Accepted Manuscript Synthesis and Biological Evaluation of Chalcones as Potential Antileishmanial Agents Shweta Gupta , Rahul Shivahare , Venkateswarlu Korthikunta , Rohit Singh , Suman Gupta , Narender Tadigoppula PII:

S0223-5234(14)00448-6

DOI:

10.1016/j.ejmech.2014.05.034

Reference:

EJMECH 6990

To appear in:

European Journal of Medicinal Chemistry

Received Date: 13 December 2013 Revised Date:

9 May 2014

Accepted Date: 11 May 2014

Please cite this article as: S. Gupta, R. Shivahare, V. Korthikunta, R. Singh, S. Gupta, N. Tadigoppula, Synthesis and Biological Evaluation of Chalcones as Potential Antileishmanial Agents, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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Antileishmanial Agents

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Synthesis and Biological Evaluation of Chalcones as Potential

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Synthesis and Biological Evaluation of Chalcones as Potential Antileishmanial Agents Shweta Guptaa, Rahul Shivahareb, Venkateswarlu Korthikuntaa, Rohit Singha, Suman Guptab and Narender Tadigoppulaa,* Division of Medicinal and Process Chemistry, bDivision of Parasitology,

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*Corresponding author: Tel.: +91 522 2612411; fax: +91 522 2623405.

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CSIR-Central Drug Research Institute, Lucknow-226 031, India

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E-mail: [email protected] or [email protected] (T. Narender)

Abstract

Antileishmanial activities of thirty five synthetic chalcones have been examined. Among them, ten compounds (4, 6, 16, 22, 23, 24, 25, 29, 35 and 37) exhibited potent in vitro

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activity (IC50 range from 1.70 to 8 µM) against extracellular promastigotes and intracellular amastigotes form of Leishmania donovani. Two promising compounds 22 and 37 were tested in vivo in L. donovani / hamster model. Chalcone 37 showed 83.32% parasite inhibition at a dose of 50 mg/kg for 10 days whereas, 75.89% parasite inhibition at 100 mg/kg dose for 5

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days by intraperitoneal route at day 7 post-treatment. Keywords: Chromenochalcones; Chromenodihydrochalcones; Antileishmanial; Leishmania

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donovani / Hamster model 1. Introduction

Leishmaniasis is a group of disease transmitted by the bite of Leishmania-infected

female sand flies. It is classified as cutaneous, muco-cutaneous and visceral leishmaniasis (VL) depending on the parasite species and cellular immune system of the patient [1,2]. This disease has been recognized as an increasing health problem worldwide by the World Health Organization (WHO) [3]. Many parts of Asia and Africa are vulnerable to leishmaniasis [4]. The first line treatment options for the visceral form of leishmaniasis are limited and involve

ACCEPTED MANUSCRIPT the administration of pentavalent antimonials (sodium stibogluconate (SSG) and meglumine antimoniate) and amphotericin B [5]. Second line drugs include, pentamidine, paromomycin and miltefosine, but these drugs have not experienced widespread use due to the severe toxicities, parenteral administration and resistance issues [5]. Presently, more than 60% VL patients in Bihar (India) are unresponsive to the antimonials [6]. Amphotericin B and its

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formulations are quite effective for VL; however, these are very expensive, highly toxic and have a longer half-life [7]. Pentamidine presents several side effects, including renal and hepatic toxicities, pancreatitis, hypotension and cardiac abnormalities [8]. Paromomycin has limited use for the treatment of VL [7]. Miltefosine, an orally effective drug also suffers from

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nephrotoxicity, hepatotoxicity and teratogenicity [7]. So far, no vaccine has been clinically approved for human use [9]. Therefore, there is an urgent need for the development of new, low-cost, effective and safe drugs for the treatment of VL. The discovery of new lead

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compounds for this disease is a pressing concern for global health programs. In this respect, several synthetic or natural plant / marine product inspired compounds have shown promising efficacy for the treatment of visceral leishmaniasis [10].

Chalcones, or 1,3-diaryl-2-propen-1-ones (Figure 1), are prominent secondary metabolite precursors of flavonoids and isoflavonoids in plants. Natural and synthetic

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chalcones are described in the literature with different pharmacological profiles, such as antiinflammatory [11], antibacterial [12], antiviral [13], antimalarial [14], anticancer [15], antileishmanial [16], antituberculosis [17], anti-HIV [18] and antifungal activities [19]. A thorough assessment of structural requirements for antileishmanial activities of chalcones is

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vital to develop and designing of novel drug like candidate [20]. Licochalcone A (II), is an oxygenated chalcone (Figure 1), isolated from Chinese licorice, efficiently inhibits

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proliferation of Leishmania donovani and L. major promastigotes and amastigotes in vitro by interfering with the function of the parasite mitochondria. As a part of our drug discovery program on antileishmanial agents from Indian medicinal plants, we have reported the isolation of three chromenodihydrochalcones, crotaramosmin (III), crotaramin (IV) and crotin (V) (Figure 1) from Crotalaria ramosissima [21], their synthesis and in vitro antileishmanial activity of III–V and analogues thereof [22,23]. Our previous work demonstrated that synthetic analogues, chromenochalcones enhanced the in vitro and in vivo antileishmanial activity [23] over the natural products, in which the benzopyran core and substitutions on ring A played an important role. Therefore, we enthusiastically moved to further work on chromenochalcones and analogues thereof with the goal of enhanced

ACCEPTED MANUSCRIPT antileishmanial activity and improved cytotoxic profiles. A series of chalcones with various structural features were synthesized and evaluated for their in vitro antileishmanial activity. Several of these compounds were found active in in vitro screening; few of them were further evaluated for in vivo antileishmanial efficacy in L. donovani / hamster model. Herein, we report a comprehensive assessment of antileishmanial activity and structure-activity

Figure 1 2. Results and Discussion

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2.1. Chemistry

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relationship (SAR) analyses of promising chalcones.

