FIELD OF THE INVENTION The invention is a method of synthesizing a tetra- hydropteroyl-poly-L-glutamic acid derivative starting froπ a tetrahydropteroyl-mono-L-glutamic acid derivative.

BACKGROUND OF THE INVENTION Folinic acid (also known as Leucovorin or Citrovorum factor) is an N-substituted derivative of L- glutamic acid bearing the chemical designation, N-[4 [ (2- amino-5-for y1-1 , 4 , 5 , 6 , 7 , 8-hexahydro-4-oxc-6-pteri- dinyl)methyl]amino]benzoylj-L-glutamic acid or 5-fcrmy tetrahydrofolic acid. Folinic acid has two chiral cen¬ ters; one at the α-position of its glutar.ic aci group, and, the other at the C-6 carbon atom of its tetrar.ydrop- teroyl ring. Folinic acid differs in configuration at only one of these chiral centers because the glutar.ic acid group is derived exclusively from the L-forir of thiε subεtituent. When prepared chemically using a reducing agent such as diethyla ine borane [Forsch, R.A. , Rosowsky, A., J. Orcr. Che . 50, 2582-2583 (1985)], folinic acid consists of a 1:1 mixture of 6R and 6S forms. However, when isolated from biological sources, fclinic acid occurs only in the (6S)-form and, generally, contains 1 tc 15 additional 7-linked L-glutamic acid residues.

A number of reports [Kal an, T.I., Cher. Biol . Pteridines, Int. Symp. Pteridines Folic Acid Deriv. : Chem., Biol. Clin. Aspects, 8th, 1986, 763-766, Edited by Cooper, B.A. , Whitehead, V.M., de Gruyter, Berlin, Fed. Rep. Ger.; Schirch, V. et al., ibid, 887-898; Mackenzie, R.E., Baught, C ., Adv. Exp. Med. Bicl. , 163: Poiyl and Anti-Folyl Polyglutamates, 1983, 19-34, Edited by Golαman, I.D., Chabner, B.A. , Bertino, J.R., Fienur Press, New York, U.S.A.; Baggott, J.E.. Krumdieck. C.I... Cher . Bid . Pteridines: Dev. Bioche . , 4 _. , 6th, 1978 (Put. 1979, , 34 ^" - 351, Edited by Kiεliuk, R.L., Brown, G.M., Elsevie / ortn- Holland, New York, U.S.A. : indicate that tetrahydropteroylpoly-L-glutamic acid derivatives nave σreatlv ennanced suostrate and/or nr.uc tcr activity wr.er

assayed against several folic acid enzymes compared vitr. the mono-L-glutamic acid forms. The availability of these derivatives has been limited by the difficulty encountered in producing large quantities of the (6S)-form of tetra- hydrofolic acid [Reeε, L. et al., Tetrahedron, 42 , 117-136 (1986)]. Until now, no direct purely chemical method existed for converting tetrahydropteroylmono-L-glutamic acid derivatives to their poly-L-glutamic acid forms.

SUMMARY OF THE INVENTION An objective of this invention was to design a simple procedure for the production of pure (6R) and SSI- tetrahydropteroylpoly-L-glutamic acid derivatives or mixtures thereof starting from the :'6R) and (6S,--tetra- hydropteroylmono-L-glutamic acid derivatives. A εeccnd objective of this invention was the chemical intermediates produced by and used in the procedure.

DETAILED DESCRIPTION OF THE INVENTION The compounds that can be made by the method of the invention have the formulas (IA) and (IB)

OlNli-

1/1

:_,κ wherein x is 0 to 14 ,

R, and R _: are independently H, C. - C. alkyl, C. - c aiky_- carbonyl, C, - C ₆ ^• alkoxycarbonyl, C - C , arylcarbonyl , C« - C, ₀ aryloxycarbonyl, or -CHO, each of which (except the -CHO) may be substituted by halogen, C. - C alkoxy c: phenyl; e.g. acetyl, t-butyl, 2, 2-dichloroacetyl, cer.zov t-butoxy caroonyi, benzyloxy carbonyi; cr F. ar.c together form a one caroor. nrid e πe weer tnε r. trcc

