J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 95 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Synthesis and Comparison of In Vitro Leishmanicidal Activity of 5- (Nitroheteroaryl)-1, 3, 4-Thiadiazols Containing Cyclic Amine of Piperidin-4-ol at C-2 with Acyclic Amine Analogues against Iranian Strain of Leishmania major (MRHO/IR/75/ER) *Azar Tahghighi 1,2, Alireza Foroumadi 2, Susan Kabudanian-Ardestani 3, Seyed Mohammad Amin Mahdian 1 1Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran 2Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran 3Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran (Received 2 Nov 2014; accepted 6 Feb 2016) Abstract Background: Cutaneous Leishmaniasis (CL) is endemic in many tropical and subtropical regions of the world. Due to the prolonged duration of therapy, adverse effect and resistance to current drugs in the treatment of CL, the dis- covery of novel, efficient, and safe leishmanicidal drugs is required. The aims of the present study was to synthesis of new compounds based on the active compounds of 5-(5-nitrofuran-2-yl)- and 5-(5-nitrothiophen-2-yl)-1,3,4-thia- diazole bearing the linear amino alcohol of 3-aminopropan-1-ol in the C-2 position of thiadiazole ring and evaluation of their activity against the promastigote and amastigote forms of Leishmania major. Methods: Reaction between the solution of 5-(5-nitro heteroaryl)-2-chloro-1, 3, 4-thiadiazole and piperidin-4-ol in absolute ethanol was performed and the resulting products were evaluated against promastigotes form of L. major with MTT assay and amastigote form of L. major in murine peritoneal macrophages. In addition, the toxicity of these compounds was assessed against mouse peritoneal macrophages with MTT assay. Results: New synthetic compounds 5a-b showed moderate in vitro antileishmanial activity against L. major pro- mastigotes with IC50 values of 68.9 and 27µ M, respectively. These compounds have also demonstrated a good antiamastigote activity in terms of amastigote number per macrophage, the percentage of macrophage infectivity and infectivity index. Conclusion: Novel cyclic compounds 5a-b were synthesized and exhibited less antipromastigote and antiamastigote activity compared to linear analogues. Keywords: Leishmania major, Thiadiazole, Nitrofuran, Nitrothiophen, Superimpose Introduction Leishmaniasis is an endemic parasitic dis- ease and a major public health problem in more than 90 countries around the world (CDC 2013). It is estimated that 1.3 million people are affected by this disease annually and the overall population at risk is 310 million peo- ple (WHO 2010). There are four forms of leish- maniasis: Visceral Leishmaniasis (VL) or kala azar, which is typically lethal if left untreat- ed, Mucocutaneous Leishmaniasis (MCL), which is a mutilating disease, Diffuse Cuta- neous Leishmaniasis (DCL) related to defec- tive immune system and Cutaneous Leish- maniasis (CL) that produces serious skin le- sions (Singh and Sivakumar 2004). The cur- rent chemotherapy against all forms of leish- maniasis including parental pentavalent an- timonial compounds remains the primary *Corresponding authors: Dr Azar Tahghighi, Email: atahghighi2009@gmail.com J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 96 http://jad.tums.ac.ir Published Online: March 14, 2017 therapy, followed by making use of other drugs like amphotericin B, paramomycin, pentamidine and orally miltefosine (Monzote 2009). However, making use of them is lim- ited owing to toxicity and the high cost of treatment (Chawla and Madhubala 2010, Tiuman et al. 2011). In addition, the devel- opment of clinical resistance and an increas- ing incidence of leishmaniasis/ AIDS co-in- fection in some regions is a serious health problem (Molina 2003). As such, the design of new, efficient, cheap and safe drugs for the treatment of leishmaniasis is imperative. 