Journal of Medicinal Chernistr?, 197c3,Vol. 26, No. 12 1389
Notes
Notes NH,
Platelet Aggregation Inhibitors. 7.l S-Substituted 2-Thioadenosine 5’-Monophosphates
I
Kiyomi Kikugawa,* Hideo Suehiro, and Motonobu Ichino
Research Laboratorie+.K o h j i n (.‘ompan?,. L t d . . 51 K o m i y a - c h o . Hachiouji Cit?, To/i?,o,J a p a n . Received J u n e E . 1973
Aggregation of blood platelets or formation of platelet thrombi is of primary importance in arterial thrombogene&.* Adenosine 5’-diphosphate (ADP) is known to induce and in i ~ i i ~ oAdenosine . platelet aggregation both in ~1iti-o~ is a powerful inhibitor of ADP-induced platelet aggregat i ~ n and , ~ certain derivatives of adenosine such as 2-chloroadenosine,6 N6-substituted a d e n o ~ i n e s , and ~ , ~ S-substituted 2-thioadenosinesl are also potent inhibitors. Adenosine 5’-monophosphate (AMP) has been known to be a less effective i n h i b i t ~ r ,and ~ 2-chloroadenosine 5’-mOnOphosphateg and certain N6-substituted adenosine 5’-monophosphatess are almost inactive. 2-Methylthioadenosine 5’-monophosphate, however, is much more potent than the original nucleoside, 2-methylthioadenosine, and has been evaluated by Michael, et al.,1° as an antithrombotic agent without undesirable side effects. In the present study, some additional S-substituted 2-thioadenosine 5 ’ monophosphates were prepared and tested as inhibitors of platelet aggregation mediated by ADP and collagen. 2-n- Amylthioadenosine ( l a ) , 2-benzylthioadenosine ( i b ) , and 2-allylthioadenosine (IC) which were representative of the S-substituted 2-thioadenosines reported in a previous paper1 were directly phosphorylated a t their 5 ’ hydroxyl functions by reaction with aqueous PoC13 in the presence of pyridine and acetonitrile.ll The structures of the products isolated as barium salts (yield, 10-20%) were identified as 2-n-amylthioadenosine 5’-monophosphate (2-n-amylthio-AMP, 2a), 2-benzylthioadenosine 5’-monophosphate (2-benzylthio-AMP, 2b), and 2-allylthioadenosine 5‘-monophosphate (2-allylthio-AMP, 2c) by ultraviolet absorption spectrum, paper chromatography, paper electrophoresis (Table I), elemental analysis, and dephosphorylation by Crotalus atrox 5’-nucleotidase. 2-n-Amylthio-AMP (2a), 2-benzylthio-AMP (2b), and 2-allylthio-AMP (2c) were tested as inhibitors of ADPinduced platelet aggregation on samples of buffered platelet-rich plasma prepared from rabbit citrated blood and platelet-rich plasma from human citrated blood. Inhibition percentages of platelet aggregation by 2a-c compared to those by adenosine and AMP are listed in Table 11. On
HO
OH 1
NH1 I
0-
H6
OH
2
R = CH,(CH,),b, R = C6H5CH2c, R = CH2=CHCH2rabbit platelet aggregation ammonium salts of 2a and 2c dissolved in saline showed potent inhibitory effect comparable to adenosine a t any concentration; these exhibited 20-8070 inhibition a t 10-F-10-4 M . whereas the ammonium salt of 2b showed 50% inhibition a t M and was M . Thus, the phosphorylated nuinactive at below cleosides 2a and 2c were more effective inhibitors of ADPinduced aggregation than the original nucleosides la and ic.1 Considerable species variation exists in the response of platelet aggregation to inhibition by 2-methylthio-AMP;lO it was 2.4 times more potent than adenosine against sheep platelet aggregation while it was 8.6 times less potent than adenosine against human platelet aggregation. When 2a and 2c were tested as inhibitors of human platelet aggregation, the inhibitory effect of these compounds was also comparable to that of adenosine. Hence, 2a and 2c might be considered more powerful inhibitors of ADP-induced aggregation than 2-methylthio-AMP with respect to human species. Compounds 2a and 2c were preincubated with buffered rabbit platelet-rich plasma a t 37“ for the longer intervals a,
Table I. Uv Spectra, Paper Chromatography, and Paper Electrophoresis of S-Substituted 2-Thioadenosine 5’-Monophosphates
Uv, max, nm ( e X __. PH 1 HzO
Compound 2-n-Amylthio-AMP (2a)barium salt 2-Benzylthio-AMP (2b) barium salt 2-Allylthio-AMP (2c) barium salt 2-n-Amylthioadenosine (la)d 2-Benzylthioadenosine (lb)d 2-Allylthioadenosine (1c)d AMP Adenosine 2 ‘,3‘-monophosphate ADP
272 (16.6) 273 (16.6) 272 (16.2) 272 (16.1) 272.5 (16.4) 272 (16 . l )
278.5 (15.5) 277 (16.2) 278 (15.4) 278 (14.6) 278 (15.9) 278 (15.0)
Paper Paper chromatography .~ eleciroRrrlj“ R f r S ) h phoresisc ~
pH 13 278.5 (15.5) 278 (15.4) 278 (16.4) 279 (14.7) 278 (15.9) 278 (14.9)
0.75
0.67
0.66 0.56 0.93
0.56 0.38
0.91 0.86
0.29 ~
C0.49 +0.48
+0.48
0.93 0.91 0.87 0.20 0.28
+1.00
0.34
0.21
0.13
+1.27
~~
“Solvent: n-PrOH-concentrated NH40H-H20 (20 :10 :3). *Solvent: i-PrOH-concentrated NH40H-H?O (7 :1:2). CMobility relative t o that of AMP. dReference 1.
