CA2016829A1 - 8,10-dideazatetrahydrofolic acid derivatives - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
8,10-Dideazatetrahydrofolic acid derivatives of the formula:

Description

~0 8,10-DIDEAZATETRAHYDROFOLIC ACIl) DERIVATIVES

ORIGIN OF ~VENTION
The invention described herein was in part made in the course of work under a grant or award from the National Institute OI Health, Department of Health, Education and Welfare.

SPECIFICATION ~ iR~
Mead, ~. S. Patent No. 3, 856, 959, patented December 24, 1974, provides a method of inhibiting methotrexate-sensitive leukemia L1210 and methotrexate-5 resistant leukemias derived from L1210, such as variantsL1210/FR-8, L1210/M-46-R, L1210/C-95, and $1210/M-66-3A
in mice, employing 5-methyltetrahydrohomofolate utilized as an injectable in a dosage regimen of 12. 5- 1600 mg/kg/inj/diem.
Mead notes that In modem kukemb Iher py the ~nd~rd dru~ or compul on ror ~ever l ~enn hu been methotre~te (~mcthopte in) The modu~ oS ction In ~rivo of thl~
compound in en~m-lb re~clion b lo ;nh;bil the ~ctbn of dihydrotobb Kductuc ~nd thi~ nd imibt trup constitut!n~4~minonn~10pe~o rOlic cid K l~no~vn u the 4 mino ntirobtes It h-~ Kccntly been K~li~d th~t ~rheK bukemi~ tron~l~r re i~nt to the 4 mino ndrobter nnd i~ ch~r~cte~cd by h4h level~ or dihydrofo1~te ~educt e cert h homorobto dcriv~
t;ve~ ~ho~ o ~ub-t~nti~l nnlileuitemlc efrcct--J ~ R
Mc d ~nn NY ~ Sel 156 5l4 S15 Nov 1971 It h~ been ho~n th-t tetnbyd ohomorobte b o po tent Inhibitor In vjt~o in ~he ensgm~tk re~ction of thy midybte ~nthetue cnd the en~yme i met bol;c~lly 2 5 beyont or beb~ ~he ~ction of the dihydrotd~te reduc-tue Thu- i~ b con~ide ed pouible th~ dih~
drohomorobte nd po~ibly homotol~e by conver~bn in vivo ~o o ~pocific inhibitor ol ~hymid~l-to ynthe ir mlgh~ bloclt the ~rowth of ~mcthopterlnmi~t~nt celb h~vin~ hi~h kveb ot dih~drorobte Kduct~ o--J /~
R Mc d et nl C~ r R J ~rh 2374-2379 ~1966) Whereas the known compounds utllized have been active at (2) in Mead's Chart 1, the tetrahydrohomofolates are active at (3), and Mead believed 5-methyltetrahydrohomofolate (5-MeTHHF) is active at (4) relative to the action oi methionine synthetase.

- ~ -CIL RT t t~
rl ab, ~D
r ,O~ r ab l~D

a~ -~ bOb .1~ .
~tt rbo~ t n oh ~9 trnco-~t~rl alu 10~8~ab ~8CNtt--8~ bab 1118CO--~rbalu e ~otw~o~ , Il,r~al, .
~ ConJu~
Dlhgd orobto rcductt~Jo Thymldybto t,ynthett~
~D Methhnlne ~ynthett e ~ Formyl~lut m le tottnyl It n~bn, c 0 N~ N" methyknetett3hydtorobte teductuc G~ L Serino byttosymethyl ~n~ letue ~D Fotmimlno~lu~ rn-te Fo m;m;no Tr ndcr- e N"-tormyltettt,hydtorobto qnthetue ~ N~ btm~lte~tthydtoroble Itomett, ~ N~, N~ melhylenclelr~hydtorobte dchydro ~ent e ~ N~ fotmlmlnotet~ hydtofiob~e cyclode-mint,~e N~, N" methenyît ttt,hydtorobte eyclohydrob~e ~! 5 Am;no-4 lmid~ole ctrbo~ mide ribonuclc otide tttntlfotmylt~e ' Glycin mide tibonuckotide tttn lotmylt, e , 5-Methyl tetrahydrohomofolate has the formula :~ .
oonl~
1I N C~l~C~k 11 11 O 11 ~1 c_~ n p~ oo ' . " ~Ha :