5-(nitroheteroaryl)-1, 3, 4-thiadiazole con- taining a cyclic (a) and acyclic (b) amine in C-2 position, depending upon the type of substituent, have shown a promising an- tileishmanial activity (Fig. 1). Furthermore, different substitutions in C-2 position in 1, 3, 4-thiadiazole ring have been able to affect potency and physicochemical properties. On this basis, to achieve a novel anti-leishmanial agent, the new derivatives were synthesized to introduce other groups on C-2 amine of thiadiazole ring such as, 4-aroylpiperazine segment (c), piperazinyl-linked benzami- dines substituent (d), N-[(1-benzyl-1H-1,2,3- triazol-4-yl)methyl] moiety (e) and various 2-thioacetamides substituent (f) (Fig. 1). These different attachments to 1, 3, 4-thiadia- zole rings causes changes the bioresponses, depending upon the type of substituent and position of attachment (Foroumadi et al. 2008, Tahghighi 2011, 2012, 2013, Marznaki 2013, Vosooghi 2014). Recently, given the im- portance of this position, several 5-(5-ni- trofuran-2-yl)- and 5-(5-nitrothiophen-2-yl)-1, 3, 4-thiadiazoles possessing acyclic amines in the C-2 position of thiadiazole ring were synthesized and theirs in vitro activity was evaluated against the promastigotes and amastigotes forms of L. major. Two com- pounds 1a-b with linear substitution of 1- propanol amine exhibited a promising an- tileishmanial activity against L. major pro- mastigotes (Fig. 1) (Tahghighi et al. 2013). Concerning these findings and simple re- action for preparation these compounds, the aim of this study was to synthesize cyclic an- alogues of these linear amino alcohols, ex- amine anti-leishmanial activity against pro- mastigote and amastigote forms of L. major and evaluate their cytotoxicity activity on macrophages. Materials and Methods Chemistry All chemical reagents and solvents were purchased from Merck Company and used without further purification. The Key inter- mediate 2-chloro-1, 3, 4-thiadiazole 4a-b was prepared by starting from 5-nitrofurfurilidine diacetate or 5-nitrothiophene-2-carboxalde- hyde (Tahghighi et al. 2011, 2012, 2013, Marznaki 2013, Vosooghi 2014). The melt- ing points of compounds were determined using a Kofler hot-stage apparatus. The IR spectra were obtained on a Shimadzu 470 spectrophotometer using KBr dicks. 1H NMR spectra were recorded on a Varian unity 500 and 400 spectrometer and chemical shifts (δ) were reported in parts per million (ppm) rel- ative to tetramethylsilane (TMS) as an inter- nal standard. The mass spectra were run on an Agilent 6410 LC-MS or a FiniganTSQ-70 spectrometer (Finigan, USA) at 70 eV. Merck silica gel 60 F254 plates were used for an- alytical TLC. Compounds 1a-b has been de- scribed in our previous work (Tahghighi et al. 2013). General procedure for the synthesis of compounds 5a-b A solution of 2-chloro-1, 3, 4-thiadiazole 4a-b (1.1mmol) and appropriate cyclic amine of piperidin-4-o l (1mmol) in absolute etha- nol (7ml) was refluxed until the reaction was completed (4h). Then, the solvent was evap- orated under reduced pressure and the residue was purified using silica gel column chro- J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 97 http://jad.tums.ac.ir Published Online: March 14, 2017 matography, eluting with appropriate solvent (Fig. 2). 1-(5-(5-nitrofuran-2-yl)-1, 3, 4-thiadiazol-2- yl)piperidin-4-ol (5a) The resulting product was purified using a short silica gel column eluting with CH3 COOC2H5 followed by CH3COOC2H5 con- taining 2% methanol. The compound was obtained as a yellow solid (Yield: 78%). Mp 147.7–149 °C. IR (KBr, cm-1): 3414, 1734, 1535, 1497 and 1344. 1H NMR (500 MHz, DMSO-d6) δ: 7.84 (d, 1H, J= 3.25 Hz, fu- ran), 7.35 (d, 1H, J= 3.25 Hz, furan), 3.80 (m, 3H, CH2 and CH), 3.41 (t, 2H, CH2), 3.33 (brs, 1H, OH), 1.85 (m, 2H, CH2), 1.51 (m, 2H, CH2). MS (ESI): 296.8 [M + H +]. 1-(5-(5-nitrothiophen-2-yl)-1, 3, 4-thiadiazol- 2-yl)piperidin-4-ol (5b) The resulting product was purified using a short silica gel column eluting with CH3 COOC2H5 followed by CH3COOC2H5 con- taining 2% methanol. The compound was obtained as an orange solid (Yield: 73%). mp160–162 °C. IR (KBr, cm-1): 3298, 1736, 1536, 1494 and 1354. 