Sonali S. Bharate

Modulation of Thermoreceptor TRPM8 by Cooling Compounds

ThermoTRPs, a subset of the Transient Receptor Potential (TRP) family of cation channels, have been implicated in sensing temperature. TRPM8 and TRPA1 are both activated by cooling. TRPM8 is activated by innocuous cooling (<30 °C) and contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia and is a receptor for menthol and icilin (mint-derived and synthetic cooling compounds, respectively). TRPA1 (Ankyrin family) is activated by noxious cold (<17 °C), icilin, and a variety of pungent compounds. Extensive amount of medicinal chemistry efforts have been published mainly in the form of patent literature on various classes of cooling compounds by various pharmaceutical companies; however, no prior comprehensive review has been published. When expressed in heterologous expression systems, such as Xenopus oocytes or mammalian cell lines, TRPM8 mediated currents are activated by a number of cooling compounds in addition to menthol and icilin. These include synthetic p-menthane carboxamides along with other class of compounds such as aliphatic/alicyclic alcohols/esters/amides, sulphones/sulphoxides/sulphonamides, heterocyclics, keto-enamines/lactams, and phosphine oxides. In the present review, the medicinal chemistry of various cooling compounds as activators of thermoTRPM8 channel will be discussed according to their chemical classes. The potential of these compounds to emerge as therapeutic agents is also discussed.

Non-enzymatic browning in citrus juice: chemical markers, their detection and ways to improve product quality

Citrus juices are widely consumed due to their nutritional benefits and variety of pharmacological properties. Non-enzymatic browning (NEB) is one of the most important chemical reactions responsible for quality and color changes during the heating or prolonged storage of citrus products. The present review covers various aspects of NEB in citrus juice viz. chemistry of NEB, identifiable markers of NEB, analytical methods to identify NEB markers and ways to improve the quality of citrus juice. 2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is one of the promising marker formed during browning process with number of analytical methods reported for its analysis; therefore it can be used as an indicator for NEB process. Amongst analytical methods reported, RP-HPLC is more sensitive and accurate method, which can be used as analytical tool. NEB can be prevented by removal of amino acids/ proteins (via ion exchange treatment) or by targeting NEB reactions (e.g. blockage of furfural/ HMF by sulphiting agent).

Discovery of a marine-derived bis-indole alkaloid fascaplysin, as a new class of potent P-glycoprotein inducer and establishment of its structure-activity relationship.

The screening of IIIM natural products repository for P-gp modulatory activity in P-gp over-expressing human adenocarcinoma LS-180 cells led to the identification of 7 natural products viz. withaferin, podophyllotoxin, 3-demethylcolchicine, agnuside, reserpine, seseberecine and fascaplysin as P-gp inducers. Fascaplysin (6a), a marine-derived bis-indole alkaloid, was the most potent among all of them, showing induction of P-gp with EC50 value of 25 nM. P-gp induction is one of the recently targeted strategy to increase amyloid-β clearance from Alzheimer brains. Thus, we pursued a medicinal chemistry of fascaplysin to establish its structure-activity relationship for P-gp induction activity. Four series of analogs viz. substituted quaternary fascaplysin analogs, D-ring opened quaternary analogs, D-ring opened non-quaternary analogs, and β-carbolinium analogs were synthesized and screened for P-gp induction activity. Among the total of 48 analogs screened, only quaternary nitrogen containing analogs 6a-g and 10a, 10h-l displayed promising P-gp induction activity; whereas non-planar non-quaternary analogs 9a-m, 13a-n, 15a-h were devoid of this activity. The P-gp induction activity of best compounds was then confirmed by western-blot analysis, which indicated that fascaplysin (6a) along with 4,5-difluoro analog of fascaplysin 6f and D-ring opened analog 10j displayed 4-8 fold increase in P-gp expression in LS-180 cells at 1 μM. Additionally, compounds 6a and 6f also showed inhibition of acetylcholinestease (AChE), an enzyme responsible for neuronal loss in Alzheimers disease. Thus, fascaplysin and its analogs showing promising P-gp induction along with AChE inhibition at 1 μM, with good safety window (LS-180: IC50 > 10 μM, hGF: 4 μM), clearly indicates their promise for development as an anti-Alzheimer agent.

