Activity of nitrogen-containing and non-nitrogen-containing bisphosphonates on tumor cell lines

We synthesized and tested three series of bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines: MCF-7 (breast), NCI-H460 (lung), and SF-268 (CNS). The first series of compounds consisted of 49 nitrogen-containing bisphosphonates, the most active species being a tetrakispivaloyloxymethyl (POM) ester, having an (average) IC(50) of 6.8 microM. The second series of compounds consisted of nine terphenylbisphosphonates, the most active species also being a POM ester, having an IC(50) of 2.2 microM. The third series of compounds consisted of seven halogen or cyanophenylbisphosphonates, the most active species again being a POM ester, having an IC(50) of 500 nM. Taken together, these results are of interest because they show that bisphosphonate esters can have potent activity against a variety of tumor cell lines, with the most active terphenyl- and halophenyl-containing species having IC(50) values approximately 10-40x lower than the most potent commercially available bisphosphonates.     

Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy

We have investigated the effects in vitro of a series of bisphosphonates on the proliferation of Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum. The results show that nitrogen-containing bisphosphonates of the type used in bone resorption therapy have significant activity against parasites, with the aromatic species having in some cases nanomolar or low-micromolar IC(50) activity values against parasite replication (e.g. o-risedronate, IC(50) = 220 nM for T. brucei rhodesiense; risedronate, IC(50) = 490 nM for T. gondii). In T. cruzi, the nitrogen-containing bisphosphonate risedronate is shown to inhibit sterol biosynthesis at a pre-squalene level, most likely by inhibiting farnesylpyrophosphate synthase. Bisphosphonates therefore appear to have potential in treating parasitic protozoan diseases.     

Activity of bisphosphonates against Trypanosoma brucei rhodesiense

We report the results of a comparative molecular field analysis (CoMFA) investigation of the growth inhibition of the bloodstream form of Trypanosoma brucei rhodesiense trypomastigotes by bisphosphonates. A quantitative three-dimensional structure-activity relationship CoMFA model for a set of 26 bisphosphonates having a range of activity spanning approximately 3 orders of magnitude (minimum IC(50) = 220 nM; maximum IC(50) = 102 microM) yielded an R(2) value of 0.87 with a cross-validated R(2) value of 0.79. The predictive utility of this approach was tested for three sets of three compounds: the average pIC(50) error was 0.23. For the nitrogen-containing bisphosphonates, in general, the activity was aromatic- > aliphatic-containing side chains. The activity of aromatic species lacking an alkyl ring substitution decreased from ortho to meta to para substitution; halogen substitutions also reduced activity. For the aliphatic bisphosphonates, the IC(50) values decreased nearly monotonically with increasing chain length (down to IC(50) = 2.0 microM for the n-C(11) alkyl side chain species). We also show, using a "rescue" experiment, that the molecular target of the nitrogen-containing bisphosphonate, risedronate, in T. b. rhodesiense is the enzyme farnesyl pyrophosphate synthase. In addition, we report the LD(50) values of bisphosphonates in a mammalian cell general toxicity screen and present a comparison between the therapeutic indices and the IC(50) values in the T. b. rhodesiense growth inhibition assay. Several bisphosphonates were found to have large therapeutic indices (> or =200:1) as well as low IC(50) values, suggesting their further investigation as antiparasitic agents against T. b. rhodesiense.     

Synthesis and evaluation of antiparasitic activities of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives

A series of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives, prepared by two synthetic routes, were in vitro assayed against three Trypanosoma strains, Leishmania donovani, and Plasmodium falciparum K1. Seven out of 17 compounds showed moderate to very good activity against blood stage T. b. rhodesiense, with 10 and 17 exhibiting highest potency (IC50 of 1.0 and 1.1 microM, respectively). Interestingly, the beta-diketone precursors 1-3 had good antitrypanosomal activity toward the insect stage, with IC50 values of 1.0-3.4 microM. Among different compounds with moderate activity against T. cruzi, compound 17 showed the lowest IC50 value of 9.5 microM; thus, the series seemed to act selectively toward the different Trypanosoma parasites. Eight compounds were moderately active against L. donovani, with 2, 3, and 12 being the most promising ones (IC50 values of 2.3-5.2 microM), whereas compound 14 was the only derivative with good activity against P. falciparum (IC50 of 3.7 microM).     

