N-(2-amino-5,6,7,8-tetrahydropyrido [2,3-b] pyridin-6-yl- acyl)-l-glutamic acid dericatives, their preparation and pharmaceutical compositions containing them and some new intermediates therefor

THE TRUSTEES OF PRINCETON UNIVERSITY and

ELI LILLY AND COMPANY

C: 77502 ^,ί

The present invention pertains to diastereomers and to the diastereomeric tamic acid derivatives of the formula:

R⁴NH

Η H

<img file="IL90179A_D0001.tif" />

N H or

R¹

<img file="IL90179A_D0002.tif" />

H H

C&lt;

R⁴NH ' N in

R¹

R²

R³

R⁴ the individual mixture of gluch₂ch₂-r^j-cnhchch₂ch₂cor²

H ch₂ . ——— ———2

COOR² , II * II . I ^CH₉CH₉-R^j-CNHCHCH₉CH₉C0R ch₂^{2 2} I ^{2 2}

COOR²

IB which:

is -0H or -NH₂;

is hydrogen or a carboxy protecting group;

is a carbon-carbon bond, alkylene of 1 to 4 carbon atoms, cyclohexylene, or _r5

<img file="IL90179A_D0003.tif" />

Q is hydrogen or an amino protecting group

Q is -S- or -0-; 90179/3

R⁵ is hydrogen, chloro or fluoro; and the configuration about the carbon atom designated * is S.

The compounds of Formula IA and IB have an inhibitory effect on one or more enzymes which utilize folic acid, and in particular metabolic derivatives of folic acid, as a substrate. The compounds thus can be used, alone or in combination, to inhibit the growth of those neoplasms which otherwise depend upon the enzymes so inhibited.

Israel patent No. 78059 discloses 5,10-dideazatetrahydrafolic acid. As in the case of folic acid, the side chain of these compounds contains a 1,4-phenylene group. The present compounds on the other hand are characterized by distinctive groups defined by R ; that is, either a direct carbon-carbon bond, an alkylene chain, a cyclohexanediyl group, or a 5-membered heterocylic ring containing oxygen or sulfur as the hetero atom.

The invention also pertains to the pharmaceutically acceptable salts of the compounds of Formula IA and IB, to processes for the preparation of these compounds and their salts, and to pharmaceutical compositions containing these compounds or their salts.

The term alkylene as used herein denotes a straight or branched divalent aliphatic group of from 1 to 4 carbon atoms including methylene, ethylene, trimethylene, tetramethylene, 1,1-propylidene, 2,2-propylidene, 1,2-propanediyl, 2,3-butanediyl, etc. Analogously, cyclohexylene denotes a divalent cycloalkane group of 6 carbon atoms including 1,2-cyclohexylene,

1,3-cyclohexylene, and 1,4-cyclohexylene.

The protecting groups designated by R² and R⁴ and’ utilized herein denote groups which generally are not found in the final therapeutic compounds but which are intentionally introduced during a portion of the synthesis to protect a group which otherwise might react in the course of chemical manipulations, thereafter being removed at a later stage of the synthesis. Since compounds bearing such protecting groups thus are of importance primarily as chemical intermediates (although some derivatives also exhibit biological activity), their precise structure is not critical. Numerous reactions for the formation and removal of such protecting groups are described in a number of standard works including, for example, Protective Groups in Organic Chemistry, Plenum Press, London and New York, 1973; Greene, Th. W. Protective Groups in Organic Synthesis, Wiley, New York, 1981; The Peptides. Vol. I, Schroder and Lubke, Academic Press, London and New York, 1965; Methoden der organischen Chemie, Houben-Weyl, 4th Edition, Vol.15/1, Georg Thieme Verlag, Stuttgart 1974.

A carboxy group can be protected as an ester group which is selectively removable under sufficiently mild conditions not to disrupt the desired structure of the molecule, especially a lower alkyl ester such as methyl or ethyl and particularly one which is branched at the 1-position such as t.-butyl; and such lower alkyl ester substituted in the 1- or 2-position with (i) lower alkoxy, such as for example, methoxymethyl, 1-methoxyethyl, and ethoxymethyl, (ii) lower alkylthio, such as for example methylthiomethyl and 1-ethylthioethyl;

(iii) halogen, such as 2,2,2-trichloroethyl, 2-bromoethyl, and 2-iodoethoxycarbonyl; (iv) one or two phenyl groups each of which can be unsubstituted or mono-, dior tri-substituted with, for example lower alkyl such as tert.-butyl, lower alkoxy such as methoxy, hydroxy, halo such as chloro, and nitro, such as for example, benzyl, 4-nitrobenzyl, diphenylmethyl, di-(4-methoxyphenyl)methyl; or (v) aroyl, such as phenacyl. A carboxy group can also be protected in the form of an /2 organic silyl group such as tri-lower alkylsilyl, as for example trimethylsilyloxycarbonyl.

