Design, synthesis and anti-plasmodial evaluation in vitro of new 4-aminoquinoline isatin derivatives

A new class of 4-aminoquinoline derivatives based on the natural product isatin scaffold were designed and synthesized for biological evaluation against three strains of the malaria parasite Plasmodium falciparum. These derivatives showed anti-plasmodial IC(50) values in the ranges of 1.3-0.079 and 2.0-0.050muM against a chloroquine-sensitive (D10) and two resistant (K1 and W2) strains of P. falciparum, respectively. In order to determine potential targets for this class of compounds in P. falciparum, selected compounds were also tested against the parasitic cysteine protease falcipain-2. In terms of further development of this class of isatin derivatives, two of the compounds based on a flexible alkyl chain linker and a thiosemicarbazone moiety warrant further investigation as potential anti-plasmodial leads. These two derivatives showed good in vitro activity against K1 and W2 with IC(50) values of 51 and 54nM, respectively, while retaining potency against the D10 strain with IC(50) values of 79 and 95nM, respectively. Generally speaking, the inhibitory potency of all compounds in the series against the parasites did not strongly correlate with inhibitory potency against falcipain-2 for selected compounds tested, which at best was weak to moderate, suggesting other mechanisms of inhibition may also be involved or compounds may be selectively taken up by Plasmodium falciparum.     

Anti-plasmodial activity of Dicoma tomentosa (Asteraceae) and identification of urospermal A-15- O-acetate as the main active compound

ABSTRACT: BACKGROUND: Natural products could play an important role in the challenge to discover new anti-malarial drugs. In a previous study, Dicoma tomentosa (Asteraceae) was selected for its promising anti-plasmodial activity after a preliminary screening of several plants traditionally used in Burkina Faso to treat malaria. The aim of the present study was to further investigate the antiplasmodial properties of this plant and to isolate the active anti-plasmodial compounds. METHODS: Eight crude extracts obtained from D. tomentosa whole plant were tested in vitro against two Plasmodium falciparum strains (3D7 and W2) using the p-LDH assay (colorimetric method). The Peters' four-days suppressive test model (Plasmodium berghei-infected mice) was used to evaluate the in vivo anti-plasmodial activity. An in vitro bioguided fractionation was undertaken on a dichloromethane extract, using preparative HPLC and TLC techniques. The identity of the pure compound was assessed using UV, MS and NMR spectroscopic analysis. In vitro cytotoxicity against WI38 human fibroblasts (WST-1 assay) and haemolytic activity were also evaluated for extracts and pure compounds in order to check selectivity. RESULTS: The best in vitro anti-plasmodial results were obtained with the dichloromethane, diethylether, ethylacetate and methanol extracts, which exhibited a high activity (IC50 [less than or equal to] 5 mug/ml). Hot water and hydroethanolic extracts also showed a good activity (IC50 [less than or equal to] 15 mug/ml), which confirmed the traditional use and the promising anti-malarial potential of the plant. The activity was also confirmed in vivo for all tested extracts. However, most of the active extracts also exhibited cytotoxic activity, but no extract was found to display any haemolytic activity. The bioguided fractionation process allowed to isolate and identify a sesquiterpene lactone (urospermal A-15-O-acetate) as the major anti-plasmodial compound of the plant (IC50 < 1 mug/ml against both 3D7 and W2 strains). This was also found to be the main cytotoxic compound (SI =3.3). While this melampolide has already been described in the plant, this paper is the first report on the biological properties of this compound. CONCLUSIONS: The present study highlighted the very promising anti-plasmodial activity of D. tomentosa and enabled to identify its main active compound, urospermal A-15-O-acetate. The high antiplasmodial activity of this compound merits further study about its anti-plasmodial mechanism of action. The active extracts of D. tomentosa, as well as urospermal A 15-Oacetate, displayed only a moderate selectivity, and further studies are needed to assess the safety of the use of the plant by the local population.     

In vitro anti-malarial activity of N6-modified purine analogs

A library of N6-hydroxy-, methoxy-, or amino-adenosine analogs was prepared and screened for anti-malarial properties. We found three compounds that possess anti-plasmodial activity in the low micromolar range against the multi-drug resistant VS1 strain, namely N6-hydroxy-9H-purin-6-amine (IC50 5.57 micro M), 2-amino-N6-amino-adenosine (IC50 12.2 micro M), and 2-amino-N6-amino-N6-methyladenosine (IC50 0.29 micro M). More importantly, the compounds were non-toxic, with 2-amino-N6-amino-N6-methyladenosine showing a selectivity index of 5008.     