2.1.1. Synthesis of Chromenochalcones

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A series of chromenochalcones were prepared to evaluate their antileishmanial activity. The acetyl chromene 2 was synthesized using pyridine-catalyzed condensation between 2,4-dihydroxyacetophenone (1) and 3-methyl-but-2-enal [24]. The resultant acetyl chromene 2 and substituted aromatic aldehydes or p-hydroxybenzene-1,3-dicarbaldehyde [25] were subjected to Claisen-Schmidt condensation using either aqueous KOH in ethanol

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or NaH in dry THF at room temperature to furnish the corresponding chromenochalcones 3– 18 in good yields (Scheme 1, and Table 1) [23]. To improve the bioavailability of compounds, the alkylated amine groups were introduced in chromenochalcone core. The synthesis of alkylated amines containing-chromenochalcones (19–24) were accomplished by

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the replacement of hydrogen from phenolic hydroxyl group of the chromenochalcone 12 (Table 1) with the alkyl part of the corresponding amines using K2CO3 in dry acetone

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(Scheme 1) [26].

Scheme 1

2.1.2. Synthesis of Chromenochalcones with Hetero Atoms in Ring-A. Chromenochalcones 25–29 which have hetero atoms in ring-A (Scheme 2) were

synthesized using Claisen-Schmidt condensation from acetyl chromen [24] 2 and various heteroaryl aldehydes to afford the compounds 25–29 in good yields (Scheme 2).

Scheme 2

ACCEPTED MANUSCRIPT 2.1.3. Synthesis of O-Prenyl/Geranyl/Farnesyl/Alkylamino Chalcones To study the role of the alkenyl (prenyl, geranyl, farnesyl) and alkylated amino groups on hydroxyl group of normal chalcones, O-prenyl, -geranyl, -farnesyl and alkylated amines containing chalcones 35–42 were prepared as shown in scheme 3. The hydroxychalcones 31– 34 were synthesized through Claisen-Schmidt condensation of 4-hydroxy acetophenone (30)

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and corresponding aromatic aldehydes. The resulting chalcones 31–34 were then smoothly alkylated with various alkenyl/aminoalkyl halides using K2CO3 in dry acetone under reflux to furnish the desired O-alkenyl/alkylated amines containing chalcones 35–42 in good yields [26].

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Scheme 3

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2.2. Biological Activity

In our efforts to develop new antiparasitic agents, we had earlier reported the in vitro and in vivo antileishmanial activity of natural and synthetic chromenochalcones [22,23]. Our previous work had shown that nature of substitutions on ring A and B are very crucial for the potent antileishmanial activity. On the basis of these optimistic results, we wanted to find out the influence of various substituents on ring A and B with the goal of synthesizing the various

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synthetic chalcones with enhanced antileishmanial activity and improved cytotoxic profiles. A series of chalcones were prepared and evaluated for antileishmanial activity against WHO reference strain (MHOM/IN/80/Dd8) of promastigotes (expressing luciferase firefly reporter gene) at 25 µM. The compounds which exhibited more than 70 % inhibition were screened

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for intracellular amastigote form (expressing luciferase firefly reporter gene) of L. donovani and their IC50 were determined. In parallel, the cytotoxicity of the compounds, which have

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IC50 < 20 µM against amastigotes, was also tested using mammalian kidney fibroblast cells (Vero cell line). Among these, two promising compounds were further evaluated against the MHOM/IN/80/Dd8 strain of L. donovani in hamster model. Standard antileishmanials, miltefosine and sodium stibogluconate (SSG) were included in this study as reference drugs. 2.2.1. In Vitro Antileishmanial Activity of Chromenochalcones In continuation of our work [22,23], chromanochalcones 3–18 were synthesized and evaluated for their in vitro activity. Chalcones 3−16, which have the electron-donating groups (EDGs), electron-withdrawing groups (EWGs) and halogen groups on ring-A, appear to be more potent against both promastigotes (70–100 % inhibition at 25 µM) and amastigotes

ACCEPTED MANUSCRIPT (IC50 = 5.1–20 µM) with good selectivity, with the exception of 3 and 10 (IC50 > 20 µM). While the introduction of aldehyde group as in 17 and fused hetero cycles as in 18 were also showed poor activity against amastigotes (IC50 > 20 µM). It is noteworthy to mention that the analogues, 4−16 have shown improved antileishmanial activity and selectivity (Table 1).

2.2.2.

In

Vitro

Antileishmanial

Activity

Chromenochalcones

of

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Table 1 Alkylated

amines

containing-

To improve the bioavailability of chromenochalcones, alkylated amines containing-

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chromenochalcones 19–24, which have an alkylated amine substituents either on ring A and B or on both rings were synthesized (Scheme 1) and evaluated for antileishmanial activity as

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shown in Table 2. All these mono- and di-alkylated amino substituted analogues showed 99.9–100% inhibition of promastigotes at 25 µM concentration. Conversely, the di-alkylated amino substituted analogues 22–36 have outstanding activity (IC50 = 2.5–4 µM) and therapeutic profiles (SI = 31–105) (Table 2). The introduction of alkylated amino substituents on ring A and B (22–24: IC50 = 2.5–4.0 µM versus 3; IC50 > 20 µM against amastigotes) significantly increased the activity profile due to better bioavailability than their parent

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compounds. Remarkably, the di-alkylated amino substituted analogues 22–24 were more effective than standard drugs, miltefosine (IC50 = 8.40 µM), and SSG (IC50 = 49.7 µM) or

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other tested compounds (Table 2).

Table 2

A.

