WO1998030550A1

WO1998030550A1 - 2,4-diaminopyrimidine compounds as anti-cancer agents

Info

Publication number: WO1998030550A1
Application number: PCT/GB1998/000111
Authority: WO
Inventors: Malcolm Francis Graham Stevens; Roger John Griffin
Original assignee: BTG International Ltd; British Technology Group Ltd
Current assignee: BTG International Ltd
Priority date: 1997-01-14
Filing date: 1998-01-14
Publication date: 1998-07-16
Anticipated expiration: 1999-07-14
Also published as: GB9700664D0; CA2275566A1; ZA98306B; AU5569098A; JP2001509149A; EP0961772A1

Abstract

Use of a compound of general formula (I) wherein R?1 and R2¿ are independently selected amino groups; R3 is C¿1-6? alkyl or haloalkyl; R?4¿ is selected from alkoxy, aralkoxy, haloalkyl, halogen and amino; and R5 is selected from nitro, amino, azido or a group -N=X-NR6R7 where R?6 and R7¿ are independently C¿1-6? alkyl or are aralkyl or together with the nitrogen to which they are attached form a heterocyclic ring with the proviso that one or both R?6 and R7¿ or the heterocyclic ring carries or includes or carries and includes at least one oxygen or sulphur atom, and X is N or CH or CR?9; or R4 and R5¿ together with the benzene ring to which they are attached form a group of formula (II), wherein X is N, CH or CR?9, and R8¿ is C¿1-6? alkyl or is aralkyl; or R?4 and R5¿ together with the benzene ring to which they are attached form a group of formula (III) wherein R9 is aryl or aralkyl; or a salt or N-oxide of any such compound, for the manufacture of a medicament for the treatment of mutant-ras gene associated cancer.

Description

2,4-DIAMINOPYRIMIDINE COMPOUNDS AS ANTI-CANCER AGENTS

This invention relates to the use of 2,4-diaminopyrimidine compounds in the treatment of certain cancers associated with ras mutations.

Antifolate agents, such as methotrexate, have been used as antitumour agents for many years and in 1954 metoprine (compound of formula A where R'=C1, R²=C1, R³=Me) entered clinical trials.

Although severe toxicity associated with this agent precluded further evaluation, the use of pyrimethamine (A. R'=C1 R²=H R³=Et) has been explored and diaminopyrimidine compounds have also been developed which have inherent species selectivity as antibacterial and antimalarial agents. WO 88/04293 (US 4992444) discloses compounds of this class with inhibition of dihydrofolate reductase (DHFR) comparable to or greater than metoprine but which are relatively less toxic. These compounds are described as of interest as antiproliferative agents useful in the treatment of tumours, psoriasis and bacterial, malarial and trypanosomal infections and are noted to act via DHFR inhibition. Certain of these compounds were also disclosed as intermediates in WO 84/04746 which relates to azido-substituted pyrimidine derivatives. WO 94/02469 discloses other related compounds, where R² is -N=N-NR⁸R⁹ for use in treatment of inter alia AIDS associated infections. The present inventors have now identified a group of these diaminopyrimidine compounds which have a previously unidentified property in so far as they have activity against mutant-ras associated tumours independent of tumour DHFR inhibitor activity. The Ras, R o, Rac and Rab proteins make up a large family of monomeric GTPases. The Ras proteins are anchored to the cytoplasmic face of the plasma membrane and relay signals from receptor tyrosine kinases to the cell nucleus in order to stimulate cell proliferation or differentiation.

These Ras proteins are produced by ras genes, mutants of which promote cancer by disrupting this control of cell proliferation and differentiation. It is believed that about 30% of human cancers involve mutant-ras genes. A review of this mechanism by F. McCormick is to found on pages 125-145 of 'Oncogenes and the Molecular Origins of Cancer" Edited by R. A. Weinberg and published b\ Cold Spring I Iarbour Laboratory Press (1989). Further details of the biochemistry of this mechanism are to be found in 'Molecular Biology of the Cell, by Alberts et al. 3rd Edition (1994) published by Garland Publishing Inc. New York and London: see pages 763-767 and 1276-1289. Other relevant publications include those by Feig.. Journal of the National Cancer fnstitute ( 1993) Vol 85. No 16: 1266-1268: Feig and Schaffhausen.. Nature (1994) 1 8: 508-9; Mars, Science (1993) Vol 260: 1588-90: Pretlow et al., Journal of the National Cancer fnstitute (1993) Vol 85 No 24:2004-2007: and Fearon.. Journal of the National Cancer Institute (1993) Vol 85 No 24: 1978-1980.

