Peganine hydrochloride dihydrate an orally active antileishmanial agent q Tanvir Khaliq a , Pragya Misra b , Swati Gupta b , K. Papi Reddy a , Ruchir Kant c , P. R. Maulik c , Anuradha Dube b, * , T. Narender a, * a Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226 001, UP, India b Parasitology Division, Central Drug Research Institute, Lucknow 226 001, UP, India c Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow 226 001, UP, India article info Article history: Received 3 December 2008 Revised 12 February 2009 Accepted 5 March 2009 Available online 16 March 2009 Keywords: Peganinehydrochloridedihydrate In-vivo and In-vitro antileishmanial activity Apoptosis Topoisomerase inhibition abstract Protozoic infections caused by genus Leishmania pose an enormous public health threat in developing countries, compounded by the toxicity and resistance to current therapies. Under the aegis of our ongoing program on drug discovery and development on antileishmanial agents from plants, we carried out bio- assay guided fractionation on Peganum harmala seeds which resulted in the isolation of 1 as an antileish- manial agent. 2D-NMR spectral data and single crystal X-ray crystallography data indicated 1 as peganine hydrochloride in dihydrated form. The compound 1 exhibited in-vitro activity against both extracellular promastigotes as well as intracellular amastigotes residing within murine macrophages in Leishmania donovani. Furthermore, 1 also exhibited in-vivo activity, 79.6 (±8.07)% against established VL in hamsters at a dose of 100 mg/kg b.wt. Ó 2009 Elsevier Ltd. All rights reserved. Visceral leishmaniasis (VL or Kala-azar) is the most devastating form of leishmaniasis among complex leishmaniasis and is caused by the invasion of the reticuloendothelial system (spleen, liver and bone marrow) by the haemoflagellate protozoan parasite Leish- mania donovani. More than 12 millions individuals were infected by leishmaniasis around the world with 40000 new cases every year. The disease is generally restricted to areas, heavily infested by the sandfly (Phlebotomus spp.), the vector of this disease, which is widely distributed in the Indian subcontinent and south-west Asia. 1,2 In India, high incidence has been reported from the states of Bihar, Assam, West Bengal and Eastern Uttar Pradesh where resistance and relapse are on the increase. Since the 1940s, penta- valent antimonial compounds have been constituted the first-line treatment for all forms of leishmaniasis. In case of therapeutic resistance to these compounds, amphotericin B desoxycholate, liposomal amphotericin B and miltefosine may also be used. 3 How- ever, most of these drugs are expensive, toxic and have side effects and complicated by the fact that they are given parenterally. 4 Moreover, cases of drug resistance are on the rise. 5 The lack of an effective antileishmanial drug has caused a renewed interest in the study of medicinal plants as source of new chemotherapeutic compounds with better activities and few- er side effects. Peganum harmala Linn, commonly known as ‘har- mal’ belonging to the family Zygophyllaceae, is one of the most important medicinal plants of India. 6 Its different parts are used in traditional systems of medicine for the treatment of variety of human ailments. 7–16 We carried out bioassay guided fractionation of P. harmala seeds which resulted in the isolation of 1 as an antile- ishmanial agent. 2D-NMR spectral data and single crystal X-ray crystallography data indicated 1 as peganine hydrochloride in dihydrated form (Figs. 2 and 3). 17,18 Recently we reported the in-vi- tro antileishmanial activity of 1 and its programmed cell death (apoptosis) and topoisomerase inhibiting activity in L. donovani. 19 Compound 1 induces apoptosis-like cell death in L. donovani. Strong binding interactions between 1 and DNA topoisomerase I in molecular modeling (docking) studies and our experimental studies against DNA topoisomerase of L. donovani was similar to some other natural as well as synthetic quinazoline alkaloiods 2- 6 (Fig. 1). The apoptosis like cell death appears to be consistent to L. donovani’s topoisomerase I inhibition by 1. To further validate the 1’s antileishmanial activity in animal studies we carried out in- vivo experiments with 1. Here, we report its in vitro and in vivo antileishmanial efficacy in hamster model. The concentration of 1 at which nearly 50% death of L. donovani promastigotes would occur, was calculated using log phase trans- genic Green Fluorescent Protein (GFP) expression of promastigotes by flow cytometry. The% cell death was measured by decrease in Mean Fluorescence Intensity (MFI) values on treatment with drug. Very rapid and dose-dependent cell death occurred with 1 at con- centrations between 25 and 100 lg/ml, reaching approximately 90% at around 75 lg/ml (IC 90 ). IC 50 was calculated to be 38 (±1.23) lg/ml. The reference drug, miltefosine, exhibited nearly 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.03.039 q CDRI Communication 7606. * Corresponding authors. Tel.: +91 522 2612411x4440; fax: +91 522 2623405 (T.N.). E-mail addresses: anuradha_dube@rediffmail.com (A. Dube), tnarender@rediff mail.com (T. Narender). Bioorganic & Medicinal Chemistry Letters 19 (2009) 2585–2586 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl