Synthesis and molecular modeling of novel tetrahydro-β-carboline derivatives with phosphodiesterase 5 inhibitory and anticancer properties

New derivatives based upon the tetrahydro-β-carboline-hydantoin and tetrahydro-β-carboline-piperazinedione scaffolds were synthesized. All compounds were evaluated for their ability to inhibit PDE5 in vitro, and numerous compounds with IC(50) values in the low nanomolar range were identified including compounds derived from l-tryptophan. Compounds with high potency versus PDE5 were then evaluated for inhibitory activity against other PDEs to assess isozyme selectivity. Compound 5R,11aS-5-(3,4-dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)dione 14 showed a selectivity index of >200 for cGMP hydrolysis by PDE5 versus PDE11. Meanwhile, 6R,12aR-6-(2,4-dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4dione 45 demonstrated strong potency for inhibition of PDE11 with an IC(50) value of 11 nM, representing the most potent PDE11 inhibitor thus far reported. Docking experiments differentiated between active and inactive analogues and revealing the conformational, steric, and lipophilic necessities for potent PDE5 inhibition. Many derivatives, including potent PDE5 inhibitors, were able to inhibit the growth of the MDA-MB-231 breast tumor cell line with low micromolar potency.     

Synthesis and pharmacological evaluations of sildenafil analogues for treatment of erectile dysfunction

The 5-[2-ethoxy-5-(4-methylpiperazin-1-ylsulfonyl)phenyl]-1-methyl-3-propyl-1,6-dihydro-7 H-pyrazolo[4,3-d]pyrimidin-7-one, sildenafil, is a cGMP-specific phosphodiesterase-5 (PDE5) inhibitor used for penile erectile dysfunction. In the search for more potent and selective PDE5 inhibitors, new sildenafil analogues (6a-v), characterized by the presence on the sulfonyl group in the 5' position of novel N-4-substituted piperazines or ethylenediamine moiety, were prepared by traditional and microwave-assisted synthesis and tested in rabbit isolated aorta and corpus cavernosum. Similarly to sildenafil, several analogues showed IC50 values in the nanomolar range. In the in vitro studies, all the tested compounds caused concentration-dependent relaxations in both rabbit isolated aorta and corpus cavernosum. All sildenafil analogues potentiated the nitric oxide-dependent vasodilation in endothelium-intact rabbit aorta. Compound 6f exhibited great pEC50 value in corpus cavernosum, and compounds 6r and 6u in isolated aorta were found as potent as sildenafil for inhibiting PDE5. Because several analogues were significantly more lipophilic than sildenafil, these compounds may offer a new lead for development of new sildenafil analogues.     

Synthesis and complement inhibitory activity of B/C/D-ring analogues of the fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy- 2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran

This paper reports the synthesis and the bioassay of 4-methoxy- and 4-hydroxyspiro[benzofuran-2(3H)-cyclohexane] partial analogues (5) of the complement inhibitory sesquiterpene fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy-2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a, K-76) and its silver oxide oxidized product (1b, K-76COOH). The described target compounds represent spirobenzofuran B/C/D-ring analogues lacking the A-ring component of the prototype structure. The target compounds were evaluated by the inhibition of total hemolytic complement activity in human serum. It was observed that the structurally simplified analogue 4-methoxyspiro[benzofuran-2(3H)-cyclohexane]-6-carboxylic acid (5a) exhibited an IC(50) = 0.53 mM similar to the IC(50) = 0.57 mM that was observed for the natural product derivative 1b. Exhibiting an IC(50) = 0.16 mM, the three-ringed partial structure 6-carboxy-7-formyl-4-methoxyspiro[benzofuran-2(3H)-cyclohexane] (5k)was found to be the most potent target compound. Like the natural product, 5k appears to inhibit primarily at the C5 activation step and inhibits both the classical and alternative human complement pathways. Several other analogues inhibited complement activation in vitro at concentrations similar to those required for inhibition by the natural product 1b.     

