January 1965
ANALOGS OF TETRAHYDROFOLIC ACID. XVII
was triturated with acetone. The insoluble ammonium chloride was removed by filtration and evaporation of the filtrate gave the oily product; yield, 112 mg. The crude product was dissolved in ether and a small amount of methanol. Dry HC1 was bubbled through the chilled solution, and the white salt was collected by filtration; yield, 65 mg. (32.47,); m.p. 180-183" (oil bath); Amax, m p ( E X p H 1, 254 (9.35); pH '7, 25'7 (0.35); pH 13, 263 (10.0); ii, em.-' (KBr): 3400 (OH), 30002700 (NHI"), 1690 (C=N+H), 1570 (C=C). -Anal. Calcd. for C8H12ClXjO: C, 41.83; H, 5.26; C1, 15.44. Found: C, 41.65; H, 5.06; C1, 15.23. Reagents and Assay Procedure.-Adenosine and adenosine deaminase were purchased from the Sigma Chemical Company. The general method of assay has been described by Kaplang and involves measuring the rate of disappearance of the absorption band of adenosine at 265 m p . -411 enzymatic reactions were performed in 0.05 211 phosphate buffer a t pH 7.6 and 25'. The substrate and the stock solutions of all reagents were prepared in 0.05 JI phosphate buffer a t pH 7.6. For the assay, the cell contained a total volume of 3.1 ml. which was 0.066 m X with respect to adenosine. To study inhibition, appropriate amounts of buffer were excluded from the cells and were replaced by an equal volume of a solution of the inhibitor in phosphate buffer.
Results and Discussion Previously it was found4 that V and VI were competitive inhibitors of adenosine deaminase with K, values (9) Pi. 0. Iiaplan in ".\lethods in Enzymology," Vol. 11. S. P. Colowick and N. 0. Kaplan, Ed., Acadeiiiic Press Inc., S e w York. N. Y., 1955, I). 473.
35
of 3.0 X ill and 9.8 X low5111, respectively. Enzymatic evaluation of compounds VII-XI revealed that they were all essentially rioninhibitory a t concentrations 2-3 times that of the substrate. These results establish that adenosine deaminase has little bulk tolerance for groups on the G-amino group of the purine nucleus. For example, the replacement of the 6amino group by a 6-methylamino group increased the K , by a factor greater than 3. Thus, it would appear that it will not be feasible to prepare active-site-directed irreversible inhibitors a t the 6-position of the purine nucleus. It might be suggested that even though the enzyme has little bulk tolerance for branch chain groups a t the 6position of the purine nucleus, it might tolerate straightchain groups. Therefore, the 6-n-propylamino analog (XII) was synthesized, and it, too, was essentially noniiihibitory a t concentrations 2-3 times that of the substrate. Consequently, adenosine deaminase has little bulk tolerance for either branched or unbranched groups on the 6-amino group of the purine nucleus. Finally, it was found that 7- (3-hydroxy propyl)-6-amhopurine (XIII) was noninhibitory against adenosine deaminase. This fact may be rationalized if it is assumed that the enzyme has little bulk tolerance for a group a t the 7 position of the purine nucleus or that an essential binding group a t the 9-position is absent. At the present time, however, it is not possible to answer this question unambiguously.
Analogs of Tetrahydrofolic Acid. XVII.1'2 On the Mode of Binding of the p-Aminobenzoyl Moiety of N-(2-Amino-4-hydroxy-6-methyl-5-pyrimidplpropyl)p-aminobenzoyl-L-glutamic Acid to Dihydrofolic Reductase B. R . BAKERAND JOHANXES H. JORDAAN Uepartttient of Xedicinal Chenzistry, School of Pharmacy, State University of iVew York at Buflalo, Buffalo 1.4, ,Yew York Received August 20, 1964 The binding of 2-aniino-5-(3-anilinopropyl)-6-methyl-4-pyrimidinol(IV) to dihydrofolic reductase is tightened by introduction of carboxyl ( V ) or carboxyglycyl groups (VI) in the para position. Since the benzene ring of IV probably binds to the enzyme in a charge-transfer complex with the benzene ring being an electron acceptor, the electron-withdrawing p-carbonyl group of V and VI could tighten binding by making the benzene ring a better electron acceptor. Strong evidence to support this hypothesis has now been obtained by comparison of IV-VI and the p-(4-chloro-3-oxo-l-butenyl) (X) and p-(4-chloro-3-oxobutyl) (VIIIb) derivatives of IV- as inhibitors of dihydrofolic reductase.
The pyriinidyl analog of tetrahydrofolic acid (111) binds to folic reductase ( K i = 2.0 X better than the substrate, folic acid ( K , = 10 X lo+). Removal of the carboxy-1-glutamate residue as in IV4 increases Ki to 63 X lo+; it can be calculat'ed readily from - AF = RT In K that the loss in free energy of binding by removal of the carboxy-L-glutamate is only 22YG of 111. -$bout one-half of the binding of the carboxyL-glutamate residue is due to the p-carboxyl as shown (1) This work was supported in p a r t b y Grants CA-05867 a n d CA-06624 from the National Cancer Institute, G. S. Public Health Service. J. H . Jordaan is indebted t o the Atomic Energy Board of the Republic of South Africa for a fellowship. (2) For the previous paper of this series, see B. R. Baker and B.-T. Ho, J . Pharm. Sci., 63,1137 (1964). (3) Papers V I and VI1 of this series: 13. R. Baker and C . E. hforreal, ibid., 61, 596 ( 1 9 6 2 ) ; ibid., 63,840 (1963). (1) Paper X of this series: J3. R . Haker, I). V. Santi, P. I. Alinauls, and W. C. Werkheiser, J . X e d . Chem., 7 , 24 (1964).
by K i = 13 X for V.4 The a-carboxyl of I11 mould appear not to contribute to binding since the Ki of VI is also 13 X thus the other half of the binding of the carboxy-L-glutamate residue of I11 is due to the y-~arboxyl.~Evidence mas presented that both the p- and y-carbonyls were probably coniplexed to the enzyme by hydrogen bonding4 It was pointed out4 that it should be possible to obtain an active-site-directed irreversible inhibitor5sh of folic reductase if the proper type of functional group for formation of a covalent bond could be placed on I11 positioned where the p- or y-carbonyl normally occur. Several chloromethyl ketones (VI11 and X) were synthesized to evaluate this possibility, since halomethyl ketones have been previously used for (5) 13. R. I h k e r , Caiicer Chemotherapy Rept., No. 4 , 1 (1959). (6) B. R. Baker, J . Pharm. Scz., 65, 347 (1@64), a review.
SI
,/
XI1
n
Xv
r - i
1 0
ASALOGSOF TETRAHYDROFOLIC ACID. XVII
January 1965
37
TABLE I OF ( DIHYDRO)FOLIC KEDGWASE BY INHIBITION OH
-----IXhydrofolic
Conipci.
111
R
-CONHCHCOOH
Inhibitor concn., inM
0.10
R inhibition
50
reductase'-Inhibitor: substrate for SOL70 inhibition
16
b'ollc reductaseh Ki X 106
2 0
CHZCHzCOOH -CONHCHZCOOH 0.16 50 2i 13 4.1; -CONH( CHz),COOH -COOH 0.07i 50 13 13 11H 0.60"-e 43 130 63 VIIIa -COCHzCl 0.0i5".' 29 30 YIIIb -( CHz)zCOCH&l 0 . 090dse 43 20 S'IIIC -( CH,),COCHzCl 0 , 023d 50 3.6 VIIId -( CHz)&!OCHzCl 0. lod," 35 32 Y -CH=CHCOCHICl 0. 50 3.0 XYIII -C4Hg-% 0 .O i d + 0 >46 a Dihydrofolic reductase from pigeon liver was a 45-9053 saturated ammonium sulfate fraction prepared and assayed us previously described'l by noting the rate of change in optical density at 340 mp [AI. J. Osborne and F. &I.Huennekens, J . Riol. Chem., 233, 968 (l%S)] with a Cary 11 recording spectrophotometer on a 0-0.1 optical density slide wire; cuvette concentrations of dihydrofolate (S. Futterman, ibid., 228, 1031 (1957)] and TPNH were 6 and 12 M.W, respectively, in Tris Bufier pH 7.4. Value previously reported4 Cuvette also contained 10(yoN,N-diwith rat liver folic reductase using folic acid as substrate at pH 6.1. c Previously reported.'* Maximum concentration allowing full light transmission. methylformamide. e Near saturation concentration.
VI XIS V
'
pure in good yield. That the compound was the styryldioxolane (XV) arid not the pheriylethyldioxolane was shown readily by short acid hydrolysis to reiiiove the dioxolane group. The resultant p-aniiiiostyryl chloroiiiethyl ketone showed a new peak of the conjugated system a t 363 nip; if the double bond had been reduced, no absorption peak a t longer wave length than the 286-nip peak of XV could be expected since the ketone arid aniiriophenyl groups would not be conjugated. Reductive conderisatiori of XV with I proceeded to crystalline IX in 80% yield; short acid hydrolysis of IX afforded the desired styryl chloromethyl ketone analog (X) which had no iiielting point below 350" but showed the conjugated carbonyl peak at 402 nip. When the hydrogenation of the riitrostyryldioxolane (XIVb) was continued after the reduction of the nitro group, a fourth inole was gradually absorbed over 48 hr. The resultant aniiriopheriethyldioxolane (XT'Ib) was an oil that was uniforni 011 thin layer chroniatography and gave proper combustion values; it was reductively condensed with I to give the crystalline pyriniidyldioxolane (VIIb) in 30y0 yield. When VIIb was treated with hot 0.1 N HCl, the dioxolarie blocking group was removed; although a long wavelength peak in the ultraviolet was not formed in the ketone of VIIIb due to lack of conjugation with the ariilino group, that the reaction had proceeded could be shown by the loss of the dioxolane C-0-C band a t 9.61 p and generation of ketone C=O a t 5.78 pin the infrared. Similarly, p-riitrocinnanialdehyde (XIb) and 4(p-nitrophenyl)-2,4-pentadienal(XIc) mere converted to crystalline XIIc and XIId, then to the desired pyriniidyl chloroniethyl ketones, VIIIc and VIIId, respectively . Enzymic Evaluation.-In Table I is listed a summary of the results of inhibition of dihydrofolic reductasr
from pigeon liver by the compounds in question, using dihydrofolate a t pH i . 4 as the substrate; also listed are the results previously obtained with some of the compounds with a different assay, that is, folic reductase from rat liver using folic acid as substrate a t pH 6.1. Except for the p-carbo.;y analog (Y), the same order of effectiveness was noted for the two assays; Y is relatively more potent as a n inhibitor in the dihydrofolate assay which niight be attributable to the pH difference sirice the carboxyl of V would be fully ionized at pH 7.4, but not a t pH 6.1. Comparison of the ketone VIIIb with the parerit anilino compound (IV) shows that the -(CH&COCHzCl group tightens the binding from 130 to 20 for the ratio of inhibitor :substrate (see Table I ) ; the most likely explanation is that the CO group of VIIIb hydrogen bonds with the enzyme. h previous coniparison4 of the parent aniliiio coiiipound (IV) with its p-carboxy (V) and p-carboxyglycyl (VI) derivatives indicated that the glycyl carboxyl did not contribute to binding sirice the latter two coiiipourids had K , = 13 X in the folic acid assay a t pH 6.1; however, in the dihydrofolic assay, these two repeatedly gave a twofold difference in binding with V being superior (Table I). When the results with IV, T.', T'T, and TTIIb are considered together, the best current rationalization is as follows. (a) Both the glycine carboxyl of VI and the ketone CO of T'IIIb can hydrogen bond to the eiizynie even though their chain lengths differ by one atoni. (b) The p-carboxylate of V makes the anilino group a better electron acceptor than the pcarboxaiiiido group of VI even though the Hamniet indicate the reverse when a p-SH is not present. (11) L. P.H a i n m r t t , "Physical Organic theinistry," 1 I c G r a a - H i l l I h o k Co., Inc., S e w r o r k , N . T., 1 W O . pp. 184-207.
Experimental llvl~iiigpoiiits IVC'L'C' take11 iii c,apill;try tubcbs ill ;L AIel-'l'c~ir~~i Iiioc*k: :ill melting points below 2'30" itre rorrerted. Infrared spertra were determined in KBr disks with a I'erkin-Elmer 137H s~)ect,rophot,oriieterunless otherwise indicated. Ultraviolel spectra were determined with a Perkin-Elmer 202 spectrophotometrr. Thin layer r*hromatograniswere run on Brinknianii silica gel G . 2-(p-Aminophenyl)-2-chIoromethyl-l,3-dioxolane (XVIa). ?'[I :t sohiticin of I .7 7. (10 nlnioles) of 4-ainitio-ol-rhloro:tc.etl,pherione in 50 nil. ( i f 1)eiiaerie was d d e d U.70 g. ( 1 1 ~iiinoles)o f (Athylriir: glyi~)l:md I .9 g. of p-tolueriesiilfonic:)ni~ :icitl. The niixtiire \vas refluxed hvitli rii:tgnetir stirring under :t I>e:in -8t:tl.k +,rap for 3 hi,. \\-her1 i i o inore water cwllected. The inixturcs W R S shaken with 120 itil. ( i f ic-e-cold 0.1 . Y HC1. The separattd benzene layer was w:ished with two 5O-nil. porlioris of wat,er, ilried with JIgSOA, theii spin evaporated in uuctio. The crystalline xesidue \vas rwrystiillized froin methanol-water; yield,
AXALOGS OF TETRAHYDROFOLIC ACID. XVII
,January 196.5
39
TABLE I1 PHYSICAL CONSTANTS OF AROMATIC PRECURSORS Compd.
m
n
Method"
% yield
M.P.,
%-
-Calcd., C
oc.
70-
-Found,
N
H
c
H
N
57.5 60.7
4.12 4.21
5.5Sd 4.91
306 (19.1) 224(7.3),345(30) 281(7.2),379(46.4)
53.5 57.0 60.1
4.61 4.94 5 22
5.13 4.6i 4.47
222(9.4),302(13.1) 242(16.4),337(18.8) 2S0(18.8),372(20.8)
XEtOH
mai,
mr
(e
x
0
NO, XIIb XIIc XIId
1 2 3
... ... ...
ilb A A'
85 64 62
116-118' 148-150 178-179
II
0
\ / (CH=CH 1 57.3 60.6
CCHZC1 557 S.05
4.00 4.36
0 0 I/
(CH=CH 1 CCH,Cl
X I S ~ ~I XIVC
XIVd
2 3
.,. , . . ,..
B B R
81 91 61
155-156 148-149 146-147
53.4 56.9 59.7
NO,
4.49 4.77 5.01
m
0 \
5.19 4.74 4.36
P
(CH,),,CCH,CI
XVIb ... 2 C 93 Oil' 59.6 6.67 XVIC ... 4 C 80 Oil' 62.3 7.47 XVId ... 6 C 94 Oil' 64.5 8.12 See Experimental. Refluxed 3 hr. in benzene. ' Lit.10 m.p. 116". MeOH. Moved as one spot on t.1.c. with 10% methanol in benzene. 2.88 2.95, 3.04, 6.03 (NH), 1.70 g. (79y0); m.p. 97-98'; , , ,A 6.22, 6.62, 12.03 (phenyl), 9.87 p (C-0-C); 242 mp ( E 12,300). Anal. Calcd. for CloH1ZClSO2: C, 56.2; H, 5.66; N, 6.55. Found: C, 56.1; H, 5.52; X, 6.29. 5-(4-Nitrophenyl)-2,4-pentadien-l-al(XIc).-A suspension of 6 g. (34 mmoles) of 4-nitrocinnamaldehyde (XIb) in 20 ml. of acetaldehyde was cooled in an ice bath a t 0" and stirred magnetically. Then 0.5 ml. of 25y0 methanolic NaOH solution was added dropwise, and stirring continued for 30 min. The reaction flask was fitted with a downward condenser, 15 ml. of acetic anhydride was added, and the mixture was heated a t 120" for 1 hr. It was cooled and 60 ml. of water carefully was added, followed by 15 ml. of 4.35 N HCl. The mixture was refluxed for 30 min., cooled, and left overnight. The product was filtered off, washed with 20 ml. of water, and recrystallized from ethanolwater; yield of yellow crystals, 6.10 g. (887,); m.p. 105-106'; , ,A, 5.96 (C=O), 6.16 (C=C), 6.62 p (KOz); 339 mp ( E 13,940). Anal. Calcd. for C11H&O3: C, 65.0; H, 4.46; N, 6.90. Found: C,64.8; H,4.46; N,6.79. Similarly, p-nitrobenzaldehyde was converted to p-nitrocinnamaldehyde (XIb) in 57y0yield, m.p. 141-142'. Chloromethyl 4-(4-Nitrophenyl)-l,3-butadienylKetone (XIIc). -A mixture of 5.32 g. (30 mmoles) of X I b and 10.59 g. (30 mmoles) of triphenyl chloroacetonyl phosphorane was dissolved in 50 ml. of methanol and heated at 50' with stirring for 36 hr. The mixture was cooled, and the crystalline product was collected on a filter and washed with 10 ml. of methanol. Recrystallization from benzene gave 4.82 g. (647,) of yellow crystals, m.p. 148-150"; Amax 5.89 (C=O), 6.23 (C=C), 6.62, 7.46 c./ (NO,); " : :A 224 mp ( c 29,770). Anal. Calcd. for C12HloClN03: C, 57.3; H, 4.00; C1, 14.1; N , 5.57. Found: C,57.5; H,4.12; C1, 14.1; X, 5.58. Other ketones prepared in this way are listed in Table I1 under method A. 2-Chloromethyl-2-(p-nitrostyryl)-l,3-dioxolane(XIVb).A mixture of 2.9 g. (12.8 mmoles) of p-nitrostyryl chloromethyl ketone (XIIb). 20 ml. of benzene, 10 ml. of ethylene glycol, and 20 mg. of p-toluenesulfonic acid was refluxed overnight; the water formed in the reaction was continuously removed with a Dean and Stark trap. The cooled mixture was washed with two 50-ml. portions of water and dried (PvlgSOr). Removal of the solvent in vacuo gave a yellow crystalline residue which was recrystallized from ethanol; yield, 2.8 g. (8lY0); m.p. 155-156; XEtOH mar 222 mp ( e 9426), 302 mp ( E 13,090);, , ,A 6.29, 6.78 (phenyl), 6.68, 7.50 (NOz), 9.7-9.8 p (C-0-C). Anal. Calcd. for CI2H12C1NO4:C, 53.4; H, 4.49; N, 5.19. Found: C, 53.5; H, 4.61; N, 5.13.
5.79 5.19 4.70 %alcd:
5.61 59.8 6.89 4.75 61.i 7.21 4.59 6 4 . 7 8.30 C1, 14.1. Found: C1,
238(10.1),276(8.3) 241(13.4),274(14.9) 236(9.8),291(1.5) 14.1. e Refluxcd 4 hr. in
Other dioxolanes prepared in this way are listed in Table I1 under method B. 2-(p-Aminostyryl)-2-chloromethyl-l,3-dioxolane(XV).A solution of 2.4 g. (8.9 mmoles) of XIVb in 200 ml. of ethyl acetate was shaken with hydrogen at 2-3 atm. in the presence of 60 mg. of platinum oxide catalyst. Approximately 3 moles of hydrogen per mole was consumed in 15 min.; the reaction then slowed down considerably and was stopped. The filtered solution was concentrated in uacuo. Recrystallization of the product from benzene-petroleum ether (6O-llOO) gave nearly 2.88, white crystals; yield, 1.65 g. (777,); m.p. 94-96'; , , A, 2.95, 3.06 (XH), 6.21, 6.60 (phenyl), 9.5-9.8 p (C-0-C); A E z 286 mp ( E 33,700), 280 mp ( E 33,700). Anal. Calcd. for CllH14C1N02: C, 60.0; H, 5.89; C1, 14.8; S, 5.84. Found: C, 60.0; H, 5.73; C1,14.8;S, 5.83. 2 4 p-Aminophenethyl)-2-chloromethyl-l,3-dioxolane (XVIb).To a solution of 2 g. (7.47 mmoles) of 2-chloromethyl-2-(4nitrostyryl)-1,3-dioxolane(XIVb) in 50 ml. of ethyl acetate and 150 ml. of ethanol was added 60 mg. of platinum oxide catalyst and the mixture was shaken with hydrogen a t 2-3 atm. until hydrogen consumption ceased (about 48 hr.). The filtered mixture was concentrated in vacuo. The remaining oil was dissolved in 25 ml. of ethanol and refluxed with 1 g. of activated charcoal for 1 hr. The mixture was filtered and removal of the solvent in vacuo yielded a colorless oil which could not be crystallized; yield, 1.68 g. (937,); Ai1: 2.86, 2.93, 3.08 (KH), 6.16, 6.61 p (phenyl); 238 mp ( E 10,100), 276 mp ( B 8300). Anal. Calcd. for C12H16ClSO: C, 59.6; H, 6.67; K, 5.79. Found: C, 59.8; H , 6.89; iY,5.61. Other reductions carried out in the same way are listed in Table I1 under method C. N- [l-(2-Amino-4-hydroxy-6-methyl-5-pyrimidyl)-3-propyl] p-amino-a-chloroacetophenoneEthylene Ketal (VIIa).-A mixture of 223 mg. (1 mmole) of XVIa and 213 mg. ( 1 mmole) of 2-acetami d o - 4 - h y d r o x y-6-me t h yl-5-p y r i m i d y l p r o p i o naldehyde3 was dissolved in 25 ml. of methanol and sodium borohydride (500 mg.) immediately was added in small portions to the stirred solution. Stirring was continued for 18 hr., then the precipitated product was collected on a filter. The product was recrystallized from ethanol-water; yield, 240 mg. (61y0); m.p. 172-173'; 253 mp ( E 18,930);, , ,A 2.9-3.5 (broad NH, CH, OH), 9.80 (C-0-C), 12.20 p (P-CBHI). Anal. Calcd. for CleH28C1N403: C, 57.1; H, 6.12; N, 14.8. Found: C, 56.8; H, 5.96; K,14.7. Other related reductive condensations carried out in the same way are listed in Table I11 under method D. N-[ 1-(2-Amino-4-hydroxy-6-methyl-5-pyrimidyl)-3-propyl] p-aminochloroacetophenone (VIIIa).-To a solution of 0.76 g. ( 2 mmoles) of VIIa in 75 ml. of ethanol was added 25 ml. of 0.1
-
-
-1 -
1-
I.
I-
I.
,January 196.5
SMOOTH
I\ IUSCLE
STIMULAKT
FATTY ACIDS
41
Other compounds prepared by a similar hydrolysis are listed 'Y HC1. The mixture was refluxed fur I hr., then diluted with 100 nil. of water, cooled in an ice bath, arid made slightly alkain Table 111under method F. 2-Amino-5-( p-n-butylanilinopropyl)-6-methyl-4-pyrimidinol line with 0.1 AVS a O H solution. Filter aid ( 3 g.) was stirred into (XVIII).-A solution of 1.49 g. (10 mmoles) of p-n-butylanilinele the mixture and left for 0.5 hr. The precipitate was collected and washed well with water. The filter cake was then extracted and 2.23 g. (10 mmoles) of I in 100 nil. of methanol was allowed with five 40-nil. portions of boiling ethanol. The alcohol was to react for 30 min. With magnetic stirring 2 g. of sodium bororemoved in vacuo and the yellow residue was recrystallized from hydride was added in portions over a period of 30 min. After being stirred overnight, 20 ml. of ST aqueous NaOH was added aqueous ethanol; yield, 0.62 g. (93%); m.p. above 300" (der.): and the methanol was removed zn z ~ u c m . The solution was A,,,, 2.95-3.60 (broad OH, S H , CH), 12.30 (p-C6H4),12.80 p diluted with 100 nil. of water and the pH was adjusted t o 9 with (C-Cl). See Table 111for other data. 2- { N- [ 1-( 2-Amino-4-hydroxy-6-methyl-5-pyrimidyl)-3- 5% HCI. The precipitated product was collected and recrystallized from methanol-water; yield, 2.3 g. ( 7 3 5 ) ; m.p. 223-225"; propyl]-I-aniinophenyl] ethyl Chloromethyl Ketone (VIIIb).A,, 2.90-3.60 (broad OH, XH, CH), 6.17, 6.58 (KH, C=K, A solution of 350 mg. (0.86 minole) of S V I b in 25 nil. of 0.1 -Y aqueous HC1 was heated on a steam bath for 1 hr. The pH 265 mp ( E 9300); 240 m p C=C), 12.38 p (CsH,-); ( E 15,600), 291 mp ( E 6300); Az:x'3 240 mp ( e 17,200), 280 mp of the solution was then adjusted to 9 with 0.1 1V aqueous XaOH. iE 79001. The product, was collected on a filt'er, washed with 10 ml. of ' Anal: Calcd. for C18H2eN40:C, 68.8; H, 8.33; N, 17.8. water, recrystallized from ethanol-water, and gave light yellow Found: C, 68.5; H, 8.50; S , 17.7. 2.9Cb3.40 crystals; yield, 200 mg. (64%); m.p. >300°; A,, (broad OH, S H , CH), 5.79 (C=O), no C-0-C band near 9.6 p; 240 mp ( e 12,700),279 mp ( e 6800);;:A: 217 mp ( e 12,800), 266 mp ( e i100). ( 1 6 ) R . R . Read and D. 13. lfullin, .I. A m . C h ~ mS. o r . , SO, 1763 (1928).