Synthesis and anti-proliferative activities of new derivatives of embelin.

Embelin (1), a benzoquinone isolated from Embelia ribes is known to possess variety of biological activities. Despite of several promising biological activities, preclinical efforts on embelin were hampered because of its poor aqueous solubility. In order to address the solubility issue, herein, we have synthesized a series of Mannich products of embelin by treating it with various secondary amines. The synthesized compounds were screened for antiproliferative and antimicrobial activities. In cytotoxicity screening, the benzyl-piperidine linked derivative 8m was found to possess better antiproliferative activity compared to parent natural product embelin against a panel of cell lines including HCT-116, MCF-7, MIAPaCa-2 and PC-3 with IC50 values of 30, 41, 34 and 36 μM, respectively. The mechanistic study of compound 8m revealed that it exhibits cytotoxicity via induction of apoptosis and mitochondrial membrane potential loss. Further, the compounds were tested for antimicrobial activity where dimethylamino- 8a and piperidine linked derivative 8b displayed antibacterial activity against Staphylococcus aureus with MIC values of 8 and 16 μg/mL, respectively. Mannich derivatives did now show improved aqueous solubility, however their hydrochloride salts 8a·HCl, 8b·HCl and 8m·HCl showed significantly improved aqueous solubility without affecting biological activities of parent Mannich derivatives.

Determining Partition Coefficient (Log P), Distribution Coefficient (Log D) and Ionization Constant (pKa) in Early Drug Discovery.

An early prediction of physicochemical properties is highly desirable during drug discovery to find out a viable lead candidate. Although there are several methods available to determine partition coefficient (log P), distribution coefficient (log D) and ionization constant (pKa), none of them involves simple and fixed, miniaturized protocols for diverse set of compounds. Therefore, it is necessary to establish simple, uniform and medium-throughput protocols requiring small sample quantities for the determination of these physicochemical properties. Log P and log D were determined by shake flask method, wherein, the compound was partitioned between presaturated noctanol and water phase (water/PBS pH 7.4) and the concentration of compound in each phase was determined by HPLC. The pKa determination made use of UV spectrophotometric analysis in a 96-well microtiter plate containing a series of aqueous buffers ranging from pH 1.0 to 13.0. The medium-throughput miniaturized protocols described herein, for determination of log P, log D and pKa, are straightforward to set up and require very small quantities of sample (< 5 mg for all three properties). All established protocols were validated using diverse set of compounds.

Impact of preformulation on drug development

Introduction: Preformulation assists scientists in screening lead candidates based on their physicochemical and biopharmaceutical properties. This data is useful for selection of new chemical entities (NCEs) for preclinical efficacy/toxicity studies which is a major section under investigational new drug application. A strong collaboration between discovery and formulation group is essential for selecting right NCEs in order to reduce attrition rate in the late stage development. Areas covered: This article describes the significance of preformulation research in drug discovery and development. Various crucial preformulation parameters with case studies have been discussed. Expert opinion: Physicochemical and biopharmaceutical characterization of NCEs is a decisive parameter during product development. Early prediction of these properties helps in selecting suitable physical form (salt, polymorph, etc.) of the candidate. Based on pharmacokinetic and efficacy/toxicity studies, suitable formulation for Phase I clinical studies can be developed. Overall these activities contribute in streamlining efficacy/toxicology evaluation, allowing pharmacologically effective and developable molecules to reach the clinic and eventually to the market. In this review, the magnitude of understanding preformulation properties of NCEs and their utility in product development has been elaborated with case studies.

Thermodynamic equilibrium solubility measurements in simulated fluids by 96-well plate method in early drug discovery☆

An early prediction of solubility in physiological media (PBS, SGF and SIF) is useful to predict qualitatively bioavailability and absorption of lead candidates. Despite of the availability of multiple solubility estimation methods, none of the reported method involves simplified fixed protocol for diverse set of compounds. Therefore, a simple and medium-throughput solubility estimation protocol is highly desirable during lead optimization stage. The present work introduces a rapid method for assessment of thermodynamic equilibrium solubility of compounds in aqueous media using 96-well microplate. The developed protocol is straightforward to set up and takes advantage of the sensitivity of UV spectroscopy. The compound, in stock solution in methanol, is introduced in microgram quantities into microplate wells followed by drying at an ambient temperature. Microplates were shaken upon addition of test media and the supernatant was analyzed by UV method. A plot of absorbance versus concentration of a sample provides saturation point, which is thermodynamic equilibrium solubility of a sample. The established protocol was validated using a large panel of commercially available drugs and with conventional miniaturized shake flask method (r(2)>0.84). Additionally, the statistically significant QSPR models were established using experimental solubility values of 52 compounds.