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2.2.3. In Vitro Antileishmanial Activity of Chromanochalcones with Hetero Atoms on Ring

After studying the effect of various substitutions nature and their positions on ring A and benzopyran moiety (Ring B) of chalcones (Table 1–2), we further moved to work on chromenochalcones with hetero atoms on ring A. We synthesized some chromenochalcones 25–29 with hetero atom in ring A (Scheme 2) and evaluated for in vitro antileishmanial activity (Table 3). Among these, most of the analogues exhibited potent activity against

ACCEPTED MANUSCRIPT promastigotes (99.8–100% inhibition at 25 µM), with the exception of chromenochalcone 27 (62.2% inhibition at 25 µM) (Table 3). On the other hand, all analogues 25–29, with exception of 26 (IC50 = 22.2 µM) and 28 (IC50 = 12.1 µM), were found extremely potent

Table 3

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(IC50 < 5.4 µM) against amastigotes and also have good selectivity index.

2.2.4. In Vitro Antileishmanial Activity of O-Alkylated Chalcones

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Finally, we wanted to study the role of the O-alkenyl (prenyl, geranyl and farnesyl) and alkylated amine substituent on ring A and B of normal chalcones, therefore various

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lipophilic monoterpenes (prenyl, geranyl, farnesyl), and hydrophilic alkyl amino substituents containing chalcones 35–42 were generated (Scheme 3). In this series, all compounds (35– 42) showed good activity against promastigotes (Table 4). The analogues 35−39, in which the ring B was substituted with O-alkenyl groups showed excellent activity (IC50 = 4.1–9.3 µM), with the exception of 38 (IC50 > 20 µM) and 39 (toxic to cells) against amastigotes (Table 4). While the introduction of alkylated amine substituents either on ring A and B significantly

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decreased the activity, with the exception of 41 (IC50 > 9.3 µM) (Table 4). Table 4

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2.2.5. In Vivo Efficacy of Chalcones in L. donovani / Hamster Model Large number of compounds (4, 6, 16, 22, 23, 24, 25, 29, 35 and 37) exhibited potent

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in vitro activity against extracellular promastigote and intracellular amastigote (IC50 1.70 to 8 µM) form of L. donovani (Table 1–4). Among these, compounds 22 (IC50 = 2 µM and SI = 162.5; table 2) and 37 (IC50 = 3.1 µM and SI = 47.2; table 4), which have potent antiamastigote activity with high selectivity index were chosen for further in vivo studies and tested against the MHOM/IN/80/Dd8 strain of L. donovani in hamster model [27]. The aqueous suspension of tested compounds was administered for 5 to 10 consecutive days at 50 and/or 100 mg/kg/day dose by intraperitoneal (IP) route. The post treatment splenic biopsies were done on day 7 after the last dose administration and amastigote counts were assessed by Giemsa staining. Compound 22 showed 48.54 ± 10.55 % inhibition on day 7 post treatment (p.t.) at 50 mg/kg/day by IP route (Table 5), where as the chalcone 37 exhibited 83.32 ±

ACCEPTED MANUSCRIPT 12.37 % suppression of parasites at 50 mg/kg/day for 10 days treatment and 75.89 ± 10.78% at 100 mg/kg/day for 5 days treatment by IP route (Table 5). Table 5

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3. Conclusion In conclusion, a series of chalcone analogues with various structural features such as chromenochalcones and O-prenyl/geranyl/farnesyl/aminoalkyl groups containing chalcones were prepared based on natural product lead and tested for their antileishmanial activity.

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Among all, ten compounds (4, 6, 16, 22, 23, 24, 25, 29, 35 and 37) found to be significantly more active than the standard antileishmanial drugs, miltefosine and sodium stibogluconate (SSG) in in vitro evaluation against Leishmania amastigotes. The in vivo studies of promising

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compounds 22 and 37 were performed in L. donovani / hamster model, in which compound 37 showed good antileishmanial activity (83.32 % parasite inhibition at the dose of 50 mg/kg x 10 days and 75.89% parasite inhibition at 100 mg/kg x 5 days by IP route). 4. Experimental Section

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4.1. General Methods.

Melting points were recorded on Buchi-530 capillary melting point apparatus and are uncorrected. IR spectra were recorded on Perkin-Elmer AC-1 spectrometer. 1H NMR spectra were recorded on Bruker Avance DPX 200 FT, Bruker Robotics and Bruker DRX 300,

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spectrometers at 200, 300 MHz (1H) and 50, 75 MHz (13C). Experiments were recorded in CDCl3, CD3OD, and DMSO-D6 at 25ºC. Chemical shifts were given in parts per million

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(ppm) downfield from internal standard Me4Si (TMS). ESI mass spectra were recorded on JEOL SX 102/DA-6000. Chromatography was executed with silica gel (60–120 or 230–400 mesh) using mixtures of ethyl acetate and hexane as eluants. Reactions, which required the use of anhydrous, inert atmosphere techniques, were carried out under an atmosphere of nitrogen. 1, 4-Dioxane was distilled over sodium. Commercially available reagents, solvents and starting materials were used without further purification. 4.1.1. Representative Procedure for the Synthesis of 1-(5-Hydroxy-2, 2-dimethyl–2Hchromene-6-yl)-ethanone (2). To a magnetically stirred solution of 1 (12.0 g, 79 mmol) in dry pyridine (8.04 mL) was added gradually 3-methyl-but-2-enal (6.8 g, 79 mmol) at rt. The

ACCEPTED MANUSCRIPT reaction mixture was refluxed for 4 h at 150oC, additional equivalent of 3-methyl-but-2-enal (6.8 g, 79 mmol) was added and refluxed for further 6 h. Excess pyridine, in the reaction mixture was evaporated by rotary evaporator under reduced pressure. The crude product was subjected to silica gel column chromatography using hexane/ethyl acetate as mobile phase to afford the desired compound 2.

