WO1998000426A1

WO1998000426A1 - 5,6,7,8-TETRAHYDROPYRIMIDO[4,5-b]AZEPINE DERIVATIVES

Info

Publication number: WO1998000426A1
Application number: PCT/US1996/011160
Authority: WO
Inventors: Edward C. Taylor; James E. Dowling
Original assignee: The Trustees Of Princeton University
Priority date: 1996-06-28
Filing date: 1996-06-28
Publication date: 1998-01-08
Also published as: AU6344396A

Abstract

Glutamic acid derivatives in which the amino group is substituted with a 2-amino-5,6,7,8-tetrahydropyrimido[4,5-b]azepin-6-yl)ethyl-Z-carbonyl group, in which Z is phenylene or thienediyl are inhibitors of glycinamide ribonucleotide formyltransferase and thus have use as antineoplastic agents. A typical embodiment is N-{4-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-b]azepin-6-yl)ethyl]-benzoyl}-L-glutamic acid.

Description

5,6,7,8-TET.RAnHYDROPYRIMIDO[4,5-b]AZEPINEDE VATIVES

This invention relates to 5,6,7,8- tetrahydropyrimido[4,5-tV)azepines of the formula:

in which R¹ is -OH or -NH₂,

R² is -OH or an a carboxylic acid protecting group, R³ is -H or an amino protecting group, and Z is phenylene or thienediyl, and the configuration about the carbon atom designated * is L.

The present invention also pertains to the pharmaceutically acceptable salts of the 5,6,7,8-tetrahydropyrimido[4,5-i]azepines of Formula I.

In addition, the invention pertains to a method of inhibiting neoplastic growth in a mammal in which the growth is dependent on folic acid, or a metabolic derivative of folic acid as a substrate (such as 10-formyltetrahydrofolate, the formyl donor to 5- aminoimidazole-4-carboxamide ribonucleotide in the de novo purine synthesis pathway, and N⁵,N^I0-methylenetetrahydrofolate, the methyl donor for deoxyuridylate yielding thymidylate in the synthesis of pyrimidines). The method comprises administering, in a single or multiple dose regimen, an effective amount of a compound according to the invention to a mammal in need of such therapy.

The present invention also pertains to the treatment of arthritis through administration of a compound of Formula I.

Finally, the invention pertains to pharmaceutical compositions which can be employed in the above indications in a mammal and which comprise a compound of Formula I in combination with a pharmaceutically acceptable carrier. The compounds of this invention are named herein as derivatives of the pyrimido[4,5- δ]azepines fused ring system which is numbered as follows:

The compounds of Formula I can be employed in the form of the free dicarboxylic acid, in which case both R² groups are hydrogen. Alternatively, the compounds often can be employed in the form of a pharmaceutically acceptable salt, in which case the hydrogen atom when R² is hydroxy is replaced by a pharmaceutically acceptable cation. Such salt 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 example the sodium, potassium, lithium, calcium, magnesium, aluminum, zinc, ammonium, trimethylammonium, triethanolammonium, pyridinium, and substituted pyridinium salts. The mono and diso- dium salts, particularly the disodium salt, are advantageous. It will be appreciated that the pyrimido[4,5-£]azepines of Formula I are the tautomeric equivalent of the corresponding 3-H-4-oxo or 3-H-4-imino structures. For simplicity's sake, the compounds are depicted herein as 4-hydroxy and 4-amino compounds, it being understood the corresponding and tautomeric keto and imino structures, respectively, are fully equivalent. In addition to the center of chirality about the carbon atom on the glutamic acid designated *, a second chiral center is present in the 6-position of the 5,6,7,8-tetrahydro- pyrimido[4,5-6]azepines ring system. Both the therapeutically active diastereomeric mixtures and the individual diastereomers are included in the scope of this invention. When both individual diastereomers are formed, they can be separated mechanically as by chro- matography or chemically by forming salts with a chiral acid, such as the individual enan- tiomers of 10-camphorsulfonic acid, camphoric acid, α-bromocamphoric acid, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic 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 substantially free of the other; i.e., in a form having an op- tical purity of >95%. The protecting groups designated by R² and R³ utilized herein denote groups which generally are not found in the final therapeutic compounds but which are intentionally introduced at some stage of the synthesis in order to protect groups which otherwise might be altered in the course of chemical manipulations. Such protecting groups are removed at a later stage of the synthesis and compounds bearing such protecting groups thus are of importance primarily as chemical intermediates (although some derivatives also exhibit biological activity). Accordingly the precise structure of the protecting group 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 Thie e Verlag, Stuttgart 1974, the disclosures of which are incorporated herein by reference. With respect to R², 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 of 1 to 12 carbon atoms such as methyl or ethyl and particularly one which is branched at the 1- or α position such as tert. -butyl; and such lower alkyl ester substituted in the 1- or 2-position with (/) lower alkoxy, such as for example, methoxymethyl, 1-methoxy ethyl, and ethoxymethyl, (//^') lower alkylthio, such as for example methylthiomethyl and 1-ethylthioethyl; (in) halogen, such as 2,2,2-trichloro- ethyl, 2-bromoethyl, and 2-iodoethoxycarbonyl; (J^'V) one or two phenyl groups each of which can be unsubstituted or mono-, di- or 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 also can be protected in the form of an organic silyl group such as trimethylsilylethyl or tri-lower alkylsilyl, as for example tri- methylsilyloxycarbonyl.