;

~o~

DeGraw, Jr. etal, U.S. PatentNo. 4,369,319, patented January 18, 1983, provides a process and composition employing 10-deazaminopterin and 10-aLkyl derivatives thereof for the treatment of leukemia, as well as other tumor systems including 5 those of ascitic character, and also a process for preparing 10-deazaminopterin compounds.
These 10-deazam inopterin compounds have the structure:

NH2 ~ N
0 N ~ ~ 1 HJN N N Cll~
COOH
In lhe compound lO de~un;noplerin, Rl nd R2 ~ e bolh hydrogen. tn Ihe ~Ikyl deriv tive, dlhct or bolh Or Rl nd R2 cnn bc nlkyl l~vlng trom one lo ~bout e;ght, pret~r bly one or two cubon ~toma When only onc Or Rl nd R2 ;- ~Ikyl, the olher b hydrogen.
Temple et al, U. S. Patent No. 4, 431, 805, patented 20 February 14, 1984, report that the 5-deaza analogs of folic acid, Nl-methylfolic acid, aminopterin, methotrexate and the diethyl ester of aminopterin,inhibit the growth of human epidermoid carcinoma cells No, 2 and are active against leukemia in laboratory animals. The 5-deaza analogs of 25 folic acid, Nl-methylfolic acid, aminopterin and methotrexate have the following structures:

Jl_ CH~Nl~;}CONl HN
U~N N N
~
NH~ U
N J~CH~NII_~I CH~CH~U
H~NJ~ N N
wheran R i~ ei~her hydro3en or melhyl DeGraw et al, U. S, Patent No. 4, 460, 591, patented July 17, 1984, and 4, 532, 241, patented July 30, 1985, provide 10-a~yl-8, 10-dideazaminopterins of the formula:

NN~ . b N J~ ~CH~I--~C ~--OH
H~N N

where R b hyd roten or ~yl Or I lo ~t I c~rbon nd thdr c-rbosyble nd Id ddltbc -lt ue de cribed ~e r~pa~ a~hb1t nndnoopb~;c ~clivity Ihet b d~lbr lo b~lt more e~tecll~ Ih~ n~clhotr~tc DeGraw et al note that ~minop~erin ~nd ;u N 10-melhyl derlvdlve melho ue~e h ve lon~ been ~eco~nked u power ul uldneo 2 5 ~ plu~ic egen~ Me1holre~le bu e~oyed rome Ihirly ye~ of cccpt nce u clinic ily u etui enti~ncer dru~ The dru~r ue ntimet bolller inhibltin~ dlhy drotobte reduct--e (DHFR) They ttecl both neopl~
lic nd norm l ho~t ~ue ~Iter tion~ or ~he p~eridine nn~ ot folic cid ~e i o bn hve~iLt~ed The ryn~ nd entifobte c~ y Or t de~folic rcld wu eportod by J I Dc Gnw R L IC61iult Y O-umont ~ C M ~u~h M~ C~k~ 17 470 (197~) ~nd t lO~ide~fiolic dd wu reponed h J 1 DeGnw R L lU liuL V. ~
Elrown nd Y. G-umont. C n~ cr 6 219 ~19~9) 8dh of ~he~e comp ~ were ~cth~e en~ito bt~ bul did not rignific nlly nnect DHFR. ~ Sriniv~
~ n nd ~ D Elroom J 0~ Cb~ m ~19tl) reponed ~he preper~lion ot t~euuninopterin nd t-de~methotrel-le but dld not repon blolotlc l ctlv i~y tor ~he compund~

Temple et al, UO S. Patents Nos. 4, 526, 964, patented July 2, 1985, and 4, 536, 575, patented August 20, 1985, indicate that The 5 de~ ul~log~ ot tolic cid, NIQsub~tituted fiolic cid ~minop~erin, Nl ~ub~tituted ~minop~erin end ~he dic~hjl e ter of eminopterln Inhibit tne ~rowth Or hum-n epldetmold cuclnom~ celb No. 2 end ~te ~ctive ~einst leukemb In l-borntory ~nlm~b The ~ deD~D
n~iogs Or tollc cid ~nd N' sub~tituted fiolic ~cld re 0 fierred to herein h~ve the fiollowin6 s~uctute HN J CH~N~3CONHCHCO~H
H~NJ~ N N CHlCHICOlH
end the ~ de~ en~lo~s Or ~minoplerin ~nd NIQsu~
stilutet ~minop~etin reterred to herein h~ve ~he fiollow ing s~ructure:

