WO1993013102A1 - Deazaaminopterins - Google Patents

Abstract

5,10-Diakyl substituted 5,10-dideazaaminopterin and a cyclized derivative thereof are disclosed as potent antineoplastic agents. Also disclosed in an improved process for preparation of 10-ethyl-10-deazaaminopterin using the intermediate methyl 4-[[2-(2,4-diamino-6-pteridinyl)-1-ethyl]ethenyl]benzoate.

Description

DEAZAAMINOPTERINS

The present invention relates to 5,10-diakyl substituted 5, 10-dideazaaminopterin and a cyclized derivative thereof and to the use of such a compound as antineoplastic agents. Another aspect of this invention relates to an improved process for the preparation of 10-ethyl-10-deazaaminopterin that is simplier and easier to carry out than tose previously reported.

Methotrexate (MTX) remains the only classical antifolate in established clinical use, and its use has continued to expand as new methods of administering the drug have been introduced and as other tumor types have been added to the list of those now being treated. MTX usage, however, suffers major limitations due to toxic side effects and the development of resistance by tumor cells. Some tumors are naturally resistant to MTX while others acquire resistance after a period of response. Three factors known to contribute to drug resistance are (a) loss of the active- transport system by which MTX enters cells, (b) increased levels of dihydrofolate reductase (DHFR), the intracellular target of MTX, and (c) the presence of structurally altered DNA having lower affinity for MTX. Another explanation of resistance may be offered in the recent description of a structurally altered DHFR from an MTX-resistant mutant cell line with unaltered affinity for MTX, but with greater capacity to reduce dihydrofolate than the DHFR from the parent MTX-sensitive cell lines. MTX and aminopterin (AMT) have the following structures:

AMT: R = H MTX: R = CH₃

As part of a program aimed toward the identification of new antϊfolate agents that exert greater therapeutic

effectiveness against a broader spectrum of tumors than agent now available, antifolates having favorably altered transport characteristics but still possessing tight binding affinity for DHFR are being sought. In studies aimed toward greater understanding of transport properties, differences have been observed between tumor and normal prolϊferative tissue in mediated cellular membrane transport of antifolates and in the intracellular γ-polyglutamylation of the agents. These

biochemical parameters appear to be critical determinants for selective antitumor activity. In studies that document these differences, positions 5 and 10 on the classical antifo late-type molecular structure have been identified as sites where modification does not reduce binding to DHFR but does influence transport efficacy to favor inward flux into tumor cells and also intracellular γ-polyglutamylation resulting in greater

accumulation in tumor cells than in normal cells.

The above findings were exploited in the design of the 10- deazaaminopterin series of antifolates [J. Med. Chem.17:552 (1974); and US Patent 4,393,964.]. This series, particularly 10- ethyl-10-deazaaminopterin [J. Med. Chem. 29:1056 (1986)], exhibited markedly enhanced therapeutic selectivity compared to MTX in both animal solid tumor models and human tumor

xenografts (Cancer Treat. Rep 69:551 (1985)]. In on-going clinical trials, 10-ethyl-10-deazaaminopterin has shown significant therapeutic activity against non-small cell lung cancer [J. Clin. Oncol. 6:446 (1988)].

Current studies with the 5-deaza analogues of AMT and MTX [J. Med. Chem. 29:1080 (1986), and US Patent 4,725,687] have revealed that 5-alkyl-5-deaza analogues of AMT and MTX are more active in vivo than MTX against four murine tumor models [Cancer Res. 48:5686 (1988)].

Neither 5,10-Dideazaaminopterin [J. Med. Chem. 28:914 (1985)] nor 5-Methyl-5,10-dideazaaminopterin [J. Med. Chem. 31 :2164 (1988)]. shows evidence of biological activity

significantly greater than that of MTX.

U. S. Patent 4,684,653 to Taylor et al discloses compounds having the formula:

wherein R is defined as hydrogen, methyl or ethyl as

antineoplastic agents.

It has now been discovered that 5,10-diakyf substituted 5,10-dideazaaminopterin and a cyclized derivative thereof are especially valuable antineoplatic agents. These compounds have the structures:

wherein R₅ and R _{1 0} represent alkyl groups containing from 1-6 carbon atoms. It will be appreciated that Structure II is similar to Structure I in that R₅ and R_{1 0} taken together represent an ethylene (-CH₂CH₂-) group.

An improved process for the production has also been discovered which 10-ethyl-10-deazaaminopterin has also been discovered which involves the production and use of the

intermediate rnethyl-4-[[2-(2,4-diamino-6-pteridinyl)-1 - ethyl]ethenyl]-benzoate. In the description which follows, reference will be made to Arabic numbers which identify compounds shown by structural formulas in Schemes I and II. Scheme I is as follows:

Synthesis of 10-ethyl-5-methyl-5,10-dideazaaminoterin (7 in Scheme I above which corresponds to general Structure I where R₅ = CH₃ and R₁₀ = CH₂CH₃) was achieved as shown below beginning with 6-(bromomethyl)-2,4-diamino-5-methyl-pyrido[2,3-d]pyrimidine hydrobromide (1). (The bromomethyl compound 1 was described by Piper et al in J. Med. Chem., 29, 1080 (1986) and in U. S. Patent 4,725,687). The bromomethyl

compound 1 was treated with tributylphophine (BU₃P) in dimethyl sulfoxide (Me₂SO), and the resulting tributylphosphonium salt was treated in situ with sodium hydride to give the ylide

intermediate compound 2 which remained in solution in Me₂SO. Methyl 4-(propionyl) benzoate was then added to the solution, and the desired Wϊttig reaction to produce the olefinic intermediate product 3 (see Example 1) occurred as expected. The product 3 was isolated and purified by column chromatography on silica gel. Hydrogenation of the olefinic bridge of compound 3 followed.