With respect to R³, an amino group can be protected as an amide utilizing an acyl group which is selectively removable under mild conditions, especially formyl, a lower alkanoyl group which is branched in 1- or α position to the carbonyl group, particularly tertiary alkanoyl such as pivaloyl, or a lower alkanoyl group which is substituted in the position α to the carbonyl group, as for example trifluoroacetyl.

Particularly preferred compounds are those wherein R¹ and R² are both hydroxy, R³ is hydrogen, and Z is phenylene , especially /κzrα-phenylene. Typical compounds include N-{4-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-^]azepin-6-yl)ethyl]-benz- oyl}-L-glutamic acid, N-{3-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5- 6]azepin-6-yl)ethyl]-benzoyl}-L-glutamic acid, N-{5-[2-(2-amino-4-hydroxy-5,6,7,8- tetrahydropyrimido[4,5-6]azepin-6-yl)ethyl]-thien-3 -yl } -L-glutamic acid, N- { 4-[2-(2- amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-6]azepin-6-yl)ethyl]-thien-3-yl}-L-glut- amic acid, N-{ 5-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-6]azepin-6-yl)- ethyl]-thien-2-yl}-L-glutamic acid, and N-{4-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydro- pyrimido[4,5-Z>]azepin-6-yl)ethyl]-thien-2-yl}-L-glutamic acid.

The compounds can be prepared by first conducting a Wittig reaction utilizing an aldehyde of Formula π, infra, in which R⁵ is a hydroxy protecting group such as trimethylsilyl, and a salt of a phosphine of Formula III in which R^δ is lower alkoxy and Z is as defined above. Conveniently this can be done under conditions which effect in situ hydrolysis of the trimethylsilyl enol ether. Thereby produced is a compound of Formula IV in which the two X*s together are a carbon-carbon bond, i.e., a 4-[3-(R⁶COZ)-prop-2- en-l-yl]cyclohexanone:

π. m. iv.

A compound of Formula IV in which the two Xs together are a carbon-carbon bond is then reduced, as through catalytic hydrogenation, to yield a compound of Formula IV in which each X is hydrogen. The compound of Formula IV in which each X is hydrogen is then subjected to a

Beckmann rearrangement, as for example with hydroxyla ine O-sulfonic acid, to yield a substituted azepine of Formula V, infra, in which R⁷ is hydrogen, each X is hydrogen, R⁶ is lower alkoxy and Z is as defined above. After protecting the ring nitrogen of the azepine so that R⁷ is an amino protecting group, as for example through formation of the N-tfcT/.-butoxycarbonyl derivative, the compound of Formula V is treated with lithium hexamethyldisilazide and Mander"s reagent (see Tetrahedron Lett., 1983, 24,5425) to effect methoxycarbonylation and yield a compound of Formula VI in which R⁷ is an amino protecting group, each X is hydrogen, R⁶ is lower alkoxy and Z is as defined above:

V. VI.

Removal of the amino protecting group then yields the lactam of Formula VI in which R⁷ is hydrogen. This is then treated with phosphorus pentasulfide to yield the corresponding thiolactam VII:

VIA. VI I.

When thiolactam VH is treated with guanidine free base under salt-free conditions, followed by acidification, there is obtained the pyrimido[4,5-i]azepine VLTI in which Rl is hydroxy, R3 is hydrogen, R2' is guanidino, and Z is as defined above.

vπ. vm.

Hydrolysis of the guanidine group in the pyrimido[4,5-6]azepine of Formula VLII with sodium hydroxide yields the corresponding compound in which R¹ and R^r are both hydroxy, R³ is hydrogen, and Z is as defined above. This can be converted to a compound of Formula I through coupling with a protected glutamic acid derivative, such as di-(/eτt.- butyl) glutamate, in the manner described in U.S. Patent No. 4,684,653, the disclosure of which is incorporated herein by reference. This involves using conventional condensation techniques for forming peptide bonds such as dicyclohexylcarbodiimide, diphenylchlorophosphonate, or 2-chloro-4,6-dimethoxy-l,3,5-triazine. Following this coupling reaction, any remaining protecting groups are removed as discussed below.

According to the foregoing processes, compounds of Formula I in which R^l is hydroxy are obtained. When a compound of Formula I in which R¹ is amino is desired, a compound in which R^l is hydroxy can be treated with 1,2,4-triazole and (4-chloro- phenyl)dichlorophosphate and the product of this reaction then treated with concentrated ammonia.