~_CHIN~CONHCHCO~H
H Nl N 1 N J CH`CH CO H

wherein R b hydro~en CH~, CH3CH~ CH~CH2CH~
CH2=CHCH2 or CH_CCH~ ~ ~
Taylor et al, UDS. Patent No. 4, 684, 653, patented August 4, 1987, provides (i~) pytidol2,3 d]r yrimidine~ Or ~h~ fiotrnub ~

'' CH CH~CONN--C~--CHICH~COON

3 0 H~N N N

wh rcln Rl h ~mino or hydro~y ~nd R~ hl hydro~en. methyl or elhyl; Ihe conrgur-lbn ~boul Iho c~rbon ~lom da4n ~ed being L;
(ib~ ~ 6 7 ~ ~eu~hydrorytidol2 3 dl pyrimidinn Or Ihe fiormul~

cH~--c~ CO-lH--CH--CH~CH~COt~H
0 ~IN N
H

wherein Rl is ~mino or hydro~y; ~d R~b hydrogen me~hyl orelhyl Iheconfigun~ion ~bou~ ~he c rbon n~om Wgneted being L:
(ii) ~hc ~u~omcric hrm- thereot; nnd (iii) thc ph~rm ceu~ic~lly ccept~ble ell-ll met l ~1k~1inc c~nh met l non ~o%ic met l ~mmonium ~nd rub~ uled ~mmonlum s lts ~hcreot These compounds are antineoplastic agents, and have an effect on one or more enzymes which utilize folic acid, and in particular metabolic derivatives of folic acid, as a substrate, including dihydrofolate reductase, thymidylate syntheiase and folate polyglutamate syntheiase.
2 5 .N (~l2~2~mlno~hydrolty ~ telnhy dropyridol2,3J]pyrimidln~yl)~ethyl~ben~oyl)~i~
glu~mic cid in penicubt is ~ ~mique n~lmet~boli~c While rn int~ining good ctivi~y ng~insl L-l~10 leuke mb whkh ;- comj~ bl lo methotre~te the com-pound Is ~ we~k inhibitor ot dihydrorol te reductue~
indi^~ing probrble ~c~ivity ~lns~ ~he rol~e-reb~ed en-yme ~rrFel- other Ih-n DltF~

In accordance with the present invention, 8,10-dideazatetrahydrofolic acid derivatives are provided of the formula:

N~ ~ 2 0 H2~`N (CH2)2 ICI-OH

where Rl and R2 are selected from hydrogen and aL~cyl having from one to about eight carbon atoms and the carboxylates and acid addition salts thereof.
These compounds, unl~ke prior folic acid derivatives, 15 such as 2, 4-diamino antifolates, and 5-methyltetrahydrohomo-folate, they show only insignificant inhibition of dihydrofolate reductase and thymidylate synthase enzymes, and it is accordingly surprising that they show strong inhibition of L1210 murine leukemia cells in culture, and modest inhibition 20 of glycinamide ribotide transformylase and aminoimidazole carboxamide ribotide transferase.
Exemplary 8,10-dideazatetrahydro$olic acid derivatives falling within the invention include:

2 5 H2~ ~N

ICo~H

- 9 - ~

OH CH9 ~H
N~CH2~H~3C~ I H
Hg~ N (Cl ~)H

O

~--CH2~H~ NI~II

C~H
, 11 O

15 and the carboxylate and acid addition salts thereof.
In accordance with the invention, the 8,10-dideazatetra-hydrofolic acid derivatives of Formula I are provided as new compounds, with distinctive and valuable properties, for example, significantly effective in the inhibition of growth of 20 L1210 murine leukemia cells.
Also in accordance with this invention, a synthesis is provided that affords 8,10-dideazatetrahydro folic acid derivatives of Formula I.
Accordingly, these 8,10-dideazatetrahydro folic acid 25 derivatives are expected to have utility in the treatment of human cancer.
The invention accordingly also provides a process of treating leukemia and ascitic tumors, which comprises administering to a warm-blooded animal having an abnormal proportion of leukocytes or other evidences of the malignancy, a therapeutic nontoxic amount of an 8,10-dideazatetrahydro folic acid derivative of Formula I, as such or in the form of a pharmaceutically acceptable carboxylate or acid addition salt 5 thereof. The carboxylates are formed by neutralization of one or both COOH groups, and the addition salts are formed with one or more free NH2 groups of the 8, 10-dideazatetrahydro folic acid derivative.
The process of the invention for the preparation of the 10 8,10-dideazatetrahydro folic acid derivative is a synthesis including the following steps, exemplified by R, as methyl CH~ and R2 as hydrogen.

5ta9~ I H2N~}CO~H
~I) ¦ 10% NaOH
HO t St'F 2~ ~--~}C02H
H2N N (2) I

2 5 HO 002CH2CH~
St 9 ~ ~I~Jx~}CONHCH
H2N N ~CC2CH2CH~
¦ I N N~l HO ~ 02H
H2Nl~}CoNH

i H2-PtO2-HCHO
HO CH~ ICO2H

H2NJ~X~}CONHCH

As the synthesis scheme shows, hydrolysis of the 2,4-diamino-4-deoxy-8, 10-dideazapteroic acid (1) with hot lO~a NaOH effects displacement of the 4-NH~ group to 4-OH
yielding 8,10-dideazapteroic acid (2). Compound 2 is coupled with diethyl L-glutamate with activation of the benzoate carboxyl group as the mixed anhydride formed by isobutyl chloroformate to afford diethyl 8,10-dideazafolate (3).
Saponification of the diester (3) in 2-methoxyethanol containing lN NaOH yields 8, 10-dideazafolic acid (4). Hydrogenation of 4 over a platinum catalyst in the presence of an equivalent of formaldehyde in an acidic medium afford 5-N-methyl-tetrahydro-8, 10-dideazafolic acid (5).
Hydrogenation of the folic acid intermediate (4) in the absence of formaldehy3e yields tetrahydro-8, 10-dideazafolic acid itself, R l and Rs = H.
Substitution of other alkyl groups for Rl and R~ in the above 6cheme are apparent to thoYe skilled in the art, with combinations of either hydrogen and alkyl or different al}cyls as R, and R2.
In Stage 1, the hydrolysis of the 4-amino group of the pyrimldine ring may be carried out by treatment with any ~pproprlate allcall metal hydro~dde at temperatures of 50-150C.
Tbe hydrolysis may also be conducted in strongly acidic media ~uch a6 mineral acids at similar temperatures. If 10-alkyl substituted analogs of formula (1), where Rs =allcyl, are employed tbe products at Stage 1 and subsequent stageg will bear the 10-alkyl ~ubstituent.

~01~

In Stage 2, the benzoic acid moiety is activated by conversion to a mixed anhydride by treatment of its triethylamine salt with isobutyl chloroformate in a polar organic solvent.
Other organic tertiary amine bases such as tributylamine, N-methyl morpholine, collidine can be employed as can other esters of chloroformic acid. The mixed anhydride is allowed to react with diethyl L-glutamate at ordinary temperatures and the product i8 best purified by chromatography on silica gel.
In Stage 3, hydrolysis of the esterifying groups is carried out with aqueous aL~ali at room temperature or above. The diester can be dissolved in a suitable solvent, such as 2-methoxyethanol, and held in the presence of the aqueous aL'sali until hydrolysis is complete. The hydrolysis product folic acid analog can then be precipitated by addition of acid such as acetic or hydrochloric. The precipitate can be recovered, washed and dried.
In Stage S, the substrate folic acid analog is hydrogenated at room temperature and atmospheric pressure in an aqueous acid medium, preferably containing acetic acid as a cosolvent, A catalyst such as platinum oxide or palladium may be employed.
To obtain a 5-N-alkyl product via reductive aL~cylation it is necessary to incorporate an equivalent of the appropriate aldehyde such as formaldehyde, acetaldehyde, etc. If the aldehyde reagent is deleted one obtains the target compounds containing an NH at the 5-position.
The following is illuskative of the procedure:

EXAMPLE
H2N 1L~

Stage I H2Nl~--{} C02H
( I ) ¦ 102 NaOH
HO
N~ ~C02H
Stage 2H2N~NJ~J
t2) 0 HO C02CH2CH~
N~ ~ CONHCH
Stage3 H2N~NJ~J C02CH2CH3 I N NaOH

N~ 9--CONHCH
Stage 4H2NJ~NJ~J C02H

¦ H2-PtO2-HCHO

HO CH~ C02H
N~ ~ CONHCH
Stage 5 H2N~'bNJ~J ( 5 ) C02H

8,10-Dideazapteroic Acid (2).
2, 4-Diamlno-4-deoxy-8, 10-dldeazapteroic acid ~1) 0, 88 g, 2.8 mmol, was heated to 85-90 with 15 ml of 10%
NaOH solution under an argon atmosphere. HPLC analysis of the reaction indicated maximum conversion after 5 hours. The reaction mixture was cooled to 5 for 48 ~hours. The resulting ppt. (di-sodium salt) was collected and washed with a small amount of ice-cold 10% NaOH solution. The ppt. was stirred with a little H ,O and the pH adjusted to 5 by addition of HOAc.
The suspended solid was stirred for 10 minutes, then collected and washed with a little H~O to yield 0. 52 g of light grey solid S after drying. Mass spectrum, m/e 310 (calc'd. 310). Anal.
calc'd. forCl~HuN403 0.5H~O:C, 60.2; H, 4.42; N, 17.5.
Found: C, 60.2; H, 4.51; N, 17.5.
8,10-Dideazafolic Acid, Diethyl Ester (3).
8, 10-Dideazapteroic acld (2) 0. 52 g, 1. 7 mmol was stirred with 15 ml of dry DMF under an argon atmosphere.
Triethylamine (0. 69 g, 6. 8 mmol) was added and the mi~ture stirred for 15 minutes, then isobutyl chloroformate 10. 87 g, 6. 4 mmol) was added. After 1 hour only partial solution was obtained. The reaction was sequentia}ly treated with triethylamine (0. 017 g, 1. 7 mmol) and isobutyl chloroformate (0. 23 g, 1. 7 mmol). Complete solution occurred after 20 minutes. Triethylamine (0. 81 g, 8.1 mmol) follo~ved by diethyl-L-glutamic acid diethyl ester in 10 ml of ~MF were added and the resulting solution was stirred for 20 hours.
The solvent was removed at 40~ (0.1 mm) and the residue was dissolved in CHCI~ then washed wlth H~O and dilute NH,OH, dried and the solution evaporated in vacuo. The gummy residue was chromatographed on silica gel with CHCla and CH,OH-CHCl~ elutio~, The product, 110 mg of yellow solid, waselutedwithCH~O~:CHCls, 2:98.,Anal. calc'dfor C2sH2qNsO3-0~5H2O C, 59.5; H, 5.99; N, 13.9. Found:
C, S9. 3; H, 5. 80; N, 13. 6.

-15~ ;R~
8,10-Dideazafolic Acid (4).
8,10-Dideazafolic acid diethyl ester (3) 110 mg, 0. 22 mmol, was treated with 1 ml of lN NaOH solution.
and 1 ml of 2-methoxyethanol. The solution was stirred for 15 hours then the solvents were removed at 40 (0.1 mm).
The residue was treated with 1 ml of H,O and acidified to pH
5. 5 with HOAc. The resulting ppt. was collected, washed with H2O and dried to yield 74 mg of product as a yellow solid.
Anal. calc'd. for C"H"lNsO6~ 2H,O: C, 53. 1; H, 5. 30;
N, 14. 7. Found: C, 53. 4; H, 4. 67; N, 14. 5.
5-Methyl-8, 10-dideaza-5, 6, 7, 8-tetrahydrofolic Acid (5).
8,10-Dideazafolic acid (4) 74 mg, 0.17 mmol was dissolved in 8 ml of acetic acid and 2 ml of H20 containing 0. 24 mmol of HCl. PtO, (24 mg) was added and the mixture was hydrogenated at atmospheric pressure. Hydrogen uptake cessed after uptake of 15 ml Hl (theory 12. 4 ml) and formaldehyde (0. 018 ml, 0. 20 mmol, of 35~0 formaldehyde) was added. Continued hydrogenation resulted in the rapid uptake of 4 ml of H, (theory 3. 8 ml). The mixture was filtered through Celite and the solvent removed at 40 (0.1 mm). The residue was twice treated with 2 ml of HIO and taken to dryness each time to leave 90 mg of white solid. Mass spectrum, m/e (TMS) 457 (calc'd. 457). Anal. calc'd for C22Hl~,N506Cl H~O:
C, 51.6; H, 5.90; N, 13.6. Found: C, 51.4; H, 5.82; N, 13.2.

