X i4
6- Substitu tecl 2,4-Dianzino-5-(4-carbethoxyy>henylazo)l~yri1iii~i~ie~ as Potential Precursors of Tetrahycll.oi,tericfine Antimetabolite+'
2 , 4 - 1 ~ i a m i i i o - : j - ( i - c ~ ~1ior;Spheiiyla~o)pyrimidiiies r~)~t beariiig tiie 2-:iiiiliniJ-2-liyd opylariiiiio or 3-ziiiiliiio2-chloropropylamino substituents in the 6 positioii have beeii synthesized altd co d in biological systenis with 9,4,6-triamino-3-( -L-c.aibethoxypheiiSla~o)pyrimidinet o teit t h e hppot hesis the former compountii might undergo ail in z'iw coiivei,aiori to a tetrahydropteridiiie. So evidence t o siipport this hypothesis could be found, arid it waz demonstrated that azopyrirnidiiies arc inhibitors, rat,hrr than substrates, of rut ::zo
L i b part of a continuing progrnni3-*jof +ynthc cbv:tluation of wbitituted pyrimidine- :ih foli tllitagoriists, n e have con-itlcretl the> pob-ibihty of -ynthebizing coin pound^ that :trc potential prerursorof folic acid tmtagoniqti ~rliic~li nwy be converted 211 wo t o :tc.tivc :mtagoriisth. Tlw advantagci of t h y type of approavh t o tlie dcsigri of c~l.iemotherapeutic:illv c.fi'ectivr coinpourids have been discussed 3evernl tinies.b-y The c o n v e r - i o n of a i 1 iiiac~tiveto aii :tc.tir.c' ronipound by cnzyinatic ac%ivntion occwring preferont idly in tlie tumor c d l offer- to c:~iiccr cheniothtwpy poisibillties of increavd s(>lctti\ity of :\(tion. rcdiicwl *ystcInic. toxicity, and po;.-iblc rc~tluc~tion111 t h c (le,velopnient of reyirtance to tlic tlrug Our preliminary approach to thi-, problciii ha- bctw to design conipounds that may kw caonvcrtccl in I Y ~ to foliv acid :mtagoniit- of tlic 2,~-di:ti~iinoptcri~liii~~ of wtiirh the bebt knou 11 nicmber I. aiiicthop1 erin (I) (+ec Scheme I). Thc1 conipoiiiidh .clectcel \V(W approprintely -,ubbtitutcd .i-arylazopyriniidiii(,b ( jr ~ ~ n TI) t l TI hicli, conceivably. c~)uldcyc'lizc~ to J t c~truhydropteridirie(VIII) after reductive fission of t h c :tzo liriliagc.lfJ ( ' o n i p o u i i c l T'II TI a\ ~nc~ludccl ab : Lcoiit ivl in which t l i i h rcduc.ti\rc. (*y*liz:xtioii c u d t l not
S( 1IE\lb I
I n
0
0
0
rn
TI
IY
V
OCrUr.
Results and Discussion 'l'hc results prescnt,cd i n T:tbl(>1hlion- t'liat. c w n i p o u i i t l ~ \?-VI1 arc irihibit,ors of folic acid reductasr, biiidiiig xomcwhat lcw efficiently than folic wid. 'l'lic effrvt, of introducing the G - S substituents of mmpouiitls V-VI1 into ~~,.l,(i-trinnii~io-~i-(-2-c;lrbethoxypeiiyl:~~o~(I) This work was supported by I'ublic Health Berxice Grant S o . (.'.\ 06645-03-04 from tiie Xational (Zancer Institute. (2) Theoretical 13lologg t-nit. Scl~oolof Piiarmarg. ( 3 ) A . 11.Triygle and D. J. Triggle. .I. l ' l i a , m . Sci., 54, iY; (1965). (4) J . Hampshire, P. Hebborn, .&. 11. Triggle, I). J. Triggle, and S. Vickers, .I. M e d . Chem., 8 , 745 (1965). ( 5 ) ,J. Hampshire, P. Hebhorn, A . h i . Triygle, and I). .J. Trigyle, J . I'harn. Sci., 56, 453 (1966). (6) P. Hebborn and .J. I'. I>anieili, B i o ~ A ~ i I'harmarol., n. 1, 19 (1958). ( i )J. >I. Jolinbon a n r l F. Hergel in "1Ietabolic Inhibitors," Vol. 11, It. demic Press Inc., New York, K . I-., 1983, p l i 3 , nc,e~,"1'1. .\. Plattner, Ed., Elsevier
( 9 ) \V. C. .l. Rubs, "i2iulo~iral. i l k ~ L i i ~ n.g (I>ublisliers),Lid.. London. l ~ l K 3 . (10) G . 11. Itamape iind G . 'Tr:*{)pe 1.1. C ' howe\-er, noted that rediiction of 4-(~-~iil~iruPti~?.1):ixninO-R-nitrupS rimidlnw and subsequent reHrixing in Ptliiinolic boluuun ,welded ~ l y o i u l i n o p ? . r i m i d i i i e ~ , formed by cycliratiun onto a ring nitrogem, railier than pteridines o r tetriiI iyclropteridines.