atoms at positions 5 and 10. The chiral caroon atom at position 6 can take the R or the s configuration or mixtures of the R and S configurations (especially equal molar mixtures) . The structure shown in Formula (IA) shows the poly-L-glutamic acid moiety being completely 7-linked, i.e., each glutamic acid moiety is linked to the next one through its 7-carboxylic acid moiety rather than the α-acid. However, the method of the invention includes the synthesis of tetrahydropteroylpoly-L-glutamic acid derivatives wherein the poly-L-glutamic acid moiety also includes some α-linked glutamic acid moieties. The α-linked glu atic acid moieties could be anywhere in the poly-L-glutamic acid except the a ino end. Sucπ ccr.pounds exist in nature (Ferone, et al., J. B o . Cner... _____ _ 16356-16362, 1986; and Ferone, et al. , J. Biol. Chem.. 261, 16363-16371, 1986, both of which are hereby incorpo¬ rated by reference) . Most particularly the tetranydro- pteroylpoly-L-glutamic acid derivatives α-linked glutamic acids have the structure of formula (IB)

wherein y is 2 to 15, more particularly 2 to 6 ana most preferably 4? R, and R are defined the same as for the compound of formula (IA) . The more pre erred compounds cf formulae (IA) and (IB) have the (6S) configuration.

Three reports guided the development c: tr.is invention: i trie observation that ir. tπe resεr.:-: _: ::.-:

equivalent of base, the α-carboxyl group of an N-subεti- tuted L-glutamic acid derivative ionizes approximately 70 to 100 times more than the -y-carboxyl group; hence, reaction with an alkyl or aryl halide should result in the preferential formation of an α-monoester of folinic acid [Nef ens, G.H.L. , Peptides, Proc. European Symp. , 5th, 1962 (Pub. 1963), 39-40, Oxford; Nefkens, G.H.L. , Nivard, R.J.F., Rec. Trav. Chem. , 83 , 199-207 (1964), which are both incorporated by reference] ; ii) the ease with which α, 7~diesterε of folinic acid are formed with alkyl/aryl halides, and excess base in DMSO [Roεowεky, A., Y_, C-S., Chem. Biol. Pteridine: Dev. Bioche . , __, 6th, lS ε (Pub. 1979), 273-277, Edited by Kiεliuk, R.L., Brown, G.K., Elsevier/North-Holland, New York, U.S.A., which Lε hereby incorporated by reference in its entirety]; iii) the development of a large scale fractional crystallization method for the reeolution of the (6R) and (6S) -forms of folinic acid, calcium εalt [Mueller, H.R., et al. , Int. Pat. PCT Int. Appl. WO 88/08,844 (Cl. C07D475/04) , 15 Ma 1987, which is incorporated by reference in its entirety].

It has been found in thiε invention that tetra- hydropteroylpoly-L-glutamic -acid derivativeε can be prepared following conversion to their α-monoester form and subsequent 7-coupling with mono- or poly-L-giutamyl εubεtituents. The invention provides a process for the preparation of pure (6R) or (6S)-tetrahydropteroylpoly-L- glutamic acid derivatives or mixtures thereof. The invention comprises the steps of: 1) attachment of an alkyl, aryl or trialkylεilyl subεtituent preferentially on the α-carboxyl group of a tetrahydropteroylmono-L-glutamic acid derivative to produce an α-monoeεteri ied protected derivative; 2) direct coupling of the tetrahydrcpteroyi α-monoester with either mono- or poly-L-glutamic acid fin suitably protected form) or εalt or eeter to the 7-car- boxyl group using standard methods of making peptide condε [see The Peptides: Major Method of Peptide Bond Forma¬ tion, 1979, 1 , 1-495, Edited by Gross, E., Meienhcfer.. J. , Academic Preεε, New York, U.S.A. , vn cπ ι_ nerεr