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Yield: 65%; MP: 102-104 °C; IR (KBr cm-1): 3435, 2862, 1633, 1486, 1333, 1109,751; 1H NMR (CDCl3, 300 MHz) δ: 12.90 (s, 1H), 7.52 (s, d, J = 8.8 Hz, 1H), 6.72 (d, J = 10.1Hz, 1H), 6.35 (d, J= 8.8 Hz, 1H), 5.57 (d, J= 10.0 Hz, 1H), 2.55 (s, 3H), 1.47(s, 6H); MS(ESI):

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m/z: 219 (M+H)+.

4.1.2. Representative Procedure for the Synthesis of (E)-1-(5-hydroxy-2,2-dimethyl-2Hchromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (3). To a stirred solution of 2 (500 mg,

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2.2 mmol) in aqueous KOH solution in ethanol (5 mL) was added 4-hydroxybenzaldehyde (279 mg, 2.2 mmol). The reaction mixture was stirred for 48 h at rt, and quenched in ice-cold water, acidified with 1 N HCl, extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with water, brine solution, dried over anhydrous Na2SO4 and the solvent was evaporated under reduced pressure. The crude product was subjected to silica gel

chromenochalcone 3.

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column chromatography using hexane/ethyl acetate as mobile phase to afford the

Yield 35%; mp 201–205 oC; FT-IR (KBr, cm-1) 3436, 1635; 1H NMR (CDCl3, 200 MHz) δ 13.76 (s, 1H), 7.83 (d, J = 15.8 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.54 (d, J = 8.6 Hz, 2H),

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7.42 (d, J = 15.8 Hz, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.39 (d, J = 9.0 Hz, 1H), 6.38 (d, J = 8.6 Hz, 2H), 5.58 (d, J = 10.0 Hz, 1H), 1.46 (s, 6H); MS (FAB) m/z: 323 (M + H)+.

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4.1.3. Representative Procedure for the Synthesis of 1-(5-Hydroxy-2, 2-dimethyl-2Hchromen-6-yl)-3-(4-nitro-phenyl)-propenone (16). To a stirred solution of 2 (575 mg, 2.6 mmol) in anhydrous THF (5 mL) was added portion wise NaH (121 mg, 5.2 mmol) and stirred for 20 min at rt under nitrogen. Then, p-nitro-benzaldehyde (398 mg, 2.2 mmol) in 2 mL of THF was added to the reaction mixture and stirred for 8 h at rt. The mixture was poured into ice-cold water, acidified with 1N HCl and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with water, brine solution, dried over anhydrous Na2SO4, and evaporated under reduced pressure. The crude product was subjected

ACCEPTED MANUSCRIPT to silica gel column chromatography using hexane/ethyl acetate as mobile phase to afford the chromenochalcone 16. Yield 57%; FT-IR (KBr, cm-1) 3425, 1625; 1H NMR (CDCl3, 200 MHz) δ 13.29 (s, 1H), 8.28 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 15.4 Hz, 1H), 7.78 (d, J = 8.7 Hz, 2H), 7.70 (d, J = 8.8 Hz, = 10.0 Hz, 1H), 1.47 (s, 6H); MS (FAB) m/z: 352 (M + H)+.

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1H), 7.65 (d, J = 15.4 Hz, 1H), 6.74 (d, J = 10.0 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 5.61 (d, J

4.1.4. Representative Procedure for the Synthesis of 1-[2,2-Dimethyl-5-(2-piperidin-1-ylethoxy)-2H-chromen-6-yl]-3-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-propenone

(24).

To

a

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stirred solution of chalcone, 3 (250 mg, 1.0 mmol) in dry acetone (20 mL) were added anhydrous K2CO3 (2.85 g, 20.8 mmol), 1-(2-Chloro-ethyl)-piperidine hydrochloride (953 mg, 5.2 mmol), and the reaction mixture was refluxed for 5 h. The mixture was filtered off under

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suction and solvent was evaporated under reduced pressure. The crude product was subjected to silica gel column chromatography using hexane/ethyl acetate as mobile phase to afford the compound 24.

Yield: 47%; FT-IR (neat, cm−1) 1652; 1H NMR (CDCl3, 200 MHz) δ 7.67 (d, J = 14.7 Hz, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 14.7 Hz, 1H), 6.91 (d, J =

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8.6 Hz, 2H), 6.78 (d, J = 10.0 Hz, 1H), 6.63 (d, J = 8.7 Hz, 1H), 5.67 (d, J = 10.0 Hz, 1H), 4.13 (t, J = 5.9 Hz, 2H), 3.93 (t, J = 5.7 Hz, 2H), 2.78 (t, J = 5.9 Hz, 2H), 2.62 (t, J = 5.7 Hz, 2H), 2.53–2.34 (m, 8H), 1.61–1.25 (m, 18H); 13C NMR (CDCl3, 50 MHz) δ 191.3, 161.1, 157.6, 155.6, 143.2, 131.5, 130.7, 130.5, 128.2 (2C), 126.6, 124.5, 117.3, 115.4, 115.3 (2C),

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113.0, 77.1, 73.9, 66.4, 58.9, 58.1, 55.4 (2C), 55.2 (2C), 28.4 (2C), 26.2 (4C), 24.6, 24.5; MS

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(FAB) m/z: 545 (M + H)+.

4.2. Experimental Section for Biology 4.2.1. Promastigote Growth Inhibition Assay The antileishmanial activity of tested compounds against the promastigote form of L.

donovani (strain: MHOM/IN/80/Dd8) was assessed as described earlier [23]. The late log / stationary phase of promastigotes (expressing firefly luciferase gene) were seeded with complete M-199 medium at 5 x 105/mL/100µL/well in 96-well plates and incubated with tested compounds in a 24ºC incubator for 96 h. Miltefosine and SSG were used as standard drugs. After 96 h of incubation, 50 µL of promastigote suspension was pipette out from each

ACCEPTED MANUSCRIPT well in to another 96-well plate and mixed with an equal volume of Steady Glo® reagent (Promega) and luminescence was measured by using a luminometer. The values were expressed as relative luminescence unit (RLU). The inhibition of parasitic multiplication is determined by comparison of the luciferase activity of compound treated parasites with that of untreated control.