In vitro activity of aurones against Plasmodium falciparum strains K1 and NF54.

A series of naturally occurring aurones (e. g., Rubiaceae, Cyperaceae) was synthesized and tested for the ability to inhibit erythrocytic stages of Plasmodium falciparum strains in vitro. Some of the these compounds exhibited antiplasmodial activity in the micromol range, determined as fifty percent-inhibitory concentrations (IC(50)). Drug activity was not associated with cytotoxicity for the mammalian tumor cell lines KB and SKMel (IC(50) > 3.0 microM). The most active compound was 4,6,4'-triacetyl-3',5'-dimethoxy-2-aurone with IC(50) values of 0.007 microM and 0.18 microM for the P. falciparum strains K1 and NF54, respectively. Interestingly, the multiple drug-resistant P. falciparum strain K1 was more sensitive to tested aurones than the drug-susceptible strain NF54.     

Activity of sulfonium bisphosphonates on tumor cell lines

We investigated three series of sulfonium bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines. The first series consisted of 6 cyclic sulfonium bisphosphonates, the most active species having an (average) IC50 of 89 microM. The second consisted of 10 phenylalkyl and phenylalkoxy bisphosphonates, the most active species having an IC50 of 18 microM. The third series consisted of 17 n-alkyl sulfonium bisphosphonates, the most active species having an IC50 of approximately 240 nM. Three QSAR models showed that the experimental cell growth inhibition results could be well predicted. We also determined the structures of one sulfonium bisphosphonate bound to farnesyl diphosphate synthase, finding that it binds exclusively to the dimethylallyl diphosphate binding site. These results are of interest since they show that sulfonium bisphosphonates can have potent activity against a variety of tumor cell lines, the most active species having IC50 values much lower than conventional nitrogen-containing bisphosphonates.     

New 4-aminobicyclo[2.2.2]octane derivatives and their activities against Plasmodium falciparum and Trypanosoma b. rhodesiense.

A series of new 2-substituted 4-dialkylaminobicyclo[2.2.2]octane derivatives was prepared and the compounds were investigated for their activity against causative organisms of tropical diseases. The tests were performed as microplate assays using the K1 strain of Plasmodium falciparum (resistant to chloroquine and pyrimethamine) and Trypanosoma brucei rhodesiense (STIB 900). The results were compared to the activities of former tested compounds of the bicyclo[2.2.2]octane series and to known drugs. Most of the 4-amino-6,7-diphenylbicyclo[2.2.2]octan-2-one thiosemicarbazones were compounds with attractive antimalarial potency (IC(50)=0.84-0.99microM, chloroquine: IC(50)=0.12microM). One of the bicyclo[2.2.2]octan-2-yl 4-tert-butylbenzenesulfonates showed the highest antitrypanosomal activity (IC(50)=0.68microM) of the so far prepared 4-amino-6,7-diarylbicyclo[2.2.2]octane derivatives, but is distinctly less active than suramin (IC(50)=0.0075microM).     