The prior art ascribes the activity of the aforesaid 2,4-diaminopyrimidine compounds to their dihydrofolate reductase (DF1FR) inhibiting activity, and particularly suggests their use in treatment of malignancies of the central nervous system and as antipsoriatic, antibacterial and antimalarial agents. Activity against a wide variety of murine tumour cell lines is demonstrated including lymphocytic leukaemia P388 and

L1210, melanotic melanoma B 16, colon 38. TLX5 lymphoma, W3129 myeloma, Walker 256 and M5076 reticulum cell sarcoma.

The present inventors have now determined that these compounds have an activity independent of that of their DHFR inhibitor activity which is specific against mutant-/ « cancer cells and, in particular those of non-small cell lung cancer (NSCL), colon cancer and pancreatic cancer. Such activity is of particular use where it is suspected or confirmed that a mutant ras cancer is present.

Thus, in a first aspect of the present invention there is provided the use of a compound of general formula (I)

wherein R¹ and R² are independently selected amino groups R³ is C,_₆ alkyl or haloalkyl

W is selected from alkoxy, aralkoxy. haloalkyl, halogen and amino and R⁵ is selected from nitro, amino. azido or a group -N=X-NR⁶R⁷ where R⁶ and R⁷ are independently C,_₆ alkyl or are aralkyl or together with the nitrogen to which they are attached form a heterocyclic ring with the proviso that one or both R⁶ and R⁷ or the heterocyclic ring carries or includes or carries and includes at least one oxygen or sulphur atom, and X is N or CH or CR⁹ or R⁴ and R⁵ together with the benzene ring to which they are attached form a group of formula II

wherein X is N, CH or CR⁹, and R⁸ is C,.₆ alkyl or is aralkyl or R⁴ and R⁵ together with the benzene ring to which they are attached form a group of formula III

wherein R⁹ is aryl or aralkyl or a salt or N-oxide of any such compound for the manufacture of a medicament for the treatment of mutani-r-/.y gene associated cancer.

More preferably, where R⁴ and R⁵ form a group of formula II or III with the benzene ring to which they are attached, that group is of formula IV or V

A particularly preferred medicament for which the compounds of formula I may be used in manufacture is for the treatment of mutant-ray gene associated cancers selected from non-small lung cell (NSCL), colon and pancreatic cancers, although it will be understood that other forms of mutant-ra^y associated cancer will also be treatable with such compounds.

Particularly preferred compounds for the use of the invention are those of formula VI

wherein R¹, R² and R³ are as defined for formula I

R¹⁰ and R" are selected from haloalkyl, halogen, amino, nitro, azido or a group -N=X-NR⁶R⁷ or, together with the benzene ring to which they are attached, form a group of formula IV or V, w ith the proviso that if one of R¹⁰ and R" is selected from haloalkyl and halo then the other is selected from amino, nitro, azido or a group -N=X-NR⁶R⁷- or a salt or N-oxide of any such compound. In all of the formulae I to VI hereinabove the term amino includes primary, secondary, tertiary and quaternary amino groups, but preferred groups are unsubstituted, mono or disubstituted amino groups, including those amino groups forming part of a heterocyclic ring, particularly a ring consisting of from 3 to 7 carbon, nitrogen, sulphur and/or oxygen atoms or as defined for -NR⁶R⁷ above, more preferably being of carbon and nitrogen atoms only. Amino groups are generally substituted by one or two alkyl or aralkyl groups in which the alkyl group or moiety preferably contains 1 -6 carbon atoms. R' and R ² are preferably unsubstituted primary amino groups or secondary or tertiary amino substituted by C alkyl groups. Where one or more of R⁴, R⁵, R^lϋ or R" is an amino group, the following groups are of particular interest: C_M alkyl amino, di-C alkyl amino, C _ή.l0aralkyl amino. C alkyl C₆._l() aralkyl amino, di-C₆.₁₀ aralkyl amino.