incorporated by reference]; and, 2 ) deprotection to give the desired tetrahydropteroylpoly-L-glutamic acid derivative or εalt thereof. The advantage of the process over the prior art is that it is a wholly chemical cro- cess, with no enzymatic step. The prior art method entails (see D'Ari et al., Methods of Enzymology, Vol. 113 , pages 169-182, 1985, which is incorporated by refer¬ ence) using a poly-L-glutamate derivative which must undergo enzyme-mediated reduction in order to form the desired (6S)-tetrahydropteroylpoly-L-glutamate. At this stage of the art this requires: (i) purification cf the enzyme, dihydrofolate reductase, from a biological source; fii, a reducing cofactor, nicotmar.ide adenme dmuclec- tide phosphate reduced form (NADPH , to act as a hydrogen donor; and, (iii) in the case of a large scale (gra s) preparations, an enzymatic method (e.g., iεocitrate dehydrogenase, glucose-6-phosphate dehydrogenase _/ creatme kinase etc.) for recycling the oxidized cofactor, mcotin- a ide adenine dinucleotide phosphate, (NADP-, see Whiteside, G.M., Wong, C.H. , Aldrichi ica acta, 16, 27-34, 1983; Whitesides, G.M. Wong, C-H Angew. Chemie, (Int. Ed.) 24, 617-718, 1985). The use of enzymes and cofactors is expensive and limits the quantity cf product that can be made. The εtepε m the εynthesiε are now written cut ;r. more detail and in structural terms:

STEP 1: Synthesis of the α-Monoeεterifiec Inter¬ mediate

A compound of the formula (II)

wherein P.. and R are the same as in formula ;'I, acove, is ester fiec at the α-carboxyl position tc fcrr t.ne α-mcnoesteπfiεα intermediate ccr.pc nα cr _crr.'__- III

R ₃ is _. any aliphatic or aromatic moiety which protects the α-carboxylic acid group in STEP 2, and which iε easily removed at the end of the reaction under conditions which do not adversely effect the rest of the molecule. More specifically, R ₃ can be 'C, - C alkyl, subεtituted C _: - C. alkyl, C ₅ - C ₆ cycloalkyl, subεtituted C, - C,, cycloalkyl , C ₆ - C _1C) aryl (phenyl and naphthyl) , subεtituted C, - C, aryl, C ₆ - C _κ , aryl C, - C _k alkyl, eubstituted C - C.. aryl C. - C alkyl, diphenylmethyl, substituted diphenyimetr.y. and tri- C _j - C, alkylεilyl. ^■ The substituentε can be any organic or inorganic moiety which do not adversely effect the R, group's ability to function as an easily removable protecting group. Examples of the εubstituents, which can number up to three, are methyl, ethyl, C. - C, alkoxy, C, - C ₃ thiol and halogen. By halogen, it is meant fluorine, chlorine, bromine or iodine, preferably chlorine or bromine. By alkyl, it iε meant a etraight chain saturated hydrocarbon moiety. Preferably R _? is C, - C _ft alkyl, substituted C - C. alkyl, C, - C„ cycloalkyl, phenyl, substituted pnenyl, benzyl, substituted benzyl, diphenylmethyl and triεthyi- εilyl.

Most preferably R ₃ iε methyl, ethyl, propyl, butyl, sec-butyl, t-butyl, pentyl, hexyl, cyclopentyl, cyclohexyl, methoxymethyl, ethoxymethyl, ethyl-thio- ethyl, 2, 2, 2-trichloroethyl, 2-bromoethyi, phenyl,. benzyl, 2-chlorobenzyl, 2 , 6-dichlorobenzyl, diphenylmethyl and triethylsilyl. The esterification reaction can be carried cut by any means know" in the art, (see, for example Nefkens et al. , Rec. Trav. Chem.. 83, 199-207, 1964, which iε nerecy incorporated by reference) . Most preferably the reaction is carried out bv reacting a compound cf the form i≥ ?. -: ^■ .

wherein R, iε the same as deεcribed above, and X ε halo¬ gen, preferably bromine, with the compound cf formula (II) . The reaction iε carried out in the presence cf base in a polar aprotic solvent. Suitable bases are carbon- ates, bicarbonates, hydroxides, hydrides, amides and amines (both aliphatic and aromatic) . The base should be strong enough to quantitatively remove the α-carboxyl hydrogen. Examples of suitable bases include NaHCO,, Na,C0 ₃ , NaH, NaNH ₂ , KHC0 ₃ , K _: C0 ₃ , LiHCO,, Li,CO _? , NaOH, KOH, LiOH, triethylamine, tetrabutylammonium hydroxide and the like. The molar ratio of base tc the compound of formula (II) should be in the ratio of 1.5 tc G.5, preferably about 1.0. Suitable polar aprctic solvents arc DMA (dimethylacetamide) , DMF (dimethyifcrmamide , DMSC fd - methylsulfoxide) and HMPT (hexamethylphosphoramidej . The molar ratio of R ₃ -X to compound (II) ie in the range of 1.5 to 0.5 and preferably iε about 1. The temperature range for the reaction with R _? -X iε from about -50°C to about +150° C, preferably about -t-25°C. The reaction is preferably carried out at atmospheric presεure. The esterification reaction results in mostly the desired α- monoesterified product, however, a small (about 10%) of the product formed iε the 7-monoeεterified product. The α-monoesterified product iε purified ioy high pressure liquid chromatography, column chromatography or fractional crystallization.

