Synthesis of 4-substituted ethers of benzophenone and their antileishmanial activities

Leishmaniasis is a vector-borne protozoan disease; it mainly originates from the bite of sandfly and initiated when parasite is transmitted to human at metacyclic flagellated promastigote form. In the current study, a synthesis of a series of 4-substituted benzophenone ethers 1–20 was carried out in good yields and their in vitro antileishmanial activities were also screened. Among synthetic derivatives, 15 compounds 1, 3, 5–12, 15 and 17–20 showed antileishmanial activities against promastigotes of Leishmania major with IC50 values in the range of 1.19–82.30 µg ml−1, and the values were compared with those of the standard pentamidine (IC50 = 5.09 ± 0.09 µg ml−1). Our study identified a series of new antileishmanial molecules as potential leads. Structures of these synthetic compounds were deduced by different spectroscopic techniques, such as 1H and 13C nuclear magnetic resonance, electron impact and high-resolution electron impact mass spectrometry and IR.

Leishmaniasis is among the neglected diseases and according to the surveys of the World Health Organization, 350 million people are suffering from this. Leishmaniasis is also responsible for a high mortality rate worldwide [18,19]. It is a vector-borne protozoan disease, mainly originated from the bite of sandfly. Leishmaniasis is initiated when parasite is transmitted to human at metacyclic-flagellated promastigote form. The main site of action involves reticulo-endothelial system of the host. Based on symptoms, leishmaniasis appears in diffused, cutaneous, mucosal and visceral (Kala Azar) forms [20,21].
In the light of a previous report on antileishmanial activities of benzophenone ethers [24], structure of pentamidine which possesses ether functionality (figure 1), and in continuation of our search for antileishmanial agents [25][26][27], we have synthesized a library of functionalized benzophenone ethers and evaluated their antileishmanial activities in vitro. To the best of our knowledge, compounds 1 and 2 were previously reported, while remaining compounds are new [28,29]

Results and discussion
2.1. Chemistry 4-Hydroxybenzophenone (2 mmol), varyingly substituted aryl halide or phenacyl halide (2 mmol), and potassium carbonate (2 mmol) in the presence of catalytic amount of tetrabutylammonium bromide (TBAB) in dichloromethane (15 ml) were refluxed for 6 h. Progression of reaction was studied by thin layer chromatography (TLC). Reaction mixture was cooled to room temperature and a solid material was obtained. The solid was filtered and washed with hexane followed by drying resulting in the desired compounds in good yields (scheme 1). The characterization of synthetic compounds was carried out by 1 H and 13 C nuclear magnetic resonance (NMR), electron impact mass spectrometry (EI-MS), high-resolution EI-MS (HREI-MS) and IR spectroscopy.
High-resolution mass spectrum of compound 18 displayed the M + at m/z 384.0321 with a composition of C 21 H 14 Cl 2 O 3 (calcd 384.0320). The per cent abundance of isotopic [M + 4] + 10%, [M + 2] + 49% and molecular ion peak M + 76% at m/z 388, 386 and 384, respectively, confirmed the presence of two chlorine atoms in a molecule. Cleavage of carbon-carbon bond from α-carbonyl group of ether resulted in respective methylene benzophenone ether which appeared at m/z 211, and remaining acylium ion appeared as base peak at m/z 173. Fragment at m/z 198 was due to benzophenone fragment. It was further fragmented into respective acylium ion at m/z 121. Fragments at m/z 105 and 77 were due to benzyl acylium ion and benzene radical cation, respectively (figure 4).
In the Fourier transform IR (FT-IR) spectrum, vibrational frequencies at 1710 and 1628 cm −1 correspond to the carbonyl (C=O) functionality. However, vibrational frequencies of aromatic (C=C) bond and ether (C-O) appeared at 1557 and 1309 cm −1 (figure 5), respectively. These are spectroscopic observations of proposed structure for compound 18. Structures of all other compounds were deduced in a similar manner.

4-Substituted ether derivatives of benzophenone
In 4-substituted ether derivatives, compound 8 having chloro group at meta position of aryl part was found to be the second most active member of the series with IC 50 = 13.11 ± 0.42 µg ml −1 . Nevertheless, the introduction of chloro substituent to para position, as in 9 (IC 50 = 17.02 ± 0.70 µg ml −1 ), showed a slight decreased activity. When a dichloro substituent was present at meta and para positions of aryl part, as in compound 5, a sharp decline (IC 50 = 63.3 ± 3.30 µg ml −1 ) in activity was observed (figure 9).
However, the presence of a chloro group at ortho and a fluoro group at para as in compound 6 demonstrated a weak inhibitory activity having an IC 50 value of 65.0 ± 5.00 µg ml −1 . Moreover, a          The presence of methoxy group at meta position as in molecule 11 made it fairly active (IC 50 = 30.43 ± 0.50 µg ml −1 ). Replacement of methoxy substituent with a methyl substituent at meta position of aryl part as in derivative 10 displayed a good activity (IC 50 = 13.59 ± 0.28 µg ml −1 ). However, when switching the methyl group to para position of aryl part as in analogue 7 (IC 50 = 77.5 ± 2.50 µg ml −1 ), a weak inhibitory activity was observed ( figure 11).    To study the effect of carbon load, compound 1 having a propyl group at ether part was screened and found to have a weak activity with an IC 50 value of 70.6 ± 2.3 μg ml −1 . But, remaining derivatives were found to be inactive ( figure 12).

Conclusion
This study deals with the synthesis of 20 4-substituted ethers of benzophenone derivatives and their antileishmanial activities were screened. Fifteen compounds displayed antileishmanial activity having IC 50 values within the range of 1.94-82.30 μg ml −1 . Compound 18 was found to be the most active compound (IC 50 = 1.94 µg ml −1 ) of this series. These compounds seemingly have potential to develop powerful antileishmanial agents.
Mass spectra were recorded with a Finnigan MAT-311A (Germany) mass spectrometer. 1 H-and 13 C-NMR spectra were recorded with Bruker Avance AM 300 and 400 MHz spectrometers. Melting points of the compounds were determined using a Stuart ® SMP10 melting point apparatus, and are uncorrected. IR spectra (KBr discs) were recorded with a FTS 3000 MX, Bio-RAD Merlin (Excalibur Model) spectrophotometer. 10 min and at the same speed and washed time three times with saline. Parasites were diluted to a final density of 1 × 10 6 cells ml −1 with a fresh culture medium.

Antileishmanial assay protocol
The assay was carried out in a 96-well micro-titre plate; the medium was added in different wells. The test compound (20 µl) was added in the medium and serially diluted. Parasite culture (100 µl) was added in all wells. Two rows were left for positive and negative controls. In positive controls, different quantities of standard antileishmanial drug pentamidine (ICN Biomedical Inc, USA) were present, while negative controls contained only medium. The plate was incubated for 72 h at 22-25°C. The culture was microscopically examined on Neubauer counting chamber. IC 50 values were calculated by software Ezfit 5.03 (Perella Scientific, USA). All tests were carried out three times [30].

General procedure for the synthesis of compounds 1-20
Differently substituted benzophenone ethers were synthesized by refluxing a mixture of 4hydroxybenzophenone, potassium carbonate, TBAB, differently substituted phenacyl halide and aryl/alkyl halide in dichloromethane as solvent. The reaction was examined by TLC. Subsequently, the reaction mixture was filtered, and cooled until precipitates became visible. These precipitates were sieved and rinsed with hexane. Yield of all the synthetic compounds was moderate to high. 4.4. Spectral data of synthetic compounds 1-20 methanone (1)