addition of N-acetylcysteine (NAC) (10 mM, 10.6 μL, 106 nmol) followed by stirring the solution for 20 min. The quenched conjugation reaction was purified by desalting using Sephadex G25 NAP-5 columns in PBS buffer. The final target product ADC1 was concentrated to a final concentration of 3.9 mg/mL by UV to yield 370 µL (1.44 mg, 9.6 nmol, 72%) of ADC solution. HIC HPLC was performed to determine the percent conjugation reaction (94%).

Preparation of antibody-drug conjugate ADC2 with Trastuzumab and compound DL2.

(a) Receiving Trastuzumab.

Trastuzumab (purchased from Roche as a white lyophilized powder for concentrated solution for infusion) was dissolved in 5 ml of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns in phosphate buffer (50 mM, pH 8.0). Trastuzumab concentration (17.1 mg/ml) was determined by measuring absorbance at 280 nm.

(b) Partial reconstitution of Trastuzumab to yield partially reconstituted Trastuzumab.

Trastuzumab solution (0.5 mL, 8.55 mg, 57 nmol) was diluted to a concentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partial reduction of disulfide bonds in the antibody was performed by adding 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP) solution (34.2 μL, 171 μmol, 3 equiv). The reduction reaction mixture was stirred for 90 min at 20°C. Ellman's assay was performed immediately after reduction, yielding a free thiol to antibody ratio (FTAR) of 6.7.

(c) Obtaining ADC2.

To a solution of partially reduced Trastuzumab (171 μL, 1.71 mg, 11.4 nmol) was added DMA (33.6 μL), followed by freshly prepared DL2 solution (10 mM in DMA, 9.1 μL, 91 nmol, 8 equiv). The conjugation reaction was stirred for 30 min at 20 °C. Excess drug was quenched by adding N-acetylcysteine (NAC) (10 mM, 9.1 μL, 91 nmol), followed by stirring the solution for 20 min. The quenched conjugation reaction was purified by desalting using Sephadex G25 NAP-5 columns in PBS buffer. The final target product ADC2 was concentrated to a final concentration of 5.14 mg/mL by UV to yield 300 µL (1.5 mg, 10 nmol, 87%) of solution. HIC HPLC was performed to determine the percent conjugation reaction (75%).

Preparation of antibody-drug conjugate ADC3 with Trastuzumab and compound DL1.

(a) Receiving Trastuzumab.

Trastuzumab (purchased from Roche as a white lyophilized powder for concentrated solution for infusion) was dissolved in 5 ml of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns in phosphate buffer (50 mM, pH 8.0). Trastuzumab concentration (16.5 mg/ml) was determined by measuring absorbance at 280 nm.

(b) Reaction of Trastuzumab with 2-iminothiolane (Troth reagent) to produce thiol-activated Trastuzumab.

Trastuzumab solution (0.5 mL, 8.25 mg, 55 nmol) was diluted to a concentration of 10 mg/mL using

- 85 048715 phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8). 14 mM Trott's reagent solution (47.1 μl, 660 nmol, 12 equiv) was added and the reaction mixture was stirred for 2 h at 20°C. The mixtures were buffer exchanged using two Sephadex G25 NAP-5 columns in PBS buffer and concentrated to a volume of 0.85 ml (9.7 mg/ml). Ellman's analysis was performed immediately afterwards, yielding a free thiol to antibody ratio (FTAR) of 5.5.

(c) Obtaining ADC3.

To a solution of thiol-activated Trastuzumab (200 μL, 1.94 mg, 12.9 nmol) was added DMA (38 μL) followed by freshly prepared DL1 solution (10 mM in DMA, 12 μL, 120 nmol, 9.3 equiv). The conjugation reaction mixture was stirred for 2 h at 25 °C and purified by desalting using a Sephadex G25 NAP-5 column in PBS buffer. The final target product ADC3 was concentrated to a final concentration of 1.48 mg/mL by UV to yield 340 μL (0.5 mg, 3.3 nmol, 25%) of ADC solution.

Preparation of antibody-drug conjugate ADC4 with Trastuzumab and DL2.

(a) Receiving Trastuzumab.

Trastuzumab (purchased from Roche as a white lyophilized powder for concentrated solution for infusion) was dissolved in 5 ml of phosphate buffer (50 mM, pH 8.0) and purified by desalting using Sephadex G25 PD-10 columns in phosphate buffer (50 mM, pH 8.0). Trastuzumab concentration (17.1 mg/ml) was determined by measuring absorbance at 280 nm.

(b) Reaction of Trastuzumab with 2-iminothiolane (Troth reagent) to produce thiol-activated Trastuzumab.

Trastuzumab solution (0.85 mL, 14.5 mg, 96.6 nmol) was diluted to a concentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 8). 14 mM Trot reagent solution (69 μL, 966 nmol, 10 equiv) was added and the reaction mixture was stirred for 2 h at 20°C. The mixtures were buffer exchanged using two Sephadex G25 NAP-5 columns in PBS buffer and concentrated to a volume of 1.45 mL (10 mg/mL). Ellman's assay was performed immediately thereafter, yielding a free thiol to antibody ratio (FTAR) of 3.7.

