RU2319482C2 - Method for identification of anti-tumor target enzymes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to identification of enzyme used for production of preparation for anti-tumor agent delivery, wherein said agents are selectively converted to active substances in tumor tissue or cells. Primarily gene expression levels in healthy tissues or normal cells are compared with gene expression levels in human tumor tissue or tumor originated cells. Further enzyme is selected which has expression level in tumor tissue or cells exceeding expression level in healthy tissues or normal cells by two or more time. Preparation for anti-cancer agent delivery is prepared. Namely cells are cultivated in presence or in absence of tested compound wherein cDNA of identified enzyme is included into abovementioned cells. Then ability of tested agent to selectively convert into active substance in tumors is determined.

EFFECT: method for tumor treatment without side effects.

14 cl, 50 ex

Description

The present invention relates to a method for identifying enzymes that are predominantly expressed in certain tumor tissues compared to rapidly growing normal cells or tissues, the use of these enzymes for the development of a compound intended for the selective formation of an active anticancer substance in tumor tissue, compounds based on these enzymes , their pharmaceutically acceptable salts, as well as pharmaceutical compositions based on them.

One of the most serious and most important problems in the field of medicinal medicine are side effects. Side effects of drugs are mainly caused by their nonspecific effect; drugs interact and affect not only target molecules, but also other molecules that play an important role in maintaining normal physiological processes. Another important cause of side effects is the non-specific distribution of drugs in many tissues; drugs are introduced not only into damaged tissues, but also into other tissue, which should not be affected to maintain normal physiological functions. Target molecules of most anti-cancer drugs are ubiquitously expressed in many tissues and are not specific to a particular tissue. On the other hand, the disease is usually caused by a dysregulation of certain molecules in certain tissues. Thus, in order to eliminate side effects, it is necessary to establish methods by which drugs affect certain molecules only in the specific tissue responsible for the disease, and provide drugs developed using the above methods.

Among many diseases in the treatment of cancer patients, the side effects of drugs are of particular importance. Cytotoxic drugs are widely used to treat cancer, and their regular use for chemotherapeutic treatment of cancer will continue for at least the next decade. However, the use of cytotoxic drugs is limited by their lack of effectiveness and severe side effects. In tumor tissues, many cytotoxic drugs, for example 5-FU, 2'-deoxycytidines, methotrexate, camptothecins and taxanes, affect tumor cells located in the S or M phase of the cell cycle, i.e. during DNA synthesis or mitosis. However, growing tumor cells of the tumor tissue are in various stages of the cell cycle, and only a small fraction of the tumor cells are in S or M phases. Therefore, the duration of action of an ideal drug should be at least longer than the time required to complete one cell cycle (lasting 20-40 hours), and the sequential daily or continuous treatment aimed at influencing cytotoxic drugs is the ideal dosage regimen all cancer cells, presence in tumor tissue. However, treatment with cytotoxic drugs in such dosing regimens has a severe toxic effect on rapidly growing healthy cells, especially blood-forming progenitor cells and intestinal crypt cells. Myelosuppression resulting from toxic effects on hematopoietic progenitor cells is the most common side effect of cytotoxic drugs and often leads to a deterioration of the host immune response and fetal infection. When symptoms of myelosuppression occur, it usually takes 2-3 weeks to recover from myelotoxicity, and this fact is the main reason for using cytotoxic drugs once every 3-4 weeks. However, this intermittent dosage regimen is responsible for the insufficient effectiveness of most existing cytotoxic drugs.

Some new antitumor agents with a new type of action are being developed. However, they also have problems associated with safety of action due to insufficient antitumor selectivity. Apparently, myelotoxicity and skin ulcers, respectively, are the main toxicity factors of farnesyl transferase inhibitors and tyrosine kinase inhibitors of the epidermal growth factor receptor (EGF). Apparently, this is due to the fact that the target enzyme or protein is overexpressed not only in the tumor tissue, but also in other healthy tissues, such as bone marrow and skin.

On the other hand, capecitabine (oral fluoropyrimidine) is a cytotoxic drug that is sequentially converted into the active 5-FU drug by enzymes that are intensively expressed in the liver and tumors, but not in the growing bone marrow cells [Miva. M. et al. Design of oral fluoropyrimidine carbamate, capecitabine, which generates 5-furuoloyracil selectiviely in tumors by enzymes concentrated in human liver and cancer tissue. Eur. J. Cancer 34, 1274-1281 (1998)]. As a result, the drug in question provides high concentrations of 5-FU directly in the tumor tissue and shows improved efficacy profiles compared to 5-FU profiles. In addition, the drug in question has low myelotoxicity. Such characteristics allow the use of such a drug in daily treatment with high dosages, even for a long time. Currently, this drug is prescribed for the treatment of breast cancer, colorectal cancer and other types of cancer. Despite this, it is still difficult to identify anticancer drugs with increased efficacy and safe use limits like capecitabine, since no approach has been developed to accurately identify enzymes and / or proteins among those expressed in various tissues.

The present invention relates to methods for detecting enzymes for constructing compounds capable of selectively being converted into active substances in tumors but not in healthy cells (hereinafter referred to as antitumor target cytotoxicants, (TTCs)), in particular granulocyte precursors that are predominantly present in bone marrow . Antitumor cytotoxicants should selectively affect the tumor with only minor myelotoxicity. Such compounds should be safely prescribed in high doses for a long time, providing higher safety and efficacy than existing cytotoxicants. The subject compounds can reduce hospitalization associated with the manifestation of side effects, and can be safely assigned to outpatients. Other advantages of antitumor target cytotoxicants are that they allow for individual healing procedures (adaptation therapy) as a result of measuring the expression levels of their activation enzymes (TTC activation enzymes). Individual tumors expressing high levels of TTC-activating enzymes can efficiently generate active drugs from antitumor cytotoxicants, and therefore they can be highly sensitive to antitumor cytotoxicants.

The purpose of the present invention is to develop methods for detecting enzymes for the production of anticancer compounds capable of selectively being converted into active substances in tumors, and such methods include measuring expression levels of genes and / or proteins in human tissue and / or cells of normal or tumor origin, comparing the measured levels expression and selection of enzymes in which the levels of mRNA and / or proteins in the tumor tissue are more than two times higher than in normal growing cr vetvornyh predecessors, the intestine and / or the skin.

Another objective of the present invention is to develop methods for identifying anticancer compounds capable of selectively converting into active substances in tumors, including the steps of generating cells expressing an enzyme with more than twice as much protein in tumor tissue as compared to healthy cells and tissues, and determining activities growth factors of these anti-cancer compounds.

Another objective of the present invention is to develop anti-cancer compounds of the formula (I)

X-Y-Q (I),

wherein

X is a profragment designed to generate an active anticancer substance (Q-Y-H) directly in tumors under the influence of the enzymes of the present invention;

Q-Y- is a radical derived from an active anti-cancer substance (Q-Y-H), in which Y is

-O-, -S- or -N-,

and their pharmaceutically applicable salts.

Another objective of the present invention is to provide anti-cancer compounds represented by the formula (II),

wherein

Q and Y have the above meanings,

R ⁰ represents a side chain of a natural or synthetic amino acid,

Z represents (C1-C3) alkylene or a group —O — CH (R ³ ) -, in which R ³ represents hydrogen or straight chained (C1-C4) alkyl,

R ¹ represents hydrogen or methyl, and

R ² represents hydrogen, branched (C3-C10) alkyl or (C3-C8) cycloalkyl,

or their pharmaceutically applicable salts.

Another objective of the present invention is to develop anti-cancer compounds represented by formula (III),

wherein

R ⁰ has the above meanings,

R ⁴ represents benzoyl or tert-butoxycarbonyl,

R ⁵ represents hydrogen or acetyl,

or their pharmaceutically applicable salts.

Another objective of the present invention is to provide anti-cancer compounds represented by the formula (IV),

wherein

R ⁰ , R ¹ , R ² and R ³ have the above meanings,

R ⁶ represents hydrogen, fluorine, hydroxyl or cyano,

R ⁷ represents hydrogen, fluorine or hydroxy,

or R ⁶ and R ⁷ together form methylidene or fluoromethylidene,

R ⁸ represents hydrogen or ethynyl,

R ⁹ represents hydrogen, fluorine, vinyl or ethynyl, and

R ¹⁰ represents hydrogen or hydroxy

or their pharmaceutically applicable salts.

Another objective of the present invention is to provide anti-cancer compounds represented by formula (V),

in which m is an integer equal to 2 or 3,

R ⁰ , R ² , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ have the above meanings,

and their pharmaceutically applicable salts.

Another objective of the present invention provides anticancer compounds represented by formula (VI),

wherein

m is an integer in the range of 1-3,

n is an integer equal to 0 or 1,

R ⁰ has the above meanings,

R ¹¹ represents hydrogen or fluorine,

R ¹² represents hydrogen, fluorine, methyl or hydroxy,

R ¹³ represents hydrogen, amino, nitro or (dimethylamino) methyl,

R ¹⁴ represents hydrogen, (C1-C4) alkyl, 4-methylpiperazinylmethyl, tert-butoxyiminomethyl, or R ¹³ and R ¹⁴ or R ¹¹ and R ¹² taken together can form a five- or six-membered ring, which may contain one or two heteroatoms and may optionally be substituted with (C1-C8) alkyl, amino, (C1-C8) alkylamino and di- (C1-C4) alkylamino,

or their pharmaceutically applicable salts.

As used herein, the term “(C1-C3) alkylene” refers to a biradical branched or unbranched hydrocarbon chain containing 1 to 3 carbon atoms, for example methylene, ethylene, propylene and trimethylene, most preferably ethylene.

As used herein, the term “—O — CH (R ³ ) -” refers to the groups —O — CH ₂ -, —O — CH (CH ₃ ) -, —O — CH (CH ₂ CH ₃ ) -, —O —CH (CH ₂ CH ₂ CH ₃ ) -, —O — CH (CH ₂ CH ₂ CH ₂ CH ₃ ) -; preferably —O — CH ₂ -, —O — CH (CH ₃ ) - and most preferably, —O — CH (CH ₃ ) -.

The term “acetyl” refers to the group CH ₃ CO—.

The term “cycloalkyl” means a saturated, cyclic hydrocarbon group containing 3-7 carbon atoms, preferably 4-7 carbon atoms, more preferably 4-6 carbon atoms, for example cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and the like.

The term “heteroatom” refers to oxygen, nitrogen, and sulfur atoms.

The term “mono- and dialkylamino” refers to the following amino groups substituted with alkyl or dialkyl, for example, alkyl-NH— and dialkyl-N—.

The term “(C1-C8) alkylamino” refers to methylamino, ethylamino, propylamino, isopropylamino, butylamino, tert-butylamino, pentylamino, hexylamino, heptylamino and octylamino; preferably to butylamino and pentylamino groups.

The term “di- (C1-C4) alkylamino” refers to dimethylamino, diethylamino, dipropylamino, dibutylamino; preferably dimethylamino and diethylamino groups.

In the definition of R ^{0 of} formula (II), the term “natural amino acid side chain” preferably means a natural amino acid side chain such as methyl, isopropyl, 2-methylpropyl, 1-methylpropyl, benzyl, indol-3-ylmethyl, 2- (methylthio) ethyl and 4-aminobutyl, 3-aminopropyl; preferably denotes a side chain of a natural lipophilic amino acid, for example methyl, 2-methylpropyl, benzyl and indole-3-methyl.

The term “synthetic amino acid side chain” preferably means (C5-C12) alkyl, cycloalkylmethyl, substituted or unsubstituted arylmethyl, (cycloalkylthio) methyl, alkylthio (CH ₂ ) _r -, where r is an integer of 1 or 2, and etc.

In the above text, the term “(C5-C12) alkyl” means an unbranched or branched alkyl chain containing 5-12 carbon atoms; more preferably, a straight chain (C8-C12) alkyl chain, for example n-octyl, nonyl, decyl, undecyl and dodecyl.

The term “alkylthio- (CH ₂ ) _r -” means alkylthiomethyl or alkylthioethyl containing a straight or branched alkyl chain of 2-10 carbon atoms, for example ethylthiomethyl, ethylthioethyl, n-propylthiomethyl, n-butylthiomethyl, n-pentylthiomethyl, n-octylthiomethyl n-nonylthiomethyl, n-decylthiomethyl, tert-butylthiomethyl and the like; more preferably ethylthioethyl, n-propylthiomethyl and n-butylthiomethyl.

The term “substituted or unsubstituted arylmethyl” preferably means 4-phenylbenzyl, naphth-2-ylmethyl, [4- (4-hydroxyphenoxy) phenyl] methyl and (4-lower alkoxyphenyl) methyl, wherein in this term the combination “lower alkoxy” means alkyl a normal or isostructure chain containing 1-6 carbon atoms; preferably methoxy, ethoxy, propoxy, butoxy and isopropoxy. The most preferred meanings of the term “substituted or unsubstituted arylmethyl” are 4-phenylbenzyl, naphth-2-ylmethyl, (4-methoxyphenyl) methyl and [4- (4-hydroxyphenoxy) phenyl] methyl.

As used in the definition of R ^{2 of} formula (II), the term “branched (C3-C10) alkyl” means a branched alkyl chain containing 3-6 carbon atoms, preferably isopropyl, 2-butyl, 3-pentyl, neopentyl and the like; more preferably isopropyl and 3-pentyl. The term “(C3-C8) cycloalkyl” means a carbon ring consisting of 3-8 carbon atoms, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like; more preferably cyclopentyl and cyclohexyl.

In the definition of R ^{3 of} formula (II), the term “branched (C1-C4) alkyl” means a branched alkyl group containing 1-4 carbon atoms, preferably methyl, ethyl and n-propyl.

The term "pharmaceutically applicable salt" refers to salts that preserve the biological effectiveness and properties of the free bases and free acids, which are acceptable in a biological or other way. The salts in question are obtained by reacting with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and with such organic acids as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, etc. In addition, the salts in question can be prepared by attaching an inorganic or organic base to a free acid. Salts derived from an inorganic base include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, etc. Organic base derivative salts, without particular restrictions, include salts of primary, secondary and tertiary amines, substituted amines, including naturally occurring amines, cyclic amines and basic ion exchange resins, for example isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyimine resins and the like. Preferred salts are hydrochlorides. Salt-free compounds can be prepared by methods known in the art.

In the context of the present invention, the phrase “pro-fragment (X)” means a leaving group that is cleaved in tumors by the action of the aforementioned enzyme after application of a compound of formula (I) or (II), for example X is a group of the formula:

Used in the text of the invention, the term "taxanes" means taxol [Front. Biotechnol. Pharm. (2000), 1, 336-348], taxoret [J.Med. Aromat. Sci. (2001), 22 / 4A-23 / 1A 4-5], IDN 5109 [Chirality, (2000), 12 (5/6), 431-441], BMS 188797 [Clinical Cancer Research. 5 (suppl.), 3859, Nov 1999], BMS184476 [L. Clinical Oncology 19: 2493-2503, 1 May 2001].

The term "camptothecins" [(a) Cancer Chemotherapy and Biotherapy: Principle and Practice, 2 ^nd Ed., Lippincott-Ravenmeans, page 463-484, (b) Biochim. Biophys. Acta (1998), 1400 (1-3), 107-119] refers to any compounds with camptothecin structure, for example camptothecin, topotecan, SN-38, 9-aminocamptothecin, 9-nitrocamptothecin, lurtotecan [Br. J. Cancer (1998), 78 (10), 1329-1336], DX-8951f [Ann. NY Acad. Sci. (2000), 922 (Camptotecins), 260-273], BN-80915 [Anti-cancer Drugs (2001), 12 (1), 9-19], etc.

The term "anti-cancer nucleosides" means a cytidine derivative [Cancer Chemotherapy and Biotherapy: 2 ^nd Ed., Lippincott-Ravenmeans, page 213-233], for example, DFDC (gemcitabine), DMDC [Clin. Cancer Res. (2000), 6 (6), 2288-2294], FMDC [Curr.Opin. Invest. Drugs (PharmaPress Ltd.) (2000), 1 (1), 135-140], Ara-C, decitabine [IDrugs (2000), 3 (12), 1525-1533] troxacitabine [Clin.Cancer Res. (2000), 6 (4), 1574-1588], 2'-cyano-2'-deoxycytidine (CNDAC), 3'-ethynylcytidine (TAS106) [Jpn.J. Cancer Res. (2001), 92 (3), 343-351], 5-fluoro-5'-deoxycytidine [Bioorg. Med. Che. Lett., (2000), 8, 1697-1706], 5-vinyl-5'-deoxycytidine or an adenosine derivative [Cancer Chemotherapy and Biotherapy: Principle and Prectice, 2 ^nd Ed., Lippincott-Ravenmeans, page 235-252], e.g. fludarabine, cladribine, and the like.

The term "dolastatin" means dolastatin 10 [Curr. Pharm. Des. (1999), 5 (3), 139-162], dolastatin 14, TZT1027 [Drugs Future (1999), 24 (4), 404-409], cemadotine, and the like.

The term “anthracyclines” [Cancer Chemotherapy and Biotherapy: Principle and Prectice, 2 ^nd Ed., Lippincott-Ravenmeans, page 409-434] means adriamycin, daunomycin, idarubicin, and the like.

The term “farnesyl transferase inhibitors” means R115777 [Cancer Res. (2001), 61 (1), 131-137], etc.

The term “tyrosine kinase inhibitors of the EGF receptor” means ZD 1839 [Drugs (2000), 60 (Suppl. 1), 33-40], CP 358774 (OSI-774) [J. Pharmacol Exp. Thr. (1999), 291 (2), 739-748], PD 158780 [J. Mad Chem. (2001), 44 (3), 429-440], GW2016, etc.

The following symbols and abbreviations used in the description text refer to the following compounds:

a) Taxol means

6,12b-bis (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10,11,12,12a, 12b-dodecahydro-4,11-dihydroxy-4a, 8 , 13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodex [3,4] benz [1,2-b] oxet-9-yl ether [2aR- [2aα, 4β, 4αβ, 6β , 9α (αR *, βS *), 11α, 12α, 12aα, 12bα] -β- (benzoylamino) -α-hydroxybenzenepropanoic acid,

b) taxotere means

12b- (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10,11,12,12a, 12b-dodecahydro-4,6,11-trihydroxy-4a, 8, 13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclododec [3,4] benz [1,2-b] oxet-9-yl ether [2aR- [2aα, 4β, 4aα, 6β, 9α (αR *, βS *, 11α, 12α, 12aα, 12bα)] - β - [[(1,1-dimethylethoxy) carbonyl] amino) -α-hydroxybenzenepropanoic acid,

c) IDN 5109 means

(3aS, 4R, 7R, 8aS, 9S, 10aR, 12aS, 12bR, 13S, 13aS) -7,12a-bis (acetyloxy) -13-benzyloxy) -3a, 4,7,8,8a, 9,10, 10a, 12,12a, 12b, 13-dodecahydro-9-hydroxy-5.8a, 14.14-tetramethyl-2,8-dioxo-6,13a-methano-13aH-oxeto [2 ", 3": 5 ' 6 '] benzo [1', 2 ': 4,5] cyclodec [1,2-d] -1,3-dioxol-4-yl ether (2R, 3S) -3 - [[(1,1- dimethylethoxy) carbonyl] amino] -2-hydroxy-5-methyl-4-hexenoic acid,

d) BMS 188797 means

(2aR, 4S, 4aS, 6R, 9s, 11S, 12S, 12aR, 12bS) -6- (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10,11, 12,12a, 12b-dodecahydro-4,11-dihydroxy-12b - [(methoxycarbonyl) oxy] -4a, 8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodec [3,4 ] benz [1,2-b] oxet-9-yl ester of (2R, 3S) -β- (benzoylamino) -α-hydroxybenzenepropanoic acid, and

e) BMS 184476

(2aR, 4S, 4aS, 6R, 9S, 11S, 12S, 12aR, 12bS) -6,12b-bis (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10 , 11,12,12a, 12b-dodecahydro-11-hydroxy-4a, 8,13,13-tetramethyl-4 - [(methylthio) methoxy] -5-oxo-7,11-methano-1H-cyclodec [3, 4] benz [1,2-b] oxet-9-yl ester of (2R, 3S) -β- (benzoylamino) -α-hydroxybenzenepropanoic acid,

f) camptothecin means

4 (S) -ethyl-4-hydroxy-1H-pyrano [3 ', 4': 6,7] indolisino [1,2b] quinoline-3,14 (4H, 12H) -dione,

g) topotecan means

monohydrochloride (4S) -10 - [(dimethylamino) methyl] -4-ethyl-4,9-dihydroxy-1H-pyrano [3 ', 4': 6,7] indolisino [1,2-b] quinolin-3, 14 (4H, 12H) dione,

h) DX-8951f means

(1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyrano [ 3 ', 4': 6,7] indolisino [1,2-b] quinoline-10,13-dione,

i) BN-80915 means

5 (R) -ethyl-9,10-difluoro-1,4,5,13-tetrahydro-5-hydroxy-3H, 15H-oxepino [3 ', 4': 6,7] indolisino [1,2-b ] quinoline-3.15-dione,

j) 9-aminocamptothecin means

(S) -10-amino-4-ethyl-4-hydroxy-1H-pyrano [3 ', 4': 6,7] indolisino [1,2-b] quinolin-3,14 (4H, 12H) -dione ,

j) 9-nitrocamptothecin means

4 (S) -ethyl-4-hydroxy-10-nitro-1H-pyrano [3 ', 4': 6,7] -indolisino [1,2-b] quinolin-3,14 (4H, 12H) -dione ,

l) DFDC means

2'-deoxy-2 ', 2'-difluorocytidine,

m) DMDC means

2'-deoxy-2'-methylidene cytidine,

n) FMDC means

(E) -2'-deoxy-2 '- (fluoromethylene) cytidine,

o) Ara-C means

1- (β-D-arabinofuranosyl) cytosine,

p) decitabine means

4-amino-1- (2-deoxy-β-D-erythropentofuranosyl) -1,3,5-triazin-2 (1H) -one,

q) troxacitabine means

4-amino-1 - [(2S, 4S) -2- (hydroxymethyl) -1,3-dioxolan-4-yl] -2 (1H) -pyrimidinone,

r) fludarabine means

2-fluoro-9- (5-O-phosphono-β-D-arabinofuranosyl) -9H-purin-6-amine,

s) cladribine means

2-chloro-2'-deoxyadenosine,

t) dolastatin 10 means

N, N-dimethyl-L-valyl-N - [(1S, 2R) -2-methoxy-4 - [(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo -3 - [[(1S) -2-phenyl-1- (2-thiazolyl) ethyl] amino] propyl] -1-pyrrolidinyl] -1 - [(1S) -1-methylpropyl] -4-oxobutyl] -N methyl L-valinamide,

u) dolastatin 14 means

cyclo [N-methylalanyl- (2E, 4E, 10E) -15-hydroxy-7-methoxy-2-methyl-2,4,10-hexadecatrienoyl-L-valyl-N-methyl-L-phenylalanyl-N-methyl- L-valyl-N-methyl-L-valyl-L-prolyl-N2-methylsparaginyl],

v) dolastatin 15 means

(1S) -1 - [[(2S) -2,5-dihydro-3-methoxy-5-oxo-2- (phenylmethyl) -1H-pyrrol-1-yl] carbonyl] -2-methylpropyl ether N, N -dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline,

w) TZT 1027 means

N, N-dimethyl-L-valyl-N - [(1S, 2R) -2-methoxy-4 - [(2S) -2- [1R, 2R) -1-methoxy-2-methyl-3-oxo- 3 - [(2-phenylethyl) amino] propyl] -1-pyrrolidinyl] -1 - [(1S) -1-methylpropyl] -4-oxobutyl] -N-methyl-L-valinamide,

x) cemadotine means

N, N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-N- (phenylmethyl) -L-prolinamide,

y) adriamycin means

hydrochloride (8S, 10S) -10 - [(3-amino-2,3,6-tridesoxy-L-dioxohexopyranosyl) oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxynaphthacene-5,12-dione,

z) daunomycin means

8-acetyl-10 hydrochloride - [(3-amino-2,3,6-trideoxy-L-dioxohexopyranosyl) oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl ) -1-methoxynaphthacene-5,12-dione,

aa) idarubicin means

(7S, 9S) -9-acetyl-7 - [(3-amino-2,3,6-tridesoxy-L-dioxohexopyranosyl) oxy] -7,8,9,10-tetrahydro-6,9,11-trihydroxynaphthacene -5,12-dion

bb) ZD 1839 means

N- (3-chloro-4-fluorophenyl) -7-methoxy-6- [3- (4-morpholinyl) propoxy] -4-quinazolinamine,

ss) CP 358774 means

N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) -4-quinazolinamine,

dd) PD 158780 means

N ⁴ - (3-bromophenyl) -N6-methylpyrido [3,4-d] pyrimidin-4,6-diamine,

her) GW 2016 means

N- (3-chloro-4 - ((3-fluorobenzyl) oxy) phenyl) -6- (5 - (((2-methylsulfonyl) ethyl) amino) methyl) -2-furyl) -4-quinazolinamine,

ff) R 115777 means

6- [1-amino-1- (4-chlorophenyl) -1- (1-methylimidazol-5-yl) methyl] -4- (3-chlorophenyl) -1-methylquinolin-2 (1H) -one.

According to the present invention, enzymes that are preferably expressed in tumor tissue by selectively activating the compounds are identified by analyzing mRNA and / or protein levels in human tissue. Compounds are then constructed from known and / or new cytotoxic drugs by adding fragments that mask the biological activities of the cytotoxic drugs, but are recognized and selectively removed by the indicated enzymes in the target tumor tissues.

Normal and tumor human tissues used for analysis include tissue from the brain, esophagus, heart, lungs, breast, stomach, liver, pancreas, gall bladder, intestines, colon, rectum, kidney, ovary, uterus, testicle, prostate glands, skin, bone, bone marrow and blood. Preferably, granulocyte precursors as normal cells are used to compare expression levels of genes and / or proteins in tumor and normal tissue, and to select genes and / or proteins that are preferably expressed in tumor tissue. After resection of human tissue during a surgical operation, it is immediately frozen in liquid nitrogen or acetone containing dry ice, with or without the addition of O.C.T. (Sakura-Seiki, Tokyo, Japan, Catalog No. 4583) and stored at a temperature below -70 ° C or -80 ° C until subsequent use.

If the tumor tissue contains a significant amount of normal cells, the tumor cells are isolated from tissue embedded in the OCT prodrug using laser-excited microdesection (Ohyama H. et al. Laser capture microdessection - generated target sample for high density oligonucleotide array hybridization. Biotechniques 29, 530-536 (2000), Leethanakul C. et al., Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries. Oral Oncol 36, 474-83 (2000)). For microdesection, frozen sections with a thickness of 6 and 10 micrometers are fixed with a 70% Mayer hematoxylin stained ethanol solution, and the loan is dehydrated in the presence of an ethanol and xylene gradient. Microdesection of tumor cells is carried out using a laser capture microdesection device (Olympus, Tokyo, Japan, Model LM200) and RNA in tumor cells is extracted using a commercially available kit (Micro RNA Isolation Kit, Stratagene, La Jolla, CA, USA).

The precursors of human granulocytes, which are most susceptible to cytotoxic drugs, were prepared by multiplying CD34-positive mononuclear cells in murine stromal cells in the presence of certain cytokines, including the Flt3 ligand, stem cell factor (SCF) and thrombopoietin (TPO). CD34-positive mononuclear cells in human umbilical cord blood or in bone marrow were incubated and bound to anti-CD34 antibody conjugated to magnetic beads and purified by magnetic field classification of cells (MACS) (Miltanyi, et al. In: Hematopoietic stem cells : The mulhouse manual, 201-213, AlphaMed press, Dayton (1994)). Purified CD34 mononuclear cells, retaining the ability to differentiate into different types of hematopoietic cells, were propagated on culture dishes and the percentage of granulocyte precursors in the culture was confirmed by studying the expression of CD34 after staining the cells with a fluorescently conjugated anti-CD34 antibody. Typically, more than 90% of the cells in culture become the precursors of CD34-positive granulocytes after propagation. The ability of these granulocyte precursors to differentiate into myeloblasts and then into myelocytes and granulocytes was tested by treatment with granulocyte colony stimulation factor (G-CSF) or interleukin 3 (IL3) in combination with granulocyte macrophage colony stimulation factor (GM-CSF) and G- CSF The line and stages of cell differentiation were studied by monitoring cell surface antigens such as CD11, CD13 and CD15, using fluorescence cell classification (FACS) using FACSCalibur (Becton Dickinson, Franklin Lakes, New Jersey, USA) and / or microscopy after staining the cells with dye according to Giemsa (Diff-Quick) (Midori-Juji, Co. Osaka, Japan, Catalog No. 16920) or Leishman dye (Merck, Darmstadt, Germany, Catalog No. 105387 0500). FACS data was analyzed using the FACSCalibur CELLQuest software, in accordance with the FACSCalibur manual, FACSStation ver.1.1. (Becton-Dickinson, Franklin Lakes, New Jersey, USA).