Table 11. Inhihition of ADP-Induced Platelet Aggregation by S-Substituted 2-Thioadenosine 5'-Munophosphates
'7 in hi bit ion', Compound
___
~.
~
__
2-n-Amylthio-AMP (2a) a m m o n i u m salt 2-Benzylthio-AMP (2b) a m m o n i u m salt, 2-Allylthio-AMP ( 2 c ) a m m o n i u m salt A M P disodium salt Adenosine _________
Molar concn ~
8
x lo--;
8 8
( 10 -6 9 x 10-7 10--1 10 --j 10 -I' 10 -- i 10 10 --"
____
-
~
.
50 8
:I1
C)
80
59 37 52 35
--____..
5 71 61 a7
86 31
94
41
"Buffered platelet-rich plasma from citrated r a b b i t blood or platelet-rich plasma from citrated h u m a n blood (1.0 ml) was incubated at 37' for 3 m i n with 10 g1 of a solution of each of t h e test compounds in saline a n d challenged with A D P in saline. P e r cent inhibition by a t e s t compound was calculated b y dividing t h e m a x i m u m deflection of t h e optical density curve by t h a t observed with control solvent (saline). As t h e sensitivity of platelets t o A D P varied from preparation t o preparation, more t h a n two experiments were carried out with different preparation of platelet-rich plasma in t h e cases of rabbit a n d h u m a n a n d inhibition percentages listed in t h e table were representative ones under t h e same conditions.
0
40
50
120
TIRE ( M I N ) BEFORE ADP A D D I T I O N
Figure 1. Effect of preincubation of S-substituted 2-thioadenosine 5'-monophosphates with rabbit platelet-rich plasma on .4DPinduced platelet aggregation. Buffered platelet-rich plasma ( 1.0 m l ) was incubated at 37" with the test sample in saline and after the indicated period it was challenged with 10 .I4 ADP. 2-nAmylthio-AMP ( E a ) ammonium salt, 8 X 10 J I ( 0 ) ; 2-allylthio-AMP (212)ammonium salt. 9 X 1W6 Av( A i : AMP. .Vl ( 0 ) : 2-n-amylthioadenosine. ( u): 2-all>-lthioadenosine. 10 ~5 M ( A ) :and adenosine. M (0). ( u p to 120 m i n ) before t h e addition of' A D P (Figure 1). At 10 AT inhibition percentages of 2a a n d 2c were m a i n tained around 5070. whereas those of t h e dephosphorylated nucleosides la a n d le gradually increased for 120 m i n b u t not exceeded 30% a n d t h a t of adenosine gradually d e creased below 10%. T h e results indicated t h a t 2a a n d 2c were powerful inhibitors characterized by long-lasting activity. Inhibition percentages of A M P a t 10 M increased as t h e preincubation proceeded. a n d t h e results agreed with t h a t of Born a n d C r o d who used h u m a n plateletrich plasma. T h e r e have been some discussions about t h e inhibitory effect 01 AMP; some reported t h a t it was a n inhibitor by itself12,13 while others reported t h a t t h e inhibitory effect of A M P was d u e to t h e hydrolyzed product. adenosine. l 4 . I 5 In our assay systems it was not known whether A M P was a n inhibitor by itself or whether the hydrolyzed product. adenosine. was a n a c t u a l inhibitor. C o m p o u n d s 2a a n d 2c could be dephosphorylated by pia!;m a phosphatase or 5'-nucleotidase. b u t t h e dephosphor~
ylated nucleosides la arid I C were less potent a t L O .II t h a n 2a a n d 2c. T h i s suggested t h a t ?a a n d 2c m a y act directly on platelet without dephosphorylation to la a n d lc or m a y be more rapidly t a k e n u p into platelet m e m brane t h a n l a a n d le thereby giving greater inhibition upon subsequent dephosphorylation. S - S u b s t i t u t e d 2-thio-AMP's 2a-c were also tested a s inhibitors of collagen-induced r a b b i t platelet aggregation a n d were found inactive. T h u s , a m m o n i u m salts of 2a-c preincubated for 3 min