(c) Obtaining ADC4.

To a solution of thiol-activated Trastuzumab (290 μL, 2.9 mg, 19.3 nmol) was added DMA (57.1 μL) followed by freshly prepared DL2 solution (10 mM in DMA, 15.4 μL, 154 nmol, 8 equiv). The conjugation reaction mixture was stirred for 2 h at 25 °C and purified by desalting using a Sephadex G25 NAP-5 column in PBS buffer. The final target product ADC4 was concentrated to a final concentration of 3.82 mg/mL by UV to yield 315 μL (1.2 mg, 8.0 nmol, 41%) of ADC solution.

Example 5. In vitro bioassays for detecting antitumor activity of medicinal products according to the invention.

The purpose of this assay is to evaluate in vitro the cytostatic (ability to inhibit or stop the growth of tumor cells) or cytotoxic (ability to destroy tumor cells) activity of the test samples.

Cell lines

Name N ATCC Types Textile Characteristics A549 CCL-185 Human Lungs lung carcinoma (NSCLC) NT29 NTV-38 Human Large intestine colorectal adenocarcinoma MDA-MB-231 NTV-26 Human Breast breast adenocarcinoma PSN1 CRM- CRL-3211 Human Pancreas gland adenocarcinoma of the pancreas

Evaluation of cytotoxic activity using srb colorimetric assay.

The Sulforhodamine B (SRB) colorimetric assay is adapted to allow quantitative measurement of cell growth and viability (following the procedure described by Skehan et al., J. Nail. Cancer Inst. 1990, 82, 1107-1112).

These assays utilize 96-well microplates for cell culture (Faircloth et al., Methods in Cell Science, 1988, 11(4), 201-205; Mosmann et al., Journal of Immunological Methods, 1983, 65(1-2), 55-63). All cell lines used in this study were obtained from the American Type Culture Collection (ATCC) and were derived from different types of human cancer.

Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptokinase.

- 86 048715 mycin at 37°C, 5% CO2 and 98% humidity. For experiments, cells were harvested from subconfluent cultures using trypsinization and resuspended in fresh medium before counting and plating.

Cells were seeded in 96-well microplates at 5x103 cells/well in 150 ml aliquots and allowed to adhere to the plate surface for 18 h (overnight) in drug-free medium. One control (untreated) plate for each cell line was then fixed (as described below) and used for reference at time zero. Culture plates were then treated with test compounds (50 ml aliquots of 4x stock solutions in complete medium plus 4% DMSO) using ten serial dilutions (concentrations ranged from 10 to 0.00262 μg/ml) and triplicate cultures (1% final concentration in DMSO). After 72 h of treatment, the antitumor effect was measured using the SRB assay: Briefly, cells were washed twice with PBS, fixed for 15 min in 1% glutaraldehyde at room temperature, washed twice in PBS, and stained with 0.4% SRB for 30 min at room temperature. Cells were then washed several times with 1% acetic acid and air dried at room temperature. SRB was then extracted in 10 mM trizma base, and absorbance was measured in an automated spectrophotometric plate reader at 490 nm. Effects on cell growth and survival were assessed using the NCI algorithm (Boyd MR and Paull KD. Drug Dev. Res. 1995, 34, 91-104).

Using the mean ± standard deviation of three cultures, a dose-response curve was automatically constructed using nonlinear regression analysis. Three reference parameters (NCI algorithm) were calculated by automatic interpolation: GI ₅₀ - the concentration of the compound causing 50% inhibition of cell growth compared to control cultures; TGI - complete inhibition of cell growth (cytostatic effect) compared to control cultures; and LC ₅₀ - the concentration of the compound providing 50% of the total cytotoxic effect killing cells.

In vitro cytostatic (ability to delay or stop the growth of tumor cells) or cytostatic (ability to kill tumor cells) activities of compounds 1, 2, 3 and ET722 and other payloads are disclosed in WO 2003066638 (compounds 64, 60, 59 and 63, respectively, on pp. 149-151).

Tables 3-6 show the biological activity data of the medicinal products of the present invention together with the biological activity of the compounds of the closest prior art.

Table 3

Biological activity (molar)

Ro=H, Ri=CN

Ro=H, Ri=OH

A549 HT29 MDA- MB-231 PSN1 A549 HT29 MDA- MB-231 PSN1 GIso 8,79E-09 8,52E-09 6,66E-09 9,72E-09 TGI 14 l,73E-08 9D9E-O9 1DZE-O8 1DZE-08 LCso >l,33E-07 >l,33E-07 2,93E-08 2,26E-08 GIso l,21E-08 8,76E-09 7D4E-09 9,98E-09 ET- 1D5E-09 1D5E-09 8,91E-10 l,48E-09 TGI 15 2,56E-08 9,57E-09 1D6E-08 l,48E-08 722 1D5E-09 l,48E-09 l,48E-09 2D6E-09 LCso >l,35E-07 >l,35E-07 2DZE-O8 4,58E-08 >l,35E-07 >l,35E-07 2,56E-09 HEALTHY-O9

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Table 4

Biological activity (molar)