8, 10-Dideaza-5, 6, 7, 8-tetrahydroiolic Acid.
8, 10-Dideazafolic acid (4) 100 mg, 0, 23 mmol dissolved in 10 ml of HOAc, 9 ml H~O, and 2 ml (0. 24 mmol) of HCl solution was hydrogenated at atmospheric pressure over 25 mg 5 of PtO" After 1 hour, 15 ml of H, (theory lS ml) had been taken up. The mixture was filtered through Celite then taken to dryness at 0.1 mm pressure (40). The residue was twice treated with 10 ml H20 and taken to dryness each time to yield 100 mg of white solid. Mass spectrum m/e 443 (calc'd. 443).
The 8,-10-dideazatetrahydro folic acid can be administered e~ ~, or in association with a pharmaceutically acceptable diluent or carrier~ The invention accordingly also provides a pharmaceutical composition in dosage unit form comprising from 0.1 to about 500 mg of 8,10-dideazatetrahydro folic acid 15 per dosage unit, together with a pharmaceutically acceptable nonta~ic inert carrier or diluent therefor.
The 8,10-dideazatetrahydro folic acid can be used as such, or in the form of their carbox~lates.or an a~id 2~

addition salt. The carboxylates are formed by neutralization of one or two of the COOH groups with an aL~ali metal or alkaline earth metal or ammonia or a lowe. aliphatic amine.
The acid addition salts are formed with one or more free N~
groups of the tetrahydro folic acid molecule.
The ac~d addUion SaltS are preîerably the pharmaceutically acceptable, nontacic addltion s~t9 wUh suitable acids, such as those with inorgantc acids, for example, hydrochlorlc, hydrobromic, nitrlc, sulphurlc and phosphoric acldS, and with organic acids, such as organ~c carboxyllc ac~ds, for example, glycolic, maletc, hydra~cymalelc, mallc, tartaric, citric, salicyclic, o-acetyla~cy-benzoic, nicotinic and isonicotinic acid, and organic sulphonic acidS, for example, methanesulphon~c, ethanesulphonic, 2-hydrco~yethane-sulphonic, toluene-p-sulphonic, and naphthalene-2-sulphonic acid.
An acid addUion Salt can be co~erted into the free compound according to known methods, for example, by treating it with a base, such as witb a metal hydracide or aD~aclde, for e1cample, an aLcali metal or alkaline earth metal hydraocide, for exampIe, lithium - hydr~ide, sodiumhydrac~de, potassiumhydr~cideorcalcium hydrcoclde; with a metal carbonate, such as an alkall metal or an alkaline earth metal carbonate or hydrogen carbonate, for example, sod~um, pOtassiUm or calclum carbanate or hydrogen carbonate;
w~th ammonia; or with a hydra~tyl ion exchange resin, or witb any other suitable reagent.
An ~cid add~on salt may also be converted into anOther acid addltion Salt according to l~nown methcds; for example, a salt with an Inorganlc acid may be treated with a metal sa~, for example a sodium, barium or si}rer salt~ Q~ an ac~d in a su~table dlluent, ~n whlch a resulting tnorgantc salt is insoluble and i9 thus removed from the reactton medium. An acid addition salt may also be converted lnto another acid addition salt by treatment with an anion exchange preparat~on The 8,10-dideazatetrahydro folic acid or carboxylate or salt thereof can be administered to the animal by any available route, including oral and parenteral (intravenous, tntraperitoneal, subcutaneous, and tntramuscular) adminlstratlon.
The amount admtnlstered l9 sufficlent to amellorate the leukemia 10 or the ascltlc tumor, and will depend upon the type of leukemia, the species of animal, and the welght of the anlmal. For example, in human admlnlStratlon, a dosage aE 8,10-dideazatetrahydro folic acid within the range from about 0.1 mg/kg or about 500 mg/kg 15 per day should be suffictent. Dosages in the higher part o~ the range, approachlng 500 mg~kg, are normally administered ~ conjunctlon wlth leucovoran (dl-5-formyl tetrahydrofolate) to reduce toxlcity.
Tn the treatment of lower test an~mals, a slmilar dosage range is therapeutlc. The upper llmtt af dosage l9 that imposed by tc~lc 20 stde e~ects, antl can be determ~ned by trlal and error for the anlmal to be treated, lncludlng humans.
To facll~tate admtnl9tratlon, the 8, 10-dideazatetrahydro folic acid or carboxylate or salt thereof can be prov~ded tn compo9lt~0n ~orm, and preferably ln dosage unlt 25 form. Whlle the compound can be adm~n~stered per se, ~t ~s 20~