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4.2.2. Antiamastigote Assay

Mouse macrophage cell line (J-774A.1) infected with promastigotes (expressing luciferase firefly reporter gene) was used for the assessment of the activity of compounds

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against the amastigote form of Leishmania parasite. J-774A.1 cells were seeded in a 96-well plate (4 x 104/mL/100µL/well) in complete RPMI-1640 medium (containing 10% heat

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inactivated foetal calf serum) and the plates were incubated at 37ºC in a 5% CO2 incubator. After 24 h, the medium was replaced with fresh medium containing stationary phase promastigotes (4 x 105/mL/100µL/well). Promastigotes were phagocytized by the macrophages and inside the phagolysosomes; they were transformed into amastigotes form. Each well of the plate was washed with plain RPMI medium after 24 h of incubation to remove the un-internalized promastigotes. The test compounds were added at dilutions up to

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7 points starting from 40 µM concentration in complete RPMI medium and the plates were incubated at 37ºC in a CO2 incubator for 72 h. After incubation, the drug containing medium was aspirated and 50 µL phosphate buffer saline (PBS) was added in each well and mixed with an equal volume of Steady Glo® reagent. After gentle shaking for 2 min, the reading was

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taken in a luminometer [23]. The values are expressed as RLU and data were transformed into a graphic program (Excel). IC50 value of each compound was calculated by nonlinear

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regression analysis of the concentration response curve using the four parameter Hill equations.

4.2.3. Cytotoxicity Assessment Assay The cytotoxicity of the compounds was determined by following the method of

Mosmann [28] with some modifications. As described previously [23], mammalian kidney fibroblast cells (Vero cell line) (1x 105/mL/100µL/well) were incubated with test compounds (in 7 concentrations starting from 400 µM) at 37ºC in a CO2 incubator. After 72 h of incubation, 25 µL of MTT (3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide)

ACCEPTED MANUSCRIPT reagent (5mg/mL) in PBS medium was added to each well and incubated at 37°C for 2 h. At the end of the incubation period, the supernatant were removed and 150 µL of pure DMSO was added to each well for solubilizing the formazan crystal. After 15 min of shaking, the readings were recorded as absorbance at 540 nm on a micro plate reader. Fifty percent cytotoxic concentration (CC50) values were estimated as described by Huber and Koella [29].

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The selectivity index (SI) for each compound was calculated as ratio between cytotoxicity (CC50) in Vero cells and activity (IC50) against Leishmania amastigotes. 4.2.4. In Vivo Evaluation in L. donovani / Hamster Model

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The in vivo antileishmanial activity was determined in golden hamsters (Mesocricetus auratus) infected with MHOM/IN/80/Dd8 strain of L. donovani as described by Gupta et al

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[27]. Golden hamsters of both sexes were infected intracardiacally with 1 x 107 amastigotes per animal. After establishment of infection in 15 days, pre-treatment splenic biopsies were performed to assess the degree of infection in all the animals. The animals with +1 grade infection (5-10 amastigotes/ 100 spleen cell nuclei) were included in the chemotherapeutic studies. Five infected animals were used for each test compound. Drug treatment in different dose regimen by intraperitoneal (IP) or per oral (PO) route was initiated after 2 days of pre-

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treatment biopsies and continued for 5-10 consecutive days. Miltefosine and SSG were used as reference drugs. Post-treatment biopsies were done on day 7 after the last dose administration and amastigote counts are assessed by Giemsa staining [23]. Intensity of infection in both, treated and untreated animals, and also the initial parasite count in treated

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animals was compared and the efficacy was expressed in terms of percentage inhibition (PI) using the following formula:-

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PI = 100- [(ANAT x 100) / (INAT x TIUC)] Where PI is Percent Inhibition of amastigotes multiplication, ANAT is Actual Number of Amastigotes in Treated animals, INAT is Initial Number of Amastigotes in Treated animals and TIUC is Time Increase of parasites in Untreated Control animals. Acknowledgements We are grateful to the Director, CSIR-CDRI, Lucknow, for the constant encouragement for the program on antiparasitic drug discovery, SAIF Division for spectral data and Department of Science and Technology (DST), New Delhi for financial support in

ACCEPTED MANUSCRIPT the form of ad-hoc project (SR/S1/OC-58/2008). The transgenic L. donovani promastigotes were originally procured from Dr. Neena Goyal, Division of Biochemistry, CSIR-CDRI, Lucknow, India. Appendix. Supplementary information

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Supplementary data associated with this article can be found in the online version at doi: References

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chalcones bioisosteric replacement of the 4′-hydroxy group, Bioorg. Med. Chem. 12

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Joussef, M. Steindel, C.M.O. Simões, A.M.T. de Souza, U.O. Magalhaes, I.F. Afonso, C.R. Rodrigues, R.J. Nunes, H.C. Castro, Synthesis, biological evaluation and SAR of sulfonamide 4-methoxychalcone derivatives with potential antileishmanial activity, Eur. J. Med. Chem. 44 (2009) 755-763. (b) P. Boeck, C.A. Bandeira Falcão, P.C. Leal, R.A.