3-D QSAR investigations of the inhibition of Leishmania major farnesyl pyrophosphate synthase by bisphosphonates

We report the activities of 62 bisphosphonates as inhibitors of the Leishmania major mevalonate/isoprene biosynthesis pathway enzyme, farnesyl pyrophosphate synthase. The compounds investigated exhibit activities (IC(50) values) ranging from approximately 100 nM to approximately 80 microM (corresponding to K(i) values as low as 10 nM). The most active compounds were found to be zoledronate (whose single-crystal X-ray structure is reported), pyridinyl-ethane-1-hydroxy-1,1-bisphosphonates or picolyl aminomethylene bisphosphonates. However, N-alicyclic aminomethylene bisphosphonates, such as incadronate (N-cycloheptyl aminomethylene bisphosphonate), as well as aliphatic aminomethylene bisphosphonates containing short (n = 4, 5) alkyl chains, were also active, with IC(50) values in the 200-1700 nM range (corresponding to K(i) values of approximately 20-170 nM). Bisphosphonates containing longer or multiple (N,N-) alkyl substitutions were inactive, as were aromatic species lacking an o- or m-nitrogen atom in the ring, or possessing multiple halogen substitutions or a p-amino group. To put these observations on a more quantitative structural basis, we used three-dimensional quantitative structure-activity relationship techniques: comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA), to investigate which structural features correlated with high activity. Training set results (N = 62 compounds) yielded good correlations with each technique (R(2) = 0.87 and 0.88, respectively), and were further validated by using a training/test set approach. Test set results (N = 24 compounds) indicated that IC(50) values could be predicted within factors of 2.9 and 2.7 for the CoMFA and CoMSIA methods, respectively. The CoMSIA fields indicated that a positive charge in the bisphosphonate side chain and a hydrophobic feature contributed significantly to activity. Overall, these results are of general interest since they represent the first detailed quantitative structure-activity relationship study of the inhibition of an expressed farnesyl pyrophosphate synthase enzyme by bisphosphonate inhibitors and that the activity of these inhibitors can be predicted within about a factor of 3 by using 3D-QSAR techniques.     

Oxoaporphine alkaloids and quinones from Stephania dinklagei and evaluation of their antiprotozoal activities

Bioactivity-guided fractionation of Stephania dinklagei yielded six compounds including, two zwitterionic oxoaporphine alkaloids, N-methylliriodendronine, and 2-O,N-dimethylliriodendronine, two oxoaporphine alkaloids, liriodenine, and dicentrinone, one aporphine alkaloid, corydine, and one anthraquinone, aloe-emodin. Apart from corydine, the isolates have not been reported as constituents of S. dinklagei. N-Methylliriodendronine, and 2-O,N-dimethylliriodendronine are reported for the first time as natural products. All isolated compound were tested for antiprotozoal activity and cytotoxic activities in vitro. N-Methylliriodendronine was the most active against L. donovani amastigotes (IC50 = 36.1 microM). Liriodenine showed the highest activity against Leishmania donovani, and Plasmodium falciparum with IC50 values of 26.16 and 15 microM, respectively. Aloe-emodin was the only compound active (IC50 = 14 microM) against T. b. brucei.     

Anti-plasmodial and anti-leishmanial activity of conformationally restricted pentamidine congeners

A library of 52 pentamidine congeners in which the flexible pentyldioxy linker in pentamidine was replaced with various restricted linkers was tested for in-vitro activity against two Plasmodium falciparum strains and Leishmania donovani. The tested compounds were generally more effective against P. falciparum than L. donovani. The most active compounds against the chloroquine-sensitive (D6, Sierra Leone) and -resistant (W2, Indochina) strains of P. falciparum were bisbenzamidines linked with a 1,4-piperazinediyl or 1, 4-homopiperazinediyl moiety, with IC50 values (50% inhibitory concentration, inhibiting parasite growth by 50% in relation to drug-free control) as low as 7 nM based on the parasite lactate dehydrogenase assay. Seven piperazine-linked bisbenzamidines substituted at the amidinium nitrogens with a linear alkyl group of 3-6 carbons (22, 25, 27, 31) or cycloalkyl group of 4, 6 or 7 carbons (26, 32, 34) were more potent (IC50<40 nM) than chloroquine or pentamidine as anti-plasmodial agents. The most active anti-leishmanial agents were 4,4'-[1,4-phenylenebis(methyleneoxy)]bisbenzenecarboximidamide (2, IC50 approximately 0.290 microM) and 1,4-bis[4-(1H-benzimidazol-2-yl)phenyl] piperazine (44, IC50 approximately 0.410 microM), which were 10- and 7-fold more potent than pentamidine (IC50 approximately 2.90 microM). Several of the more active anti-plasmodial agents (e.g. 2, 31, 33, 36-38) were also potent anti-leishmanial agents, indicating broad antiprotozoal properties. However, a number of analogues that showed potent anti-plasmodial activity (1, 18, 21, 22, 25-28, 32, 43, 45) were not significantly active against the Leishmania parasite. This indicates differential modes of anti-plasmodial and anti-leishmanial actions for this class of compounds. These compounds provide important structure-activity relationship data for the design of improved chemotherapeutic agents against parasitic infections.     