This step was achieved in glacial acetic acid promoted by

palladium-on-carbon (Pd/C) catalyst under hydrogen at ambient conditions in a gas burette. The desired hydrogenated product 4 (see Example 2) was separated from unwanted coproducts and unchanged compound 3 by silica gel chromatography.

Saponification of the ester group of product 4 in Me₂SO

containing a slight excess of aqueous sodium hydroxide followed. Following removal of the Me₂SO, the product 5 (see Example 3) was isolated by acidification of an aqueous solution of its sodium salt. Coupling of compound 5 with diethyl L-glutamate followed; the reaction was promoted by diethyl cyanophosphonate in Me₂SO containing N-methylmorpholine. The coupled product 6 (see

Example 4) was purified by chromatography on silica gel.

Saponification of the ester groups of product 6 was carried out at room temperature in aqueous methanol containing a small excess of sodium hydroxide to give the disodium salt of the target compound, and acidification of the solution caused precipitation of target compound 10-ethyl-5-methyl-5,10-dideazaaminopterin compound 7 (see Example 5).

The synthetic route used to prepare 7 is similar in principle to one described for synthesizing 10-deazaaminopterin [J. Med. Chem. 23:320 (1980]) and U. S. Patent 4,172,200]. Three

important differences in the syntheses are: first, the route to 10-deazaaminopterin made use of a triphenylphorphorance intermediate for a Wittig reaction with an aldehyde co-reactant in N,N-dimethylacetamide, whereas the present method required the less sterically bulky tributylphosphorane for Wittig reaction with a keto co-reactant and required Me₂SO as solvent. Second, the key Wittig reaction, which afforded intermediate 3, gave better results using methyl 4-(propionyl)benzoate than when diethyl N-[(4-propionyl)benzoyl]-L-glutamate was used, whereas in the earlier synthesis of 10-deazaaminopterin, the use of the complete glutamte-bearing side chain proved satisfactory. Thus, the glutamic acid moiety had to be introduced after the Wittig conversion in the synthesis of 7. The third difference is that in the hydrogenation step during synthesis of 10-deazaaminopterin, platinum catalyst is used as the catalyst. This catalyst promotes hydrogenation, not only of the olefinic bridge, but also the pyrazine-ring moiety affording a tetrahydro derivative of the intended product; however, the aromaticity of the pyrazine-ring moiety is easily regenerated by mild oxidation with aqueous hydrogen peroxide. In the 5-deaza analogue, however, reversal of hydrogenation of the pyrido-ring moiety could not be done easily, if at all. It is therefore important to avoid hydrogenation other than in the olefinic bridge. For this reason, in the hydrogenation of the olefin precursor to this reason, in the hydrogenation of the olefin precursor to 7, conditions were selected to try to minimize hydrogenation within its pyrida-ring moiety.

In working out the conditions that made the key Wittig condensation with methyl 4-(propionyl)benzoate effective use was made of available 6-(bromomethyl)-2,4-pteridine-diamine hydrobromide [J. Org. Chem. 42:208 (1977), and US Patent Nos. 4,077,957 and 4,079,056] as a model or prototype for 6- (bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidme hydrobromide (compoundl ). The full-pteridine analogue was used in the experiments that led to the findings that Me₂SO was the clear solvent of choice and that tributylphosphine would allow the Wittig conversion to occur, whereas triphenyiphosphine would not. The conversion shown below to produce methyl 4-[[2-2,4- diamϊno-6-pteridinyl)-1 -ethyl]ethenyI]benzoate (compound 10, see Example 6) then served as a guide model for the preparation of the 5-methyI-5-deaza analogue of compound 3. Very similar results were obtained from both conversions.

Compound 10 may then be converted to 10-ethyl-10- deazaaminopterin in the manner previously described for the conversion of Compound 3 to 5-methyl-10-ethyl-5,10- dideazaaminopterin, compound 7. Thus, another preparation of 10- ethyl-10-deazaaminopterin by hydrogenating compound 10, saponifying the ester group from said hydrogenated compound, coupling said saponified product with diethyl-1-glutamate, and saponifying the ester groups from the resultant product to yield 10- ethyl-10-deazaaminopterin.

With regard to this aspect of the present invention, the invention relates to a new process for preparing 4-[1 -[(2,4- diamino-6-pteridinyl)-methyl]propyl]benzoic acid (4-amino-4- deoxy-10-ethyl-10-deazapterioc acid, that is compound 27, that is simpler and easier to carry out than those previously reported. Compound 27 is readily and efficiently converted to N-[4-[1 - [(2,4-diamino-6-pteridinyl)methyl]propyl]benzoyl]-L-glutamic acid (10-ethyl-10-deazaaminopterin, 10-EDAM). Thus, a

synthetic route affording improved access to derivative 27 is tantamount to a better method for the synthesis of glutamate bearing 10-EDAM, a compound now undergoing clinical trial as an anticancer agent. The structure of 10-EDAM is

wherein Y is CCH₂CH₃

The separate C-10 diastereomers of 10-EDAM are

equivalent in efficacy against L1210 leukemia in mice, and the diastereomers are essentially comparable with respect to biochemical tests and cellular membrane transport. In advanced clinical trials comparing 10-EDAM with MTX, 10-EDAM has given significantly better results against nonsmall cell lung cancer. In clinical combination chemotherapy studies, combinations including 10-EDAM have produced improved response rates. In a recent review results from clinical studies of 10-EDAM, a conclusion was presented that continued advanced clinical evaluation of 10-EDAM, particularly against nonsmall cell lung cancer, was warranted.