Medicine

Reference connection

1st

6-SR ₀ = H, R] = CN

7-SR ₀ =H, R OH

A549 HT29 MDA- MB-231 PSN1 A549 HT29 MDA- MB-231 PSN1 GIso 6-S 8,08E-09 3,33E-09 2,95E-09 3,72E-09 TGI 1D2E-08 3,59E-09 6,03E-09 5,77E-09 LCso >l,28E-07 >l,28E-07 2D4E-08 l,09E-08 GIso 7-S 8D7E-09 3,37E-09 2,85E-09 ZD1E-O9 ET722 1D5E-09 1D5E-09 8,91E-10 1D8E-09 TGI 1D8E-08 3,63E-09 4,28E-09 4D5E-09 l,35E-09 1D8E-09 1D8E-09 2D6E-09 LCso >l,30E-07 >l,30E-07 6,88E-09 6,62E-09 >l,35E-07 >l,35E-07 2,56E-09 HEALTHY-O9

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Table 5

Biological activity (molar)

Drug Reference Compound

10-S Ro=H, Ri=CN

11-S Ro=H, Ri=OH

May

Ro=H, Ri=CN

Ro=H, Ri=OH

10-R Ro=H, Ri=CN

11-R Ro=H, Ri=OH

A549 HT29 MDA- MB-231 PSN1 A549 HT29 MDA- MB-231 PSN1 GIso 10-S 4,32E-08 l,23E-08 l,20E-08 8,64E-09 2 l,28E-08 5DZE-09 5,00E-09 2,05E-09 TGI l,05E-07 l,23E-08 l,23E-08 l,48E-08 l,28E-08 5,64E-09 5,26E-09 3,08E-09 LCso >l,23E-06 >l,23E-06 l,36E-08 >l,23E-06 >l,28E-06 >l,28E-06 5,77E-09 5,00E-09 GIso 10-R 4D2E-09 8,64E-10 6.79E-10 7,53E-10 TGI l,20E-08 l,61E-09 l,21E-09 l,48E-09 LCso >l,23E-07 >l,23E-07 3,09E-09 >l,23E-07 GIso 11-S 6,62E-08 1D7E-08 l,05E-08 l,62E-08 1 l,82E-09 1D8E-09 7.52E-10 1D2E-09 TGI >l,25E-07 2,50E-08 l,87E-08 2,37E-08 3,37E-09 1DZE-O9 1D0E-09 l,69E-09 LCso >l,25E-07 >l,25E-07 4,50E-08 >l,25E-07 7,78E-09 >l,30E-07 2,46E-09 2D4E-09 GIso 11-R l,50E-08 2,00E-09 l,62E-09 2D2E-09 TGI 4,50E-08 3,62E-09 2,87E-09 3,62E-09 LCso >l,25E-07 >l,25E-07 7,24E-09 l,50E-08

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Table 6

Biological activity (molar)

Medicine

Reference connection

18-S Ro=H, Ri=CN

19-S Ro=H, Ri=OH

A549 HT29 MDA-MB231 PSN1 A549 HT29 MDA- MB-231 PSN1 GIso 18- S 8,08E-09 3,33E-O9 2,95E-09 3,72E-09 TGI 1D2E-08 3,59E-09 6,03E-09 5,77E-09 LCso >l,28E-07 >l,28E-07 2,44E-08 l,09E-08 GIso 19S 8D7E-09 3,37E-09 2,85E-09 ZD1E-O9 ET- 722 l,35E-09 l,35E-09 8,91E-10 l,48E-09 TGI l,28E-08 3,63E-09 4,28E-09 4D5E-09 l,35E-09 l,48E-09 l,48E-09 2D6E-09 LCso >l,30E-07 >l,30E-07 6,88E-09 6,62E-09 >l,35E-07 >l,35E-07 2,56E-09 HEALTHY-O9

Example 6. Demonstration of cytotoxicity of antibody-drug conjugates of the present invention.

Bioassays for determination of antitumor activity.

The purpose of this assay was to evaluate in vitro the cytostatic (ability to delay or stop the growth of tumor cells) or cytotoxic (ability to kill tumor cells) activity of the test samples.

Cell lines and cell culture.

The following human cell lines were obtained from the American Type Culture Collection (ATCC): SK-BR-3 (ATCC HB-30), HCC-1954 (ATCC CRL-2338) (breast cancer, HER2+); MDA-MB-231 (ATCC HTB-26), and MCF-7 (ATCC HTB-22) (breast cancer, ^HER2- ). Cells were maintained at 37°C, 5% CO2, and 95% humidity in Dulbecco's modified Eagle's medium (DMEM) (for SK-BR-3, MDA-MB-231, and MCF-7 cells) or RPMI-1640 (HCC-1954), all media supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, and 100 units/ml penicillin and streptomycin.

Cytotoxicity assay.

