WO1998024789A1

WO1998024789A1 - PROCESS FOR MAKING η-METHYLENE-10-DEAZAAMINOPTERIN (MDAM)

Info

Publication number: WO1998024789A1
Application number: PCT/US1997/021743
Authority: WO
Inventors: Frederick H. Hausheer; Dasharatha Reddy
Original assignee: Bionumerik Pharmaceuticals, Inc.
Priority date: 1996-12-02
Filing date: 1997-12-02
Publication date: 1998-06-11
Also published as: AU7624498A

Abstract

A process to synthesize the dihydrofolate reductase inhibitor, 4-amino-4-deoxy-10-deazaaminopterin (MDAM). The process synthesizes the key intermediate, pteroic acid in 5 steps, and uses commonly available starting materials. The steps include the formation of a 6-substituted pteridine, followed by alkylation, hydrogenation, and hydrolysis steps to form pteroic acid from the starting material which is tetraamino pyrimidine, or a salt thereof.

Description

PROCESS FOR MAKING γ-METHYLENE - 10 -DEAZAAMINOPTERIN (MDAM)

FIELD OF THE INVENTION

This invention relates to a novel and improved

process for synthesizing the useful antitumor agent γ-

methylene-10-

deazaaminopterin (MDAM) .

BACKGROUND OF THE INVENTION

A. Brief History of MDAM

MDAM is a potent inhibitor of dihydrofolate

reductase (DHFR) . The therapeutic value of MDAM as an

antitumor agent has been well documented in recent

literature, as have its increased specificity and reduced

toxicity, particularly when compared to analogs, such as

methotrexate (MTX) . MDAM is currently undergoing Phase I

clinical trials at Johns Hopkins University as an

antitumor compound.

MDAM and certain derivatives thereof, are the

subject of United States Patents 4,996,207 and 5,073,554,

and is also disclosed and claimed in several overseas

patents in Europe, Canada, Mexico and Japan. MDAM has

the following structure I:

SUBSTITUTE SHEET (RULE 28) (I)

The general formula for known active derivatives of

MDAM is seen below as Formula II:

(ID

wherein R_x is hydrogen or lower alkyl;

R₂ is hydrogen or hydroxy;

R₃ is hydrogen or methylene; and

the dashed line indicates a single bond or a

double bond.

MDAM is similar in structure to the known antitumor

and anti-inflammatory drug methotrexate seen below as

Formula III:

(III)

SUBSTITUTE SHEET (RULE 28) MDAM specifically inhibits dihydrofolate reductase

to a far greater extent than MTX. Further, MDAM does not

undergo the polyglutamylation common to MTX and

derivatives, thereby reducing the toxicity of MDAM far

below that of MTX. The greater efficacy and reduced

toxicity of MDAM (compared to MTX and derivatives

thereof) has been well established for many years now.

B. Current Synthetic Procedures For Making MDAM

The current process for making MDAM involves a

complex 11 step procedure to make the key intermediate,

plus an additional three steps to produce the key

intermediate diethylγ-methylene glutamate) , and a final

coupling step, followed by the hydrolysis of the ester to

MDAM. The prior processes to make the key intermediate,

and/or MDAM or derivatives are outlined in the above

mentioned U.S. Patents, and in several publications known

in the literature, the most notable being Nair, J^". Org .

Chem . , 50:1879, 1985, which publication recites the

process shown below as Scheme I .

Further, the previous process for preparing MDAM

required the nitration of 2 , 4-diamino-6-chloro-pyrimidine

(a necessary reagent), which was a difficult, time-

SUBSTITUTE SHEET (RULE 2β) consuming, step in the process. A schematic depiction of

the prior process is shown below as Scheme 1, and is

intended to illustrate the complexity and difficulty

previously associated with synthesizing MDAM.

Scheme 1

CHβCHJP'iPtή BT

H_jCH_j ^■ COjMe

N

II

HjN- CH_jCCH_jCH_j- -o NO,

COgMe CCHfeCH_j C0₂Me

UN

SUBSTITUTE SHEET (RULE 2 As shown in Scheme 1, the prior art process for

synthesizing MDAM requires a number of awkward and time-

consuming maneuvers to produce the desired product. The

most awkward and time-consuming step is the required

nitration of 2 , 4-diamino-6-chloro-pyrimidine, which is

necessary for the eighth step in the process, the

addition of the pyrimidine ring with the 5- and 6-

positions having attached nitrogen groups to enable its

later conversion to the required pteridine fused ring

group .