Enzymes and proteins expressed in some tumor tissues were determined by measuring their mRNA and / or proteins in human tissues and cells. The mRNA expression levels were determined by such known methods as microarray DNA analysis (Schena, M. Et al. Quantitative minitoring of gene expression patterns with complementary DNA microarray, Science 270, 467-470 (1995), and Lipshutz, RJ et al. High density synthetic oligonucleotide arrays. Nature Genetics 21, 20-24 (1999)), reverse transcription polymerase reaction (hereinafter RT-PCR) (Weis, JH et al. Detection of rare mRNAs via quantitative RT-PCR, Trend Genetics 8, 263 -264 (1992), Bustin, SA Absolute quantification of mRNA using real-time reverse transcription polymerase chain traction assays, J. Mol. Endocrinol. 25, 169-193 (2000)), Northern blotting and in situ hybridization (Parker, RM, Barnes NM mRNA: detection in situ and northern hybridization, Methods Mol. Biol. 106, 247-283 (1999), protection assay RNA (Hod, YA Samplified ribonuclease protection assay, Biotechniques 13, 8520854 (1992), Saccomanno, CF et al. A faster ribonuclease protection assay, Biotechniques 13, 846-850 (1992)), Western blotting (Towbin, H. et al. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, Proc. Natl. Acad. Sci. US A 764350-4354 (1979), Burnette, W, N, Western blotting: Electrophoretic transfer of proteins form sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radioiodinated protein A, Anal. Biochem. 112, 195-2-3 (1981)), ELISA (Engvall E, Perlman, P. Enzyme-linked immunosorbent assay (ELISA): Quantitative assay of immunoglobulin G, Immunochemistry 8: 871-879 (1971) and protein analysis for matrices (Merchant, M, Weinberger, SR Review: Recent advancements in surface-enhanced laser desorption | ionization-time of light-mass spectrometry, Electrophoresis 21, 1164-1177 (2000), Paweletz, CP et al. Rapid protein display profiling of cancer progression directly from human tissue using a protein biochip, Drug Development Research 49, 34-42 (2000). For high-throughput and quantitative analysis of mRNA expression, it is preferable to use methods such as microarray DNA analysis and RT-PCR, respectively.

For DNA analysis on RNA microarrays, RNAs are cut from small pieces of tissue and / or cells that were frozen in liquid nitrogen or an acetone-dry ice system and stored at a temperature of -70 or -80 ° C. Tissues or cells are homogenized and RNA in the tissue and cell homogenates are extracted with chloroform and precipitated with isopropyl alcohol. DNA contaminated RNA preparation was digested with DNase I and RNA was further purified by gel filtration column chromatography. The conclusion about the quality of RNA is made from the ratio between 28S and 18S ribosomal RNA after agarose gel electrophoresis and RNA staining with ethidium bromide.

Using complete RNA as a template, cDNA synthesis is performed in the presence of an oligo-dT primer (Sawady Technology, Tokyo, Japan), which contains the T7 promoter and reverse transcriptase sequences. The resulting cDNA was extracted with a mixture of phenol and chloroform and separated from the short oligonucleotides by gel filtration column chromatography.

Using cDNA as a template, cRNA is synthesized using T7 polymerase, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate (UTP), Bio-11CTP and Bio-16 UTP (ENZO Diagnostics, Farming , USA, Catalog No. 42818 and 42814, respectively) at 37 ° C. for 6 hours. The resulting cRNA is separated from nucleotides by gel filtration column chromatography. The conclusion about the quality of cRNA is made from the length of cRNA after electrophoresis on an agarose gel and staining of the cRNA with ethidium bromide.

Microarray DNA analysis is performed in the presence of high density oligonucleotide chips (HuGeneFL matrix, Affymetrix, Santa Clara, USA, Catalog No. 510137) (Lipshutz, RL et al. Nature Genet. 21, 20-24 (1999)) in accordance with the manufacturer's instructions . Fragmentation of cRNA at 95 ° C, hybridization and washing are carried out in accordance with the manufacturer's instructions. Each pixel is recorded using a laser scanner (Affymetrix, Santa Clara, USA) and the expression levels of each cDNA and the reliability of the determination (request for presence / absence) are calculated using Affymetrix GeneChip ver.3 software. and Affymetrix Microarray Suite ver. 4.0.

In addition to DNA analysis on microarrays, other methods including RT-PCR (Weis, JH et al. Detection of rare mRNAs via quantitative RT-PCR, Trend Genetics 8, 263-264 (1992), and Bustin, SA Absolute quantification of mRNA using real-time reverse transcription polymerase chain traction assays, J. Mol. Endocrinol. 25, 169-193 (2000)), Northern blotting and in situ hybridization (Parker, RM, Barnes NM mRNA: detection in situ and northern hybridization, Methods Mol. Biol. 106, 247-283 (1999)), differential mapping (Zhu, W, and Liang, P. Detection and isolation of differentially expressed genes by differential display, Methods Mol. Biol. 68, 211-20 (1997) , Liang, P., Pardee AD Differential display of euceriotic messenger RNA by means of the polymerase chain reaction. Science, 257, 967-971 (1992)), RNA protection assay (Hod, YA Samplified ribonuclease protectio n assay, Biotechniques 13, 8520854 (1992), Saccomanno, C. F. et al. A faster ribonuclease protection assay, Biotechniques 13, 846-850 (1992)), matrix protein analysis ((Merchant, M, Weinberger, S.R. Review: Recent advancements in surface-enhanced laser desorption / ionization-time of light-mass spectrometry, Electrophoresis 21, 1164-1177 (2000), Paweletz, C.P. et al. Rapid protein display profiling of cancer progression directly from human tissue using a protein biochip, Drug Development Research 49, 34-42 (2000)), Western blotting (Towbin, H. et al. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets , Proc. Natl. Acad. Sci. US A 764350-4354 (1979), Burnette, W, N, Western blotting: Electrophoretic transfer of proteins form sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radioiodinated protein A, Anal. Biochem. 112, 195-2-3 (1981)), two-dimensional electrophoresis gel (O'farrell, PH High-resolution two-dimensional electrophoresis of protein. L. Biol. Chem. 250: 4007-4021 (1975)), ELISA assay (Engwall, E., Perlman, P. Enzyme-linked immunosorbent assay (ELISA): Quantitative assay of immunoglobulin G. Immunochemistry 8: 871-879 (1971)) can also be used to determine levels of I mRNA and / or protein.

Enzymes and / or proteins that are predominantly expressed in some tumors, but not in the precursors of granulocytes and other normal tissues, are identified by comparing the levels of mRNA and proteins in the tumor tissue with the corresponding values in normal tissue. Genes and / or proteins whose expression levels in some tumors and granulocyte precursors are more than doubled are selected as candidate genes for enzymes and / or proteins suitable for TTC activation. More preferred are those genes and / or proteins that show the greatest differences in expression levels between expression in some tumors and granulocyte precursors. After that, mRNA levels, which showed high expression in some tumor tissues, but not in the precursors of granulocytes, are compared with the corresponding values in other normal tissues, in particular with a normal liver, since the liver is the main organ responsible for the metabolism of most drugs. Those mRNAs are selected whose levels in some tumor tissues are higher than the corresponding values in granulocyte precursors and other normal tissues, especially in the liver.

Additional selection is carried out among enzymes and / or proteins selected in accordance with the difference in expression levels between some tumor tissues and granulocyte precursors and other normal tissues, for example, the liver, which have a relatively broad spectrum of substrate specificity and an enzymatic reaction mechanism suitable for constructing a compound .

Such enzymes include phospholipase C, microsomal dipeptidase, aryl sulfatase A, DT-diaphorase, pyrroline 5'-carboxy reductase, dehydrodiol dehydrogenase, carbonyl reductase, lysylhydroxylase, glucosyl aztosuzolazole azluzylamine -6-phosphate dehydrogenase, stearoyl coenzyme A desaturase, epoxide hydrolase and aldolase C.

More preferred enzymes for the construction of TTC are microsomal dipeptidases, phospholipase C, DT-diaphorase, dihydrodiol dehydrogenase, pyrroline 5'-carboxy reductase, carbonyl reductase, lysyl hydrolase or matrix metalloproteinases.

Considered enzymes can be used to create anti-cancer compounds of the formula (I)

X-Y-Q (I),

wherein

X is a pro-fragment for selectively generating an active anticancer substance (Q-Y-H) in tumors using enzymes detected by the method of the present invention; (Q-Y-) is a radical derived from the active anti-cancer substance (Q-Y-H), in which Y is —O—, —S— or —N—.

The compounds of formula (I) are described in more detail below. The active anti-cancer agent (Q-Y-H) may be any anti-cancer agent. This substance can be coupled to the pro-fragment X via a -YH- group, such as an amino, hydroxy or sulfhydryl group in the structure (QYH), so that spontaneous release of the active anticancer substance can occur under the influence of an enzyme detected by the methods of the present inventions. More specifically, (Q-Y-H) is a cytotoxic agent such as taxane, camptothecin, anti-cancer nucleoside, dolastatin, as well as anthracycline and farnesyl transferase inhibitor, tyrosine kinase EGF receptor inhibitor and the like.

Preferred substances are compounds in which the active anti-cancer substance (Q-Y-H) is a taxane selected from the group consisting of

a) taxol

6,12b-bis (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10,11,12,12a, 12b-dodecahydro-4,11-dihydroxy-4a, 8 , 13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodec [3,4] benz [1,2-b] oxet-9-yl ether [2aR- [2aα, 4α, 4αβ, 6β , 9α (αR *, βS *), 11α, 12α, 12aα, 12bα] -β- (benzoylamino) -α-hydroxybenzenepropanoic acid,

b) taxotere

12b- (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10,11,12,12a, 12b-dodecahydro-4,6,11-trihydroxy-4a, 8, 13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclododec [3,4] benz [1,2-b] oxet-9-yl ether [2aR- [2aα, 4β, 4aα, 6β, 9α (αR *, βS *, 11α, 12α, 12aα, 12bα)] - β - [[(1,1-dimethylethoxy) carbonyl] amino) -α-hydroxybenzenepropanoic acid,

c) IDN 5109

(3aS, 4R, 7R, 8aS, 9S, 10aR, 12aS, 12bR, 13S, 13aS) -7,12a-bis (acetyloxy) -13-benzyloxy) -3a, 4,7,8,8a, 9,10, 10a, 12,12a, 12b, 13-dodecahydro-9-hydroxy-5.8a, 14.14-tetramethyl-2,8-dioxo-6,13a-methano-13aH-oxeto [2 ", 3": 5 ' 6 '] benzo [1', 2 ': 4,5] cyclodec [1,2-d] -1,3-dioxol-4-yl ether (2R, 3S) -3 - [[(1,1- dimethylethoxy) carbonyl] amino] -2-hydroxy-5-methyl-4-hexenoic acid,

d) BMS 188797

(2aR, 4S, 4aS, 6R, 9s, 11S, 12S, 12aR, 12bS) -6- (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10,11, 12,12a, 12b-dodecahydro-4,11-dihydroxy-12b - [(methoxycarbonyl) oxy] -4a, 8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodec [3,4 ] benz [1,2-b] oxet-9-yl ester of (2R, 3S) -β- (benzoylamino) -α-hydroxybenzenepropanoic acid,

e) BMS 184476

(2aR, 4S, 4aS, 6R, 9S, 11S, 12S, 12aR, 12bS) -6,12b-bis (acetyloxy) -12- (benzoyloxy) -2a, 3,4,4a, 5,6,9,10 , 11,12,12a, 12b-dodecahydro-11-hydroxy-4a, 8,13,13-tetramethyl-4 - [(methylthio) methoxy] -5-oxo-7,11-methano-1H-cyclodec [3, 4] benz [1,2-b] oxet-9-yl ester of (2R, 3S) -β- (benzoylamino) -α-hydroxybenzenepropanoic acid.

Preferred compounds in which the active anti-cancer agent (Q-Y-H) is camptothecin selected from the group consisting of:

a) camptothecin

4 (S) -ethyl-4-hydroxy-1H-pyrano [3 ', 4': 6,7] indolisino [1,2b] quinoline-3,14 (4H, 12H) -dione,

b) topotecan

monohydrochloride (4S) -10 - [(dimethylamino) methyl] -4-ethyl-4,9-dihydroxy-1H-pyrano [3 ', 4': 6,7] indolisino [1,2-b] quinolin-3, 14 (4H, 12H) dione,

c) DX-8951f

(1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [de] pyrano [ 3 ', 4': 6,7] indolisino [1,2-b] quinoline-10,13-dione,

d) BN-80915

5 (R) -ethyl-9,10-difluoro-1,4,5,13-tetrahydro-5-hydroxy-3H, 15H-oxepino [3 ', 4': 6,7] indolisino [1,2-b ] quinoline-3.15-dione,

e) 9-aminocamptothecin

(S) -10-amino-4-ethyl-4-hydroxy-1H-pyrano [3 ', 4': 6,7] indolisino [1,2-b] quinolin-3,14 (4H, 12H) -dione ,

f) 9-nitrocampothecin

4 (S) -ethyl-4-hydroxy-10-nitro-1H-pyrano [3 ', 4': 6,7] -indolisino [1,2-b] quinolin-3,14 (4H, 12H) -dione ,

g) (9S) -9-ethyl-9-hydroxy-1-pentyl-1H, 12Hprano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6,5] pyrido [4, 3,2-de] quinazolin-10,13 (9H, 15H) -dione,

h) (9S) -9-ethyl-9-hydroxy-2-methyl-1-pentyl-1H, 12Hpirano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

i) (9S) -9-ethyl-9-hydroxy-2-hydroxymethyl-1-pentyl-1H, 12Hprano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione.

Also preferred are those compounds in which the active anti-cancer substance (Q-Y-H) is an anti-cancer nucleoside selected from the group consisting of

a) DFDC

2'-deoxy-2 ', 2'-difluorocytidine,

b) DMDC

2'-deoxy-2'-methylidene cytidine,

c) FMDC

(E) -2'-deoxy-2 '- (fluoromethylene) cytidine,

d) Ara-C

1- (β-D-arabinofuranosyl) cytosine,

e) decitabine

4-amino-1- (2-deoxy-β-D-erythropentofuranosyl) -1,3,5-triazin-2 (1H) -one,

f) troxacitabine

4-amino-1 - [(2S, 4S) -2- (hydroxymethyl) -1,3-dioxolan-4-yl] -2 (1H) -pyrimidinone,

g) fludarabine

2-fluoro-9- (5-O-phosphono-β-D-arabinofuranosyl) -9H-purin-6-amine,

h) cladribine

2-chloro-2'-deoxyadenosine.

Preferred compounds are those in which the active anti-cancer substance Q-Y-H is dolastatin selected from the group consisting of

a) dolastatin 10

N, N-dimethyl-L-valyl-N - [(1S, 2R) -2-methoxy-4 - [(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo -3 - [[(1S) -2-phenyl-1- (2-thiazolyl) ethyl] amino] propyl] -1-pyrrolidinyl] -1 - [(1S) -1-methylpropyl] -4-oxobutyl] -N methyl L-valinamide,

b) dolastatin 14

cyclo [N-methylalanyl- (2E, 4E, 10E) -15-hydroxy-7-methoxy-2-methyl-2,4,10-hexadecatrienoyl-L-valyl-N-methyl-L-phenylalanyl-N-methyl- L-valyl-N-methyl-L-valyl-L-prolyl-N2-methylsparaginyl],

c) dolastatin 15

(1S) -1 - [[(2S) -2,5-dihydro-3-methoxy-5-oxo-2- (phenylmethyl) -1H-pyrrol-1-yl] carbonyl] -2-methylpropyl ether N, N -dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline,

d) TZT 1027

N, N-dimethyl-L-valyl-N - [(1S, 2R) -2-methoxy-4 - [(2S) -2 - [(1R, 2R) -1-methoxy-2-methyl-3-oxo -3 - [(2-phenylethyl) amino] propyl] -1-pyrrolidinyl] -1 - [(1S) -1-methylpropyl] -4-oxobutyl] -N-methyl-L-valinamide,

e) cemadotine

N, N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-N- (phenylmethyl) -L-prolinamide.

A preferred compound in which the active anticancer substance (Q-Y-H) is anthracycline is selected from the group consisting of

a) adriamycin

hydrochloride (8S, 10S) -10 - [(3-amino-2,3,6-tridesoxy-L-dioxohexopyranosyl) oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl) -1-methoxynaphthacene-5,12-dione,

b) daunomycin

8-acetyl-10 hydrochloride - [(3-amino-2,3,6-trideoxy-L-dioxohexopyranosyl) oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl ) -1-methoxynaphthacene-5,12-dione,

c) idarubicin

(7S, 9S) -9-acetyl-7 - [(3-amino-2,3,6-tridesoxy-L-dioxohexopyranosyl) oxy] -7,8,9,10-tetrahydro-6,9,11-trihydroxynaphthacene -5,12-dione.

Also preferred is a compound in which the active anti-cancer agent (Q-Y-H) is a tyrosine kinase EGF receptor inhibitor or a farnesyl transferase inhibitor.

Preferred is a compound in which the active anticancer substance (Q-Y-H) is a tyrosine kinase EGF receptor inhibitor selected from the group consisting of

a) ZD 1839

N- (3-chloro-4-fluorophenyl) -7-methoxy-6- [3- (4-morpholinyl) propoxy] -4-quinazolinamine,

b) CP 358774

N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) -4-quinazolinamine,

c) PD 158780

N ⁴ - (3-bromophenyl) -N6-methylpyrido [3,4-d] pyrimidin-4,6-diamine,

d) GW 2016

N- (3-chloro-4 - ((3-fluorobenzyl) oxy) phenyl) -6- (5 - (((2-methylsulfonyl) ethyl) amino) methyl) -2-furyl) -4-quinazolinamine.

Also preferred is a compound in which the active anti-cancer agent (QYH) is a farnesyl transferase inhibitor R 115777 of the formula 6- [1-amino-1- (4-chlorophenyl) -1- (1-methylimidazol-5-yl) methyl] -4- (3-chlorophenyl) -1-methylquinolin-2 (1H) -one.

Antitumor compounds of the formula (II) of the present invention

in which Q and Y have the above meanings; R ⁰ represents a side chain of a natural or synthetic amino acid; Z represents (C1-C3) alkylene or a group —O — CH (R ³ ) -, in which R ³ represents hydrogen or unbranched (C3-C10) alkyl or (C3-C8) cycloalkyl selectively generating active anti-cancer substances in tumors under the action of microsomal dipeptidase are illustrated below by an example of constructing a compound using an enzyme detected by the methods described above. However, the present invention is not limited to the presented example. The compounds of formula (II) also include pharmaceutically acceptable salts thereof.

A first example of the present invention relates to antitumor compounds developed using taxanes as active anti-cancer drugs and microsomal dipeptidase as an activating enzyme represented by the general formula (III):

in which R ⁰ has the above meanings, R ⁴ represents benzoyl or tert-butoxycarbonyl, and R ⁵ represents hydrogen or acetyl, as well as their pharmaceutically acceptable salts.

Preferred R ⁰ values in formula (III) are methyl, isopropyl, 2-methylpropyl, 1-methylpropyl, benzyl, indol-3-ylmethyl, and 2- (methylthio) ethyl; more preferably methyl, benzyl and 2-methylpropyl.

According to the present invention, the preferred compounds of formula (III) are the following substances:

a) 13 - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-amino-4-methyl-pentanoylamino) -5-hydroxycarbonyl] pentanoyloxy} -3-benzoylamino-3- phenylpropionyloxy) -2α-benzyloxy-4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9-one,

b) 13α - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-amino-propionylamido) -5-hydroxycarbonyl] pentanoyloxy} -3-benzoylamino-3-phenylpropionyloxy) -2α -benzyloxy-4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9-one,

c) 13 - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-amino-3-phenyl-propionylamido) -5-hydroxycarbonyl] pentanoyloxy} -3-benzoylamino-3- phenylpropionyloxy) -2α-benzyloxy-4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9-one

and their pharmaceutically acceptable salts.

Figure 1 illustrates tumor-selective activation of compounds of formula (III) by microsomal dipeptidase.

Scheme 1

Второй пример противоопухолевых соединений, разработанных с использованием производного нуклеозида в качестве активного противоракового лекарственного средства и микросомальной дипептидазы в качестве активирующего фермента, представлен формулой (IV):

A second example of antitumor compounds developed using a nucleoside derivative as an active anticancer drug and microsomal dipeptidase as an activating enzyme is represented by formula (IV):

in which R ⁰ , R ¹ , R ² and R ³ have the meanings given above for formula (II), R ⁶ represents hydrogen, fluoro, hydroxyl or cyano, R ⁷ represents hydrogen, fluoro or hydroxy, or R ⁶ and R ⁷ together form methylidene or fluoromethylidene, R ⁸ represents hydrogen or ethynyl, R ⁹ represents hydrogen, fluorine, vinyl or ethynyl, and R ¹⁰ represents hydrogen or hydroxy, the formula encompassing pharmaceutically acceptable salts of such compounds.

A preferred embodiment of the present invention relates to compounds of the above formula (IV) in which R ⁶ is hydrogen, fluoro, hydroxyl, R ⁷ is fluoro or hydroxy, or R ⁶ and R ⁷ together form a methylidene or fluoromethylidene group. According to another preferred embodiment, the present invention relates to the compounds of formula (IV) above, wherein R ⁰ is 2-methylpropyl, cyclohexylmethyl, 2-naphthylmethyl, 4-phenylbenzyl, (4-cyclohexylcyclohexyl) methyl, alkylthiomethyl, cyclohexylthiomethyl, or 4-alkoxybenzyl, and R ³ represents hydrogen or methyl.

Preferred active nucleosides contained in the compounds of formula (IV) are DFDC, DMDC, FMDC, Ara-C, decitabine, troxacitabine, 2'-cyano-2'-deoxycytidine, 3'-ethynylcytidine, 5-fluoro-5'- deoxycytidine, 5-vinyl-5'-deoxycytidine and the like; more preferably DFDC, DMDC and FMDC.

The preferred value of R ⁰ in formula (IV) is the residue of a lipophilic natural amino acid, (C8-C12) alkyl, (C3-C8) cycloalkylmethyl, substituted or unsubstituted benzyl or naphthylmethyl, (C8-C12) alkylthiomethyl, (C3-C8) cycloalkylthiomethyl, more preferably 2-methylpropyl, cyclohexylmethyl, benzyl, naphth-2-ylmethyl, 4-phenylbenzyl, methylthioethyl, cyclohexylthiomethyl and the like.

In accordance with the present invention, preferred compounds of formula (IV) may be selected from the group consisting of:

(a) (2R) - ((2S) -amino-3-cyclohexyl-propionylamino) - (3S) - [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro-furan- ( 2R) -yl) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyloxy) butyric acid,

(b) (2R) - ((2S) -amino-4-methyl-pentanoylamino) - (3S) - [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro-furan- ( 2R) -yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy) butyric acid,

(c) (2R) - ((2S) -amino-3-biphenyl-4-yl-propionylamino) - (3S) - [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro -furan- (2R) -yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy) butyric acid,

(d) (2R) - ((2S) -amino-3-biphenyl-4-yl-propionylamino) -3- {1 [4 (S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro-furan - (2R) -yl] -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyloxy) propionic acid,

(f) (2R) - ((2S) -amino-3-naphthalen-2-yl-propionylamino) - (3S) - [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro -furan- (2R) -yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy) butyric acid,

(f) (2R) - {(2S) -amino-3- [4- (4-hydroxy-phenoxy) phenyl] propionylamino} -3- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl- 3-methylene-tetrahydro-furan-2-yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy) butyric acid,

(g) (2R) - [((2S) -amino-3- (4-methoxy-phenyl) -propionylamino) - (3S) - [1 - [(4S) -hydroxy- (5R) -hydroxymethyl-3- methylene-tetrahydro-furan-2-yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy) butyric acid,

(h) (2R) - [(2S) -amino-4-ethylsulfanylbutyrylamino] - (3S) - [1 - [(4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro-furan- (2R) -yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy) butyric acid,

(i) (2R) - ((2S) -amino-3-cyclohexyl-propionylamino) - (3S) - [1- (3,3-difluoro- (4R) -hydroxy- (5R) -hydroxymethyl-tetrahydro-furan -2-yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy) butyric acid,

(j) 2 (S) - [2 (S) -amino-3-cyclohexyl-propionylamino) -3- [1- (3,3-difluoro-4 (R) -hydroxy-5 (R) -hydroxymethyl-tetrahydro furan-2 (R) -yl) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyloxy) -2 (S) methylpropionic acid,

(k) 2 (R) - [2 (S) -amino-3-cyclohexyl-propionylamino) -3- {1- (3,3-difluoro-4 (R) -hydroxy-5 (R) -hydroxymethyl-tetrahydro -furan-2 (R) -yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy) -2 (R) methylpropionic acid,

(l) (2S, 3S) -2- (2-amino-3-cyclohexyl-propionylamino) -3- [1 - {(4R, 5R) -3,3-difluoro-4-hydroxy-5-hydroxymethyl-tetrahydro -furan-2-yl} -2-oxo-1,2-dihydro-pyridin-4-ylcarbamoyloxy] -2-methylbutyric acid,

(m) (2R, 3R) -2- (2-amino-3-cyclohexyl-propionylamino) -3- [1 - {(4R, 5R) -3,3-difluoro-4-hydroxy-5-hydroxymethyl-tetrahydro -furan-2-yl} -2-oxo-1,2-dihydro-pyridin-4-ylcarbamoyloxy] -2-methylbutyric acid, and

(n) isopropyl ether (2R) - [(2S) -amino-3-cyclohexyl-propionylamino] - (3S) - [1 - [(4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro-furan - (2R) -yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy] butyric acid,

and their pharmaceutically acceptable salts.

A third example of antitumor compounds obtained using nucleosides as active anti-cancer drugs and microsomal dipeptidase as an activating enzyme is represented by formula (V):

in which m is an integer equal to 2 or 3, and R ⁰ , R ² , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ have the above meanings.

Preferred examples of the active cytidine analogs contained in formula (V) are DFDC, DMDC, FMDC, Ara-C, decitabine, troxacitabine, 2'-cyano-2'-deoxycytidine, 3'-ethynylcytidine, 5-fluoro-5'- deoxycytidine, 5-vinyl-5'-deoxycytidine and the like; more preferably DFDC, DMDC and FMDC.

Preferred R ⁰ values in formula (V) are cyclohexylmethyl, naphth-2-ylmethyl, 4-phenylbenzyl, benzyl, indol-3-ylmethyl or 4-alkoxybenzyl, for example, such (4-lower alkoxyphenyl) methyl, such as 4-methoxybenzyl, 4-ethoxybenzyl and the like.

In accordance with the present invention, the preferred compounds of formula (V) are the following substances:

a) (2R) - [(2S) -amino-3- (1H-indol-3-yl) propionylamino] -4- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2 -yl) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyl] butyric acid,

b) (2R) - ((2S) -amino-3-cyclohexylpropionylamino) -4- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2-yl) -2-oxo-1 , 2-dihydropyrimidin-4-ylcarbamoyl] butyric acid,

c) (2R) - ((2S) -amino-3-biphenyl-4-ylpropionylamino) -4- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2-yl) -2 -oxo-1,2-dihydropyrimidin-4-ylcarbamoyl] butyric acid, and

d) (2R) - ((2S) -amino-3-naphthalen-4-ylpropionylamino) -4- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2-yl) -2 -oxy-1,2-dihydropyrimidin-4-ylcarbamoyl] butyric acid

and their pharmaceutically acceptable salts.

A fourth example of antitumor compounds constructed using camptothecin as an active anti-cancer drug and microsomal dipeptidase as an activating enzyme is represented by formula (VI):

in which m represents an integer from 1 to 3, n represents an integer from 0 to 1, R ⁰ has the same meanings as indicated for formula (II), R ¹¹ represents hydrogen or fluorine, R ¹² represents hydrogen , fluoro, methyl or hydroxy, R ¹³ represents hydrogen, amino, nitro, or (dimethylamino) methyl, R ¹⁴ represents hydrogen, (C1-C4) alkyl, (4-methylpiperazinyl) methyl, (tert-butoxyimino) methyl or R ¹³ and R ¹⁴ or R ¹¹ and R ¹² , together with each other, form a 5- or 6-membered ring, which optionally contains 1-2 heteroatoms and may optionally be optionally substituted with 1-3 substituents selected from the group consisting of (C1-C8) alkyl, amino, (C1-C8) alkylamino and / or di- (C1-C4) alkylamino and their pharmaceutically acceptable salts. More preferably, the compounds of formula (VI) are characterized in that R ¹¹ is hydrogen, R ¹² is hydrogen or hydroxy, R ¹³ is hydrogen or (dimethylamino) methyl, and R ¹⁴ is hydrogen or ethyl. The preferred value of R ⁰ in the formula (VI) is 2-methylpropyl, cyclohexylmethyl, benzyl, indol-3-ylmethyl, 4-aminobutyl, 4-aminopropyl; more preferably 2-methylpropyl, cyclohexylmethyl, benzyl and indol-3-ylmethyl.

Preferred examples of the active camptothecin analog covered by formula (VI) are camptothecin, topotecan, SN-38, lurtotecan, 9-aminocamptothecin, 9-nitro-camptothecin, DX-8951f, BN-80915, (9S) -9-ethyl-9-hydroxy -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione, (9S) -9-ethyl-9-hydroxy-2-methyl-1-pentyl-1H, 12Hpirano [3 ", 4": 6 ', 7'] indolisino [1 ', 2 ': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione, and (9S) -9-ethyl-9-hydroxy-2-hydroxymethyl-1-pentyl- 1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) dion, etc.

Preferred R ⁰ values in formula (VI) are 2-methylpropyl, cyclohexylmethyl, benzyl, indol-3-ylmethyl, 4-aminobutyl, 4-aminopropyl; more preferably 2-methylpropyl, cyclohexylmethyl, benzyl and indol-3-ylmethyl.

