Don N. Harris

1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009)—a potent new inhibitor of cyclic 3′,5′-nucleotide phosphodiesterases☆

Abstract The properties of SQ 20009 [1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl] as a cyclic nucleotide phosphodiesterase inhibitor have been investigated. The phosphodiesterase preparations used in this study were ammonium sulfate-fractionated supernatants of homogenates of rat brain, rabbit brain, rat adrenal, rat lipocyte and cat heart; commercially available beef heart phosphodiesterase was also studied. The concentrations of SQ 200009 required to inhibit these phosphodiesterase activities 50 per cent were 2·0, 4·8, 20, 21, 27 and 60 μM, respectively, using 1·6 × 10−7 M cyclic AMP as substrate. SQ 20006 (the parent of SQ 20009 lacking the 4-isopropylidene moiety), theophylline and caffeine were also tested against all six enzyme preparations. Whereas SQ 20009 was more potent than SQ 20006 using the phosphodiesterase prepared from rat adrenal, the potencies were reversed when the lipocyte enzyme was used. SQ 20009 was approximately 60 and 75 times as potent an inhibitor of rat brain cyclic AMP phosphodiesterase as were theophylline and caffeine respectively. The kinetic properties of the phosphodiesterases prepared from rat brain, cat heart and beef heart were also investigated. Using the rat brain enzyme, two Km values for cyclic AMP, 4·0 × 10−6 and 1·2× 10−4 M and a single Km, 2·0 × 10−5 M, for cyclic GMP were confirmed. The Ki of SQ 20009 against the low Km cyclic AMP phosphodiesterase was 2·0 × 10−6 M and that for cyclic GMP hydrolysis was 2·4 × 10−5 M. The inhibition by SQ 20009 of the hydrolysis of both cyclic nucleotides by both the rat brain and beef heart phosphodiesterases was competitive. The cat heart cyclic nucleotide phosphodiesterase was inhibited non-competitively by SQ 20009; the Ki for cyclic AMP hydrolysis was 6·4 × 10−5 M, and the Ki for cyclic GMP hydrolysis was 3·0 × 10−5 M. The inhibition by SQ 20009 of cyclic AMP hydrolysis by both the rat brain and cat heart preparations was reversible.

Chronic antihypertensive effects of captopril (SQ 14,225), an orally active angiotensin I-converting enzyme inhibitor, in conscious 2-kidney renal hypertensive rats

Indirect systolic blood pressure (SBP) was monitored in 9 groups of 15 male conscious 2-kidney renal hypertensive rats (RHR) for over 6 months. Daily oral dosing with captopril (SQ 14,225, D-3-mercapto-2-methylpropanoyl-L-proline, 30 mg/kg), an orally active angiotensin I-converting enzyme inhibitor, lowered SBP 30--50 MM Hg during this period. Withdrawal of captopril for 5 days at 1, 3 and 6 months resulted in gradual return of SBP to control levels without overshoot. Resumption of dosage with captopril again decreased SBP. Daily oral dosing with hydrochlorothiazide (HCTZ, 6 mg/kg/day) alone for 6 months had little or no effect on SBP, but increased the antihypertensive effect of captopril. Daily oral dosing with hydralazine (6 mg/kg) caused an initial marked antihypertensive effect greater than that of captopril but almost complete tolerance developed within 4 weeks of dosing. Highest survival rates occurred in RHR treated with captopril plus HCTZ. In four other similarly treated groups of RHR and normotensive rats (NR), least cardiac hypertrophy and highest plasma renin activity occurred in captopril-treated animals compared with vehicle-treated controls. Plasma renin activity was about 2 to 4 fold higher in the rats dosed with captopril compared with vehicle-treated rats. Heart weight/body weight ratios, initially higher in the two RHR groups compared to NR, decreased only in the captopril treated group to or near those of the NR groups. These results indicate that chronic treatment with captopril decreased SBP and cardiac weights of RHR, and that HCTZ, or possibly other diuretics, can augment the antihypertensive effect of captopril while having little or no effect by themselves.

Hypotension induced by inhibition of angiotensin-converting enzyme in pentobarbital-anesthetized dogs

The mechanism of the hypotensive response produced by inhibition of the angiotensin converting enzyme was studied in pentobarbital anesthetized dogs. A recently developed potent inhibitor of the converting enzyme, SQ 14,225 (D-3-mercapto-2-methyl propanoyl-L-proline), administered i.v. to intact dogs resulted in a rapid marked decrease in blood pressure. In nephrectomized dogs, SQ 14,225 retained significant hypotensive activity, although the absolute magnitude of the decreases in blood pressure were less than had been observed in dogs with intact kidneys. SQ 14,225 also lowered blood pressure when administered to intact dogs in which angiotensin II receptors had been blocked with the receptor antagonist Sar1,Ala8-angiotensin II. This apparent ability of SQ 14,225 to decrease blood pressure in the absence of a functional renin angiotensin system was shared by a structurally dissimilar, nonapeptide, angiotensin converting enzyme inhibitor, SQ 20,881 (Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro). SQ 20,881 also produced significant decreases in blood pressure in nephrectomized dogs. These findings indicate that the angiotensin converting enzyme inhibitors, SQ 14,225 and SQ 20,881 may lower blood pressure in anesthetized normotensive dogs via a mechanism unrelated to either the renin angiotensin system or the renal kinin system.

Effect of cyclic nucleotides on activity of cyclic 3′,5′-adenosine monophosphate phosphodiesterase

Abstract A series of cyclic 2′,3′-nucleotides, cyclic 3′,5′-nucleotides and derivatives of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) with a substituent at the C-8 position were investigated as inhibitors of partially purified cyclic AMP phosphodiesterases (PDE) of cat heart and rat brain. The assays were carried out at a substrate concentration (0.06 μM) where the contribution to the total enzyme activity by phosphodiesterases with K m values for cyclic AMP above 100 μM was insignificant; consequently the activity measured was that of low K m enzymes. Cyclic 3′,5′-guanosine monophosphate (cyclic GMP) and cyclic 3′,5′-inosine monophosphate (cyclic IMP) were shown to be the most potent inhibitors of PDE of cat heart (I 50 = 1 and 2 μ M, respectively). Of the cyclic AMP derivatives tested that have a substituent at the C-8 position, 8-bromo cyclic AMP was the most potent inhibitor; next most potent were the derivatives with a sulfur atom, whereas derivatives with oxygen- or nitrogen-containing substituents were the least potent inhibitors of PDE of cat heart or rat brain. Most of the cyclic nucleotides that were tested were more potent inhibitors of the PDE of cat heart than that of PDE of rat brain. The kinetic properties of PDE of cat heart were also investigated in the presence of cyclic GMP, cyclic IMP and 8-bromocyclic AMP. All three compounds were found to be competitive inhibitors, with apparent K i values of 0.51, 2.3 and 20 μM respectively. The possible pharmacologic role of cyclic nucleotides is discussed.

Effects of SQ 14,225, an orally active inhibitor of angiotensin-converting enzyme on blood pressure, heart rate and plasma renin activity of conscious normotensive dogs.

Oral administration of SQ 14,225 (0.03--3 mg/kg) to conscious normotensive dogs caused inhibition of the pressor response to intravenously administered angiotensin I (AI), the duration of which was dose-dependent. All doses of 0.1 mg/kg or greater caused 85--95% inhibition 30 min after administration whereas 0.03 mg/kg produced only a 25% inhibition. Pressor responses to angiotensin II (AII) were not similarly inhibited. Blood pressure was moderately reduced in a dose-related manner and followed the same pattern as inhibition of the AI pressor responses. The maximum change occurred after 1.0 mg/kg with only a more rapid onset occurring after the 3.0 mg/kg dose. Heart rate was not appreciably changed. SQ 14,225 also increased plasma renin activity (PRA), the levels and duration of which were dose-related. These data indicate that SQ 14,225 is an orally effective, potent inhibitor of angiotensin I-converting enzyme (ACE) in dogs. It appears that in mongrel dogs, ACE inhibition results in a slight to moderate reduction in blood pressure and an increase in PRA.

The Effects of Captopril, Propranolol, and Indomethacin on Blood Pressure and Plasma Renin Activity in Spontaneously Hypertensive and Normotensive Rats

Summary Captopril reduced blood pressure and increased PRA in both SHR and NTR. Both propranolol and indomethacin caused significant reductions in resting PRA of both SHR and NTR but only propranolol caused an inhibition of the PRA changes caused by captopril. Furthermore, since propranolol was highly effective only in SHR and captopril decreased blood pressure much more in SHR than NTR, the increase in PRA caused by captopril in SHR was probably reflexly mediated by compensatory sympathetic activation, all of which actions were a consequence of reducing angiotensin II formation.

9α-homo-9,11-epoxy-5,13-prostadienoic acid analogues: Specific stable agonist (SQ 26,538) and antagonist (SQ 26,536) of the human platelet thromboxane receptor

A newly synthesized 9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26, 536, (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I50 value of 1.7 microM. SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH2 and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A50 value of 2.5 microM. SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a Ki value of 3 microM. Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH2-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.

Effects of SQ 27,427, a thromboxane A2 receptor antagonist, in the human platelet and isolated smooth muscle

The TxA2 receptor antagonist properties of SQ 27,427 [a cyclohexylcarbinol-7-oxabicyclo(2.2.1)heptenoic acid analog] were studied in vitro both in the human platelet and various isolated smooth muscle preparations. SQ 27,427 was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid, ADP, epinephrine, collagen and the stable TxA2 agonists 9,11-azoPGH2 and SQ 26,655. Inhibition of platelet aggregation was achieved at concentrations of SQ 27,427 which did not alter TxB2 levels. SQ 27,427 was found to weakly inhibit the formation of TxB2 from arachidonic acid and had no effect on the synthesis of PGE2 or PGI2 from arachidonic acid. SQ 27,427 was also found to be a weak stimulator of platelet adenylate cyclase, being 1000 times less potent than PGI2. In isolated smooth muscle experiments, SQ 27,427 was shown to be a potent and specific TxA2 receptor antagonist. It caused competitive antagonism of 9,11-azoPGH2-induced contractions of vascular, respiratory and gastrointestinal smooth muscles. This antagonism was specific, as responses to norepinephrine, serotonin, PGE2, PGI2, PGF2 alpha, histamine, carbachol and KCl were not altered by SQ 27,427.

Thromboxane receptor antagonist properties of SQ 27,427 in the anesthetized guinea pig

The TXA2 receptor antagonist properties of SQ 27,427 (a novel oxabicyclo[2.2.1]heptane derivative) were studied in vivo in the anesthetized guinea pig where changes in pulmonary resistance, dynamic compliance, and mean arterial blood pressure were measured. Both the bronchoconstrictor and pressor responses to arachidonic acid (AA) and to the stable TXA2 mimic 9,11-azoPGH2 (AZO) were taken as indices of in vivo TXA2 receptor activation. The administration of SQ 27,427 (0.1-1.0 mg/kg i.v., and 10.0 mg/kg p.o.) caused dose-related inhibitions of both AA- and AZO-induced bronchoconstriction. Relative specificity of this antagonism was evidenced by the failure of SQ 27,427 (1.0 mg/kg i.v.) to inhibit histamine-induced bronchoconstriction. In the same experiments the pressor response to AA was reversed to a depressor response by SQ 27,427. This reversal was abolished by indomethacin. The pressor response to AZO was antagonized by SQ 27,427, but not by indomethacin. The reversal of the pressor response to AA by SQ 27,427 may be due to the unmasking of the depressor effect of a cyclooxygenase product, i.e., prostacyclin. It is concluded that SQ 27,427 is a relatively specific TXA2 receptor antagonist in vivo in the guinea pig.

Some biochemical activities of 10,10-Difluoro-13-dehydroprostacyclin, a chemically stable analog of prostacyclin, in human blood platelets

Abstract 10,10-Difluoro-13-dehydroprostacyclin (DF 2 -PGI 2 ) is a chemically stable analog of prostacyclin (PGI 2 ). DF 2 -PGI 2 was 6 times more potent than prostaglandin E 1 (PGE 1 ), and 1/10 as potent as PGI 2 as an inhibitor of arachidonic acid (AA)-induced platelet aggregation. DF 2 -PGI 2 also inhibited platelet aggregation induced by collagen, ADP, epinephrine and two thromboxane agonists, 9,11-azoPGH 2 and SQ 24,810 (9,11-epoxy-9α-homo-5(Z), 13(E)-15β-hydroxyprostadienoic acid, racemic mixture). The antiaggregatory effects by DF 2 -PGI 2 and PGI 2 on AA-induced platelet aggregation were potentiated by etazolate (SQ 20,009), a potent inhibitor of cyclic AMP phosphodiesterase (PDE), and were blocked by SQ 22,536 (9-(tetrahydro-2-furyl) adenine), an inhibitor of adenylate cyclase (AC). DF 2 -PGI 2 was also more potent than PGE 1 , and less potent than PGI 2 as a stimulator of AC activity in platelet homogenates. DF 2 -PGI 2 - and PGI 2 -stimulated AC activity was inhibited by a series of AC inhibitors, SQ 22,536, SQ 4,647 (9-furfuryl adenine) and 2′,5′-dideoxyadenosine. Furthermore, SQ 22,536 inhibited DF 2 -PGI 2 - and PGI 2 -stimulated AC activity in a concentration-related manner, with 150 values of 150 and 300 μM, respectively. Finally, PGE 1 , DF 2 -PGI 2 and PGI 2 , at 0.35 μM induced 2-, 10- and 17-fold increases, respectively, in cyclic AMP levels of intact platelets in platelet rich plasma (PRP). The increase in cyclic AMP levels by PGI 2 and DF 2 -PGI 2 in PRP was also blocked by SQ 22,536. We conclude that DF 2 -PGI 2 , like PGI 2 and PGE 1 , inhibits platelet aggregation by elevating platelet cyclic AMP levels.