Bifunctional [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues substituted at position 3 with alkylated phenylalanine derivatives yield potent mixed mu-agonist/delta-antagonist and dual mu-agonist/delta-agonist opioid ligands

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2',6'-dimethyl-l-tyrosine) analogues, containing alkylated Phe3 derivatives, 2'-monomethyl (2, 2'), 3',5'- and 2',6'-dimethyl (3, 3', and 4', respectively), 2',4',6'-trimethyl (6, 6'), 2'-ethyl-6'-methyl (7, 7'), and 2'-isopropyl-6'-methyl (8, 8') groups or Dmt (5, 5'), had the following characteristics: (i) [Xaa3]EM-2 analogues exhibited improved mu- and delta-opioid receptor affinities. The latter, however, were inconsequential (Kidelta = 491-3451 nM). (ii) [Dmt1,Xaa3]EM-2 analogues enhanced mu- and delta-opioid receptor affinities (Kimu = 0.069-0.32 nM; Kidelta = 1.83-99.8 nM) without kappa-opioid receptor interaction. (iii) There were elevated mu-bioactivity (IC50 = 0.12-14.4 nM) and abolished delta-agonism (IC50 > 10 muM in 2', 3', 4', 5', 6'), although 4' and 6' demonstrated a potent mixed mu-agonism/delta-antagonism (for 4', IC50mu = 0.12 and pA2 = 8.15; for 6', IC50mu = 0.21 nM and pA2 = 9.05) and 7' was a dual mu-agonist/delta-agonist (IC50mu = 0.17 nM; IC50delta = 0.51 nM).     

Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity

The inhibitory potential of novel anti-platelet aggregatory cilostamide analogues on phosphodiesterase (PDE) isozyme activities was investigated with recombinant PDE isozymes expressed in a baculovirus/ Sf9 expression system. The recombinant enzymes (PDE1-PDE5 and PDE7) showed Km values and sensitivities to selective inhibitors similar to those reported previously for native enzymes purified from tissues. The cyclooctylurea derivative OPC-33540 (6-[3-[3-cyclooctyl-3-[(1R*,2R*)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone) inhibited recombinant PDE3A (IC50 = 0.32 nM) more potently and selectively than the classical PDE3 inhibitors cilostamide, cilostazol, milrinone, and amrinone. The cyclopropylurea derivative OPC-33509 [(-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone] was less potent (IC50 = 0.10 microM) than OPC-33540, demonstrating that the cyclooctyl moiety was important for a potent inhibitory effect. In platelets, OPC-33540 potentiated cyclic AMP accumulation concentration-dependently in both the absence and the presence of 3 nM prostaglandin E1 (PGE1) (doubling concentrations: 32.5 and 6.2 nM, respectively). OPC-33540 inhibited thrombin-induced platelet aggregation potently (Ic50 = 27.8 nM). The anti-platelet aggregation effect also was stimulated in the presence of 3 nM PGE1 (IC50 = 6.0 nM). There was a good correlation between the IC50 values of PDE3 inhibitors in this study for recombinant PDE3A activity and their IC50 values for thrombin-induced platelet aggregation (r = 0.998). These data demonstrated that OPC-33540 is a highly selective and potent PDE3 inhibitor and a useful probe for identification of the intracellular functions of PDE3.     

Structure-activity studies of the melanocortin peptides: discovery of potent and selective affinity antagonists for the hMC3 and hMC4 receptors

We have designed and synthesized several novel cyclic SHU9119 analogues (Ac-Nle4-[Asp5-His6-DNal(2')7-Arg8-Trp9-Lys10]-NH2) modified in position 6 with nonconventional amino acids. SHU9119 is a high affinity nonselective antagonist at hMC3R and hMC4R with potent agonist activity at hMC1R and hMC5R. We measured the binding affinity and agonist potency of the novel analogues at cloned hMC3R, hMC4R, and hMC5R receptors and identified several selective, high affinity hMC3R and hMC4R antagonists. Compound 4 containing Che substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.48 nM) with 100-fold selectivity over hMC3R antagonist. Analogue 7 with a Cpe substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.51 nM) with a 200-fold selectivity vs the hMC3R. Interestingly, analogue 9 with an Acpc residue in position 6 is a high affinity hMC3R antagonist (IC50 = 2.5 nM) with 100-fold selectivity vs the hMC4R antagonist based on its binding affinities. This compound represents the first cyclic lactam antagonist with high selectivity for the hMC3R vs hMC4R. To understand the possible structural basis responsible for selectivity of these peptides at hMCR3 and hMCR4, we have carried out a molecular modeling study in order to examine the conformational properties of the cyclic peptides modified in position 6 with conformationally restricted amino acids.     

