Mohammad Faheem Khan

Constituents from fruits of Cupressus sempervirens.

Two new phenolic glycosides (1, 2), along with fourteen known compounds (3-16) have been isolated from the fruit of Cupressus sempervirens. The structures of these compounds were determined by spectroscopic analysis and were evaluated for their inhibitory activity against glycogen phosphorylase and glucose-6-phosphatase enzymes. Compounds 14 showed a moderate inhibition against glucose-6-phosphatase and 15 against glycogen phosphorylase enzymes.

Aza-annulation on the 16-dehydropregnenolone, via tandem intermolecular aldol process and intramolecular Michael addition.

16-Dehydropregnenolone undergoes a smooth annulation with propan-1-amine and aromatic aldehydes. Several amine derivatives of 16- dehydropregnenolone were synthesized and evaluated as inhibitors of DPP-IV. The structures of compounds were confirmed by (1)H, (13)C, NMR and mass spectral analysis. Among 17 compounds evaluated only five compounds 1, 9, 13, 15 and 16 demonstrated significant inhibition of DPP. This study suggest that introduction of appropriate substituents in the 16-dehydropregnenolone plays an important role in DPP-IV inhibitory activity.

Chemical constituents of Kigelia pinnata twigs and their GLUT4 translocation modulatory effect in skeletal muscle cells.

Phytochemical investigation of the ethanolic extract of twigs of Kigelia pinnata DC. afforded one new iridoid 7-hydroxy eucommiol (1), and nine known compounds (2-10). The structure of compounds was elucidated by extensive spectroscopic methods, including 1D, 2D NMR experiments and MS analysis. All these compounds were evaluated for GLUT4 translocation modulatory effect in skeletal muscle cells. Four of the tested compounds 1, 5, 6 and 7 showed significant stimulation of GLUT4 translocation to cell surface in skeletal muscle cells without any adverse effect on cell viability. Effect of these four compounds was concentration-dependent and comparable to standard drug rosiglitazone. These findings indicate that constituents of K. pinnata may provide leads for the therapeutics for insulin resistance and diabetes.

Design and synthesis of lupeol analogues and their glucose uptake stimulatory effect in L6 skeletal muscle cells

Structure modifications of lupeol at the isopropylene moiety have been described via allylic oxidation using selenium dioxide. The antidiabetic efficacy of lupeol analogues were evaluated in vitro as glucose uptake stimulatory effect in L6 skeletal muscle cells. From all tested compounds, 2, 3, 4b and 6b showed significant stimulation of glucose uptake with respective percent stimulation of 173.1 (p <0.001), 114.1 (p <0.001), 98.3 (p <0.001) and 107.3 (p <0.001) at 10μM concentration. Stimulation of glucose uptake by these compounds is associated with enhanced translocation of glucose transporter 4 (GLUT4) and activation of IRS-1/PI3-K/AKT-dependent signaling pathway in L6 cells. Structure-activity relationship analysis of these analogues demonstrated that the integrity of α,β-unsaturated carbonyl and acetyl moieties were important in the retention of glucose uptake stimulatory effect. It is therefore proposed that naturally occurring lupeol and their analogues might reduce blood glucose, at least in part, through stimulating glucose utilization by skeletal muscles.

Synthesis of novel imbricatolic acid analogues via insertion of N-substituted piperazine at C-15/C-19 positions, displaying glucose uptake stimulation in L6 skeletal muscle cells

A new class of N-substituted piperazine analogues of imbricatolic acid have been designed and synthesized by using the appropriate synthetic routes in excellent yield. All synthesised compounds were screened for their in vitro glucose uptake stimulatory activity. Among them compounds 4b, 4e, 8b, and 8e triggered L6 skeletal muscle cells for glucose uptake at 54.73%, 40.79%, 40.90%, and 39.55% stimulation, respectively. Compound 4b has emerged as important lead compound showing potential antidiabetic activity. Illustration about their synthesis and in vitro glucose uptake activity is described.