Anti-plasmodial flavonoids from the stem bark of Erythrina abyssinica

The ethyl acetate extract of the stem bark of Erythrina abyssinica showed anti-plasmodial activity against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum with IC(50) values of 7.9+/-1.1 and 5.3+/-0.7 microg/ml, respectively. From this extract, a new chalcone, 2',3,4,4'-tetrahydroxy-5-prenylchalcone (trivial name 5-prenylbutein) and a new flavanone, 4',7-dihydroxy-3'-methoxy-5'-prenylflavanone (trivial name, 5-deoxyabyssinin II) along with known flavonoids have been isolated as the anti-plasmodial principles. The structures were determined on the basis of spectroscopic evidence.     

A tyrosinase inhibitor, Daedalin A, from mycelial culture of Daedalea dickinsii

A chromene-type compound, daedalin A (1), was isolated from mycelial culture broth of Daedalea dickinsii. Based on spectroscopic data, the structure of 1 was found to be (2R)-6-hydroxy-2-hydroxymethyl-2-methyl-2H-chromene. Daedalin A (1) strongly inhibited the activity of tyrosinase (IC(50): 194 muM). In addition, 1 also showed 1,1-diphenyl-2-picrylhydrazyl scavenging activity (IC(50): 16.9 microM) and superoxide anion scavenging activity (IC(50): 28.5 microM).     

Synthesis and biological evaluation of norcantharidin analogues: towards PP1 selectivity

Simple modifications to the anhydride moiety of norcantharidin have lead to the development of a series of analogues displaying modest PP1 inhibition (low muM IC(50)s) comparable to that of norcantharidin (PP1 IC(50)=10.3+/-1.37 microM). However, unlike norcantharidin, which is a potent inhibitor of PP2A (IC(50)=2.69+/-1.37 microM), these analogues show reduced PP2A inhibitory action resulting in the development of selective PP1 inhibitory compounds. Data indicates that the introduction of two ortho-disposed substituents on an aromatic ring, or para-substituent favours PP1 inhibition over PP2A inhibition. Introduction of a p-morphilinoaniline substituent, 35, affords an inhibitor displaying PP1 IC(50)=6.5+/-2.3 microM; and PP2A IC(50)=7.9+/-0.82 microM (PP1/PP2A=0.82); and a 2,4,6-trimethylaniline, 23, displaying PP1 IC(50)=48+/-9; and PP2A IC(5) 85+/-3 microM (PP1/PP2A=0.56). The latter shows a 7-fold improvement in PP1 versus PP2A selectivity when compared with norcantharidin. Subsequent analysis of 23 and 35 as potential PP2B inhibitors revealed modest inhibition with IC(50)s of 89+/-6 and 42+/-3 microM, respectively, and returned with PP1/PP2B selectivities of 0.54 and 0.15. Thus, these analogues are the simplest and most selective PP1 inhibitors retaining potency reported to date.     

Exploration of biguanido–oxovanadium complexes as potent and selective inhibitors of protein tyrosine phosphatases

The inhibitory effects of three biguanido-oxovanadium complexes ([VO(L(1-3))(2)]·nH(2)O: HL(1) = metformin, HL(2) = phenformin, HL(3) = moroxydine) against four protein tyrosine phosphatases (PTPs) and an alkaline phosphatase (ALP) were investigated. The complexes display strong inhibition against PTP1B and TCPTP (IC(50), 80-160 nM), a bit weaker inhibition against HePTP (IC(50), 190-410 nM) and SHP-1(IC(50), 0.8-3.3 μM) and much weaker inhibition against ALP (IC(50), 17-35 μM). Complex 3 is about twofold less potent against PTP1B, TCPTP and HePTP than complexes 1 and 2, while complex 2 inhibits SHP-1 more strongly (about three to fourfold) than the other two complexes. These results suggest that the structures of the ligands slightly influence the potency and selectivity against PTPs. The complexes inhibit PTP1B and ALP with a typical competitive type.     

The first two cantharidin analogues displaying PP1 selectivity

High pressure Diels-Alder reactions of furan and dimethylmaleate, and thiophene and maleimide resulted in two cantharidin analogues, 3 and 6 possessing PP1 selectivity (>40- and >30-fold selectivity) over PP2A. Both compounds exhibited moderate PP1 activity, 3 IC(50) 50 microM and 6 IC(50) 12.5 microM. Interestingly, the corresponding mono-ester derivatives of 3 showed no such selectivity.     