A new synthetic route according to the present invention beginning with readily accessible materials allows facile access to 10-EDAM, a compound whose clinical trial results indicate that it will likely become the second (after MTX) classical antifolate to attain the status of clinical usage. The new synthesis features the facile production and use of the intermediate compound 10, methyl 4-[[2-(2,4-diamino-6-pteridinyI)-1 -ethyl]ethenyl]- benzoate.

Scheme I outlines the new synthesis path to compound 27. With this approach, a better synthetic route to 10-EDAM is made available since the final two steps, which consist of coupling of compound 27 with diethyl L-glutamate followed by hydrolysis of the ester groups, are standard conversions. Furthermore, as will be explained later, the sequence of reactions could be rearranged such that the methods and procedures of this route could lead directly to 10-EDAM.

Scheme 1 begins the 6-(bromomethyl)-2,4-pteridinediamine hydrobromide (compound 8) treated with tributylphosphine in dimethylsulfoxide (Me₂SO), and the resulting tributylphosphonium salt being treated in situ with sodium hydride to give the ylide intermediate compound 9 which remained in solution in Me₂SO. Methyl 4-propϊonylbenzoate was then added to the solution, and the desired Wittig reaction occurred as expected to produce the olefinic intermediate compound 10. This product was easily isolated in 80 % yield and high purity without need for

purification efforts such as column chromatography.

Hydrogenation of compound 10 at 46 psi in glacial acetic acid containing PtO₂ resulted in saturation of both the olefinic bridge and the pyrazine-ring moiety to give hydrogenated product 25, which was not isolated. The hydrogenation step was followed by mild in situ oxidation in order to regenerate the full aromatic pteridine ring and afford pure methyl 4-[1 -[(2,4-diamino-6- pteridinyl)methyl]propyl]benzoate (compound 26), which may also be called methyl 4-amino-4-deoxy-10-ethyl-10deazapteroate, in 73% yield. At this point the new route intersects the initial synthetic pathway to 10-EDAM reported by DeGraw. However, the preferred procedure for hydrolysis of compound 26 to the acid 27 differs from that used by DeGraw.

In addition to the specific compound 10, modifications may be made in going from compound 9 to compound 10 to produce other intermediate compounds. For example, the compound shown by the structure 10 may have the general structure:

wherein R and R' may be a lower alkyl of 1 to 6 carbons, as for example R being an alkyl of 2 carbons and R' being an alkyl of 1 carbon for the compound 10. Such substitutions and their ultimate effects on subsequent compounds 25, 26, 27 and 10- EDAM are considered to be within the aspects of the present invention.

There are important differences in the preferred route to key precursor 27 according to the present invention compared with that described for preparing 10-deazaaminopterin using a Wittig process. The route to 10-deazaaminopterin made use of a triphenylphosphorane intermediate for a Wittig reaction with and aldehyde co-reactant in N,N-dimethylacetamide (Me₂NAc), whereas the present process required the less sterically bulky tributylphosphorane (compound 9) for Wittig reaction with a keto co-reactant and furthermore required Me₂SO as solvent. The triphenylphosphorane corresponding to tributylphosphorane 9 would not react with the keto co-reactant in either Me₂NAc or Me₂SO even at elevated temperatures, and gave only poor conversions in Me₂NAC, even at elevated temperatures. At elevated temperatures, unwanted coproducts formed, and isolation of the slight amount of compound 10 which did form required special and laborious effort. Only the use of the tributylphosphorane in Me₂SO gave smooth conversion to

compound 10 in good yield at ambient temperature. Also, the key Wittig reaction, which afforded intermediate 10, gave better results using methyl 4-propionylbenzoate than when diethyl N-[4- propionylbenzoyl]-L-glutamate was used, whereas in the earlier synthesis of 10-deazaaminopterin, the use of the complete glutamate-bearing side chain proved satisfactory. The synthesis of 10-EDAM using complete glutamate bearing side chain

intermediates can be achieved by the procedures at hand; but, the higher-yield conversion using methyl 4-propionylbenzoate and the facile, high yield conversion of eventual product 7 to 10-EDAM made the Wittig approach through compound 27 the route of choice over that using the intact glutamate-bearing side chain.

The compound of Structure II, 5,10-ethano-5,10- dideazaaminopterin, may be prepared by the following scheme:

These and other aspects of the present invention may be determined from the following examples illustrating the best mode known for carrying out this invention. In these examples, examinations by TLC were performed on Analtech precoated (259 μM) silica gel G(F) plates. Products were dried in vacuo (1 mm or less) at 22-25 °C over P₂O₅ and NaOH pellets. Final products were dried and then allowed to equilibrate under ambient condition. Mass spectra were recorded on a Varian MAT 11 A mass spectrometer in the fast-atom-bombardment mode. UV spectra were determined with a Perkin-Elmer Model Lambda 9

spectrometer. Samples were first dissolved in 0.1 N NaOH, and the solutions were diluted 10-fold with the medium given in the listing. Maximma are expressed in nanometers with molar absorbance given in parentheses. Molecular weights used in calculations conform with the compositions listed wit the elemental analysis results, the following abbreviations are used in the examples: Me₂SO is dimethylsulfoxide, Et₂O is ethylether, AcOH is acetic acid, MeOH is methyl alcohol and THF is

tetrahydrofuran.