Labda‐8(17),12,14‐trien‐19‐oic Acid Contained in Fruits of Cupressus sempervirens Suppresses Benign Prostatic Hyperplasia in Rat and In Vitro Human Models Through Inhibition of Androgen and STAT‐3 Signaling

Fruit extract of Cupressus sempervirens (CS), which is used traditionally to treat Benign Prostatic Hyperplasia (BPH)‐like urinary symptoms in patients, was scientifically validated for anti‐BPH activity. The ethanolic fruit extract of CS inhibited proliferation of human BPH‐stromal cells and the activity was localized to its chloroform‐soluble, diterpene‐rich fraction. Eight major diterpenes isolated from this fraction exhibited moderate to potent activity and the most active diterpene (labda‐8(17),12,14‐trien‐19‐oic acid) exhibited an IC50 of 37.5 μM (antiproliferative activity against human BPH‐stromal cells). It significantly inhibited activation (phosphorylation) of Stat‐3 in BPH‐stromal cells and prevented transactivation of androgen sensitive KLK3/PSA and TMPRSS2 genes in LNCaP cells. Labda‐8(17),12,14‐trien‐19‐oic acid‐rich CS fraction prevented prostatic hyperplasia in rat model and caused TUNEL labeling of stromal cells with lower expressions of IGF‐I, TGF‐ß and PCNA, and bcl‐2/bax ratio. Human BPH tissues exhibited precise lowering of stromal component after incubation in labda‐8(17),12,14‐trien‐19‐oic acid, ex vivo. We conclude that labda‐8(17),12,14‐trien‐19‐oic acid contained in CS exhibits anti‐BPH activity through inhibition of stromal proliferation and suppression of androgen action in the prostate, presenting a unique lead structure for further optimization of anti‐BPH activity. Copyright

Design and synthesis of lupeol analogues and their in vitro PTP-1B inhibitory activity

Synthetic analogues of the naturally occurring triterpenoid, lupeol by modification of A-ring and isopropylene moiety with different carboxylic acid functionalities and their biological activity were investigated. The analogues were designed by considering the incorporation of ester and amide linkages in the parent molecule. They were assayed for protein tyrosine phosphatase-1B (PTP-1B) inhibitory activity. Among them, compounds 9a, 9b, 14a, 14b and 14c showed significant dose-dependant inhibition at 10 μM concentration. Our report is marked by readily accessible synthesis, excellent yield and significant PTP-1B inhibitory effect.Graphical abstractA synthetic approach and in vitro PTP-1B inhibition of novel analogues of lupeol are described. Compounds 9a, 9b, 14a, 14b and 14a represent a new class of PTP-1B inhibitors in type 2 diabetes

Analysis of constituents of the eastern Nigeria mistletoe, Loranthus micranthus linn revealed presence of new classes of osteogenic compounds

ETHNOPHARMACOLOGICAL RELEVANCE Mistletoe extracts (decoctions) are used traditionally in eastern Nigeria for the management of bone pain, post menopausal syndrome and diabetes amongst several other ailments. While scientific evidence supporting its folkloric use as an antidiabetic agent has been documented, the age-long practice of its use in treatment of post menopausal syndrome has not been scientifically validated. Postmenopausal osteoporosis accounts for one of the prevalent disease conditions in aging population globally. This situation is exacerbated by the lack of osteogenic therapy. In search for plants of Nigerian origin with osteogenic potential, we evaluated eastern Nigerian mistletoe, having ethnotraditional claims of anti-diabetic, anti-hypertensive and anti-cancer activities as well as preventive effect in various post-menopausal syndromes. MATERIALS AND METHODS Methanolic extracts of mistletoe leaves harvested from three host tress - Kola acuminata (KM), Citrus spp (CM) and Garcinia kola (GKM) - were evaluated for osteoblast viability and osteogenic activities using primary rat calvaria culture. Lupeol (1) was isolated from the stem bark of Bombax ciba and its congener, dihydoxylupeol palmitate (2) in addition to three other compounds; 3-methoxy quercetin (3), 3,4,5-trimethoxy gallate (4), and friedelin (5) were isolated from the leaves of mistletoes species. Following their chemical characterization, the compounds were evaluated for osteogenic potential using validated models including alkaline phosphatase (ALP) assay, mineralization assay and expression of osteogenic genes - bone morphogenetic protein-2 (BMP2) and osteoblast transcription factor (RUNX2) - in primary calvarial cultures harvested from neonatal rats. Uterine estrogenicity of the extracts was tested in adult female Sprague Dawley rats. RESULTS Methanol extracts of mistletoe from three hosts exhibited increase in ALP activity (a marker of osteoblast differentiation) at lower concentrations (0.2-0.8 μg/ml) and either no or inhibitory effect at higher concentrations (1.6 and 3.2 μg/ml). None of the extract had cytotoxicity to osteoblasts at the concentrations tested. Five compounds viz. 1 from Bombax ciba, and 2-5 were isolated from the mistletoe leaves. Out of these, 5 exhibited significant loss of osteoblast viability and hence it was not considered further. All four compounds exhibited stimulatory effects on osteoblast differentiation as assessed by ALP assay and determination of osteogenic gene expression. Compound 2 was relatively more potent than its precursor, compound 1 in stimulating BMP2 upregulation. KM did not show uterine estrogenicity. CONCLUSION Methanolic extracts from the three mistletoes species possess in vitro osteogenic activity, and from these extracts three new classes of compounds have been found to promote osteoblast differentiation in vitro. In light of these findings, we propose that mistletoe species may be developed as safer alternative(s) in the management of diseases where lack of bone formation is the pathology.

