Chemotherapy of leishmaniasis part XIII: Design and synthesis of novel heteroretinoid-bisbenzylidine ketone hybrids as antileishmanial agents

Accepted Manuscript Chemotherapy of leishmaniasis part XIII: Design and synthesis of novel heteroretinoid-bisbenzylidine ketone hybrids as antileishmanial agents Avinash Tiwari, Santosh Kumar, Rahul Shivahare, Padam Kant, Suman Gupta, S.N. Suryawanshi PII: DOI: Reference:

S0960-894X(14)01034-8 http://dx.doi.org/10.1016/j.bmcl.2014.09.078 BMCL 22044

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

7 May 2014 15 September 2014 17 September 2014

Please cite this article as: Tiwari, A., Kumar, S., Shivahare, R., Kant, P., Gupta, S., Suryawanshi, S.N., Chemotherapy of leishmaniasis part XIII: Design and synthesis of novel heteroretinoid-bisbenzylidine ketone hybrids as antileishmanial agents, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl. 2014.09.078

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Chemotherapy of leishmaniasis part XIII: Design and synthesis of novel heteroretinoid-bisbenzylidine ketone hybrids as antileishmanial agents+ Avinash Tiwaria, Santosh Kumara, Rahul Shivahareb, Padam Kantc, Suman Guptab and S. N. Suryawanshi*a a

Division of Medicinal and Process Chemistry and bDivision of Parasitology, CSIR-Central Drug

Research Institute, Lucknow-226 031, India c

Department of Chemistry, University of Lucknow, Lucknow-226 001, India

Abstract Some novel heteroretinoid-bisbenzylidine ketone hybrids have been synthesized and evaluated for their in vitro antileishmanial activity against intramacrophagic amastigotes of Leishmania donovani. Among all the nine synthetic compounds, five compounds (7c, 7d and 7f-h) have shown significant (less than 7 µM) activity against intramacrophagic amastigotes. The IC50 values of these compounds were found better than the reference drugs sodium stibogluconate (SSG) and miltefosine. This study helped us in identifying the new class of compounds that could be exploited as potent antileishmanial agents.

Keywords: Heteroretinoid, Bisbenzylidine ketone, Leishmania donovani, In vitro activity.

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CSIR-CDRI Communication No.:

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Corresponding author Tel.: 91-522-2771-940; Fax: 91-522-2771-941

E-mail address: [email protected]

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Leishmaniasis is a neglected disease characterized by high morbidity, deeply linked to malnutrition, humanitarian emergencies and environmental changes that affect vector biology. Leishmaniasis is caused by several species of protozoan parasites, Leishmania, and is transmitted to humans through the bite of infected female sandflies which are very small insect vectors with a wide range of habitats. The disease classified in three clinical forms: cutaneous, mucocutaneous and visceral. The first two result in severe skin or muco-membranous lesions and high morbidity, and consequently high DALYs (Disability Adjusted Life Years). Visceral Leishmaniasis (VL), also known as Kala azar, rarely results in long term illness; however, if left untreated, patients have a fatality rate of 100% within two years. The situation has become complicated because of the emergence of Post kala-azar Dermal Leishmaniasis (PKDL), which appears in 0-6 months after the successful curing of VL.1 According to the World Health Organization (WHO), leishmaniasis currently affects some 12 million people in 88 countries and there are 2 million new cases per year. Moreover, it is estimated that approximately 350 million people live at risk of infection with Leishmania parasites.2 Visceral Leishmaniasis (VL) occurs in 65 countries3 and more than 90% of the VL cases worldwide are registered in India, Bangladesh, Nepal, and Sudan. Leishmania/HIV co-infections have increased in Mediterranean countries, where up to 70% of potentially fatal VL cases are associated with HIV infection and up to 9% of AIDS cases suffer from newly acquired or reactivated VL.3 WHO recently classified leishmaniasis as a category I: emerging or uncontrolled disease.4 Leishmaniasis control relies on chemotherapy since there are no licensed vaccines available in the market. Available drugs are limited in number and suffer from several limitations such as high cost, toxicity, parenteral administration, emergence and spread of drug resistance. Antimonials are the first line of treatment options for VL, which were discovered almost 70 years ago. These suffer from major side effects including cardiac arrhythmia and pancreatitis. Besides their toxicity, treatment failure with antimonials use has increased; sometimes, as high as 62% in some of the regions.1 Second line treatment options for VL include pentamidine and amphotericin B but their widespread use is limited because of 2

