Synthesis and Evaluation of Chirally Defined Side Chain Variants of 7-Chloro-4-Aminoquinoline To Overcome Drug Resistance in Malaria Chemotherapy.

A novel 4-aminoquinoline derivative [(S)-7-chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

Design and synthesis of 3-[(7-chloro-1-oxidoquinolin-4-ylamino)alkyl]-1,3-thiazolidin-4-ones as antimalarial agents.

A new series of quinoline analogs have been synthesized and found active against P. falciparum in vitro and P. yoelli in vivo. Compounds 8, 10 and 11 exhibited superior in vitro activity compared to chloroquine. Selected compounds 8, 10 and 11 exhibited significant suppression of parasitaemia in vivo assay. These analogs form a complex with hematin and inhibit the ß-hematin formation, suggesting that this class of compounds act on a heme polymerization target. Further this study confirms that quinoline ring nitrogen is essential for both transportation of the molecule across the membrane as well as for tight binding to hematin.

Design, synthesis of 4-aminoquinoline-derived thiazolidines and their antimalarial activity and heme polymerization inhibition studies.

The present study describes the synthesis of a series of new 4-aminoquinoline-derived thiazolidines and evaluation of their antimalarial activity against a NF-54 strain of Plasmodium falciparum in vitro and N-67 strain of Plasmodium yoelii in vivo. Among the series, two compounds, 2-(4-chloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (14) and 2-(2,6-dichloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (22) exhibited significant suppression of parasitaemia in the in vivo assay. All the analogues were found to form strong complex with haematin and inhibited the ß-haematin formation in vitro. These results suggest that these compounds act on heme polymerization target.

CpG oligodeoxynucleotide 2006 and miltefosine, a potential combination for treatment of experimental visceral leishmaniasis.

In view of the severe immunosuppression in visceral leishmaniasis (VL), a rational approach to effectively combat the parasitic scourge would be to enhance the immune status of the host. Use of CpG oligodeoxynucleotide (CpG-ODN) against leishmaniasis has previously been reported, especially as an immunomodulator and adjuvant with various immunogens. In the present study, experiments were carried out with BALB/c mice and hamsters infected with Leishmania donovani. Immunostimulating class B bacterial CpG-ODN namely, ODN-2006, was administered at various doses by the intraperitoneal (i.p.) route. The dose of CpG-ODN-2006 (1 nM/single dose) showing the most antileishmanial activity was given as free and liposomal forms with different doses of miltefosine, namely, 5 and 10 mg/kg of body weight, for 5 days in mice and hamsters, respectively. Among the various groups, mice coadministered liposomal CpG-ODN and miltefosine (5 mg/kg) showed the best inhibitory effect (97% parasite inhibition) compared with free CpG-ODN plus miltefosine and miltefosine, free CpG-ODN, and liposomal CpG-ODN given separately. Similar responses were observed in the case of hamsters, where the combination of liposomal CpG-ODN with miltefosine (10 mg/kg) gave 96% parasite inhibition. Promising antileishmanial efficacy was observed in animals treated with liposomal CpG-ODN and miltefosine.

CpG oligodeoxynucleotide augments the antileishmanial activity of miltefosine against experimental visceral leishmaniasis.

OBJECTIVES: To evaluate the combination of CpG oligodeoxynucleotide (CpG ODN) and miltefosine for the treatment of experimental visceral leishmaniasis (VL). METHODS: The experiments were carried out using BALB/c mice and hamsters, infected with Leishmania donovani. CpG ODN was administered at various doses by the intraperitoneal (ip) route. The dose of CpG ODN (1 nM/single dose) showing best antileishmanial activity was given as free and liposomal forms with a subcurative dose of miltefosine, namely 2.5 and 5 mg/kg x 5 days in mice and hamsters, respectively. RESULTS: Among the various groups of mice, co-administered liposomal CpG ODN and miltefosine showed the best inhibitory effect (85% inhibition) compared with free CpG ODN and miltefosine, and miltefosine, free CpG ODN and liposomal CpG ODN separately. Production of Th1 cytokines, nitric oxide (NO), reactive oxygen species (ROS) and H(2)O(2) was enhanced. A remarkable increase in the phagocytosis index was also observed, indicating overall immunological support to antileishmanial activity of miltefosine by CpG ODN. Similar responses were observed in hamsters. CONCLUSIONS: Promising antileishmanial efficacy was observed in animals treated with liposomal CpG ODN and miltefosine, strongly supported by enhancement of Th1 cytokines as well as NO, ROS and H(2)O(2) levels. The correlation of experimental findings in both the models (mouse and hamster) strengthens the potential of CpG ODN as an immunomodulator in combination with miltefosine against VL.