Design, synthesis, and biological evaluation of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides as cyclooxygenase isozyme inhibitors

A group of N-acetyl-2-(or 3-)carboxymethylbenzenesulfonamides, possessing either a F or a substituted-phenyl ring substituent (4-F, 2,4-F2, 4-SO2Me, 4-OCHMe2) attached to its C-4 or C-6 position, was prepared using a palladium-catalyzed Suzuki cross-coupling reaction for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. Although N-acetyl-3-carboxymethyl-6-fluorobenzenesulfonamide [14, COX-1 IC50 = 2.26 microM; COX-2 IC50 = 0.012 microM; COX-2 selectivity index (SI) = 188] and N-acetyl-3-carboxymethyl-6-(4-isopropoxyphenyl)benzenesulfonamide (20c, COX-1 IC50 >100 microM; COX-2 IC50 = 0.15 microM; COX-2 SI >667) exhibited potent in vitro COX-2 inhibitory activity and high COX-2 selectivity, both compounds were inactive anti-inflammatory agents in a carrageenan-induced rat paw edema assay. In contrast, the less potent and less selective COX-2 inhibitors N-acetyl-2-carboxymethyl-4-fluorobenzenesulfonamide (12, COX-1 IC50 = 4.25 microM; COX-2 IC50 = 0.978 microM; COX-2 SI = 4.3), N-acetyl-2-carboxymethyl-4-(2,4-difluorophenyl)benzenesulfonamide (17c, COX-1 IC50 = 1.02 microM; COX-2 IC50 = 1.00 microM; COX-2 SI = 1.02), and N-acetyl-3-carboxymethyl-6-(4-methanesulfonylphenyl)benzenesulfonamide (20e, COX-1 IC50 = 0.109 microM; COX-2 IC50 = 1.14 microM; COX-2 SI = 0.095) exhibited moderate anti-inflammatory activity where a 75 mg/kg oral dose reduced inflammation 26%, 14%, and 20%, respectively, at 3 h postdrug administration relative to the reference drug aspirin where a 50 mg/kg oral dose reduced inflammation by 25% at 3 h postdrug administration.     

Anti-protozoal and plasmodial FabI enzyme inhibiting metabolites of Scrophularia lepidota roots

The ethanolic root extract of Scrophularia lepidota, an endemic plant of the Turkish flora, has been investigated for its anti-protozoal and inhibitory effect towards plasmodial enoyl-ACP reductase (FabI), a key enzyme of fatty acid biosynthesis in Plasmodium falciparum. Chromatographic separation of the extract yielded 10 iridoids (1-10), two of which are new, and a known phenylethanoid glycoside (11). The structures of the new compounds were determined as 3,4-dihydro-methylcatalpol (8) and 6-O-[4'-O-trans-(3,4-dimethoxycinnamoyl)-alpha-L-rhamnopyranosyl]aucubin (scrolepidoside, 9) by spectroscopic means. The remaining metabolites were characterized as catalpol (1), 6-O-methylcatalpol (2), aucubin (3), 6-O-alpha-L-rhamnopyranosyl-aucubin (sinuatol, 4), 6-O-beta-D-xylopyranosylaucubin (5), ajugol (6), ajugoside (7), an iridoid-related aglycone (10) and angoroside C (11). Nine isolates were active against Leishmania donovani, with the new compound 9 being most potent (IC50 6.1 microg/ml). Except for 4, all pure compounds revealed some trypanocidal potential against Trypanosoma brucei rhodesiense (IC50 values 29.3-73.0 microg/ml). Only compound 10 showed moderate anti-plasmodial (IC50 40.6 microg/ml) and FabI enzyme inhibitory activity (IC50 100 microg/ml). 10 is the second natural product inhibiting the fatty acid biosynthesis of Plasmodium falciparum.     

N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea and N'-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]-thiourea as potent inhibitors of multidrug-resistant human immunodeficiency virus-1.