1H NMR (400 MHz, CDCl3) δ: 7.86 (d, 1H, J= 4.4 Hz, thiophen), 7.16 (d, 1H, J= 4.4 Hz, thiophen), 4.05 (brs, 1H, OH), 3.91 (m, 3H, CH2 and CH), 3.49 (t, 2H, J= 9.3 CH2), 2.03 (m, 2H, CH2), 1.73 (m, 2H, CH2). MS (ESI): 312.8 [M+ H +]. Antileishmanial activity against Leishma- nia major promastigotes The antileishmanial activity of compounds 5a-b was performed using MTT assay (Dutta et al. 2005). The promastigote form of para- site (vaccine strain MRHO/IR/75/ER, ob- tained from Pasteur Institute, Tehran, Iran) was grown in blood agar cultures at 25 °C. The growth curve of the L. major strain was determined daily under light microscope and counting in a Neuberger’s chamber. Then, parasites (2× 106/ml) in the logarithmic phase were incubated with different concentrations (12.5, 25, 50 and 75µ g/ml) of test com- pounds for 24 h at 25 °C. A negative control (DMSO without any test compounds with culture medium), and positive control (with Glucantime and Fluconazole) were used on same plate. After incubation, the media was renewed with 100μg/well of MTT (0.5mg/ml) and plates were further incubated for 4 h at 37 °C. Then, the plates were centrifuged (2000rpm× 5min× 4 °C) and the obtained pellets were dissolved in 200μL of DMSO. The optical density (OD) of samples due to cleavage of the tetrazolium salt MTT [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazo- lium bromide] into a colored product forma- zan by the parasite was measured by ELISA plate reader at a wavelength of 492nm. Two or more independent experiments in tripli- cate were performed for each compound. The IC50 values (the concentration required to inhibit 50% growth of promastigotes after 24h) were calculated by linear regression analysis, expressed in mean ± SD. Antileishmanial activity against Leishma- nia major amastigotes Compounds 5a-b was evaluated for their activity against amastigote form of L. major in murine peritoneal macrophages. Briefly, the abdominal cavity of BALB/c mice was torn then washed to inject 5 ml of phosphate buffered saline (PBS) by a sterile syringe and collected cells. The cell suspension was cen- trifuged (2000rpm× 10min× 4 °C). Mouse per- itoneal macrophages were plated in RPMI 1640 supplemented with 10% of heat-in- activated fetal bovine serum, 2mM gluta- mine, 100U/ml penicillin (Sigma) and 100µ g/ ml streptomycin. Macrophages were placed on sterile glass cover slips in 24-well plates (1× 106/well). After 1h, non-adherent cells were removed by washing with RPMI 1640, the stationary phase promastigotes in RPMI 1640 were added (2×106 parasites/well, three para- sites/ macrophage) to macrophage monolay- er and the plates were kept at 37 °C in a CO2 J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 98 http://jad.tums.ac.ir Published Online: March 14, 2017 incubator for 2h. Extracellular parasites were removed by washing and then new media containing IC50 concentration of the drug were added. Two sets of experiments were carried out for each compound at 24h. Fol- lowing these procedures, cells were fixed with methanol, stained with Giemsa stain (Sigma) and the infectivity index was deter- mined by multiplying the percentage of mac- rophages that had at least one intracellular parasite by the average number of intracellu- lar parasites per infected macrophage (100 cells were examined/well) (Tanaka et al. 2007). Toxicity against macrophages The toxicity of compounds 5a-b was as- sessed against mouse peritoneal macrophag- es plated in 96-well plates at 2× 105 cells/ well. After cell adherence, the medium was removed and replaced by the media contain- ing different concentrations of each com- pounds. The plates were incubated for 24 h at 37 °C in a humidified incubator with 5% CO2. DMSO without any test compounds was used as a negative control. Cell viability was determined by MTT colorimetric assay (Kiderlen et al. 1990). Two independent ex- periments in triplicate were performed for determination of toxicity of each compound. The