Synthesis and antimalarial activity of novel side chain modified antimalarial agents derived from 4-aminoquinoline.

Malaria is one of the foremost public health problems in developing countries affecting nearly 40% of the global population. Apart from this, the past two decade's emergence of drug resistance has severely limited the choice of available antimalarial drugs. Furthermore, the general trend emerging from the SAR-studies is that chloroquine resistance does not involve any change to the target of this class of drugs but involves compound specific efflux mechanism. Based on this premise a number of groups have developed short chain analogues of 4-aminoquinoline, which are active against CQ-resistant strains of P. falciparum in in vitro studies. However, these derivatives undergo biotransformation (de-alklyation) significantly affecting lipid solubility of the drug. In view of this background information, we thought that it would be interesting to study the effect of additional lipophilicity and cationic charge at the lateral side chain of 4-aminoquinoline. This prompted us to explore the cationic amino acid conjugates namely, lysine and ornithine of 4-aminoquinoline with a view to achieve improved antimalarial activity and to the best of our knowledge such amino acid conjugates have not been hitherto reported in the literature in the case of 4-aminoquinolines. In the present study, a new series of side-chain modified 4-aminoquinolines have been synthesized and found active against both susceptible and multidrug resistant strains of P. falciparum in vitro and P. yoelli in vivo. The seminal finding of the present study is that a new series of compounds having significantly more activity against CQ resistant parasites has been identified.

Synthesis and antimalarial activity of side chain modified 4-aminoquinoline derivatives.

A new series of side-chain modified 4-aminoquinolines have been synthesized and found active against P. falciparum in vitro and P. yoelli in vivo. Compounds 6, 11, 12, and 19 exhibited superior in vitro activity compared to chloroquine. Selected compounds 6, 12, and 19 exhibited significant suppression in the in vivo assay. These analogs form a complex with hematin and inhibit the beta-hematin formation, suggesting that this class of compounds act on a heme polymerization target.

Design, synthesis and antimalarial activity of a new class of iron chelators.

Iron is crucial for many biochemical reactions involved in the growth and multiplication of the malaria parasite Plasmodium falciparum. There are many reports indicating that the iron chelators have antimalarial activity in vitro, in vivo and in human studies. However, these compounds suffer from a number of serious problems such as limited membrane permeability, short half-life and require long subcutaneous infusions. To circumvent these drawbacks we have designed a new class of iron chelators, wherein EDTA is tethered to 4-aminoquinoline. Here 4-aminoquinoline scaffold is used as a carrier to penetrate biological membrane and facilitate targetting the compounds to acidic food vacuole of the parasite. This study describes the synthesis of novel iron chelators and their in vitro antimalarial activity against P. falciparum strain of NF-54. The calculated LogP values of these compounds suggest the importance of lipophilicity for the antimalarial activity. The EDTA esters are more active than the corresponding acids. The biophysical studies suggest that these compounds may inhibit the parasite growth by iron chelation mechanism.

Tuftsin-bearing liposomes as antibiotic carriers in treatment of macrophage infections.

Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) that specifically binds monocytes, macrophages, and polymorphonuclear leukocytes and potentiates their natural killer activity against tumors and pathogens. The antimicrobial activity of this peptide is significantly increased by attaching at the C-terminus a fatty acyl residue through the ethylenediamine spacer arm. This activity is further augmented by incorporating the modified tuftsin in the liposomes. The tuftsin-bearing liposomes not only enhance the host's resistance against a variety of infections but also serve as useful vehicles for the site-specific delivery of drugs in a variety of macrophage-based infections, such as tuberculosis and leishmaniasis.

Immunomodulatory activity of analog of muramyl dipeptide and their use as adjunct to chemotherapy of Leishmania donovani in hamster.

In search of a potent immunomodulator to be used as an immunoprophylactic agent and as adjunct to chemotherapy against Leishmania infection, two analogs of muramyl dipeptide, viz. N.Ac-norMur-MeVal-D-isoGln (86/448) and N.AcMur-Acc-D-isoGln (89/729) were evaluated for desired activity. Effect of these peptides on cell mediated and humoral immunity was studied by immunizing the peptide treated mouse with sheep red blood cells (SRBC) and determining HA-titer, plaque forming cells assay and delayed type of hypersensitivity (DTH) response after 4-5 days. Both the peptides stimulated cell mediated immunity (CMI), humoral response as well as macrophage function in terms of super oxide anion (O2-) and nitric oxide (NO) generation. Mitogen induced lymphocyte proliferation and production of IL-2 and INF-gamma increased while that of IL-4 and IL-10 decreased by both the peptides showing a typical Th1 type response. After establishing the immunostimulatory activity, these peptides were evaluated for immunoprophylactic efficacy as well as for use as adjunct to chemotherapy with stibanate (SSG) against Leishmania donovani infection in golden hamster. These peptides were found quite effective in both the modes. In adjunct use the treatment may require lower dose of SSG and thereby reduce the chances of drug toxicity.