For SK-BR-3, HCC-1954, MDA-MB-231 and MCF-7 cells, the Sulforhodamine B (SRB) colorimetric assay was adapted for quantitative measurement of cell growth and cytotoxicity as described by V. Vichai and K. Kirtikara. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols, 2006, 1, 1112–1116. Briefly, cells were seeded in 96-well microtiter plates and incubated for 24 h in drug-free medium, then treated with vehicle or test substances only for 72 h. For quantification, cells were washed twice with phosphate-buffered saline (PBS), fixed for 15 min in 1% glutaraldehyde, washed twice with PBS, stained in 0.4% (w/v) SRB with 1% (v/v) acetic acid for 30 min, washed several times with 1% acetic acid, and air-dried. SRB was then extracted into 10 mM Trizma base and the absorbance was measured at 490 nm in a microplate reader.

Cell survival was expressed as a proportion of the survival of untreated control cells. All assessments were performed in triplicate and the data were fitted by nonlinear regression to a four-parameter logistic curve, from which the IC ₅₀ value (the concentration of the compound causing 50% cell death compared to the survival of control cells) was calculated.

Bioactivity example 1. Cytotoxicity of ADC1 conjugate and related reagents against HER2 positive and negative breast cancer cells.

In vitro cytotoxicity of ADC1 together with parent cytotoxic compounds 1 and Trastuzumab was assessed against four different human breast cancer cell lines, overexpressing or not the HER2 receptor, including SK-BR-3, HCC-1954 (HER2-positive cells), and MDA-MB-231 and MCF-7 (HER2-negative cells). Standard dose-response curves were generated.

- 90 048715 (DZ) for 72-hour incubation with test compounds.

Trastuzumab cytotoxicity.

In vitro cytotoxicity of Trastuzumab was assessed against different tumor cell lines by performing triplicate D3 curves of 10-point, 2.5-fold dilutions from 50 to 0.01 μg/mL (3.33E-078.74E-11 M). Trastuzumab was completely inactive, did not achieve IC ₅₀ in any of the cell lines tested, regardless of their HER2 status, as shown in Table 7, which presents the geometric mean IC ₅₀ values obtained in three independent experiments.

Table 7

Brief description of in vitro cytotoxicity of Trastuzumab___________

HER2 positive HER2 negative SK-BR-3 NSS-1954 MDA-MB-231 MCF-7 > 50 > 50 > 50 > 50 > 3.4E-07 > 3.4E-07 > 3.4E-07 > 3.4E-07

Cytotoxicity 1.

The cytotoxicity of payload 1 was assessed against different tumor cell lines by obtaining triplicate DZ curves of 10-point, 2.5-fold dilutions from 100 to 0.03 ng/mL (1.26E-073.3E-11 M).

As shown in Table 8, which presents the geometric mean IC ₅₀ values obtained from three independent experiments, the cytotoxicity of this compound was similar in all tumor cell lines regardless of their HER2 expression, with IC ₅₀ values in the low nanomolar range, from 8.82E-10 to 1.95E-09 M. The geometric mean IC ₅₀ values across the entire cell panel were 1.32E-9 M.

Table 8

Brief description of in vitro cytotoxicity 1 HER2 positive HER2 negative SK-BR-3 NSS-1954 MDA-MB-231 MCF-7 1C ₅ 0, mcg/ml 8,60E-04 1,50E-03 6,80E-04 1,20E-03 IC50, M 1D2E-09 1,95E-09 8.82E-10 1,56E-09

Cytotoxicity of ADC1.

The cytotoxicity of ADC1 was assessed against different tumor cell lines by performing triplicate D3 curves of 10-point, 2.5-fold dilution from 100 μg/mL to 26 ng/mL (6.67E-07 - 1.75E-10 M). The assessment was performed in three independent experiments and Table 9 summarizes the results corresponding to the geometric mean IC ₅₀ values obtained in three independent experiments. As shown in Table 9, ADC1 showed cytotoxicity similar to the parent drug 1 only in HER2-positive cells. However, in HER2-negative cells, such toxicity was significantly lower: about 8-fold lower according to the selectivity index obtained by dividing the mean IC ₅₀ value in HER2-negative cells by that in HER2-positive cells. This selectivity suggests that the ADC1 conjugate acts through interaction of the antibody with the membrane-associated HER2 receptor on tumor cells, followed by intracellular delivery of the cytotoxic drug.

Table 9

Brief description of in vitro activity of ADC1

HER2 positive HER2 negative IC50B HER2+ (cpgeom.) 1C ₅₀ in HER2(cpgeom. Index select. SK-BR-3 NSS- 1954 MDA- MB-231 MCF-7 IC50 (mcg/ml) 9,00E-01 1.00E+00 5.70E+00 1.00E+01 9,49E-01 7.55E+00 8.0 IC50 (M)) 6,00E-09 6,67E-09 3,80E-08 6,67E-08 6,ZZE-09 5,OZE-08

Bioactivity example 2: Cytotoxicity of ADC2 conjugate and related reagents against HER2 positive and negative breast cancer cells.

In vitro cytotoxicity of ADC2 together with the parent cytotoxic compound 2 was assessed against four different breast cancer cell lines, overexpressing or not the HER2 receptor, including

- 91 048715 XK-BR-3, HCC-1954 (HER2 positive cells), and MDA-MB-231 and MCF-7 (HER2 negative cells) teas. Standard dose-response (DRP) curves were obtained for 72 h of incubation with the test compounds. The results were also compared with the monoclonal antibody Trastuzumab described above.