Amino groups similarly can be protected as an amide utilizing an acyl group which is selectively 5 removable under mild conditions, especially formyl, a lower alkanoyl group which is branched at the 1-position, particularly tertiary alkandyl such as pivaloyl, or a lower alkanoyl group which is substituted in the 1-position, as for example trifluoroacetyl.

A first group of preferred compounds are those wherein R¹ is -0H and each of R² and R⁴ is hydrogen. A first preferred subgroup within this group are those compounds in which R³ is a carbon-carbon bond, methylene, ethylene, trimethylene, tetramethylene, or 1,415 cyclohexylene. Preferred species within this subgroup include the (R,S) and (S,S) diastereomers of N-[3-(2amino-4-hydroxy-5,6,7,8-tetrahydropyrido [ 2,3-d] pyrimidin-6-yl) propionyl ]-L-glutamic acid; N-[42)־ammo-4-hydroxy-5,6,7,8-tetrahydropyrido [ 2,3-d] 20 pyrimidin-6-yl)butyryl]-L-glutamic acid; N-[5-(2-amino4-hydroxy-5,6,7,8-tetrahydropyrido [2,3-d] pyrimidin-6yl)pentanoyl]-L-glutamic acid; N-[6-(2-amino-4-hydroxy-

5,6,7,8-tetrahydropyrido [ 2,3-d] pyrimidin-6yl)hexanoyl]-L-glutamic acid; N-[7-(2-amino-4-hydroxy25 5,6,7,8-tetrahydropyrido [ 2,3 -d ] pyrimidin-6- yl)heptanoyl]-L-glutamic acid; and N-[4-(2-(2-amino-4hydroxy-5,6,7,8-tetrahydropyrido [2,3-d] pyrimidin-6yl} ethyl) cyclohex-l-ylcarbonyl ] -L-glutamic acid.

A second preferred subclass are those compounds in which R³ is:

<img file="IL90179A_D0004.tif" />

Q where R⁵ is hydrogen and Q is -S-. Preferred species within this subgroup include the (R,S) and (S,S) diastereomers of N-[5-(2-{2-amino-4-hydroxy-

5,6,7,8-tetrahydropyrido[.2,3-d]pyrimidin-6-yl}ethyl) thien-2-ylcarbony]-L-glutamic acid; N-[5-(4-{2-amino-4hydroxy-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-6-yl)ethyl)thien-2-ylcarbony]-L-glutamic acid; N-[5-(2-{2amino-4-hydroxy-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-6-yl}ethyl)thien-3-ylcarbony]-L-glutamic acid; N[4-(2-{2-amino-4-hydroxy-5,6,7,8-tetrahydropyrido[2,3d]pyrimidin-6-yl}ethyl)thien-3-ylcarbony]־L-glutamic acid; N-[3-(2-{2-amino-4-hydroxy-5,6,7,8-tetrahydro- pyrido[2,3-d]pyrimidin-6-yl}ethyl)thien-2-ylcarbony]-Lglutamic acid; N-[2-(2-{2-amino-4-hydroxy-5,6,7,8tetrahydropyrido[2,3-d]pyrimidin-6-yl}ethyl) thien-3ylcarbony]-L-glutamic acid; and N-[5-(2-{2-amino-4hydroxy-5,6,7,8-tetrahydropyrido [ 2,3-d] pyr imid in-6yl)ethyl)fur-2-ylcarbony]-L-glutamic acid.

The compounds of the present invention often can be employed advantageously in the form of a pharmaceutically acceptable salt. Such forms, including hydrates thereof, are often crystalline and advantageous for forming solutions or formulating pharmaceutical compositions. Pharmaceutically acceptable salts with bases include those formed from the alkali metals, alkaline earth metals, non-toxic metals, ammonium, and mono-, di- and trisubstituted amines, such as for exampie the sodium, potassium, lithium, calcium, magnesium, aluminum, zinc, ammonium, trimethylammonium, triethan olammonium, pyridinium, and substituted pyridinium salts. The mono and disodium salts, particularly the disodium salt, are advantageous.

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The compounds of this invention in which R is alkylene or

Q can be prepared through catalytic hydrogenation of a compound of the formula:

R⁴׳NH

C=C-R³' -cnhchch₂ch₂cor־

COOR²' in which:

Z¹ and Z² taken individually are each hydrogen or taken together are a carbon-carbon bond;

R¹ is as herein defined;

, . 1 ר

R is a carboxy protecting group;

R³' is a carbon-carbon bond _t alkylene of 1 to 4 carbon atoms, or and

R⁴' is an amino protecting group /2

Suitable hydrogenation catalysts include noble metals and noble metal oxides such as palladium or platinum oxide, rhodium oxide, and the foregoing on a support such as carbon or calcium oxide.

There is obtained a mixture of diastereomers of Formulas IA and IB in which R²’ is a carboxy protecting group, and R⁴' is an amino protecting group. These protecting groups can then be removed through acidic or basic hydrolysis, as for example with sodium hydroxide, _e o to yield the compounds of Formula I in which each of R and R⁴ is hydrogen.