Bioavailability, plasma protein binding and metabolic stability studies of a ALDH2 activator, alda-1, using a validated LC-ESI-MS/MS method in rat plasma

Alda-1 is an activator of the enzyme ALDH2. It has been suggested as a novel therapeutic for cardiovascular implications such as myocardial infarction, coronary bypass surgery, heart transplantation, peripheral artery disease, ischemia reperfusion injury, angina and alcoholic cardiomyopathy. Despite its widespread experimental use, no reports are available on its pharmacokinetics or bioanalytical quantification. In the present study, a simple, precise and reliable LC-ESI-MS/MS method has been developed and validated for the first time for quantification of alda-1 in plasma. Alda-1 was analyzed on a C18 column using methanol and 0.1% formic acid (60 : 40, v/v) as the mobile phase at a flow rate of 0.7 mL min−1. The method was found to be linear within the concentration range of 1–500 ng mL−1. The intra- and inter-day precision and accuracy were within acceptable limits. For the first time, the preclinical oral and intravenous pharmacokinetics of alda-1 were conducted. Alda-1 was found to be a rapidly absorbed, high clearance and poorly bioavailable compound in rats. Its plasma protein binding was found to be 82–86%. In view of the new regulatory guidelines, incurred sample reanalysis was also performed and all the samples were found within 15% of the mean value. From the in vitro microsomal incubation studies, it was found to be a high extraction compound. The data presented here provide important information to support the in vivo efficacy of alda-1 and would be helpful in its further development as a therapeutic agent and synthesis of its analogs with better systemic exposure and disposition properties.

Osteogenic activity of natural diterpenoids isolated from Cupressus sempervirens fruits in calvarial derived osteoblast cells via differentiation and mineralization

The aim of the present study was to investigate the antiosteoporotic activity of four structurally related diterpenoids: sugiol (1), trans-communic acid (2), 15-acetoxy imbricatolic acid (3) and imbricatolic acid (4). Their osteogenic effect was evaluated by using validated models including alkaline phosphatase (ALP) assay, mineralization assay and expression of osteogenic genes-bone morphogenetic protein-2 (BMP-2) and osteoblast transcription factor (RUNX2) - in primary calvarial cultures harvested from neonatal mice. Among them, compound 1 at a dose of 1.0 mg/kg body weight exhibited significant osteoprotective effects and did not show uterine estrogenicity at the same dose. Additionally, compound 1 treatment led to improved biomechanical properties as exhibited by increased power, energy and stiffness in femoral bones compared to untreated Ovx animals. Since osteoporotic compression fracture correlates with the mechanical characteristics of trabecular bone, so that it could effectively reduce the risk of this type of fracture by improving trabecular micro architecture in postmenopausal women. Therefore, our findings proposed that diterpenoids may be useful new chemical agents in the treatment of diseases associated with bone loss.