Synthesis and antimalarial activity of sulfonamide chalcone derivatives

A series of sulfonamide chalcone derivatives were synthesized and investigated for their abilities to inhibit beta-hematin formation in vitro and their activity against cultured Plasmodium falciparum parasites. Inhibition of beta-hematin formation was minimal in the aromatic ring of the chalcone moiety as it appeared for compounds 4b, 4d-f, and greatest with compounds 4g (IC50 0.48 microM) and 4k (IC50 0.50 microM) with a substitution of 3,4,5-trimethoxyl and 3-pyridinyl, respectively. In this study, the most active compound resulted 1[4'-N(2',5'-dichlorophenyl) sulfonyl-amidephenyl]-3-(4-methylphenyl)-2-propen-1-one 4i, effective as antimalarial by the inhibition of cultured P. falciparum parasites (1 microM). These studies open up the novel possibility of development of sulfonamide derivatives as antimalarials that target beta-hematin formation and the inhibition of the development of cultured P. falciparum parasites, which should help delay the rapid onset of resistance to drugs acting at only a single site. Results with these assays suggest that chalcones exert their antimalarial activity via multiple mechanisms.     

Antimalarial activity of crambescidin 800 and synthetic analogues against liver and blood stage of Plasmodium sp

Structural features associated with the antimalarial activity of the marine natural product crambescidin 800 were studied using synthetic analogues of the related compound ptilomycalin A. The study suggests that the guanidine moiety is cytotoxic, whereas the spermidine-containing aliphatic chain increases activity. The most active analogue, compound 11, had in vitro activity against Plasmodium falciparum strain 3D7 (IC50=490 nM) that was stronger than the in vitro activity against murine L5178Y cells (IC50 = 8.5-59 microM). In vitro growth inhibition of liver stages of P. yoelii yoelii in mouse hepatocytes was observed (IC50 = 9.2 microM). The compound did not significantly prolong median survival time after a single subcutaneous administration of 80 mg/kg in P. berghei-infected mice. Compound 11 did not cause DNA fragmentation in an in vitro micronucleus assay.     

Parallel solution-phase synthesis of conformationally restricted congeners of pentamidine and evaluation of their antiplasmodial activities

Conformationally restricted bisbenzamidines and related congeners have been synthesized and evaluated for activity against two Plasmodium falciparum strains. The most active compounds, bisbenzamidines linked by a 1,4-piperazinediyl core, had IC(50) values between 3 and 18 nM against both chloroquine-susceptible and -resistant parasites and IC(50) values for cytotoxicity greater than 5 microM, using the A549 human lung epithelial cell line. DNA binding affinity, as estimated by DeltaT(m), did not correlate with either antiparasite effects or cytotoxicity. Each of the active bisbenzamidines interfered with the formation of hemozoin in cell-free systems.     

Synthesis and structure-activity relationships of 4-pyridones as potential antimalarials

A series of diaryl ether substituted 4-pyridones have been identified as having potent antimalarial activity superior to that of chloroquine against Plasmodium falciparum in vitro and murine Plasmodium yoelii in vivo. These were derived from the anticoccidial drug clopidol through a systematic study of the effects of varying the side chain on activity. Relative to clopidol the most active compounds show >500-fold improvement in IC50 for inhibition of P. falciparum in vitro and about 100-fold improvement with respect to ED50 against P. yoelii in mice. These compounds have been shown elsewhere to act selectively by inhibition of mitochondrial electron transport at the cytochrome bc1 complex.     