All of the aforesaid groups may be substituted; e.g. by halogen or C alkyl, particularly by chloro. Where the group includes a benzene ring. e.g. an aralkyl amino group, that ring may be substituted one or more times, preferably 1 or 2 times, and most preferably substituted with halogen, e.g. chloro.

Particularly preferred amino groups at R⁴. R ⁵, R ¹⁰ and R "are selected from -NHCH,. -NHCT L. -NH(nC₄H₉), -NHCH₂CH,PhenyI. -NHCI LPhcnyl. -N(CH,)CH,Phenyl. -N(CH,Phenyl)₂, -N(C₂H₅)CH₂Phcnyl and -NHCH(CH,)Phenyl.

Substituents on aryl rings may conveniently be halogen, C,_₆ alkyl or haloalkyl, C₂. alkenvl or haloalkenyl, C,_₆ alkoxy or haloalkoxy. nitro, or -C0₂R¹² where R'² is h> drogen. C,.₆ alkyl or C, _ή alkoxyalkyl. It is found that a -CONHCH-, substitution of these phe 1 groups at the 4 position is not desirable as it appears to lead to loss of anti-ra.s activit} . However, substitution with halo. e.g. chloro, especially 3.4 dichloro. results in best activity, e.g. see compound 1 of the Examples.

Thus where a group is designated as aralkyl that is preferably a C,.₄ alkylene group in association with a benzene ring, eg. phenylmethyl, 2-phenylethyl, 3-phenylpropyl or

4-phenylbutyl. Where a group is designated as haloalkyl it is preferably trifluoromethyl. When R⁴ is an alkoxy group it is preferably a C,_₆ alkoxy group. OQ-L,. OC₂H, and

0-nC I I₉ being particularly preferred.

The group R³ is preferably C,.₆ alkyl. preferably methyl or ethyl.

Most preferred groups R'° are mono or disubstituted amino groups such as -NR'²R^π groups where R'² is H or C alkyl and R¹³ is aralkyl, most preferably unsubstituted or halo substituted aralkyl and most particularly R¹⁰ is -N(CH₃)CH₂Phenyl, -N(CH,)-CH₂-(3,4 dichloroPhenyl), -NH-CH,-Phenyl or -NH-CH₂-(3.4-dichloroPhenyl).

R\ and R", are most preferably nitro. although azido also leads to good activit} . Where one of R¹⁰ and R¹ ' is nitro and the other halogen, particularly chloro. activity is good regardless of position. Similar position independent activity is expected with azido The compounds used in the manufacture as described by the present invention are all accessible by methods known in the art with all the examples described herein being known compounds. The compounds described in WO 88/04293 (US 4992444) are cited herein as particular compounds for use in the present manufacture, as are those of WO 94/02469 and WO 84/0446 where they fall within the formula disclosed above.

Details of preparative methods for obtaining compounds for use of the invention are to be found in the aforesaid patent documents as well as the following papers: Griffin et al (1985) J. Chem. Soc, Perkin Trans. I :2267; Griffin et al (1989) J. Medicinal Chem 32: 2468-2474; Griffin et al ( 1990) Anti-Cancer Drug Design, 5: 210-21 1. In a second aspect of the present invention there is provided a method of treatment of a patient having mutant-ra.y associated cancer, preferably of the NSCL. colon or pancreatic types, comprising administering to them an effective dose of a compound as described according to formula I or formula VI above.

The amount of these compounds used in the manufacture or method of the invention w ill of course vary with the type of cancer to be treated and the individual concerned.

Dosage forms and route of administration are more fully considered in the PCT application publications referred to above and their associated literature, as are forms such as salts suitable for administration. (See e.g. WO 94/02469 pages 6-9). The corresponding US applications US 4992444 and Serial No 08/374508 are incorporated herein by reference for that purpose and for their teaching of preparation of compounds for use in the method of the invention. The doses of such compounds may be conveniently assessed by reference to the relative efficacy evidenced against ras and wild type cancer cells shown in the Examples below.