CC50 (cytotoxic concentration for 50% inhibition) were calculated by linear regres- sion analysis. Overlay study of compounds The 3D structures of the ligands of Table 1 were drawn using HyperChem software (version 7.0) and subsequently energy mini- mized using semiemperical method, AM1 level of theory. Then, energy minimized mol- ecules were superimposed using atoms selec- tion. In this way, one of the molecules was opened and atoms selected, then the second molecule was opened and the same atoms selected and tow molecules were superim- posed by Overlay option of HyperChem program. Results Chemistry The intermediate 2-chloro-1, 3, 4-thiadia- zole 4a-b was obtained from 5-nitrofurfuri- lidine diacetate or 5-nitrothiophene-2-carbox- aldehyde [7–12]. The target compounds 5a-b were synthesized in high yield by the reac- tion of 5-(5-nitro heteroaryl)-2-chloro-1, 3, 4-thiadiazole 4a-b and appropriate piperidin- 4-ol in refluxing absolute ethanol (Fig. 2). All of the synthesized compounds were char- acterized by IR, 1H NMR and LC-MS. Biological activity We synthesized novel 5-(nitroheteroaryl)- 1, 3, 4-thiadiazols containing acyclic amines in C-2 and evaluated antileishmanial activity of these compounds against the promastigote and amastigote stage of L. major. The com- pounds of 3-(5-(5-nitrofuran-2-yl)-1, 3, 4- thiadiazol-2-ylamino) propan-1-ol 1a and 3- (5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2- ylamino) propan-1-ol 1b showed IC50 values of 18±0.2 and 3±0.41µ M, respectively, against the promastigotes form of L. major and , in contrast, the analogue of 5-nitrothio- phen 1b showed 6-fold more potent than its 5-nitrofuran counterpart 1a. This finding in- dicated that different responses take place due to O/S replacement in the scaffold. The previous compounds 1a-b was also evaluated against the amastigotes form of L. major. These compounds decreased the num- ber of intracellular amastigotes per macro- phage, the percentage of macrophage infec- tivity and infectivity index compared with the control group. The in vitro cytotoxic ac- tivity of the compounds 1a-b demonstrated toxicity against mouse peritoneal macro- phages (CC50 values of 62.33 and 42.16µ M, respectively). Furthermore, the compound 1b displayed the highest selectivity index (SI= 14.05) (Table 1). J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 99 http://jad.tums.ac.ir Published Online: March 14, 2017 Moreover, we synthesized two cyclic com- pounds 5a-b similar to these linear compounds and evaluated their activity against the pro- mastigote and amastigote forms of L. major. Fluconazole and Meglumine antimonate (Glu- cantime®) were used as reference drugs. The- se compounds have a medium anti-pro- mastigote activity with IC50 values less than 70μM. Furthermore, these compounds were evaluated for their activity against amastigote form of L. major in murine peritoneal mac- rophages (Fig. 3) and thiophen analogues 1b and 5b exhibited higher activity against amastigotes as showed by amastigote num- ber per macrophage, the percentage of mac- rophage infectivity and infectivity index com- pared with furan analogues 1a and 5a (Fig. 3A, 3B and 3C, respectively). In addition, the CC50 values for these compounds 5a-b against mouse peritoneal macrophages were determined using MTT assay (Table 1). They were toxic to macro- phages (CC50< 80 µ M) and that the com- pound 1a had the highest selectivity index (SI= 14.05). Overlay study The superimposition studies of the target compounds 5a-b and lead molecules 1a-b in their energy-minimized conformations and without hydroxyl group's selection revealed that the thiophen or furan and thiadiazole ring, nitro group and amine group in C-2 position of thiadiazole ring at these com- pounds overlaid completely on each other whereas hydroxyl groups located in different positions (Fig. 4 A1, B1). The superimposi- tion of these compounds with hydroxyl group selection showed that there was a weak overlay between target and lead molecules (Fig. 4 A2, B2). X