Augmentation of human natural killer cells by splenopentin analogs.

Splenopentin, Arg-Lys-Glu-Val-Tyr (SP-5) and its synthetic analogs; Arg-D-Lys-Glu-Val-Tyr (pentapeptide 1), Lys-Lys-Glu-Val-Tyr (2), D-Lys-Lys-Glu-Val-Tyr (3), Arg-Lys-Gly-Val-Tyr (4), and Arg-Lys-Gln-Val-Tyr (5) have been examined for augmentation of human natural killer (NK) cell activity and human T-cell transformation response. Pentapeptides 2 and 3 were found to significantly augment the in vitro human NK cell activity. However, none of them had any effect on lymphocyte proliferative responses.

Thymopentin and splenopentin as immunomodulators. Current status.

Splenopentin (SP-5, Arg-Lys-Glu-Val-Tyr) and thymopentin (TP-5, Arg-Lys-Asp-Val-Tyr) are synthetic immunomodulating peptides corresponding to the region 32-34 of a splenic product called splenin (SP) and the thymic hormone thymopoietin (TP), respectively. TP was originally isolated as a 5-kDa (49-amino acids) protein from bovine thymus while studying effects of the thymic extracts on neuromuscular transmission and was subsequently observed to affect T cell differentiation and function. TP I and II are two closely related polypeptides isolated from bovine thymus. A radioimmunoassay for TP revealed a crossreaction with a product found in spleen and lymph node. This product, named splenin, differs from TP only in position 34, aspartic acid for bovine TP and glutamic acid for bovine splenin and it was called TP III as well. Synthetic pentapeptides (TP-5) and (SP-5), reproduce the biological activities of TP and SP, respectively. It is now evident that various forms of TPs were created by proteolytic cleavage of larger proteins during isolation. cDNA clones have been isolated for three alternatively spliced mRNAs that encodes three distinct human T cell TPs. The immunomodulatory properties of TP, SP, TP-5, SP-5 and some of their synthetic analogs reported in the literature have been briefly reviewed.

A solid phase approach to substituted pyrimidines and their conversion into condensed heterocycles for potential use in combinatorial chemistry.

A novel general synthesis of substituted pyrimidine 3 has been carried out on solid support. The C-atoms carring the cyano, amino, carboxamido, as well as anchoring site have exploited to generate libraries of compounds 6-8, 10, 13, 15, 17, 19, 21, 23, 25 and 27. A novel strategy to cleave the resin to resin-site unsubstituted system has been developed and it provides 5,6-disubstituted pyrimidines 6-8. In addition, synthesis of 2,5,6-trisubstituted pyrimidines of prototype 10 were carried out by nucleophilic displacement of the anchor by various amines. Further investigations were directed toward the solid phase synthesis of pyrimido[4,5-d]pyrimidines 12, 16, 20 and 24 in which C-atoms carring the oxo, thio, amino, anchoring site as well as NH could be introduced as center of diversity to generate libraries of compounds for potential use. 4-Aminopyrimido[4,5-d]pyrimidines 13 and 17 were obtained from fusion of 3a with urea or thiourea followed by cleavage of support while 3-phenylpyrimido[4,5-d]pyrimidines 21 and 27 were synthesized from cyclisation of 4 with phenyl isocyanate or isothiocyanate followed by release of resin. 7-substituted pyrimido[4,5-d]pyrimidines 15, 19, 23 and 27 were obtained by oxidation of 12, 16, 20 and 24 followed by aminolytic cleavage of support.

Structure-activity relationship studies of dynorphin A and related peptides.

An up-to-date review is presented covering all the available information concerning the isolation, discovery, synthesis, conformation, receptor binding characteristics, pharmacological properties and SAR studies of dynorphin A and related peptides. The potential of dynorphin A and its analogs has yet to be fully realized.

Stimulation of nonspecific resistance by thymopentin and its analogs against Leishmania donovani infection in hamsters.

Thymopentin and its analogs have been synthesized by the solution phase method of peptide synthesis and evaluated for their prophylactic efficacy against L. donovani infection in hamsters. Thymopentin and some of the analogs were found to stimulate nonspecific resistance of the host against Leishmania donovani infection in hamsters.

Antisense peptide interactions studied by electrospray ionization mass spectrometry.

Non-covalent interactions between met- and leu-enkephalins and their antisense peptides were studied by electrospray ionization mass spectrometry. Mixtures of sense and antisense peptides gave both the corresponding homodimers and heterodimers. The relative abundance ratios of the heterodimer to that of the homodimer of the sense peptide and the relative stability constants of the heterodimers were compared with the corresponding values from mixtures of the sense peptides and a control peptide. The results show that there is a preferential interaction between the sense and antisense peptides compared with that between the sense and control peptides.