Cytotoxicity 2.

The cytotoxicity of intermediate 2 was assessed against different tumor cell lines by performing triplicate 10-point, 2.5-fold dilution D3 curves from 100 to 0.03 ng/mL (1.26E-07 to 3.3E-11 M). As shown in Table 10, which presents the geometric mean IC50 values obtained from three independent experiments, the cytotoxicity of this compound was similar in all tumor cell lines regardless of their HER2 expression, with IC50 values in the low nanomolar range, from 8.85E-10 to 2-31E-09 M. The geometric mean IC50 values across the entire cell panel were 1.53E-09 M.

Table 10

Brief description of in vitro cytotoxicity 2

ICso, mcg/ml

ICso, M

HER2 positive HER2 negative SK-BR-3 NSS-1954 SK-BR-3 NSS-1954 9,60E-04 1,80E-03 6,90E-04 1,70E-03 1,23E-09 2,31E-09 8.85E-10 2,18E-09

ADC2 cytotoxicity.

The cytotoxicity of ADC2 was assessed against different tumor cell lines by performing triplicate D3 curves of 10-point, 2.5-fold dilution from 100 μg/mL to 26 ng/mL (6.67E-07 - 1.75E-10 M). The assessment was performed in three independent experiments, and Table 11 summarizes the results corresponding to the geometric mean IC50 values obtained in three independent experiments. As shown in Table 11, ADC2 showed cytotoxicity similar to parent drug 2 only in HER2-positive cells. However, in HE112-negative cells, such toxicity was significantly lower as determined by the selectivity index obtained by dividing the mean IC50 value in HER2-negative cells by that in HER2-positive cells. This selectivity suggests that ADC2 acts through interaction of the antibody with the membrane-associated HER2 receptor on tumor cells, followed by intracellular delivery of the cytotoxic drug.

Table 11

Brief description of in vitro cytotoxicity of ADC2

HER2 and positive HER2 negative ICso in HER2+ (avg. geom.) ICso in HER2(avg. geom.) Index select. SK-BR-3 NSS-1954 SK-BR-3 NSS- 1954 1C ₅₀ (mcg/ml) 8.50E+00 1.80E+01 >1.0E+02 >1.0E+02 1.24E+01 >1.0E+02 >8.09 1C ₅₀ (M) 5,67E-08 1,20E-07 >6.67E-07 >6.67E-07 8,25E-08 >6.67E-07

Bioactivity example 3. Cytotoxicity of ADC3 conjugate and related reagents against HER2 positive and negative breast cancer cells.

In vitro cytotoxicity of ADC3 was assessed against four different human breast cancer cell lines, overexpressing or not the HER2 receptor, including SK-BR-3, HCC-1954 (HER2-positive cells), and MDA-MB-231 and MCF-7 (HER2-negative cells). Standard dose-response curves (DRCs) were generated for 72-hour incubations with test compounds.

ADC3 cytotoxicity.

The cytotoxicity of ADC3 was assessed against different tumor cell lines by performing triplicate D3 curves of 10-point, 2.5-fold dilution from 100 μg/mL to 26 ng/mL (6.67E-07 - 1.75E-10 M). The assessment was performed in three independent experiments and Table 12 summarizes the results corresponding to the geometric mean IC50 values obtained in three independent experiments. As shown in Table 12, ADC3 showed cytotoxicity similar to the parent drug 1 only in HER2-positive cells. However, in HER2-negative cells, such toxicity was significantly lower: about 56-fold lower according to the selectivity index obtained by dividing the mean IC50 value in HER2-negative cells by that in HER2-positive cells. This selectivity allows us to conclude that this conjugate acts through the interaction of the antibody with the membrane-associated HER2 receptor on tumor cells, followed by

-

Claims (2)