The compounds of this invention have an effect on one or more enzymes which utilize folic acid, and in particular metabolic derivatives of folic acid, as a substrate. The action of the compounds appear to be similar in this regard to that of 6-(R)-5,10-dideazatetrahydro- folic acid which is described in U.S. Patent No. 4,684,653. The IC₅₀ value against human T-cell derived lymphoblastic leukemia cells (CCRF-CEM) for N-{4-[2-(2-amino-4- hydroxy-5,6,7,8-tetrahydropyrimido[4,5-6]azepin-6-yl)ethyl]-benzoyl}-L-glutamic acid is 0.047 μ/mL.

The compounds exhibit particularly strong inhibitory activity against the enzyme glycinamide ribonucleotide formyltransferase. As can be seen from the following data for N-{4-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-i]azepin-6-yl)ethyl]- benzoyl}-L-glutamic acid, this activity is not reversed by thymidine (which would indicate the compound acts against thymidylate synthetase) but is reversed by the addition of hypoxanthine, AICA, and a combination of hypoxanthine and thymidine, indicating the inhibition involves the purine de novo synthesis:

Reversal Comp. Cone. Inhibition

none 0.047μM 0.0215 μg/mL thymidine 5μM 0.0328μM 0.015 μg/mL hypoxanthine lOOμM 36.5μM >16.7 μg/mL

AICA 300μM 22.94μM 10.496 μg/mL thymidine + 5μM hypoxanthine lOOμM >36.5μM 16.7 μg/mL The compounds can be used, under the supervision of qualified professionals, to inhibit the growth of neoplasms including choriocarcinoma, leukemia, 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, arthritis, 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, intra- thecal, 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. Concomitant administration of folic acid as a rescue therapy also may be indicated. 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 illustrate specific aspects of the present invention and are not intended to limit the scope thereof in any respect and should not be so construed. 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.

EXAMPLE 1

Methyl /r /ts-4-[2-(4-Oxocyclohexyl)ethenyl]benzoate To a stirred suspension of l-trimethylsilyloxycyclohexene-4-carboxaldehyde (4.0 g,

20 mmol) and 4-methoxycarbonylbenzyltriphenylphosphonium bromide (10.9 g, 22 mmol) in 35 mL of tetrahydrofuran was added diazadicycloundecane (3.7 g, 24 mmol) neat, via syringe. The resulting bright yellow suspension was stirred at room temperature for 2 hours to permit the initial mild exotherm to dissipate and then heated at reflux for 20 hours. The cooled reaction mixture was partitioned between ethyl acetate (25 mL) and water (25 mL) and the aqueous phase reextracted with ethyl acetate (25 mL). The combined organic phases were washed with water (2 x 25 mL), dried over magnesium sulfate, filtered and evaporated in vacuo to give a brown, solid residue. Chromatography on silica using 25% ethyl acetate in hexanes as eluent gave 3.8 g (75%) of a 7:1 trans.cis mixture of olefins, as determined by integration of the signals at 5.60 (dd, cis isomer) and 6.32 (dd, trans isomer) in the 1 H NMR spectrum. Recrys- tallization of a portion of the mixture from ether/hexanes gave the trans isomer of methyl 4-[2-(4-oxocyclohexyl)ethenyl]benzoate as a fluffy white solid, mp 101 °C. 1 H NMR (CDCI3, 300 MHz) δ 1.75 (m, 2H), 2.18 (m, 2H), 2.46 (m, 4H), 2.68 (m, 1 H), 3.91 (s, 3H), 6.32 (dd, 1 H, J = 16, 6.7 Hz), 6.51 (d, 1 H, J = 16 Hz), 7.41 (d, 2H), 7.98 (d, 2H); 13C NMR (CDC1₃, 75.6 MHz) δ 32.45, 39.43, 40.60, 52.17, 126.06, 128.60, 128.83, 130.04, 136.03, 141.87, 166.96, 211.08; IR (KBr) 1718, 1700 cm-1; MS m e (relative intensity) 258 (81), 227 (23), 162 (43), 129 (100); HRMS calcd for C₁₆H ,₈O₃: 258.1256. Found: 258.1255. Anal. Calcd for CiβH ,₈O₃: C, 74.40; H, 7.02. Found: C, 74.36; H, 7.20.

Methyl 5-[2-(4-oxocyclohexyl)ethenyl]thien-2-ylcarboxylate and methyl 5-[2-(4- oxocyclohexyl)ethenyI]thien-3-ylcarboxylate are obtained in a similar fashion from 2- methoxycarbonylthien-5-ylmethylltriphenylphosphonium bromide and 3-methoxycarbonyl- thien-5-ylmethylltriphenylphosphonium bromide, respectively.

