CA1111028A

CA1111028A - N-benzyl anthracyclines

Info

Publication number: CA1111028A
Application number: CA313,173A
Authority: CA
Inventors: Helen Y. Wu; Thomas H. Smith; David W. Henry
Original assignee: SRI International Inc
Current assignee: SRI International Inc
Priority date: 1977-10-17
Filing date: 1978-10-12
Publication date: 1981-10-20
Also published as: GB2007645A; IT1109281B; IT7851494A0; DE2844993A1; FR2405957B1; FR2405957A1; JPS6320839B2; GB2007645B; JPS5470255A

Abstract

ABSTRACT OF DISCLOSURE

N-Benzyl and N,N-dibenzyl derivatives of the anthracycline compounds adriamycin and dauno-mycin, said derivatives having anti-cancer properties and being active against leukemia p-388 in mice, for example. The N,N-dibenzyl derivatives have the unique quality of exhibit-ing a loss of DNA-binding properties.

Description

ORIGIN OF INVENTION
.

The invention described herein was made in the course of or under a contract with the U.S.
Department of Health, Education, and Welfare.

SUMMARY OF I~VENTION

The present invention relates to the provision of i 15 novel N-benzyl and N,N-dibenzyl derivatives of :: ~ . adriamycin and daunomycin and to compositions containing the same, said derivatives having i utility as anti-cancer chemicals and exhibiting the structure CH30 Ho ~0 ~ B -2-., ~

.

' ~ ' ' -wherein R represents -COCH3, -CHOH-CH3, -COCH2OH
- or -CHOH-CH2OH and wherein X represents -NHCH2Ph or -N(CH2Ph)2 in which "Ph" represents a phenyl group, and their pharmaceutically acceptable acid addition salts.

For ease of identification, the eight compounds falling wi~hin the scope of the present invention are enumerated (I through VIII) in Table l below.
', Table 1 :
X -- NHCH2Ph X = N(CH2Ph)2 R = COCH3 I V
R = CHOH-CH3 II VI
R = COCH2OH III VII
R = CHOH-CH2OH IV VIII
. .
The preparation of the foregoing compounds is des-cribed in detail by the examples. However, it may be noted here that reductive alkylation of dauno-mycin (IX) with benzaldehyde in the presence of sodium cyanoborohydride for prolonged reaction periods at room temperature produces good yields of the N,N-dibenzyl compound (V), plus the 13-dihydro derivative (VI) as byproduct. With shorter reaction periods, the mono-N-benzyldaunomycins I
and II predominate. Either N-benzylation or N,N-dibenzylation can be favored by choosing the stoichiometry. N,N-Dibenzyladriamycin (VII) was similarly obtained by treat:ing adriamycin (X) with benzylaldehyde and sodium cyanoborohydride under conditions that gave predominantly mono-N-benzylation, compound tIII)~ Since reductive alkylation is accompanied by ketone reduction, four products (I, II, V, VI) were observed in the benzylation of daunomycin and four more - (III, IV, VII, VIII) from the benzylation of adriamycin. Compound VIII, though identified in analytical procedures, was not isolated.
However, it is believed to have anti-cancer activity paralleling that of the other compounds of this invention.

. The compounds of the present invention can be prepared either in the free base or acid addition salt form.
. The salts are soluble in water and aqueous propylene glycol, for example, while the compounds in free base form are soluble in selected organic solvents : such as chloroform) methylene chloride and ethyl acetate, for example. The salts are thus particularly well adapted in the treatment of animals, including humans, since they may be used in aqueous, includ-ing saline, solution form. These acid addition salts (prepared here as those of HCl) are prefer-ably the pharmaceutically acceptable, nontoxic ~ $ ~

addition salts with suitable acids such as thosewith inorganic acids, for example, hydrochloric, .- hydrobromic, nitric, sulphuric and phosphoric ` acids, and with organic acicls, such as organic carboxylic acids, for exampl.e, glycolic, maleic, ~ hydroxymaleic, malic, tartaric, citric, salicylic :~ acids, and organic sulphonic acids, for example, methanesulphonic and toluene-p-sulphonic acids.