EXAMPLE 1

Methyl 4-[[2-(2,4-Diamino-5-methylpyrido[2,3-d]- pyrimidin-6-yl)-1 -ethyl]ethenyl]benzoate

compound 3

A solution of the bromomethyl compound 1 (3.13 g, 8.24 mmol) and tributylphosphine (5.00 g, 24.7 mmol) in Me₂SO (200 ml) was kept 20 hours at 20-23°C, then gradually warmed during 90 minutes to 55°C for 30 minutes, and cooled to 20-23°C.

Methyl 4-(propϊonyl) benzoate (1.58 g, 8.22 mmol) was then added, followed by sodium hydride (660 mg of 60 % dispersion in oil, 16.5 mmol). Complete solution occurred readily. After 44 hours at 20-23°C, the solution was heated at 70-75°C for 64 hours. Removal of the Me₂SO by distillation in vacuo (less than 1 mm, bath to 55°C) followed. The syrupy residue was stirred with Et₂O until the yellow solid that formed dispersed. The solid was collected with the aid of Et₂O, air dried, then stirred thoroughly with H₂O before it was again collected and dried in vacuo (78°C) over NaOH pellets with P₂O5). Crude 3 (2.31 g, 77 % crude yield) was combined with a sample from another run and the combined batch was purified by flash chromatography on silica gel (230- 400 mesh) using elution by CHCl₃-MeOH (9:1). Fractions found by TLC to be homogeneous (UV detection) were pooled and evaporated to give pure 3. The purified material amounted to 50 % recovery of material applied to the column. Spectral data: mass, m/z 364, (MH+) for C₂₀H₂₁N₅O₂.

Example 2

Methyl 4-[1 -[(2,4-Diamino-5-methylpyrido[2,3-d]- pyrimidin-6-yl)methyl]propyl]benzoate

compound 4

Compound 3 (9710 mg, 2.51 mmol) in glacial AcOH was treated with stirring with 30% Pd/C (120 mg) for 10 minutes, then 5% Pd/C (1.40 g) was added. Hydrogenation at atmospheric pressure (gas burette) followed. After 2 hours, uptake of hydrogen had reached 100 ml and the rate had slowed from a peak rate of 1 ml per minute to about 1 ml per 8 minutes. Examination at this point by TLC indicated two unwanted coproducts formed by hydrogenation in the pyrido ring moiety. Hydrogenation was discontinued, and workup was begun. The catalyst was removed by filtration and extracted on the funnel by stirring with

methanol. The methanol washings of the catalyst were combined with the original acetic acid filtrate. The MeOH-AcOH solution was then combined with that from another run with similar experiences (on 360 mg of 3 with an uptake of 53 ml of hydrogen). The clear pale yellow solution was evaporated under reduced pressure (H₂O aspirator, rotary evaporator, bath to 55°C) until nearly all the AcOH had been removed. The concentrated yellow oil that remained was treated with Et₂O to cause separation of crude 4 as a beige solid. The mixture was stirred until the solid was well dispersed before it was collected. Examination by TLC (CHCl₃-MeOH, 4:1 ) of the Et₂O-insoluble solid and the ethereal filtrate revealed the solid to be considerably less contaminated by two faster-moving coproducts (from ring reduction) and unchanged starting material (spot barely above that due to desired product) than the ethereal filtrate. The solid (670 mg, 66% crude yield) required further purification. (Unchanged 3 proved more difficult to remove than the excessively hydrogenated coproducts. This experience suggests that some degree of ring hydrogenation during the preparation of 4 would be acceptable provided conversion of starting 3 is complete). The ethereal filtrate was evaporated, and the residual viscous yellow oil was stirred with H₂O and treated with saturated NaHCO₃ solution to produce pH 8. The yellow solid which formed was collected, dried in vacuo, and examined by TLC. This portion of the product mixture consisted mostly of the two excessively hydrogenated coproducts plus some unchanged 3 and some 4. This crude fraction was applied in CHCl₃MeOH (1 :1) solution to a preparative TLC plate which was developed using CHCL₃-MeOH (4:1) to give a band which consisted mostly of 4 plus some unconverted 3. Extraction of this band afforded 60 mg of material of about the same purity as the Et₂O-insoluble portion isolated earlier. The two portions (670 mg plus 60 mg) were combined for flash-column chromatography. Two column runs followed, the first using CHCl₃-MeOH (4:1) and the second using CHCl₃-MeOH (7:1). The column fractions which appeared

essentially homogeneous in 4 according to TLC were combined and evaporated to give 370 mg (34%), but a sizeable portion of the desired product was left in unresolved fractions contaminated by unconverted 3. Spectral data for 4: mass, m/z 366, MH+ for C₂₀H₂₃N₅O₂. EXAMPLE 3

4-[1 -[(2,4-Diamino-5-methylpyrido[2,3-d]pyrimidin-6- yl)methyl]propyl]benzoic Acid

compound 5

A solution of 4 (190 mg, 0.521 mmol) in Me₂SO (17 ml) was treated with 1 N NaOH (0.75 ml). After 20 hours at 20-23°C, the solution was found by TLC (CHCl₃-MeOH, 4:1 ) examination to contain a small amount of unchanged 4. More 1 N NaOH (0.50 mL) was added to the solution, kept at 20-23°C, and 2 hours later TLC showed completed disappearance of 4. The Me₂SO was then removed by distillation in vacuo (less than 1 mm, bath to 45°C).