Antimalarial, antitrypanosomal, and antileishmanial activities and cytotoxicity of bis(9-amino-6-chloro-2-methoxyacridines): influence of the linker

Forty bis(9-amino-6-chloro-2-methoxyacridines), in which acridine moieties are joined by alkanediamines, polyamines, or polyamines substituted by a side chain, were synthesized and tested for their in vitro activity upon the erythrocytic stage of Plasmodium falciparum, trypomastigote stage of Trypanosoma brucei, and amastigote stage of Trypanosoma cruzi and Leishmania infantum as well as for their cytotoxic effects upon MRC-5 cells. Results clearly showed the importance of the nature of the linker and of its side chain for antiparasitic activity, cytotoxicity, and cellular localization. Among several compounds devoid of cytotoxic effects at 25 microM upon MRC-5 cells, one displayed IC(50) values ranging from 8 to 18 nM against different P. falciparum strains while three others totally inhibited T. brucei at 1.56 microM.     

Bisphosphonate inhibitors of Toxoplasma gondi growth: in vitro, QSAR, and in vivo investigations

We have investigated the activity of 60 bisphosphonates against the replication of Toxoplasma gondii in vitro and of three of the most active compounds, in vivo. The two most active compounds found were n-alkyl bisphosphonates containing long (n = 9 or 10) hydrocarbon chains, not the nitrogen-containing species used in bone resorption therapy. The target of all of the most active bisphosphonates appears to be the isoprene biosynthesis pathway enzyme farnesyl pyrophosphate synthase (FPPS), as indicated by the correlations between T. gondii growth inhibition and FPPS (human and Leishmania major) enzyme inhibition and by the fact that a T. gondii strain engineered to overexpress FPPS required considerably higher levels of bisphosphonates to achieve 50% growth inhibition, while the IC(50) for atovaquone (which does not inhibit FPPS) remained the same in the overexpressing strain. The phosphonate inhibitor of the non-mevalonate pathway, fosmidomycin, which inhibits the enzyme 1-deoxyxylulose-5-phosphate reductoisomerase, had no effect on T. gondii growth. To investigate structure-activity relationships (SARs) in more detail, we used two three-dimensional quantitative SAR methods: comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), to investigate all 60 bisphosphonates. Both the CoMFA and CoMSIA models indicated a 60-70% contribution from steric interactions and a 30-40% contribution from electrostatic interactions and using four N = 55 training sets for each method, we found on average between a factor of 2 and 3 error in IC(50) prediction. The three most active compounds found in vitro were tested in vivo in a Smith-Webster mouse model and the two most active bisphosphonates were found to provide up to an 80% protection from death, a considerable improvement over that found previously with nitrogen-containing bisphosphonates. This effect may originate in the much higher therapeutic indices of these alkyl bisphosphonates, as deduced from in vitro assays using LD(50) values for growth inhibition of a human cell line. Overall, these results indicate that alkyl bisphosphonates are promising compounds for further development as agents against Toxoplasma gondii growth, in vivo.     

Antiprotozoal activities of new bicyclo[2.2.2]octan-2-imines and esters of bicyclo[2.2.2]octan-2-ols.