` An acid addition salt can be converted into the - 10 free compound according to known methods, for example, by treating it with a base, such as with a metal hydroxide or alkoxide, for example, . . .
an alkali metal or alkaline earth metal hydroxide, for example, lithium hydroxide, sodium hydroxide, potassium hydroxide or calcium hydroxide; with a metal carbonate, such as an alkali metal or an alkaline earth metal carbonate or hydrogen car-bonate, for example sodium, potassium or calcium carbonate or hydrogen carbonate; with ammonia;
or with a hydroxyl ion exchange resin, or with any other suitable reagent.

''' : An acid addition salt may a:Lso be converted into another acid addition salt according to known methods; for exampleJ a salt with an inorganic acid may be treated with a metal salt, for example a sodium, barium or silver salt, of an acid in a suitable diluent, in which a resulting inorganic salt is insoluble and is thus removed from the reaction medium. An acid addition salt - may also be ccnverted into another acid addition salt by treatment with an anion exchange preparation.
' The preparation of each of the foregoing compounds ....
: is described in the following examples:
' ` E~LE 1 N-Benzyldaunomycin (I), N-Benzyl-13-dihydrodaunomycin (II) and N,N-Dibenzyldaunomycin (V), and HCl salts Daunomycin HCl (2~26 g, 4.0 mmol) and benzaldehyde ` (8.2 mL, 80.0 mmol) were placed in 3:1 acetonitrile~H2O
(116 mL) and stirred at 23 for 0.5 hour. NaCNBH3 (0.75 g, 12.0 mmol) was added and stirring was con-tinued for 0.5 hour. The reaction was diluted with H2O (150 ~L) and extracted with CHC13 (3 x 100 mL).
The extracts were combined, washed with H2O (30 mL) and the aqueous phase reextracted with CHCl3 (50 mL).
The organic solutions were combined and extracted with cold 0.1 N HOAc (6 x 100 mL). The extracts were combined, immediately basified with NaHCO3 and extracted with CHC13 (3 ~ 300 mL). The extracts were combined, evaporated and the residue chroma-~ographed (1.3 kg silica ge:L -50 mm x 7 ft dry co~umn - 10:1 CHC13/MeOH) to afford 1~06 g of N-benzyldaunomycin (I) (51%) and 0.17 g of N-benzyl-13-dihydrodaunomycin (II) (7%) as the free bases. The organic solution after the HOAc extrac-tion was dried and evaporated. The residue was chromatographed (560 g silica gel -50 mm x 3 ft dry column - 10:1 CHC13/MeOH) to afford 0.18 g (6%) of N,N-dibenzyldaunomycin (V) free base. The HCl salts were prepared by reacting a CHC13 solution - of each compound with an equivalent amount of methanolic HCl followed by precipitation with ether to afford;
1-28 g (49%) of (I); [~]D + 252 (c .05 95% EtOH);
tlc (10:1 CHC13/MeOH) Rf 0.60, ' or C34H35NOlo HCl H2O
C H N Cl 60.76 5.70 2.08 5.28 Found: 60.88 5.59 1.99 5.40;

0.16 g (6%) of (II); [~]D + 207, (c 0.047, 95%
EtOH); tlc (10:1 CHC13/MeOH) Rf 0.4, 34H37Nol0 HCl 1.25 H2O
C H N Cl 60.17 6.02 2.06 5.22 Found: 60.07 5.79 2.26 5.38;

~$~

and 0.16 g (5%) of (V); [~]D ~ 283 (c 0.048, 95%
EtOH); tlc (10:1 CHC13/MeOH) Rf 0.90, Anal- calcdfor C41H41Nl0 HCl 75 H2O:
C H N Cl 5 64.99 5.79 1.85 ~.68 . Found:65.08 5~69 1.77 4.45 ; EXAMPLE 2 N-Benzyladriamycin (III), N,N-Dibenzyladriamycin (VII), and N-Benzyl-13-dihydroadriamycin (IV2, and HCl salts.