Nitrofuranyl Methyl Piperazines as New Anti-TB Agents: Identification, Validation, Medicinal Chemistry, and PK Studies.

Whole-cell screening of 20,000 drug-like small molecules led to the identification of nitrofuranyl methylpiperazines as potent anti-TB agents. In the present study, validation followed by medicinal chemistry has been used to explore the structure-activity relationship. Ten compounds demonstrated potent MIC in the range of 0.17-0.0072 μM against H37Rv Mycobacterium tuberculosis (MTB) and were further investigated against nonreplicating and resistant (Rif(R) and MDR) strains of MTB. These compounds were also tested for cytotoxicity. Among the 10 tested compounds, five showed submicromolar to nanomolar potency against nonreplicating and resistant (Rif(R) and MDR) strains of MTB along with a good safety index. Based on their overall in vitro profiles, the solubility and pharmacokinetic properties of five potent compounds were studied, and two analogues, 14f and 16g, were found to have comparatively better solubility than others tested and acceptable pharmacokinetic properties. This study presents the rediscovery of a nitrofuranyl class of compounds with improved aqueous solubility and acceptable oral PK properties, opening a new direction for further development.

Biphenyl-4-carboxylic Acid [2-(1H-Indol-3-yl)-ethyl]-methylamide (CA224), a Nonplanar Analogue of Fascaplysin, Inhibits Cdk4 and Tubulin Polymerization: Evaluation of in Vitro and in Vivo Anticancer Activity

Biphenyl-4-carboxylic acid-[2-(1H-indol-3-yl)-ethyl]-methylamide 1 (CA224) is a nonplanar analogue of fascaplysin (2) that specifically inhibits Cdk4-cyclin D1 in vitro. Compound 1 blocks the growth of cancer cells at G0/G1 phase of the cell cycle. It also blocks the cell cycle at G2/M phase, which is explained by the fact that it inhibits tubulin polymerization. Additionally, it acts as an enhancer of depolymerization for taxol-stabilized tubulin. Western blot analyses of p53-positive cancer cells treated with compound 1 indicated upregulation of p53, p21, and p27 proteins together with downregulation of cyclin B1 and Cdk1. Compound 1 selectively induces apoptosis of SV40 large T-antigen transformed cells and significantly reduces colony formation efficiency, in a dose-dependent manner, of lung cancer cells. It is efficacious at 1/10th of the MTD against human tumors derived from HCT-116 and NCI-H460 cells in SCID mouse models. The promising efficacy of compound 1 in human xenograft models as well as its excellent therapeutic window indicates its potential for clinical development.

Pyrano-isochromanones as IL-6 inhibitors: synthesis, in vitro and in vivo antiarthritic activity.

Bergenin (1), a unique fused C-glycoside isolated from Bergenia species, possesses interesting anti-inflammatory and antipain activities. To study SAR of this scaffold, first-generation derivatives were synthesized and evaluated for inhibition of lymphocyte proliferation and production of pro-inflammatory cytokines. The C-7 substituted derivatives showed inhibition of IL-6 as well as TNF-α production. Bergenin and its most potent IL-6 inhibitor derivatives 4e and 4f were then investigated in a panel of in vitro and in vivo inflammation/arthritis models. These compounds significantly decreased the expression of NF-kB and IKK-β in THP-1 cells. In in vivo study in BALB/c mice, a dose-dependent inhibition of SRBC-induced cytokines, reduction in humoral/cell-mediated immunity, and antibody titer was observed. The CIA study in DBA/1J mice indicated that compounds led to reduction in swelling of paws, cytokine levels, and anticollagen IgG1/IgG2a levels. The significant in vivo immunosuppressive efficacy of pyrano-isochromanones demonstrates the promise of this scaffold for development of next-generation antiarthritic drugs.