We have replaced the pyridyl ring of trovirdine with an alicyclic cyclohexenyl, adamantyl or cis-myrtanyl ring. Only the cyclohexenyl-containing thiourea compound N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]- thiourea (HI-346) (as well as its chlorine-substituted derivative N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]- thiourea/HI-445) showed RT inhibitory activity. HI-346 and HI-445 effectively inhibited recombinant RT with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cell-free RT inhibition assays was: HI-346 (IC50 = 0.4 microM) > HI-445 (IC50 = 0.5 microM) > trovirdine (IC50 = 0.8 microM) > MKC-442 (IC5 = 0.8 microM) = delavirdine (IC50 = 1.5 microM) > nevirapine (IC50 = 23 microM). In accord with this data, both compounds inhibited the replication of the drug-sensitive HIV-1 strain HTLV(IIIB) with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cellular HIV-1 inhibition assays was: HI-445 = HI-346 (IC50 = 3 nM) > MKC-442 (IC50 = 4 nM) = AZT (IC50 = 4 nM) > trovirdine (IC50 = 7 nM) > delavirdine (IC50 = 9 nM) > nevirapine (IC50 = 34 nM). Surprisingly, the lead compounds HI-346 and HI-445 were 3-times more effective against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V,41L, and 215Y) than they were against HTLV(IIIB) with wild-type RT. HI-346 and HI-445 were 20-times more potent than trovirdine, 200-times more potent than AZT, 300-times more potent than MKC-442, 400-times more potent than delavirdine, and 5000-times more potent than nevirapine against the multidrug resistant HIV-1 strain RT-MDR. HI-445 was also tested against the RT Y181C mutant A17 strain of HIV-1 and found to be >7-fold more effective than trovirdine and >1,400-fold more effective than nevirapine or delavirdine. Similarly, both HI-346 and HI-445 were more effective than trovirdine, nevirapine, and delavirdine against the problematic NNI-resistant HIV-1 strain A17-variant with both Y181C and K103N mutations in RT, although their activity was markedly reduced against this strain. Neither compound exhibited significant cytotoxicity at effective concentrations (CC50 >100 microM). These findings establish the lead compounds HI-346 and HI-445 as potent inhibitors of drug-sensitive as well as multidrug-resistant stains of HIV-1.     

Bromophenols as inhibitors of protein tyrosine phosphatase 1B with antidiabetic properties

A series of bromophenol derivatives were synthesized and evaluated as protein tyrosine phosphatase 1B (PTP1B) inhibitors in vitro and in vivo based on bromophenol 4e (IC(50)=2.42 μmol/L), which was isolated from red algae Rhodomela confervoides. The results showed that all of the synthesized compounds displayed weak to good PTP1B inhibition at tested concentration. Among them, highly brominated compound 4g exhibited promising inhibitory activity against PTP1B with IC(50) 0.68 μmol/L, which was approximately fourfold more potent than lead compound 4e. Further, compound 4g demonstrated high selectivity against other PTPs (TCPTP, LAR, SHP-1 and SHP-2). More importantly, in vivo antidiabetic activities investigations of compound 4g also demonstrated inspiring results.     

Synthesis, biological evaluation and molecular modelling studies of methyleneimidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17). Part I: Heterocyclic modifications of the core structure

Novel chemical entities were prepared via Suzuki and S(N) reaction as AC-ring substrate mimetics of CYP17. The synthesised compounds 1-31 were tested for activity using human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against hepatic CYP enzymes (3A4, 2D6, 1A2, 2C9, 2C19, 2B6). Two potent inhibitors (27, IC50 = 373 nM/28, IC50 = 953 nM) were further examined in rats regarding their effects on plasma testosterone levels and their pharmacokinetic properties. Compound 28 was similarly active as abiraterone and showed better pharmacokinetic properties (higher bioavailability, t(1/2) 9.5 h vs 1.6 h). Docking studies revealed two new binding modes different from the one of the substrates and steroidal inhibitors.     

Safety, efficacy and anti-inflammatory activity of rho iso-alpha-acids from hops

A defined mixture of rho iso-alpha-acids (RIAA), a modified hop extract, was evaluated for anti-inflammatory efficacy and safety. RIAA inhibited LPS-stimulated PGE(2) formation with >200-fold selectivity of COX-2 (IC(50)=1.3 microg/ml) over COX-1 (IC(50)>289 microg/ml). This occurred only when RIAA was added prior to, but not post, LPS stimulation. Consistent with this observation, RIAA produced no physiologically relevant, direct inhibition of COX-1 or COX-2 peroxidase activity. This suggests that RIAA inhibits inducible but not constitutive COX-2. In support, we found RIAA showed minimal PGE(2) inhibition (IC(50)=21mug/ml) relative to celecoxib (IC(50)=0.024 microg/ml), aspirin (IC(50)=0.52 microg/ml) or ibuprofen (IC(50)=0.57 microg/ml) in the AGS gastric mucosal model, where COX-1 and -2 are expressed constitutively. Taken together these results predict RIAA may have lower potential for gastrointestinal and cardiovascular toxicity observed with COX enzyme inhibitors. Following confirmation of bioavailable RIAA administered orally, gastrointestinal safety was assessed using the fecal calprotectin biomarker in a 14-day human clinical study; RIAA (900 mg/day) produced no change compared to naproxen (1000 mg/day), which increased fecal calprotectin 200%. Cardiovascular safety was addressed by PGI-M measurements where RIAA (1000 mg) did not reduce PGI-M or affect the urinary PGI-M/TXB(2) ratio. Drug interaction potential was evaluated against six major CYPs; of relevance, RIAA inhibited CYP2C9. Toxicity was assessed in a 21-day oral, mouse subchronic toxicity study where no dose dependent histopathological effects were noted. Clinically, RIAA (1000 mg/day) produced a 54% reduction in WOMAC Global scores in a 6-week, open-label trial of human subjects exhibiting knee osteoarthritis.     