intracellular delivery of a cytotoxic drug. Table 12 In vitro activity of ADC3 HER2 positive HER2 negative ICso in HER2+ (cpgeom.) ICso in HER2(cpgeom.) Index select. SK-BR-3 НСС- 1954 MDA- MB-231 MCF-7 1C ₅₀ (µg/ml) 2.50E-01 2.70E-01 1.40E+01 1.70E+01 2.60E-01 1.94E+00 55.7 1C ₅ 0 (M) 1.67E-09 1.80E-09 9.33E-08 1.00E-07 1.73E-09 9.66E-08 Bioactivity Example 4: Demonstration of in vivo efficacy of antibody-drug conjugates of the present invention. In vitro cytotoxicity of ADC4 alone was assessed against four different human breast cancer cell lines, HER2 receptor overexpressing or not, including SK-BR-3, HCC-1954 (HER112-positive cells), and MDA-MB-231 and MCF-7 (HER2-negative cells). Standard dose-response curves (DRCs) were generated for 72-hour incubations with test compounds. Cytotoxicity of ADC4. The cytotoxicity of ADC4 was assessed against different tumor cell lines by performing triplicate D3 curves of 10-point, 2.5-fold dilution from 100 μg/mL to 26 ng/mL (6.67E-07 - 1.75E-10 M). The assessment was performed in three independent experiments and Table 13 summarizes the results corresponding to the geometric mean IC ₅₀ values obtained in three independent experiments. As shown in Table 13, ADC4 showed cytotoxicity similar to the parent drug 2 only in HER2-positive cells. However, in HER2-negative cells, such toxicity was significantly lower: about 14-fold lower according to the selectivity index obtained by dividing the mean IC ₅₀ value in HER2-negative cells by that in HER2-positive cells. This selectivity suggests that the ADC4 conjugate acts through interaction of the antibody with the membrane-associated HER2 receptor on tumor cells, followed by intracellular delivery of the cytotoxic drug. Table 13 In vitro activity of ADC4 HER2 positive HER2 negative ICso in HER2+ (avg. geom.) ICso in HER2(avg. geom.) Selective index SK-BR-3 НСС-1954 MDA- MB-231 MCF-7 1C ₅₀ (µg/ml) ZD0E-01 6,ZOE-01 7.00E+00 5.40E+00 4.42E-01 6D5E+00 13.91 1C ₅ o (M) 2.07E-09 4.20E-09 4.67E-08 3.60E-08 2.95E-09 4D0E-08 CLAUSE OF THE INVENTION 1. A drug conjugate comprising a drug moiety covalently attached to the remainder of the drug conjugate, having the formula [D-(X)b-(AA) _w -(T) _g -(L)-]n-Ab, where D is a moiety of a drug of the following formula (IH), or a pharmaceutically acceptable salt or stereoisomer thereof - 93 048715 where the wavy line denotes the point of covalent attachment to (X) _b if present, or to (AA) _w if present, or to (T)g if present, or to (L); Y represents -NH- or -O-; R1 represents -OH or -CN; R ₂ represents the group -C(=O)R _a ; R ₃ represents hydrogen or the -OR _b group; R4 is selected from hydrogen, -CH2OH and -CH ₂ NH ₂ ; Ra is unsubstituted C ₁ -C ₁₂ -alkyl; R _b is unsubstituted C ₁ -C ₁₂ -alkyl; X represents an extending group selected from: -COO-(C ₁ -C ₆ -alkylene)NH-; -COO-CH ₂ -(unsubstituted phenylene)-NH-; -COO-(C ₁ -C ₆ -alkylene)NH-COO-CH ₂ -(unsubstituted phenylene)-NH-; -COCH2NH-COCH2-NH-; -COCH2NH-; -COO-(C ₁ -C ₆ -alkylene)S-; -COO-(C1-C6-alkylene)NHCO(C1-C6-alkylene)S-; T is an extension group selected from the group consisting of -CO-(C ₁ -C ₆ -alkylene)-NH-, -CO-(C ₁ -C ₆ -alkylene)-[O-(C ₂ -C ₆ alkylene)]j-NH-, -COO-(C ₁ -C ₆ -alkylene)-[O-(C ₂ -C ₆ alkylene)]j-NH-; where j is an integer from 1 to 25; (AA) _w is represented by formula (II) ^L 21 (II) where the wavy lines represent the point of covalent attachment to (X) _b , if present, or to the drug moiety (wavy line on the left) and to (T)g, if present, or to the linker (wavy line on the right); and R ₂₁ in each case is selected from the group consisting of hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, -CH2CH2COOH, (CH)3NHC(=NH)NH2, -(CH2bNH2, -(CH2^NHC(=NH)NH2, > 10 -(CH ₂ ) ₄ NH ₂ , (CH ₂ ) ₃ NHCONH ₂ , -(CH ₂ ) ₄ NHCONH ₂ , phenyl, cyclohexyl, / „ and w is an integer in the range from 0 to 6; L is a linker group selected from the group consisting of where the wavy lines represent the point of covalent attachment to Ab (wavy line on the right) and to - 94 048715 (T)g if present, or (AA) _w if present, or (X) _b if present, or D (wavy line on the left); R19 is unsubstituted -C ₁ -C ₁₂ -αalkylene-; R30 represents a -C ₁ - ₆ -alkylene- group; M is unsubstituted -C ₁ - ₆ -alkylene-; b is equal to the integer 0 or 1; g is equal to the integer 0 or 1; Ab is an anti-HER2 antibody or antigen-binding fragment thereof; and n is the ratio of the [D-(X) _b -(AA) _w -(T) _g -(L)-] group to Ab and ranges from 1 to 20. 2. A drug conjugate according to claim 1, wherein D is a compound of the formula or a pharmaceutically acceptable salt thereof. 3. A drug conjugate according to claim 1, wherein D is a compound of the formula or a pharmaceutically acceptable salt thereof. 4. A drug conjugate according to any one of claims 1 to 3, wherein the salt is selected from hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate, para-toluenesulfonate, sodium, potassium, calcium, ammonium, ethylenediamine, ethanolamine, N,N-diα-alkyleneethanolamine, triethanolamine and basic amino acids. 5. A drug conjugate according to any one of claims 1 to 4, selected from formulas (IV), (V) and (VI) ABOUT. D-(X) _b -(AA) _w —(T) _g -C—R19-N. (V) D-(X) _b -(AA) _w —(T) _d O Ab (IV) D-(X) _b -(AA) _w —(T)gC—R19-I Ab (VI) where X, T, AA, w, b, g, D, Ab and n are as defined in any of paragraphs 1-4; R ₁₉ is unsubstituted -C ₁ -C ₈ -alkylene-; R ₃₀ represents a -C ₂ -C ₄ -alkylene- group; and M is unsubstituted -C ₁ -C ₃ -αalkylene-; or selected from formulas (IV), (V) and (VI) - 95 048715 (VI) where X, T, AA, w, b, g, D, Ab and n are as defined in any of paragraphs 1-4; R19 is unsubstituted -C ₁ - ₆ -αalkylene-; R30 represents a -C ₂ -C ₄ -alkylene- group; and M is unsubstituted -C ₁ -C ₃ -alkylene-. 6. A drug conjugate according to any one of claims 1 to 5, wherein (AA) _w is represented by formula (II), where R21 in each case is selected from the group consisting of hydrogen, methyl, isopropyl, sec-butyl, benzyl, _{indolylmethyl} , -( _CH2 ) _3NHCONH2 , -(CH2) _4NH2 , -(CH2) _3NHC (-NH)NH2 and -( _CH2 ) _4NHC (=NH) _NH2 ; or where w is 0 or 2, and when w is 2, then (AA) _w is represented by formula (III): η ^н ? ²² Ч ₄ ΑχΝ-Α _Ν Α I II н R ₂ 3 O (III) , where the wavy lines represent the point of covalent attachment to (X) _b , if present, or to the drug moiety (wavy line on the left) and to (T) _g , if present, or to the linker (wavy line on the right); R ₂₂ is selected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl; and R23 is selected from methyl, -(CH ₂ ) ₄ NH ₂ , -(CH ₂ ) ₃ NHCONH ₂ and -(CH ₂ ) ₃ NHC(=NH)NH ₂ . 7. A drug conjugate according to any one of claims 1 to 6, wherein X is a stretching group selected from the group consisting of the following groups: -COO-(C ₂ -C ₄ -αalkylene)NH-; -COO-CH ₂ -phenylene-NH-; -COO-(C ₂ -C ₄ -alkylene)NH-COO-CH ₂ -(phenylene)-NH-; -COCH2NH-COCH2-NH-; -COO-(C ₂ -C ₄ -alkylene)S-; -COO-(C ₂ -C ₄ -alkylene)NHCO(C ₁ -C ₃ -alkylene)S-; and b is 0 or 1; or where X is an extending group selected from the group consisting of the following groups: -COO-CH ₂ -phenylene-NH-; -COO(CH ₂ ) ₃ NHCOOCH ₂ -phenylene-NH-; -COO(CH2)3NH-; -COO(CH2)3-S-; -COO(CH2)3NHCO(CH2)2S-; and b is 0 or 1. 8. A drug conjugate according to claim 7, where b is equal to 1. 9. A drug conjugate according to any one of claims 1 to 8, wherein T is a stretching group selected from the group consisting of groups -CO-(C ₁ -C ₄ alkylene)NH-, -CO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-, -COO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-, where j is an integer from 1 to 10; and g is 0 or 1; or where T is an extending group selected from the group consisting of groups - 96 048715 -CO-(C ₁ -C ₄ alkylene)NH-, -CO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-, -COO-(C ₁ -C ₄ alkylene)-[O-(C ₂ -C ₄ alkylene)]j-NH-; where j is an integer from 1 to 5; and g is 0 or 1. 10. A drug conjugate according to any one of claims 1 to 9, wherein D is a drug moiety of formula (IH), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1 represents CN or OH; R2 is C(=O)R _a , where R _a is unsubstituted C ₁ -C ₆ alkyl; R3 represents hydrogen or an -ORb group, where Rb represents an unsubstituted C _1-6 alkyl group, R4 is selected from hydrogen, -CH2OH and -CH2NH2; and Y represents -NH- or -O-; or where D is a moiety of a drug of formula (IH), or a pharmaceutically acceptable salt or stereoisomer thereof, where R ₁ represents CN or OH; R2 is acetyl; R ₃ represents hydrogen or methoxy; R ₄ is hydrogen or -CH ₂ OH; and Y represents -NH- or -O-; or where D is a moiety of a drug of formula (IH), or a pharmaceutically acceptable salt or stereoisomer thereof, where R ₁ represents CN; R2 is acetyl; R3 is methoxy; R ₄ represents hydrogen; and Y represents -NH- or -O-. 11. A drug conjugate according to claim 10, wherein R3 is methoxy. 12. A drug conjugate according to claim 10, wherein Y is -NH-. 13. A drug conjugate according to any one of claims 1 to 12, wherein D is selected from O; AsO O; AcO or a pharmaceutically acceptable salt or stereoisomer thereof, where the wavy line denotes the point of covalent attachment to (X)b if present, or to (AA) _w if present, or to (T) _g if present, or to (L). 14. A drug conjugate according to any one of claims 1 to 13, wherein the Ab moiety is trastuzumab, or an antigen-binding fragment or immunologically active portion thereof. 15. A drug conjugate according to claim 1, which is an antibody-drug conjugate selected from the group consisting of: - 97 048715 - 98 048715 where n is from 2 to 6, and each is an aHTu-HER2 antibody such as trastuzumab, or an antigen-binding fragment or immunologically active portion thereof. 16. A drug conjugate according to claim 15, wherein each is trastuzumab, or its antigen-binding fragment, or an immunologically active portion. 17. A drug conjugate according to claim 15, wherein n is 3, 4 or 5. 18. A drug conjugate according to any one of claims 1 to 17, which is an antibody-drug conjugate in isolated or purified form. 19. A compound of the formula D-(X) _b -(AA) _w -(T) _g -L ₁ or the formula D-(X) _b -(AA) _w -(T) _g -H, where L1 is a linker group: where the wavy line denotes the point of covalent attachment to (T) _g if present, or to (AA) _w if present, or to (X) _b if present, or to D; R19 is unsubstituted -C ₁ -C ₁₂ -alkylene-; b is equal to the integer 0 or 1; g is equal to the integer 0 or 1; w is an integer in the range 0 to 6; where, for compounds of the formula D-(X-) _b -(AA) _w -(T) _g -H, b+w+g^0; each of D, X, T and AA is as defined in any one of paragraphs 1-18. 20. The compound according to claim 19 of the formula 21. A compound of formula D-(X) _b -(AA) _w -(T) _g -L1 or formula D-(X) _b -(AA) _w -(T) _g -H, wherein each of D, X, AA, T, L1, b, g and w is as defined in any one of claims 1 to 18; but additionally wherein if the compound is a compound of formula D-(X) _b -(AA) _w -(T) _g -H, then b+w+g^0. 22. A drug conjugate according to any one of claims 1 to 18, wherein b+g+w is not equal to 0; or where b+w is not equal to 0; or where, when w is not 0, then b is 1; - 99 048715 or where when w is 0 then b is 1. 23. Use of a drug conjugate according to any one of claims 1 to 18 as a drug. 24. The use according to claim 23, wherein the medicinal product is a medicinal product for the treatment of a cancer selected from HER2 positive lung cancer, including HER2 positive NSCLC, HER2 positive gastric cancer, HER2 positive colorectal cancer, HER2 positive breast cancer, HER2 positive pancreatic carcinoma, HER2 positive endometrial cancer, HER2 positive bladder cancer, HER2 positive cervical cancer, HER2 positive esophageal cancer, HER2 positive gallbladder cancer, HER2 positive uterine cancer, HER2 positive salivary duct cancer and HER2 positive ovarian cancer. 25. The use according to claim 24, wherein the medicinal product is a medicinal product for the treatment of a cancer selected from HER2 positive cancer, selected from HER2 positive breast cancer, HER2 positive ovarian cancer and HER2 positive gastric cancer. 26. The use according to claim 25, wherein the medicinal product is a medicinal product for the treatment of HER2 positive breast cancer. 27. A pharmaceutical composition comprising a drug conjugate according to any one of claims 1-18 and a pharmaceutically acceptable carrier. 28. A method for the prevention or treatment of cancer, comprising administering to a patient in need thereof an effective amount of a drug conjugate according to any one of claims 1 to 18; wherein the cancer is selected from HER2 positive lung cancer, including HER2 positive NSCLC, HER2 positive gastric cancer, HER2 positive colorectal cancer, HER2 positive breast cancer, HER2 positive pancreatic carcinoma, HER2 positive endometrial cancer, HER2 positive bladder cancer, HER2 positive cervical cancer, HER2 positive esophageal cancer, HER2 positive gallbladder cancer, HER2 positive uterine cancer, HER2 positive salivary duct cancer, and HER2 positive ovarian cancer. 29. The method according to claim 28, wherein the cancer is a HER2 positive cancer selected from HER2 positive breast cancer, HER2 positive ovarian cancer and HER2 positive gastric cancer. 30. The method of claim 29, wherein the cancer is HER2 positive breast cancer. 31. A drug conjugate according to any one of claims 1 to 18, wherein n is in the range of 1-12, 1-8, 3-8, 3-6, 3-5, is 1, 2, 3, 4, 5 or 6. 32. A drug conjugate according to claim 31, wherein n is 3, 4 or 5. 33. A drug conjugate according to claim 32, where n is 4. 34. A method for producing an antibody-drug conjugate according to any one of claims 1 to 18, comprising conjugating a moiety Ab containing at least one antigen-binding site and a drug D, wherein Ab and D are as defined in any one of claims 1 to 18; where obtaining an antibody-drug conjugate of formula (G) or (G'): (θ’), includes the following stages: (1) interaction of the drug D-H of the formula (IH)-H: - 100 048715 where the substituents in definitions (III)-H are as defined in any of paragraphs 1-18, with a compound of formula (D') or (E): to obtain a compound of formula (F) or (F'), respectively: (2) partial reduction of one or more disulfide bonds in the antibody to be conjugated to yield a reduced antibody Ab-SH having free thiol groups: AЬ reduction / \ --------► Ab-SH s---s disulfide bonds (3) reaction of the partially reduced antibody Ab-SH having free thiol groups with the compound of formula (F) or (F') obtained in step (1) to obtain the desired antibody-drug conjugate of formula (G) or (G'), respectively: - 101 -