with buffered platelet -rich plasma inhibited less t h a n 10L70of collagen-induced aggregation at 10 * whereas adenosine inhibited 93% a t ALf and a n d A M P inhibited 2070 at 10 -2. Nucleosides la a n d IC were effective a t 10 .V against both A D P - a n d collagen-induced aggregation.l whereas t h e n u cleotides ? a a n d 2c u-ere ineffective a t t h e same concentration against collagen-induced aggregation. This seemed interesting to us since most agents t h a t inhibit ADP-induced aggregation inhibit collagen-induced aggregation.2 I n conclusion. 2-n-amylthio-AMP (2a) a n d 2-allylthio. AMP ( 2 c ) were found t o be potent inhibitors oi A?il)P-in duced rabbit and h u m a n platelet aggregation. T h e degree of inhibition by 2a a n d 2c was comparable tci t h a t of adenosine in b o t h species a n d t h e inhibitory etf'cct 'icab Characterized by long-lasting activity.
Experimental Section Xlethods. LVhere analyse- were indicated only 1, svmljolz (it ;he rlernentr. malytic,a! results ohtainr within &0,4% oi the theoretical values. w i t h a Hitachi recording specrrophotom niatopraphy IVR' pertormed using To>o Rnahi So. 5 l A p a p e r In :in ascending technique nirh .olvcnt system? 1 1 i '7 -1'rOt-I rim( trateti SH4OH-H2O l?():i()::iJ ) i-PrOH-concenl r2itc.d P was carried o u t in 0.05 NH40H-H20 \::]:?I. Pap !.I phoaphare I i u t t e r IpH 10 cm and tor 1 hr. anti t h e mobilit c. represen -e value t n that of ALII'. Ikterminat ion of whole phosphorus !vas done arcording T o f tic. method of .411e11"~ and that of inorganic phcxphat e wah accordin:: t o The method of F'iske and Suhbarou..'; Sodium metaperiodate \mi used t o confirm the prehence of the ~511-
hosphoryiariiin i J ) i-niicieiitidai.e was periormed a- fililo\v* g 01 the test sample dissolved in 5 0 . 2 , I ml oi 0.05 .2/ Tris-HVI 0.2 rnl cii 0.i)lri 51 41gClz. and 0 . 2 in1 of a solution 01 ( o + r ' , r .'n,,'r? !Sit!mki ('hemicai ( ~ 1 - l . . L t d . ) 1,O.d nig ml of 'l'ris tfCi
Notes
Journal of Medicinal Chemistry, 1973, vel. 16, No. 12 1391
mol (theoretical, 1.00 mol). The sodium metaperiodate-benzidine buffer) was incubated at 37“ for 15 hr and was added to 0.2 ml of test confirmed the presence of the 2’,3’-cis-glycol function. The 55% cold trichloroacetic acid. After centrifugation a t 3000 rpm for products corresponding to the bands, Rt 0.35 and 0.18, must be 10 min, the supernatant (1 ml) was submitted to the inorganic the 5’-diphosphate analog and 2’,3’-phosphorylated analog(s) exphosphate determination. Under the conditions, AMP and ADP amined by paper chromatography, paper electrophoresis, and the liberated 0.95 and 1.96 mol of inorganic phosphate, respectively, sodium metaperiodate-benzidine test. while adenosine 2’,3’-monophosphate liberated less than 0.04 mol Platelet Aggregation Test. Platelet aggregation studies were of inorganic phosphate. performed on buffered platelet-rich plasma from rabbit citrated 2-n-Amylthioadenosine 5’-Monophosphate (2a). 2-n-Amylblood and platelet-rich plasma from human citrated blood, using thioadenosine (la),l 92.5 mg (0.25 mmol), was suspended in 0.25 the optical density method reported before.’ Platelet-rich plasma ml of acetonitrile and 0.10 ml (1.2 mmol) of pyridine, and the from a male rabbit was immediately buffered with an equal volmixture was cooled to 0”. To the suspension was added 0.10 ml ume of isotonic barbital buffer (pH 7.3), and the mixture was (1.1 mmol) of POC13 and 0.010 ml (0.5 mmol) of H20, and the used as buffered platelet-rich plasma. homogeneous mixture was stirred at 0” for 2 hr. It was poured As the barium salts of 2a-c were insoluble in saline, ammointo 5 ml of ice-water and was adjusted to pH 1 with 2 N HCI to nium salts were used in platelet aggregation studies. The ammoafford a white precipitate. The precipitate was separated by cennium salt of each nucleotide to be tested was prepared from the trifugation a t 2000 rpm for 10 min and was redissolved in 0.5 ml filtrate through the millipore filter of the NHJOH extract of the of concentrated NH4OH and submitted to preparative paper spot corresponded to the nucleotide. Concentration of each nuclechromatography using solvent 1. Three bands ( R , 0.51, 0.42, and otide in saline was determined by optical density. The extract of 0.29) were detected on the chromatogram by uv ray. The fastest filter paper by dilute NH40H did not contain any substances moving major band ( R , 0.51) was cut out and extracted with 0.05 that affect platelet aggregation, and the inhibitory effect of the iV NH40H and the extract was evaporated to a small volume. In N H 4 0 H extract of 2a-c on paper chromatograms could be attriborder to eliminate minor contaminants and impurities it was puuted to the ammonium salts of 2a-c. rified again by the same preparative chromatography. The single band was extracted by 0.05 N ”4OH and was evaporated to Acknowledgments. The authors wish to express their dryness and redissolved in about 2.0 mi of H20, which was subsesincere thanks to Mrs. R. Toyoshima for her technical asquently filtered through a millipore filter (0.22 I * ) . To the filtrate sistance of the platelet aggregation studies and to the labwas added 35 mg (0.08 mmol) of BaIz dissolved in 4 ml of EtOH. oratory staffs for collection of their blood. The white precipitate was separated by centrifugation a t 3000 rpm for 10 min and washed with HZO-EtOH (1:2) twice and fiReferences nally with EtOH. It was dried in cacuo at 50“ for 24 hr over PzO5. 2-n-Amylthio-AMP (2a) barium salt was obtained in a yield of (1) K. Kikugawa, H. Suehiro, and M.Ichino, J . Med. Chem., 13.5% (21.0 mg) based on la. Anal. ( C ~ ~ H Z Z O ~ N ~ S P B C, ~ . ~ H Z O )16,1381 (1973) (paper6). H, N, P. The content of inorganic phosphate was less than 0.01 (2) A. Gaarder, J . Jonsen, S. Laland, A. Hellem, and P. A. mol. Labile phosphate by 5’-nucleotidase was 0.995 mol (theoretiOwren, Nature (London), 192,531 (1961). cal, 1.00 mol). The sodium metaperiodate-benzidine test con(3) G. V. R. Born and M. J . Cross, J. Physiol.. 168.178 (1963). firmed the presence of the 2’,3’-cis-glycol function. (4) G. V. R. Born, A. J. Honour, and J . R. A. Michell, Nature 2-Benzylthioadenosine 5’-Monophosphate (2b). 2-Benzyl(London), 202,761 (1964). thioadenosine ( l b ) , l 105.5 mg (0.25 mmol), was suspended in 0.25 (5) G. V. R. Born and M. J. Cross, h o c . Phys. Soc.. London, 29 ml of acetonitrile and 0.10 ml of pyridine. The mixture was cooled (1962). to 0” and to this was added 0.10 ml of POCIS and 0.010 ml of (6) G. V. R. Born, Nature (London), 202,95 (1964). H20. After the mixture was stirred at 0” for 2 hr, it was mixed (7) K . Kikugawa, K. Iizuka, Y. Higuchi, H. Hirayama, and M. with 5 ml of ice-water and subsequently with 0.5 ml of 2 N HCI. Ichino, J. Med. Chem., 15,387 (1972). The white precipitate which separated was dissolved in concen(8) K. Kikugawa, K. Iizuka, and M. Ichino, ibid., 16,358 (1973). trated NH40H and submitted to preparative paper chromatogra(9) G. Gough, M. H. Maguire, and F. Michael, ibid., 12, 494 phy as described above. Three bands (Rt 0.49, 0.40, and 