Design, synthesis, and biological evaluation of novel (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids as selective inhibitors for AKR1B1

New substituted (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids were designed as the inhibitor of AKR1B1 based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa, and twenty derivatives were synthesized and evaluated. The most active compound of the series was (2-benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7m), which showed comparable inhibitory activity for AKR1B1 (IC(50)=0.15μM) with clinically used epalrestat (IC(50)=0.1μM). In the view of activity and selectivity, the most potent compound was (2-benzyl-6-carboxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7t), which showed strong inhibitory effect (IC(50)=0.17μM) and very high selectivity for AKR1B1 against AKR1A1 (311:1) and AKR1B10 (253:1) compared with epalrestat.     

Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica

Temporins are small antimicrobial peptides isolated from North American and Eurasian ranid frogs that are particularly active against Gram-positive bacteria. To date, no temporins have been characterized from North African frog species. We isolated three novel members of the temporin family, named temporin-1Sa (FLSGIVGMLGKLF(amide)), -1Sb (FLPIVTNLLSGLL(amide)), and -1Sc (FLSHIAGFLSNLF(amide)), from the skin of the Sahara frog Pelophylax (Rana) saharica originating from Tunisia. These temporins were identified by a combined mass spectrometry/molecular cloning approach. Temporin-1Sa was found to be highly active against Gram-positive and Gram-negative bacteria, yeasts and fungi (MIC=2-30muM). To our knowledge, this is the first 13-residue member of the temporin family with a net charge of +2 that shows such broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC=10muM) and Pseudomonas aeruginosa (MIC=31muM). Moreover, temporin-1Sa displays significant antiparasitic activity (IC(50) approximately 20muM) against the promastigote and amastigote stages of Leishmania infantum.     

Synthesis and cytotoxic activity of N-[(alkylamino)alkyl]carboxamide derivatives of 7-oxo-7H-benz[de]anthracene, 7-oxo-7H-naphtho[1,2,3-de]quinoline, and 7-oxo-7H-benzo[e]perimidine

7-Oxo-7H-naphtho[1,2,3-de]quinoline-11-carboxamides and analogues were prepared and evaluated for in vitro and in vivo antitumor activity. Chromophore variations included 'deaza' (7-oxo-7H-benz[de]anthracene) and 'diaza' (7-oxo-7H-benzo[e]perimidine) analogues, and side chain variations included chiral alpha-methyl compounds. The naphthoquinolines were the most cytotoxic, with IC(50) values of 5-20 nM, and showed the strongest DNA binding, with high selectivity for G-C rich DNA. The chiral alpha-methyl analogues were 10-20-fold more cytotoxic than the parent des-methyl compound. Both enantiomers provided substantial growth delays against s.c. colon 38 tumors in mice, with the R-enantiomer more active than the S (tumor growth delays of >35 and 12 days, respectively).     

Synthesis, biological evaluation, and molecular modeling studies of methylene imidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17)--part II: Core rigidification and influence of substituents at the methylene bridge

Thirty-five novel substituted imidazolyl methylene biphenyls have been synthesized as CYP17 inhibitors for the potential treatment of prostate cancer. Their activities have been tested with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against CYP11B1, CYP11B2, and hepatic CYP enzymes 3A4, 1A2, 2B6 and 2D6. The core rigidified compounds (30-35) were the most active ones, being much more potent than Ketoconazole and reaching the activity of Abiraterone. However, they were not very selective. Another rather potent and more selective inhibitor (compound 23, IC(50)=345 nM) was further examined in rats regarding plasma testosterone levels and pharmacokinetic properties. Compared to the reference Abiraterone, 23 was more active in vivo, showed a longer plasma half-life (10h) and a higher bioavailability. Using our CYP17 homology protein model, docking studies with selected compounds were performed to study possible interactions between inhibitors and amino acid residues of the active site.