10 Adriamycin HCl (2.90 g, 5~0 mmol) and benzaldehyde (10.5 mL, 100 mmol) were placed in 2:1 acetonitrile/H2O (150 mL) and stirred at 23 for 20 minutes. The solution was added dropwise over 15 minutes to a solution of NaCNBH3 (0.94 g, 15.0 mmol) and benzaldehyde (10.5 mL) in 2:1 acetonitrile/H2O (60 mL). Stirring was continued for 30 minutes and the reaction was diluted with H2O (150 mL) and extracted with CHC13(4 x 100 mL). The extracts were combined, washed with saturated NaCl (2 x 100 mL), dried and evaporated. The residue was chromatographed ~.3 kg silica gel- 50 mm x 7 ft dry column - 40:10:1 CHC13/MeOH/H2O) to afford 0.10 g (3%) N,N-dibenzyl-adriamycin (VII), 1.02 g (30%) of N-benzyladriamycin (III), and 1.?0 g (33%) of N-benzyl-13-dihydroadriamycin (IV) as the free bases. Eluting after (VII) was a small amount of material, never completely purified, but tentatively identified as N,N-dibenzyl-13-dihydro-adriamycin (VIII). These were converted to the HCl salts ; by the procedure described in the previous example . .
to afford 0.07 g (2%) of (VII); [~]D + 275 (c 0.051, 85% EtOH); tlc (10:1 CHC13/MeOH) Rf 0.85, .

C41H4lNOll HCl 0.6 H2O:
C H N Cl 63.86 5.64 1.81 4.59 Found: 63.88 5.57 1.83 4.45;

0.90 g (28%) of (III); [~]D + 209 (c. 0.048, 95%
EtOH); tlc (10:1 CHC13/MeOH) Rf 0.55, C34H35Nll HCl H2O:
-~ C H N Cl 59.34 5.56 2.03 5.15 Found: 59.46 5.40 2.01 5.37;

and 1.00 g (30%) of (IV); [~]D + 204 (c 0.050, 95% EtOH); tlc (10:1 CHC13/MeOH~ Rf 0.25, Anal- calcd for C34H37Nll-HCl--8 H2O
C H N Cl ~- 59.48 5.87 2.04 5.16 Found: 59.43 5.81 2.05 5.40.

N,N-Dibenzyldaunomycin (V), N,N-Dibenzyl-13-dihydrodaunomycin (VI), N-BInzyldaunomycin (I) and N-Benzyl-13-dihydrod_unomycin (II), and HCl salts.

Daunomycin HCl (2.54 g, 4.5 mmol) and benzaldehyde (9.2 mL, 90.0 mmol) were placed in 2:1 acetonitrile/
H20 (90 mL) and stirred at 23 for 20 minu~es. The solution was added dropwise over 20 minutes to a stirred solution of NaCNBH3 (0.57 g, 9.0 mmol) in acetonitrile (20 mL) and stirring was continued for 14 days. The reaction was diluted with H20 (150 mL) and extracted with CHC13 (5 x 100 mL). The extracts were combined, washed with H20 (100 mL) and the aqueous layer extracted with CHC13 (50 mL).
The organic solutions were combined, dried and evaporated and residue chromatographed (190 g silica gel -2.8 x 64 cm column - CHC13 to 9:1 ` CHG13/MeOH) to afford in order of elution 1.19 g (38%) of (V), 0.41 g (13%) of N,N-diben2yl-13-dihydrodaunomycin (VI), 0.30 g (11%) of (I) and 0.10 g (4%) of (II) as the free bases. These were converted to HCl salts by the above procedure to afford 1.20 g (35%) of (V) and 0.37 (11%) of (VI);
[~]D + 247 (c 0.049, 95% EtOH), tlc (10:1 CHC13/
MeOH) Rf 0.85.

`:~
`Anal calcd for C41H43NlO-HCl-l 5 ~2;
C H N Cl ~`63.68 6.13 1.81 4.58 Found: 63.66 5.75 1.78 4.86; and 0.23 g (8%) of (I) and 0.07 g (2%) of (II), as HCl Salts.

Thecompounds of this invention, including the salts thereof, can be administered to the animal - by any available route, including oral and parenteral ; (intravenous, intraperitoneal, subcutaneous, and intramuscular) administration. The amount ad-ministered is sufficient to ameliorate the leukemia or other type of cancer against which the compounds hereof may prove to be effective, and will depend upon the type of cancer, the species of animal, and the weight of the animal. For example, in human administration, a dosage of a compound of the present invention within the range from about 0.1 mg/kg to about 500 mg/kg per day should be sufficient : to ameliorate leukemia. In the treatment of lower test animals, a similar dosage range is therapeutic. The upper " limit dosage is that imposed by toxic side effects, and can be determined by trial and error for the animal to be treated, including humans.

To facilitate administration, the compounds of this invention, including the salts thereof, - ~ : . '~
,' ` ' " ~ ' ' ' ~ , .