The prior art processes to synthesize MDAM were

further limited in their adaptability to bulk

manufacturing. Cost effectiveness, yield and purity are

significant considerations in the bulk manufacture of any

pharmaceutical product, and the prior processes employed

to synthesize MDAM were neither cost effective nor did

they generate acceptable yields of product .

SUMMARY OF THE INVENTION

The process described by this invention delivers a

highly efficient, convergent process for the synthesis of

MDAM and useful derivatives thereof. The inventive

SUBSTITUTE SHEET (RULE 28» process reduces the number of steps to produce the

pteroic acid intermediate from 11 to 5, and further,

significantly increases the yield and purity of the end

product when compared with prior processes employed to

synthesize these useful compounds.

As stated above, the inventive process involves a

five step process to produce the pteroic acid

intermediate, which is then coupled to an ester of γ-

methylene glutamic acid, and finally hydrolyzed to form

the desired end product in a highly pure form.

The process begins with the conversion of 2,4,5,6-

tetraaminopyrimidine to the corresponding pteridine

intermediate. After bromination and alkylation of this

6-hydroxymethyl pteridine intermediate to form a 9,10-

dehydro pteroic acid ester, reduction of the 9,10-double

bond produces pteroic acid ester, and then hydrolyzing

the ester to form pteroic acid (the key intermediate) ,

coupling with γ-methylene glutamic acid ester and

hydrolyzing the resulting compound (the diester of MDAM)

yields MDAM in very high yield.

The schemes and specific examples for carrying out

the inventive process are illustrated in the detailed

description set forth below.

SUBSTITUTE SHEET (ROLE 28i It is an object of this invention to provide for' a

highly efficient and convergent process for synthesizing

MDAM and useful derivatives thereof.

Another object is to provide for a process for

synthesizing MDAM and derivatives, which process is

readily adaptable to GMP conditions.

Another object is to provide for a process for

synthesizing MDAM which is time efficient, cost

effective, and produces MDAM in sufficiently high yield.

Other objects will become apparent from a reading of

the following detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred embodiments herein described are not

intended to be exhaustive or to limit the invention to

the precise details disclosed. They have been chosen and

described to explain the principles of the invention, and

the application and practical use thereof, so that those

skilled in the art may understand its teachings.

The process of this invention is designed to

produce, in substantially pure form, the antitumor agent

γ-methylene-10-deazaaminopterin (MDAM) . The general

UBSTITUTE SHEET (RULE 26) process described by this invention is shown below as

Scheme II

As shown in Scheme II, the starting material is

2 , 4, 5, 6-tetraamino pyrimidine (a) . The starting material

is preferably obtained as a salt of the actual compound.

Most preferred is the sulfate salt, which compound is

available commercially from Aldrich Chemical Company and

others .

This initial step in the process is carried out

according to published conditions in Baugh, et al , The

Journal of Organic Chemistry, Volume 29: p 3610 (1969) .

SUBSTITUTE SHEET (flic 26) The intermediate 6-hydroxymethyl pteridine (b) is the

resultant compound, which is formed by a closure of the

fused ^λB' ring of the pteridine molecule by condensation

of 2 , 4 , 5 , 6-tetraamino pyrimidine and dihydroxy acetone.

The preferred reagents are those which support these

conditions and the ring formation, preferably ketols, and

in the most preferred case, 1 , 3-dihydroxy-2 -propanone .

The 6-hydroxymethyl pteridine (b) is then brominated

to facilitate the alkylation step of the process. The

bromination is achieved by reacting the 6-hydroxymethyl

intermediate (b) with an excess of a brominating reagent

which replaces the terminal hydroxy moiety with a bromine

atom. Preferred reagents for the brominating step are

identified in Piper, et al , The Journal of Organic

Chemistry, Volume 42:208 (1977). The most preferred

reagent is dibromotriphenylphosphine, and subsequent work

up and recrystallization from acetic acid and isopropanol

yields the hydrogen bromide isopropanol salt of 6-

bromomethyl pteridine (c) in high yield.

This intermediate salt (c) is then reacted with

carbonyl compounds under Wittig reaction conditions to

form the 9,10-dehydro pteroate ester intermediate (d) .

The most preferred intermediate (d) is shown as the methyl ester of 9,10-dehydro pteroic acid. Preferred

reagents used in the reaction are triphenylphosphine and

methyl -4 -formyl benzoate, in a strong base such as sodium

methoxide, to form the intermediate (d) .