The present invention will now be illustrated further by reference to the following non-limiting examples. Further embodiments falling within the scope of the invention will occur to those skilled in the art in the light of these. EXAMPLES

A number of known DHFR inhibitor compounds were selected for screening for anti-tumour activity using the NCI-In Vitro Anticancer Drug Discovery Screen. This is described in detail in a number of publications, particularly by Boyd and Paul in Drug Development Research (1995) 34:91 -109. These compounds are set out in Table I below in terms of their values R¹ to R" of formula VI. Et in Table 1 represents ethyl.

In brief, the screen measures effect of a given compound on a cell line through to optical density changes which it interprets as changes in percent growth, growth inhibition (GI) and LC_5ϋ. Non-small cell lung cancer lines included are A 549/ATCC, EKVX, HOP-62.

HOP-92. NCI-H226. NCI-H23. NCI-H322M. NCI-H460, NCI-H522. Colon cancer cell lines include COLO-205. HCC-2998, HCT-1 16. HCT-15, HT-29. KM- 12 and SW-620. On analysis of screening results it was found that the compounds 1 to 13 of Table 1 above gave the Pearson coefficients shown in Table 2 below as a measure of positive correlation of effect with anti ras-mutant seed cell efficacy for both NSCL and Colon ras types, the results in that table being at Log concentration -4 M. These correlations compare to values of less than 0.3 at the same concentration for a full 59 cell line screen.

The anti-ra.y activity of the compounds for the use of the present invention is illustrated further in Table 3 where individual ra.y v. wild type (Wt) cell efficacies are given and in Table 4 where activity against pancreatic ras lines is given.

TABLE 1

Compound R¹ R² R³ R¹ R¹¹

1 -NH₂ -NH₂ Et 4-(N-(3',4'-dichlorobenzyl)arnino) 3-NO₂

2 -NH₂ -NH₂ Et 4-(N-benzyl N-methylamino) 3-NO₂

3 -NH₂ -NH₂ Et 4-C1 3-NO₂ n c 4 -NH₂ -NH₂ Et 4-NO₂ 3-C1

DO O)

-4 5 -NH₂ -NH₂ Et formula V where R⁹ is phenyl

H

C 6 -NH₂ -NH₂ Et 4-C1 3-N, H m

(Λ I 7 -NH₂ -NH₂ Et 4-N-methylamino 3-NO₂

I m I m 8 -NH₂ -NH₂ Et 4-N-(2-phenylethyl)amino 3-NO₂

H

9 -NH₂ -NH₂ Et 4-(4-methylpiperazin- 1 -yl) 3-NO₂ c m 10 -NH₂ -NH₂ Et 4-C1 3-N=CH-NMe₂ r σ> 11 -NH₂ -NH₂ Et 4-N-benzylamino 3-NO₂

12 -NH₂ -NH₂ Et formula IV wherein X is N and R⁸ is benzyl

13 -NH₂ -NH₂ Et 4-NO₂ 3-N-benzylamino

14 -NH₂ -NH₂ Et 4-NO₂ 3-(N-benzyl,N-methylami

15 -NH₂ -NH, Et 4-C1 3-NH,

TABLE 2

PARTIAL RAS SEED - NSCL AND COLON

POSITIVE CORRELATION IS FOR RAS MUTANT SEED CELLS

WITH SENSITIVE CELLS FROM DATABASE

TABLE 3

ω c σ cn

H

H C H m ω z m m

H c r m r σ>

TABLE 4

Actiλ'ity of compounds agaisnt the pancreatic tumour cell line M l - mutant Ki-Ras gene 7 day assay.

Claims

Use of a compound of general formula (I)

wherein

R^] and R² are independently selected amino groups R¹ is C,.₆ alkyl or haloalkyl 5 R⁴ is selected from alkoxy, aralkoxy. haloalkyl, halogen and amino and

R^s is selected from nitro, amino. azido or a group -N=X-NR⁶R⁷ where R⁶ and R⁷ are independently C,_₆ alkyl or are aralkyl or together with the nitrogen to which they are attached form a heterocyclic ring with the proviso that one or both R⁶ and R⁷ or the heterocyclic ring carries or includes or carries and includes at least one oxygen or sulphur

[ 0 atom, and X is N or CH or CR⁹ or R'¹ and R⁵ together with the benzene ring to which they are attached form a group of formula II

wherein X is N, CH or CR⁹, and R^s is C,_₆ alkyl or is aralkyl

l J- or R⁴ and R³ together with the benzene ring to which they are attached form a group of formula III

wherein R⁹ is aryl or aralkyl or a salt or N-oxide of any such compound for the manufacture of a medicament for the treatment of mutant-ras gene associated cancer.