- can be provided in composition form, and preferably in dosage unit form. While any compound selected . can be adminis~ered per se, it is normally administered in conjunction with a pharmaceutically acceptable carrier therefor, which dilutes the compound and facilitates handling~ The term "pharmaceutically acceptable" means that the carrier (as well as the resulting composition) is sterile and nontoxic.

: ' The carrier or diluent can be solid, semisolid, or liquid, and can serve as a vehicle, excipient, or medium for the anti-cancer agent. Exemplary diluents and carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan, mono-laurate, methyl- and propyl-hydroxybenzoate, talc or magnesium stearate.

For convenience in handling, the compounds hereof and the carrier or diluent can be enclosed or encapsulated in a capsule, sachet, cachet, gelatin, paper or other container, especially when intended for use in dosage units. The dosage units can for exarnple take the form of tablets, capsules, suppositories or cachets.

' :`

The following examples illustrate various forms of dosage units in which the HCl salt of compound V can be prepared.

EX~MPLE: 4 Tablet formulationM~/tablet Compound V 15 : Lactose 86 Cornstarch (dried) 45.5 Gelatin 2.5 Magnesium stearate l.0 . .
Compound V is powdered and passed through a mesh ~; sieve and well mixed with the lactose and 30 mg .~. o~ the cornstarch, both passed through a sieve.
., The mixed powders are massed with a warm gelatin solution, prepared by stirring the gelatin in water and heating to form a 10% w/w solution.
The mass is granulated by passing through a sieve, and the moist granules dried at 40C.

The dried granules are regranulated by passing through a sieve and the balance of the starch and the magnesium stearate is added and thoroughly mixed.

The granules are compressed to produce table~s each weighing lS0 mg.

EX~LE 5 Tablet formulationMg/tablet Compound V 100 Lactose 39 Cornstarch (dried) 80 Gelatin 4.0 Magnesium stearate 2.0 . . .
The method of preparation is identical with that of Example 4 except that 60 mg of starch is used in the granulation process and 20 mg during tableting.

Capsule formulation Mg/capsule : 15 Compound V 250 Lactose lS0 Compound V and lactose are passed through a sieve and the powders well mixed together before filling into hard gelatin capsules of suitable size, so that each capsule contains 400 mg of mixed powders.
.

, ': . , Suppositories Mg/suppository Compound V 50 Oil of Theobroma 950 Compound V is powdered and passed through a sieve and triturated with molten oil of theobroma at 45C
to form a smooth suspension.

: The mixture is well stirred and poured into molds, . each of nominal 1 g capacity, to produce suppositories.

. Cachets Mg/cachet Compound V 100 Lactose 400 Compound V is passed through a mesh sieve, mixed with lactose previously sieved and fitted into cachets of ~; suitable size so that each contains 500 mg.

Intramuscular injection (sterile suspension in aqueous vehicle?_ Mg Compot:md V 10 Sodiu~l citrate 5.7 Sodium carboxymethycellulose (low viscosity grade) 2.0 Methyl para-hydroxybenzoate 1.5 Propyl para-hydroxybenzoate 0.2 Water for injection to 1.0 ml - :
.. .
- ~ ~

:
Example 10 : Intraperitoneal,intravenous or subcutaneous injection : (sterile solu~ion in aqueous carrier system) - Compound V, hydrochloric acid ~: addition salt 15 ` Sodium citrate 5-7 - Sodium carboxymethylcel:Lulose (low viscosity grade) 2.0 Methyl para-hydroxybenzoate 1.5 Propyl para-hydroxybenzoate 0.2 : Water for injection to 1.0 ml ` The other compounds of this invention could be :. 15 preparedin dosage unit form in the same general fashion as that described above for compound V.
,:

; '' BIOLOGICAL TESTS

Sufficient samples of all but compound (VIII) have been obtained for biological screening, and recent results of tests both in vitro and ; 5 in vivo are shown in Table 2 below. It will be seen that every compound was active against leukemia P388 in mice, with N,N-dibenzyldaunomycin `` (compound VI) being clearly the best. The in vitro results are surprising and appear to be highly significant in that there is an abrupt and unexpected loss of DNA-binding properties in going from N-benzyl to N,N-dibenzyl compounds.
~Tm is the most direct measure of DNA-binding but the inhibition of nucleic acid synthesis is also lost. Most of the anthracyclines bind to DNA, presumably by an intercalation mechanism and are mutagenic. In these N,N-dibenzyl compounds the anti-tumor activity in the mouse screen is retain~d but the DNA-binding properties are lost.
This suggests the N,N-dibenzyl compounds may be useful and effective anti-cancer agents, but may act by a different mechanism from most of the other anthracyclines, and thus may avoid the serious cardiotoxic side effects common to the anthracyclines now in use.