The ester (d) is then reduced to saturate the C9-C10

bond. The preferred process involves reacting the ester

intermediate (d) with palladium/carbon palladium on

alumina in an organic solvent to produce intermediate

methyl pteroate ester (e) .

The methyl pteroate ester (e) is then converted to

the key pteroic acid intermediate (f) , 4-amino-4-deoxy-

10-deaza pteroic acid. Preferably the hydrolysis is

conducted in a strong base, most preferably in aqueous

sodium hydroxide solution along with an organic solvent

2-methoxyethanol .

The pteroic acid intermediate (f) is then activated,

and coupled (as the mixed anhydride) to an ester of γ-

methylene glutamic acid to form the esterified form of

MDAM, and then hydrolyzed to the active compound (g) of

Formula I . Scheme I la

e1

In an alternative alkylation of intermediate (c) ,

the bromomethyl pteridine salt (c) is pH neutralized,

then reacted with a diester of homoterephthallic acid (4-

carboxybenzoic acid) to form the esterified form of the

10-carboxy intermediate (el) . Intermediate (el) is then

decarboxylated and hydrolyzed the other ester group with

a strong nucleophile to form the key pteroic acid

intermediate (f) . Preferred nucleophilic agents include

sodium cyanide, most preferably dissolved in

dimethylsulfoxide (DMSO) .

The following specific examples illustrate the best

mode for carrying out the inventive process. The

examples are disclosed for illustrative purposes only,

and are not to be considered as limiting the invention to

the precise details set forth. All NMR data are expressed in parts per million

(ppm) , and the resonances expressed as follows: s,

singlet; d, doublet; t, triplet; br.s., broad singlet;

sep, septet; c, complex set of signals, the center of

which is provided.

Example 1

2.4-diamino-6-hydroxymethyl pteridine

Tetraaminopyrimidine sulfate (10 mmoles) was taken

up in 40 mL of water and barium chloride (10 mmoles) in

10 mL of water was added. The mixture was placed in a

boiling water bath for 10 min. After cooling, the barium

chloride was removed by filtration and the solid was

washed on the filter with about 10 mL of water. The

combined washings and filtrate were made up to 50 mL with

water. The solution was added to a solution of 150 mL of

4 M sodium acetate containing dihydroxy acetone (30

mmoles) and cysteine hydrochloride monohydrate (10 mmoles

in a 1 L Erlenmeyer flask and was placed on a rotary

shaker at room temperature for 24 hours. After this

period, the flask was placed in the cold for several

12

SUBSTITUTE SHEET (RULE 26} hours, the precipitate was then collected by suction

filtration and washed with cold water. The precipitate

was resuspended in 100 mL of water and heated to boiling;

if necessary drops of IN sodium hydroxide solution were

added to effect the solution. Norit (0.5 g) was added and

the hot solution thoroughly mixed, filtered hot through

heated funnel. After cooling to room temperature, the pH

was adjusted to 6.0 with 1 N HC1 and the flask was placed

in the cold for several hours . The precipitate was

collected by filtration, washed with cold water, ethanol,

(50:50) ethanol-ether, and finally with ether, then dried

in vacuo. The yield after an additional recrystallization

from water is 55%. The compound exhibited following

proton NMR signals in DMSO-d6; (ppm) 8.8 (s, 2H,

aromatic), 7.6 and 6.6 (broad singlets, 4H, Amino), 5.5

(broad singlet, 1H, hydroxy) and 4.65 (s, 2H, benzylic) .