urn
pyriniidinr (111 [Sjdi,= O . O 1 l ) l 15 to produccb t i c v * i c ~ : i ~ t ~ . 111 biiidirig c*ap:icity of approximately 100-foltl. 111 tlic work of Baher L L I Shnpiro1I ~ i t is alm evidc~iitt h t introduction of two hydrophobic substitueiitb i n t o 2,Jdianiinopyrimidiiies does riot produce an :i(ldit I Y P illcrease in binding capacity toward dihydrofolir ruliicatase. Thus, repluccinent of the 6-methyl group of ~,,4-diami1io-j-(4-pl~enylbutyl)-(i-methylpyrimid11ie by phenyl leads to an approxiniately 50-fold loss in effwtiveness of birding, although 2,4-dianrino-(i-phcnylpyrimidine binds to diliydrofolic reductaw wv(~11 times more effertively than 2,4-diamino-fi-iiic~tl~yIpyrimidine. Oiie explanation of these finding- 13 th:tt
875
UXIOH
NO.
TABLEI ACTIVITY O F 6-SUBSTITUTED ~ , 4 - ~ I . i ~ ~ l ~ O - ~ - ( 4 - C . l ~ t B E ~ H O ~ Y P H E S ~ ~ L . I Z O ) P Y R I J l I U I
( [ I l l [sl)saa
1.5 3.75
blouse toxicity,b mdkg
Rat toxicity,c mg/kg qd 5-9
>1600 300
>400 140
Dose, mgjkg qd 5-Sd
570 body wt changee
T/Cf
400 +6 0.5 \'I 100 +3 0.67 50 19 0.0 1-11 1.2 1100 ,550 400 -9 0.40 a Folic acid redrictase iiihibit,ion. lbatio of coiicentratiori of irihibitor reqiiired for X ( ' ~ L inhibitioii of enzyme activity to coiiceiitrittioii Approximate acute LDSO(see Experimeiital Section). Approximate LDm, five daily doses or1 of sribstrate, [folate] = 8 x 10-3 JI. Against established Jlurphy-dt'urm lymphosarcoma. e Weight of rats on day 5 taken as 100yo. days 5-9 (see Experimental Sectioii). 1 Ratio of volume of t,reated arid control tumors estimated on day 12. \'
the binding site of folic and dihydrofolic reductase contains only one hydrophobic bonding area. Introduction of two hydrophobic groups into the 2,4diaminopyrimidine structure may cause one of these groups to interact a t a nonhydrophobic binding area with resulting desorption of the molecule from the enzyme. Compounds 1'-VII are relatively nontoxic and exert little or no antitumor action even at dose levels approaching the toxic range (Table I). The broad similarity in toxicities and antitumor activities of compounds V-VI1 does not lend any support to the idea that V and VI may be converted in civo to the pteridine, VIII, which might be regarded as a potentially more cytotoxic agent. Confirmatory evidence for this view was obtained by in vitro studies carried out to determine the ease of reduction of the azo linkage in azopyrimidines arid azobenzenes. R a t liver homogenate fortified with an YADPH-generating system as recommended by llueller and lIillerl2 was employed as the reducing medium. -4s a n t i ~ i p a t e d , ' ~substituted azobenzenes (2-X,S-diethylaminoazobenzene, 2-carboxy-2'-S,Sdiethylaminoazobenzene, and 2-carbomethoxy-2'-S,Kdiethylaminoazobenzenej were reduced relatively rapidly (100-400 mpmoles/min per g of tiisue wet weight), but the 3-arylazopyrimidines V-1-11, 2,4,Gtriamino-5-phenylazopyrimidine, 2,4,Ci-trianiino-3-(4carbethoxyphenylazo)pyrimidine, and 2,4,6-triamino5-(4-carboxy-L-glutaniylpheriylazo j pyrimidine were vompletely resistant to reduction even after a 2-hr incubation period. Furthermore, it was established that reduction of 2-carboxy-2'-S,S-diethylaminoazobenzene was inhibited by 2,4,G-triamino-5-(4-carboxyL-glutaniylpheny1azo)pyrimidine with the latter exhibiting an [I]/[Slj0ratio of 1.5. It i b thus probable that 3-arylazopyrimidines are inhibitors, rather than substrates, of liver azo reductases.
Experimental SectionI4 N-( 2-Hydroxy-3-anilino)propylphthalimide (III).-X-( 2,sc~)oxypropyl)phthalimidelj(11, 10 g, 0.05 mole) aiid aniline (4.0 g, 0.05 mole) in methaiiol (100 ml) were refluxed for 12 hr. The solution waa cooled, arid the precipitate was filtered, v ashed with cold methanol (20 ml), and recrystallized from methanol to give 111, mp 151-132', in >90% yield. Anal. Calcd for C17Hl&20a: C, 68.95; H, 5.44; X, 9.46. Found: C, 69.06; H, 5.46; N,9.51. (12) G. C. .Mueller and J. A. Miller, J . B i d . Chem., 180, 1125 (1949). (13) W.C. J. Ross and G . P. Karmick, J. Chem. Soc.. 1364 (1956). (14) Melting points were recorded on a Thomas-Kofler hot stage and are
corrected. Analyses are by Galbraith Laboratories, Inc., Knoxville, Tenn., and hy I)r. A . 13. T3erntiardt. hlullieim, Ruhr, Germany. (15) hl. \Vcizuiann and S. LIalkoiva, Cornpt. R e n d . , 190, 1Y5 (1930).