The compound referred to herein as daunomycin (IX) is otherwise known as daunorubicin or rubidomycin.

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. .

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a compound of the formula:

I

wherein R is -COCH3, -CHOH-CH3, -COCH2OH and -CHOH-CH2OH and X is -NHCH2Ph and -N(CH2Ph)2, wherein Ph represents a phenyl group, and their pharmaceutically acceptable acid addition salts, the process comprising reacting a compound of the formula:
II

wherein R is as defined above, or its acid addition salts, with benzaldehyde in the presence of sodium cyanoborohydride and separating the resulting compound of formula I, and, if desired, converting the resulting compound of formula I into its pharmaceutically acceptable acid addition salt, or, if desired, converting an acid addition salt of the compound of formula I into a pharmaceutically acceptable acid addition salt thereof.

2. The process of claim 1 wherein the compound of formula II is daunomycin and the resulting compound is N-benzyl-daunomycin, N-benzyl-13-dihydrodaunomycin, N,N-dibenzyldaunomycin or N,N-dibenzyl-13-dihydrodaunomycin and mixtures thereof.

3. The process of claim 2 which includes separating N-benzyldaunomycin from the mixture.

4. The process of claim 2 which includes separating N-benzyl-13-dihydrodaunomycin from the mixture.

5. The process of claim 2 which includes separating N,N-dibenzyldaunomycin from the mixture.

6. The process of claim 2 which includes separating N,N-dibenzyl-13-dihydrodaunomycin from the mixture.

7. The process of claim 1 wherein the compound of formula II is adriamycin and the resulting compound is N-benzyladriamycin, N-benzyl-13-dihydroadriamycin, N,N-dibenzyladriamycin or N,N-dibenzyl-13-dihydroadriamycin and mixtures thereof.

8. The process of claim 7 which includes separating N-benzyladriamycin from the mixture.

9. The process of claim 7 which includes separating N-benzyl-13-dihydroadriamycin from the mixture.

10. The process of claim 7 which includes separating N,N-dibenzyladriamycin from the mixture.

11. The process of claim 7 which includes separating N,N-dibenzyl-13-dihydroadriamycin from the mixture.

12. A compound of the formula:

wherein R is -COCH3, -CHOH-CH3, -COCH2OH and -CHOH-CH2OH and X is NHCH2Ph and N(CH2Ph)2 and their pharmaceutically acceptable acid addition salts whenever prepared or produced by the process of claim 1 or by their obvious chemical equivalents.

13. N-benzyldaunomycin and its pharmaceutically acceptable acid addition salts whenever prepared or produced by the process of claim 3 or by it's obvious chemical equivalent.

14. N-benzyl-13-dihydrodaunomycin and it's pharmaceutically acceptable acid addition salts whenever prepared or produced by the process of claim 4 or by its obvious chemical equivalent.

15. N,N-dibenzyldaunomycin and its pharmaceutically acceptable acid addition salts whenever prepared or produced by the process of claim 5 or by its obvious chamical equivalant.

16. N,N-dibenzyl-13-dihydrodaunomycin and its pharmaceutic-ally acceptable acid addition salts whenever prepared or produced by the process of claim 6 or by its obvious chemical equivalent.

17. N-benzyladriamycin and its pharmaceutically acceptable acid addition salts whenever prepared or produced by the process of claim 8 or by its obvious chemical equivalent.

18. N-benzyl-13-dihydroadriamycin and its pharmaceutically acceptable acid addition salts whenever prepared or produced by the process of claim 9 or by its obvious chemical equivalent.

19. N,N-dibenzyladriamycin and its pharmaceutically acceptable acid addition salts whenever prepared or produced by the process of claim 10 or by its obvious chemical equivalent.

20. N,N-dibenzyl-13-dihydroadriamycin and its pharmaceutic-ally acceptable acid addition salts whenever prepared or produced by the process of claim 11 or by its obvious chemical equivalent.