normally admini9tered in conjunction with a pharmaceutically acceptable carr~er therefor, which dilute9 the compound and f aclllta~es handling. The term "pharmaceutlcally acceptable"
means that the carrier (as well as the resulting composition) ~s 5 sterile and nonta~clc.
The carrier or dUuent can be solid, semisolld, or liquid, and can serve as a vehicle, excipient, or medlum for the 8,10-dideazatetrahydro folic acid.
Exemplaly diluents and carriers include lactose, dextrose, 10 sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginateS, tragacanth, gelatln, syrup, methyl cellulose, polyo.xyethylene sorbitan monolaurate, methyl-and propyl-hyd~oxybenzoate, talc or magnesium stearate.
For convenience in handling, the 8,10-dideazatetrahydro folic acid and carrier or diluent can be enclosed or encapsulated in a capsule, sachet, cachet, gelatin, paper or other container, especiall~r when lntended for use in dosage unlts The dOsage unlts can ~r example take the form 20 of tablets, capSUles~ supposltor~es or cachets.
The following Examples illustrate var~ous forms of dosage unlts In whlch the 8,10-dideazatetrahydro folic acid or carboxylates cr salt~ thereof can be prepare~d.

-20- 2~
Tablet formulation M~/tablet .
8,10-dideazatetrahydro folic acid 15 Lactose 86 5 Corn starch (dried) 45 . 5 Gelatin 2. 5 Magnesium stearate 1. 0 The q, 10-dideazatetrah~dro folic acld is po~dered and passed through a mesh sieve and 10 well mixed with the lactose and 30 mg of the corn starch, both passed through a sieve.
The mbced powaers are ~3ed w ith a warm gelatin solution, prepared bg stirring the gelat~ in water and heating to form a 109 $ ~/~ solution. The mass is granulated by passing through a sieve, 15 and the moist granules dried at 40C.
The dried granules are regranulated by passing through a sieve and the balance ~ the starch and the magnesium stearate Is added and thoroughly m~ced.
The gra~ules are compressed to produce tablets each 20 weighlng 150 mg.
ExamPle 2 Tablet formulat~on Mg~tablet 8,10-dideaz~tetrahydro folic acid 100 2~ ~actose 39 Corn starch td~ted) 80 Ge~tn 4 0 Magnesium stearate 2. 0 The raethod af preparation is identical with that of Example 1 except that 60 mg of starch is used in the granulation process and 20 mg during tabletting.
Example 3 Capsule formulation Mg/capsule 8,10-dideazatetrahydro folic acid 250 LactoSe 150 The 8,10-dideazatetrahydro folic acid 10 and lactose are passed through a sieve and the powders well mixed together before filling into hard gelatin capsules of suitable size, so that each capsule contains 400 mg of mixed powders.
Example 4 15 Suppositories Mg/suppositories 8, 10-dideazatetrahydro folic acid 50 Oil of Theobroma 950 The 8,10-dideazatetrahydro folic acid 20 is powdered and passed through a sieve and triturated wlth molten oll of theobroma at 45~C to form a smooth suspension .
The mlxture is well stirred and poured lnto molds, each oE nominal 1 g capaclty, to produce supposU~ies.