EXAMPLE 2

Methyl 4-[2-(4-OxocycIohexyl)ethyl]benzoate

A 250 mL round bottom flask was charged with a mixture of cis- and trans- methyl 4-[2-(4-oxocyclohexyl)ethenyl] benzoate (3. 00 g, 11.6 mmol), 30 mg of 1 0% Pd on carbon and 40 mL of ethyl acetate and stirred at room temperature under a balloon of hydrogen for 8 hours. The reaction mixture was filtered through Celite with the aid of additional ethyl acetate and the filtrate concentrated in vacuo to give a pale yellow oil which solidified on standing (3.02 g, quantitative). Recrystallization from ether/hexanes gave methyl 4-[2-(4-oxocyclohexyl)ethyl]benzoate as white flakes, mp 55-56°C. 1 H NMR (CDCI₃, 300 MHz) δ 1.36 (m, 2H), 1.60 (m, 3H), 2.01 (m, 2H), 2.25 (m, 4H), 2.65 (t, 2H), 3.82 (s, 3H), 7.17 (d, 2H), 7.88 (d, 2H); 1 3C NMR (CDC1₃, 75.6 MHz) δ 32.66, 33.71, 35.59, 37.04, 40.80, 52.11, 127.99, 128.43, 129.88, 147.86, 167.15, 212.04; 1 R (KBr) 171 8, 1706 cm-1; MS m e (relative intensity) 260 (38), 229 (63), 163 (30). Methyl 5-[2-(4-oxocyclohexyl)ethyl]thien-2-ylcarboxylate and methyl 5-[2-(4- oxocyclohexyl)ethyl]thien-3-ylcarboxylate are obtained in a similar fashion from methyl 5- [2-(4-oxocyclohexyl)ethenyl]thien-2-ylcarboxylate and methyl 5-[2-(4-oxocyclohexyl)- ethenyl]thien-3-ylcarboxylate. EXAMPLE 3 Methyl 4- [2-(2-Oxohexahydroazepin-5-yl)ethyl] benzoate

To a solution of methyl 4-[2-(4-oxocyclohexyl)ethyl]benzoate (983 mg, 3.78 mmol) in 6 mL formic acid was added a solution of hydroxylamine O-sulfonic acid (640 mg, 5.67 mmol) in 3 mL of formic acid over 10 min. The resulting solution was heated at 95°C for 6 hours, cooled with the aid of an ice water bath and diluted with 25 mL of ice water. Neutralization with 30 mL of 3N sodium hydroxide resulted in the appearance of voluminous white precipitate. The suspension was transferred to a separatory funnel with the aid of methylene chloride (25 mL) and extracted with additional methylene chloride (4 X 25 mL). The combined organic phases were dried over sodium sulfate, filtered and evaporated in vacuo to give a brown oil which was purified by radial chromatography (4 mm plate) using 5% methanol in methylene chloride as eluent. The pale brown solid which was obtained (747 mg) was recrystallized from hexanes/tetrahydrofuran to give methyl 4-[2-(2-oxohexahydroazepin-5-yl)ethyl]benzoate as a white solid (720 mg), 71 %, mp 124°C. 1 H NMR (CDC1₃, 270 MHz) δ 1.25 (m,2H), 1.55 (m, 3H), 1.85 (m, 2H), 2.41 (dd, 2H), 2.65 (t, 2H), 3.17 (m, 2H), 3.85 (s, 3H), 6.92 (bit, 1 H), 7.19 (d, 2H), 7.91 (d, 2H); 13C NMR (CDC1₃, 75.6 MHz) δ 29.24, 33.20, 35.27, 35.95, 38.42, 41.19, 41.64, 51.99, 128.08, 128.38, 129. 86, 147.81, 167.10, 178.77; IR (KBr) 3295, 1710,1654, 1604 cm-1; MS m/e (relative intensity) 275 (77), 244 (34), 215 (38), 113 (100); HRMS calcd for Ci6H₂,NO₃: 275.1521. Found: 275.1519. Anal. Calcd for C₁₆H₂ιNO₃: C, 69.78; H, 7.69; N, 5.09. Found: C, 69.74; H, 7.72; N, 5.34.

Methyl 5-[2-(2-oxohexahydroazepin-5-yl)ethyl]thien-2-ylcarboxylate and methyl 5- [2-(2-oxohexahydroazepin-5-yl)ethyl]thien-3-ylcarboxylate are obtained in a similar fashion from methyl 5-[2-(4-oxocyclohexyl)ethyl]thien-2-ylcarboxylate and methyl 5-[2- (4-oxocyclohexyl)ethyl]thien-3-ylcarboxylate.