+
N-Phenyl-2-hydroxy-l,3-propanediamine (IV).-111 (10 g, 0.035 mole) arid I-ICI (100 ml, d 1.2) were refluxed for 6 hr. The mixture was diluted with water (50 ml) and left overnight a t 5". The calculated quantity of phthalic acid m s filtered and the filtrate m s spin evaporated in vacuo to give a residue which was recryst,allized from methanol (charcoal) to give the dihydrochloride of IV, mp 177-179" (yield 6.2 g, 79j'; j. Anal. Calcd for C9HI6Cl2N2O:C, 45.20; H, 6.74; C1, 29.63; S, 11.7. Found: C, 45.04; H, 6.70; C1, 29.75; K, 11.4. 2,4,6-Triamino-5-(4-carbethoxyphenylazo)-6-N-(2-hydroxy-3ani1ino)propylpyrimidine (V).-Compound I V (3.32 g, 0.02 mole as free base from IT' '2HC1 and NaOEt) aiid 2,4-diamino-5-(4carbethoxyphenylazo)-6-chloropyrimidine4(2.85 g, 0.01 mole) suspeiided in ethanol (100 ml) were heated a t 100" to complete solution. On cooling, L- crystallized as orange needles with mp 201-203" (4.0 g, 907;). An analytical sample (from methanol) had mp 202-204". Anal. Calcd for C22H26N6O3:C, 58.25; H, 5.78; N, 24.7. Found: C, 57.95; H, 5.84; N, 24.59. 2,4,6-Triamino-5-( 4-carbethoxyphenylazo)-6-N-( 2-chloro-3ani1ino)propylpyrimidine (VI).-Compound V (2.25 g, 0.005 mole) was finely powdered and suspended in dry ether (50 ml) a t 0". Thionyl chloride (0.9 g, 0.0075 mole) was added with vigorous stirring, and the mixture was maintained at 35" for 1.5 hr. Excess thionyl chloride was destroyed with ethanol (2 ml), and the product was filtered, washed with ether, and recrpst,allized (ethanol) to give V I as the hydrochloride in yellow plates, mp 146-150", in near quantitative yield. Despite repeated recrystallization, it was impossible to obtain a completely satisfactory elemeiital analysis for this material. Anal. Calcd for C2?H26C12S802: C, 52.25; H, 6 . 2 ; C1, 14.02; N, 22.17. Calcd for C2?H?,Cl?~&h.2H20;C, 48.8; H, 4.83; C1, 13.1; N,20.7. Found: C, 48.75; H, 4.50; C1, 12.34; N, 19.84. 2,4,6-Triamino-5-(4-carbethoxyphenylazo)-6-N-( 3-ani1ino)propylpyrimidine (VII).-?;-Pheny1-1,3-propanediamine1~ (2.9% g, 0.02 mole) and 2,4-diamino-5-(4-carbethoxyphenylazo)-6chloropj-rimidine (2.85 g, 0.01 mole) suspended in ethanol (100 nil) were heated a t 100" to complete solution. On cooling, VI1 crystallized as orange needles, m p 178-181' (3.8 g, 88y0). An analytical sample (from methanol) had m p 182-184'. Anal. Calcd for C22H26NS02: C, 60.85; H, 6.04; N, 25.8. Found: C, 60.54; H, 5.91; S, 25.131. Inhibition of Folic Acid Reductase."A high-speed, superiiataiit fraction of rat liver homogenate was used as the source of folic acid reductase.17 The assay procedure for folic acid reductase activity was similar to that described by Werkheiser.18 Reduction of Azo Compounds.12-A 10% suspension of rat sucrose conliver homogeiiate was prepared in ice-cold, 0.25 taining 0.01 JI phosphate buffer, p H 7.4. The incubation mixtiire contaiiied 15.2 pmoles of fructose 1,G-diphosphate, 200 pg of NAD, 200 pg of NAIIP, 120 pmoles of nicotinaniide, 10 pmoles of JIgCI?, 200 pnioles of KC1, 50 yrnoles of phosphate buffer, pH i.4, mid 0.5 nil of rat liver homogenate in a total volume of 3.0 ml. Azo compound, dissolved in dimethyl sulfoxide or 0.02 ,TI NaOH was added to a final concentration of 75 mpmoles. Blank samples contained all constituents except the azo compound. Duplicate mixtures were incubated at 37" with shaking and, (16) A. Goldenring, Ber., 23, 1169 (1890). (17) S. F.Zakrewski and C. A. Nichol, J. Pharmacol. SzptZ. Thernp., 137, 162 (1962).
(18) \V. C. \\erkheiser, J . Diol. Chem., 236, 888 (1961).