S NN N HNO2 X S NN NHR NO2 X = O, S R = alkyl, aminoalkyl, oxyalkyl, .... X S NN NO2 X = O, S Y = CH2, NH, NR, O n = 1, 2 N Y na b 1a-b a: X = O b: X = S O S NN NO2 R = alkyl, morpholine, cyclic amidine, .... X S NN NO2 X = O, S R = halophenyl, halothiophen N c d N Ar O N N NH NHR O S NN NO2 R = alkyl, halide, .... e N H N N N R O S NN NO2 R = halophenyl, nitrophenyl, isoxazolyl, benzothiazolyl, .... S O H N R ( ) n f OH ( ) 3 ( ) Fig. 1. General structures of antileishmanial compounds (a) bearing a cyclic amine (b) containing acyclic amine (c- f) with aromatic substitutions and (1a-b) containing hydroxypropyl amine at the C-2 position of thiadiazole ring J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 100 http://jad.tums.ac.ir Published Online: March 14, 2017 X S NN N OH NO2 X S NN Cl NO2 X S NN NH2 NO2 X CH(OCOCH3)2 NO2 a, b c d 2a-b a: X = O b: X = S 3a-b a: X = O b: X = S 4a-b a: X = O b: X = S 5a-b a: X = O b: X = S Fig. 2. Synthetic route to compounds 5a-b. Reagents and conditions: a) thiosemicarbazide, EtOH, HCl, reflux, (b) NH4Fe(SO4)2.12H2O, H2O, reflux; c) NaNO2, HCl, Cu, d) piperidin-4-ol, EtOH, reflux Table 1. In vitro antileishmanial activity of thiadiazole derivatives 1a-b and 5a-b against promastigote form of Leishmania major X S NN R NO2 Compounds R X Anti-promastigote activity IC50 (µM) Cytotoxicity CC50 (µM) a SIb 1a HN OH O 18 ± 0.2 62.33 3.46 1b HN OH S 3 ± 0.41 42.16 14.05 5a N OH O 68.9 ± 0.107 78.54 1.14 5b N OH S 27 ± 0.12 63.55 2.35 Glucantime - - 68.44 c - - Fluconazole - - 941.1 ± 4.98 - - aCytotoxicity was evaluated against mouse peritoneal macrophages. bSelectivity index (SI) CC50/ IC50. cThe IC50 of Glucantime was in mM. J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 101 http://jad.tums.ac.ir Published Online: March 14, 2017 Fig. 3. The in vitro activity of selected compounds against intramacrophage amastigotes of Leishmania major. (A) The mean number of amastigotes per macrophage after treatment with selected compounds for 24h. (B) The per- centage of infected macrophages after treatment. (C) Infectivity index of macrophages cultured 24 h in presence of selected compounds. The infectivity index was determined by multiplying the percentage of macrophages that had at least one intracellular parasite by the average number of intracellular parasite per infected macrophage (100 cells were examined/well). A B C J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 102 http://jad.tums.ac.ir Published Online: March 14, 2017 Fig. 4. A1: Overlay between compounds 1a and 5a without hydroxyl selection. A2: Overlay between compounds 1a and 5a with hydroxyl selection. B1: Overlay between compounds 1b and 5b without hydroxyl selection. B2: Overlay between compounds 1b and 5b with hydroxyl selection. Discussion The cyclic analogues of 5-(5-nitrofuran-2- yl)- and 5-(5-nitrothiophen-2-yl)-1, 3, 4-thia- diazole-2-amines bearing piperidin-4-ol at the C-2 position of thiadiazole ring were syn- thesized and evaluated in vitro against pro- mastigote and amastigote forms of L. major. These novel compounds, which were com- pared with their linear analogues, exhibited less antileishmanial activity against pro- mastigote form of L. major. Our previous studies demonstrated that the C-2 substituent in 5-(nitroheteroaryl)-1, 3, 4-thiadiazoles is the most flexible site for chemical change and is an area where it de- termines the potency and physicochemical properties of these synthetic compounds. Ac- cordingly, several series of these derivatives were synthesized by our research group with different linkers and substitution groups con- nect to these linkers. All of the compounds were evaluated against L. major with MTT assay. It seems that in these compounds c-f (Fig. 1), the type of aryl ring and the linker are the important factors for their antileishma- nial activity. In the compound (c), aryl group with a piperazinyl methanone linker were the most suitable functions for antileishmanial activity. The best compound in this series has 