Inhibitory effects of quercetin derivatives on phosphodiesterase isozymes and high-affinity [<Superscript>3</Superscript><Emphasis Type="Italic">H</Emphasis>]-rolipram binding in guinea pig tissues

Rolipram has high (PDE4(H)) and low (PDE4(L)) affinities for phosphodiesterase (PDE)-4, respectively. In general, it is believed that inhibitions by PDE4(H) and PDE4(L) are respectively associated with an adverse response and with anti-inflammatory and bronchodilating effects. This has provided a rational basis for designing new compounds with high PDE4(H)/PDE4(L) ratios. In the present study, we attempted to determine the PDE4(H)/PDE4(L) ratios of quercetin (1), qercetin-3-O-methylether (3-MQ, 2), quercetin-3,7,4'-O-trimethylether (ayanin, 3), quercetin-3,7,3',4'-O- tetramethylether (QTME, 4), quercetin-3,5,7,3',4'-O-petamethylether (QPME, 5), quercetin-3,5,7,3',4'-O-pentaacetate (QPA, 6), and quercetin-3-O-methyl-5,7,3',4'-O-tetraacetate (QMTA, 7). The activities of PDE1 approximately 5, which were partially separated from homogenates of guinea pig lungs and hearts, were measured by a two-step procedure using adenosine 3',5'-cyclic monophosphate (cAMP) with [(3) H]-cAMP or guanosine 3',5'-cyclic monophosphate (cGMP) with [(3) H]-cGMP as substrates. The IC(50) values of all of these compounds except quercetin (1), 3-MQ (2), and QMTA (7) on PDE1 approximately 5 inhibition were determined. The anti-inflammatory effects of PDE4 inhibitors were reported to be associated with inhibition of PDE4 catalytic activity. Therefore, these IC(50) values for PDE4 inhibition were taken as the PDE4(L) values. The effective concentration (EC(50)), at which one half of the [(3) H]-rolipram bound to high-affinity rolipram binding sites (HARBSs) of brain cell membranes was replaced, was defined as the PDE4(H) value. In the present results, the PDE4(H)/PDE4(L) ratios of quercetin (1), ayanin (3), and QPME (5) were >30, >19, and 11, respectively (Table 1), which are higher than or equal to that of AWD12-281, the selective PDE4 inhibitor with the greatest potential currently undergoing clinical trials for treating asthma and chronic obstructive pulmonary disease.     

1-Arylnaphthalene lignan: a novel scaffold for type 5 phosphodiesterase inhibitor

1-Arylnaphthalene lignan, which had been reported as a PDE4 inhibitor by Iwasaki, was disclosed as a new structural class of PDE5 inhibitors. The structural requirements for potent and specific PDE5 inhibition were revealed in a 1-arylnaphthalene lignan series, in which 1-(3-bromo-4, 5-dimethoxyphenyl)-5-chloro-3-[4-(2-hydroxyethyl)-1-piperazinylcarbon yl]-2-(methoxycarbonyl)naphthalene hydrochloride (27q) showed the most potent and specific inhibition (PDE5 inhibition IC50 = 6.2 nM, selectivity for PDE5 against PDE1, -2, -3, and -4 >16 000). It is noteworthy that 27q has the best selectivities against PDE isoforms among PDE5 inhibitors so far reported. Compound 27q exhibited almost the same relaxant effects on rat aortic rings as sodium 1-[6-chloro-4-[(3, 4-methylenedioxybenzyl)amino]quinazolin-2-yl]piperidine-4-ca rboxylate (35) (27q, EC50 = 0.10 microM; 35, EC50 = 0.20 microM) and was selected for further biological evaluation.     

Ring substituted and other conformationally constrained tyrosine analogues of [D-Pen2,D-Pen5]enkephalin with delta opioid receptor selectivity.

The conformationally restricted, cyclic disulfide-containing delta opioid receptor selective enkephalin analogue [D-Pen2,D-Pen5] enkephalin (DPDPE) was modified by 2' (CH3) and 3' (I, OCH3, NO2, NH2) ring substitutions and by beta-methyl conformationally constrained beta-methyltyrosine derivatives in the 1 position. The potency and selectivity of these analogues were evaluated by bioassay in the mouse vas deference (MVD, delta receptor assay) and guinea pig ileum (GPI, mu receptor assay) assays and by radioreceptor binding assays in the rat brain using [3H]CTOP (mu ligand) and [3H][p-ClPhe4]DPDPE (delta ligand). The analogues showed highly variable potencies in the binding assays and in the bioassays. Aromatic ring substituents with positive Hammett constants had decreased potency, while substituents with negative Hammett constraints has increased potency for the opioid receptor. The most potent and most selective compound based on the binding was [2'-MeTyr1]DPDPE (IC50 = 0.89 nM and selectivity ratio 1310 in the binding assays). The 6-hydroxy-2-aminotetralin-2-carboxylic acid-containing analogue, [Hat1]DPDPE, also was highly potent and selective in both assays, demonstrating that significant modifications of tyrosine in enkephalins are possible with maintenance of high potency and delta opioid receptor selectivity. Of the beta-methyl-substituted Tyr1 analogues, [(2S,3R)-beta-MeTyr1]DPDPE was the most potent and the delta receptor selective. The results with substitution of beta-MeTyr or Hat instead of Tyr also demonstrate that topographical modification in a conformationally restricted ligand can significantly modulate both potency and receptor selectivity of peptide ligands that have multiple sites of biological activity.     