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Siegel, M.L. Bolognesi, Privileged structure-guided synthesis of quinazoline derivatives as inhibitors of trypanothione reductase, Bioorg. Med. Chem. Lett. 19 (2009) 3031-3035. J.K. Kumar, T. Narender, M.S. Rao, P.S. Rao, G. Toth, B. Balazs, H.J. Duddeck,

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Table 1. In vitro antileishmanial activity of chromenochalcones against promastigotes and amastigotes of L. donovani and their cytotoxicity Table 2. In vitro antileishmanial activity of alkylated amines containing-chromenochalcones

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against promastigotes and amastigotes of L. donovani and their cytotoxicity Table 3. In vitro antileishmanial activity of chromenochalcones with hetero atoms on ring A against promastigotes and amastigotes of L. donovani and their cytotoxicity

amastigotes of L. donovani and their cytotoxicity

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Table 4. In vitro antileishmanial activity of O-alkylated chalcones against promastigotes and

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Table 5. In vivo efficacy of chalcones against L. donovani / hamster model Figure 1. General skeleton and naturally occurring antileishmanial chalcones Scheme 1. Synthesis of Chromenochalcones (3–18) and Alkylated amine containingchromenochalcones (19–24)

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Scheme 2. Synthesis of chromenochalcones containing the hetero atoms in ring A (25–29)

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42)

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Scheme 3. Synthesis of O-prenyl/geranyl/farnesyl/alkylatedamines containing chalcones (35–

ACCEPTED MANUSCRIPT Table 1. In vitro antileishmanial activity of chromenochalcones against promastigotes and

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amastigotes of L. donovani and their cytotoxicity

In vitro activity Antipromastigote Antiamastigote (% inhibition at IC50 (µM)b a 25 µM)

Index (SI)d

> 20

NDe

-

5.1

215

42.1

97.1

19.8

> 400

> 20.2

H

98.7

6.1

278.5

45.6

OMe

H

98.2

11.9

266.6

22.4

H

F

H

99.0

16.7

158.4

9.5

H

H

Cl

H

99.9

17.9

92.2

5.1

10

H

H

Br

H

100

> 20

ND

-

11

H

F

F

H

99.9

17.0

145.7

8.6

12

H

Cl

Cl

H

99.9

13.9

141.7

10.2

13

Cl

H

Cl

H

99.9

9.7

130.9

13.5

14

H

15

H

R2

R3

R4

3

H

H

OH

H

70.2

4

H

H

OEt

H

88.9

5

H

H

NMe2

H

6

H

H

NHAc

7

H

OMe

8

H

9

17 18

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F

H

99.9

12.1

184.9

15.3

H

CN

H

99.6

8.2

80.2

9.8

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16

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R1

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Cytotoxicity CC50 (µM)c

Compd

H

H

NO2

H

97.4

5.2

106.2

20.4

OH

H

H

CHO

94.5

> 20

ND

-

H

91.2

> 20

ND

-

H

SSG

-

-

-

-

No inhibition

49.7

> 400

> 8.0

Miltefosine

-

-

-

-

100

8.4

52.5

6.2

a

Values are represented as average of at least duplicate measurements (SD ± 2%). bIC50 and cCC50 values are represented as average of at least duplicate measurements (SD ± 10%), dSI (selectivity index) = CC50/ IC50. eND: not determined.

ACCEPTED MANUSCRIPT Table 2. In vitro antileishmanial activity of alkylated amines containing-chromenochalcones

Index (SI)d

341.2

22.9

> 20

NDe

ND

100

17.7

> 400

> 22.6

22

100

2.0

325.4

162.5

23

99.9

2.5

258.1

103.2

100

2.8

86.5

30.9

No inhibition

49.7

> 400

> 8.0

100

8.4

52.5

19

H

100

20

H

100

21

H

-

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SSG Miltefosine

R2

14.9

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R1

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Cytotoxicity CC50 (µM)c

Compd

24

a

In vitro activity Antipromastigote Antiamastigote (% inhibition at IC50 (µM)b a 25 µM)

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19–24 against promastigotes and amastigotes of L. donovani and their cytotoxicity

-

b

6.2 c

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Values are represented as average of at least duplicate measurements (SD ± 2%). IC50 and CC50 values are represented as average of at least duplicate measurements (SD ± 10%), dSI (selectivity index) = CC50/ IC50, eND: not determined.

ACCEPTED MANUSCRIPT Table 3. In vitro antileishmanial activity of chromenochalcones with hetero atoms on ring A

a

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against promastigotes and amastigotes of L. donovani and their cytotoxicity

Cytotoxicity CC50 (µM)c

Index (SI)d

2.0

20.5

10.2

22.2

8.6

0.4

NDe

ND

-

100

12.1

256.8

21.2

99.8

5.3

399.4

75.3

No inhibition

49.7

> 400

> 8.0

100

8.4

52.5

6.2

R2

X

Y

25

-

-

N

CH

99.9

26

-

-

CH

N

100

27

H

H

-

-

62.2

28

Cl

H

-

-

29

Cl

Me

-

-

SSG

-

-

-

-

Miltefosine

-

-

-

-

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R1

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In vitro activity Antipromastigote Antiamastigote IC50 (µM)b (% inhibition at 25 µM)a

Compd

b

c

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Values are represented as average of at least duplicate measurements (SD ± 2%). IC50 and CC50 values are represented as average of at least duplicate measurements (SD ± 10%), dSI (selectivity index) = CC50/ IC50. eND. not determined.

ACCEPTED MANUSCRIPT Table 4. In vitro antileishmanial activity of O-alkylated chalcones against promastigotes and amastigotes of L. donovani and their cytotoxicity R2 R1O

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O 35-42

R1

R2

NMe2

84.2

36

OH

95.2

37

OMe

93.0

3.1

146.5

47.2

e

-

Toxic for cells

ND

-

99.9

>20

ND

-

99.9

9.3

251.3

27.0

99.9

>20

ND

-

OEt

41

NMe2

-

1.4

100

40

Miltefosine

12.9

ND

42 -

No inhibition

49.7

> 400

> 8.0

-

100

8.4

52.5

6.2

EP

Inhibition at 25 µM and values are represented as average of at least duplicate measurements (SD ± 2%). bIC50 and cCC50 values are represented as average of at least duplicate measurements (SD ± 10%),ND = not determined, dSI (selectivity index) = CC50/ IC50. eND. not determined.