Compounds of Formula II can be prepared utilizing procedures analogous to those described in US Patent 4,818,819.

Thus in a first embodiment, a compound of the formula:

r⁴'nh in which X is bromo or iodo and R⁴' is as herein defined, is allowed to react with an unsaturated compound of the formula:

/2 in which Z^x, Z^z, R^z , and R are as herein defined, in the presence of a palladium/tr!substituted phosphine catalyst of the type described by Sakamoto, Synthesis, 1983, 312 et seq.

The compounds of this invention in which R³ is cyclohexylene can be prepared through initial catalytic hydrogenation of a compound of the formula:

N in which R⁴' is an amino protecting group and R¹is a herein defined. Compounds of Formula V in which R¹ is -OH are prepared analogously to the methods described in US patent 4,818,819 and the corresponding compounds in which R¹ is -NH₂ are generated therefrom in the manner described above.

Suitable catalysts for the hydrogenation of compounds of Formula V include noble metals and noble metal oxides such as palladium or platinum oxide. Thus obtained is a compound of the formula:

ch-ch₂ch₂-c₆h₁₀-cooh /2

Compounds of Formula VI then are coupled with a protected glutamic acid derivative of the formula:

HnNCHCHnCHnCOR²² | . ²

COOR²

VII

י 0 . ״ in which R is a carboxy protecting group, in the manner generally described in PCT application WO 86/05181, utilizing conventional condensation techniques for forming peptide bonds, such as activation of the carboxy group through formation of a mixed anhydride, treatment with DCC, or use of diphenyl15 chlorophosphonate. Protecting groups designated by R² ’ and R⁴ then are removed in the manner described above.

In a third embodiment, compounds of Formula II can be prepared utilizing procedures analogous to those described in US Patent 4,818,819. Thus an unsaturated compound of the formula:

<img file="IL90179A_D0005.tif" />

in which Z¹, Z², allowed to react with

VIII and R⁴ are as herein defined, is compound of the formula:

// ' \ ¹¹ * יל X—# TT־^CNHCHCH2^CH2^COR

Q ^Z COOR²'

IX

1 c in which X is bromo or iodo and R , R , and Q are as herein defined, in the presence of a palladium/trisubstituted phosphine catalyst of the type described by Sakamoto, Synthesis, 1983, 312 et seq.

There is obtained according to the foregoing processes a compound of Formula II in which R^ is -OH. When a compound in which R¹ is -NH2 is desired, this product can be treated with 1,2,4-triazole and (4chlorophenyl)dichlorophosphate and the product of this reaction then treated with concentrated ammonia.

Compounds of Formula IX are prepared by coupling a compound of the formula:

Q

X in which X, R⁵, and Q are as herein defined, with a protected glutamic acid derivative of formula VII in the manner described above utilizing conventional condensation techniques for forming peptide bonds.

The mixture of the individual diastereomers depicted by Formulas IA and IB can be used therapeutically as such or can be separated mechanically as by /2 chromatography. Alternatively, the individual diastereomers can be separated by forming diastereomeric salts with a chiral acid such as the individual enantiomers of 10-camphorsulfonic acid, camphoric acid, 5 alpha-bromocamphoric acid, :nienthoxyacetiC! acid, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5carboxylic acid, and the like, and then freeing one or both of the individual diastereomeric bases, optionally repeating the process, so as obtain either or both sub10 stantially free of the other; i.e., in a form having an optical purity of >95%. This separation can be effected before or after removal of any protecting groups.

As noted, the active compounds of this invention have an 15 effect on one or more enzymes which utilize folic acid, and in particular metabolic derivatives, of folic acid, as a substrate. The compounds can be used, under the supervision of qualified professionals, to inhibit the growth of neoplasms including choriocarcinoma, leu20 kemia, adenocarcinoma of the female breast, epidermid cancers of the head and neck, squamous or small-cell lung cancer, and various lymphosarcomas. The compounds can also be used to treat mycosis fungoides and psoriasis.

The compounds can be administered orally but preferably are administered parenterally, alone or in combination with other therapeutic agents including other anti-neoplastic agents, steroids, etc., to a mammal suffering from neoplasm and in need of treatment.

Parenteral routes of administration include intramuscular, intrathecal, intravenous and intra-arterial. Dosage regimens must be titrated to the particular neoplasm, the condition of the patient, and the response but generally doses will be from about 10 to about 100 mg/day for 5-10 days or single daily administration of 250-500 mg, repeated periodically; e.g. every 14 days. While having a low toxicity as compared to other antimetabolites now in use, a toxic response often can be eliminated by either or both of reducing the daily dosage or administering the compound on alternative days or at longer intervals such as every three days. Oral dosage forms include tablets and capsules containing from 1-10 mg of drug per unit dosage. Isotonic saline solutions containing 20-100 mg/ml can be used for parenteral administration.

The following examples will serve to further illustrate the invention. In the NMR data, *s denotes singlet, d denotes doublet, t denotes triplet, q denotes quartet, ״m denotes multiplet, and br denotes a broad peak.