39
January 1970 TABLEI v Yield, Compound
l-A\Iethy1-4,.5-diphenj-lpiperazine (3) 2-Methyl-1,3,4,13b-tetrahydro-2H-pyrazino[ 1,2-j]phenanthridine (4) 2-lIethyl-1,2,3,4,10,14b-hexah~-dropyrazino [ 1,231niorphanthridine ( 5 ) 2-PIIethyl-1,3,4,10,1l,l.jb-hesahydro-2H-dibenz [c,g]pyrazino[l,2-a]azocine (6)
Salt
%
BIp,
2HC1
85
224-226
CiiHz2CnNz
C, H, N
HCl
92
236-240
CiiHISClNn
C,H, N, C1
HCI
90
282-284
CisHziClS2
C, H, N, C1
HCl
43
305-310
CigHZ,Cl?jz
C, H, N, C1
heated during 43 min from room temperatiire to 140". The temperat,ure was then raised to 180"in 1.5 min and kept at this level for 0.5 hr. Diiring the reaction about 50 ml of EtOH distilled off. The reaction mixtiire was cooled and treated with 200 nil of CsH6. The crystals were filtered, xashed with Et,O, and dried in vacuo to give the diketopiperazino compounds listed in Table 111. Diborane Reduction of the Substituted Diketopiperazines 11, 15, 21, and 29 to the Piperazino Compounds 3-6.-To a suspension of 120 g ( 3 . 2 moles) of NaBH4 in 350 ml of dry T H F was added dropwise a solntion of 320 ml of BFI etherate during 2 hr. The generated BZHG was introduced directly with stirring into a
o c
Formula
Analyses
suspeiiaion of 100 g of the diketopiperazino compound. The entire manipulation was carried out. under S2. The mixture was then refluxed for 1 hr. Excess BZH,was decomposed by adding slowly 330 ml of 96Yc Et,OH, and the solution was evaporated to dryness. The vitreous residue was dissolved in 1800 ml of 187, aqueous HCl and heated on a steam bath for 1 hr, cooled, made alkaline with 30CC NaOH, and extracted with CHsCl?. The extract m-as n-abhed with H20, dried (Na2S04),and evaporated to dryness to give 90-9.54;c of the oily piperazino compound. The product wa* converted to its hydrochloride with alcoholic HCl tallized from EtOH. In this way the compounds listed in Table 11- were obtained.
Effect of Eight Prostaglandins on Platelet Aggregation' N. CHAXDRA SEKHAH~ Cui diovascular Daseases Research, The Upjohn Company, Kalainazoo, Jliclzigan
49001
Receiued A p r i l 14, 1969 Eight prostaglandins, PGE1, PGE2, PGAI, PGAz, PGFI,, PGFZ,, PGF16, and PGFq, were t,ested for their effect on platelet aggregation-adhesion induced by ADP, thrombin, and collagen. All compounds inhibited aggregation in platelet-rich rat plasma and human plasma t'o varying degrees. PGE, was the most active compound in the group. In addition, PGEI showed very pot'ent thrombolytic effect against ADP-induced platelet thrombi in uilro. h single intravenous injection of 3 mg of PGE1,'kg in rats inhibited platelet aggregation in blood samples withdrawn from animals 30 min following the injection. Platelet aggregation was also inhibited significant,ly in rats given infusions of PGE, at 1.8 mg/kg per day for 30 days.
Prostaglandins are powerful vasoactive compounds which occur in human seminal plasma, sheep seminal vesicles, and other tissues. Born and coworkers3have reported that a number of vasoactive compounds show a corresponding ability to affect platelet behavior. Since prostaglandin E1 (PGE1) is knolm to be a potent vasodilator, it was of interest to investigate the influence of PGEl and other prostaglandins on platelet aggregation. I1200
Time at which lysis occurred
>1200
401.0 i 33.0 182.0 i 6 . 7 ( - 85% (-67'%Ib i) 172.8 7.0 330.0 i 4 . 7 (-86%)' ( - 71%)b 1 136.8 =!c 3 . 9 340.3 i 17.4 ( --87%Ib (-72%)b 0.1 144.5 i 8 . 5 366.5 Z!C 31.0 ( - 88% Ib ( -69%Ib 0.05 235.0 i 1 6 . 5 715.0 f 50.2 ((-40%) 0.01 453.5 i 179.1 113.8 f 54.2 (-62%) (-7%) >1200 0.003 757.0 Z!C 15.1 (-37%) 0.001 1078.0 zt 45.6 > 1200 (-10%) a PGEl is added 30 sec after addition of ADP; lysis time in seconds is expressed as mean =tstandard error; four samples for each concentration of PGEI. * St'atistically significant; p < 0.05 by St,udent's t t,est; control value is taken as 1200 for calculating percentage reduction in experimental samples.