STEP 2: Coupling Reaction

The compound of formula (III) produced in Step 1 is coupled to compound (IVA) or (IVB) :

--. 'i.

wherein x is 0 to 1 , and R ₄ is a carboxyl protecting group which can be chosen from the same moietiee aε R ₃ above. Preferably R ₄ is methyl, ethyl, propyl, butyl, sec-butyl, t-butyl, pentyl or hexyl; and most preferably

R _< is ethyl or t-butyl.

wherein y is 1 to 15, preferably 1 to 6 and most preferably 3; and R ₄ is the same aε defined above. The coupling reaction produces compounds of formulae (VA) or (VB) :

C > .

wherein R,, R ₂ , R ₃ , R ₄ , x and y are defined as above. Tne coupling reaction can be any of the standard methodε known in the art of peptide synthesis, see Groεε et al., εupra, particularly pageε 106-309. Examples include the activa- tion of the carboxyl group with reagents such as DCC (N', N'-dicyclohexylcarbodimide) , EEDQ (N-ethoxycarbonyl- idiimidazole, diphenylchlorophosphate and the like. Co poundε of formulae (IVA) and (IVB; are commercially available; also, the D 'Ari et al., reference fsupra) teaches how to make these compounds. Methods of making the protected specieε of formulae (IVA) and (IVB) are alεc taught by Krumidieck, et al., Biochem. , £ _. , 1568-1572, 1969; Meienhofer, J., et al., J. Ore . Cher,. , 35 , 4137- 4140, 1970; Drey, C.N.C., et al., Z . Cher. Soc. , Cher.. Comm.. 144-145, 1977; Krumdieck, C.L., et al. , Meth. Enzvmol. , __ , 523-229, 1980; Silks, V.F., Ph.D. theεiε, Univ. of S. Carolina, 1-147, November 1989; all of which are hereby incorporated by reference. STEP 3: Deprotection The compounde of formula (VA or VB) are treated with a reagent which removes the R-. and P.- protecting groups to produce the compoundε of formula (IA or IB; . Suitable reagents are well known in the art of peptide syntheεiε (Greene, Th. W. , Protective Groups in Organic Synthesiε, Wiley, New York, (1981) , pages 152-187, which pageε are incorporated by reference) . Inorganic or organic baεeε and inorganic or organic acidε are suitable. Examples of the bases include aqueous alkaline earth metal or alkali metal hydroxides, or tertiary or quaternary C; - C ₄ alkyl ammonium hydroxides; specifically magnesium, calcium, sodium and potassium hydroxides and trimethyl, triethyl, tetramethyl or tetraethyi ammonium hydroxides. Examples of the acids include trifluoroacetic acid, formic acid, aqueous HC1 and HBr, and optionally in a water miscible solvent such aε methanol, ethanol, iεcpropancl, acetonitrile, tetrahydrofuran, dioxanε, dimethylforma ide (DMF) and the like.

Suitable tetrahydrcptercyimcnc—l-g_ tami acid

derivatives for the preparation of their α-monoesterε in STEP 1 include tetrahydrofolic acid, 5-methyl tetrahydro¬ folic acid, 5, 10-methylene tetrahydrofolic acid, 5,10- methenyl tetrahydrofolic acid (with a suitable counterion selected from chloride, bromide, iodide, fluoride, for¬ mate, trifluoroacetate, acetate, sulfate, phosphate and the like), 5-formyl tetrahydrofolic acid, 5, 10-diformyl tetrahydrofolic acid, 5-t-butoxy carbonyl-tetrahydrofolic acid and 5, 10-di-t-butoxy carbonyl tetrahydrofolic acid. Folinic acid, however, iε the preferred derivative of tetrahydropteroylmono-L-glutamic acid for the purpose cf thiε invention. Most of the starting compounds are commercially available, also, see Methods of Enzvmology, Vol. 6 _. , pageε 802-815, 1963, which iε hereby incorporated by reference. Thiε reference teaches how to make several of these compoundε.