toxicity and cost. Perhaps the most significant recent advancement has been the effective oral treatment of VL by using miltefosine. Despite its great efficacy, miltefosine is also not free from toxicity and shows teratogenic effects in pregnant women.5 New antileishmanial drugs are required in view of the shortcomings associated with the existing drugs. Currently, efforts are being made to search new molecules from the natural sources6 and in this endeavor diaryl heptanoids and aryl chalcones represent the useful lead molecules in the area of anticancer and antileishmanial drug development. Efforts are also being made to design multi-targetdirected ligands to develop new lead molecules for neglected tropical diseases.7 In this regard two or more small molecules are being covalently linked to act on two or more different targets. These kinds of hybrid molecules are under investigation and the results are quite promising.8,9 In recent years, in depth information is being generated on the biochemical targets involved in the chemotherapy of cancer as compared to most neglected tropical diseases like leishmaniasis, malaria, filarial, and Chagas disease. Biologically, most of the biochemical targets involved in the proliferation mechanism and pathogenesis of cancer and leishmaniasis have lots of similarities and as a result, clinically active anticancer drug miltefosine is quite effective in chemotherapy of leishmaniasis. In view of this, a number of biologically active anticancer natural products (curcumin, licochalcone etc.) are acting as very good leads in the design and development of antileishmanial agents. As a part of our research program, we have been designing antileishmanial agents on the basis of anticancer natural products curcumin and licochalcone (Figure 1).10 Recently we have reported the synthesis and antileishmanial activity of some novel heteroretinoids.11 In continuation of our efforts in this context, we have covalently linked heteroretinoid moiety with bisbenzylidine ketones and the resulting chemically novel hybrid molecules were analyzed for their in vitro antileishmanial activity. Some of the hybrid prototypes displayed good in vitro antileishmanial profile and the results are part of this document.

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Please insert Figure 1 here… The overall strategy for the synthesis of novel heteroretinoid-bisbenzylidine ketone hybrids is depicted in Scheme 1. Briefly, (E)-5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carboxylic acid (4) was synthesized as described in our previous article. 11 Compound 4 was reacted with oxalyl chloride to furnish acid chloride (5) which was next coupled with piperidone hydrochloride to give (E)-1(5-(2-(2,6,6-trimethylcyclohex-2-enyl)vinyl)isoxazole-3-carbonyl)piperidin-4-one (6). Finally, compound 6 was reacted with various substituted benzaldehydes to obtain the desired compounds (7a-i) in moderate to good yield. The structures of all the synthetic compounds were determined on the basis of their spectroscopic data and microanalysis. The IR spectra of compounds (7a-i) exhibited characteristic absorption bands in the range of 1657-1634 cm-1 and 1599-1577 cm-1 displaying C=O and C=N stretching respectively. The ESI-MS (mass spectra) of the all the synthetic compounds showed molecular ion peak at [M+1]+. The presence of two carbonyl carbons in the synthetic hybrids can easily be detected by observing the resonance at δ 186 (C=O of bisbenzylidine ketone function) and 169 (C=O of heteroretinoid moiety) in their 13C NMR spectra. Spectral data for all the synthetic compounds are given in the Supplementary data. Please insert Scheme 1 here… The compounds selected for study (7a-i) were evaluated in vitro against intracellular L. donovani amastigotes12 and cytotoxicity responses13 were assessed using vero cell line. Standard antileishmanials, sodium stibogluconate and miltefosine were used as reference drugs. Cell viability was determined using the MTT assay.13 Podophyllotoxin was used as a reference drug for cytotoxicity assay. IC50 values of compounds were calculated by nonlinear regression analysis of the concentration-response curve using the four parameter Hill equations. CC50 values were estimated through the preformed template as described by Huber and Koella.14 The Selectivity index (SI) is defined as the ratio of CC50 on vero cells to