In accordance with the present invention, preferred compounds of formula (VI) are the following substances:

a) 20-O - [(S) -tryptophil-γ- (S) -glutamyl] -20- (S) -camptothecin,

b) 20-O - [(S) -valyl-γ- (S) -glutamyl] -20- (S) -camptothecin,

c) 20-O - [(S) -phenylalanyl-γ- (S) -glutamyl] -20- (S) -camptothecin,

d) 20-O - [(S) -leucyl-γ- (S) -glutamyl] -20- (S) -camptothecin,

e) 20-O - [(R) -leucyl-γ- (S) -glutamyl] -20- (S) -camptothecin,

f) 20-O - [(R) -phenylalanyl-γ- (S) -glutamyl] -20- (S) -camptothecin,

g) 20-O - [(S) -tryptophil-γ- (R) -glutamyl] -20- (S) -camptothecin,

h) 20-O - [(R) -tryptophil-γ- (R) -glutamyl] -20- (S) -camptothecin,

i) 20-O - [(S) -phenylalanyl-γ- (R) -glutamyl] -20- (S) -camptothecin,

j) 20-O - [(S) -leucyl-γ- (R) -glutamyl] -20- (S) -camptothecin,

k) 20-O - [(R) -leucyl-γ- (S) -glutamyl] -20- (S) -camptothecin,

l) 20-O - [(R) -phenylalanyl-γ- (R) -glutamyl] -20- (S) -camptothecin,

m) 20-O - [(R) -leucyl-γ- (R) -glutamyl] -20- (S) -camptothecin,

n) 7-ethyl-10-hydroxy-20-O - [(R) -tryptophil- (R) -homoglutamyl] -20 (S) -camptothecin,

o) 7-ethyl-10-hydroxy-20-O - [(R)-tryptophil-γ- (R) -glutamyl] -20 (S) -camptothecin,

p) 7-ethyl-10-hydroxy-20-O - [(S) -phenylalanyl-γ- (R) -glutamyl] -20 (S) -camptothecin,

q) 7-ethyl-10-hydroxy-20-O - [(S) -phenylalanyl-γ- (S) -aspartyl] -20 (S) -camptothecin,

r) 7-ethyl-10-hydroxy-20-O - [(S) -leucyl-γ- (S) aspartyl] -20 (S) -camptothecin,

s) 20-O - [(S) -tryptophil-β- (R) -aspartyl] -20 (S) -camptothecin,

t) 20-O - [(S) -phenylalanyl-β- (R) -aspartyl] -20 (S) -camptothecin,

u) 20-O - [(R) -phenylalanyl-β- (R) -aspartyl] -20 (S) -camptothecin,

v) 20-O - [(S) -phenylalanyl-β- (S) -aspartyl] -20 (S) -camptothecin,

w) 20-O - [(S) -leucyl-β- (R) -aspartyl] -20 (S) -camptothecin,

x) 20-O - [(S) -valyl-β- (R) -aspartyl] -20 (S) -camptothecin,

y) 7-ethyl-10-hydroxy-20-O - [(S) -cyclohexylalanyl- (R) -glutamyl] -20 (S) -camptothecin,

z) 7-ethyl-10-hydroxy-20-O - [(S) -cyclohexylalanyl- (S) -glutamyl] -20 (S) -camptothecin,

aa) 20-O - [(S) -lysyl-γ- (S) -glutamyl] -20 (S) -camptothecin,

bb) 20-O - [(S) -orinityl-γ- (S) -glutamyl] -20 (S) -camptothecin,

ss) (9S) -9-ethyl-9 - [(L) -tryptophil- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

dd) (9S) -9-ethyl-9 - [(L) -cyclohexylalanyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

e) (9S) -9-ethyl-9 - [(L) -phenylalanyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

ff) (9S) -9-ethyl-9 - [(L) -leucyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

gg) (9S) -9-ethyl-9 dihydrochloride - [(L) -lysyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

hh) (9S) -9-ethyl-9 - [(L) -valyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

ii) (9S) -9-ethyl-9 dihydrochloride - [(L) -oryninyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

jj) methanesulfonic acid salt of (9S) -9-ethyl-9 - [(L) -leucyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7 '] indolisino [1', 2 ': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

kk) (9S) -9-ethyl-9 dihydrochloride - [(D) -lysyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

ll) (9S) -9-ethyl-9 - [(L) -phenylalanyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

mm) (9S) -9-ethyl-9 - [(L) -cyclohexylalanyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

nn) (9S) -9-ethyl-9 - [(L) -cyclohexylalanyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

oo) (9S) -9-ethyl-9 - [(L) -tryptophil- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

pp) (9S) -9-ethyl-9 dihydrochloride - [(L) -ornithyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

qq) (9S) -9-ethyl-9 - [(L) -leucyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

rr) (9S) -9-ethyl-9 - [(L) -valyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

ss) (9S) -9-ethyl-9 - [(L) -leucyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

tt) (9S) -9-ethyl-9 - [(L) -cyclohexylglycyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

uu) (9S) -9-ethyl-9 - [(D) -cyclohexylalanyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

vv) (9S) -9-ethyl-9 dihydrochloride - [(L) -lysyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

ww) (9S) -9-ethyl-9 - [(L) -tryptophil- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

xx) (9S) -9-ethyl-9 - [(L) -leucyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

yy) (9S) -9-ethyl-9- [glycyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

zz) (9S) -9-ethyl-9 - [(L) -alanyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

aaa) (9S) -9-ethyl-9 - [(L) -phenylalanyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

non-salt compounds; and other pharmaceutically acceptable salts of these compounds.

More preferred examples of the compounds of formula (VI) are the following substances:

a) 20-O - [(S) -tryptophil-γ- (S) -glutamyl] -20 (S) -camptothecin,

b) 20-O - [(S) -leucyl-γ- (S) -glutamyl] -20 (S) -camptothecin,

c) 20-O - [(S) -tryptophil-γ- (R) -glutamyl] -20 (S) -camptothecin,

d) 20-O - [(S) -leucyl-γ- (R) -glutamyl] -20 (S) -camptothecin,

e) 7-ethyl-10-hydroxy-20-O - [(S) -phenylalanyl-β- (R) -glutamyl] -20 (S) -camptothecin,

f) 7-ethyl-10-hydroxy-20-O - [(S) -phenylalanyl-β- (S) -aspartyl] -20 (S) -camptothecin,

g) 20-O - [(S) -phenylalanyl-β- (S) -aspartyl] -20 (S) -camptothecin,

h) 7-ethyl-10-hydroxy-20-O - [(S) -cyclohexylalanyl- (R) -glutamyl] -20 (S) -camptothecin,

i) 7-ethyl-10-hydroxy-20-O - [(S) -cyclohexylalanyl- (S) -glutamyl] -20 (S) -camptothecin,

j) (9S) -9-ethyl-9 - [(L) -tryptophil- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

k) (9S) -9-ethyl-9 - [(L) -cyclohexyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

l) (9S) -9-ethyl-9 - [(L) -phenylalanyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

m) (9S) -9-ethyl-9 - [(L) -leucyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

n) (9S) -9-ethyl-9 dihydrochloride - [(L) -lyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

o) (9S) -9-ethyl-9 - [(L) -valyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

p) (9S) -9-ethyl-9 dihydrochloride - [(L) -oryninyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

q) a methanesulfonic acid salt of (9S) -9-ethyl-9 - [(L) -leucyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7 '] indolisino [1', 2 ': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

r) (9S) -9-ethyl-9 dihydrochloride - [(D) -lyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

s) (9S) -9-ethyl-9 - [(L) -phenylalanyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

t) (9S) -9-ethyl-9 - [(L) -cyclohexylalanyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

u) (9S) -9-ethyl-9 - [(L) -cyclohexylalanyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

v) (9S) -9-ethyl-9 - [(L)-tryptophil- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

w) (9S) -9-ethyl-9 dihydrochloride - [(L) -ornithyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

x) (9S) -9-ethyl-9 - [(L) -leucyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

y) (9S) -9-ethyl-9 - [(L) -valyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

z) (9S) -9-ethyl-9 - [(L) -leucyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

aa) (9S) -9-ethyl-9 - [(L) -cyclohexylglycyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

bb) (9S) -9-ethyl-9 - [(D) -cyclohexylalanyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

ss) (9S) -9-ethyl-9 dihydrochloride - [(L) -lysyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

dd) (9S) -9-ethyl-9 - [(L) -tryptophil- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

e) (9S) -9-ethyl-9 - [(L) -leucyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

ff) (9S) -9-ethyl-9- [glycyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

gg) (9S) -9-ethyl-9 - [(L) -alanyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

hh) (9S) -9-ethyl-9 - [(L) -phenylalanyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7' ] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione,

non-salt compounds; and other pharmaceutically acceptable salts of these compounds.

The most preferred example of the compounds of formula (VI) is (9S) -9-ethyl-9 - [(L) -isyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano dihydrochloride [3 ", 4 ": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione, the compound is not in the form of a salt and its other pharmaceutically acceptable salts.

The compound of formula (I) can be obtained by the condensation reaction of compound QYH with a reactive derivative X. Such reactions are known in the art: for example, a compound of formulas (II), (III), (V) and (VI) can be obtained by a condensation reaction of a compound of formula (VII), and a compound of formula (IV) can be prepared by a condensation reaction of a compound of formula (VIII) as described above.

The compound of formula (II), (III), (V) and (VI) can be obtained by the condensation reaction of the compounds of formula (VII):

in which P ₁ and P ₂ are amino and carboxy-protecting groups, respectively; R ⁰ and m are as defined above, and of a suitably protected anticancer compound, for example, paclitaxel, cytidine derivatives or camptothecins, with a condensing agent such as dicyclohexylcarbodiimide, BOP, HBTU, TNTU, PyBroP ^™ , TBTU, TSTU, HOBt [commercially available crosslinkers Agents: The Combinatirial Chemistry Catalog, Feb., 1997; Novabiochem.] And the like, followed by deprotection.

The above amino and carboxy protecting groups P ₁ and P ₂ , as well as the condensation reaction, are known to those skilled in the art [The Practice of Peptide Synthesis, M. Bodansky and A. Bodansky. 2 ^nd ed., 1994 (Springer-Verlag)].

Compounds of formula (IV) can be prepared by a condensation reaction of a compound of formula (VIII):

in which P ₁ , P ₂ , R ⁰ , R ¹ and R ³ are as defined above, and an appropriately protected cytidine derivative with a condensation agent such as 4-nitrophenyl chloroformate and triphosgene, followed by deprotection.

The reaction can be carried out in a solvent such as methylene chloride, pyridine, N, N-dimethylformamide, N-methylpyrrolidone, acetonitrile, etc., in the presence or absence of a base such as triethylamine, diisopropylethylamine, pyridine, N, N -dimethylaminopyridine and the like, at a temperature in the range from -20 ° C to + 50 ° C, preferably at 0-25 ° C.

The removal of the amino-protecting group, when using the amino- and / or carboxy-protected dipeptide for the condensation reaction, can be carried out by known methods, for example, by treatment with trifluoroacetic acid in the case of the Boc group, piperidine in the case of the Fmoc group, or tetrabutylammonium fluoride in the case of 2- (trimethylsilyl) ethoxycarbonyl (Teoc), trimethylsilylethyl and tert-butyldimethylsilyl groups, as well as by catalytic hydrogenolysis in the case of the Cbz group.

The amino acid derivatives used to prepare the dipeptide derivatives in formulas (VII) and (VIII) are commercially available compounds or can be prepared by known methods described in the literature (e.g., J. Am. Chem. Soc. 20000, 122, 762- 766; J. Org. Chem. 1998 5240; Tetrahedron Asymmetry 1995, 1741; Tetrahedron Asymmetry 1998, 4249). S-alkyl-cysteine derivatives are prepared by S-alkylating an amino / carboxy-protected cysteine derivative in the presence of an alkylating agent, or by replacing the hydroxy-group of the amino / carboxy-protected serine derivatives with a bromine atom, followed by a substitution reaction using a thiol derivative. O-alkyl tyrosine derivatives are prepared by O-alkylation of amino / carboxy-protected tyrosine derivatives with an alkylating agent.

Dipeptide derivatives can be obtained by traditional methods of peptide chemistry known to the person skilled in the art [The Practice of Peptide Synthesis, M. Bodansky and A. Bodansky. 2 ^nd ed., 1994 (Springer-Verlag)].

TTCs are then tested for their selective activation by a specific enzyme using recombinant enzymes and / or cell extracts that express or do not express significant amounts of TTC-activating enzymes. Precursors of human hematopoietic cells are also used as cells that do not express TTC-activating enzymes or express them only in small quantities. Recombinant proteins for TTC-activating enzymes can be produced by expression of cDNA for enzymes in bacteria or other cells, including insect cells or mammalian cells. Cell lines that constitutively express high levels of TTC-activating enzymes are also produced by transfection of a plasmid in which the cDNA of the TTC-activating enzyme is cloned by a strong constitutive promoter, including the cytomegalovirus (CMV) promoter (Foecking, MK and Hofstetter, H. Powerful and versatile enhancer -promotor unit for mammalian expression vectors. Gene. 45, 101-105 (1986)). Thus, transcription of TTC-activating enzymes in transfectants is controlled by a strong constitutive promoter. TTC activation is studied by incubating TTC in the presence of recombinant TTC activating enzymes and / or cell extracts capable of expressing or not expressing TTC activating enzymes, and by measuring the amount of TTC and active drugs by HPLC and / or LCMS.

In addition to cells carrying additional copies of cDNA for TTC-activating enzymes, extracts of various human and animal tissues can also be used to confirm tumor-specific activation of TTC. Tumors and normal tissues used for analysis include brain, heart, lung, stomach, intestine, colon, liver, kidney, blood and bone marrow tissues of mice, rats, monkeys, and humans.

The selective effect of TTC is additionally confirmed by comparing the inhibition of cell growth under the influence of TTC using cells expressing high levels of TTC activating enzymes and cells expressing very low levels of TTC activating enzymes. Inhibition of cell growth is determined by quantitative measurement of living cells after their cultivation in the presence or absence of TTC.

The conclusion that the activation of the compound is mediated by microsomal dipeptidase is based on the inhibitory activity of the compounds with respect to the growth of cells expressing low levels of microsomal dipeptidase, cells expressing high levels of microsomal dipeptidase and granulocyte precursors that multiply ex vivo. The human colon cancer cell line, HCT116 (American Type Culture Collection No. CCL-247), and granulocyte precursors are used as cells expressing only a low level of microsomal dipeptidase. A stable transfectant, HCT116 / S5, into which the human microsomal dipeptidase cDNA (hereinafter referred to as the MDP) transfected with the CMV promoter is transfected, is used as cells expressing a high level of microsomal dipeptidase. Dipeptidase cDNA (Saton et al. Biotechnol. Prog. 10 (2), 134-140 (1994) and other references) and the aforementioned cloning methods are known in the art. HCT116, HCT116 \ S5 and granulocyte precursors are cultured in the absence or presence of drugs and IC50 values, which are the drug concentrations required for 50% inhibition compared to cells cultured in the absence of the drug, are determined and compared in systems containing HCT116, HCT116 \ S5 and granulocyte precursors. Although the duration of drug exposure on the cells may vary depending on the type of cells and drugs, it is usually 24, 96, or 168 hours. In the case when the cells are cultured in the presence of drugs for 24 hours, the drugs are removed from the culture medium by replacing it and the cells are further incubated for 72 hours before measuring the IC50 values of the drug.

Biological data on the compounds used in each of examples 4, 16, 17, 31, 39, 49-1, 49-2, 49-3, 49-4 and 49-11.

Cytoxicity (IC ₅₀ in nm)

[duration of exposure to the drug: 24 hours]

Compound HCT116 HCT116 / S5 CFU-GM Pactitaxel 2,5 2,4 16 Example 4 51 5.1 54 Camptothecin 19 5,6 6.1 Example 31 300 eighteen 170 SN38 3,7 2.2 1.8 Example 39 23 2,8 twenty Example 49-1 33 3.3 61 Example 49-2 eighteen 2.6 31 Example 49-4 fifteen 2.1 54 Example 49-3 > 50 2.9 250 Example 49-11 > 50 13 120

Cytotoxicity (IC ₅₀ in nm)

[duration of exposure to the drug: 96 hours]

[duration of exposure to the drug: 168 hours]

Compound HCT116 HCT116 / S5 CFU-GM DMDC 0.2 0.3 0,07 Example 16 1.7 0.23 2,8 Example 17 0.99 0,098 1,1

HCT116: human colon cancer cell line; HCT116 / S5: HCT116 transfected human microsomal dipeptidase cDNA; CFU-GM: the precursors of human hematopoietic cells.

Thus, it can be expected that, under clinical conditions, significantly improved efficacy and safety characteristics of TTC can be achieved, especially with regard to myelotoxicity, compared with the characteristics of known cytotoxicants.

Another embodiment of the present invention relates to pharmaceutical compositions containing the compound described above. Such compositions are particularly suitable for oral and parenteral use.

As noted above, drugs containing a compound of formula I are also an object of the present invention, as is a method for their preparation, moreover, such a method consists in converting one or more compounds of formula I and, if desired, one or more other therapeutically valuable substances to galenic form.

The pharmaceutical compositions can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions and suspensions. Such formulations can also be used rectally, for example, in the form of suppositories; locally or transdermally, for example, in the form of ointments, creams, gels or solutions; or parenterally, for example, using injectable solutions.

To obtain pharmaceutical preparations (tablets, coated tablets, dragees, or hard gelatine capsules), formulations of the subject compounds and therapeutically inert, inorganic or organic carriers can be formulated. As carriers for tablets, coated tablets, dragees, and hard gelatine capsules, lactose, maize starch or derivatives thereof, talc, stearic acid or its salts can be used. Suitable carriers for soft gelatin capsules may be vegetable oils, waxes, fats, semi-solid or liquid polyols. Depending on the nature of the active substance, in the case of soft gelatin capsules, the use of carriers is usually not required. Suitable carriers for the production of solutions and syrups are water, polyols, sucrose, invert sugar and glucose. Suitable carriers for injection solutions include water, alcohols, polyols, glycerin and vegetable oils. Suitable candle carriers are natural or solidified oils, waxes, fats and semi-liquid polyols.

The subject pharmaceutical preparations may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweetening agents, coloring agents, perfumes, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They may also contain other therapeutically valuable substances.

Dosage can vary widely, and it is assumed that it must meet individual requirements in each case. As a rule, for oral or parenteral administration in adults, a suitable daily dosage is 5-500 mg / m ² . If appropriate, the upper limit indicated may be exceeded.

The daily dosage may be used as a single dose or in divided doses, and in the case of oral or parenteral administration, continuous infusion may be used.

Another embodiment of the present invention relates to the use of the anti-cancer agents described above for the manufacture of medicaments, preferably for the treatment of cell proliferation disorders, for example for the treatment of cancer, for example colorectal cancer, lung cancer, breast cancer, stomach cancer, cervical cancer and bladder cancer.

The present invention also relates to a method for treating cell proliferation disorders, for example cancer, for example, a solid tumor, colorectal cancer, lung cancer, breast cancer, stomach cancer, cervical cancer, and bladder cancer, the method comprising administering to a patient in need of treatment a therapeutically effective amount anti-cancer compound described above.

The present invention also relates to the compounds described above for therapeutic use.

The following examples are provided solely for the purpose of illustrating preferred methods for identifying enzymes and / or proteins suitable for activating the compounds with the indicated enzymes and for preparing the compounds of the present invention, the scope of the invention being not limited to the presented examples.

EXAMPLES

Example 1:

Measurement of different levels of mRNA in human and normal tissue tumors using an oligonucleotide microprobe and RT-PCR

1-1. Tissue RNA Extraction

Small pieces of samples consisting of 41 human colorectal tumors, 30 gastric tumors, 41 non-small cell lung carcinomas, 24 breast tumors, 15 ovarian tumors, 53 hepatocellular carcinomas and 15 non-tumor liver tissues (in each case about 125 mm ³ ) and 10 ⁷ ex vivo propagated granulocyte precursors were rapidly frozen in liquid nitrogen. For RNA extraction, tissues and cells were suspended in TRIZOL (Life Technologies, Gaithersburg, USA, Catalog No. 15596-018) or Sepasol-RNAI (Nacalai tesque, Kyoto, Japan, Catalog No. 306-55) and homogenized twice in Polytron (Kinematica, Littau , Switherland) (5 seconds at maximum speed). After addition of chloroform, the tissue homogenizer was centrifuged at 15,000 × g for 10 minutes and the aqueous phases containing RNA were collected. Total cellular RNA was precipitated with isopropyl alcohol, washed with 70% ethanol and suspended in DEPC-treated water (Life Technologies, Gaithersburg, USA, Catalog No. 10813-012). After treating RNA with 1.5 units of DNase I (Life Technologies, Gaithersburg, USA, Catalog # 18068-015), RNA was re-extracted with the TRIZOL / chloroform system, precipitated with ethanol and dissolved in DEPC-treated water. After that, the low molecular weight nucleotides were removed using the RNeasy Mini Kit (QIAGEN, Hilden, Germany, Catalog No. 74104) using the manufacturers instructions. Electrophoresis of RNA on agarose gel and staining with ethidium bromide revealed 28S and 18S ribosomal RNA, and fluorescence of ethidium bromide bound to 28S RNA was higher than that of 18S RNA. Purified whole RNA was stored at -80 ° C in a 70% ethanol solution until used in cDNA synthesis.

1-2. Synthesis of cDNA and labeled cRNA probes

cDNA was synthesized using the reverse SuperScript Choise System (Life Technologies, Gaithersburg, USA, Catalog No. 18090-019) in accordance with the manufacturer's instructions. Five micrograms of purified total RNA was hybridized with an oligo-dT primer (Sawady Technology, Tokyo, Japan) containing T7 promoter sequences and 200 SuperScriptII reverse transcriptase units, and the resulting system was incubated at 42 ° C for 1 hour. The resulting cDNA was extracted with phenol / chloroform and purified using Phase Lock Gel ^™ Light (Eppendorf, Hamburg, Germany, Catalog No. 0032 005.101).

cRNA was also synthesized using the MEGAscript T7 kit (Ambion, Austin, USA, Catalog No. 1334) and cDNA as template in accordance with the manufacturer's instructions. Approximately 5 μg cDNA was incubated in the presence of 2 μl of the enzyme mixture containing T7 polymerase, 7.5 M adenosine triphosphate (ATP) and guanosine triphosphate (GTP), 5.625 mm cytidine triphosphate (CTP) and uridine triphosphate (UTP), 1.875 m B -11-CTP and Bio-16-UTP (ENZO Diagnostics, Farmingdale, USA, Catalog No. 42818 and 42814, respectively) at 37 ° C for 6 hours. Mononucleotides and short nucleotides were removed by column chromatography on a CHRPMA SPIN + STE-100 column (CLONTECH, Palo Alto, USA, Catalog No. K1302-2) and cRNA in the eluates was precipitated by the addition of ethanol. After isolating 0.1 micrograms of cRNA by agarose gel electrophoresis and staining with ethidium bromide, the cRNA had a length in the range from 300 bases to 3 thousand bases. The purified cRNA was stored at a temperature below -80 ° C in a 70% ethanol solution until subsequent use.

1-3. Analysis of the tumor and normal tissue for gene expression

Samples of the gene expression of primary human tumors taken from patients with liver cancer were examined with a high density oligonucleotide microarray (HuGeneFL array, Affymetrix, Santa Clara, USA, Catalog No. 510137) (Lipshutz, RL et al. Nature Genet. 21, 20-24 ( 1999)). For hybridization with oligonucleotides on cRNA chips, they were fragmented at 95 ° C for 35 minutes in a buffer containing 40 mM Tris system (Sigma, St. Louis, USA, Catalog No. T1503) -acetic acid (Wako, Osaka, Japan, Catalog No. 017 -00256) (pH 8.1), 100 mM potassium acetate (Wako, Osaka, Japan, Catalog No. 160-03175), and 30 mM magnesium acetate (Wako, Osaka, Japan, Catalog No. 130-00095). Hybridization was carried out in 200 μl of a buffer containing 0.1 M 2- (N-morpholino) ethanesulfonic acid (MES) (Sigma, St. Louis, USA, Catalog No. M-3885), (pH 6.7), 1 M NaCl (Nacalai tesque, Tokyo, Japan, Catalog No. 313-20), 0.01% polyoxyethylene (10) octylphenyl ether (Wako, Osaka, Japan, Catalog No. 168-11805), 20 μg herring sperm DNA (Promega, Madison, USA, Catalog No. B181B), 100 μg of bovine serum acetylated albumin (Sigma, St. Louis, USA, Catalog No. B-8894), 10 μg of fragmented cRNA and biotinylated control oligonucleotides, Biotin-5′-CTGAACGGTAGCATCTTGAC-3 ′ (Sawady technology, Tokyo, Japan) at 45 ° C for 12 hours. After washing the chips with a buffer containing 0.01 M MES (pH 6.7), 0.1 M NaCl, 0.001% polyoxylene (10) octylphenyl buffer, the chips were incubated with biotinylated anti-streptavidin antibody (Funakoshi, Tokyo, Catalog No. BA0500) and stained streptavidin with R-phycoerythrin (Molecular Probes, Eugene, USA Catalog No. S-866) to amplify hybridization signals in accordance with manufacturers instructions (Affymetrix, Santa Clara, USA), after which the expression levels of each cDNA and the reliability of the experiment (presence / absence) was calculated using Affymetrix GeneChip ver.3 software. and Affymetrix Microarray Suite ver. 4.0. As a result of the experiments, the expression of approximately 6,000 genes was determined in an array of 41 human colorectal tumors, 30 gastric tumors, 41 small cell lung carcinomas, 24 breast tumors, 15 ovarian tumors, 53 hepatitocellular carcinomas, and 15 non-tumor liver tissues and 10 batches independently cultured granulocyte precursors (10 ⁷ cells in each batch).

Example 2:

The selection of enzymes, the expression of which is carried out preferably in tumors, but not in the precursors of granulocytes and in the liver

2-1. Reproduction of ex vivo granulocyte precursors

CD-34 mononuclear cells obtained from human umbilical cord and bone marrow blood were purchased from Veritas (Veritas Co., Tokyo, Japan, Catalog No. CB009F, ABM019F) and cultured on the confluent monolayer MS5 (Itoh, K., et al. Reproducible establishment of hematopoietic supportive stromal cells from murine bone marrow. Exp. Hematol. 17, 145-153 (1989)) stromal cell lines in alpha MEM medium (Life Technologies, Gaithersburg, USA, Catalog No. 12571-0063) supplemented with 10% ( v / v) horse serum (HS) (Stem Cell Technologies, Vancouver, Canada, Catalog No. 06750), 10% (v / v) fetal calf serum (FBS) (Stem Cell Technologies, Vancouver, Canada, Catalog No. 06450), 50 ng / ml Flt3 ligand (Pepro Tec EC., London, UK., Catalog 3 300-19), 100 n g / ml SCF (Pepro Tec EC., London, UK., Catalog No. 300-07) and 50 ng / ml TPO (Pepro Tec EC., London, UK., Catalog No. 300-18) at 37 ° C in the atmosphere humid air containing 5% CO ₂ . Surfaced hematopoietic cells were harvested and stained with monoclonal bodies against PerCP anti-CD34 (BD pharMingen, SanDiego, USA, Catalog No. 340430), PE-anti-CD13 (BD pharMingen, SanDiego, USA, Catalog No. 30525X) and FITC-anti-15 (BD pharMingen, SanDiego, USA, Catalog No. PM30525). Five microliters of each antibody was added in 50 μl of cell suspension and the mixture was incubated for 25 minutes at 4 ° C. After washing with PBS containing 10% (v / v) FCS, expression of CD antigens was detected using FACSCalibur (Becton Dickinson, Franklin Lakes, New Jersey, USA) according to the FACSCalibur Traing manual (FACStation ver1.1. Becton Dickinsojn, Franklin Lakes , New Jersey, USA). According to the FACS analysis, it was found that more than 90% of the mononuclear cells expressed the marker of the precursor CD34 after propagation under the above conditions. After the considered CD-34-positive cells were treated with 50 ng / ml G-CSF (Souza, LM, et al. Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 232, 61-65 (1986 ). Pepro Tech EC, London, UK. Catalog No. 300-23), over 7 days more than 80% of the cells differentiated into CD-34-negative, CD13- and CD15-positive myeloblasts and myelocytes and 14 days into neutrophils after adding G -CSF.

2-2. cDNAs, preferably expressing in tumors, but not in precursors of granulocytes and other non-tumor tissues

As a result of experiments with a DNA chip, several hundred cDNAs were obtained in which mRNA was absent (in accordance with the request for absence), or its expression proceeded at very low levels (as judged by the average difference value below 50) in the precursors of granulocytes and liver, while expression was observed (in accordance with a request for presence) with noticeable levels (as judged by the average difference above 200) in tumors of the breast, liver, stomach, colorectal region, pancreas, or egg Cove in more than 50% of the total patients. More than 150 cDNAs encoding proteins with known catalytic activity were selected from such cDNAs. Such enzymes include phospholipase C, microsomal dipeptidase, arylsulfatase A, DT-diaphorase, 5'-pyrroline karboksireduktazu, degidrodioldegidrogenazu, karbonilreduktazu, lysyl hydroxylase, prolidase, dihydropyrimidinase, gamma glyutimiltranspeptidazu, glutamine: fructose-6-fosfatamidotransferazu, UDP-galaktozakeramidgalaktoziltransferazu, lysyl oxidase, enolase, glucose-6-phosphate dehydrogenase, uridine phosphorylase, stearoyl coenzyme desaturase, epoxide hydrolase, aldolase C.

2-3. Kinetic RT-PCR analysis

The mRNA levels in the cDNA of the TTC-activating enzyme were also checked by studying the kinetics of RT-PCR. The kinetics of RT-PCR was studied by real-time fluorescence in a PCR system. PCR amplification using the LightCycler system (Roche Diagnostics, Mannheim, Germany, Catalog No. 2011468) was performed in 20 μl of a reaction mixture consisting of a starting mixture containing Taq DNA polymerase, reaction buffer, dNTP mixture and SYBR Green I dye (LightCycler - DNA Master SYBR Green I, Roche Diagnistics, Mannheim, Germany, Catalog # 2158817), 4 mM magnesium chloride (Nacalai rescque, Tokyo, Japan, Catalog No. 7791-18-6), 10 picomoles PCR primers (Sawady Technology, Tokyo, Japan) and 2 μl of template cDNA in a LightCycler capillary (Roche Diagnostics, Mannheim, Germany, Catalig No. 1909339). Human microsomal dipeptidase cDNA primers had the sequence ATCGACTTGGCTCACGTGTCTGTGG and TGTGATCCAGATGGTCGGCCACTTG. Amplification was carried out in LightCycler, carrying out 40 incubation cycles at 95 ° C for a period of time from 0 seconds to denaturation, at 57-60 ° C for 3-10 seconds for the purpose of annealing and at 72 ° C for 10 seconds for the purpose of building up, at a temperature gradient of 20 ° C / sec. Real-time PCR monitoring was provided by measuring fluorescence signals at the end of the denaturation phase in each amplification cycle. cDNAs from normal lungs, heart, liver, kidneys, intestines, colon, skin, and brain were synthesized using RNA supplied by Srtategene (Srtategene, La Jolla, USA, Catalog No. B6030-01 for the brain, D6050-01 for the colon, D6064- 01 for the heart, D6065-01 for the small intestine, D6070-01 for the kidneys, D6080-01 for the liver, D6115-01 for the skin).

Kinetic RT-PCR analysis for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using hybridization probes was also performed to qualitatively assess the integrity of the isolated RNA and normalize the number of copies of the target sequences. External standards for target mRNA and mRNA for GAPDH were prepared by 10-fold serial dilutions (from 10 ³ to 10 ⁸ ) of plasmid DNA. Quantification of mRNA in each sample was carried out automatically, referring to a standard curve plotted at each time point using LightCycler software (LightCycler software version 3, Roche Diagnostics, Mannheim, Germany). GAPDH cDNA amplification primers had the following sequences:

TCTCCAGAACATCATCCCTGCCTCTAC and

TGCTGTAGCCCAAATTCGTTGTCATACC.

Although microsomal dipeptidase mRNA was detected in the kidneys and small intestines, it was absent in the lungs, heart, stomach, colon, and liver. However, the levels of microsomal dipeptidase mRNA found in 12 colorectal tumors were significantly higher than in the kidneys and small intestines.

Microsomal dipeptidase mRNA levels in human tissues.

Fabrics MRNA level (relative to mRNA GAPDH)
Microsomal dipeptidase mRNA / GAPDH mRNA Colorectal tumors 2.6 Granulocyte Precursors 0.02 Colon 0.06 Leather <0.01 Brain <0.01 Heart <0.01 Liver 0,03 Kidney 0.58 Small intestine 0.37

Example 3:

Formate 13α - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-amino-4-methylpentanoylamino) -5-hydroxycarbonyl] pentanoyloxy} -3-benzoylamino-3-phenylpropionyloxy-2α -benzyloxy-4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9-one

a) A mixture of 2α-benzyloxy-13α - ((2R, 3S) -3-benzoylamino-2-hydroxy-3-phenylpropionyloxy) -4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11 en-9-one (taxol) (50.6 mg), 1- [3- (dimethylamino) propyl] ethylcarbodiimide hydrochloride (13.9 mg), dimethylaminopyridine (1.0 mg) and 1-benzyl ether (2S ) -2 - ((2S) -2-benzyloxycarbonylamino-4-methyl-pentanoylamino) hexanoic acid (31.9 mg) in dichloromethane (2.0 ml) was stirred at room temperature for 22 hours. The reaction mixture was cooled with water (3 ml) and the organic layer was separated. The aqueous layer was extracted twice with dichloromethane. The combined organic layer was washed with brine and dried over anhydrous sodium sulfate, and then concentrated in vacuo. The resulting mixture was purified by silica gel column chromatography, eluting with dichloromethane-ethyl acetate (2: 1), to obtain 13α - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-benzyloxycarbonylamino 4-methylpentanoylamino) -5-hydroxycarbonyl] pentanoyloxy} -3-benzoylamino-3-phenylpropionyloxy-2α-benzyloxy-4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9- it is in the form of a pale yellow solid (74.8 mg, 97.6%).

b) A mixture consisting of the above 13α - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-benzyloxycarbonylamino-4-methylpentanoylamino) -5-hydroxycarbonyl] pentanoyloxy} -3- benzoylamino-3-phenylpropionyloxy-2α-benzyloxy-4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9-one (31.3 mg), 10% Pd / C ( 7.1 mg) and formic acid (0.42 ml) in methanol (6.0 ml) were stirred in the presence of H ₂ at room temperature for 6.5 hours, the resulting mixture was filtered and the filtrate was concentrated in vacuo to give 13α formate - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-amino-4-methylpentanoylamino) -5-hydroxycarbonyl ] pentanoyloxy} -3-benzoylamino-3-phenylpropionyloxy-2α-benzyloxy-4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9-one as a pale yellow solid 23.6 mg, 91%).

¹ H-NMR (CDCl ₃ ): δ 0.8 ~ 0.9 (6H, m), 0.98 (3H, s), 1.20 (3H, s), 1.48 (3H, s), 1.2 ~ 1.6 (2H, m), 1.55 ~ 1.8 (7H, m), 1.75 (3H, s), 2.16 (3H, s), 2.3 ~ 2, 5 (6H, m), 2.25 (3H, s), 3.50 (1H, br), 3.80 (1H, m), 3.95 (2H, m), 4.08 (1H, m ), 4.7 ~ 4.9 (3H, m), 5.32 (1H, d, J = 11 Hz), 5.39 (1H, d, J = 8 Hz), 5.50 (1H, t , J = 9 Hz), 5.78 (1H, dt, J = 8.8 Hz), 6.29 (1H, s), 7.17 (1H, m), 7.4 ~ 7.8 (11H , m), 7.87 (2H, m), 7.98 (2H, m), 9.28 (1H, d, J = 11 Hz); ESI-MS: m / z 1110 (M ⁺ -CO ₂ H).

The compounds of examples 4 and 5 were obtained according to the procedure of Example 3 from 1-benzyl ester of (2S) -2 - ((2S) -2-benzyloxycarbonylamino-3-phenylpropionylamino) hexanedioic acid or 1-benzyl ester (2S) -2 - ((2S ) -2-benzyloxycarbonylamino-propionylamino) hexanedioic acid.

Example 4:

Formate 13α - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-amino-propionylamino) -5-hydroxycarbonyl] pentanoyloxy} -3-benzoylamino-3-phenylpropionyloxy-2α-benzyloxy -4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9-one

¹ H-NMR (CDCl ₃ ): δ 1.04 (3H, s), 1.10 (3H, s), 1.20 (3H, d, J = 11 Hz), 1.49 (3H, s) 1.5 ~ 1.8 (6H, m), 1.75 (3H, s), 2.14 (3H, s), 2.3 ~ 2.5 (6H, m), 2.25 (3H , s), 3.50 (1H, m), 3.80 (1H, m), 3.98 (2H, m), 4.12 (1H, m), 4.7 ~ 4.9 (3H, m), 5.32 (1H, d, J = 11 Hz), 5.42 (1H, d, J = 8 Hz), 5.52 (1H, t, J = 9 Hz), 5.80 (1H , drt, J = 8.8 Hz), 6.39 (1H, s), 7.18 (1H, m), 7.4 ~ 7.8 (11H, m), 7.87 (2H, m) 7.98 (2H, m); 9.31 (1H, d, J = 11 Hz); ESI-MS: m / z 1068 (M ⁺ -CO ₂ H).

Example 5:

Formate 13α - ((2R, 3S) -2 - {(5S) - [5 - ((2S) -2-amino-3-yenyl-propionylamino) -5-hydroxycarbonyl] pentanoyloxy} -3-benzoylamino-3-phenylpropionyloxy -2α-benzyloxy-4α, 10β-diacetoxy-1β, 7β-dihydroxy-5β, 20-epoxy-tax-11-en-9-one

¹ H-NMR (CDCl ₃ ): δ 0.98 (3H, s), 1.00 (3H, s), 1.47 (3H, s), 1.4 ~ 1.8 (6H, m), 1.75 (3H, s), 2.08 (3H, s), 2.3 ~ 2.5 (8H, m), 2.20 (3H, s), 3.60 (1H, br), 3 , 80 (1H, m), 3.98 (2H, m), 4.10 (1H, m), 4.70 (1H, br.), 4.9 (2H, br), 5.31 (1H d, J = 11 Hz), 5.40 (1H, d, J = 8 Hz), 5.53 (1H, t, J = 9 Hz), 5.79 (1H, dt, J = 8.8 Hz), 6.29 (1H, s), 7.15 ~ 7.30 (6H, m), 7.4 ~ 7.8 (11H, m), 7.87 (2H, m), 7.98 (2H, m), 9.31 (1H, d, J = 11 Hz); ESI-MS: m / z 1144 (M ⁺ -CO ₂ H).

Example 6:

(2R) - ((2S) -amino-3-cyclohexyl-propionylamino) - (3S) - [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydrofuran- (2R) -yl) -oxy-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy] butyric acid.

a) To a stirred solution of 2.5 g (8.09 mmol) of BOC-D-Thr (Bzl) -OH in 200 ml of CH ₂ CCl ₂ (dehydrated) was added 1.3 ml (8.9 mmol) of 2- (trimethylsilyl) ) ethanol, 0.5 g (4.04 mmol) of DMAP and 2.3 g (12.13 mmol) of WSC HCl. The resulting mixture was stirred for 5 hours in an Ar atmosphere at room temperature. The reaction mixture was cooled by adding water and the organic layer was separated. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with water and brine. The extract was dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on SiO ₂ (eluent: 20% EtOAc / Hexane) to give (3S) -benzyloxy- (2R) -tet-butoxycarbonylamino-butyric acid 2-trimethylsilanylethyl ester as a colorless viscous oil (2.66 g , 79%).

¹ H-NMR (270 MHz, CDCl ₃ ) δ 0.02 (9H, s), 0.90 (2H, dd, J = 6.6, 3.3, 2.6 Hz), 1 23 (3H, d, J = 6.3 Hz), 1.43 (9H, s), 4.03-4.26 (4H, m), 4.34 (1H, d, J = 12.0 Hz, AB), 4.54 (1H, d, J = 12.0 Hz, AB), 5.26 (1H, d, J = 9.6 Hz), 7.18-7.33 (5H, m ); MS: (LCMS) m / z 410 [M + H] ⁺ , 432 [M + Na].

b) To a stirred solution of 2.68 g (6.55 mmol) of (3S) -benzyloxy- (2R) -tet-butoxycarbonylamino-butyric acid 2-trimethylsilanyl ethyl ester in 50 ml of CH ₂ Cl ₂ (dehydrated) was added TFA at room temperature. The resulting mixture was stirred for 7 hours and concentrated under reduced pressure to obtain 2-trimethylsilanylethyl ethyl ester (2R) -amino (3S) -benzyloxy butyric acid trifluoroacetate as a viscous pale yellow oil (in the amount of 3,555 g). The resulting product was used in the next step without further purification.

¹ H-NMR (270 MHz, CDCl ₃ ) δ 0.03 (9H, s), 0.88 (2H, dd, J = 11.9, 5.9, 5.3 Hz), 1 36 (3H, d, J = 6.3 Hz), 3.94 (1H, d, J = 3.3 Hz), 4.11 (2H, m), 4.23 (1H, dd, J = 10.2, 6.9 Hz), 4.37 (1H, d, J = 11.9 Hz, AB), 4.61 (1H, d, J = 11.9 Hz, AB), 5, 26 (1H, d, J = 9.6 Hz), 7.18-7.34 (5H, m); MS: (LCMS) m / z 310 [M + H] ⁺ .

c) To a stirred solution of 181.6 g (0.439 mmol) of 2-trimethylsilanylethyl ethyl ester (2R) -amino (3S) -benzyloxy butyric acid in 5 ml of CH ₂ Cl ₂ (anhydrous preparation) was added 131 mg (0.484 mmol) of N -BOC- (L) - cyclohexylalanine and 170 mg (0.878 mmol) WSC + HCl at room temperature.

The resulting mixture was stirred for 18 hours in an Ar atmosphere at room temperature. The reaction mixture was cooled by adding water and the organic layer was separated. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with water and brine. The extract was dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on SiO ₂ (eluent: 10% EtOAc / Hexane) to give (3S) -benzyloxy- (2R) - ((2S) -tetbutoxycarbonylamino-3-cyclohexyl-propionylamino butyric acid 2-trimethylsilanylethyl ether as a colorless viscous oil (63.1 mg, 26%).

¹ H-NMR (270 MHz, CDCl ₃ ) δ 0.02 (9H, s), 0.91 (2H, dd, J = 10.2, 7.5, 6.9 Hz), 0 24-1.82 (13H, m), 1.21 (3H, d, J = 6.3 Hz), 1.43 (9H, s), 4.03-4.26 (3H, m), 4.37 (1H, d, J = 11.9 Hz, AB), 4.57 (1H, d, J = 11.9 Hz, AB), 4.59 (1H, dd, J = 9, 6, 2.3 Hz), 4.83 (1H, m), 6.77 (1H, d, J = 8.9 Hz), 7.21-7.64 (5H, m); MS: (LCMS) m / z 563 [M + H] ⁺ .

d) To a stirred solution of 61.3 mg (0.109 mmol) of (3S) -benzyloxy- (2R) - ((2S) -tetbutoxycarbonyl-amino-3-cyclohexylpropionylamino-butyric acid 2-trimethylsilanylethyl ester in 10 ml of CH ₂ Cl ₂ ( dehydrated) was added 1.0 ml of TFA at room temperature. The resulting mixture was stirred for 1 hour and then concentrated under reduced pressure to obtain 2-trimethylsilanylethyl ethyl ester trifluoroacetate (2R) - ((2S) -amino-3-cyclohexyl-propionylamino) -3-benzyloxybutyric acid as a colorless viscous oil ( in the amount of 77.9 mg). The resulting product was used in the next step without further purification.

¹ H-NMR (270 MHz, CDCl ₃ ): δ 0.02 (9H, s), 0.88 (2H, m), 0.8-1.74 (13H, m), 1.19 (3H, d, J = 6.3 Hz), 4.00-4.21 (3H, m), 4.11 (2H, m), 4.35 (1H, d, J = 11.9 Hz, AB), 4.54 (1H, dd, J = 8.2, 2.3 Hz), 4.57 (1H, d, J = 11.9 Hz, AB), 7.20-7.35 (5H, m); MS: (LCMS) m / z 463 [M + H] ⁺ .

f) To a stirred solution of (2R) - ((2S) amino-3-cyclohexyl-propionylamino-3-benzyloxybutyric acid 2-trimethylsilanyl ethyl ester trifluoroacetate in 5.0 ml of THF, 130 ml of 1 mol / l was added dropwise at room temperature NaOH. The pH of the reaction mixture was adjusted to 7. Thereafter, 74 mg (0.262 mmol) of 2- (trimethylsilyl) ethyl p-nitrophenyl carbonate was added to the reaction mixture, and the resulting mixture was heated to 60 ° C. in an oil bath. After stirring for 1 day at 60 ° C., the mixture was cooled to room temperature and diluted with EtOAc (20 ml) and water (20 ml) and the organic layer was separated. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with water and brine. The extract was dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on SiO ₂ (eluent: 10% -15% EtOAc / hexane) to give 2-trimethysilanyl ether (3S) -benzyloxy- (2R) - [3-cyclohexyl- (2S) - (2- trimethylsilanyl-ethoxycarbonylamino) propionylamino] butyric acid as a colorless viscous oil (49.6 mg, 62%).

¹ H-NMR (270 MHz, CDCl ₃ ) δ 0.02 (18H, s), 0.87-1.04 (6H, m), 1.10-1.28 (2H, m), 1.21 (3H, d, J = 6.3 Hz), 1.37-1.57 (3H, m), 1.67-1.83 (6H, m), 4.03-4.23 (4H, m ), 4.36 (1H, m), 4.38 (1H, d, J = 11.9 Hz, AB), 4.58 (1H, d, J = 11.9 Hz, AB), 4.59 (1H, dd, J = 9.2, 2.3 Hz), 4.97 (1H, m), 6.68 (1H, d, J = 9.2 Hz), 7.23-7, 36 (5H, m); MS: (LCMS) m / z 607 [M + H] ⁺ , 629 [M + Na] ⁺ .

f) To a solution of (3S) -benzyloxy- (2R) - [3-cyclohexyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) propionylamino-butyric acid 2-trimethysilanyl ester in 10 ml EtOAc was added 10% Pd / C . The reaction mixture was thoroughly mixed in an atmosphere of H ₂ . After stirring for 2 hours, the mixture was filtered by passing through a Celite thin layer column. The filtrate was concentrated under reduced pressure to obtain (2R) - [3-cyclohexyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) propionylamino] - (3S) hydroxybutyric acid 2-trimethisilanyl ether as a colorless viscous oil. The resulting product was used in the next step without further purification.

¹ H-NMR (270 MHz, CDCl ₃ ): δ 0.04 (9H, s), 0.05 (9H, s), 0.09-1.07 (6H, m), 1.11-1, 30 (2H, m), 1.22 (3H, d, J = 6.3 Hz), 1.37-1.56 (3H, m), 1.67-1.82 (6H, m), 2 09 (1H, d, J = 5.3 Hz), 4.15-4.36 (7H, m), 4.54 (1H, dd, J = 8.9, 2.6 Hz), 4.95 (1H, d, J = 7.6 Hz); 6.76 (1H, d, J = 8.6 Hz); MS: (LCMS) m / z 517 [M + H] ⁺ , 539 [M + Na] ⁺ .

g) To a stirred solution of 39.1 mg (0.075 mmol) of 2-trimethysilanyl ether (2R) - [3-cyclohexyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) propionylamino] - (3S) hydroxybutyric acid in 5.0 30 mg (0.151 mmol) of 4-nitrophenyl chloroformate and 1.0 ml of pyridine were added at room temperature in ml of CH ₂ Cl ₂ (dehydrated).

After stirring for 3 hours, the reaction mixture was cooled with water and the organic layer was separated. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with water and brine. The extract was dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on SiO ₂ (eluent: 20% EtOAc / Hexane) to give 2-trimethylsilanylethyl ether (2R) - [3-cyclohexyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) propionylamino] - ( 3S) - (4-nitrophenoxycarbonyloxy) -butyric acid as a colorless solid (64.7 mg).

¹ H-NMR (270 MHz, CDCl ₃ ): δ 0.02 (9H, s), 0.04 (9H, s), 0.97-1.04 (6H, m), 1.17-1, 38 (3H, m), 1.41 (3H, d, J = 6.3 Hz), 1.47-1.83 (8H, m), 4.16-4.33 (5H, m), 4 84 (1H, dd, J = 9.2, 2.6 Hz), 4.90 (1H, m), 7.38 (2H, d, J = 9.2 Hz), 8.2 ( 2H, d, J = 9.2 Hz); MS: (LCMS) m / z 681 [M + H] ⁺ .

h) To a stirred solution of 62.2 mg (0.09 mmol) of 2-trimethylsilanylethyl ether (2R) - [3-cyclohexyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) propionylamino] - (3S) - (4- nitrophenoxycarbonyloxy) -butyric acid in 5 ml of THF (dehydrated) was added 85 mg (0.182 mmol) of 3 ', 5'-di-tert-butyldimethisilyl-DMDC at room temperature. The reaction mixture was heated to 60 ° C in an oil bath. After stirring for 4 days, the mixture was cooled to room temperature and concentrated under reduced pressure. The oily residue was dissolved in EtOAc and washed with saturated NaHCO ₃ , water and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on SiO ₂ (eluent: 20-30% EtOAc / Hexane) to give 2-trimethylsilanylethyl ether (2R) [3-cyclohexyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) propionylamino] - (3S) - {1 - [(4S) - (tert-butyldimethylsilanyloxy) - (5R) - (tert-butyldimethylsilanyloxymethyl) -3-methylene-tetrahydrofuran- (2R) -yl] -2-oxo-1,2-dihydro-pyrimidin-4 -alkarbamoyloxy} -butyric acid as a colorless solid (38.8 mg, 50%, 2 steps).

¹ H-NMR (270 MHz, CDCl ₃ ) δ 0.00 (12H, s), 0.01 (9H, s), 0.02 (9H, s), 0.92 (9H, s), 0, 95 (9H, s), 0.86-1.81 (13H, m), 1.33 (3H, d, J = 6.3 Hz), 3.82 (2H, m), 4.17 (4H , m), 4.40 (1H, br s), 4.78 (2H, m), 5.32 (3H, m), 5.66 (1H, br s), 6.79 (1H, broad s), 7.1 (1H, broad d, J = 6.9 Hz), 7.65 (1H, broad s), 8.16 (1H, broad d, J = 6.9 Hz ), 9.80 (1H, broad s); MS: (LCMS) m / z 1010 [M + H] ⁺ .

i) To a stirred solution of 37.3 mg (0.037 mmol) of (2R) [3-cyclohexyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) propionylamino] - (3S) - {1 - [(4S) 2-trimethylsilanylethyl ether ) - (tert-butyldimethylsilanyloxy) - (5R) - (tert-butyldimethylsilanyloxymethyl) -3-methylene-tetrahydrofuran- (2R) -yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy} -butyric acid in 5.0 ml of THF (dehydrated) was added 220 ml of n-tetrabutylammonium fluoride (1 mol / L in THF) at room temperature.

After stirring for 1 hour, the solvent was removed under reduced pressure, the oily yellowish residue was purified by preparative HPLC (C18) * to give (2R) - ((2S) amino-3-cyclohexylpropionylamino) - (3S) - {1 - (( 4S) -hydroxy-pyrimidin-4-ylcarbamoyl] -butyric acid as a colorless solid (12.1 mg, 61%).

* Conditions for analysis by HPLC: column, 2 × 25cm (TSK-gel 80-TS ODS), eluent; 5% MeCN / H ₂ O to 100% MeCN (30 minutes, linear gradient), gas flow rate 9 ml / min, detection: photodiode array.

¹ H-NMR (270 MHz, CD ₃ OD): δ 0.95-1.82 (13H, m), 2.65 (3H, d, J = 6.6 Hz), 3.79 (2H, m ), 3.95 (2H, m), 4.46 (1H, d, J = 4.3 Hz), 4.68 (1H, m), 5.42 (1H, dd, J = 6, 6, 4.3 Hz), 5.47 (2H, t, J = 2.0 Hz), 6.67 (1H, d, J = 1.3 Hz), 7.26 (1H, d, J = 7.6 Hz), 8.20 (1H, d, J = 7.6 Hz); MS: (FABMS) m / z 538 [M + H] ⁺ .

The following compounds of Examples 7-13 were obtained from DMDC using dipeptide (threonine) derivatives of formula (VIII), following a procedure similar to that of Example 6.

Example 7

(2R) - ((2S) -Amino-4-methyl-pentanoylamino) - (3S) - [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydrofuran- (2R) -yl) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyloxy] butyric acid was obtained from 2-trimethylsilanylethyl ether (3S) -hydroxy- (2R) - [4-methyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) pentanoylamino] butyric acid.

¹ H-NMR (270 MHz, CD ₃ OD) δ 0.98 (6H, d, J = 4.9 Hz), 1.31 (3H, d, J = 6.3 Hz), 1.63-1 76 (3H, m), 3.77-3.99 (4H, m), 4.46 (1H, d, J = 4.0 Hz), 4.73 (1H, m), 5.41 ( 1H, m), 5.46 (2H, s), 6.66 (1H, s), 7.26 (1H, d, J = 7.3 Hz), 8.19 (1H, d, J = 7 , 3 Hz); MS: (FABMS) m / z 498 [M + H] ⁺ .

Example 8:

(2S) - ((2S) -Amino-3-biphenyl-4-yl-propionylamino) - (3S) - [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydrofuran- (2R ) -yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy] butyric acid was obtained from trimethylsilanylethyl ether (2R) - [3-biphenyl-4-yl- (2S) - (2-trimethylsilanyl- ethoxycarbonylamino) propionylamino] - (3S) hydroxybutyric acid.

¹ H-NMR (270 MHz, CD ₃ OD) δ 0.92 (3H, d, J = 6.6 Hz), 3.20 (2H, m), 3.76 (2H, m), 3.84 (1H, m), 4.26 (1H, t, J = 7.56 Hz), 4.35 (1H, d, J = 3.3 Hz), 4.68 (1H, m), 5.28 (1H, dd, J = 6.3, 3.0 Hz), 5.47 (2H, m), 6.54 (1H, d, J = 1.6 Hz), 7.11 (1H, d, J = 7.6 Hz), 7.23-7.60 (9H, m), 8.13 (1H, d, J = 7.6 Hz); MS: (FABMS) m / z 608 [M + H] ⁺ .

Example 9:

(2S) - ((2S) -Amino-3-biphenyl-4-yl-propionylamino) -3- {1- [4 (S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydrofuran- (2R) -yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy] propionic acid was obtained from trimethylsilanylethyl ether (2R) - [3-biphenyl-4-yl- (2S) - (9H-fluoren-9 -ylmethoxycarbonylamino) propionylamino] -3-hydroxypropionic acid.

¹ H-NMR (270 MHz, CD ₃ OD) δ 3.13 (2H, ddd, J = 13.2, 8.9, 8.2 Hz), 3.76 (1H, m), 3.87 ( 2H, m), 4.13 (1H, dd, J = 8.2, 6.9 Hz), 4.21 (1H, dd, J = 10.9, 3.3 Hz), 4.35 (1H dd, J = 10.9, 5.3 Hz), 4.59 (1H, dd, J = 5.2, 3.0 Hz), 4.70 (1H, m), 5.48 (2H, d, J = 2.3 Hz), 6.62 (1H, d, J = 1.3 Hz), 6.98 (1H, d, J = 7.6 Hz), 7.20-7.57 ( 9H, m), 8.04 (1H, d, J = 7.6 Hz); MS: (FABMS) m / z 594 [M + H] ⁺ .

Example 10

(2R) - ((2S) -amino-3-naphthalen-2-yl-propionylamino) 2-trimethylsilanylethyl ether - (3S) - [1- (4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro -furan- (2R) -yl) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyloxy] -butyric acid was obtained from 2-trimethylsilanylethyl ether of (3S) -hydroxy- (2R) - [3-naphthalene-2- yl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) propionylamino] butyric acid.

¹ H-NMR (270 MHz, CD ₃ OD) δ 0.69 (3H, d, J = 6.6 Hz), 3.20 (2H, m), 3.80 (1H, m), 3.90 (2H, m), 4.29-4.35 (2H, m), 4.67 (1H, m), 5.24 (1H, dd, J = 6.6, 3.0 Hz), 5.46 (2H, s), 6.65 (1H, d, J = 1.7 Hz), 7.12 (1H, d, J = 7.5 Hz), 7.38-7.47 (3H , m), 7.81-7.87 (4H, m), 8.13 (1H, d, J = 7.5 Hz); MS: (FABMS) m / z 582 [M + H] ⁺ .

Example 11:

(2R) - {(2S) -Amino-3- [4- (4-hydroxyphenoxy) phenyl] propionylamino} -3- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylene- tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyloxy] butyric acid was obtained from 2-trimethylsilanyl ethyl ester of (3S) -hydroxy- (2R) - [3- {4- [4- ( tert-butyl-dimethyl-silanoyloxy) phenoxy] phenyl} - (2S) - (2-trimethylsilyl-ethoxy-carbonylamino) -propionylamino] butyric acid.

¹ H-NMR (CD ₃ OD): δ 0.95 (3H, d, J = 6.2 Hz), 2.89-3.08 (2H, m), 3.74-3.98 (3H, m), 4.34 (1H, d, J = 2.9 Hz), 4.66 (1H, m), 5.30 (1H, m), 5.44 (2H, s), 6.65 ( 1H, s), 6.72 (2H, d, J = 7.1 Hz), 6.80 (2H, d, J = 7.0 Hz), 6.82 (2H, d, J = 8.5 Hz), 7.18 (2H, d, J = 8.6 Hz), 7.22 (1H, d, J = 7.6 Hz), 8.13 (1H, d, J = 7.6 Hz) ; MS: LC-MS m / z 640.0 [M + H] ⁺ .

Example 12:

(2R) - {(2S) -Amino-3- [4methoxyphenyl] propionylamino} - (3S) - [1 - [(4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydrofuran-2-yl] -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyloxy] butyric acid was obtained from 2-trimethylsilanyl-ethyl ester of (3S) -hydroxy- (2R) - [3- (4-methoxyphenyl) - (2S) - ( 2-trimethylsilanyl-ethoxy-carbonylamino) propionylamino] butyric acid.

¹ H-NMR (CD ₃ OD) δ 0.93 (3H, d, J = 6.6 Hz), 2.91-3.14 (2H, m), 3.72 (s, 3H), 3, 73-3.95 (3H, m), 4.11 (1H, t, J = 6.7 Hz), 4.34 (1H, br), 4.65 (1H, m), 5.31 (1H , m), 5.45 (2H, s), 6.65 (1H, s), 6.86 (2H, d, J = 6.9 Hz), 7.17 (2H, d, J = 7, 0 Hz), 7.21 (1H, d, J = 8.6 Hz), 8.15 (1H, d, J = 6.9 Hz); ESI-MS m / z 561.9 [M + H] ⁺ , 434, 297, 150.

Example 13:

(2R) Trimethylsilanylethyl ether - [(2S) -Amino-4-ethylsulfanyl-butyrylamino] - (3S) - [1 - [(4S) -hydroxy- (5R) -hydroxymethyl-3-methylene-tetrahydro-furan- (2R ) -yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyl] butyric acid was obtained from trimethylsilanylethyl ether (2R) - [4-ethynylsulfanyl- (2S) - (3-trimethylsilanyl-propionylamino) butylamino ] - (3S) -hydroxybutyric acid.

H-NMR (400 MHz, CD ₃ OD) δ 1.22 (3H, t, J = 7.6 Hz), 1.34 (3H, d, J = 6.4 Hz), 2.05 (1H, m), 2.14 (1H, m), 2.52-2.62 (4H, m), 3.80 (2H, m), 3.93 (1H, m), 4.08 (1H, t , J = 6.6 Hz), 4.49 (1H, d, J = 4.0 Hz), 4.68 (1H, m), 5.42 (1H, dd, J = 6.4, 4, 0 Hz), 5.47 (2H, broad), 6.67 (1H, s), 7.25 (1H, d, J = 7.2 Hz), 8.20 (1H, d, J = 7, 6 Hz); MS: (FAB-MS) m / z 530 [M + H] ⁺ .

Example 14:

(2R) - {(2S) -Amino-3- (1H-indol-3-yl) propionylamino] -4- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2-yl ) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyl] butyric acid.

(a) A mixture of Teoc-L-Trp-OH (25 g), hydrochloride of 5-benzyl ester-1- (2-trimethylsilanylethyl ether (2R) -aminopentanedioic acid (23 g), obtained according to the literature method (Pacofsky, Gregory J; J. Med. Chem., 41, 11, 1998, 1894-1908), WSCI (14 g), and diisopropylethylamine (25 ml) in dichloromethane (250 ml) was stirred at room temperature in an Ar atmosphere for 22 hours. The reaction mixture was cooled with water and the organic layer was separated.The aqueous layer was extracted with dichloromethane.The combined organic layer was washed with brine and dried over anhydrous sodium sulfate, after which was concentrated in vacuo.

The crude residue was purified by silica gel column chromatography, eluting with n-hexane-ethyl acetate (2: 1) to give 5- benzyl ester of 1- (2-trimethylsilanylethyl) ether (2R) - [3- (1H-indole-3 -yl) -2S- (2-trimethylsilanylethoxycarbonylamino) propionylamino] pentanedioic acid as a colorless oil (39 g, 93.2%).

¹ H-NMR [270 MHz: CDCl ₃ ]: δ 0.023-0.005 (18H, m), 0.88-0.98 (4H, m), 0.6-2.0 (4H, m), 3, 07-3.15 (1H, m), 3.25-3.30 (1H, m), 4.06-4.17 (4H, m), 4.4-4.6 (2H, m), 5.08 (2H, s), 5.2-5.3 (1H, br s), 6.18 (1H, d, J = 7.6 Hz), 6.96 (1H, s), 7 , 0-7.25 (3H, m), 7.3-7.4 (5H, m), 7.66 (1H, d, J = 7.3 Hz), 7.81 (1H, br s ); FAB-MS: m / z 668 [M + H] ⁺ .

(b) A mixture of 5- benzyl ester of 1- (2-trimethylsilanylethyl) ether (2R) - [3- (1H-indol-3-yl) -2S- (2-trimethylsilanylethoxycarbonylamino) propionylamino] pentanedioic acid (36 g) and 10% Pd-C (3.6 g) in ethyl acetate (350 ml) was stirred in the presence of gaseous H ₂ at room temperature for 22 hours.

The reaction mixture was filtered and the filtrate was evaporated in vacuo to give 1- (2-trimethylsilanylethyl) ether (2R) - [3- (1H-indol-3-yl) -2S- (2-trimethylsilanylethoxy carbonylamino) propionylamino] pentanedioic acid as a colorless oils (32 g).

¹ H-NMR: [270 MHz: CDCl ₃ ]: δ 0.018-0.01 (18H, m), 0.85-1.0 (4H, m), 0.6-2.1 (4H, m) , 3.1-3.4 (2H, m), 4.0-4.2 (4H, m), 4.4-4.6 (2H, m), 5.3-5.4 (1H, br.s), 6.5-6.6 (1H, br.s), 7.0-7.2 (3H, m), 7.33 (1H, d, J = 7.6 Hz), 7 61 (1H, d, J = 8.2 Hz), 8.33 (1H, br s) LS-MS: m / z 578 [M + H] ⁺ .

(c) Mixture of 1- (2-trimethylsilanylethyl) ether (2R) - [3- (1H-indol-3-yl) -2S- (2-trimethylsilanylethoxy carbonylamino) propionylamino] pentanedioic acid (29 g), 3 ', 5 '-bis-O- (tert-butyldimethylsilyl) -2'-deoxy-2'-methylidenecytidine (24 g), BOP reagent (27 g) and diisopropylethylamine (12 ml) in dichloromethane (500 ml) was stirred at room temperature in the atmosphere Ar for 19 hours. The reaction mixture was cooled with water and the organic layer was separated. The aqueous layer was extracted with dichloromethane. The combined organic layer was washed with brine and dried over anhydrous sodium sulfate, and then concentrated in vacuo.

The crude residue was purified by silica gel column chromatography, eluting with n-hexane / acetone (3: 1), to give 1- (2-trimethylsilanylethyl) ether 4- [1- (4S-tert-butyldimethylsilanyloxy-5R-tert-butyldimethylsilanyloxymethyl-3-methyl-3-methyl -2-yl) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyl] -2R- [3- (1H-indol-3-yl) -2S- (2-trimethylsilanylethoxycarbonylamino) propionylamino] butyric acid as a colorless amorphous solid (45 g, 86.4%).

¹ H-NMR: [270 MHz: CDCl ₃ ]: δ 0.01-0.13 (30H, m), 0.8-1.0 (22H, m), 1.6-2.1 (4H, m), 3.1-3.3 (2H, m), 3.78-3.85 (2H, m), 4.0-4.2 (5H, m), 4.35-4.45 ( 1H, m), 4.45-4.65 (1H, m), 4.77-4.79 (1H, m), 5.33-5.34 (1H, m), 5.44 (1H, d, J = 7.6 Hz), 5.6-5.7 (1H, m), 6.51 (1H, d, J = 7.9 Hz), 6.78 (1H, d, J = 1 , 3 Hz), 7.07-7.23 (4H, m), 7.35-7.38 (1H, m), 7.64 (1H, d, J = 7.3 Hz), 8.17 (1H, d, J = 7.6 Hz), 8.63 (1H, br s), 8.86 (1 H, br s); FAB-MS: m / z 1027 [M + H] ⁺ .

d) Mixture of 4- [1 - ((4S-tert-butyldimethylsilanyloxy- (5R) -tert-butyldimethylsilanyloxymethyl-3-methylenetetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidamo-4-yl ] - (2R) - [3- (1H-indol-3-yl) - (2S) - (2-trimethylsilanylethoxycarbonylamino) propionylamino] butyric acid (2 g) and TBAF [1 mol / L in THF] (39 ml) in tetrahydrofuran (20 ml) was stirred at room temperature in an Ar atmosphere for 23 hours. The reaction mixture was evaporated in vacuo. The crude residue was purified by ion exchange chromatography [Amberlite®CG-50], eluting with methanol m, and then by preparative reverse-phase HPLC, eluting with a mixture of H ₂ O-acetonitrile (85:15) to obtain (2R) - [(2S) amino-3- (1H-indol-3-yl) propionylamino ] -4- [1β - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyl] butyric acid as a white solid (449 mg, 41.6%).

¹ H-NMR: [270 MHz: CD ₃ OD]: δ 1.6-2.0 (4H, m), 3.17 (1H, dd, J = 7.3, 14.2 Hz), 3, 2-3.4 (1H, m), 3.76-3.83 (2H, m), 3.93 (1H, dd, J = 3.3, 13.2 Hz), 4.06-4, 17 (2H, m), 4.66-4.69 (1H, m), 5.44-5.47 (2H, m), 6.67 (1H, d, J = 1.7 Hz), 6 , 98-7.08 (2H, m), 7.17 (1H, s), 7.29-7.32 (2H, m), 7.58-7.61 (1H, m), 8.16 (1H, d, J = 7.6 Hz); FAB-MS: m / z 555 [M + H] ⁺ .

The following substances in examples 15-18 were obtained from DNDC using various dipeptide (glutamic acid) derivatives of formula (VII) using methods analogous to the method of Example 14.

Example 15:

(2R) - ((2S) -Amino-3-cyclohexylpropionylamino) -4- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2-yl) -2-oxo-1,2 -dihydropyrimidin-4-ylcarbamoyl] butyric acid was obtained from 1- (2-trimethylsilanylethyl) ether (2R) - [3-cyclohexyl- (2S) - (2-trimethylsilanylethoxycarbonylamino) propionylamino] pentanedioic acid.

¹ H-NMR: [270 MHz: DMSO-d ₆ ]: δ 0.7-1.0 (2H, m), 1.0-1.8 (11H, m), 1.8-2.0 ( 2H, m), 2.3-2.5 (2H, m), 3.5-3.8 (4H, m), 4.0 (1H, m), 4.51-4.53 (1H, m), 5.31 (1H, s), 5.34 (1H, s), 6.55 (1H, s), 7.20 (1H, d, J = 7.6 Hz), 8.10 ( 1H, d, J = 7.3 Hz); FAB-MS: m / z 522 [M + H] ⁺ .

Example 16:

(2R) - ((2S) -Amino-3-biphenyl-4-ylpropionylamino) -4- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2-yl) -2-oxo -1,2-dihydropyrimidin-4-ylcarbamoyl] butyric acid was obtained from 1- (2-trimethylsilanylethyl) ether (2R) - [3-biphenyl-4-yl- (2S) - (2-trimethylsilanylethoxycarbonylamino) propionylamino] pentanedioic acid.

¹ H-NMR: [270 MHz: DMSO-d ₆ ]: δ 1.7-1.8 (1H, m), 1.9-2.0 (1H, m), 2.2-2.4 ( 2H, m), 2.76-2.80 (1H, m), 3.0-3.04 (1H, m), 3.5-4.0 (4H, m), 4.08 (1H, m), 4.52-4.54 (1H, m), 5.12 (1H, br s), 5.32 (1H, s), 5.35 (1H, s), 5.74 (1H broad s), 6.55 (1H, s), 7.17 (1H, d, J = 8.0 Hz), 7.28-7.40 (5H, m), 7.55-7, 60 (4H, m), 8.09 (1H, d, J = 7.5 Hz), 8.11 (1H, br s), 11.0 (1H, br s); FAB-MS: m / z 592 [M + H] ⁺ .

Example 17:

(2R) - ((2S) -Amino-3-naphthalen-2-ylpropionylamino) -4- [1 - ((4S) -hydroxy- (5R) -hydroxymethyl-3-methylenetetrahydrofuran-2-yl) -2-oxo -1,2-dihydropyrimidin-4-ylcarbamoyl] butyric acid was obtained from 1- (2-trimethylsilanylethyl) ether (2R) - [3-nphthalen-2-yl- (2S) - (2-trimethylsilanylethoxycarbonylamino) propionylamino] pentanedioic acid.

¹ H-NMR: [270 MHz: DMSO-d ₆ ]: δ 1.7-2.0 (2H, m), 2.3-2.35 (2H, m), 2.89 (1H, dd, J = 8.6, 13.5 Hz), 3.19 (1H, dd, J = 5.3, 13.5 Hz), 3.5-4.0 (4H, m), 4.0-4 , 1 (1H, m), 4.51-4.54 (1H, m), 5.31 (1H, s), 5.34 (1H, s), 6.55 (1H, s), 7, 19 (1H, d, J = 7.6 Hz), 7.39-7.47 (3H, m), 7.73 (1H, s), 7.80-7.84 (3H, m), 8 11 (1H, d, J = 7.6 Hz); 8.20 (1H, br s); FAB-MS: m / z 566 [M + H] ⁺ .

Example 18:

(2R) - ((2S) -Amino-3-cyclohexyl-propionylamino) - (3S) - [1- (3,3-difluoro- (4R) -hydroxy- (5R) -hydroxymethyl-tetrahydro-furan-2- il) -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyl] butyric acid was obtained from DFDC and 2-trimethylsilanylethyl ether (2R) - [3-cyclohexyl- (2S) - (2-trimethylsilanyl-ethoxycarbonylamino) - propionylamino] - (3S) -hydroxybutyric acid, following the procedure of Example 6.

¹ H-NMR: (270 MHz: CD ₃ OD): δ 0.89-1.05 (2H, m), 1.16-1.25 (2H, m), 1.40-1.82 (9H , m), 1.32 (3H, d, J = 6.3 Hz), 3.80 (1H, dd, J = 2.9, 12.5 Hz), 3.93-4.05 (3H, m), 4.30 (1H, dq, J = 4.3, 8.3 Hz), 4.44 (1H, d, J = 3.9 Hz), 5.42 (1H, dt, J = 2 0, 4.3 Hz), 6.24 (1H, t, J = 6.5 Hz), 7.31 (1H, d, J = 7.6 Hz), 8.33 (1H, d, J = 7.6 Hz); LC-MS: m / z 561.9 [M + H] ⁺ .

Example 19:

(S) - [2 (S) -Amino-3-cyclohexyl-propionylamino) -3- [1- (3,3-difluoro-4 (R) -hydroxy-5 (R) -hydroxymethyl-tetrahydro-furan-2 - (R) -yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy] -2- (S) methylpropionic acid.

a) To a stirred solution of 255.1 mg (0.514 mmol) of 2 (S) - [2 (S) -benzyloxycarbonylamino-3-cyclohexyl-propionylamino] -3-hydroxy-2 (S) methylpropionic acid benzyl ester in 10.0 ml CH ₂ Cl ₂ (dehydrated) was added 207 mg (1,028 mmol) of 4-nitrophenyl chloroformate and 83 μl of pyridine at room temperature.

After stirring for 1.5 hours, the reaction mixture was cooled by adding water and the organic layer was separated. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with water and brine. The extract was dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure.

The crude product was purified by flash chromatography on SiO ₂ (eluent: 20% EtOAc / Hexane) to give 2 (S) - [2) S) benzyl ether-benzyloxycarbonylamino-3-cyclohexyl-propionylamino] -2 (S) -methyl- 3- (4-nitro-phenoxycarbonyloxy) propionic acid as a yellow solid (342.2 mg, quantitative yield; including a small amount of p-nitrophenol).

¹ H-NMR: (270 MHz, CDCl ₃ ) δ 0.97 (2H, m), 1.13-1.50 (5H, m), 1.61 (3H, s), 1.62-1, 77 (6H, m), 4.20 (1H, m), 4.66 (1H, d, J = 10.9 Hz, AB), 4.92 (1H, d, J = 10.9 Hz, AB ), 4.98 (1H, broad d), 5.08 (2H, m), 5.22 (2H, m), 6.92 (1H, broad s), 7.30 (2H, d, J = 9.6 Hz), 7.34 (10H, s), 8.22 (2H, d, J = 9.6 Hz); MS: (LCMS) m / z 662 [M + H] ⁺ .

b) To a stirred solution of 337 mg (0.509 mmol) of 2 (S) - [2) S) -benzyloxycarbonylamino-3-cyclohexyl-propionylamino] -2 (S) -methyl-3- (4-nitro-phenoxycarbonyloxy) propionic benzyl ester acid in 5 ml of THF (dehydrated preparation) was added 310 mg (0.611 mmol) of 3 ', 5'-di-O-tert-butyldimethylsilyl-DFDC at room temperature.

The reaction mixture was heated to 60 ° C in an oil bath. After stirring for 18 hours, the mixture was cooled to room temperature and concentrated under reduced pressure. The oily residue was dissolved in EtOAc and washed with saturated NaHCO ₃ solution, water and brine. The organic layer was dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on SiO ₂ (eluent: 20-40% EtOAC / hexane) to give the crosslinked product as a colorless solid (437.7 mg, 85%). The resulting product (106 mg; 0.105 mmol) was dissolved in 10 ml of THF (anhydrous preparation) and then added to 200 ml of n-tetrabutylammonium fluoride (1 mol / L in THF) at room temperature.

The stirring field for 2 hours, the solvent was removed under reduced pressure, the oily yellowish residue was purified by flash chromatography on SiO ₂ (eluent: 70% EtOAc - 100% EtOAc) to give 2 (S) - [2 (S) -benzyloxycarbonylamino benzyl ester -3-cyclohexyl-propionylamino) -3- [1- (3,3-difluoro-4 (R) -hydroxy-5 (R) -hydroxymethyl-tetrahydrofuran-2 (R) -yl) -2-oxo-1, 2-dihydropyrimidin-4-ylcarbamoyloxy] -2 (S) methylpropionic acid as a colorless solid (67.9 mg, 82%).

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.75 (2H, m), 1.03-1.37 (4H, m), 1.43 (3H, s), 1.52-1 62 (7H, m), 3.79 (2H, m), 3.85 (1H, m), 4.13 (1H, m), 4.19 (2H, m), 4.80 (1H, br.s), 4.90 (1H, d, J = 12.5 Hz, AB), 4.99 (1H, d, J = 12.5 Hz, AB), 5.02 (2H, s), 6.16 (1H, dd, J = 7.9 6.9 Hz), 7.19 (1H, d, J = 7.6 Hz), 7.20 (5H, s), 7.21 (5H, s), 8.21 (1H, d, J = 7.6 Hz); MS: (LCMS) m / z 786 [M + H] ⁺ .

c) To a solution of 62.1 mg (0.079 mmol) of 2 (S) - [2 (S) -benzyloxycarbonylamino-3-cyclohexyl-propionylamino) -3- [1- (3,3-difluoro-4 (R)] benzyl ester -hydroxy-5 (R) -hydroxymethyl-tetrahydro-furan-2 (R) -yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy] -2 (S) methylpropionic acid in 5 ml of MeOH 10% Pd / C was added.

The reaction mixture was thoroughly mixed in a hydrogen atmosphere. After stirring for 15 minutes, the mixture was filtered through a short-bed column of Celite. The filtrate was concentrated under reduced pressure and the crude product was purified by preparative HPLC (ODS) to give 2 (S) - [2 (S) amino-3-cyclohexyl-propionylamino) -3- [1- (3,3-difluoro-4 (R) -hydroxy-5 (R) -hydroxymethyl-tetrahydrofuran-2 (R) -yl) -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy] -2 (S) -methylpropionic acid as a colorless solid material (35.8 mg, 81%).

Conditions GHUR analysis: column 5 × 30 cm (TSK-gel 80-TS ODS), eluent; 5% MeCN / H ₂ O - 100% MeCN (40 minutes, linear gradient), flow rate 50 ml / min, detector: photodiode array.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.90 (2H, m), 1.01-1.34 (4H, m), 1.50 (3H, s), 1.54-1 75 (7H, m), 3.76 (2H, m), 3.95 (2H, m), 4.27 (1H, dd, J = 12.2, 8.2 Hz), 4.48 ( 1H, d, J = 10.9 Hz, AB), 4.92 (1H, d, J = 10.9 Hz, AB), 6.25 (1H, dd, J = 7.6, 6.9 Hz ), 7.28 (1H, d, J = 7.6 Hz), 8.30 (1H, d, J = 7.6 Hz); MS: (LCMS) m / z 562 [M + H] ⁺ .

The following compounds of Examples 20-22 were obtained from BABS using various dipeptide derivatives of formula (VIII), following the procedure of Example 19.

Example 20:

2 (R) - [2 (S) -Amino-3-cyclohexyl-propionylamino) -3- {1- [3,3-difluoro-4 (R) -hydroxy-5 (R) -hydroxymethyl-tetrahydro-furan- 2 (R) -yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy} -2 (R) methylpropionic acid was obtained from DFDC and 2 (R) - [2 (S) -benzyloxycarbonylamino benzyl ester -3-cyclohexyl-propionylamino] -3-hydroxy-2 (R) methylpropionic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.90 (2H, m), 1.02-1.45 (4H, m), 1.55 (3H, s), 1.62-1 75 (7H, m), 3.80 (1H, m), 3.93 (3H, m), 4.21 (1H, m), 4.27 (1H, d, J = 10.5 Hz, AB), 5.00 (1H, d, J = 10.5 Hz, AB), 6.24 (1H, dd, J = 7.6, 7.3 Hz), 7.27 (1H, d, J = 7.6 Hz), 8.28 (1H, d, J = 7.6 Hz); MS: (LCMS) m / z 562 [M + H] ⁺ .

Example 21:

(2S, 3S) -2- (2-amino-3-cyclohexyl-propionylamino) -3- [1 - {[4R, 5R) -3,3-difluoro-4-hydroxy-5-hydroxymethyl-tetrahydro-furan- 2-yl} -2-oxo-1,2-dihydropyridin-4-ylcarbamoyloxy] -2-methylbutyric acid was obtained from DFDC and (2S, 3S) -2- (2-benzyloxycarbonylamino-3-cyclohexyl-propionylamino) - benzyl ester 3-hydroxy-2-methylbutyric acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.91-1.70 (13H, m), 1.34 (3H, d, J = 6.3 Hz), 1.56 (3H, s ), 3.73-3.94 (4H, m), 4.25 (1H, td, J = 12.2, 8.6 Hz), 5.50 (1H, q, J = 6.6 Hz) 6.20 (1H, t, J = 7.3 Hz); 7.19 (1H, d, J = 7.6 Hz); 8.25 (1H, d, J = 7.6 Hz); MS: (LC-MS) m / z 576 [M + H] ⁺ .

Example 22:

(2R, 3R) -2- (2-amino-3-cyclohexyl-propionylamino) -3- [1 - {[4R, 5R) -3,3-difluoro-4-hydroxy-5-hydroxymethyl-tetrahydro-furan- 2-yl} -2-oxo-1,2-dihydro-pyridin-4-ylcarbamoyloxy] -2-methylbutyric acid was obtained from DFDC and (2R, 3R) -2- (2-benzyloxycarbonylamino-3-cyclohexyl-propionylamino benzyl ester ) -3-hydroxy-2-methylbutyric acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.77-1.75 (13H, m), 1.42 (3H, d, J = 6.6 Hz), 1.63 (3H, s ), 3.77-3.99 (4H, m), 4.27 (1H, td, J = 12.2, 8.3 Hz), 5.54 (1H, q, J = 6.6 Hz) 6.26 (1H, dd, J = 7.6, 7.3 Hz), 7.31 (1H, d, J = 7.6 Hz), 8.29 (1H, d, J = 7.6 Hz); MS: (LC-MS) m / z 576 [M + H] ⁺ .

Example 23:

2R- (2S-amino-3-cyclohexyl-propionylamino) -3S- [1- (4S-hydroxy-5R-hydroxymethyl-3-methylene-tetrahydro-furan-2R-yl) -2-oxo-1,2 isopropyl ester -dihydro-pyrimidin-4-ylcarbamoylosi] -butyric acid.

a) To a stirred solution consisting of 5.5 g (17.8 mmol) of BOC-D-Thr (Bzl) -OH, 280 mg (2.3 mmol) of DMAP and 2.7 ml (35.6 mmol) of 2 propanol in 50 ml of dichloromethane (dehydrated), 4.41 g (23.1 mmol) of WSC HCl was added at 0 ° C. The resulting mixture was stirred for 5 hours in an Ar atmosphere at ambient temperature. The reaction mixture was cooled with 300 ml of water and the organic layer was separated. The aqueous layer was extracted with EtOAc (300 ml × 2). The combined organic layer was washed with water (300 ml) and brine (300 ml), dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was removed under reduced pressure. The crude product was purified on a silica gel column (100 g, eluent: 20% EtOAc / Hexane) to give 3S-benzyloxy-2R-tert-butoxycarbonylaminobutyric acid isopropyl ester as a colorless syrup (6.372 g, quantitative yield).

¹ H-NMR: (270 MHz, CDCl ₃ ) δ 1.10-1.30 (9H, m), 1.43 (9H, s), 4.03-4.26 (2H, m), 4, 34 (1H, d, J = 11.6 Hz), 4.51 (1H, d, J = 11.6 Hz), 4.99 (1H, heptet, J = 6.6 Hz), 5.24 ( 1H, broad d, J = 8.9 Hz), 7.11-7.35 (5H, m); MS: (LCMS) m / z 373.9 (M + Na).

b) In a solution containing 6.372 g (18.1 mmol) of 3S-benzyloxy-2R-tert-butoxycarbonylaminobutyric acid isopropyl ester in 200 ml of EtOAc, 10% Pd / C was suspended and the resulting system was stirred vigorously for 3 hours under H ₂ atmosphere. The catalyst was filtered off and washed thoroughly with EtOAc. The filtrate was concentrated under reduced pressure to obtain 2R-tert-butoxycarbonylamino-3S-hydroxybutyric acid isopropyl ester as a colorless syrup (4.74 g, quantitative yield). The resulting product was used in the next step without further purification.

¹ H-NMR: (270 MHz, CDCl ₃ ) δ 1.16 (6H, d, J = 6.3 Hz), 1.23 (3H, d, J = 6.3 Hz), 1.43 (9H , s), 2.05 (1H, br s), 4.14-4.25 (2H, m), 5.06 (1H, heptet, J = 6.3 Hz), 5.27 (1H, d, J = 4.3 Hz); MS: (LCMS) m / z 262.1 [M + H] ⁺ .

c) To a solution of 4.74 g (18.1 mmol) of 2R-tert-butoxycarbonylamino-3S-hydroxybutyric acid isopropyl ester in 50 ml of EtOAc was added 18 ml of 4 N. HCl in EtOAC at room temperature. The resulting mixture was stirred for 14 hours and concentrated under reduced pressure to obtain 2R-amino-3S-hydroxybutyric acid isopropyl ester hydrochloride as a colorless syrup (3.60 g, quantitative yield). The resulting product was used in the next step without further purification.

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 1.21 (6H, d, J = 6.6 Hz), 1.25 (3H, d, J = 6.3 Hz), 3.83 (1H, d, J = 4.0 Hz), 4.05-4.15 (1H, m), 5.00 (1H, heptet, J = 6.3 Hz), 5.65 (1H, d, J = 5.3 Hz), 8.40 (3H, br s); MS: (LCMS) m / z 162.0 [M + H] ⁺ .

d) A solution consisting of 3.7 g (17.8 mmol) of 3-cyclohexyl-2S-amino-propionic acid hydrochloride, 6.3 g (18.7 mmol) of FmocOSu and 2.47 ml (21.5 mmol) triethylamine in 30 ml of dioxane and 15 ml of water was stirred for 18 hours at ambient temperature. The reaction mixture was concentrated under reduced pressure, and the residue was partitioned between EtOAc (200 ml) and 0.1 N. water citrate. The aqueous layer was extracted with EtOAc (200 ml). The combined organic layer was washed with water (100 ml), dried over anhydrous Na ₂ SO ₄ and filtered through a glass filter. The filtrate was concentrated under reduced pressure and the remaining solid material was treated with 20% EtOAc / n-hexane (100 ml) to give 3-cyclohexyl-2S- (9H-fluoren-9-ylmethoxycarbonylamino) propionic acid as colorless crystals (6.8 g , 97%).

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.76-0.96 (2H, m), 1.10-1.20 (4H, m), 1.25-1.35 (1H , m), 1.50-1.70 (6H, m), 4.00 (1H, dd, J = 8.9, 5.6 Hz), 4.21-4.30 (2H, m), 7.32 (2H, t, J = 7.6 Hz), 7.41 (2H, t, J = 7.6 Hz), 7.64 (1H, d, J = 8.3 Hz), 7, 90 (2H, d, J = 7.3 Hz), 12.5 (1H, s); MS: (LCMS) m / z 393.9 [M + H] ⁺ .

f) To a stirred suspension of 6.8 g (17.3 mmol) of 3-cyclohexyl-2S- (9H-fluoren-9-ylmethoxycarbonylamino) propionic acid and 2.0 g (17.3 mmol) of N-hydroxysuccinimide in 60 ml of a 50% dioxane-EtOAc system, 3.92 g of dicyclohexylcarbodiimide was added in one portion at 0 ° C. The reaction mixture was stirred for 6 hours at room temperature. The resulting precipitate was filtered through a glass filter and washed thoroughly with EtOAc. The filtrate was concentrated under reduced pressure to give crude N-succinimide ether. The residue was dissolved in 100 ml of dichloromethane, and 3.52 g (17.8 mmol) of 2R-amino-3S-hydroxybutyric acid isopropyl ester hydrochloride and 5.18 ml (37.4 mmol) were added to the system, and the resulting mixture was stirred for 9 hours. at ambient temperature. The reaction mixture was cooled with 0.1 N. aqueous citrate (100 ml) and the organic layer was separated. The aqueous layer was extracted with EtOAc (200 ml × 2) and the combined organic layer was washed with brine (100 ml), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The remaining material was recrystallized from 20% EtOAc / n-hexane to give 2R- [3-cyclohexyl-2S- (9H-fluoren-9-ylmethoxycarbonylamino) propionylamino] -3S-hydroxymalic acid isopropyl ester as colorless crystals (8.432 g, 91%).

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.86-0.96 (2H, m), 1.03 (3H, d, J = 6.3 Hz), 1.00-1, 40 (11H, m), 1.50-1.75 (6H, m), 4.08-4.30 (5H, m), 4.90 (1H, heptet, J = 5.6 Hz), 4 97 (1H, d, J = 5.6 Hz), 7.28-7.53 (4H, m), 7.60-7.78 (3H, m), 7.90 (2H, d, J = 7.3 Hz); MS: (LCMS) m / z 537.0 [M + H] ⁺ .

f) To a stirred solution of 8.40 g (15.7 mmol) of isopropyl ether 2R- [3-cyclohexyl-2S- (9H-fluoren-9-ylmethoxycarbonylamino) propionylamino] -3S-hydroxymalic acid in 200 ml of dichloromethane (dehydrated) 8.2 g (4.1 mmol) of 4-nitrophenyl chloroformate and 3.29 ml of pyridine were added at room temperature.

After stirring for 2 hours, the reaction mixture was cooled by adding water and the organic layer was separated. The aqueous layer was extracted with EtOAc (200 ml), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude product was recrystallized from EtOAc and n-hexane to give 2R- [3-cyclohexyl-2S- (9H-fluoren-9-ylmethoxycarbonylamino) propionylamino] -3S- (4-nitro-phenoxycarbonyloxybutyric acid isopropyl ester as colorless crystals ( 6 g, 96%).

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.86-0.96 (2H, m), 1.29 (3H, d, J = 6.3 Hz), 1.00-1, 40 (11H, m), 1.50-1.75 (6H, m), 4.21-4.35 (5H, m), 4.68 (1H, dd, J = 4.3, 8.6 Hz), 4.93 (1H, heptet, J = 6.3 Hz), 5.26 (1H, m), 7.29-7.55 (6H, m), 7.60-7.78 (2H , m), 7.90 (2H, d, J = 7.3 Hz), 8.31 (2H, dd, J = 2.3, 6.9 Hz), 8.54 (1H, d, J = 8.6 Hz); MS: (LCMS) m / z 702.1 [M + H] ⁺ .

g) A solution consisting of 5.0 g (7.0 mmol) of 2R- [3-cyclohexyl-2S- (9H-fluoren-9-ylmethoxycarbonylamino) propionylamino] -3S- (4-nitro-phenoxycarbonyloxy-butyric oil isopropyl ester acid and 3.8 g (8.12 mmol) of 3 ', 5'-di-tert-butyldimethylsilyl-DMDC in 40 ml of THF (anhydrous) were stirred for 2 days at 70 ° C. The resulting mixture was concentrated under reduced pressure. The oily residue was partitioned between EtOAc (150 ml × 2) and saturated NaHCO _3. combine the organic layer was washed with water (100 mL) and brine (100 ml × 2), dried over anhydrous Na ₂ SO ₄ and concentrated etc. The crude product was purified on a silica gel column (eluent: 25% EtOAc / Hexane) to give 2R- [3-cyclohexyl-2S-) 9H-fluoren-9-ylmethoxycarbonylamino) -propionylamino] -3S- {1- [4S- (tert-butyldimethylsilanyloxy) -5R- (tert-butyldimethylsilanyloxymethyl) -3-methylene-tetrahydro-furan-2R-yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy} butyric acid colorless amorphous material (6.6 g, 90%).

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.06 (3H, s), 0.06 (3H, s), 0.07 (6H, s), 0.83 (9H, s) , 0.88 (9H, s), 0.83-1.81 (22H, m), 3.70-3.80 (2H, m), 3.82 (1H, d, J = 6.8 Hz ), 4.15-4.25 (4H, m), 4.54 (1H, dd, J = 4.3, 8.6 Hz), 4.74 (1H, d, J = 5.3 Hz) 4.86 (1H, heptet, J = 6.3 Hz), 5.23 (1H, m), 5.29 (1H, s), 5.37 (1H, s), 6.57 (1H, s), 6.94 (1H, d, J = 5.0 Hz), 7.29 (2H, t, J = 7.3 Hz), 7.39 (2H, t, J = 7.3), 7.61 (1H, d, J = 8.3 Hz), 7.73 (2H, dd, J = 3.3, 7.6 Hz), 7.87 (2H, d, J = 7.3) 7.98 (2H, m); MS: (LCMS) m / z 1030.3 [M + H] ⁺ .

h) To a solution consisting of 200 mg (0.194 mmol) of 2R- [3-cyclohexyl-2S- (9H-fluoren-9-ylmethoxycarbonylamino) propionylamino] -3S- {1- [4S- (tert-butyldimethylsilanyloxy) isopropyl ester -5R- (tert-butyldimethylsilanyloxymethyl) -3-methylene-tetrahydrofuran-2R-yl] -2-oxo-1,2-dihydropyrimidin-4-ylcarbamoyloxy} butyric acid in 3 ml THF (anhydrous), was added at room temperature 323 ml (1.941 mmol) of triethylamine HF (98%). After stirring for 14 hours, the reaction mixture was concentrated under reduced pressure, and the residue was purified on a silica gel column (eluent: 6.25% methanol / dichloromethane) to give 2R- [3-cyclohexyl-2S- (9H-fluorene-9-) isopropyl ether ylmethoxycarbonylamino) propionylamino] -3S- [1- [4S-hydroxy-5R-hydroxymethyl-3-methylene-tetrahydrofuran-2R-yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy} butyric acid as a colorless amorphous material (145.7 mg, 94%).

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.80-0.99 (2H, m), 1.10-1.81 (20H, m), 3.55-3.80 (3H , m), 4.15-4.30 (4H, m), 4.50 (1H, m), 4.58 (1H, dd, J = 3.3, 8.9 Hz), 4.86 ( 1H, heptet, J = 6.3 Hz), 5.01 (1H, m), 5.20 (1H, m), 5.30 (1H, s), 5.34 (1H, s), 5, 66 (1H, brd), 6.53 (1H, s), 6.90 (1H, d, J = 7.6 Hz), 7.30 (2H, t, J = 7.3), 7 39 (2H, t, J = 7.2 Hz), 7.65 (1H, d, J = 8.2 Hz), 7.72 (2H, dd, J = 3.3, 7.6 Hz) 7.88 (2H, d, J = 7.3 Hz), 7.94 (1H, d, J = 8.2 Hz), 8.10 (1H, d, J = 7.6 Hz); MS: (LCMS) m / z 802.0 [M + H] ⁺ .

i) To a solution of 136 mg (0.17 mmol) of 2R- [3-cyclohexyl-2S- (9H-fluoren-9-ylmethoxycarbonylamino) propionylamino] -3S- [1- [4S-hydroxy-5R-hydroxymethyl- 3-methylene-tetrahydrofuran-2R-yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy} butyric acid in 1 ml of DMF (anhydrous) was added 100 ml of piperidine at room temperature.

After stirring for 3 hours, the solvent was removed under reduced pressure. The yellowish residue was purified on a silica gel column (eluent: 10% methanol / dichloromethane) to give 2R- (2S-amino-3-cyclohexyl-propionylamino] -3S- [1- [4S-hydroxy-5R-hydroxymethyl-3-] isopropyl ester methylene-tetrahydrofuran-2R-yl] -2-oxo-1,2-dihydro-pyrimidin-4-ylcarbamoyloxy} butyric acid as a colorless solid (28.6 mg, 29%).

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.75-0.95 (2H, m), 1.12-1.80 (20H, m), 3.55-3.80 (3H , m), 4.50 (1H, m), 4.60 (1H, m), 4.88 (1H, heptet, J = 6.3 Hz), 5.03 (1H, m), 5.30 -5.35 (4H, m), 5.70 (1H, brd), 6.53 (1H, s), 6.93 (1H, d, J = 7.6 Hz), 8.06 ( 1H, br s), 8.10 (1H, d, J = 7.6 Hz); MS: (LCMS) m / z 579.9 [M + H] ⁺ .

Reference example 2.1:

Preparation of (20S) -9-nitrocampothecin-N-oxide 20-acetate

To a solution of 9-nitro-camptothecin 20-acetate (8.62 g, 19.8 mmol) in trifluoroacetic acid (65 ml), a urea complex with hydrogen peroxide (3.11 g, 33.1 mmol) was added at room temperature. After stirring for 4 hours at room temperature, the resulting mixture was concentrated under reduced pressure to half volume and poured into a mixture of ice water. The resulting residue was collected by filtration, washed with distilled water and dried in vacuo to give the title compound (8.35 g, 93% yield).

¹ H-NMR: (270 MHz) δ (CDCl ₃ ) 0.98 (t, J = 7.6 Hz, 3H), 2.08-2.33 (m, 2H), 2.23 (s, 3H) ), 5.38 (s, 2H), 5.40 (d, J = 17.7 Hz, 1H), 5.67 (d, J = 17.7 Hz, 1H), 7.96 (s, 1H ), 7.96 (dd, J = 7.6 and 7.8 Hz, 1H), 8.67 (s, 1H), 9.16 (d, J = 7.6 Hz, 1H); MS m / z (ES) 452 (M ⁺ +1).

Reference example 3.1:

Preparation of (20S) -7-chloro-9-nitrocamptothecin 20-acetate

To a solution of (20S) -9-nitrocampothecin-N-oxide of 20-acetate (10.88 g, 24.1 mmol) from Reference Example 2.1 in N, N-dimethylformamide (196 ml) at 0 ° C. was added oxalyl chloride (4 , 2 ml, 48.2 mmol) and the resulting mixture was stirred for 3 hours at 15 ° C. The mixture was poured into ice water (500 ml) and extracted with ethyl acetate (500 ml × 1, 250 ml × 2). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1/1) to obtain the title compound (5.54 g, 49%) as a yellow solid.

¹ H-NMR: (270 MHz) δ (CDCl ₃ ) 0.99 (t, J = 7.6 Hz, 3H), 2.07-2.33 (m, 2H), 2.23 (s, 3H) ), 5.33 (s, 2H), 5.41 (d, J = 17.8 Hz, 1H), 5.69 (d, J = 17.8 Hz, 1H), 7.20 (s, 1H ), 7.87-7.95 (m, 2H), 8.44 (dd, J = 2.3 and 7.6 Hz, 1H); MS m / z (ES) 470 (M ⁺ +1).

Reference example 4.1:

Preparation of (20S) -9-nitro-7- (pentylamino) camptothecin 20-acetate

To a suspension of (20S) -7-chloro-9-nitro-camptothecin 20-acetate (2.58 g, 5.49 mmol) from Reference Example 3.1 in 1,4-dioxane (29 ml) was added n-amylamine (25 ml, 21 , 96 mmol) and the resulting mixture was stirred for 2 hours at 80 ° C, followed by concentration under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane / acetone = 30 / 1-20 / 1) to give the title compound (1.80 g, 63%) as a brown oil.

¹ H-NMR: (270 MHz) δ (CDCl ₃ ) 0.86-1.01 (m, 6H), 1.22-1.59 (m, 4H), 1.60-1.78 (m, 3H), 2.03-2.37 (m, 5H), 3.57-3.68 (m, 2H), 5.02 (br, 1H), 5.40 (d, J = 17.2 Hz , 1H), 5.47 (s, 2H), 5.67 (d, J = 17.2 Hz, 1H), 7.13 (s, 1H), 7.66 (dd, J = 2.0, 7.9 Hz, 1H), 7.71 (t, J = 7.9 Hz, 1H), 8.23 (dd, J = 2.0, 7.9 Hz, 1H); MS (ES) m / z 521 (M ⁺ +1).

Reference example 4.15:

Preparation of (20S) -7-butylamino-9-nitrocamptothecin 20-acetate

The title compound was prepared from (20S) -7-chloro-9-nitro-camptothecin 20-acetate of Reference Example 3.1 and butylamine in accordance with a method similar to that described in Reference Example 4.1.

¹ H-NMR: (270 MHz) δ (CDCl ₃ ) 0.97 (t, J = 7.6 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H), 1.43- 1.52 (m, 2H), 1.63-1.71 (m, 2H), 2.13-2.32 (m, 2H), 2.22 (s, 3H), 3.62-3, 69 (m, 2H), 5.02 (br t, 1H), 5.40 (d, J = 17.2 Hz, 1H), 5.47 (s, 2H), 5.66 (d, J = 17.2 Hz, 1H), 7.14 (s, 1H), 7.65-7.74 (m, 2H), 8.23 (dd, J = 1.6 and 7.9 Hz, 1H) ; MS m / z (ES) 507 (M ⁺ +1).

Reference example 5.1:

Preparation of (20S) -9-amino-7- (butylamino) camptothecin 20-acetate

(20S) -7-butylamino-9-nitro-camptothecin 20-acetate (156 mg, 0.31 mmol) from Reference Example 4.15 was dissolved in MeOH (10 ml) and 1N. aqueous solution of HCl (2 ml). 5% Pd-C (15 mg) was added and hydrogenated in a H ₂ atmosphere from a balloon at room temperature for 1 hour. After removal of Pd-C by filtration, the filtrate was concentrated under reduced pressure to obtain the product (137 mg, 87% yield).

¹ H-NMR: (270 MHz) δ (CDCl ₃ ) 0.95 (t, J = 7.6 Hz, 3H), 1.01 (t, J = 7.3 Hz, 3H), 1.48- 1.60 (m, 2H), 1.68-1.78 (m, 2H), 2.10-2.31 (m, 2H), 2.20 (s, 3H), 3.60-3, 67 (m, 2H), 3.90 (br s, 2H), 5.39 (d, J = 17.0 Hz, 1H), 5.41 (s, 2H), 5.66 (d, J = 17.0 Hz, 1H), 6.85 (d, J = 7.3 Hz, 1H), 7.11 (s, 1H), 7.45 (dd, J = 7.3 and 8.3 Hz , 1H), 7.64 (d, J = 8.3 Hz, 1H), 8.77 (br s, 1H); MS (ES) m / z 477 (M ⁺ +1).

Reference example 5.14:

Preparation of (20S) -9-amino-7- (pentylamino) camptothecin 20-acetate

The specified compound was obtained from (20S) -9-nitro-7- (pentylamino) camptothecin 20-acetate from Reference example 4.1, using a technique similar to that of Reference example 5.1.

MS (ES) m / z 491 (M ⁺ +1).

Example 1.1

Preparation of (9S) -1-butyl-9-ethyl-9-hydroxy-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4 , 3,2-de] quinazolin-2,10,13 (3H, 9H, 15H) -trione.

The production method includes the following two stages in which the compound (a) is used.

(a) (9S) -9-acetoxy-1-butyl-9-ethyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-2,10,13 (3H, 9H, 15H) -trion

(20S) -9-amino-7- (butylamino) -9-nitrocamptothecin 20-acetate hydrochloride (123 mg, 0.24 mmol) from Reference Example 5.1 was dissolved in dry CH ₂ Cl ₂ (5 ml) and cooled in a bath ice. DIEA (390 μl, 2.3 mmol) and triphosgene (67 mg, 0.23 mmol) were successively added and the resulting mixture was stirred for 1 hour in an ice bath. The reaction mixture was cooled 1 N. aqueous HCl to 0 ° C and was extracted with CH ₂ Cl ₂ (20 ml). The CH ₂ Cl ₂ layer was washed with brine, dried over MgSO ₄ and evaporated under reduced pressure. The resulting residue was purified by column chromatography (dichloromethane / acetone = 15 / 1-7 / 1) to give a pure product (70 mg, 56%).

¹ H-NMR: (270 MHz) δ (CDCl ₃ ) 0.98 (t, J = 7.7 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H), 1.43- 1.59 (m, 2H), 1.66-1.77 (m, 2H), 2.07-2.35 (m, 2H), 2.23 (s, 3H), 4.12-4, 18 (m, 2H), 5.36 (s, 2H), 5.40 (d, J = 17.4 Hz, 1H), 5.68 (d, J = 17.4 Hz, 1H), 6, 76 (dd, J = 1.5 and 6.7 Hz, 1H), 7.16 (s, 1H), 7.56-7.67 (m, 2H), 9.24 (s, 1H); MS (ES) m / z 503 (M ⁺ +1).

(b) (9S) -1-butyl-9-ethyl-9-hydroxy-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-2,10,13 (3H, 9H, 15H) -trion

To a solution of (9S) -9-acetoxy-1-butyl-9-ethyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [ 4,3,2-de] quinazolin-2,10,13 (3H, 9H, 15H) -trione (11.5 mg, 0.023 mmol) in MeOH (3 ml), cooled in an ice bath, anhydrous hydrazine ( 100 μl). The resulting mixture was warmed to room temperature and stirred for 1 hour. To acidify the reaction mixture, 1N was added dropwise. aqueous HCl and the resulting mixture was stirred at room temperature for 1 hour. After concentration under reduced pressure, the obtained residue was extracted with CH ₂ Cl ₂ (20 ml × 3). The combined CH ₂ Cl ₂ solution was washed with brine, dried over MgSO ₄ and evaporated. The residue was purified by column chromatography (dichloromethane / methanol = 30/1) to obtain the pure product (6.1 mg, 58%).

¹ H-NMR (400 MHz) δ (DMSO) 0.87 (t, J = 7.2 Hz, 3H), 0.96 (t, J = 7.6 Hz, 3H), 1.39-1, 47 (m, 2H), 1.64-1.70 (m, 2H), 1.81-1.91 (m, 2H), 4.03-4.07 (m, 2H), 5.42 ( s, 2H), 5.43 (s, 2H), 6.51 (s, 1H), 6.77 (d, J = 7.2 Hz, 1H), 7.24 (s, 1H), 7, 41 (d, J = 7.6 Hz, 1H), 7.61 (dd, J = 7.2 and 7.6 Hz, 1H), 11.15 (br s, 1H); MS (ES) m / z 461 (M ⁺ +1).

Example 1.14:

Preparation of (9S) -9-ethyl-9-hydroxy-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4 , 3,2-de] quinazolin-2,10,13 (3H, 9H, 15H) -trione

The title compound was prepared from (20S) -9-amino-7- (pentylamino) camptothecin 20-acetate from Reference Example 5.14 in accordance with a procedure similar to that described in Example 1.1, using a two-step process using compound (a) .

(a) (9S) -9-acetoxy-9-ethyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-2,10,13 (3H, 9H, 15H) -trion

¹ H-NMR: (270 MHz) δ (CDCl ₃ ) 0.92 (t, J = 6.9 Hz, 3H), 0.98 (t, J = 7.6 Hz, 3H), 1.29- 1.53 (m, 4H), 1.65-1.76 (m, 2H), 2.12-2.30 (m, 5H), 3.75-4.17 (m, 2H), 5, 36 (s, 2H), 5.40 (d, J = 17.5 Hz, 1H), 5.68 (d, J = 17.5 Hz, 1H), 6.75 (dd, J = 1.7 , 6.9 Hz, 1H), 7.15 (s, 1H), 7.58 (dd, J = 1.7, 6.9 Hz, 1H), 7.64 (dd, J = 6.9, 8.6 Hz, 1H); 8.88 (broad, 1H); MS (ES) m / z 517 (M ⁺ +1).

(b) (9S) -9-ethyl-9-hydroxy-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-2,10,13 (3H, 9H, 15H) -trion

¹ H-NMR: (270 MHz) δ (DMSO-d ₆ ) 0.85-0.93 (m, 6H), 1.36-1.38 (m, 4H), 1.69-1.88 ( m, 4H), 4.05 (m, 2H), 5.43 (s, 4H), 6.49 (s, 1H), 6.78 (d, J = 8.0 Hz, 1H), 7, 25 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 11.13 (br, 1H); MS (ES) m / z 475 (M ⁺ +1).

Example 2.1:

Preparation of (9S) -1-butyl-9-ethyl-9-hydroxy-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4 , 3,2-de] quinazolin-10,13 (9H, 15H) -dione

The production method includes the following two steps using compound (a).

(a) (9S) -9-acetoxy-1-butyl-9-ethyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione

To a solution of (20S) -9-amino-7- (butylamino) camptothecin 20-acetate hydrochloride (14.9 mg, 0.029 mmol) from Reference Example 5.1 in dry CH ₂ Cl ₂ (5 ml) was added trimethyl chloroformate (100 μl) and p-toluenesulfoxylate monohydrate (5 mg). The resulting mixture was washed successively with 1% aqueous NaHCO ₃ solution and brine, dried over MgSO ₄ and concentrated under reduced pressure. The obtained residue was purified by column chromatography (eluent: dichloromethane / methanol = 20/1) to obtain a pure product (12.6 mg, 89%).

¹ H-NMR (400 MHz) δ (CDCl ₃ ) 0.96 (t, J = 7.6 Hz, 3H), 1.01 (t, J = 7.4 Hz, 3H), 1.49-1 58 (m, 2H), 1.74-1.82 (m, 2H), 2.09-2.17 (m, 2H), 2.21 (s, 3H), 2.24-2.31 (m, 1H), 3.84 (t, J = 7.4 Hz, 2H), 5.22 (d, J = 17.8 Hz, 1H), 5.25 (d, J = 17.8 Hz , 1H), 5.39 (d, J = 17.2 Hz, 1H), 5.65 (d, J = 17.2 Hz, 1H), 7.10 (s, 1H), 7.16 (d , J = 7.2 Hz, 1H), 7.40 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.68 (dd, J = 7.2 and 8, 4 Hz, 1H); MS (ES) m / z 487 (M ⁺ +1).

(b) (9S) -1-butyl-9-ethyl-9-hydroxy-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione

To a solution of (9S) -9-acetoxy-1-butyl-9-ethyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [ 4,3,2-de] quinazolin-10,13 (9H, 15H) dione (6.1 mg, 0.013 mmol) in MeOH (2 ml), cooled in an ice bath, anhydrous hydrazine (100 μl) was added and the resulting the mixture was stirred for 1 hour at room temperature. To acidify the reaction mixture, 1N was added dropwise. an aqueous HCl solution and the resulting mixture was stirred at room temperature for 1 hour. After concentration under reduced pressure, the resulting residue was extracted with CH ₂ Cl ₂ (30 ml) and the CH ₂ Cl ₂ solution was washed with brine, dried over MgSO ₄ and evaporated. The residue was purified by column chromatography (dichloromethane / methanol = 20/1) to give a pure product (3.9 mg, 70%).

¹ H-NMR (400MHz) δ (DMDO-d ₆₎ 1.02 (t, J = 7,2 Hz, 6H), 1.50-1.59 (m, 2H), 1,76-1, 93 (m, 4H), 3.82 (t, J = 7.2 Hz, 2H), 3.88 (br s, 1H), 5.21 (s, 2H), 5.27 (d, J = 16.2 Hz, 1H), 5.70 (d, J = 16.2 Hz, 1H), 7.11 (dd, J = 1.6 and 7.4 Hz, 1H), 7.37 (s , 1H), 7.51 (s, 1H), 7.59-7.67 (m, 2H); MS (ES) m / z 445 (M ⁺ +1).

Example 2.15:

Preparation of (9S) -9-ethyl-9-hydroxy-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4 , 3,2-de] quinazolin-10,13 (9H, 15H) dione

The title compound was prepared from (20S) -9-amino-7- (pentylamino) camptothecin 20-acetate of Reference Example 5.14 according to a similar procedure to that used in Example 2.1 using a two-step process using compound (a).

(a) (9S) -9-acetoxy-9-ethyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione

¹ H-NMR (270 MHz) δ (CDCl ₃ ) 0.91-0.99 (m, 6H), 1.26-1.58 (m, 4H), 1.74-1.82 (m, 2H ), 2.09-2.31 (m, 5H), 3.83 (t, J = 7.3 Hz, 2H), 5.23 (s, 2H), 5.39 (d, J = 17, 2 Hz, 1H), 5.65 (d, J = 17.2 Hz, 1H), 7.09 (s, 1H), 7.17 (dd, J = 1.5, 6.9 Hz, 1H) 7.40 (s, 1H), 7.62 (dd, J = 1.5, 8.6 Hz, 1H); 7.68 (dd, J = 6.9, 8.6 Hz, 1H);

(b) (9S) -9-ethyl-9-hydroxy-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione

¹ H-NMR (270 MHz) δ (DMSO-d ₆ ) 0.85-0.92 (m, 6H), 1.35-1.38 (m, 4H), 1.75-1.93 (m 4H), 3.89-3.94 (m, 2H), 5.29 (s, 2H), 5.40 (s, 2H), 6.46 (s, 1H), 6.99 (dd, J = 1.0, 7.4 Hz, 1H), 7.18 (s, 1H), 7.47 (dd, J = 1.0, 8.6 Hz, 1H), 7.62 (dd, J = 7.4, 8.6 Hz, 1H); 7.86 (s, 1H); MS (ES) m / z 459 (M ⁺ +1).

Example 2.28:

Preparation of (9S) -9-ethyl-9-hydroxy-2-methyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5 ] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione

The title compound was prepared from (20S) -9-amino-7- (pentylamino) camptothecin 20-acetate of Reference Example 5.14 and trimethylorthoacetate in accordance with a method similar to that used in Example 2.1 using a two-step process using compound (a).

(a) (9S) -9-acetoxy-2-methyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione

¹ H-NMR (270 MHz) δ (CDCl ₃ ) 0.94 (t, J = 6.9 Hz, 3H), 0.97 (t, J = 7.3 Hz, 3H), 1.30-1 56 (m, 4H), 1.65-1.89 (m, 2H), 2.05-2.35 (m, 2H), 2.21 (s, 3H), 2.49 (s, 3H) ), 3.79-4.01 (m, 2H), 5.24 (br s, 2H), 5.39 and 5.66 (q, J = 17.2 Hz, 1H × 2), 7, 04-7.12 (m, 1H), 7.08 (s, 1H), 7.52-7.71 (m, 2H); MS (ES) m / z 515 (M ⁺ +1).

(b) (9S) -9-ethyl-9-hydroxy-2-methyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6 , 5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione

¹ H-NMR (270 MHz) δ (DMSO-d ₆ ) 0.87 (t, J = 7.3 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H), 1.30 -1.60 (m, 4H), 1.66-1.94 (m, 4H), 2.45 (d, J = 2.6 Hz, 3H), 3.93 (broad, 2H), 5, 23-5.44 (m, 2H), 5.41 (br s, 2H), 6.50 (br s, 1H), 6.89-7.00 (m, 1H), 7.19 ( d, J = 2.3 Hz, 1H), 7.38-7.49 (m, 1H), 7.62 (dt, J = 3.6 and 7.9 Hz, 1H); MS (FAB) m / z 473 (M ⁺ +1).

Example 3.1:

Preparation of (9S) -9-ethyl-9-hydroxy-2-hydroxymethyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5 ] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione.

The production method includes the following two steps using compound (a).

(a) (9S) -9-acetoxy-2-acetoxymethyl-9-ethyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6 , 5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione

To a solution of (20S) -9-amino-7- (pentylamino) camptothecin 20-acetate hydrochloride (1.61 g, 3.07 mmol) from Reference Example 5.14 in dry dichloromethane (120 ml), cooled in an ice bath, sequentially acetoxyacetyl chloride (4.3 ml) and diisopropylethylamine (1.07 ml) were added. After complete addition, the resulting mixture was warmed to room temperature and stirred overnight. Water (50 ml) was added and the mixture was extracted with dichloromethane (100 ml). The dichloromethane layer was washed with brine, dried over MgSO ₄ and concentrated under reduced pressure. The obtained residue was purified by column chromatography (eluent: ethyl acetate / hexane = 8/1) to obtain a pure product (1.72 g, 98%).

¹ H-NMR (400 MHz) δ (CDCl ₃ ) 0.91 (t, J = 7.3 Hz, 3H), 0.97 (t, J = 7.5 Hz, 3H), 1.31-1 48 (m, 4H), 1.70-1.82 (m, 2H), 2.08-2.30 (m, 2H), 2.22 (s, 3H), 2.25 (s, 3H) ), 3.86 (t, J = 7.9 Hz, 2H), 5.04 (s, 2H), 5.26 (s, 2H), 5.39 (d, J = 17.1 Hz, 1H ), 5.66 (d, J = 17.1 Hz, 1H), 7.13 (s, 1H), 7.19 (dd, J = 2.0 and 6.6 Hz, 1H), 7.63 -7.73 (m, 2H); MS (ES) m / z 573 (M ⁺ +1).

(b) (9S) -9-ethyl-9-hydroxy-2-hydroxymethyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6 , 5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione

To a solution of (9S) -9-acetoxy-2-acetoxymethyl-9-ethyl-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6, 5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione (34 mg, 0.059 mmol) in methanol (3 ml), cooled in an ice bath, anhydrous hydrazine (100 μl) was added and the resulting mixture was stirred for 2 hours at room temperature. To acidify the reaction mixture, 1N was added dropwise. HCl solution (5 ml) and the resulting mixture was stirred at room temperature for 1 hour. The mixture was extracted with dichloromethane (50 ml) and the dichloromethane layer was washed with brine, dried over MgSO ₄ and evaporated. The residue was purified by column chromatography (dichloromethane / methanol = 25/1) to give pure product (19 mg, 65%).

¹ H-NMR (400 MHz) δ (DMSO-d ₆ ) 0.87 (t, J = 7.6 Hz, 3H), 0.90 (t, J = 6.9 Hz, 3H), 1.32 -1.45 (m, 4H), 1.74-1.90 (m, 4H), 4.04 (m, 2H), 4.43 (d, J = 5.6 Hz, 2H), 5, 36 (s, 2H), 5.41 (s, 2H), 5.79 (t, J = 5.6 Hz, 1H), 6.50 (s, 1H), 7.03 (dd, J = 1 , 0 and 7.3 Hz, 1H), 7.20 (s, 1H), 7.50 (dd, J = 1.0 and 8.6 Hz, 1H), 7.66 (dd, J = 7, 3 and 8.6 Hz, 1H); MS (ES) m / z 489 (M ⁺ +1).

Example 24:

Preparation of 20-O - [(S) -tryptophil-γ- (S) -glutamyl] -20- (S) -camptothecin hydrochloride

a) To a stirred solution of 2.5 g (7.58 mmol) of α-tert-butyl-γ-benzyl diester of L-glutamic acid hydrochloride in 75 ml of dichloromethane was added 3.65 g (9.10 mmol) of N-α-Boc -L-tryptophan hydroxy succinimide in 1.59 ml (9.10 mmol) of N, N-diisopropylethylamine. The resulting mixture was stirred overnight under nitrogen at room temperature. The reaction mixture was cooled by the addition of a saturated solution of ammonium chloride and the organic layer was separated. The aqueous layer was extracted with dichloromethane. The combined organic layer was washed with water and brine. The extract was dried over anhydrous magnesium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by medium pressure liquid chromatography with Lobar LiChroprep Si-60 Grobe C (eluent: ethyl acetate / dichloromethane = 1/1) to give 1-tert-butyl ether 5-benzyl ether 2 (S) - [2 (S) - tert-butoxycarbonylamino-3- (1H-indol-3-yl) -propionylamino] -pentanedioic acid as a white amorphous substance (4.38 g, quantitative yield).

¹ H-NMR: (270 MHz, CDCl ₃ ) δ 1.30-1.49 (15H, m), 1.62 (3H, s), 1.73-1.95 (1H, m), 2, 02-2.25 (3H, m), 3.13 (1H, dd, J = 14.5, 6.3 Hz), 3.27-3.43 (1H, m), 4.33-4, 56 (2H, m), 4.90-5.15 (3H, m), 5.08 (2H, s), 6.52 (1H, d, J = 7.3 Hz), 7.00 (1H d, J = 2.3 Hz), 7.03-7.28 (3H, m), 7.30-7.47 (5H, m), 7.59 (1H, dd, J = 5.6 , 1.7 Hz), 7.90 (1H, br s); MS (LCMS) m / z 580 [M + H] ⁺ .

b) To a stirred solution of 4.33 g (7.47 mmol) of 1 tert-butyl ether 2 (S) - [2 (S) -tert-butoxycarbonylamino-3- (1H-indol-3-yl) -propylamino] - pentanedioic acid 5-benzyl ether in 80 ml of ethyl acetate was added a catalytic amount of palladium on carbon. The mixture was stirred overnight in a hydrogen atmosphere at room temperature. The mixture was filtered to remove the catalyst and the solvent was removed under reduced pressure to obtain 2 (S) - [2 (S) -tert-butoxycarbonylamino-3- (1H-indol-3-yl) -propionylamino] -pentadiol 1-tert-butyl ester acid in the form of a white amorphous substance (3.70 g, quantitative yield). This product was used in subsequent reactions without further purification. MS: (LCMS) m / z 490 [M + H] ⁺ .

c) To a stirred solution of 3.70 g (7.56 mmol) of 1-tert-butyl ether of 2 (S) - [2 (S) -tertbutoxycarbonylamino-3- (1H-indol-3-yl) -propionylamino] -pentanedioic acid in 1.83 g (14.9 mmol) of 4- (dimethylamino) pyridine, 5.73 g (29.9 mmol) of 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride and 1.73 were added to 200 ml of dichloromethane. g (4.98 mmol) camptothecin. The resulting mixture was stirred for 2 hours under nitrogen at room temperature. The reaction was stopped by adding water and the organic layer was separated. The aqueous layer was extracted with dichloromethane. The combined organic layer was washed with 0.5 N. hydrochloric acid solution, saturated sodium bicarbonate solution, water and brine. The extract was dried over anhydrous magnesium sulfate and filtered. The solvent was removed under reduced pressure. The crude product was purified by medium pressure liquid chromatography with Lobar LiChroprep Si-60 Grobe C (eluent: ethyl acetate / dichloromethane = 20/1) to give 5- (4 (S) -ethyl-3,13-dioxo-3 1-tert-butyl ether. 4,12,13-tetrahydro-1H-2-oxa-6,12a-diazadibenzo [b, h] fluoren-4-yl ester 2 (S) - [2 (S) -tert-butoxycarbonylamino-3- (1H- indol-3-yl) -propionylamino] -pentanedioic acid as a light yellow amorphous material (3.95 g, 97%) MS:. (LCMS) m / z 820 [M ⁺ H +].

d) To a stirred solution of 3.95 g (4.82 mmol) of α5- (4 (S) -ethyl-3,13-dioxo-3,4,12,13-tetrahydro-1H-2-oxa 1-tert-butyl ester -6,12a-diazadibenzo [b, h] fluoren-4-yl ester of 2 (S) - [2 (S) -tert-butoxycarbonylamino-3- (1H-indol-3-yl) -propionylamino] -pentanedioic acid in 20 ml of ethyl acetate were added 40 ml of 1N hydrochloric acid and 20 ml of trifluoroacetic acid.The mixture was stirred overnight under nitrogen at room temperature. The reaction mixture was added to 800 ml of ethyl acetate and the resulting precipitate was filtered off with hydrochloride. 20-O - [(S) -tryptophil-γ- (S) -glutamyl] -20 (S) -camptothecin as a reddish solid (3.2 g, 95%).

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.03 (3H, t, J = 7.5 Hz), 2.02-2.39 (4H, m), 2.68-2.83 (2H, m), 3.06-3.23 (2H, m), 3.27-3.35 (m), 3.38-3.50 (2H, m), 4.17-4.33 (1H, m), 4.42-4.57 (1H, m), 4.79-4.97 (m), 5.20-5.38 (2H, m), 5.55 (2H, dd , J = 38.6, 16.8 Hz), 6.97 (1H, t, J = 7.6 Hz), 7.11 (1H, t, J = 7.9 Hz), 7.19 (1H , s), 7.36 (2H, d, J = 8.3 Hz), 7.58 (1H, s), 7.56 (1H, d, J = 7.9 Hz), 7.76 (1H , t, J = 6.9 Hz), 7.83-7.93 (1H, m), 8.11 (1H, d, J = 8.6 Hz), 8.25 (1H, d, J = 8.3 Hz), 8.77 (1H, s); MS (LCMS) m / z 664 [M + H] ⁺ .

The following compounds in examples 25-49 were obtained from camptothecin or SN38 using various dipeptide derivatives of formula (VII), following a procedure similar to that used in Example 24.

Example 25:

20-O - [(S) -valyl-γ- (S) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (S) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 3-methyl-butyrylamino] -pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.97-1.14 (9H, m), 1.93-2.38 (6H, m), 2.63-2.92 (2H, m), 3.27-3.34 (m), 3.81 (1H, d, J = 5.3 Hz), 4.48-4.59 (1H, m), 4.80-4.97 (m), 5.38 (2H, br s), 5.55 (2H, dd, J = 35.6, 17.2 Hz), 7.69 (1H, s), 7.80 (1H, t, J = 8.3 Hz), 8.00 (1H, td, J = 8.6, 1.3 Hz), 8.18 (1H, d, J = 8.3 Hz), 8.33 ( 1H, d, J = 8.6 Hz), 8.88 (1H, s); MS (LCMS) m / z 577 [M + H] ⁺ .

Example 26:

20-O - [(S) -phenylalanyl-γ- (S) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (S) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 3-phenyl-propionylamino] pentanedioic acid.

¹ H-NMR (270 MHz, CD ₃ OD) δ 0,99-1,10 (3H, m), 1.33-1.42 (1H, m), 1,97-2,42 (4H, m), 2.73 (1H, t, J = 9.6 Hz), 2.93-3.12 (1H, m), 3.19-3.40 (m), 3.52-3.82 (2H, m), 4.02-4.26 (1H, m), 4.40-4.60 (1H, m), 4.71-4.91 (m), 5.28-5.39 (2H, m), 5.56 (2H, dd, J = 38.3, 17.2 Hz), 7.14-7.23 (1H, m), 7.25-7.43 (6H, m ), 7.73 (1H, t, J = 6.9 Hz), 7.88 (1H, t, J = 8.6 Hz), 8.09 (1H, d, J = 8.3 Hz), 8.13-8.22 (1H, m), 8.64 (1H, d, J = 8.3 Hz); MS: (LCMS) m / z 625 [M + H] ⁺ .

Example 27:

20-O - [(S) -leucyl-γ- (S) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (S) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 4-methyl-pentanoylamino] -pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.89-1.12 (11H, m), 1.58-2.41 (8H, m), 2.60-2.99 (2H, m), 3.23-3.36 (m), 3.51-3.79 (7H, m), 3.87 (1H, br s), 4.00-4.11 (1H, m) 4.47-4.63 (1H, m), 4.74-5.00 (m), 5.39 (2H, br s), 5.54 (2H, dd, J = 36.0, 17.5 Hz), 7.78-7.94 (2H, m), 7.98-8.11 (1H, m), 8.24 (1H, d, J = 8.3 Hz), 8, 42 (1H, d, J = 8.6 Hz), 8.99 (1H, s); MS: (LCMS) m / z 591 [M + H] ⁺ .

Example 28:

20-O - [(R) -leucyl-γ- (S) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (S) - [2 (R) -tert-butoxycarbonylamino 1-tert-butyl ester 4-methyl-pentanoylamino] -pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.83-1.12 (10H, m), 1.60-1.82 (3H, m), 1.90-2.40 (3H, m), 2.54-2.80 (2H, m), 3.17-3.40 (m), 3.51-3.80 (3H, m), 3.81-3.97 (1H, m), 4.43-4.60 (1H, m), 4.62-5.00 (m), 5.32 (2H, br s), 5.54 (2H, dd, J = 37, 3, 17.0 Hz), 7.38 (1H, s), 7.71 (1H, t, J = 7.3 Hz), 7.87 (1H, t, J = 7.8 Hz), 8 06 (1H, d, J = 7.6 Hz), 8.16 (1H, d, J = 8.4 Hz), 8.63 (1H, s); MS (LCMS) m / z 591 [M ⁺ H] +.

Example 29:

20-O - [(R) -phenylalanine-γ- (S) -glutamyl] -20 (S) -camptothecin trifluoroacetate was obtained from 2 (S) - [2 (R) -tert-butoxycarbonylamino 1-tert-butyl ester 3-phenyl-propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.05 (3H, t, J = 7.6 Hz), 1.70-1.99 (1H, m), 2.02-2.47 (5H, m), 2.95-3.22 (2H, m), 3.23-3.41 (m), 4.09 (1H, t, J = 7.9 Hz), 4.43 ( 1H, dd, J = 9.2, 4.6 Hz), 4.70-4.90 (m), 5.29 (2H, d, J = 3.3 Hz), 5.54 (2H, dd , J = 38.3, 17.2 Hz), 7.19-7.49 (6H, m), 7.72 (1H, td, J = 6.9, 1.3 Hz), 7.88 ( 1H, td, J = 6.9, 1.3 Hz), 8.08 (1H, d, J = 7.3 Hz), 8.17 (1H, d, J = 8.6 Hz), 8, 62 (1H, s); MS (LCMS) m / z 625 [M + H] ⁺ .

Example 30:

20-O - [(S) -tryptophil-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (R) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 3- (1H-indol-3-yl) propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.03 (3H, t, J = 7.6 Hz), 1.69-1.98 (1H, m), 2.05-2.36 (5H, m), 3.14 (2H, d, J = 36.6 Hz), 3.22-3.39 (m), 4.09 (1H, t, J = 6.9 Hz), 4 , 33-4.46 (1H, m), 4.72-5.02 (m), 5.03 (2H, dd, J = 42.2, 18.8 Hz), 5.53 (2H, dd , J = 45.5, 16.8 Hz), 6.80-6.98 (2H, m), 7.10 (1H, t, J = 7.3 Hz), 7.29 (1H, d, J = 7.9 Hz), 7.32-7.41 (2H, m), 7.69 (1H, td, J = 13.9, 6.9 Hz), 7.78-7.89 (1H , m), 8.00 (1H, d, J = 7.9 Hz), 8.10 (1H, d, J = 8.6 Hz), 8.44 (1H, s); MS: (LCMS) m / z 664 [M ⁺ H] +.

Example 31:

20-O - [(R) -tryptophil-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (R) - [2 (R) -tert-butoxycarbonylamino 1-tert-butyl ester 3- (1H-indol-3-yl) propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.03 (3H, t, J = 7.3 Hz), 1.94-2.40 (6H, m), 2.73 (2H, t , J = 7.3 Hz), 3.00-3.17 (1H, m), 3.19-3.48 (m), 3.53-4.78 (2H, m), 4.08- 4.23 (1H, m), 4.45-4.58 (1H, m), 4.72-5.00 (m), 5.21 (2H, dd, J = 40.9, 19.8 Hz), 5.52 (2H, dd, J = 40.9, 16.8 Hz), 6.94-7.19 (3H, m), 7.32-7.40 (2H, m), 7 60 (1H, t, J = 7.9 Hz), 7.70 (1H, td, J = 8.3, 1.3 Hz), 7.87 (1H, td, J = 8.6, 1 , 3 Hz), 8.08 (1H, d, J = 7.3 Hz), 8.17 (1H, d, J = 8.6 Hz), 8.59 (1H, s); MS: (LCMS) m / z 664 [M + H] ⁺ .

Example 32:

20-O - [(S) -phenylalanyl-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (R) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 3-phenyl-propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.97 (3H, t, J = 7.3 Hz), 1.60-2.38 (7H, m), 2.98 (2H, d , J = 7.9 Hz), 3.14-3.29 (m), 4.00 (1H, t, J = 7.3 Hz), 4.28-4.39 (1H, m), 4 70-4.89 (m), 5.29 (2H, d, J = 4.6 Hz), 5.54 (2H, dd, J = 40.3, 16.8 Hz), 7.02- 7.32 (7H, m), 7.62 (1H, td, J = 7.3, 1.0 Hz), 7.77 (1H, td, J = 8.6, 1.7 Hz), 7 99 (1H, d, J = 8.2 Hz), 8.05 (1H, d, J = 8.6 Hz), 8.52 (1H, s); MS: (LCMS) m / z 625 [M + H] ⁺ .

Example 33:

20-O - [(S) -leucyl-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was obtained from 2 (R) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 4-methyl-pentanoyl] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.91-1.09 (10H, m), 1.60-1.81 (2H, m), 1.94-2.41 (3H, m), 2.70 (2H, d, J = 8.6 Hz), 3.23-3.38 (m), 3.82-3.98 (1H, m), 4.43-4.54 (1H, m), 4.79-4.97 (m), 5.32 (2H, br s), 5.54 (2H, dd, J = 38.9, 16.8 Hz), 7, 40 (1H, s), 7.71 (1H, td, J = 7.3, 1.3 Hz), 7.82-7.93 (1H, m), 8.08 (1H, d, J = 7.3 Hz), 8.18 (1H, d, J = 8.6 Hz), 8.64 (1H, s); MS: (LCMS) m / z 591 [M + H] ⁺ .

Example 34:

20-O - [(R)-tryptophil-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (S) - [2 (R) -tert-butoxycarbonylamino 1-tert-butyl ester 3- (1H-indol-3-yl) propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.97-1.10 (3H, m), 1.68-2.48 (9H, m), 2.62-2.78 (1H, m), 2.94-3.47 (m), 3.93-4.36 (3H, m), 4.46-4.58 (1H, m), 4.80-4.98 (m) 5.02-5.29 (2H, m), 5.52 (2H, dd, J = 40.3, 17.2 Hz), 6.85 (1H, s), 6.96-7.24 (5H, m), 7.34 (2H, s), 7.39 (1H, s), 7.44 (2H, t, J = 7.6 Hz), 7.62 (1H, d, J = 7.9 Hz), 7.71 (2H, t, J = 6.9 Hz), 7.78-7.90 (2H, m), 8.00-8.18 (4H, m), 8, 49 (1H, s); 8.58 (1H, s); MS: (LCMS) m / z 664 [M + H] ⁺ .

Example 35:

20-O - [(R) -phenylalanyl-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was obtained from 2 (R) - [2 (R) -tert-butoxycarbonylamino 1-tert-butyl ester 3-phenyl-propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.03 (3H, t, J = 7.6 Hz), 1.90-2.38 (4H, m), 2.65-2.78 (2H, m), 2.94-3.08 (1H, m), 3.22-3.34 (m), 4.10-4.22 (1H, m), 4.48-4.57 (1H, m), 4.73-4.98 (m), 5.30 (2H, br s), 5.54 (2H, dd, J = 40.3, 16.8 Hz), 7, 21-7.43 (6H, m), 7.70 (1H, t, J = 7.9 Hz), 7.81-7.92 (1H, m), 8.08 (1H, d, J = 8.2 Hz), 8.17 (1H, d, J = 8.6 Hz), 8.62 (1H, s); MS: (LCMS) m / z 625 [M + H] ⁺ .

Example 36:

20-O - [(R) -leucyl-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (R) - [2 (R) -tert-butoxycarbonylamino 1-tert-butyl ester 4-methyl-pentanoylamino] -pentanedioic acid.

¹ H-NMR (270 MHz, CD ₃ OD) δ 0,91-1,09 (9H, m), 1,58-2,39 (8H, m), 2.68-2.80 (2H, m), 3.25-3.34 (m), 3.86-3.98 (1H, m), 4.49-4.60 (1H, m), 4.79-4.97 (m) 5.31 (2H, br s), 5.54 (2H, dd, J = 38.9, 16.8 Hz), 7.40 (1H, s), 7.68-7.75 (1H , m), 7.83-7.93 (1Н, m), 8.08 (1Н, d, J = 8.2 Hz), 8.18 (1Н, d, J = 8.3 Hz), 8 64 (1H, s); MS: (LCMS) m / z 591 [M + H] ⁺ .

Example 37:

7-ethyl-10-hydroxy-20-O - [(R)-tryptophil-γ- (R) -homoglutamyl] -20 (S) -camptothecin hydrochloride was obtained from 2 (R) - [2-tert-butyl ether (R) -tert-butoxycarbonylamino-3- (1H-indol-3-yl) propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.04 (3H, t, J = 7.6 Hz), 1.39 (3H, t, J = 7.6 Hz), 1.60- 2.34 (6H, m), 2.53-2.70 (2H, m), 3.02-3.49 (m), 4.09-4.19 (1H, m), 4.38- 4.52 (1H, m), 5.16-5.33 (2H, m), 5.52 (2H, dd, J = 41.2, 17.3 Hz), 6.92-7.48 ( 8H, m), 7.65 (1H, d, J = 8.1 Hz), 7.98-8.08 (1H, m); MS: (LCMS) m / z [M + H] ⁺ .

Example 38:

7-ethyl-10-hydroxy-20-O - [(R)-tryptophil-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was obtained from 1 (t) -butyl ether 2 (R) - [2 (R) -tert-butoxycarbonylamino-3- (1H-indol-3-yl) propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.03 (3H, t, J = 7.3 Hz), 1.36 (3H, t, J = 7.6 Hz), 2.01- 2.36 (4H, m), 2.73 (2H, t, J = 7.3 Hz), 3.02-3.18 (3H, m), 3.26-3.44 (m), 4 15 (1H, dd, J = 8.9, 5.4 Hz), 4.52 (1H, dd, J = 9.2, 4.6 Hz), 4.82-4.98 (m), 5.09 (2H, dd, J = 44.6, 18.6 Hz), 5.52 (2H, dd, J = 44.0, 16.7 Hz), 6.99-7.15 (4H, m), 7.29 (1H, s), 7.34-7.43 (3H, m), 7.60 (1H, d, J = 8.1 Hz), 8.00 (1H, d, J = 9.7 Hz); MS: (LCMS) m / z 708 [M + H] ⁺ .

Example 39:

7-Ethyl-10-hydroxy-20-O - [(S) -phenylalanyl-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was obtained from 1 (t) -butyl ether 2 (R) - [2 (S) -tert-butoxycarbonylamino-3-phenyl-propionylamino] pentanedioic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.05 (3H, t, J = 7.4 Hz), 1.38 (3H, t, J = 7.6 Hz), 1.86 ( 1H, m), 2.05-2.40 (5H, m), 3.04 (2H, brd), 3.14 (2H, brq), 4.08 (1H, t, J = 7.4 Hz), 4.42 (1H, m), 5.16 (1H, d, J = 18.8 Hz), 5.26 (1H, d, J = 18.8 Hz), 5.46 (1H, d, J = 16.8 Hz), 5.62 (1H, d, J = 16.8 Hz), 7.13 (2H, m), 7.28 (4H, m), 7.42 (2H, m), 8.00 (1H, d, J = 7.3 Hz); MS: (FABMS) m / z 669 [M + H] ⁺ .

Example 40:

7-Ethyl-10-hydroxy-20-O - [(S) -phenylalanyl-β- (S) -aspartyl] -20 (S) -camptothecin hydrochloride was prepared from 1 (t) -butyl ether 2 (S) - [2 (S) -tert-butoxycarbonylamino-3-phenyl-propionylamino] succinic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.04 (3H, t, J = 7.4 Hz), 1.43 (3H, t, J = 7.6 Hz), 2.22 ( 2H, m), 2.97-3.35 (6H, m), 4.15 (1H, m), 4.83 (1H, m), 5.38 (2H, s), 5.50 (1H d, J = 17.2 Hz), 5.63 (1H, d, J = 17.2 Hz), 7.30 (5H, m), 7.57-7.68 (3H, m), 8.23 (1H, m); MS: (FABMS) m / z 655 [M + H] ⁺ .

Example 41:

7-Ethyl-10-hydroxy-20-O - [(S) -leucyl-β- (S) -aspartyl] -20 (S) -camptothecin hydrochloride was obtained from 2 (S) - [2-tert-butyl ether (S) -tert-butoxycarbonylamino-4-methyl-pentanoylamino] succinic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.94-1.08 (9H, m), 1.42 (3H, t, J = 7.6 Hz), 1.61-1.83 (3H, m), 2.12-2.31 (2H, m), 3.18-3.35 (4H, m), 3.88 (1H, m), 4.85 (1H, m), 5.38 (2H, s), 5.50 (1H, d, J = 17.2 Hz), 5.63 (1H, d, J = 17.2 Hz), 7.50-7.61 (3H , m) 8.16 (1H, d, J = 9.2 Hz); MS: (FABMS) m / z 621 [M + H] ⁺ .

Example 42:

20-O - [(S) -tryptophil-β- (S) -aspartyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (R) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 3- (1H-indol-3-yl) -propionylamino] succinic acid.

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.87 (3H, t, J = 7.6 Hz), 2.18 (2H, m), 2.70-3.03 (4H, m), 3.84 (1H, m), 4.34 (1H, d, J = 19.2 Hz), 4.70 (1H, m), 4.98 (1H, d, J = 19.2 Hz), 5.49 (2H, s), 6.56 (1H, t, J = 7.5 Hz), 6.77 (1H, s), 6.93 (1H, t, J = 7.2 Hz), 7.07 (1H, d, J = 7.9 Hz), 7.14 (1H, s), 7.25 (1H, d, J = 7.9 Hz), 7.70 (1H, t, J = 7.0 Hz), 7.84 (4H, m), 8.01 (1H, d, J = 7.6 Hz), 8.11 (1H, d, J = 8.6 Hz) 8.25 (1H, s); 9.05 (1H, d, J + 8.6 Hz); 10.8 (1H, s); MS: (FABMS) m / z 650 [M + H] ⁺ .

Example 43:

20-O - [(S) -phenylalanyl-β- (R) -aspartyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (R) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 3-phenyl-propionylamino) succinic acid.

¹ H-NMR: (270 MHz, DMSDO-d ₆ ) δ 0.90 (3H, t, J = 7.3 Hz), 2.18 (2H, m), 2.55-3.05 (4H, m), 3.95 (1H, m), 4.68 (1H, m), 4.74 (1H, d, J = 19.3 Hz), 5.13 (1H, d, J = 19.3 Hz), 5.50 (2H, s), 6.76 (2H, d, J = 7.3 Hz), 6.99 (2H, t, J = 7.5 Hz), 7.13 (1H, t, J = 7.6 Hz), 7.14 (1H, s), 7.73 (1H, t, J = 7.6 Hz), 7.88 (1H, t, J = 7.3 Hz) 7.94 (3H, m), 8.14 (2H, br t), 8.53 (1H, s), 9.03 (1H, d, J = 8.6 Hz; MS: (FABMS) m / z 611 [M + H] ⁺ .

Example 44:

20-O - [(R) -phenylalanyl-β- (R) -aspartyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (R) - [2 (R) -tert-butoxycarbonylamino 1-tert-butyl ester 3-phenyl-propionylamino) succinic acid.

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.91 (3H, t, J = 7.5 Hz), 2.18 (2H, m), 2.87-3.09 (4H, m), 3.99 (1H, m), 4.67 (1H, m), 5.26 (2H, br s), 5.50 (2H, br s), 7.16-7.27 (6H, m), 7.74 (1H, br t), 7.88 (1H, br t), 8.03 (3H, m), 8.16 (2H, br t), 8, 69 (1H, s), 9.12 (1H, d, J = 7.9 Hz); MS: (FABMS) m / z 611 [M + H] ⁺ .

Example 45:

20-O - [(S) -phenylalanyl-β- (S) -aspartyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (S) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 3-phenyl-propionylamino) succinic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 1.04 (3H, t, J = 7.4 Hz), 2.13-2.32 (2H, m), 2.96-3.37 (4H, m), 4.13 (1H, m), 4.85 (1H, m), 5.36 (2H, s), 5.49 (1H, d, J = 17.2 Hz), 5 62 (1H, d, J = 17.2 Hz), 7.30 (5H, br s), 7.50 (1H, s), 7.76 (1H, br t), 7.93 ( 1H, br t), 8.14 (1H, d, J = 7.6 Hz), 8.22 (1H, d, J = 8.6 Hz), 8.76 (1H, s); MS: (FABMS) m / z 611 [M + H] ⁺ .

Example 46:

20-O - [(S) -leucyl-β- (R) -aspartyl] -20 (S) -camptothecin hydrochloride was obtained from 2 (R) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 4-methyl-propionylamino) succinic acid.

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.67 (3H, d, J = 7.4 Hz), 0.68 (3H, d, J = 7.4 Hz), 0.89 (3H, t, J = 7.6 Hz), 1.43-1.62 (3H, m), 2.15 (2H, m), 3.03 (2H, m), 3.78 (1H, m), 4.64 (1H, broad s), 5.48 (2H, broad s), 7.17 (1H, s), 7.74 (1H, broad) t), 7.89 (1H, brt), 8.16 (5H, m), 8.72 (1H, s), 8.95 (1H, d, J = 7.3 Hz); MS: (FABMS) m / z 577 [M + H] ⁺ .

Example 47:

20-O - [(S) -valyl-β- (R) -aspartyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (R) - [2 (S) -tert-butoxycarbonylamino 1-tert-butyl ester 3-methyl-butyrylamino) succinic acid.

¹ H-NMR: (270 MHz, CD ₃ OD) δ 0.66 (3H, d, J = 6.9 Hz), 0.70 (3H, d, J = 6.9 Hz), 1.03 ( 3H, t, J = 7.5 Hz), 1.98 (1H, m), 2.22 (2H, m), 3.22 (2H, m), 3.64 (1H, d, J = 5 , 3 Hz), 4.85 (1H, m), 5.38 (2H, br s), 5.48 (1H, d, J = 16.8 Hz), 5.61 (1H, d, J = 16.8 Hz), 7.47 (1H, br t), 7.94 (1 H, br t), 8.13 (1 H, br d), 8 22 (1H, broad d), 8.75 (1H, s); MS: (FABMS) m / z 563 [M + H] ⁺ .

Example 48:

7-ethyl-10-hydroxy-20-O - [(S) -cyclohexylalanyl-γ- (R) -glutamyl] -20 (S) -camptothecin hydrochloride was obtained from 2 (R) - [2-tert-butyl ester (S) -tert-butoxycarbonylamino-3-cyclohexyl-propionylamino] pentanedioic acid

¹ H-NMR (270 MHz, DMSO-d ₆₎ δ 0,81-2,66 (22H, m), 0.92 (3H, t, J = 7,3 Hz), 1.29 (3H, t, J = 7.6 Hz), 3.02-3.17 (2H, m), 3.76-3.88 (1H, m), 4.27-4.39 (1H, m), 5 30 (1H, s), 5.49 (1H, s), 7.00 (1H, d, J = 8.2 Hz), 7.43-7.47 (2H, m), 8.03 ( 1H, d, J = 3.0, 8.2 Hz), 8.22 (2H, br s), 8.94 (1H, d, J = 4.9 Hz); MS: (FABMS) m / z 675 [M + H] ⁺ .

Example 49:

7-ethyl-10-hydroxy-20-O - [(S) -cyclohexylalanyl-γ- (S) -glutamyl] -20 (S) -camptothecin hydrochloride was prepared from 2 (S) - [2-tert-butyl ether (S) -tert-butoxycarbonylamino-3-cyclohexyl-propionylamino] pentanedioic acid

¹ H-NMR: (270 MHz, DMSO-d ₆ ) δ 0.77-2.85 (22H, m), 0.92 (3H, t, J = 7.3 Hz), 1.29 (3H, t, J = 7.6 Hz), 3.01-3.17 (2H, m), 3.76-3.90 (1H, m), 4.30-4.45 (1H, m), 5 31 (1H, s), 5.50 (1H, s), 7.02 (1H, d, J = 4.6 Hz), 7.40-7.50 (2H, m), 8.04 ( 1H, d, J = 9.6 Hz), 8.22 (2H, br s), 8.78 (1H, d, J = 7.6 Hz); MS: (FABMS) m / z 675 [M + H] ⁺ .

The compounds listed in Examples 49-1 to 49-25 were obtained from (9S) -9-ethyl-9-hydroxy-1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6,5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) dione using various peptide derivatives of formula (VII), in accordance with a procedure similar to that as described in Example 24. The dipeptides are listed in the following table:

Example Dipeptide fragment 49-1 HCl. (L) Trp- (L) -γ-Glu 49-2 HCl. (L) Cyclohexylalanyl- (D) -γ-Glu- 49-3 HCl. (L) Phe- (D) -γ-Glu- 49-4 HCl. (L) Leu- (D) -γ-Glu- 49-5 2HCl. (L) Lys- (L) -γ-Glu- 49-6 HCl. (L) Val- (D) -γ-Glu- 49-7 2HCl. (L) Orn- (L) -γ-Glu- 49-8 MsOH. (L) Leu- (D) -γ-Glu- 49-9 2HCl. (D) Lys- (L) -γ-Glu- 49-10 HCl. (L) Phe- (L) -β-Asp- 49-11 HCl. (L) Cyclohexylalanyl- (D) -β-Asp- 49-12 HCl. (L) Cyclohexylalanyl- (L) -β-Asp- 49-13 HCl. (L) Trp- (L) -β-Asp- 49-14 2HCl. (L) Orn- (D) -γ-Glu- 49-15 HCl. (L) Leu- (D) -β-Asp- 49-16 HCl. (L) Val- (D) -β-Asp- 49-17 HCl. (L) Leu- (L) -β-Asp- 49-18 HCl. (L) Cyclohexylglycyl- (L) -γ-Glu- 49-19 HCl. (D) Cyclohexylalanyl- (L) -γ-Glu- 49-20 2HCl. (L) Lys- (D) -γ-Glu- 49-21 HCl .. (L) Trp- (D) -γ-Glu- 49-22 HCl. (L) Leu- (L) -γ-Glu- 49-23 HCl.Gly- (D) -γ-Glu- 49-24 HCl. (L) Ala- (D) -γ-Glu- 49-25 HCl. (L) Phe- (D) -γ-Glu-

Example 49-1

(9S) -9-ethyl-9 - [(L)-tryptophil- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 774 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.98 (t, J = 7.0 Hz, 3H), 1.03 (t, J = 7.6 Hz, 3H), 1.39-2 32 (m, 10H), 2.71-3.47 (m, 4H), 4.10-4.23 (m, 2H), 4.35 (m, 2H), 5.28 (d, J = 17.2 Hz, 1H), 5.38 (d, J = 17.2 Hz, 1H), 5.48 (d, J = 17.2 Hz, 1H), 5.63 (d, J = 17 , 2 Hz, 1H), 6.83 (t, J = 7.3 Hz, 1H), 7.05 (br t, 1H), 7.11 (s, 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.92 (t, J = 7 6 Hz, 1H), 7.97 (brd, 1H), 8.00 (s, 1H), 8.27 (s, 1H).

Example 49-2

(9S) -9-ethyl-9 - [(L) -cyclohexylalanyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 741 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.99 (t, J = 7.3 Hz, 3H), 1.04 (t, J = 7.3 Hz, 3H), 1.00-2 36 (m, 23H), 2.70-2.85 (m, 2H), 2.95 (br t, J = 7.8 Hz, 2H), 4.01 (m, 1H), 4, 24 (br t, 2H), 4.42 (m, 1H), 5.48 (d, J = 17.5 Hz, 1H), 5.51 (s, 2H), 5.63 (d, J = 17.5 Hz, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.83 (s, 1H), 7.95 (d, J = 7.6 Hz, 1H), 8.06 (br t, 1H); 8.36 (s, 1H).

Example 49-3

(9S) -9-ethyl-9 - [(L) -phenylalanyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 735 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.99 (t, J = 7.3 Hz, 3H), 1.06 (t, J = 7.3 Hz, 3H), 1.39-1 60 (m, 4H), 1.86-2.31 (m, 6H), 2.34-2.74 (m, 2H), 3.05-3.25 (m, 2H), 4.15 -4.25 (m, 3H), 4.42 (m, 1H), 5.46 (s, 2H), 5.48 (d, J = 17.2 Hz, 1H), 5.62 (d, J = 17.2 Hz, 1H), 7.20-7.31 (m, 5H), 7.50 (d, J = 7.6 Hz, 1H), 7.82 (s, 1H), 7, 90 (d, J = 8.3 Hz, 1H), 8.00 (br t, 1H), 8.32 (s, 1H).

Example 49-4

(9S) -9-ethyl-9 - [(L) -leucyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 701 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.94-1.08 (m, 12H), 1.39-2.35 (m, 13H), 2.65-2.85 (m, 2H ), 3.98 (m, 1H), 4.23 (br t, 2H), 4.42 (m, 1H), 5.49 (d, J = 17.5 Hz, 1H), 5.50 (s, 2H), 5.63 (d, J = 17.5 Hz, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.79 (s, 1H), 7.92 (d, J = 8.3 Hz, 1H), 8.06 (br t, 1H), 8.35 (s, 1H).

Example 49-5

(9S) -9-ethyl-9 dihydrochloride - [(L) -lyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 716 (MH ⁺ ); ¹ H-NMR (400 MHz, CD ₃ OD) δ 0.99 (t, J = 7.2 Hz, 3H), 1.04 (t, J = 7.2 Hz, 3H), 1.46-2 30 (m, 16H), 2.76-2.90 (m, 2H), 2.95 (br t, J = 7.8 Hz, 2H), 4.07 (t, J = 6.6 Hz, 1H), 4.22 (br t, 2H), 4.55 (dd, J = 10.0, 4.4 Hz, 1H), 5.49 (d, J = 16.8 Hz, 1H ), 5.50 (s, 2H), 5.63 (d, J = 16.8 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H ), 7.93 (d, J = 8.0 Hz, 1H), 8.06 (t, J = 8.0 Hz, 1H), 8.30 (s, 1H).

Example 49-6

(9S) -9-ethyl-9 - [(L) -valyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 687 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.97-1.11 (m, 12H), 1.40-1.59 (m, 4H), 1.88-2.33 (m, 7H ), 2.66-2.89 (m, 2H), 3.80 (d, J = 5.6 Hz, 1H), 4.23 (br t, 2H), 4.43 (dd, J = 9.2, 4.6 Hz, 1H), 5.49 (d, J = 17.5 Hz, 1H), 5.51 (s, 2H), 5.63 (d, J = 17.5 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.80 (s, 1H), 7.93 (d, J = 7.6 Hz, 1H), 8.08 (br. t, 1H), 8.35 (s, 1H).

Example 49-7

Dihydrochloride (9S) -9-ethyl-9 - [(L) -ornithyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 702 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.99 (t, J = 7.3 Hz, 3H), 1.04 (t, J = 7.3 Hz, 3H), 1.40-2 38 (m, 14H), 2.68-3.00 (m, 2H), 3.01 (br t, 2H), 4.11 (m, 1H), 4.24 (br t, 2H) ), 4.57 (m, 1H), 5.48 (d, J = 17.2 Hz, 1H), 5.51 (s, 2H), 5.64 (d, J = 17.2 Hz, 1H ), 7.57 (d, J = 7.3 Hz, 1H), 7.93 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H), 8.11 (br t , 1H); 8.33 (s, 1H).

Example 49-8

(9S) -9-ethyl-9 - [(L) -leucyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino methanesulfonate [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 701 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.95-1.08 (m, 12H), 1.39-2.35 (m, 13H), 2.66-2.75 (m, 2H ), 2.72 (s, 9H), 3.96 (m, 1H), 4.25 (br t, 2H), 4.45 (m, 1H), 5.49 (d, J = 17, 5 Hz, 1H), 5.51 (s, 2H), 5.63 (d, J = 17.5 Hz, 1H), 7.52 (s, 1H), 7.54 (d, J = 7, 9 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), 8.09 (br t, 1H), 8.38 (s, 1H).

Example 49-9

(9S) -9-ethyl-9 dihydrochloride - [(D) -lyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 716 (MH ⁺ ); ¹ H-NMR (400 MHz, CD ₃ OD) δ 0.95-1.07 (m, 6H), 1.42-2.30 (m, 16H), 2.64-2.95 (m, 2H ), 2.99 (br t, 2H), 4.02 (br t, 1H), 4.26 (m, 2H), 4.39 (m, 1H), 5.51 (d, J = 17.2 Hz, 1H), 5.52 (s, 2H), 5.63 (d, J = 17.2 Hz, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7 96 (s, 1H), 7.99 (brd, 1H), 8.09 (br t, 1H), 8.33 (s, 1H).

Example 49-10

(9S) -9-ethyl-9 - [(L) -phenylalanyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 721 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.99 (t, J = 7.3 Hz, 3H), 1.04 (t, J = 7.3 Hz, 3H), 1.42-2 30 (m, 8H), 2.98-3.37 (m, 4H), 4.17 (m, 1H), 4.24 (br t, 2H), 4.82 (m, 1H), 5.50 (s, 2H), 5.51 (d, J = 17.2 Hz, 1H), 5.62 (d, J = 17.2 Hz, 1H), 7.31 (br s, 5H ), 7.53 (d, J = 7.3 Hz, 1H), 7.68 (s, 1H), 7.83 (d, J = 7.6 Hz, 1H), 8.08 (br t , 1H); 8.36 (s, 1H).

Example 49-11

(9S) -9-ethyl-9 - [(L) -cyclohexylalanyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 727 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.98 (t, J = 7.3 Hz, 3H), 1.04 (t, J = 7.3 Hz, 3H), 0.80-2 27 (m, 21H), 3.23 (brd, J = 6.0 Hz, 2H), 3.98 (m, 1H), 4.24 (br t, 2H), 4.85 ( m, 1H), 5.50 (d, J = 17.2 Hz, 1H), 5.52 (s, 2H), 5.63 (d, J = 17.2 Hz, 1H), 7.54 ( d, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 8.09 (t, J = 7.6 Hz, 1H), 8.36 (s, 1H).

Example 49-12

(9S) -9-ethyl-9 - [(L) -cyclohexylalanyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 727 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.98 (t, J = 7.3 Hz, 3H), 1.04 (t, J = 7.3 Hz, 3H), 0.90-2 30 (m, 21H), 3.12-3.34 (m, 2H), 3.93 (m, 1H), 4.22 (br t, 2H), 4.83 (m, 1H), 5.50 (s, 2H), 5.51 (d, J = 17.5 Hz, 1H), 5.63 (d, J = 17.5 Hz, 1H), 7.47 (brd, 1H ), 7.65 (br s, 1H), 7.82 (br d, 1H), 8.03 (br t, 1H), 8.30 (s, 1H).

Example 49-13

(9S) -9-ethyl-9 - [(L)-tryptophil- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (FAB) m / z 760 (MH ⁺ ); ¹ H-NMR (270 MHz, CD ₃ OD) δ 0.98 (t, J = 6.9 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H), 1.39-1 60 (m, 4H), 1.83-1.97 (m, 2H), 2.13-2.30 (m, 2H), 3.05-3.40 (m, 4H), 4.12 (m, 2H), 4.29 (m, 1H), 4.79 (m, 1H), 5.27 (d, J = 17.8 Hz, 1H), 5.36 (d, J = 17, 8 Hz, 1H), 5.51 (d, J = 17.5 Hz, 1H), 5.63 (d, J = 17.5 Hz, 1H), 8.68 (br t, 1H), 7 06 (br t, 1H), 7.10 (s, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H) , 7.53 (d, J = 7.3 Hz, 1H), 7.84 (s, 1H), 7.85 (d, J = 8.3 Hz, 1H), 8.06 (br t, 1H), 8.30 (s, 1H).

Example 49-14

Dihydrochloride (9S) -9-ethyl-9 - [(L) -ornithyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

MS (LCMS) m / z 702 (MH ⁺ ); ¹ H-NMR (400 MHz, CD ₃ OD) δ 0,98 (t, J = 7,0 Hz, 3H), 1.04 (t, J = 7,4 Hz, 3H), 1,46-2 21 (m, 14H), 2.77-2.81 (m, 2H), 3.01 (br t, J = 7.4 Hz, 2H), 4.03 (t, J = 6.6 Hz, 1H), 4.24 (m, 2H), 4.44 (m, 1H), 5.50 (d, J = 16.8 Hz, 1H), 5.51 (s, 2H), 5, 63 (d, J = 16.8 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.91 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 8.09 (t, J = 8.0 Hz, 1H), 8.34 (s, 1H).

Example 49-15

(9S) -9-ethyl-9 - [(L) -leucyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 0.80-0.91 (m, 6H), 0.91-1.06 (m, 6H), 1.37-1.80 (m, 7H), 2.83-2.01 (m, 2H), 2.07-2.30 (m, 2H), 3.15-3.40 (m, 2H), 3.88-4.00 ( m, 1H), 4.25 (br t, 2H), 4.74-4.93 (m, 1H), 5.50 (d, J = 17.2 Hz, 1H), 5.52 (s , 2H), 5.62 (d, J = 17.2 Hz, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.75 (s, 1H), 7.88 (d , J = 8.6 Hz, 1H), 8.10 (br t, 1H), 8.37 (s, 1H); MS (FAB) m / z 687 (MH ⁺ ).

Example 49-16

(9S) -9-ethyl-9 - [(L) -valyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 0,86 (d, J = 6,9 Hz, 3H), 0.88 (d, J = 6,9 Hz, 3H), 0,94- 1.08 (m, 6H), 1.40-1.61 (m, 4H), 1.89-2.05 (m, 2H), 2.05-2.28 (m, 3H), 3, 13-3.38 (m, 2H), 3.77 (d, J = 5.3 Hz, 1H), 4.25 (br t, 2H), 4.77-4.91 (m, 1H) , 5.49 (d, J = 17.2 Hz, 1H), 5.53 (s, 2H), 5.62 (d, J = 17.2 Hz, 1H), 7.55 (d, J = 7.3 Hz, 1H), 7.79 (s, 1H), 7.91 (d, J = 7.6 Hz, 1H), 8.10 (br t, 1H), 8.37 (s, 1H); MS (FAB) m / z 673 (MH ⁺ ).

Example 49-17

(9S) -9-ethyl-9 - [(L) -leucyl- (L) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 0,79-1,00 (m, 12H), 1.30-1.51 (m, 4H), 1.30-1.75 (m, 3H), 1.75-1.92 (m, 2H), 2.00-2.21 (m, 2H), 3.00-3.30 (m, 2H), 3.76-3.85 ( m, 1H), 4.09-4.20 (m, 2H), 4.68-4.85 (m, 1H), 5.41 (d, J = 17.3 Hz, 1H), 5.43 (s, 2H), 5.57 (d, J = 17.3 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.65 (s, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.99 (br t, 1H), 8.27 (s, 1H); MS (FAB) m / z 687 (MH ⁺ ).

Example 49-18

(9S) -9-ethyl-9 - [(L) -cyclohexylglycyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 0.92-1.07 (m, 6H), 1.7-2.30 (m, 21H), 2.62-3.09 (m, 2H), 3.87 (d, J = 5.6 Hz, 1H), 4.24 (br t, 2H), 4.40-4.51 (m, 1H), 5.48 (d, J = 17.2 Hz, 1H), 5.51 (s, 2H), 5.63 (d, J = 17.2 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.88 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 8.06 (br t, 1H), 8.32 (s, 1H); MS (FAB) m / z 727 (MH ⁺ ).

Example 49-19

(9S) -9-ethyl-9 - [(D) -cyclohexylalanyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 0.93-1.08 (m, 6H), 1.15-2.36 (m, 23H), 2.60-2.94 (m, 2H), 3.99 (br t, 1H), 4.22 (br t, 2H), 4.37-4.45 (m, 1H), 5.49 (d, J = 17.3 Hz) , 1H), 5.50 (s, 2H), 5.62 (d, J = 17.3 Hz, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.80 (s , 1H), 7.91 (d, J = 8.2 Hz, 1H), 8.05 (br t, 1H), 8.32 (s, 1H); MS (FAB) m / z 741 (MH ⁺ ).

Example 49-20

(9S) -9-ethyl-9 dihydrochloride - [(L) -lyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 0.92-1.10 (m, 6H), 1.40-1.62 (m, 6H), 1.62-1.80 (m, 7H), 1.83-2.41 (m, 8H), 2.70-2.82 (m, 2H), 2.89-3.00 (m, 2H), 3.93-4.03 ( m, 1H), 4.16-4.31 (m, 2H), 4.40-4.50 (m, 1H), 5.30 (d, J = 17.2 Hz, 1H), 5.53 (s, 2H), 5.63 (d, J = 17.2 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.87 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 8.10 (br t, 1H), 8.35 (s, 1H); MS (FAB) m / z 716 (MH ⁺ ).

Example 49-21

(9S) -9-ethyl-9 - [(L) -triptyphophil- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz, CD ₃ OD) δ 0.99 (t, J-7.3 Hz, 3H), 1.04 (t, J = 7.3 Hz, 3H), 1.40-2 39 (m, 9H), 2.19 (t, J = 7.3 Hz, 1H), 2.58-2.74 (m, 1H), 2.91-3.05 (m, 1H), 3.10 (dd, J = 15.2, 5.6 Hz, 1H), 3.40 (dd, J = 15.2, 5.6 Hz, 1H), 3.95-4.15 (m, 2H), 4.21 (br.t, J = 5.6 Hz, 1H), 4.58-4.69 (m, 1H), 5.00 (d, J = 8.3 Hz, 1H), 5.20 (d, J = 8.3 Hz, 1H), 5.48 (d, J = 7.3 Hz, 1H), 5.67 (d, J = 7.3 Hz, 1H), 6, 59 (t, J = 7.3 Hz, 1H), 6.66 (s, 1H), 6.94-7.03 (m, 2H), 7.25 (d, J = 7.9 Hz, 1H ), 7.45 (d, J = 7.6 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.92 (s, 1H), 7.97 (t, J = 7.6 Hz, 1H); 8.18 (s, 1H); MS (FAB) m / z 774 (MH ⁺ ).

Example 49-22

(9S) -9-ethyl-9 - [(L) -leucyl- (L) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 1.01-1.06 (m, 12H), 1.39-2.32 (m, 13H), 2.67-2.79 (m, 1H), 2.89-2.98 (m, 1H), 4.08 (br t, J = 5.6 Hz, 1 H), 4.23 (br t, 2 H), 4.50 (dd , J = 9.6, 4.6 Hz, 1H), 5.49 (d, J = 17.2 Hz, 1H), 5.51 (s, 2H), 5.63 (d, J = 17, 2 Hz, 1H), 7.52 (dd, J = 7.6, 1.0 Hz, 1H), 7.88 (s, 1H), 7.96 (dd, J = 8.2, 1.0 Hz, 1H), 8.05 (br t, 1H), 8.30 (s, 1H); MS (ES) m / z 701 (MH ⁺ ).

Example 49-23

(9S) -9-ethyl-9- [glycyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4" hydrochloride: 6 ', 7'] indolisino [1 ', 2 ': 6.5] pyrido [4,3,2-de] quinazolin-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 0.95-1.05 (m, 6H), 1.39-2.30 (m, 10H), 2.62-2.89 (m, 2H), 3.48 (d, J = 16.0 Hz, 1H), 3.86 (d, J = 16.0 Hz, 1H), 4.23 (br t, J = 7.7 Hz, 2H), 4.51 (dd, J = 9.4, 4.5 Hz, 1H), 5.48 (d, J = 17.2 Hz, 1H), 5.50 (s, 2H), 5, 62 (d, J = 17.2 Hz, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.77 (s, 1H), 7.91 (d, J = 7.6 Hz, 1H), 8.06 (br t, 1H), 8.31 (s, 1H); MS (ES) m / z 645 (MH ⁺ ).

Example 49-24

(9S) -9-ethyl-9 - [(L) -alanyl- (D) -γ-glutamyloxy] -1-pentyl-1H, 12H-pyrano [3 ", 4": 6 ', 7'] indolisino hydrochloride [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 0.98 (t, J = 7.3 Hz, 3H), 1.03 (t, J = 7.3 Hz, 3H), 1.56 ( d, J = 6.9 Hz, 3H), 1.39-2.34 (m, 10H), 2.64-2.85 (m, 2H), 4.03 (q, J = 6.9 Hz , 1H), 4.23 (br t, J = 7.6 Hz, 2H), 4.44 (dd, J = 9.4, 4.8 Hz, 1H), 5.48 (d, J = 17.2 Hz, 1H), 5.50 (s, 2H), 5.62 (d, J = 17.2 Hz, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7 76 (s, 1H), 7.89 (d, J = 8.2 Hz, 1H), 8.06 (br t, 1H), 8.32 (s, 1H); MS (ES) m / z 659 (MH ⁺ ).

Example 49-25

(9S) -9-ethyl-9 - [(L) -phenylalanyl- (D) -β-aspartyloxy] -1-pentyl-1H, 12H-pyrano hydrochloride [3 ", 4": 6 ', 7'] indolisino [1 ', 2': 6.5] pyrido [4,3,2-de] quinazoline-10,13 (9H, 15H) -dione

¹ H-NMR (270 MHz) δ (CD ₃ OD) 1.01 (t, J = 7.3 Hz, 3H), 1.04 (t, J = 7.3 Hz, 3H), 1.40- 2.25 (m, 7H), 2.62 (dd, J = 14.5, 9.9 Hz, 1H), 3.02 (dd, J = 14.5, 4.3 Hz, 1H), 3 , 33-3.47 (m, 2H), 4.10-4.20 (m, 3H), 4.93-5.01 (m, 2H), 5.12 (d, J = 18.5 Hz , 1H), 5.32 (d, J = 18.5 Hz, 1H), 5.48 (d, J = 17.2 Hz, 1H), 5.64 (d, J = 17.2 Hz, 1H ), 6.81-6.88 (m, 4H), 7.01 (m, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.91 (s, 1H), 7 93 (br d, 1H), 8.09 (br t, 1H), 8.24 (s, 1H); MS (FAB) m / z 721 (MH ⁺ ).

Example 50: Tumor-specific activation and antitumor activity of TTC

50-1. Generation of cells capable of constitutively expressing high levels of microsomal dipeptidase

Among the enzymes that were selected according to the oligonucleotide ranking results, full length microsomal dipeptidase (MDP) cDNA (GenBank Accesion No. J05257. Adachi, H., et.al. Primary atructure of human microsomal dipeptidase dedused from molecular cloning. J. Biol. Chem, 265, 3992-3995 (1990)) was cloned at the Hind III site of the pRC / CMV vector (Invitriogen, San Diego, USA, Catalog No. V750-20) and transfected into the HCT116 human tumor cell line (ATCC Number, CCL-247) which expresses only a slight level of microsomal dipeptidase mRNA. The pRC / CMV vector not containing any cDNA was also transfected into the same cell line in order to generate a control line of cells. Transfection of cDNA was performed using TransIT-LT2 (Pan Vera, Madison, USA, Catalog No. MIR2320) in accordance with the manufacturers instructions. The resulting transfectants were cultured in MaCoy5A medium (Sigma, St Louis, USA, Catalog No. M8403) supplemented with 10% (v / v) fetal calf serum and 1 mg / ml geneticin disulfate (Waco, Osaka, Japan, Catalog No. 535- 24624). Cells growing in the presence of 1 mg / ml G418 were harvested and cultured in MaCoy5A medium.

To determine microsomal dipeptidase activities in cells, subconfluent monolayer cultures of HCT116 treated with pRC / CMV, HCT116 bearing pRC / CMV-MDP (hereinafter referred to as HCT5) were washed with phosphate buffered saline (PBS), cells were harvested with a blade, suspended in PBS and collected by low speed centrifugation at 1000 × g for 5 minutes. Cell debris was suspended in PBS and the lysis was checked by ultrasound treatment using Polytron (5 seconds at maximum speed). Using a similar method, cell extracts of granulocyte precursors were also prepared from CD-34 positive mononuclear human umbilical cord blood cells. Floated granulocyte precursors cultured for 5 days on a fused monolayer of MS5 in the presence of 50 ng / ml Flt3 ligand, 100 ng / ml SCF and 50 ng / ml TPO, were collected, washed with PBS, suspended in PBS and lysed by homogenization using Polytron After removing cell debris by centrifugation at 15,000 × g for 15 minutes, supernatants were used in the following experiments.

After determining the concentration of proteins in cell extracts using a kit for DC protein analysis (Bio-Rad, Hercules, USA, Catalog No. 500-0116), microsomal dipeptidase activities were also determined in accordance with the method of Watanabe et al. (Watanabe, T. et al., Biochim. Biophys. Acta. 1298, 109-118 ((1996). Cell extracts were incubated for 30 minutes at 37 ° C. in 100 μl of a reaction mixture containing 25 mM Tris-HCl (pH 8.0), 10 μm ZnCl ₂ , 10 mM glycine- (D) -alanine, 20 μM FAD, 3.75 U / ml D-amino acid oxidase (Roche Diagnostics, Mannheim, Germany, Catalog No. 102 784). Then the reaction was stopped by adding 40 μl of 25% (w / v) trifluoroacetic acid After centrifugation at 10,000 × g for 5 minutes, 100 μl of the supernatant was mixed with 20 μl of 0.1% (w / v) 2,4-dinitrophenylhydrazine dissolved in 2M HCl and incubated for 15 minutes at 37 ° C. After this The solution was mixed with 3.75 M NaOH and further incubated for 10 minutes at room temperature.The amount of (D) -alanine produced from glycine- (D) -alanine by cell extracts was calculated from spectral absorption at a wavelength of 445 nm, and in comparison with standard (D) -alanine.

The enzymatic activities of one of the HCT5 clones that carry pRC / CMV-MDP provide a higher level of activity of microsomal dipeptidases (430 nmol of (D) -alanine produced per minute per mg of protein), compared with the value of the vector-transfected HCT116 ( less than 1 nmol of (D) -alanine produced per minute of mg of protein) and predecessors of granulocytes (less than 1 nmol of (D) -alanine produced per minute per mg of protein).

50-2. TTS inhibitory activity growth, dependent on microsomal dipeptidase.

Using 96-well multiplates, approximately 2 × 10 ³ cells per well of HCT116 carrying pRC / CMV and HCT116S5 carrying pRC / CMV-MDP were cultured in 200 μl of McCoy5A medium supplemented with 10% FBS in the presence or absence of these drug concentrations at 37 ° C in an atmosphere of moist air containing 5% CO ₂ . Cells were exposed to drugs for 24 hours (taxol, camptothecins and their prodrugs) or 96 hours (DMDC and its derivatives). At a drug exposure time of 24 hours, the culture medium containing the drugs was removed and the cells were washed and suspended in fresh drug-free medium, and further cultured at 37 ° C. in an atmosphere of moist air containing 5% CO ₂ in for a certain number of days. Then, 10 μl of WST-8 (Cell Counting Kit-8, Wako, Osaka, Japan, Catalog No. 343-07623) was added to the culture media, and the cells were further incubated for 1-2 hours at 37 ° C. Cell growth inhibition was calculated as IC ₅₀ values according to optical densities at 450 nm and 655 nm. To study the effect of drugs on granulocyte precursors, granulocyte precursors that multiplied on the MS-5 monolayer for 7 days were collected and washed with RPMI1640 medium.

Approximately 5,000 cells were suspended in 200 μl of RPMI medium supplemented with 10% FBS and 50 ng / ml G-CSF in the presence or absence of drugs and cultured in the presence of drugs for 24 hours (Taxol, camptothecins and their prodrugs) or 7 days (DMDC and its derivatives) at 37 ° C in an atmosphere of moist air containing 5% CO ₂ . In the case where the exposure time of the drugs to the cells was 24 hours, the drugs were removed 24 hours after they were added by washing the cells with the above medium, and the cells were further cultured for 6 days in the same medium containing no drugs. Then, 20 μl of WST-1 (Roche Diagnostics, Mannheim, Germany, Catalog No. 1644807) was added to the culture media and the cells were further incubated for 6 hours at 37 ° C. Cell growth inhibition was calculated as IC ₅₀ values according to optical densities at 450 nm and 655 nm. Growth inhibition by paclitaxel, camptothecin and DMDC did not differ significantly in the case of HCT116, HCT116 / S5 and granulocyte precursors. However, their derivatives exhibit stronger anti-proliferative activity against HCT116 / S5, which express higher microsomal dipeptidase activities than HCT116 and granulocyte precursors with very low microsomal dipeptidase activity (see biological activities in the examples).

EXAMPLE A

Tablets containing these ingredients can be obtained in the traditional way:

Ingredients Per tablet Compound of Example 4 10.0-300.0 mg Lactose 125.0 mg Maize starch 75.0 mg Talc 4.0 mg Magnesium stearate 1.0 mg

EXAMPLE B

Tablets containing these ingredients can be obtained in the traditional way:

Ingredients Per capsule Compound of Example 4 100.0 mg Lactose 150.0 mg Maize starch 20.0 mg Talc 5.0 mg

EXAMPLE C

Solutions for injection can have the following composition:

Compound of Example 4 10.0 mg Sodium chloride q.s. mg Water for injections up to 2.0 ml

Claims (14)

1. A method for identifying an enzyme used in the preparation of a delivery of anticancer compounds that selectively turn into active substances in tumor tissue or cells, wherein said method comprises the steps of comparing gene expression levels in healthy human tissue or normal cells with gene expression levels in human tumor tissue or cells of a tumor origin, and the selection of an enzyme having an expression level of more than twice as high in the tissues or cells of a tumor origin compared to normal cells or healthy tissue. 2. The method according to claim 1, in which the enzyme is identified using assays, including microarray DNA analysis, polymerase chain reaction, Northern blotting, in situ hybridization, differential substitutions, RNA protection analysis, protein analysis on matrices, Western blotting , two-dimensional gel electrophoresis, enzyme-linked immunosorbent assay. 3. The method according to claim 2, in which the enzyme is identified by DNA analysis on microarrays or polymerized chain reaction. 4. The method according to claim 1, in which normal cells or healthy tissue come from healthy growing cells. 5. The method according to claim 1, in which the source of tumor tissue or cells of tumor origin is selected from the group consisting of a tumor of the brain, lungs, esophagus, breast, stomach, pancreas, liver, colon, rectum, kidneys, ovaries, uterus, bladder, prostate, skin and blood. 6. The method according to claim 1, in which normal cells are derived from hematopoietic progenitor cells, intestinal cells or skin. 7. The method according to claim 1, in which healthy tissue comes from the intestines or skin. 8. The method according to claim 6, in which the hematopoietic progenitor cells are derived from bone marrow or cord blood. 9. The method according to claim 4, where the level of expression of the enzyme in tissues or cells of tumor origin is more than twice the level of expression in normal cells or healthy tissue selected from hematopoietic progenitor cells, as well as normal cells or healthy liver tissue. 10. A method of manufacturing a preparation for the delivery of anticancer compounds that are capable of being converted into active substances selectively in tumors, comprising the steps of (a) culturing cells in the presence or absence of a test compound, wherein cDNA of an enzyme identified by the method according to any one of claims 1 to 9 is included in said cells; and (b) determining that the test compounds are anticancer compounds that are capable of being selectively converted into active substances in tumors if inhibition of cell growth is quantified and can be compared relative to living cells in the presence and absence of test compounds. 11. The method according to p. 10, in which these enzymes are microsomal dipeptidase, arylsulfatase A, pyrroline 5'-carboxy reductase, dehydrodiol dehydrogenase, carbonyl reductase, lysylhydroxylase, prolidase, dihydropyrimidosuctose-fructose fructose fructose fructose fructose fructose lysyl oxidase, enolase, glucose-6-phosphate dehydrogenase, stearoyl coenzyme A desaturase, epoxide hydrolase or aldolase C. 12. The method of claim 10, wherein said enzymes are microsomal dipeptidase, dehydrodioldehydrogenase, pyrroline 5'-carboxy reductase, carbonyl reductase or lysyl hydroxylase, or preferably microsomal dipeptidase. 13. The method of claim 10, wherein said enzyme is a microsomal dipeptidase. 14. The use of the enzyme identified by the method according to any one of claims 1 to 9, for the delivery of anticancer compounds that are capable of being converted into active substances selectively in tumors, where the production of anticancer compounds is carried out by the method of claim 10 or 11.