Several bicyclo[2.2.2]octan-2-imines and esters of bicyclo[2.2.2]octan-2-ols were prepared. Their antitrypanosomal and antiplasmodial activities against Trypanosoma brucei rhodesiense (STIB 900) and the K1 strain of Plasmodium falciparum (resistant to chloroquine and pyrimethamine) were determined using microplate assays. Two of the synthesized bicyclo[2.2.2]octan-2-one 4'-phenylthiosemicarbazones showed the highest antitrypanosomal activity (IC(50)<0.3 microM) of the so far prepared 4-amino-6,7-diarylbicyclo[2.2.2]octane derivatives, but they are distinctly less active than suramine (IC(50)=0.0075 microM). Most of the 4'-phenylthiosemicarbazones and a single bicyclo[2.2.2]octan-2-yl benzoate exhibit attractive antimalarial activity (IC(50)=0.23-0.72 microM). Two bicyclooctanone oximes are even as active as chloroquine (IC(50)=0.08-0.15 microM, chloroquine: IC(50)=0.12 microM against sensitive strains).     

Synthesis and evaluation of new antimalarial phenylurenyl chalcone derivatives

Phenylurenyl chalcone derivatives have been synthesized and tested as inhibitors of in vitro development of a chloroquine-resistant strain of Plasmodium falciparum, activity of the cysteine protease falcipain-2, in vitro globin hydrolysis, beta-hematin formation, and murine Plasmodium berghei malaria. The most active antimalarial compound was 1-[3'-N-(N'-phenylurenyl)phenyl]-3(3,4,5-trimethoxyphenyl)-2-propen-1-one 49, with an IC(50) of 1.76 microM for inhibition of P. falciparum development. Results suggest that chalcones exert their antimalarial activity via multiple mechanisms.     

Synthesis, cytotoxicity and antimalarial activity of ferrocenyl amides of 4-aminoquinolines

Series of 4-aminoquinolines bearing an amino side chain linked to the ferrocene moiety through an amide bond were synthesized and evaluated for their antimalarial activity against both chloroquine-sensitive (D10, CQ-S) and chloroquine-resistant (Dd2, CQ-R) strains of Plasmodium falciparum. They were also tested for cytotoxicity against Chinese Hamster Ovarian (CHO) cells. Amide 12 featuring propyl side chain linked to the ferrocene ring was the most active of all tested compounds. With an IC50 value of 0.08 microg/mL, this amide showed 1.5-fold higher activity than chloroquine diphosphate (IC50 = 0.12 microg/mL) against the resistant strain, with a selectivity index of 550 indicating its high selectivity towards the parasite. Derivatives which were equipotent against both strains also showed up to ten-fold increase in activity compared to primaquine.     

Antiplasmodial activity of Cryptolepis sanguinolenta alkaloids from leaves and roots

The roots of Cryptolepis sanguinolenta have been investigated for their chemical composition since 1931 but so far no studies on the leaves have been reported although they are used in traditional medicine in Guinea-Bissau. Two new alkaloids identified as cryptolepinoic acid (1) and methyl cryptolepinoate (2) and the known alkaloids cryptolepine (4), hydroxycryptolepine (5/5a) and quindoline (6), were isolated from the ethanolic and chlorophormic leaf extracts. Aqueous and ethanolic extracts of the leaves and roots and seven alkaloids isolated from those extracts were tested in vitro against Plasmodium falciparum K1 (multidrug-resistant strain) and T996 (chloroquine-sensitive clone). All the extracts were shown to give 90% inhibition of P. falciparum K1 growth at concentrations < 23 micrograms/ml. Cryptolepine (4) was the most active alkaloid tested with IC50 values (0.23 microM to K1; 0.059 microM to T996) comparable with chloroquine (0.26 microM to K1; 0.019 microM to T996). The indolobenzazepine alkaloid cryptoheptine (7) was the second most active with IC50 values of 0.8 microM (K1) and 1.2 microM (T996). Cryptolepinoic acid (1) showed no significant activity while its ethyl ester derivative 3 was active against P. falciparum K1 (IC50 = 3.7 microM). All the indoloquinoline alkaloids showed cross-resistance with chloroquine but not the indolobenzazepine alkaloid 7. It was noticed that alkaloids with weakly basic characteristics were active whereas other structurally related alkaloids with different acid-base profiles were inactive. These observations are in agreement with the antimalarial mechanism of action for quinolines.