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IC50 on L. donovani intramacrophagic amastigotes. Any synthetic analogues with in vitro IC50 value exceeding above 15 µM was considered as inactive. The in vitro biological activities of heteroretinoid-bisbenzylidine ketone hybrids (7a-i) have shown encouraging results against L. donovani. Table 1 displays IC50 values of the synthetic hybrids against intracellular amastigotes and cytotoxicity of the compounds on vero cell line. The IC50 values of the test derivatives against amastigotes indicate that out of 9 synthetic compounds, 5 compounds (7c, 7d and 7f-h) exhibited high activity against L. donovani (IC50 = 1.83-6.10 µM), better than the reference drug sodium stibogluconate (IC50 = 53.12 µM) and miltefosine (IC50 = 8.10 µM). Please insert Table 1 here… The overall activity profile of compounds (7a-i) demonstrated that there is considerable difference in their IC50 values. Thus, the biological activity was influenced to an extent by the type of substituent present and their position in the phenyl ring. Compounds 7a-d, which have monomethoxy, dimethoxy and trimethoxy phenyl rings were found to show interesting results. Compounds having monomethoxy substitutions (7a, b) were found inactive whereas compound 7c, having 3,4-dimethoxy phenyl ring, was found to exhibit better antileishmanial activity with an IC50 value of 3.75 µM. Although to a lesser extent but on further substitution diminution of biological activity took place (7d, IC50 = 4.70 µM). Attachment of benzyloxy group at 4 position of phenyl ring rendered the molecule inactive (7e, IC50 > 40 µM). However, it was noted that the introduction of OMe group at 3 position together with 4-OBn greatly enhanced the activity (7f, IC50 = 5.02 µM). Similarly, among the methoxy derivatives (7a-d), activity of compounds 7c and 7d exhibited an increment as compared to monomethoxy derivatives (7a, b) because of the presence of an additional OCH3 group at position 3. Considering these results and activity profile of the target compounds (7a-i), we can say that OCH3 group at position 3 plays a critical role in the antileishmanial activity of these compounds. Among the vanillin nucleus containing compounds (7g and 7h), methoxy vanillin derivative (7g) having OCH3 group at position 3 was found more active than

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ethoxy vanillin derivative (7h). In addition to that it was also found that by protecting the OH group in compound 7g (IC50 = 1.83 µM, SI = 12.81) with benzyl group, activity decreased slightly but selectivity increased over 6 fold (7f, SI > 79.68). This indicates that as the hydrophilicity decreases and hydrophobicity increases, selectivity increases accordingly. The presence of p-chloro substituent has shown deleterious effect on the antiamastigote activity of compound 7i (IC50 > 40 µM) (Table 1). Within this manuscript, we present the efficient synthesis of a series of heteroretinoidbisbenzylidine ketone hybrids, which showed significant antileishmanial activity. The activity results clearly indicate that newly synthetic compounds reported herein are promising one and provide useful model for further structural and biological optimization. Compound 7f displayed not only a lower IC50 value than that of reference drugs, but also over 10- and 12- fold more selective as compared to that of standard drugs sodium stibogluconate and miltefosine, respectively. The study opens up the possibility of advancing this new class of compounds as novel antileishmanial agents. Further studies on these heteroretinoid-bisbenzylidine ketone hybrids to optimize the efficacy are in progress in our laboratory. Acknowledgements The authors are thankful to the division of Sophisticated Analytical Instrument Facility (SAIF), CSIR-CDRI for providing spectral and elemental analysis data. Financial support from U.G.C. is gratefully acknowledged. Technical assistance by Mrs. Manju is gratefully acknowledged. The transgenic L. donovani promastigotes were originally procured from Dr. Neena Goyal, Division of Biochemistry, CSIR-CDRI, Lucknow, India. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:

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References 1. Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Rijal, S.; Peeling R. W.; Alvar, J.; Boelaert, M. Nat. Rev. Microbiol. 2007, 5, 873. 2. Ashford, R. W.; Desjeux, P.; Deraadt, P. Parasitol. Today 1992, 8, 104. 3. Desjeux, P.; Clinics in Dermatology 1996, 14, 417. 4. Alvar, J.; Yactayo, S.; Bern, C. Trends Parasitol. 2006, 22, 552. 5. Croft, S. L.; Olliaro, P. Clin. Microbiol. Infect. 2011, 17, 1478. 6. (a) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461; (b) Rocha, L. G.; Almeida, J. R. G. S.; Macêdo, R. O.; Barbosa-Filho, J. M. Phytomedicine , 2005, 12, 514. 7. Cavalli, A.; Bolognesi, M. L. J. Med. Chem. 2009, 52, 7339. 8. (a) Meunier, B. Acc. Chem. Res.; 2008, 41, 69; (b) Camps, P.; Formosa, X.; Galdeano, C.; Gómez, T.; Muñoz-Torrero, D.; Scarpellini, M.; Viayna, E.; Badia, A.; Clos, M. V.; Camins, A.; Pallàs, M.; Bartolini, M.; Mancini, F.; Andrisano, V.; Estelrich, J.; Lizondo, M.; Bidon-Chanal, A.; Luque, F. J. J. Med. Chem. 2008, 51, 3588; (c) Belluti, F.; Fontana, G.; Bo, L. D.; Carenini, N.; Giommarelli, C.; Zunino, F. Bioorg. Med. Chem. 2010, 18, 3543. 9. (a) Das, B. C.; Mahalingam, S. M.; Panda, L.; Wang, B.; Campbell, P. D.; Evans, T. Tet. Lett. 2010, 51, 1462; (b) Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yánez, M.; Fraiz, N.; Alcaide, C.; Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257. 10. (a) Suryawanshi, S. N.; Chandra, N.; Kumar, P.; Porwal, J.; Gupta, S. Eur. J. Med. Chem. 2008, 43, 2473; (b) Kumar, S.; Tiwari, A.; Suryawanshi, S. N.; Mittal, M.; Vishwakarma, P.; Gupta, S. Bioorg. Med. Chem. Lett. 2012, 22, 6728; (c) Suryawanshi, S. N.; Tiwari, A.; Kumar, S.; Shivahare, R.; Mittal, M.; Kant, P.; Gupta, S. Bioorg. Med. Chem. Lett. 2013, 23, 2925. 7