5,10-Methylenetetrahydro-5-deazafolic acid and analogues: synthesis and biological activities.

The synthesis of 5,10-methylene-5-deazatetrahydrofolic acid (2), a stable, rigid analogue of 5,10-methylenetetrahydrofolate (1), is reported as a potential inhibitor of thymidylate synthase. The target compound was obtained by a Fisher-indole type cyclization of the hydrazone 16 from 2-amino-6-hydrazino-4-oxopyrimidine (10) and diethyl N-[4-(3-formyl-1-pyrrolyl)benzoyl]-L-glutamate (15) followed by catalytic reduction of the product 17. Similarly, modification of the Fisher-indole type cyclization of the appropriate hydrazone precursors 11 and 12 afforded the nonclassical analogues 3-amino-7,8,9-trimethyl-2H-pyrrolo[3',4':4,5]pyrido[2,3-d]pyrimidin-1- one (4) and 3-amino-8-benzyl-7,9-dimethyl-2H-pyrrolo[3',4':4,5]pyrido [2,3-d]pyrimidin-1-one (5), respectively. The target compound 2, its aromatic precursor 18, and the nonclassical analogue 4 were evaluated as inhibitors of the growth of Manca human lymphoma cells and also as inhibitors of human dihydrofolate reductase, human thymidylate synthase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase. Compound 18 showed weak inhibition of lymphoma cell growth (IC50 = 42 microM) and of AICAR formylTF (IC50 = 17 microM). Compounds 2 and 4 did not inhibit lymphoma cell growth or thymidylate synthase. The inactivity of 2 was attributed to its lack of flexibility leading to its inability to bind to thymidylate synthase.     

Structure-activity relationships of novel cyclic alpha-MSH/beta-MSH hybrid analogues that lead to potent and selective ligands for the human MC3R and human MC5R

It has been shown by extensive studies that alpha-MSH bioactivity is critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, however with poor selectivity for the human MC3R-MC5R. The structure-activity relationships study here is aimed at identifying lead structures or templates of this core sequence by the use of different conformational constraints that might impart changes in its topography and thus promote differences in potency and selectivity at these receptors. Our peptide library consists of a novel series of cyclic alpha-MSH analogues that have disulfide bridges between Cys or Cys-like residues at positions 4 and 10, giving rise to 23-membered rings fused at the C-terminal end with the C-terminal fragment of beta-MSH (Pro-Pro-Lys-Asp). While such constraints of the peptide backbone with disulfide bridges of different chirality affect potency and selectivity at these receptors, further changes in the hydrophobicity at position 7 with either a D-Phe or D-Nal(2') and replacement of a His with a Pro in position 6 cause additional effects. Thus, the most interesting lead compounds that emerged from this study are (1) compound 5, Ac-c[Cys-Glu-His-D-Phe-Arg-Trp-D-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 10 nM), which is the first potent and highly selective antagonist ligand for the hMC5R (560-fold vs the MC3R and 1000-fold vs the MC4R); (2) compound 7, Ac-c[Cys-Glu-Pro-D-Nal(2')-Arg-Trp-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 31 nM), which is a highly selective antagonist analogue for the MC3R (560-fold vs the hMC4R and about 3000-fold vs the hMC5R; and (3) compound 9, Ac-c[Pen-Glu-His-D-Nal(2')-Arg-Trp-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 3 nM), which is more potent than 7 at the MC3R but not as selective.     

New 2,4-diamino-5-(2',5'-substituted benzyl)pyrimidines as potential drugs against opportunistic infections of AIDS and other immune disorders. Synthesis and species-dependent antifolate activity

In a continuing effort to design small-molecule inhibitors of dihydrofolate reductase (DHFR) that combine the enzyme-binding selectivity of 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP) with the potency of 2,4-diamino-5-methyl-6-(2',5'-dimethoxybenzyl)pyrido[2,3-d]pyrimidine (piritrexim, PTX), seven previously undescribed 2,4-diamino-5-[2'-methoxy-5'-(substituted benzyl)]pyrimidines were synthesized in which the substituent at the 5'-position was a carboxyphenyl group linked to the benzyl moiety by a bridge of two or four atoms in length. The new analogues were all obtained from 2,4-diamino-5-(5'-iodo-2'-methoxybenzyl)pyrimidine via a Sonogashira reaction, followed, where appropriate, by catalytic hydrogenation. The new analogues were tested as inhibitors of DHFR from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three life-threatening pathogens often found in AIDS patients and individuals whose immune system is impaired as a result of treatment with immunosuppressive chemotherapy or radiation. The selectivity index (SI) of each compound was obtained by dividing its 50% inhibitory concentration (IC(50)) against Pc, Tg, or Ma DHFR by its IC(50) against rat DHFR. 2,4-Diamino-[2'-methoxy-5'-(3-carboxyphenyl)ethynylbenzyl]pyrimidine (28), with an IC(50) of 23 nM and an SI of 28 in the Pc DHFR assay, had about the same potency as PTX and was 520 times more potent than TMP. As an inhibitor of Tg DHFR, 28 had an IC(50) of 5.5 nM (510-fold lower than that of TMP and similar to that of PTX) and an SI value of 120 (2-fold better than TMP and vastly superior to PTX). Against Ma DHFR, 28 had IC(50) and SI values of 1.5 nM and 430, respectively, compared with 300 nM and 610 for TMP. Although it had 2.5-fold lower potency than 28 against Ma DHFR (IC(50) = 3.7 nM) and was substantially weaker against Pc and Tg DHFR, 2,4-diamino-[2'-methoxy-5'-(4-carboxyphenyl)ethynylbenzyl]pyrimidine (29), with the carboxy group at the para rather than the meta position, displayed 2200-fold selectivity against the Ma enzyme and was the most selective 2,4-diamino-5-(5'-substituted benzyl)pyrimidine inhibitor of this enzyme we have encountered to date. Additional bioassay data for these compounds are also reported.     

Inactivation of atrial natriuretic factor-stimulated cyclic guanosine 3',5'-monophosphate (cGMP) in UMR-106 osteoblast-like cells

Previous studies have suggested a role of cyclic guanosine 3', 5'-monophosphate (cGMP) in the differentiation and proliferation of osteoblasts. We studied the effect of ANF (atrial natriuretic factor) on intracellular cGMP accumulation, cGMP efflux, and cGMP-phosphodiesterase (PDE) activity in UMR-106 osteoblast-like cells. ANF rapidly increased both intracellular cGMP and cGMP efflux. ANF-stimulated intracellular cGMP peaked at 2 min in the absence and at 10 min in the presence of 0.25 mM 3-isobutyl-1-methylxanthine. Probenecid, an antagonist of anion transport, blocked the efflux of cGMP (IC(50) = 0.1 mM), ruling out simple diffusion as a mechanism of the efflux. cGMP-PDE activity was increased threefold in crude homogenates from ANF-treated cells (IC(50) = 23 nM). ANF-evoked stimulation of cGMP-PDE activity was reached simultaneously with the peak in intracellular cGMP. Separation of the PDEs by Q-Sepharose chromatography revealed three cGMP-hydrolyzing peaks. The first peak was sensitive to the PDE5 (cGMP-specific PDE) isoenzyme-selective inhibitor zaprinast (IC(50) = 0.45 microM). The second peak was stimulated fourfold by the addition of calcium/calmodulin, indicating the presence of PDE1. The third peak was sensitive to the PDE2 (cGMP-stimulated PDE) isoenzyme-selective inhibitor 9-[2-hydroxy-3-nonyl]adenine (EHNA) (IC(50) = 3 microM), and was activated by over 300% in the presence of 4 microM cGMP. Our results show that ANF-stimulated cGMP is released from UMR-106 cells by a probenecid-sensitive mechanism. ANF also stimulates cGMP hydrolysis by activating cGMP-PDE activity. Three distinct cGMP-hydrolyzing PDEs, namely PDE5, PDE1, and PDE2, are present in the studied cells.     

Novel sst(4)-selective somatostatin (SRIF) agonists. 3. Analogues amenable to radiolabeling

After our discovery that H-c[Cys-Phe-Phe-DNal-Lys-Thr-Phe-Cys]-OH (ODN-8) had high affinity and marginal selectivity for human sst(3) (part 2 of this series: Erchegyi et al. J. Med. Chem., preceding paper in this issue)(11) and that H-c[Cys-Phe-Phe-DTrp-Lys-Thr-Phe-Cys]-OH (ODT-8, 3) had high affinity and marginal selectivity for human sst(4), that H-c[Cys-Phe-Tyr-D-threo-beta-Me2Nal-Lys-Thr-Phe-Cys]-OH had high affinity for all sst's except for sst(1), and that H-c[Cys-Phe-Tyr-L-threo-beta-Me2Nal-Lys-Thr-Phe-Cys]-OH had high affinity for sst(4) (IC(50) = 2.1 nM), with more than 50-fold selectivity toward the other receptors (parts 1 and 2 of this series: Rivier et al. and Erchegyi et al. J. Med. Chem., preceding papers in this issue), we found H-c[Cys-Phe-Phe-Trp-Lys-Thr-Phe-Cys]-OH (OLT-8, 2), H-c[Cys-Phe-Phe-L-threo-beta-MeTrp-Lys-Thr-Phe-Cys]-OH (4) and H-c[Cys-Phe-Phe-D-threo-beta-MeTrp-Lys-Thr-Phe-Cys]-OH (5) to have very high affinity for sst(4) (IC(50) = 0.7, 1.8, and 4.0 nM, respectively) and 5- to 10-fold selectivity versus the other sst's. From earlier work, we concluded that an l-amino acid at position 8 and a tyrosine or 4-aminophenylalanine substitution at position 7 may lead to high sst(4) selectivity. In fact, [Tyr(7)]-2 (6) and [Tyr(7)]-3 (7) show ca. 5-fold selectivity for sst(4), and [Aph(7)]-2 (8) and [Aph(7)]-3 (9) have high sst(4) affinity (IC(50) = 1.2 and 0.88 nM, respectively) and selectivity, suggesting that indeed an l-residue at position 8 will direct selectivity toward sst(4). Unexpectedly, [Ala(7)]-2 (10) and [Ala(7)]-3 (11) have very high sst(4) affinity (IC(50) = 0.84 and 0.98 nM, respectively) and selectivity (>600- and 200-fold, respectively). The combination of Tyr(2) and dTrp(8) in analogues 14 and 22 did not affect the affinity of the analogues for sst(4) (IC(50) = 1.2 and 1.1 nM, respectively) but resulted in loss of selectivity, whereas the combination of Tyr(2) and LTrp(8) in H-Tyr-c[Cys-Phe-Aph-Trp-Lys-Thr-Phe-Cys]-OH (13) and H-Tyr-c[Cys-Phe-Ala-Trp-Lys-Thr-Phe-Cys]-OH(19) retained high affinity (IC(50) = 1.9 and 1.98 nM, respectively) and sst(4) selectivity (>50 and >250, respectively). Interestingly, the same substitutions at positions 2 and 7, with l-threo-beta-MeTrp at position 8, yielded a much less selective analogue (20). Carbamoylation of the N-terminus of most of these analogues resulted in slightly improved affinity, selectivity, or both. Other amino acid substitutions in this series, such as those with Amp (25, 26), Orn (27), or IAmp (29) at position 7, were also tolerated but with a 2- to 3-fold loss of affinity and concomitant loss of selectivity. Analogous peptides with a tyrosine at position 11 (31-36) were less selective than the corresponding peptides with a tyrosine at position 2. Several analogues in this series compared favorably with the non-peptide L-803,087 (37) in terms of affinity and selectivity. Analogues 8, 10, and 21 potently inhibited the forskolin-stimulated cAMP production in sst(4)-transfected cells, therefore acting as full agonists. Cold monoiodination of 19 yielded 21, with retention of high sst(4) selectivity and affinity (IC(50) = 3.5 nM). (125)Iodinated 19 selectively binds to sst(4)-transfected cells but not to sst(1-3)- or sst(5)-transfected cells. Binding in sst(4)-transfected cells was completely displaced by SRIF-28 or the sst(4)-selective L-803,087.     

5-Amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indoles: relationships between structure and cytotoxicity for analogues bearing different DNA minor groove binding subunits.