Example 2

2.4-diamino-6-bromomethyl pteridine

Solid hydroxymethyl pteridine (110 mmoles) was added

to a mixture of Ph₃PBr₂ (363 mmoles) and dimethylacetamide

13

UBSTITOTE SHEET (RULE 2d (360 mL) in a 2L three necked flask. The mixture was

stirred at 20-25° C for 3.5 hours. The solution that

formed was treated with drop wise during 15 minutes with

7.2 mL of ethanol and stirred for 15 minutes longer

before benzene (1.17 L) was added. A dark oil

precipitated, and the mixture was stirred for 30 minutes

longer and left to stand overnight . The clear

supernatant liquid was decanted from the semi solid

precipitate, which was dissolved with stirring in hot

glacial acetic acid (600 mL, pre heated to 100°C) . The

solution was filtered while hot, and the beige,

crystalline material that separated from the cooled

filtrate was collected after 4 hours at 20-25°C. The ether

washed solid (solvated by acetic acid) was recrystallized

from isopropanol to give yellow orange platelets, which

were washed with ether and dried to yield 50% of the

theoretical yield. The compound exhibited following

proton NMR signals in DMSO-d6; (ppm) 9.3 and 9.2 (broad

singlet, 4H, amino groups), 9.0 (s, 1H, aromatic), 4.9

(s, 2H, benzylic) , 3.8 (septet, 1H, CH of isopropanol),

and 1.0 (d, 6H, methyls of isopropanol) . Example 3

Methyl-9.10-dehydro-4-amino-4-deoxy pteroate

100 grams of the intermediate from Example 2 above

was stirred in a solution of 3.0 liters of N,N-

dimethylacetamide and one molar equivalent of

triphenylphosphine for 4 hours at 80° C. The solution was

allowed to cool to room temperature, after which two

molar equivalents of sodium methoxide and one molar

equivalent of methyl -4 -formyl benzoate were added, and

stirred for 18 more hours. The solution was then diluted

with 12.0 liters of distilled water and the precipitate

was filtered under suction and then washed successively

with 200 L of toluene and diethyl ether. The solid was

then dried under a vacuum, to yield 62.0 grams of the

title compound (75% yield) .

Η NMR: 8.85δ (s,lH); 8.0δ-7.65δ (d,4H); 7.85δ-7.5δ

(d,2H) ; 3.8δ (s,3H) .

15

SUBSTITUTE SHEET (RULE 26} Example 4

Methyl -4 -amino-4-deoxy pteroate

40 grams of the intermediate from Example 3 above

was dissolved in 10.0 liters of glacial acetic acid, and

20.0 grams of 10% palladium on carbon catalyst added and

hydrogenated at ambient temperature and pressure. The

reaction was stopped after 24 hours and the palladium

filtered out of the solution. The filtrate was then

heated to 45° C and purged with air for four hours and

evaporated the solvent or in alternate method the

filtrate was diluted with 3.0 liters of 3% hydrogen

peroxide and stirred four more hours at room temperature.

Evaporation of the solvent at reduced pressure yielded

32.0 grams of the title compound representing a yield of

80%.

Η NMR: 8.5δ (s,lH); 7.82δ-7.4δ (d,4H); 7.45δ-6.5δ

(br.s 4H) ; 3.8δ (s,3H); 3. lδ (s,4H). Example 5

4-Amino-4-deoxy pteroic Acid

20.0 grams of the intermediate from Example 4,

above, was dissolved and stirred in 500 mL of 0.5 N

sodium hydroxide and 500 mL of 2-methoxyethanol at room

temperature for 24 hours. The solution was concentrated

to 100 mL, filtered, and the filtrate acidified with

glacial acetic acid to pH 4.5. A copious yellow

precipitate formed and was refrigerated overnight, then

filtered, washed with distilled water and dried. The

crude product was determined to be 90% pure by HPLC, and

resulted in the recovery of 17.0 grams of the title

compound (90% yield) .

Η NMR: 8.45δ (s,lH); 7.8δ-7.3δ (d,4H); 7.5δ-6.7δ

(br.s, 4H) ; 3.1δ (s,4H) .

MS (FAB) : 310 Example 6

Methyl -4 -amino-4 -deoxy- 10 -methoxycarbonyl -pteroate

3.0 grams of dimethyl homoterephthallate is slowly

added to a stirred suspension of 1.64 grams of 35%

potassium hydride in mineral oil and 12 mL of dry N,N-

dimethyl formamide which has been cooled to 0° C. After

30 minutes the yellow solution was cooled to -40° C, and

1.42 grams of 6-bromomethyl pteridine hydrogen bromide

(0.33 molar equivalents) in 5 mL of dry DMF was added

over a period of 10 minutes. Alternatively, one molar

equivalent of 6-bromomethyl pteridine may be used. The

mixture was brought to room temperature and stirred for

two more hours. The solvent was then removed by vacuum,

and the residue was extracted with chloroform and then

dried over anhydrous magnesium sulfate, then concentrated

in vacuo to yield 2.0 grams (-60% yield) of the title

compound .