11. Suryawanshi, S. N.; Tiwari, A.; Chandra, N.; Ramesh, Gupta, S. Bioorg. Med. Chem. Lett. 2012, 22, 6559. 12. Shivahare, R.; Venkateswarlu, K.; Chandasana, H.; Suthar, M. K.; Agnihotri, P.; Vishwakarma, P.; Chaitanya, T. K.; Kancharla, P.; Khaliq, T.; Gupta, S.; Bhatta, R. S.; Pratap, J. V.; Saxena, J. K.; Gupta, S; Narender, T. J. Med. Chem. 2014, 57, 3342. 13. Mosmann, T. J. Immunol. Methods 1983, 65, 55. 14. Huber, W.; Koella, J. C. Acta Trop. 1993, 55, 257.

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Figure captions: Figure 1: Chemical structure of curcumin and licochalcone A. Scheme 1: Synthesis of novel heteroretinoid-bisbenzylidine ketone hybrids. Table 1: Antileishmanial activity and cytotoxicity of synthetic hybrids (7a-7i).

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Figure 1

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Scheme 1

O Diethyl oxalate O

O NH2OH.HCl

OEt

NaH, toluene

O

1

OEt

Ethanol

O

O

2 O OH O

3 O

Oxalyl chloride

NaOH, ethanol

Cl

DCM, DMF

N

O O

4

N

N

5

R1 Piperidone hydrochloride

R2

O

R3 N

DCM O 6

N

R4

R4

O

Piperidine, L-proline, Ethanol, reflux

R2

O O N

R3

R1

N

R4 O R3 R2

R1

7a-i

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Table 1 R4

R3 R2

O

R1

N

O N R4

O R3 R2

R1 7a-i

Entry

R1

R2

R3

R4

Antiamastigote activity (IC50 in µM)

Cytotoxicity (CC50 in µM)

Selectivity Index (SI)

7a

H

H

OMe

H

>40

NT

NA

7b

OMe

H

H

H

>20

NT

NA

7c

H

H

OMe

OMe

3.75 ± 0.31

45.78 ± 5.71

12.20

7d

H

OMe

OMe

OMe

4.70 ± 0.48

25.32 ± 3.40

5.38

7e

H

H

OBn

H

>40

NT

NA

7f

H

H

OBn

OMe

5.02 ± 0.49

>400

>79.68

7g

H

H

OH

OMe

1.83 ± 0.21

23.45 ± 3.82

12.81

7h

H

H

OH

OEt

6.10 ± 0.62

27.65 ± 4.1

4.53

7i

H

H

Cl

H

>40

NT

NA

Standard drug

Sodium stibogluconate

53.12 ± 4.56

>400

>7.53

Standard drug

Miltefosine

8.10 ± 0.51

52.86 ± 4.81

6.52

The Selectivity Index (SI) is defined as the ratio of CC50 (50% maximum cytotoxic concentration) on Vero cells to IC50 (50% maximum inhibitory concentration) on L. donovani intramacrophagic amastigotes; NT = Not tested; NA = Not available; IC50 and CC50 values are the average (mean ± S.D.) of three independent experiments.

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Chemotherapy of leishmaniasis part XIII: Design and synthesis of novel heteroretinoid-bisbenzylidine ketone hybrids as antileishmanial agents+ Avinash Tiwaria, Santosh Kumara, Rahul Shivahareb, Padam Kantc, Suman Guptab and S. N. Suryawanshi*a a

Division of Medicinal and Process Chemistry and bDivision of Parasitology, CSIR-Central Drug

Research Institute, Lucknow-226 031, India c

Department of Chemistry, University of Lucknow, Lucknow-226 001, India

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