2- chlorophenyl substitution (IC50 value of 10.73µ M). In the 5-nitrofuran-2-yl-1, 3, 4- thiadiazol-2-yl) piperazin-1-yl) benzamidine (d), the maximum antileishmanial activity observed with n-propyl substitution on ben- zamidine (IC50 value of 10µ M). In other series, 5-(5-nitrofuran-2-yl)-1, 3, 4-thiadiazol- 2-amines were synthesized by introducing N-[(1-benzyl-1H-1, 2, 3-triazol-4-yl)methyl] moiety as a new functionality on the C-2 amine of thiadiazole ring (e). The most ac- tive compound of this series has p-methyl substitution on phenyl ring (IC50 value of 12.2µ M). Hence, 5-(5-nitrofuran-2-yl)-1, 3, 4-thiadiazole structure bearing 2-mercaptoa- cetamide linker (f) were prepared and dis- played in vitro activity against promastigotes and amastigotes of L. major. In this series, 3, 4-Dimethoxyphenethyl substitution was caused to suitable activity against promastigote form of L. major with IC50 value of 19.1µ M. The comparison of antileishmanial activity of compounds c-f with compounds 1a-b was showed linear substitution and thiophen ring at compound 1b are the best substitutions at C-2 and C-5 positions of thiadiazole ring. J Arthropod-Borne Dis, March 2017, 11(1): 95–104 A Tahghighi et al.: Synthesis and Comparison of … 103 http://jad.tums.ac.ir Published Online: March 14, 2017 The presence of aryl group on linker is not necessary for increase antileishmanaial activ- ity. However, in the compounds with aryl substitution, type of this substitution affected the antileishmanial activity. Furthermore, the compounds without aryl substitution were synthesized with a simple reaction (Fig. 2) and their activity was com- pared with their corresponding analogues. As shown in Table 1, the substitution of the linear hydroxypropyl in the C-2 position of 1, 3, 4-thiadiazole ring increased the in vitro activity against promastigotes whereas the attachment of cyclic group of piperidin-4-ol exhibited lower antipromastigote activity. Lin- ear substitution 1a-b seems to have played an important role in the mode of action of these compounds whilst cyclic substitution 5a-b has caused a steric hindrance around the C-2 position. It may have prevented the binding of the compounds to the parasitic macromo- lecular target. However, 5-nitrothiophene de- rivatives 1b and 5b with IC50 values of 3 and 27μM were more active than the correspond- ing 5-nitofuran analogues and, as such, these findings confirmed the fact that thiophen analogues were more potent than furan ana- logues at compounds 1a-b and 5a-b. Besides, thiophen analogues exhibited bet- ter antiamastigote activity compared to furan analogues. Concerning low potency of com- pounds 5a-b compared with their linear an- alogues, it is suggested that likewise piper- idinol ring in the 2-position of thiadiazole ring may be responsible for decreased inter- action between ligand and target macromol- ecule in amastigote form. The differences in the superimposition studies can help to explain why the target compounds 5a-b have lower activity. The compounds 1a and 1b have linear substitu- tion with free rotation around single bond. However, the compounds 5a and 5b have rigid cyclic substitution (4-piperidinol) that can cause a steric hindrance around the C-2 position and, therefore, interaction between the compounds and parasitic macromolecu- lar targets affect. Conclusion Overall, the strategic positioning of sub- stitution of linear hydroxypropyl can offer additional binding interactions probably re- sulting in the better binding profile of these compounds, based on the discussion above. Finally, experimental and computational in- vestigations confirm that the cyclic deriva- tives 5a-b have lower activity than linear an- alogues 1a-b due to steric hindrance. Acknowledgements This work was supported by grants from the Research Council of Tehran University of Medical Sciences and Iran National Sci- ence Foundation (INSF). The authors declare that there is no conflict of interests. 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