The residue was dissolved in H₂O (3 ml), and the resulting clear solution was carefully treated dropwise with 1 N HCI to produce pH 5 and cause precipitation of 5 as a pale-beige solid. The mixture was chilled in a refrigerator for several hours before solid was collected and dried; yield 165 mg (82%). Anal. Calcd. for C₁₉H₂₁ N₅O₂.2H₂O; C, 58.90; H, 6.5; N, 18.08. Found: C, 59.40; H, 6.14; N, 17.63. Spectral data: mass, m/z 352, MH+ for

C_{1 9}H₂₁ N₅O₂; UV λmax 234 nm (ε 39 300), 322 (7730) at pH 1 ; 235 nm (ε 37 800), 335 (6050) at pH 7; 236 nm (ε 37 600), 346 (6730) at pH 13. EXAMPLE 4

Diethyl N-[4-[1 -[(2,4-Diamon-5-methylpyrido[2,3-d]- [yrimidin-6-yl)methyl]propyl]benzoyl]-L-glutamate compound 6

A solution of 5 . 2H₂O (155 mg, 0.400 mmol) in Me₂SO (25 ml) was treated with N-methylmorpholine (124 mg of 98%, 1.20 mmol) and diethyl cyanophopsphonate (206 mg of 95 %, 1.20 mmol). The resulting solution was kept at 20-23°C for 62 hours.

Me₂SO was removed by distillation in vacuo (bath to 45°C), and the residue was treated with H₂O (5 mL). The resulting acidic solution was treated with 10 % NaHCO₃ solution to produce pH 9 and cause precipitation of 6 as a beige solid. After a

refrigeration period (2 hours), the solid was collected and dried. Crude 6 (192 mg) thus obtained was combined with a sample (60 mg) obtained from a smaller run for purification. Initially, the material was subjected to gravity flow chromatography on a short (4 cm x 10 cm ) silica gel column (70-230 mesh) eluted with CHCl₃-MeOH (4:1). A fast-moving UV-absorbing contaminant was essentially removed before the desired 6 began eluting.

Fractions nearly pure in 6 were combined and evaporated to give 160 mg of material. Further purification was necessary, however, and this was done on a preparative TLC plate developed with CHCl₃-MeOH (4:1) after application in MeOH solution. The band due to 6 was removed and extracted with MeOH. Evaporation of the filtered solution gave pure 6 (100 mg). Spectral data:

mass, m/z 537, MH⁺ for C₂₈H₃₆N₆O₅. EXAMPLE 5

N-[4-[1 -[2,4-Diamino-5-methylpyrido[2,3-d]pyrimidin-6

yl)methyl]propyl]benzoyl]-L-glutamic Acid 10-Ethyl-5-methyl-5,1 0-dideazaaminopterin

compound 7

The extract from the excise plate band described under 6 above (100 mg) was dissolved in MeOH (15 ml) containing 1 N

NaOH (0.5 mL), and the pale-yellow solution was kept at 20-23°C for 20 hours. MeOH was then removed under reduced pressure

(H₂O aspirator, bath at 20-25°C), and the residue was dissolved in H₂O (6 ml). This solution was kept for 24 hours at 20-23°C before it was filtered to insure clarity, the filtrate was then carefully treated dropwise with 1 N HCI to produce pH 3.8 (meter) and precipitate 7 as a light-beige solid. The mixture was kept in an ice-H₂O bath for 1.5 hours before the solid was collected with the aid of cold H₂O and dried. After removal from the drying chamber, the sample was allowed to equilibrate with ambient conditions where it underwent a weight increase from 67 mg initially to 72 mg finally. This increase corresponds with that calculated for the transition of anhydrous 7 (Mol. Wt. 480.5) to its dihydrate (Mol. Wt. 516.6). The yield from 6 was 75 %. Anal. Calcd. for C₂₄H₂₈N₆O₅ · 2H₂O; C, 55.80; H, 6.24; N, 16.27. Found: C, 55.88; 55.85; H, 5.95, 6.21 ; N, 6.32, 16.27. Spectral data:

mass, m/z 481 , MH⁺; UV λmax 233 nm (ε 40 600), 321 (7890) at pH 1 ; 235 nm (ε 40 600), 334 (6100) at pH 7; 237 nm (ε 40 600), 346 (6820) at pH 13.