Synthesis and opioid activity of novel tetrapeptides analogous to sequence (1-4) of dermorphin.

Seven new tetrapeptides analogous to (1-4) sequence of dermorphin were synthesized and evaluated for their opioid activity. The peptides were synthesized by the solution phase method. Their opioid activity revealed that peptides II and V were the most potent in the analgesia test as well as in the peripheral assays. Peptide II was most active in the guinea pig ileum assay, whereas peptide VI was 2763 times more selective for mu-receptors.

Attenuation of alcohol-induced hypothermia by cyclo (His-Pro) and its analogs.

Acute administration of cyclo (His-Pro) to rats cause a dose-dependent decrease in ethanol-induced hypothermia. Bromination of the imidazole moiety of histidine in cyclo (His-Pro) resulted in a significant increase in its potency to attenuate ethanol hypothermia. In contrast, benzylation of the imidazole moiety of histidine or the substitution of one or both of the amino acids in cyclo(His-Pro) led to a total loss of its thermomodulatory activity. In conclusion, it appears from these preliminary data that it may be possible to design analogs of CHP that may be effective antagonists for ethanol hypothermia.

Properties of TAN-67, a nonpeptidic delta-opioid receptor agonist, at cloned human delta- and mu-opioid receptors.

2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino[2,3,30g]isoquinoline (TAN-67) is a nonpeptidic delta-opioid receptor agonist. This report describes its receptor binding affinity and agonist potency at human and mouse delta and mu-opioid receptors. The binding affinities of TAN-67 and the cyclic enkephalin analog, (D-Pen2, 4'-Cl-Phe4, D-Pen5]enkephalin (pCl-DPDPE) were measured by radioligand binding inhibition studies at mouse and human variants of the delta and mu-opioid receptor using [3H]Naltrindole and [3H]D-Phe-Cys-Tyr-D-Trp-Orn-Thr -Pen-Thr-NH2, respectively. TAN-67 showed high binding affinity (Ki = 0.647 nM) at the human delta-opioid receptor and high delta-opioid receptor binding selectivity ( > 1000-fold) relative to the human mu-opioid receptor. TAN-67 also showed high potency (EC50 = 1.72 nM) for the inhibition of forskolin-stimulated cAMP accumulation at human delta-opioid receptors expressed by intact Chinese hamster ovary cells but low potency (EC50 = 1520 nM) at human mu-opioid receptors expressed by intact B82 mouse fibroblast cells. The results show that TAN-67 has similar binding affinities, selectivity and potencies as pCl-DPDPE at human delta and mu-opioid receptors. These results combined with the nonpeptidic structure of TAN-67 suggest that this compound has therapeutic potential as a delta-opioid receptor agonist.

Reversal of T cell anergy in leprosy patients: in vitro presentation with Mycobacterium leprae antigens using murabutide and Trat peptide in liposomal delivery.

Mycobacterium leprae, the causative agent of leprosy resides and multiplies within the host monocytes and macrophages, thereby evading host immune system. Cell-mediated immune response (CMI) plays a vital role as evidenced from the high CMI in BT/TT (borderline and tuberculoid) patients and conversely low in BL/LL (borderline and lepromatous) patients. In the present study, an attempt was made to immunomodulate the anergized T cells of lepromatous leprosy patients by presenting the mycobacterial antigen in combination with T cell adjuvant, murabutide (active analog of muramyl' dipeptide, MDP-BE) and a Trat peptide (T cell epitope of Integral membrane protein (Trat) from Escherichia coli) in particulate form (liposomes) or soluble form (media). PBMNC of normal, BT/TT and BL/LL were stimulated in vitro with five mycobacterial antigens (Ag) in the following formulations, Ag, Ag+murabutide, Ag+murabutide+Trat peptide either in liposomes or in medium. All the five antigen(s) when delivered in liposomes containing murabutide and Trat peptide showed a very high lymphoproliferative response (p<0.001) in all the three groups. IFN-gamma and IL-2 were significantly (p<0.001) high in these culture supernatants compared to IL-10 and IL-4 confirming a shift from CD4+Th2 to Th1 response in leprosy patients with particulate mode of antigen presentation. Interestingly, PBMNC derived from lepromatous patients also showed consistent T cell proliferation with all the formulations. Further, the mechanism of liposomal processing of antigens was studied using different inhibitors that interfere at different stages of antigen presentation. Results indicate that this study may pave way for an immunotherapeutic approach for reverting the anergic T cells of lepromatous patients to proliferating T cells with the release of Th1 cytokines thereby restoring the CMI response in these patients.