Η NMR: 8.8δ (s,lH); 7.9δ-7.3δ (d,4H); 7.6δ-6.6δ

(br.s,4H); 4.62δ (t,lH); 3.5δ-3.55δ (s,6H); 3.3δ-3.5δ

(m,2H) . Example 7

4-amino-4-deoxy-pteroic acid

0.55 grams (1.44 mmoles) of the intermediate from

Example 7 above, and 0.21 grams (4.32 mmoles) of sodium

cyanide were dissolved in 10 mL of dimethylsulfoxide and

stirred for 3 hours at 175° C-180° C for 3 hours. The

dark mixture was cooled, and the solvent removed by

vacuum. The residue was dissolved in 15 mL of distilled

water, filtered, and the filtrate acidified with glacial

acetic acid. The precipitate was collected and washed

with water to obtain 0.41 grams of crude pteroic acid in

quantitative yield.

Η NMR: 8.45δ (s,lH); 7.8δ-7.3δ (d,4H); 7.5δ-6.7δ

(br.s,4H) ; 3.lδ (s,4H) .

MS (FAB) : 310

The pteroic acid intermediate is coupled with γ-

methylene glutamic acid (or an ester thereof) to form

MDAM. The coupling process is carried out as described

in any of the above referenced patents or publications. Further synthesis of Formula II compounds may be

carried out as disclosed in PCT Publication WO 91/10666,

published July 25, 1991.

The above description does not limit the invention

to the details given above, but may be modified within

the scope of the following claims.

Claims

What Is Claimed Is:

1. A process for synthesizing a compound of the

formula :

ⁱ (ID

wherein R_x is hydrogen or lower alkyl;

R₂ is hydrogen or hydroxy;

R₃ is hydrogen or methylene; and

the dashed line indicates a single bond or a

double bond;

said process comprising the steps of :

a) providing a starting reagent 2 , 4 , 5 , 6-tetraamino

pyrimidine, or a salt thereof;

b) reacting the starting reagent with 1, 3-dihydroxy-

2-propanone to form the intermediate compound, 6-

hydroxymethyl pteridine;

c) reacting the intermediate compound with a

brominating agent to form 6-bromomethyl pteridine; d) alkylating the 6-bromomethyl pteridine to form

an esterified intermediate of the formula:

(IV)

wherein R₄ is hydrogen or lower alkyl;

e) hydrolyzing the formula IV compound to form the

acid homologue thereof;

f) reacting the acid homologue of the formula IV

compound with a γ-methylene glutamate ester to form an

esterified form of the formula II compound; and

g) hydrolyzing the esterified compound of step f) ) to

form the formula II compound.

The process of Claim 1 wherein step d) includes

alkylating the 6-bromomethyl pteridine with a

homoterephthallic acid ester, then decarboxylation at the

10 position and hydrolysis of the resulting pteroate

ester to form the formula IV compound.

3. The process of Claim 1 wherein step d) includes

alkylating the 6-bromomethyl pteridine with a 4-acyl

benzoic acid ester, then reducing the resulting pteroate

ester under catalytic conditions followed by reoxidation

to the formula IV compound.

4. The process of Claim 1 wherein the starting

reagent is present as the sulfate salt thereof.

5. The process of Claim 1 wherein step c) includes

brominating the hydroxymethyl intermediate with an excess

of dibromotriphenylphosphine in an organic solvent to

form 6-bromomethyl pteridine hydrogen bromide salt.

6. The process of Claim 5 wherein step c) further

includes recrystallizing the hydrogen bromide salt in an

alcohol and acid mixture to form the hydrogen bromide

isopropanol salt of 6-bromomethyl pteridine.

7. A process for producing γ-methylene-10-

deazaaminopterin comprising the steps of:

a) providing as a starting reagent 2,4,5,6-

tetraamino pyrimidine, or a salt thereof; b) reacting the starting reagent with 1, 3 -dihydroxy-

2-propanone to form the intermediate compound, 6-

hydroxymethyl pteridine;

c) reacting the intermediate compound with a

brominating agent to form 6-bromomethyl pteridine;

d) alkylating the 6-bromomethyl pteridine to form

an esterified intermediate of the formula:

(IV)

wherein R₄ is hydrogen or lower alkyl;

e) hydrolyzing the formula IV compound to form

the acid analog thereof;

f) reacting the acid homologue of the formula IV

compound with a diethyl γ-methylene glutamate to form

an esterified form of γ-methylene-10-

deazaaminopterin; and

g) hydrolyzing the esterified compound of step

f)to form γ-methylene-10-deazaaminopterin.