- - -

EXAMPLE 6

6-Bromomethyl-2,4-pteridinediamine Hydrobromide compound 8

Br₂(147 g, 0.919 mol) was added dropwise during 0.5 h to a mechanically stirred solution of (C₆H₅)₃P (240 g, 0.915 mol) in

Me₂NAc (1.5 L) maintained at 10-15° C. 2,4-Diamino-6- pteridinemethanol 15 (53.4 g, 0.278 mol) was then added in one portion. The cooling bath was removed, and stirring was

continued for 2.5 h. Solution occurred during the first 0.5 h, and the temperature of the solution did not exceed 25°C. The dark- red reaction solution was then transferred from the 5-L three neck reaction flask to a 5-L round bottom flask and evaporated in vacuo (,1 mm, rotary evaporator, bath to 35°C). When most of the Me₂NAc had been removed, the dark residue was treated with C₆H₆ (1.5 L). The two-phase mixture was swirled for a few min, then left overnight. The upper phase was removed by decantation, and the thick dark gum was treated with another portion of C₆H₆ (0.7 L). The supernatant portion was again removed by

decantation, and the last of the C₆H₆ was removed by evaporation under reduced pressure (H₂O aspirator, rotary evaporator, bath to 30°C). The residue was then treated with hot (80°C) glacial AcOH (2L) with efficient agitation. Crystalline product began separating from the dark solution at nearly the time the gum had completely dissolved, but the mixture was kept at about 80°C and agitation was continued 5 min longer to insure that all the viscous gum had dissolved. The mixture was then allowed to cool while the crystalline product separated. After the mixture had been at about 20°C for 4 h, the product was collected, washed with a small amount of AcOH, then thoroughly with Et₂O, and dried in vacuo (20-25°C over P₂O₅ and NaOH pellets). The material obtained (solvated by AcOH and weighing 102 g) was dissolved (in a 12-L three necked flask with mechanical stirring) in boiling 2-PrOH(9L). The stirred mixture was treated while boiling with Norit, then filtered through a mat of Celite on a Buchner Funnel. The bromomethyl compound separated from the cooled filtrate as pale-yellow platelets. The mixture was kept overnight in a refrigerator at 3-5°C before the first crop was collected and washed sparingly on the funnel with cold 2-PrOH. The filtrate was set aside for isolation of a second crop. The first crop was washed copiously with Et₂O, then dried in vacuo (25-25°C over P₂O₅ and NaOH pellets); yield 51.9 g. The filtrate from this crop was evaporated under reduced pressure (H₂O aspirator, rotary evaporator, bath to 35°C) to a volume of about 2.5 L. This concentrated mixture was heated to boiling to again give a clear solution, which when allowed to cool, gave a second crop. The additional crop, collected and washed as before, amounted to 8.5 g. Thus the yield was 60.4 g. The ¹H NMR spectrum of this material in CF₃CO₂D showed the expected multiplet due to 2-PrOH at 51.3-1.5, singlets due to 6-methyI contaminant at δ 2.84(CH3) and δ 8.85(7-H), and singlets due to the 6-bromomethyl compound at δ 4.74 (CH₂Br) and 5 9.10 (7-H). Molar ratios of these components calculated on the basis of the integrated ¹ H NMR spectrum showed the molar ratio of 6- CH₂Br:6-CH₃:2-PrOH to be 1 :0.07:0.02. The run described is typical of several which gave very similar results. Elemental analysis results from other runs have confirmed the ¹ H NMR assays. In the use of this material, we treated it as being of 95% in 6-CH₂Br by weight and having formula weight 355 (calculated from the ratio given).

In another run, toluene was used in the workup in place of C₆H₆, and the first crop yield of product recrystallized from 2- PrOH was 47% of material of molar ratio 6-CH₂Br:6-CH₃:2-PrOH of 1 :0.05:0.56 (88% in 6-CH₂Br by weight); the second crop (11% yield) had a molar ratio (in some order as above) of 1 :0.03:0.26 (94% in 6-CH₂Br by weight) giving a yield of 58%, which is typical yield for the procedure.

EXAMPLE 7

Methyl 4-Propionylbenzoate.

A solution of 4-propionylbenzoic acid¹³ (3.56 g, 20.0 mmol), N,N-diisopropylethylamine (3.36g, 26.0 mmol), and methyl iodide (3.98 g, 28.0 mmol) in N,N-dimethylformamide (25 mL) was kept in a stopper flask at 20-25°C for 44 H. Evaporation in vacuo (finally at ,1 mm, bath up to 30°C) followed, and the residue was stirred with 3% Na₂CO₃ (30 ml) for 5 min. The suspended white solid was collected, washed with H₂O, and dried in vacuo (over P₂O₅ at 20-25°C) to give methyl 4-propionylbenzoate in 60% yield (2.28 g); mp 80-81 °C (reported⁴ mp 80-81 °C). Anal. Calcd for C₁₁ H₁₂O₃: C, 68.73; H, 6.30. Found: C, 68.69, 68.93; H, 6.52, 6.60. Homogeneous according to TLC (cyclohexane-EtOAc, 3:1 ). EXAMPLE 8