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a

9.3

>20

39

-

> 97.6

76.1

38

SSG

> 400

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NMe2

Index (SI)d

4.1

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35

Cytotoxicity CC50 (µM)c

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Compd

In vitro activity Antipromastigote Antiamastigote (% inhibition at IC50 (µM)b a 25 µM)

ACCEPTED MANUSCRIPT Table 5. In vivo efficacy of chalcones against L. donovani / hamster model Dose

Compd

(mg/kg)

Treated days

Route

% Inhibition ± SD at day 7 post treatment

50

5

IP

48.54 ± 10.55

37

50

10

IP

83.32 ± 12.37

37

100

5

IP

75.89 ± 10.78

SSG

40

5

IP

89.47 ± 4.72

Miltefosine

30

5

PO

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22

98.50 ± 1.10

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SD, standard deviation; IP, intraperitoneal; PO, per oral, SSG, sodium stibo-gluconate

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Figure 1. General skeleton and naturally occurring antileishmanial chalcones

Scheme 1. Synthesis of chromenochalcones (3–18) and alkylated amine containingchromenochalcones (19–24)

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ACCEPTED MANUSCRIPT

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Scheme 2. Synthesis of chromenochalcones containing the hetero atoms in ring A (25–29)

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(35–42)

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Scheme 3. Synthesis of O-prenyl/geranyl/farnesyl/alkylated amines containing chalcones

ACCEPTED MANUSCRIPT

Research Highlights

Chalcone derivatives were prepared based on the natural products lead molecules.

•

Compounds were assessed for in vitro and in vivo antileishmanial potential.

•

Ten compounds exhibited promising antileishmanial activity.

•

The potent compound showed >83% inhibition at dose 50 mg/kg x 10d in hamster

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•

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model.

ACCEPTED MANUSCRIPT

Supporting Information Synthesis and Biological Evaluation of Chalcones as Potential

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Antileishmanial Agents Shweta Gupta,a Rahul Shivahare,b Venkateswarlu Korthikunta,a Rohit Singh,a Suman Guptab and Narender Tadigoppula*a a

Division of Medicinal and Process Chemistry, b

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Division of Parasitology,

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CSIR-Central Drug Research Institute, Lucknow-226 031, India

*Corresponding author:

Tel.: +91 522 2612411; fax: +91 522 2623405.

E-mail: [email protected] or [email protected] (T. Narender) Table of Contents:

S-2

2. NMR spectra of the selected chalcones

S-18

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1. Compounds characterization data

S-1

ACCEPTED MANUSCRIPT Compounds characterization data

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(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (3):

Yield 35%; mp 201–205 oC; FT-IR (KBr, cm-1) 3436, 1635; 1H NMR (CDCl3, 200 MHz) δ

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13.76 (s, 1H), 7.83 (d, J = 15.8 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 15.8 Hz, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.39 (d, J = 9.0 Hz, 1H), 6.38 (d, J = 8.6 Hz, 2H),

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5.58 (d, J = 10.0 Hz, 1H), 1.46 (s, 6H); MS (FAB) m/z: 323 (M + H)+.

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3-(4-Ethoxy-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (4):

Yield 56%; FT-IR (KBr, cm-1) 3395, 1631; 1H NMR (CDCl3, 200 MHz) δ 13.56 (s, 1H), 7.86 (d, J = 15.8 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 15.8 Hz, 1H),

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6.92 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 10.0 Hz, 1H), 6.37 (d, J = 8.8 Hz, 1H), 5.68 (d, J = 10.0 Hz, 1H), 4.08 (q, J = 6.0 Hz, 2H), 1.60–1.46 (m, 9H); 13C NMR (CDCl3, 50 MHz) δ 192.4, 161.6,

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161.3, 160.0, 144.6, 131.0 (2C), 130.8, 128.5, 127.8, 118.0, 116.4, 115.3 (2C), 114.5, 109.8, 108.6, 78.2, 64.1, 28.8 (2C), 15.1; MS (FAB) m/z: 351 (M + H)+. (E)-3-(4-(dimethylamino)phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1one(5):

S-2

ACCEPTED MANUSCRIPT Yield 55%; FT-IR (KBr, cm-1) 3432, 1630; 1H NMR (CDCl3, 200 MHz) δ 13.80 (s, 1H), 7.64 (d, J = 14.5 Hz, 1H), 7.53 (m, 2H), 7.51 (d, J = 14.5 Hz, 1H), 7.49 (d, J = 8.9 Hz, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.82-6.66 (m, 2H), 6.48 (d, J = 8.9 Hz, 1H), 5.89 (d, J = 10.0 Hz, 1H), 3.01 (s,

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6H), 1.46 (s, 6H); MS (FAB) m/z: 350 (M + H)+. (E)-N-(4-(3-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-oxoprop-1-

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enyl)phenyl)acetamide (6):

Yield 75%; FT-IR (KBr, cm-1) 3436, 1635; 1H NMR (CDCl3, 200 MHz) δ 14.00 (s, 1H), 10.22 (s, 1H), 8.20(d, J = 8.9 Hz, 2H), 7.87(d, J = 14.0 Hz, 1H), 7.86-7.83 (m, 2H), 7.72-7.67 (m, 2H), 6.65 (d, J = 10.0 Hz, 1H), 6.46 (d, J = 8.8 Hz, 1H), 5.78 (d, J = 10.0 Hz, 1H), 2.10 (s, 3H), 1.46 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 19.4, 169.2, 160.3, 159.5, 144.5, 142.1, 132.3, 130.4(2C),

(M + H)+.