EXAMPLE 4

Methyl 4-[2-(l-ter/.-ButoxycarbonyI-2-oxohexahydroazepin-5-yl)ethyl]benzoate

A solution of methyl 4-[2-(2-oxohexahydroazepin-5-yl)ethyl]benzoate (275 mg, 1.0 mmol), di-tert. -butyl dicarbonate (436 mg, 2.0 mmol), triethylamine (202 mg, 2.0 mmol) and dimethylaminopyridine (244 mg, 2.0 mmol) in 10 mL of tetrahydrofuran was stirred at room temperature for 12 hours. The volatile components were removed in vacuo and the resulting semi-solid residue was applied to a silica gel column and chromatographed using 25% ethyl acetate in hexanes as eluent to afford methyl 4-[2-(l-tert.-butoxycarbonyl-2- oxohexahydroazepin-5-yl)ethyl]benzoate as a clear colorless oil (375 mg) in quantitative yield. 1 H NMR (CDClj, 300 MHz) δ 1.26-1.48 (m, H), 1.52 (s, 9H), 1.58 (m, 3H), 1.99 (m, 2H), 2.66 (m, 4H), 3.33 (dd, 1 H, J = 10.4, 15.3 Hz), 3.90 (s, 3H), 4.20 (dd, 1 H, J = 7.1, 15.3 Hz), 7.22 (d, 2H), 7.94 (d, 2H); 13C NMR (CDC1₃, 75.6 MHz) δ 28.14, 29.50, 33.22, 34.87, 38.04, 38.29, 39.88, 44.96, 52.08, 82.92, 128.00, 128.37, 129.86, 147.65, 152.82, 167.07, 175.24; 1 R (NaCl) 3006, 2980, 2931, 2859, 1767, 1718, 171 0, 1610 cm-1; MS m/e (relative intensity) 275 (86), 250 (20), 244 (41), 215 (43), 149 (62), 113 (1 00); Anal. Calcd for C₂ιH₂₉NO_s: C, 67.16; H, 7.79; N, 3.73. Found: C, 67.1 1; H, 7.63; N, 3.76.

Methyl 5-[2-( 1 -tert.-butoxycarbonyl-2-oxohexahydroazepin-5-yl)ethyl]thien-2-yl- carboxylate and methyl 5-[2-(l-tert.-butoxycarbonyl-2-oxohexahydroazepin-5-yl)- ethyl]thien-3-ylcarboxylate are obtained in a similar fashion from methyl 5-[2-(2-oxo- hexahydroazepin-5-yl)ethyl]thien-2-ylcarboxylate and methyl 5-[2-(2-oxohexa- hydroazepin-5-yl)ethyl]thien-3-ylcarboxylate.

EXAMPLE S Methyl 4-[2-(l-tert -Butoxycarbonyl-2-oxo-3-methoxycarbonylhexa- hydroazepin-5-yl)ethyI] benzoate

To a solution of methyl 4-[2-(l-/t?rt.-butoxycarbonyl-2-oxohexahydroazepin-5- yl)ethyl]benzoate (375 mg, 1.0 mmol) in 5 mL of tetrahydrofuran at -78°C was added a solution of lithium hexamethyldisilazide (from 275 mg, 1.6 mmol hexamethyldisilazide and 1.0 mL of 1.6. M «-butyllithium in hexanes) in 2 mL of tetrahydrofuran. After the reaction had been stirred for 30 minutes, neat methylcyanoformate (102 mg, 1.2 mmol) was added via syringe. The reaction vessel was removed from the cooling bath after 45 minutes and allowed to warm to room temperature. The reaction mixture was partitioned between ethyl acetate (5 mL) and a saturated aqueous solution of ammonium chloride (5 mL). The aqueous phase was reextracted with ethyl acetate (5 mL) and the combined organic extracts were washed with 1 0 mL of water, dried over magnesium sulfate, and filtered through a plug of silica gel. The filtrate then was evaporated in vacuo. The resulting oil was purified by radial chromatography (2mm plate) using 25% ethyl acetate and 1 % triethylamine in hexanes as eluent to give methyl 4-[2-(l-tert.-butoxycarbonyl-2-oxo-3- methoxycarbonylhexahydroazepin-5-yl)ethyl]benzoate as a clear colorless oil (280 mg, 65%).l H NMR (CDCI₃, 300 MHz) δ 1.31 (m, 1 H), 1.55 (m, 3H), 1.68 (m, 4H), 2.1 0 (m, 1 H), 2.30 (d, 1 H), 2.67 (t, 2H), 3.35 (dd, 1 H), 3.72 (partially obscured m, 1 H), 3.75 (s, 3H), 3.87 (s, 3H), 7.22 (d, 2H), 7.96 (d, 2H); IR (NaCl) 2971, 2935, 1759, 1745, 1710, 1604 cm-1. Methyl 5-[2-(l-tert.-butoxycarbonyI-2-oxo-3-methoxycarbonylhexahydroazepin-5- yl)ethyl]thien-2-ylcarboxylate and methyl 5-[2-(l-tert.-butoxycarbonyl-2-oxo-3- methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-3-ylcarboxylate are obtained in a similar fashion from methyl 5-[2-(l-/t?rt.-butoxycarbonyl-2-oxohexahydroazepin-5- yl)ethyl]thien-2-ylcarboxylate and methyl 5-[2-(l-tert.-butoxycarbonyl-2-oxohexa- hydroazepin- 5 -yl)ethyl]thien-3 -ylcarboxylate.