The preεent invention extende to the monocationic εaltε and the α-monoesterε of tetrahydropteroylmono-L- glutamic acid derivatives. The compoundε of formula- (III) are novel. Preferential εaltε being monoalkaline earth metals or alkali, monotertiary alkyl or quaternary alkyl ammonium ions, silver and lead.

The following examples eerve to delineate the invention. EXAMPLES

Example 1 - Preparation of the α-2 , 6-Dichlorobenzyl Monoeεter of (R,S) Folinic Acid

500 mg of (6R,S) folinic acid and 56 mg of Na _: C0 _; were added to 30 ml of dimethylsulfoxide (DMSO) . 317 mg of α-2, 6-dichlorobenzyl bromide iε added and the mixture stirred at room temperature for 5 hours. The reaction mixture is then concentrated in vacuc and the resulting residue flash chromatographed [Still et al. , J. Orσ. Chem. 43, 2923-2925 (1978)] over C8 (80:20 (v/v) 0.1 M aqueous acetic acid/acetonitrile) . The product containing frac¬ tions (50 ml) were combined and dried in vacuo tc yield 325 mg of an approximately 8:1 ratic of the c. -,-2,6- dichlorobenzyl monoeεtεrε of folinic acid. The twc

isomerε were resolved by high preεεure liquid chromatogra¬ phy on a CIS column (70:30 v/v) C.l K aqueouε acetic acid/acetonitrile) and dried in vacuo to yield 280 mg of the pure α-2 , 6-dichlorobenzyl monoeεter of folinic acid: p >300°; IR (KBr) wavenumber 3345, 1730, 1620, 1325, 1188, 770; 1H 500 Hz NMR (Me _: SO-d ₆ ) delta 1.97 (cm, 1H) , 2.21 (cm,2H) , 2.33 (cm,2H) , 2.80(cm,lH) , 3.07 (cm, 1H) , 3.13 (dd,J=4.1, 12.6 HZ,1H) , 3.41 (dd , J=5.1 , 12.7 , 1H) , 4.21(q,J=7.5,lH) , 4.31(cm,lH) , 4.78(cm,lH) , 5.26, 5.32(AB,J=12 Hz,2H) , 6.31 (bt,J=-5.0 Hz,lH) , 6.57fd,J=8.7 Hz,2H) , 6.69(bε,2H) , 6.97(d,lH) , .44 (cm,J=7.3 , 8.8 HZ , 1H) , 7.53 ( Cm , J=7.9 , 2H; , ~ .60 f Vt , 1 H ) , 7.64 (d,J=S.5Hz,2H) , 8.832fε,lH) , 9.12 fts , 1H, , 11.3 fcε , K ι ; MS(HRFAB) m/∑ found 632.1480 (M ^~ ) , calcd. for C _r K _: Cl.N-C 632.1424 and 34 mg of the pure ->-2, 6-dichlorobenzyl monoeεter of folinic acid: mp >300 ^C C; IR (Kbr) wavenumber 3345, 1722, 1620, 1325, 1188, 770; ^J H 500 m Hz NMR (Me _: SO- d _ή ) delta 1.90(cm,lH) , 2.20 (cm, 2H) , 2.34 (cm, 2H) , 2.86(cm,lH) , 3.06(cm,lH) , 3.12 (dd, 3=3 .3 , 12.2 H_,1H) , 3.40 (dd, =4.4 , 12.2 Hz,lH) , 4.2(q,J=7.1 H_,1H) , 4.80(cm,lH), 5.26(ε,2H), 6.32 (b ,J=-5.0 Hz,lH) , ό.34fbε 2H) , 6.60(d,J=8.5 Hz,2H) , 6.97(d,lH) , 7.43 (cm,J=7.7 , 8.4 Hz,lH) , 10.6(bε,lH) , -12 ffolded) ; MS (HRFAB) m/z found 632.1366(M~) , calcd. for C _r K _r Cl _: N-0- 632.1424. Example 2 - Preparation of the α-2 , 6-Dichlorobenzyl , d_- ^» ,- L-glutamate diethyl triester of (6R.S) Folinic acid

260 mg of the (6R,S) -α-2, 6-dichlorobenzyl monoeε¬ ter of folinic acid, 123 mg of L-glutamic acid diethyl eεter hydrochloride, 106 mg of dicyclohexyl carbodiimide (DCC) and 69 mg of 1-hydroxybenzotrιazcle (HOBt, were added to 15 ml of dimethylforma ide (DMF) containing 4: ul of 4-methylmorpholine. The reaction mixture iε εtirreά for 16 hours a room temperature and concentrated in vacuo. The resulting residue is then chromatographed (Chromatotron) over silica (10:1 v/v) methylene cnio- ride/methanol) and dried to yield 262 mg cf the α-1,6- dichlorobenzyl , di-7-glutamate diethyl trieεter cf fclinic acid: mp >300°C; IR (Kbr _. waven mcer 2225 1 ^" :: 1C25

1335, 1185, 770; Η 500 m Hz NMR delta 1.154, 1.157 (two (t) , J=7.1 Hz,6H), 1.79(cm,lH), 1.94(cm,2H), 2.05(cm,lH) , 2.26(cm,2H), 2.33(cm,2H), 2.87 (td,J=5.1, 13.5 Hz,lH), 3.07(ddd,J=6.6,8.1,13.5 Hz,lH), 3.13 (dd,J= .1, 12.5 H,1H), 3.41(dd,J=5.0,12.2, Hz,lH), 4.03,4.04 (two (q) , J=7.2 Hz, 4H) , 4.21 (ddd,J=5.4,7.5,8.8 Hz, 1H) , 4.4(cm, v=25 Hz, 1H) , 4.8 (cm,lH), 5.28,5.33 (AB,J=12.0 Hz, 2H) , 6.20(bε,2H) , 6.36(t,J=5.7 Hz,lH), 6.61 (d,J=8.7 Hz,2H) , 6.98(α,J=4.6 Hz,lH), 7.45(cm,J=7.3,8 ^' .8 Hz,lH), 7.53 (cm,J=8.0 Hz,2H), 7.66(d,J=8.7 Hz,2H), 8.23(d,J=7.4 Hz,lH), 8.45(ε,lH) , 10.22(s,lH) ; MS (FAB) relative intensity m/ ^' z 817(KH ^" ,S) . Example 3 - Preparation of the Triam oniur. Salt c: fβ?,S; Folinic di-7-L-glutamic acid

5 ml of 0.1 N NaOH iε added to 1C ml of ε 50:50 (v/v) p-dioxane/H _: 0 solution which containe 262 mg of the α-2, 6-dichlorobenzyl, di-7-L-glutamate diethyl trieεter. The reaction mixture is stirred for 6 hours at room temperature, cooled to 4°C and the pK adjuεted tc .0 with acetic acid. The solution iε then chromatographe over cellulose (60:40 (v/v) 0.5 M NH ₄ HC0 ₃ /EtOH) and dried in vacuo to yield 184 mg of the triammonium εalt of (6R,S) folinic di-7-glutamic acid: MS (FAB) relative intensity m/z 691(MNa ₄ ",23) , 669 (MNa ₃ ~ , 22) . Exampleε 1-3 are illus¬ trated in Scheme 1. Scheme 2 εhowε the method in general.

OCαC _j H _i OCOCH _: CH _: CH(NH _: )CO _: C-,H _< HCUHOBUN-matnyimoipnolint in OUF

1) 0.1 N NtOH Ir. p-αιo_*na

2) pH > |u.taβ to 7.0 wiui tcttic ic

3) Callulott column, aiuiaύ with O.S M H ₄ HCO _: . ιO

Scheme 1

- x -

Tetranyαropteroyi derivative

1 equivalent of base. R ' X

Tetranyαropteroyi derivative

1. -,-carboxyi group activation

2. mono- or poly-L-giutamic acid or salt or ester

Tetra yαropteroyl - derivative

:o,r.

Dβprotectioπ

TetranydroDteroyi poly-L-glutamic aciα or salt

Scheme