A series of 5-amino-seco-CBI compounds, designed for use as effectors for prodrugs, were prepared to study structure-activity relationships for the cytotoxicity of side chain analogues. Compounds were prepared by coupling 1-(chloromethyl)-5-nitro-1, 2-dihydro-3H-benz[e]indole to appropriate carboxylic acids, followed by nitro group reduction, or by coupling suitable 5-amino-protected indolines to alpha,beta-unsaturated acids, followed by deblocking. These AT-specific DNA alkylating agents were evaluated for cytotoxicity in a series of tumor cell lines (AA8, UV4, EMT6, SKOV3). For those analogues bearing an indolecarbonyl side chain, the 5'-methoxy derivative was the most cytotoxic (IC(50) 1.3 nM in AA8 cells, 4 h exposure), comparable to that of the parent CBI-TMI (5', 6',7'-trimethoxyindole) derivative (IC(50) 0.46 nM in the above assay). A subset of solubilized derivatives bearing O(CH(2))(2)NMe(2) substituents were about 10-fold less potent. For compounds containing an acryloyl linker in the side chain, the 4'-methoxycinnamoyl derivative proved the most cytotoxic (IC(50) 0. 09 nM in the above assay). A number of these 5-amino-seco-CBI-TMI analogues (including the solubilized compounds) are of interest both as cytotoxins and as components of amine-based prodrugs designed for tumor-specific activation.     

Studies on anti-MRSA parenteral cephalosporins. III. Synthesis and antibacterial activity of 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-alkoxyiminoacetamido-3

In the course of our exploration for a novel cephalosporin derivative having excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), we modified the C-3 linked spacers of cephem derivatives bearing a 1-methylimidazo[1,2-b]pyridazinium-6-yl group at the C-3' position and 2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-cyclopentyloxy-iminoacetyl group at the C-7 position. The optimal spacers were the (E)-2-vinyl and (E)-2-thiovinyl groups seen in 19a and 29aa, respectively. Their anti-MRSA activity was 16 to 32 times as potent as that of cefozopran (CZOP). Focusing on the (E)-2-vinyl and (E)-2-thiovinyl spacers, we further modified the alkoxyimino groups in the C-7 acyl moiety and the 1-alkylimidazo[1,2-b]pyridazinium moieties at the C-3' position and investigated the structure-activity relationships (SAR) of the derivatives. Consequently, we selected 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-fluoromethoxyiminoacetamido]-3-[(E)-2-(1-methylimidazo[1,2-b]pyridazinium-6-yl)thiovinyl]-3-cephem-4-carboxylate (29ca) as a new anti-MRSA parenteral cephalosporin candidate for further biological evaluation. The selected 29ca showed anti-MRSA activity comparable to that of vancomycin (VCM) both in vitro and in vivo, high affinity (IC50)=2.7 microg/ml) for penicillin binding protein 2' (PBP2') of MRSA and potent activity against Gram-negative bacteria as well.     

Novel, potent, and selective phosphodiesterase 5 inhibitors: synthesis and biological activities of a series of 4-aryl-1-isoquinolinone derivatives

A novel class of potent and selective phosphodiesterase 5 (PDE5) inhibitors, 4-aryl-1-isoquinolinone derivatives, which have been designed by the comparison of the structure of cGMP and a previously reported 1-arylnaphthalene lignan, was disclosed. Among these compounds, methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate dihydrochloride (36a) exhibited potent PDE5 inhibitory activity (IC(50) = 1.0 nM) with high isozyme selectivities (IC(50) ratio: PDE1/PDE5 = 1300, PDE2/PDE5 > 10 000, PDE3/PDE5 > 10 000, PDE4/PDE5 = 4700, PDE6/PDE5 = 28). Compound 36a also showed the most potent relaxant effect on isolated rabbit corpus cavernosum (EC(30) = 7.9 nM). Compound 63 (T-1032), the sulfate form of 36a, was selected for further biological and pharmacological evaluation of erectile dysfunction.     

Muscarinic cholinergic agonists and antagonists of the 3-(3-alkyl-1,2,4-oxadiazol-5-yl)-1,2,5,6-tetrahydropyridine type. Synthesis and structure-activity relationships

A series of 3-(3-alkyl-1,2,4-oxadiazol-5-yl)-1,2,5,6-tetrahydro-1-methylpyr idines (2a-q) were synthesized and tested for central muscarinic cholinergic receptor binding affinity by using [3H]oxotremorine-M and [3H]QNB as ligands and in a functional assay using guinea pig ileum. The analogues with unbranched C1-8-alkyl substituents (2a-g) were agonists, whereas the compounds with branched or cyclic substituents (2h-m) were antagonists. The alkyl ether analogues (2o-q) were also agonists but had lower receptor binding affinity than the corresponding alkyl analogues. The 3-(5-alkyl-1,2,4-oxadiazol-3-yl)-1,2,5,6-tetrahydro-1-methylpyridi ne analogues had only ver low affinity for the central muscarinic receptors and were weak antagonists in the ileum assay. A few 3-(3-butyl-1,2,4-oxadiazol-5-yl)-1,2,5,6-tetrahydro-1-methylpyr idines substituted with methyl or hydrogen in the 1-, 5-, or 6-position were synthesized and tested. N-Desmethyl analogue 7 was a potent muscarinic agonist, whereas N-desmethyl-5-methyl analogue 11 and N-methyl-6-methyl analogue 13 both were antagonists with lower muscarinic receptor affinity. The 3-(3-butyl-1,2,4-oxadiazol-5-yl)quinuclidine (17) and tropane (15) analogues were both very potent antagonists with high affinity for central muscarinic receptors. The ratio [IC50(QNB)/IC50(Oxo-M)] x 0.162 proved to be a good indicator of the efficacy of the compounds in the guinea pig ileum assay.     

Antitrypanosomal activity of 5'-deoxy-5'-(iodomethylene)adenosine and related 6-N-cyclopropyladenosine analogues

Treatment of the 6-N-cyclopropyl-2',3'-di-O-isopropylideneadenosine 5'-aldehyde with sulfone-stabilized phosphonate or fluorophosphonate reagents followed by stannyldesulfonylations and subsequent iodo- or protiodestannylation gave 6-N-cyclopropyl-5'-deoxy-5'-(iodomethylene)adenosine 8b or its 5'-fluoromethylene analogue 11. Treatment of the 5'-aldehyde with hydroxylamine or dibromomethylene- or cyanomethylene-stabilized Wittig reagents and deprotections gave the oxime 4b, 5'-cyanomethylene 5b, and 5'-dibromomethylene 13b analogues. Dehydrobromination of 13b gave acetylenic compound 14b. From the tested 6-N-cyclopropyladenosine analogues modified at the 5' carbon, the 5'-iodomethylene 8b had the most potent activity against Trypanosoma brucei in vitro with an IC50 of 12 microg/mL. The IC50 value was 19 microg/mL for both the 5'-fluoromethylene 11 and the 5'-cyanomethylene 5b compounds. The (E)-5'-deoxy-5'-(iodomethylene)adenosine 2a, a known inhibitor of AdoHcy hydrolase not modified with a cyclopropyl ring at 6-amino group, also inhibited T. brucei with an IC50 of 9 microg/mL. In contrast to some other adenosine analogues modified at C5', the 6-N-cyclopropyladenosine analogues described here do not exhibit an inhibitory effect on AdoHcy hydrolase and displayed only marginal antiviral activity.     

Synthesis and benzodiazepine receptor affinities of rigid analogues of 3-carboxy-beta-carbolines: demonstration that the benzodiazepine receptor recognizes preferentially the s-cis conformation of the 3-carboxy group

1H-Indolo[3',2':4,5]pyrido[3,2-b]-2-penten-5-olide (6) and 1H,5H-indolo[3',2'-c]-6,7-dihydro-2-pyridone (7), rigid analogues of methyl 4-ethyl-beta-carboline-3-carboxylate (8) and N-methyl-4-ethyl-beta-carboline-3-carboxamide (9), respectively, were synthesized and their in vitro binding affinities to the central type benzodiazepine receptors were compared. The IC50 values of 6 and 8 were approximately equivalent (42 and 27 nM, respectively). The amide derivative 9, for which theoretical energy calculations indicate that the s-trans carbonyl conformation is the preferred one, displayed very low affinity (IC50 greater than 10(4) nM). However, when the carbonyl group of 9 was forced to adopt the s-cis conformation as in lactam 7, binding to the benzodiazepine receptor was largely restored (IC50 = 150 nM), indicating that the s-cis carboxy conformation at C-3 of beta-carbolines is preferentially recognized by this receptor. In vivo, compound 6 showed neither convulsant, proconvulsant, nor anticonvulsant activity in mice. Moreover, 6 did not antagonize methyl beta-carboline-3-carboxylate induced convulsions in mice. This lack of activity of 6 was attributed to its inability to cross the blood-brain barrier since no significant displacement of [3H]Ro 15-1788 from mouse brain benzodiazepine receptors by 6 could be observed in vivo.