EXAMPLE 6

Methyl 4-[2-(2-Oxo-3-methoxycarbonylhexahydroazepin-5-yI)ethyl]benzoate

To a solution of methyl 4-[2-(l-tert. -butoxycarbonyl-2-oxo-3-methoxycarbonyl- hexahydroazepin-5-yl)ethyl]benzoate (583 mg, 1.34 mmol) in methylene chloride (25 mL) was added 4 mL of trifluoroacetic acid and the mixture was stirred for 4 hours. Removal of the solvent in vacuo afforded an oil which was purified by radial chromatography (2 mm plate) using 5% methanol in methylene chloride as eluent to give a methyl 4-[2-(2- oxo-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]benzoate as a colorless oil (424 mg, 95%) which solidified upon prolonged standing. 1 H NMR (CDC1₃, 300 MHz) δ 1.22 (m, 1 H), 1.61 (m, 3H), 1.89 (m, 1 H), 2.16 (m, 1 H), 2.67 (t, 2H), 3.35 (m, 2H), 3.73 (s, 3H), 3.87 (s, 3H), 7.20 (d, 2H), 7.92 (d, 2H); MS m/e (relative intensity) 333 (56), 302 (34), 273 (29), 171 (100); HRMS calcd for QgHaNOs: 333.1576. Found: 333.1577.

Methyl 5-[2-(2-oxo-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-2-ylcarb- oxylate and methyl 5-[2-(2-oxo-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-3- ylcarboxylate are obtained in a similar fashion from methyl 5-[2-(l-tert.-butoxycarbonyl-2- oxo-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-2-ylcarboxylate and methyl 5-[2- ( 1 -tert. -butoxycarbonyl-2-oxo-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-3- ylcarboxylate.

EXAMPLE 7

Methyl 4-[2-(2-Thio-3-methoxycarbonyIhexahydroazepin-5-yl)ethyI]benzoate

A suspension of phosphorous pentasulfide (400 mg, 0.90 mmol) and methyl 4-[2-(2- oxo-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]benzoate (300 mg, 0.90 mmol) in 1 0 mL of tetrahydrofuran was heated at reflux for 45 min. The reaction mixture was filtered through a glass fiber filter to remove an amorphous, white, semi-solid material which was washed with 20 mL of tetrahydrofuran. The filtrate was concentrated in vacuo and the residue was then purified by radial chromatography (2 mm plate) using 30% ethyl acetate in hexanes as eluent to afford methyl 4-[2-(2-thio-3-methoxycarbonylhexahydroazepin-5- yl)ethyl]benzoate (250 mg, 80%) as a mixture of diastereomers. 1 H NMR (CDC1₃, 300 MHz) δ 1.25 (m, 1 H), 1.56 (m, 3H), 1.88 (d, 1 H), 2.30 (d, 1 H), 2.68 (t, 2H), 3.40 (m, 2H), 3.75 (partially obscured , 1 H), 3.76 (s, 3H), 3.88 (s, 3H), 7.20 (d, 2H), 7.93 (d, 2H), 9.01 (t, 1 H); MS m/e (relative intensity) 349 (92), 318 (80), 289 (39), 256 (60), 101 (100).

Methyl 5-[2-(2-thio-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-2-ylcarb- oxylate and methyl 5-[2-(2-thio-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-3- ylcarboxylate are obtained in a similar fashion from methyl 5-[2-(2-oxo-3-methoxycar- bonylhexahydroazepin-5-yl)ethyl]thien-2-ylcarboxylate and methyl 5-[2-(2-oxo-3- methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-3-ylcarboxylate, respectively

EXAMPLE 8

4-[2-(2-Amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-*]azepin-6- yl)ethyI]benzoic Acid

A solution of guanidine, prepared from guanidine hydrochloride (153 mg, 1.6 mmol, 5 eq.) and sodium methoxide (from 37 mg, 1.6 mmol, of sodium metal) in 2 mL of methanol was filtered directly into a flask containing methyl 4-[2-(2-thio-3- methoxycarbonylhexahydroazepin-5-yl)ethyl]benzoate (1 13 mg, 0.32 mmol) in 2 mL of methanol. The flask was fitted with a vacuum adapter and the solvent gradually removed with the aid of an aspirator. The remaining oily residue was then heated under reduced pressure in an 80°C oil bath for 1 hour. After cooling to room temperature, water (2 mL) was added and the mixture was acidified with 6M hydrochloric acid. The resulting white solid was collected, redissolved in 1 N sodium hydroxide and heated for 24 hours at 60°C.