2. Use as claimed in claim 1 characterised in that R⁴ and R⁵ form a group of formula

II or III with the benzene ring to which they are attached and that group is of formula IV or V

R⁸

3. Use as claimed in claim 1 or 2 characterised in that the medicament manufactured is for the treatment of mutant-ras gene associated cancers selected from non-small lung cell (NSCL), colon and pancreatic cancers.

4. Use as claimed in any one of claims 1 to 3 characterised in that the compound is of formula VI

wherein R'° and R" are selected from haloalkyl, halogen, amino, nitro, azido or a group - N=X-NR⁶R⁷ or, together with the benzene ring to which they are attached, form a group of formula IV or V, with the provisio that if one one of R¹⁰ and R" is selected from haloalkyl and halo then the other is selected from amino. nitro, azido or a group -N=X-NR⁶R^λ or a salt or N-oxide of any such compound.

5. Use as claimed in any one of the preceding claims characterised in that the amino groups are selected from unsubstituted, mono and disubstituted amino groups, including those amino groups forming part of a heterocylic ring.

6. Use as claimed in claim 5 characterised in that the amino group comprises part of a ring consisting of from 3 to 7 carbon, nitrogen, sulphur and/or oxygen atoms.

7. Use as claimed in claim 6 characterised in that the ring is of carbon and nitrogen atoms only.

8. Use as claimed in any one of the preceding claims characterised in that when R⁴,

R³, R¹⁰ or R" are amino groups they are independently selected from C alkyl amino, di-C_M alkyl amino, C₆._l0 aralkyl amino, C,_₄ alkyl C_6.,₀ aralkyl amino or di-C₆.₁₀ alkyl amino.

9. Use as claimed in claim 8 characterised in that when R⁴, R⁵, R¹⁰ or R" are aralkyl amino groups they are halo substituted on the phenyl ring.

10. Use as claimed in any one of the preceding claims characterised in that when R⁴. R⁵. R'° or R" are amino. one or more of them are independently selected from -NHCH_V -NHC₂H₅, -NH(nC₄H₉) -NHCH,CH₂Phenyl, -NHCH₂Phenyl. -N(CH₃)CH₂Phenyl, -N(CH₂Phenyl)₂. -N(C₂H₅)CH₂Phenyl and -NHCH(CH,)Phenyl.

1 1. Use as claimed in claim 10 characterised in that the aryl group, if present, is substituted by halogen, C,_₆ alkyl or haloalkyl. C₂.₆ alkenyl or haloalkenyl, C,.₆ alkoxy or haloalkoxy, nitro, or -C0 R^]2 where R¹² is hydrogen alkyl or alkoxyalkyl or C,_₆

12 Use as claimed in any one of the preceding claims characterised in that R⁵ or R", is nitro or azido and R⁴ or R'° is amino.

13. Use as claimed in any one of the claims 4 to 12 characterised in that one of R¹⁰ and R" is nitro or azido and the other is halogen.

14. Use as claimed in claim 1 characterised in that the compound is one of 2.4-Diamino-5-[4-(3,4-dichlorobenzylamino)-3-nitrophenyl]-6-ethylpyrimidine,

2.4-Diamino-5-[4-benzylamino-3-nitrophenyl]-6-ethylpyrimidine, 2.4-Diamino-5-[4-chloro-3-nitrophenyl]-6-ethylpyrimidine, 2.4-Diamino-5-f4-nitiO-3-chlorophenyl|-6-ethylpyrimidine 2.4-Diamino-5-[4-chloro-3-azidophenyl]-6-ethylpyrimidine, and 2.4-Diamino-5-[4-N-methylamino-3-nitrophenyl]-ethylpyrimidine.

15. A method of treatment of a patient having mutant-ras associated cancer comprising administering to them a compound as defined according to fonnula I or formula VI above.

16. A method as claimed in claim 12 wherein the cancer is of the NSCL. colon or pancreatic types.