Example 5 C achets M~/cachet 8 10-dideazatetrahydro folic acid 100 5 Lactose 400 The 8 10-dideazatetrahydro folic acid is passed ~rough a mesh sieve mi~ed with lactose previously sieved and fitted Into cachets af SUitable size so that each contains 500 mg.
Example 6 Tntramuscular injectlon (ster~le suspension ~n aaueous vehlcle) Mg 8 10-dideazatetrahydro folic acid 10 15 Sodium citrate s.q Sodlum carba~ymethylcelluloSe (low vlscoslty grade) 2. 0 Methyl para-hydra cybenzoate 1. 5 Propyl para-hydra~cybeDzoate 0. 2 20 Water for injectlon to 1. 0 ml E~ample q Tntraperitoneal ~ntraveneous or subcutaneous iniectl~ (sterUe solut~on In aoueous car.rler system) M~

25 8;1a-dideazate~rahy.dro folh ac~d . 15 Scdlum citrate 5 . 7 Sodlum carba~cymethylcelluloSe (low viscosity grade) 2. 0 Methyl para-hydra cybenzoate 1. 5 Propyl para-hydra~benzoate 0. 2 30 Water ~or ~njectlon to 1 J0 ml

Claims (20)

1. 8,10-dideazatetrahydro folic acid compounds having the formula:

where R1 and R2 are selected from hydrogen and alkyl having from one to about eight carbon atoms; and the carboxylate and acid addition salts thereof.

2. 8,10-dideazatetrahydro folic acid compounds according to claim 1 in which R1 is hydrogen and R2 is alkyl.

3. 8,10-dideazatetrahydro folic acid compounds according to claim 2 in whieh the alkyl is methyl.

4. 8,10-dideazatetrahydro folic acid compounds according to claim 1 in which R1 and R2 are each hydrogen.

5, 8,10-dideazatetrahydro folic acid compounds according to claim 1 in which R1 and R2 are each alkyl.

6. A pharmaceutical composition in dosage unit form for treating leukemia or an ascites tumor comprising an amount per dosage unit within the range from about 0.1 to about 500 mg of an 8,10-dideazatetrahydro folic acid compound according to claim 1, therapeutically effective to ameliorate leukemia or the ascites tumor, together with a pharmaceutically acceptable nontoxic carrier or diluent therefor.

7. A pharmaceutical composition according to claim 6 in which the 8,10-dideazatetrahydro folic acid is in the form of a pharmaceutically acceptable acid addition salt.

8. A pharmaceutical composition according to claim 6 in which the 8,10-dideazatetrahydro folic acid is in the form of a pharmaceutically acceptable carboxylate.

9. A pharmaceutical composition according to claim 6 in tablet form.

10. A pharmaceutical composition according to claim 6 in capsule form.

11. A pharmaceutical composition according to claim 6 in suppository form.

12. A pharmaceutical composition according to claim 6 in cachet form.

13. A pharmaceutical composition according to claim 6 in sterile aqueous form.

14. A process for treating leukemia and ascites tumors which comprises administering to a warm-blooded animal having an abnormal proportion of leukocytes or other evidence of the malignancy, a therapeutic and relatively nontoxic amount of an 8,10-dideazatetrahydro folic acid compound according to claim 1, to ameliorate leukemia or ascites tumors.

15. A process according to claim 14 in which the 8,10-dideazatetrahydro folic acid compound is administered as a pharmaceutically acceptable acid addition salt thereof.

16. A process according to claim 14 in which the 8,10-dideazatetrahydro folic acid compound is administered as a pharmaceutically acceptable carboxylate.

17. A process according to claim 14 in which the 8,10-dideazatetrahydro folic acid compound is administered in an amount within the range from about 0.1 to about 500 mg per day.

18, A process according to claim 14 in which the 8,10-dideazatetrahydro folic acid compound is administered with an inert diluent or carrier.

19. A process according to claim 14 in which the 8,10-dideazatetrahydro folic acid compound is administered orally.

20. A process according to claim 14 in which the 8,10-dideazatetrahydro folic acid compound is administered parenterally.