The cooled reaction mixture was rendered acidic with 6M hydrochloric acid and 4-[2-(2- anώιo-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-6]azepin-6-yl)ethyl]benzoic acid which formed was collected and dried (40 mg, 38 %). HRMS calcd for C_!7H₂ιN₄O₃ (MH+):

329.1613. Found: 329.1614.

5-[2-(2-Amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-δ]azepin-6-yl)ethyl]thien- 2-ylcarboxylic acid and 5-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5- έ]azepin-6-yl)ethyl]thien-3-ylcarboxylic acid are obtained in a similar fashion from methyl 5-[2-(2-thio-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-2-ylcarboxylate and methyl 5-[2-(2-thio-3-methoxycarbonylhexahydroazepin-5-yl)ethyl]thien-3-ylcarboxylate. .EXAMPLE 9

Di-tert-butyl N-{4-[2-(2-Amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-fc]azepin-6- yl)ethyI]benzoyI}-L-glutamate

To a stirred suspension of 4-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5- £]azepin-6-yl)ethyl]benzoic acid (60 mg, 0.18 mmol) in 6 mL of dimethylformamide was added

N-methylmorpholine (22 mg, 0.22 mmol), neat, via syringe. After 10 minutes, 2-chloro-4,6- dimethoxy-l,3,5-triazene (39 mg, 0.22 mmol) was added and stirring was continued for 1 hour. Additional N-methylmorpholine (22 mg, 0.22 mmol) was added, followed, after 20 minutes, by L-glutamic acid di-tert. -butyl ester hydrochloride (81 mg, 0.27 mmol) and the reaction was allowed to continue for 12 hours. Removal of the solvent in vacuo afforded a residue which was purified by radial chromatography (2mm plate) using 1 0% methanol in methylene chloride as eluent to give di-tert. -butyl N-{4-[2-(2-amino-4-hydroxy-5,6,7,8- tetrahydropyrimido[4,5-t ]azepin-6-yl)ethyl]benzoyl}-L-glutamate as a tan solid (72 mg, 70%). 1 H NMR (CDC1₃, 500 MHz) δ 1.40 (m, 1 H), 1.43 (s, 9H), 1.51 (s, 9H), 1.58 (m, 2H), 1.86 (m, 1 H), 2.08 (m, 2H), 2.25 (m, 1 H), 2.44 (m, 1 H), 2.51 (t, 2H), 2.70 (m, 2H), 2.81 (d, 1 H), 3.30 (m, 1 H), 3.47 (m, 1 H), 3.97 (s, 1 H), 4.68 (ddd, 1 H), 5.70 (brs, 1 H), 6.30 (brs, 2H), 7.15 (s, 1 H), 7.23 (d, 2H), 7.72 (d, 2H); MS (FAB) 570, 514, 458, 31 1; HRMS calcd for C₃0H44N5O6 (MH+): 570.3292. Found: 570.3297.

Di-tert. -butyl N-{5-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-i]azepin-6- yl)ethyl]thien-2-ylcarboxy}-L-glutamate and di-tert. -butyl N-{5-[2-(2-amino-4-hydroxy- 5,6,7,8-tefrahydropyrimido[4,5-δ]azepin-6-yl)ethyl]thien-3-ylcarboxy}-L-glutamate are obtained in a similar fashion from 5-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5- ό]azepin-6-yl)ethyl]thien-2-ylcarboxylic acid and 5-[2-(2-amino-4-hydroxy-5,6,7,8- tetrahydropyrimido[4,5-δ]azepin-6-yl)ethyl]thien-3-ylcarboxylic acid.

EXAMPLE 10

N-[4-[2-(2-Amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-6Jazepin-6- yI)ethyl]benzoyl]-L-glutamic Acid

To a solution of

N-[4-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydro- pyrimido[4,5-^]azepin-6-yl)ethyl]benzoyl]-L-glutamate (46 mg, 81 gmol) in 5 mL of methylene chloride was added trifluoroacetic acid (1 mL) and the mixture was stirred at room temperature for 12 hours. The solvent was removed in vacuo and water (2 mL) was added to the resulting residue. The acidic solution was neutralized with 2N sodium hydroxide and reacidified with 3M hydrochloric acid to afford N-[4-[2-(2-amino-4- hydroxy-5,6,7,8-tetrahydropyrimido[4,5-δ]azepin-6-yl)ethyl]benzoyl]-L-glutamic acid as a white precipitate which was collected and dried in vacuo (29 mg, 78%). 1 H NMR (DMSO-d6, 500 MHz) δ 1.35 (m, 1 H), 1.54 (m, 2H), 1.65 (m, 1 H), 1.95 (m, 2H), 2.06 (m, 1 H), 2.21 (dd, 1 H), 2.35 (t, 1 H), 2.68 (m, 3H), 3.05 (m, 1 H), 4.37 (ddd, 1 H), 5.77 (t, 1 H), 5.91 (s, 2H), 7.29 (d, 2H), 7.78 (d, 2H), 8.48 (d, 1 H), 9.86 (s, 1 H); MS (FAB) 458, 311, 260, 204, 148; HRMS calcd for C₂₂H_2gN₅O«(MH+): 458.2039. Found: 458.2034.