+
TABLE I1 INHIBITIOX O F Huhf.&sPLATELET ,%GGILEO.ITION BY PROSTAGLANDINS in Vitroa Compd, min effective concn (pdml)
------Platelet Vh
aggregation parametersb-
ss
PH
Control, 0 PGE,, 0.05
15.8 f 0 . 3 21.4 & 0 . 4 25.8 f 0.6 33.2 i 3 . 0 d d (+llO%)C PGEZ, 20.0 23.8 f 1 . 9 41.8 i 3 . 3 53.2 f 8 . 0 (+51%7,) (+95% 1" (+106%)' PGhi, 30.0 30.5 i 4 . 5 >43' d (+937,)c PGA?, 70 . 0 23.7 f 2 . 4 4 j . 3 =t8 . 5 43.8 i 5 . 0 (+SO%) (+113%Ic (+78%jC PGFlu, 70.0 23.2 f 2 . 2 49.8 i 7 . 5 >42e (+GO%? (+133 PGFip, 100.0 33.2 4 . 6 >4@ >706 ( 106% 1" PGFq, 100.0 3 2 . 5 f 6 . 3 >42" cl (+ 106%IC PGFZ,, 100.0 2 3 . 8 i 1 . 9 41.8 f 3 . 3 53.2 f 8 . 0 (+SI%) (+95%Y (+106%P .Twelve samples for controls and six samples each for prostaglandins. ADP-induced aggregation; time in seconds expressed as mean i standard error. Statistically significant, p < 0.05 by Student's t test. d The reaction did not occur. e The reaction occurred only in some of the samples; the lon-est value obtained in the group is shonn.
+
+
Since PGE1 was the most potent inhibitor of ADPand thrombin-induced platelet aggregation, the effect of the compound against collagen-induced adhesion was examined. The results presented in Figure 1 show that even a low concentration of PGEl (0.1 pg/ml) inhibits collagen-induced adhesion and aggregation of PRHP. Similar experiments with P R R P (Figure 2 ) showed that PGEl was equally active against collageninduced rat platelet aggregation a t 0.1 pg/ml. The inhibitory action of PGEl was concentration dependent in the range 0.05-0.2 pg/ml. Concentrations of 0.2
To investigate whether PGEl would affect platelet behavior in vivo, rats (six per group) were given a single intravenous injection of 3 mg of PGE,/kg or saline. Table IV shows that platelet aggregation in PRRP was significantly inhibited up to 30 min following injection. Lower doses of PGEl showed shorter duration of activity. I t was also observed that 3 mg of PGE,/kg was too high a dose for the animals to tolerate without overt signs of discomfort. Since single injections of lower doses did not produce significant inhibition of platelet aggregation and since single injections of high doses produced undesirable acute effects, the effect of continuous infusion of a low dose of PGEl (0.18 mg/kg infused over a 5-min period, ten times each day) for 30 days was investigated in rats. Results in Table V show that VA, SS, and P H were significantly inhibited in the PGEl-infused rats. Results obtained with rats given PGE1 orally showed that the compound did not affect platelet aggregation of animals even a t massive doses of 10 mg/kg body weight.
Y
w R
25
L
8
Discussion
I t was reported ti) Iiloeze4that W E 1 inhibits SUPilrduced aggregation of rat and human platelets. Hc ; ~ l observed ~o that PGE? accelerates ADP-induced aggregation of rat platelets and that the compound showed 110 ~igiiificiiritrffect on human platelet s. Ehmons aiid cuIvorlierb8 hxve confirmed Iiloeze’s finding:, with I’( Xi :&lidhave further reported that topical applicatiorii uf PGE1 solution arid iiitraveiious irijectiori of I’GE1 temporarily inhibited “injury-induced platelet thrombi formation” in cerebral cortical arteries of rtibbits. Our irivestigatioii on the effect of prostaglandins o n platelet aggregat 11 induced by three agents (,lDP, thrombin, or collagen) showed that a11 prostaglalidiris pos:,ehsed some degree of inhibitor\ activitj , though PGE1 waq by far the most potent compound in the group. Data in Table. I arid I1 :,how that at least two of the prostaglandins (PGIS, and PGFI,) are more active in PRRI’ than in PRHI’. In addition, I’G& iq active :kt 70 pg/ml agrtiiist human and rat platelet aggrtptioii (T:hleh T and TI). whereas it is inactive agairiiit rabbit pltttelct aggregatioii even at 100 pg/ml.These ohseivatioris suggeht that tlie diff erent prostagI:ttidiiis show L: poshible :,pccie.: difference in their inhibition of platelet aggregation.
10
2
6
f’(;EL, though riot a z active j’C+l+;~,inhihitetl platelet aggregation of rat arid humaii pla~~llu:, itt conceiitratiorii of 12.3 arid 20 pg/nil, respcctiveIy ; l o n c ~ r coiicentration. ot iYiE2 ( 3 to 0.1 pg:nil) I I I O W C ~ :t slight pot eiit ia t iiig eft’ect on XDP-inducetf rat plat aggregatioii. l’(il