Methyl4-[2-(2,4-Diamino-6-pteridinyl)-1 -ethyletheneyl]benzoate compound 10

6-Bromomethyl-2,4-pteridinediamine hydrobromide (10.0 mmol) was added to a stirred solution of tributylphosphine (6.07 g, 30.0 mmol) in dry Me₂SO (150 ml). (The reaction mixture was stirred under N2 throughout the reaction period), the solution that formed was heated at 55°C (bath temperature) for 0.5 h, then allowed to cool to 25°C. Methyl 4-propionylbenzoate (1.92 g, 10.0 mmol) was added followed by NaH (0.80 g of 60% dispersion in oil, 20.0 mmol). The expected frothing due to H₂ evolution occurred while the mixture became red in color. After about 1 h, H₂ evolution had ended. The reaction mixture was left stirring at 20-25°C under N₂ in a stoppered flask. During this time the color changed to dark orange. After 21 h, examination by TLC (CHCI₃- MeOH, 3:1 ) revealed the expected major spot of R_f approximately 0.8 (Samples of the solution in Me₂SO were spotted on the analytical TLC plates which were then kept at , 1 mm in a vacuum dessicator over P₂O₅ for 45 min to remove Me₂SO before

developing.) The solution was left under N₂ at 20-25°C for another 24 h, but no further change was evident according to TLC. Most of he Me₂SO was then removed by distillation in vacuo (<1mm, bath to 40°C). The concentrated reaction mixture was stirred with Et₂O (300mL) to give Et₂O-insoluble orange solid which was collected with the aid of Et₂O. After being dried free of Et₂O, the solid was washed on the funnel several times with H₂O, then dried in vacuo (<1 mm, 20-25°C, over P₂O₅ and NaOH pellets). The dried solid, 2.82 g (80% yield), gave thin-layer chromatograms that indicated this intermediate to be of high purity. Although this material is a cis/trans mixture according to ¹ H NMR data from an earlier run, we could not detect the separate isomer by TLC as described. Further purification was not needed. The sample was successfully used in the

hydrogenation step. EXAMPLE 9

Methyl 4-[1 -[(2,4-Diamino-6-pteridinyl)methyl]propyl]benzoate compound 26

A solution of the sample of methyl 4-[2-(2,4-diamino-6- pteridinyl0-1-ethylethenyl]benzoate (4) described above (2.82 g, 8.0 mmol) in glacial AcOH (400 mL) containing PtO₂ (0.75g) was hydrogenated in a Parr shaker at 46 psi (3.2 kg/cm²) for 18 h. (Mass spectral examination showed the expected over- hydrogenated product of m/z 357, 5, MH⁺ for C₁₈H₂₄N₆O₂.) The catalyst was removed by filtration, and the filtrate was treated with 3% H₂O₂ (10mL). This solution was left stirring open to the air for 7.5 h before more 3% H₂O₂ (5mL) was added, then the solution was left stirring open to air for 16 h. The solution was then concentrated (H₂O aspirator, rotary evaporator, bath to 40°C) until most of the AcOH had been removed. The residual solution (about 25 mL) was diluted with H₂O (250 ml). The nearly clear solution that formed was stirred rapidly while it was treated with concentrated NH₄OH solution until the pH reached 7. Yellow solid precipitated, and the mixture was left in a

refrigerator for 4-5 h before the solid was collected, washed with H₂O, and dried in vacuo (<1 mm, 20-25°C, over P₂O₅ and

NaOH pellets); yield 2.06 g (73%), homogeneous according to TLC (CHCI₃-MeOH-concd NH₄OH, 4:1 :0.05; R_f approximately 0.7).

Spectral data: MS, m/z 353, MH⁺ for C₁₈H₂₀N₆O₂; 1H NMR

(Me₂SO-d₆) 50.76(t,CH₃CH₂), 1.70 (m, CH₂CH₃), 3.2 (C⁹H₂C¹⁰H), 3.80 (s, OCH₃), 6.52 (br s, NH₂), 7.36 (d, 3',5'-ArH), 7.50 (m,NH₂),

7.84 (d, 2',6'-ArH), 8.35 (s, C⁷-H). This spectral data supports the assigned structures and is in agreement with that reported by DeGraw and coworkers. EXAMPLE 10

4-[1 -1 [(2,4-Diamino-6-pteridinyl)methyl]propyl]benzoic Acid compound 27

A stirred suspension of 6 (1.30 g, 3.69 mmol) in Me₂SO (70 mL) was treated with NaOH (4.5 mL of 1 N). The resulting orange solution was kept at 20-25°C for 23 h before examination by TLC showed nearly complete disappearance of compound 26. More

NaOH (1.5 mL of 1N) was added. After 2 h, another treatment with

NaOH (0.75 mL of 1 N) followed. About 4 h later (29 h total), TLC showed disappearance of compound 26. The Me₂SO was removed by short-path distillation in vacuo (<1 mm, bath to 35°C. The residue was stirred with H₂O (45 mL), and the red-orange solution was treated with decolorizing carbon and filtered. The filtrate was acidified with 1 N HCL to pH 5.0. A yellow gel separated. The mixture was caused to freeze solid using a dry ice-acetone bath. Slow thawing in a refrigerator at about 3°C afforded 7 as a granular solid which was collected, washed with H₂O and dried in vacuo (<1 MM, 20-25°C over P₂O₅). The dried solid was then allowed to equilibrate with ambient conditions; yield 77% (1.04 g). Anal, calcd for C₁₇H₁₈N₆O₂.1.5H₂O: C, 55.88; H, 5.76; N, 23.00. Found C, 56.03, 56.10; H, 5.54, 5.54; N, 22.48, 22.53. Spectral data: UV, λmax (ε x 10^-3), 0.1 N HCL, 242 (29.5), 279(5.68), 340 (10.4); pH 7, 237 (22.2), 256 (26.2), 371 (7.32; 0.1 N NaOH, 236 (22.2), 256(26.8), 371 (7.49). EXAMPLE 11

4-(4'-Cyanophenyl)-4-hydroxycyclohexanone ethylene ketal compound 11

4-Bromobenzonitrile was treated in THF-hexane with n- butyllithium at -100°C. Cyclohexanedione monoethylene ketal was added at -100°C to give 70 % yield of 11.