N-{5-[2-(2-ammo-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-i]azepin-6-yl)ethyl]- thien-2-ylcarboxy}-L-glutamic acid and N-{5-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydro- pyrimido[4,5-6]azepin-6-yl)ethyl]thien-3-ylcarboxy}-L-glutamic acid are obtained in a similar fashion from di-tert. -butyl N-{5-[2-(2-amino-4-hydroxy-5,6,7,8-tetrahydro- pyrimido[4,5-A]azepin-6-yl)ethyI]thien-2-ylcarboxy}-L-glutamate and di-tert. -butyl N-{5- [2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrimido[4,5-6]azepin-6-yl)ethyl]thien-3- ylcarboxy}-L-glutamate.

EXAMPLE 11

Hard gelatin capsules are prepared using the following ingredients:

Quantity (mg/capsule)

N- { 3 -[2-(2-amino-4-hydroxy- 5,6,7,8-tetrahydropyrimido [4,5-6]azepin 6-yl)ethyl]- 250 benzoyl-L-glutamic acid

Starch, dried 200

Magnesium stearate JO

460 mg

EXAMPLE 12

Tablets are prepared using the ingredients below:

Quantity

(mg/capsulei N- { 3 -[2-(2-amino-4-hydroxy-

5,6,7,8-tetrahydropyrimido [4,5-ό]azepin 6-yl)ethyl]- 250 mg benzoyl-L-glutamic acid

Cellulose, microcrystalline 400 Silicon dioxide, fumed 10

Stearic acid __5

665 mg

The components are blended and compressed to form tablets each weighing 665 mg.

EXAMPLE 13

An intravenous formulation may be prepared as follows:

Quantity

N- { 3-[2-(2-amino-4-hydroxy-

5 ,6,7, 8-tetrahydropyrimido

[4, 5-tVJazepin 6-yl)ethyl]- 100 mg benzoyl-L-glutamic acid

Isotonic saline 1,000 mL

Claims

What is claimed is:

1. A compound selected from the group consisting of ( ) a pyrimidino[4,5-*]azepine of the formula:

in which R^l is -OH or -NH₂, R² is -OH or an a carboxylic acid protecting group, R³ is -H or an amino protecting group, and Z is phenylene or thienediyl; and the configuration about the carbon atom designated * is L, and (t a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 in which R¹ is -NH₂, R² is -OH, and R³ is -H.

3. A compound according to claim 2 in which Z is phenylene.

4. A compound according to claim 2 in which Z is thienediyl.

5. A compound according to claim 1 in which R¹ and R² are -OH, and R³ is -H 6. A compound according to claim 5 in which Z is phenylene. 7. A compound according to claim 5 in which Z is thienediyl. 8. The compound according to claim 1 which is N-{4-[2-(2-amino-4-hydroxy-5,

6,

7,

8- tetrahydropyrimido[4,5-δ]azepin-6-yl)ethyl]-benzoyl }-L-glutamic acid.

9. The compound according to claim 1 which is N-{3-[2-(2-amino-4-hydroxy-5,6,7,8- tetrahydropyrimido[4,5-ft]azepin-6-yl)ethyl]-benzoyl}-L-glutamic acid.

10. The compound according to claim 1 which is N-{5-[2-(2-amino-4-hydroxy- 5,6,7,8-tetrahydropyrimido[4,5-»5]azepin-6-yl)ethyl]-thien-3-yl}-L-glutamic acid.

11. The compound according to claim 1 which is N-{4-[2-(2-amino-4-hydroxy- 5,6,7,8-tetrahydropyrimido[4,5-i]azepin-6-yl)ethyl]-thien-3-yl}-L-glutamic acid.

12. The compound according to claim 1 which is N-{5-[2-(2-amino-4-hydroxy- 5,6,7,8-tetrahydropyrimido[4,5-6]azepin-6-yl)eUιyl]-thien-2-yl}-L-glutamic acid.

13. The compound according to claim 1 which is N-{4-[2-(2-amino-4-hydroxy- 5,6,7, 8-tetrahydropyrimido[4, 5-i]azepin-6-yl)ethyl]-thien-2-yl } -L-glutamic acid.

14. The method of inhibiting neoplastic growth in a mammal which growth is depend- ent on folic acid or a metabolic derivative of folic acid as a substrate, which comprises administering to the mammal in a single or multiple dose regimen an effective amount of a compound according to claim 1.

15. The method of combating arthritis in a mammal in need thereof which comprises administering to the mammal in a single or multiple dose regimen an effective amount of a compound according to claim 1.

16. A pharmaceutical composition for inhibiting neoplastic growth in a mammal which growth is dependent on folic acid or a metabolic derivative of folic acid as a substrate, which comprises an amount of a compound according to claim 1 which upon admini- stration to the mammal in a single or multiple does regimen is effective to inhibit said growth, in combination with a pharmaceutically acceptable carrier.