0.26) ( 1969). were detected on the chromatogram. The major band ( R , 0.49) (10) (a) F. Michael, M. H. Maguire, and G. Gough, Nature (Lonwas extracted with 0.05 N NH4OH and purified again by the don), 222, 1073 (1969); (b) British Patent 1,226,699 (1972); chromatography. The barium salt of 2-benzylthio-AMP (2b) was (c) U. S.Patent 3,678,162 (1972). obtained in a yield of 12.5% (20.0 mg) based on lb. Anal. (11) S. Ouchi, T. Yamada, M. Kurihara, S. Seno, and T. Sowa, (C17H1807N5SPBa.2Hz0)C, H, N , P. The content of inorganic Japanese Patent 11464 (1968); Chem. A b s t r . , 70, 4549d phosphate was less than 0.01 mol. Labile phosphate by 5’-nucleo(1969). tidase was 1.048 mol (theoretical, 1.00 mol). The sodium meta(12) M. A. Packham, N. G. Ardlie, and J . F. Mustard, Amer. J . periodate-benzidine test confirmed the presence of the 2’,3’-cisPh\siol., 217, 1009 (1969). glycol function. (13) M. A. Packham, M. A. Guccione, D. R. Perry, R. L. Kin2-Allylthioadenosine 5’-Monophosphate ( 2 c ) . 2-Allylthioadelough-Rathbone, and J . F. Mustard, ibid., 223, 419 (1972). nosine ( l c ) , l 374 mg (1.0 mmol), was suspended in 1.0 ml of ace(14) E. W. Salzman, D. A. Chambers, and L. L. Neri, Nature tonitrile and 0.39 ml (4.8 mmol.) of pyridine. The mixture was (London), 210,167 (1966). cooled to 0”and to this was added 0.40 ml (4.4 mmol) of PoC13 (15) M. C. Rozenberg and H. Holmsen, Biochirn. BiophJs. Acta, and 0.040 ml (2.0 mmol) of HzO. After the mixture was stirred a t 155,342 (1968). 0” for 4 hr, it was subsequently added to 5 ml of cold water. A (16) R. J . L. Allen, Biochem. J., 34,858 (1940). small amount of precipitate was removed by centrifugation, and (17) C. H. Fiske and Y. Subbarow, J. Biol. Chem., 66,375 (1925). the supernatant was acidified at pH 1 with 2 N HC1. It was then (18) J. A. Cifonelli and F. Smith, Anal. Chem.. 26,1132 (1954). absorbed on a column of 20 g of active carbon. The column, washed well with H20, was eluted with 300 ml of 10% KHIOHEtOH (1:l). The effluent was evaporated in cacuo to dryness and submitted to preparative paper chromatography as described above. Three bands ( R , 0.46, 0.35, and 0.18) were detected on the Synthesis of chromatogram, and the relative amounts of the products corre1,4-Bis(6-methoxy-8-quinolylaminoalkyl)piperazines as sponding to these three bands estimated by optical density (273 Potential Prophylactic Antimalarial Agents nm, pH 1) were 75, 8.5, and l6.S%, respectively. The major band ( R , 0.46) was cut out, extracted with 0.05 N NH40H, evaporated Kalidas Paul and C. DeWitt Blanton, Jr.* to dryness, and redissolved in 6.0 ml of HzO. It was subsequently filtered through a millipore filter and the filtrate was added to Department of Medicinal Chemistry, School of Pharmacy, 105 mg (0.25 mmol) of barium iodide dissolved in 12 ml of EtOH. Uniuersity of Georgia, Athens, Georgia 30602. The white precipitate which separated was collected by centrifuReceived June 25, 1973 gation. It was redissolved in 6.0 ml of HzO, filtered, and precipitated by the addition of 12 ml of EtOH. Finally it was washed Malaria continues to be a problem, despite the years of with EtOH twice and dried to afford 90.0 mg (yield, 15.5%) of 2and screening of thousands of compounds. allylthio-AMP (2c) barium salt. Anal. ( C ~ ~ H ~ ~ O T N ~research SPToday, drugs are available that interrupt the infection a t Ba.1.5Hz0) C, H, N, P. The content of inorganic phosphate was its various stages, but clinically useful prophylactic antiless than 0.01 mol. Labile phosphate by 5’-nucleotidase was 0.997