EXAMPLE 12 4-(4'-Carbomethoxyphenyl)cyclohexanone

compound 14

Compound 11 was heated in 2-methoxyethanol · 2N NaOH at

100°C to yield 12 (94%). Esterfication and deprotection in refluxing MeOH containing HCI and a small amount of H₂O gave a quantitative recovery of 13. Hydrogenation of 13 in 1%

concentrated HCI/diaxane in the presence of palladium black at

40-48° afforded 14 in a 39 % yield.

EXAMPLE 13

2-Carbomethoxy-4(4'-carbomethoxyphenyl)cyclohexanone compound 15

Compound 14 was acylated with dimethylcarbonate/NaH/- .

KH in THF to give 15 (63%). EXAMPLE 14

Methyl-4-amino-4-deoxy-5,1 0-ethano-5,10-dideazapteroate compound 18 Compound 15 was reacted with 2,4,6-triaminopyrimidine in phenyl ether at 190-205° to give 16 in 82% yield. Compound 16 was treated with 1M borane - THF affording 17 (56% yield).

Reaction of 17 with DDQ in acetic acid gave 18 in 61% yield.

EXAMPLE 15

5,10-Ethano-5,10-dideazaaminopterin diethyl ester

compound 20

Compound 18 was hydroiyzed with NaOH in 2- methoxyethanol to give 19 (80 % yield). Compound 19 was coupled with diethyl-L-glutamate-HCL affording 10 in 44% yield.

Example 11

5,10-Ethano-5,10-dideazaaminopterin

compound 21

The diester 20 was saponified with 1 N NaOH in 2- methoxyethanol to give 21 in 73% yield.

The compounds according to this invention form

pharmaceutically acceptable salts with both organic and

inorganic acids. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicyclic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic, and the like. The salts are prepared by

contacting the free base form with an equivalent amount of the desired acid in the conventional manner. The free base may be regenerated by treating the salt form with a base. For example, dilute aqueous base solutions may be utilized. Dilute aqueous sodium hydroxide, potassium carbonate, ammonia, and sodium bicarbonate solutions are suitable for this purpose. The free base form differs from its respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but the salts are otherwise equivalent to the respective free base form for purposes of this invention.

The compounds of this invention also form pharmaceutically acceptable carboxylate salts by reacting a suitable base with one or more of the free carboxy groups. Suitable bases include alkali metal or alkaline earth metal hydroxides or carbonates, for example, sodium hydroxide, potassium hydroxide, calcium

hydroxide, magnesium hydroxide, and corresponding carbonates; and nitrogen bases such as ammonia and alkylamines such as trimethylamine and triethylamine.

The novel compounds of the present invention inhibit transplanted mouse tumor growth when administered in amounts ranging from about 5 mg to about 200 mg per kilogram of body weight per day. A preferred dosage for optimum results would be from about 5 mg to about 50 mg per kilogram of body weight per day, and such dosage units are employed that a total of from about 350 mg to about 3.5 grams of the active compounds for a subject of about 70 kg of body weight are administered in a 24 hour period. This dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be

proportionally reduced as indicated by the exigencies of the therapeutic situation. A decided practical advantage is that the active compounds may be administered in any convenient manner such as by the oral, intravenous, intramuscular or subcutaneous routes.

The active compounds may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafer and the like. Such compositions and preparation should contain at least 0.1 % of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 and about 60 % of the weight of the unit. The amount of active compound in such therapeutic useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 5 and about 200 milligrams of active compound. The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to material of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.

Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparations and

formulations.

The active compounds may also be administered

parenterally or intraperitoneally. Solutions of the active

compounds as free base or pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the condition of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent of dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the

maintenance of the required particle size in the case of

dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbϊc acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the

preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such a media and agents for pharmacetically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use is the therapeutic

compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the

limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having diseased condition in which bodily health is impaired as herein disclosed in detail.

The principle active ingredient is compounded for

convenient and effective administration in effective amounts with a suitable pharmaceutically-acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principle active compound in amounts ranging from about 0.1 to about 400 mg, with from about one to about 30 mg being preferred. Expressed in proportions, the active compound is generally present in from about 0.1 to about 400 mg/ml of carrier. In the case of compositions containing

supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

Thus, while we have illustrated and described the preferred embodiment of our invention, it is to be understood that this invention is capable of variation and modification, and we therefore do not wish to be limited to the precise terms set forth, but desire to avail ourselves of such changes and

alterations which may be made for adapting the invention to various usages and conditions. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims.

Having thus described our invention and the manner and a process of making and using it in such full, clear, concise and exact terms so as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same;

Claims

We Claim:

1. The compound methyl 4-[[2-(2,4-diamino-6-pteridinyl)- 1 -ethyl]ethenyl]benzoate.

2. A process for the preparation of 10-ethyl-10- deazaaminopterin, which comprised hydrogenating the compound methyl 4-[[2-(2,4-diamino-6-pteridinyl)-1 - ethyl]ethenyI]benzoate, saponifying the ester group from said hydrogenated compound, coupling said saponifying product with diethyl-1 -glutamate, and saponifying the ester groups from the resultant product to yield 10-ethyl-10-deazaaminopterin.

3. A compound of the formula:

wherein R and R' are lower alkyl moieties of 1 to 6 carbon atoms in length.