Synthesis of Fatty Acids with Smooth Muscle Stimulant Activity E.
CIIUNI)WELL,~
i r . A. PINNEGAR,
W. TEMPLETOX
AND
J l i l ~ s - dines Krsearch Laboraturies, Stoke Poges, Buckinghamshire, England
Receiv~dAugust I S , 1964 12-Hydroxyheptadec-trans-10-enoic acid has been synthesized bv reduction of the corresponding acetylenic derivative -7th lithium in liquid ammonia a t room temperature after pretreatment with lithium hydride to avoid hydrogenolysis. This acid, which represents a fragment of a prostaglandin, has been found to be three times as active as ricinelaidic arid in stimulating the isolated hamster colon. Homologs and other derivatives, including some containing an additional 6-oxo or 6-hydroxy group, have been synthesized and their activity has been determined. The hydroxyacetylenic acids were synthesized via chloroalkynols from alk-l-yn-3-01s and were converted into hydroxyouoacet?.lenic acids via reaction of the acid chlorides with cycloalkenamines.
Interest has recently been renewed in the pharinacological potentialities of fatty acids largely because of the elucidation of the nat'ure of the prostaglandins.2 Consideration of the structure of prostaglandin E1 (1) and of the pharmacologically active acids, notably ricinelaidic acid (2), among those examined for smooth muscle stiiiiulant a ~ t ' i v i t y ,suggests ~,~ that coninion features may include a hydroxyl group a t position 12 and urisaturatioii or structural rigidit'y between positions 8 and 11 in an unbranched aliphat'ic acid.
unstated yield by a route involving the reaction of heptanal with lithiuiii 8-chlorooctyne. From work with diniethylundecynaniide (described below) there was reason to believe that higher alkynes niight be unreactive so the following route was examined. This was RCHOHC=CH +RCHOPyC=CH + 4 5 RCHOHCEC(CH,)nCl+ RCHOHCSEC(CH~),CN+
RCHO
-+
7
6
KCHOHC=C( CH&C02Me
-+
8
C,H,,CHOHCH ~ C -uHC ; C H > ) h C O ; H
C
RCHOHCH=CH(CH2),C02Me
HO
9
1
Py = 2-tetrahydropyranyl
t
CsHj,CHOHCH2CH=CH( CH?);CO,H 2 R CHOHC=C( CH2)nCOrH 3
Synthesis of acetylenic acids of type 3 was therefore investigated. One previous example (3, R = C6H13; n = 6) has been reported,5 synthesized in (1) School of Pharmacy, College of Technology, Park Road. Portsmouth, Hampshire. England. ( 2 ) S.Bergstrnm, R. Ryliage, R. Samuelsson, a n d .J. Sjijvall, A c t a Chem. Scand., 1 6 , 5 0 1 (1962). ( 3 ) N. Ambache, .I. Physiol. (London), 146, 25.5 (1859); S. .\mbaclie, M. Reynolds, and .J. AI. C. Whiting, ibid., 166,261 (1963). (4) M . S. Rlasri, I,. .I. Goldblatt, F. IleEds, and G. 0.Koliler, .I. !'harm.
Sci., SI, 899 (1962). (5) A. S. Bailey, V. G. Kendall, P. R. Liimb, .T. \Valker, J . Chem. Soc.. 3027 (1957).
r. Smith,
and C . H.
tested using the readily available hexynol (4, R = C3H7)to give the ester (8, R = C3H7; n = 6). Difficulties arose when the route was extended t o use oct1-yn-3-01 (4, R = CsHlx). This had been previously prepared6 by selenium dioxide oxidation of 1-octyne. This method is unsuitable for large-scale preparation so the familiar reaction of sodiuni acetylide with an aldehyde was used. However, it mas found that with hexanal, particularly commercial samples, the yield and purity of octynol was veiy variable and frequently only higher boiling unsaturated hydroxy ketones were obtained. Similar difficulties were found with heptanal,7 or when ethynylniagnesiuni bromide was used. (6) R . Truchet, Compt. Tend., 196, 708 (1933). (7) L. Crombie, personal communication, 1963.