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129.5, 129.2, 119.2(3C), 115.2, 114.0, 108.8, 108.4, 78.1, 28.2(2C), 24.4; MS (FAB) m/z: 364

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3-(3,4-Dimethoxy-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (7):

Yield 45%; mp 150–155 oC; FT-IR (KBr, cm-1) 3437, 1648; 1H NMR (CDCl3, 200 MHz) δ 13.62 (s, 1H), 7.84 (d, J = 15.3 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.42 (d, J = 15.3 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 7.15 (d, J = 1.6 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 10.0 Hz, 1H), 6.38 (d, J = 8.9 Hz, 1H), 5.59 (d, J = 10.0 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 1.47 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 192.3, 161.3, 160.1, 151.9, 149.7, 144.8, 130.9, 128.4, 128.2, 123.7, S-3

ACCEPTED MANUSCRIPT 118.4, 116.3, 114.5, 111.6, 110.6, 109.8, 108.5, 78.0, 56.4 (2C), 28.7 (2C); MS (FAB) m/z: 367 (M + H)+.

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3-(4-Fluoro-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (8):

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Yield 59%; FT-IR (KBr, cm-1) 3429, 1627; 1H NMR (CDCl3, 200 MHz) δ 13.50 (s, 1H), 7.83 (d, J = 15.5 Hz, 1H), 7.72–7.60 (m, 3H), 7.47 (d, J = 15.5 Hz, 1H), 7.11 (m, 2H), 6.74 (d, J = 10.0

M AN U

Hz, 1H), 6.38 (d, J = 8.9 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 192.1, 167.0, 162.0, 161.4, 160.3, 143.3, 132.0, 131.5, 131.0, 130.9, 130.7, 128.6, 120.4, 116.8, 116.3, 114.4, 109.8, 108.8, 78.3, 28.8 (2C), Note– More number of carbon signals appear due to long range coupling with Fluorine (F) substitution on phenyl ring; MS (FAB) m/z: 325

TE D

(M + H)+.

EP

3-(4-Chloro-phenyl)-1-(5-hydroxy-2, 2-dimethyl-2H-chromen-6-yl)-propenone (9):

AC C

Yield 52%; FT-IR (KBr, cm-1) 3436 1628; 1H NMR (CDCl3, 200 MHz) δ 13.61 (s, 1H), 7.73 (d, J = 15.5 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 15.5 Hz, 1H), 7.31 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 10.0 Hz, 1H), 6.30 (d, J = 8.8 Hz, 1H), 5.52 (d, J = 10.0 Hz, 1H), 1.39 (s, 6H); MS (FAB) m/z: 341 (M + H)+. 3-(4-Bromo-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (0):

S-4

ACCEPTED MANUSCRIPT

Yield 46%; mp 152–153 oC; FT-IR (KBr, cm-1) 3440, 1634; 1H NMR (CDCl3, 200 MHz) δ

RI PT

13.63 (s, 1H), 7.79 (d, J = 15.4 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.55 (d, J = 7.8 Hz, 2H), 7.53 (d, J = 15.4 Hz, 1H), 7.52 (d, J = 7.8 Hz, 2H), 6.74 (d, J = 10.0 Hz, 1H), 6.38 (d, J = 8.9 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 192.0, 161.4, 160.4, 143.2,

(2C); MS (FAB) m/z: 385 (M + H)+, 387 (M + 3H)+.

SC

134.1, 132.6 (2C), 131.1, 130.2 (2C), 128.6, 125.3, 121.3, 116.2, 114.4, 109.8, 108.8, 78.4, 28.8

M AN U

3-(3,4-Difluoro-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (11):

TE D

Yield 38%; mp 138–139 oC; FT-IR (KBr, cm-1) 3430, 1639; 1H NMR (CDCl3, 200 MHz) δ 13.54 (s, 1H), 7.89 (d, J = 15.6 Hz, 1H), 7.68 (d, J = 9.1 Hz, 1H), 7.61 (d, J = 15.6 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 6.97 (dd, J = 8.1, 7.6 Hz, 1H), 6.87 (dd, J = 9.2, 8.7 Hz, 1H), 6.75 (d, J =

EP

10.0 Hz, 1H), 6.38 (d, J = 8.8 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.40 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 191.7, 161.4, 160.5, 142.1, 132.5, 131.0, 128.6, 125.8, 121.7, 118.5, 118.1, 117.1,

AC C

116.7, 116.1, 114.3, 109.8, 108.9, 78.4, 28.8 (2C); MS (FAB) m/z: 343 (M + H)+. (E)-3-(3,4-dichlorophenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one (12):

S-5

ACCEPTED MANUSCRIPT Yield 62%; FT-IR (KBr, cm-1) 3444, 1611; 1H NMR (CDCl3, 200 MHz) δ 13.52 (s, 1H), 7.75 (d, J = 15.6Hz, 1H), 7.69(m, 2H), 7.52 (d, J = 15.6 Hz, 1H), 7.48 (m, 2H), 6.74 (d, J = 10.0 Hz, 1H), 6.39 (d, J = 8.8 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.46 (s, 6H); MS (FAB) m/z: 375 (M + H)+.

SC

RI PT

3-(2,4-Dichloro-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (13):

Yield 49%; FT-IR (KBr, cm-1) 3438, 1629; 1H NMR (CDCl3, 200 MHz) δ 13.61 (s, 1H), 8.16 (d,

M AN U

J = 15.4 Hz, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 15.4 Hz, 1H), 7.46 (m, 1H), 7.30 (m, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.37 (d, J = 8.9 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 191.7, 161.5, 160.6, 139.1, 136.9, 136.5, 132.2, 131.1, 131.6, 128.9, 128.6, 127.9, 123.7, 116.2, 114.3, 109.9, 108.9, 78.4, 28.8 (2C); MS (FAB) m/z: 375 (M + H)+.

EP

(14):

TE D

3-(3-Ethoxy-4-fluoro-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone

AC C

Yield 50%; FT-IR (KBr, cm-1) 3397, 1631; 1H NMR (CDCl3, 200 MHz) δ 13.69 (s, 1H), 7.74 (m, 2H), 7.36 (m, 3H), 6.69 (m, 1H), 6.75 (d, J = 10.1 Hz, 1H), 6.38 (d, J = 8.8 Hz, 1H), 5.59 (d, J = 10.1 Hz, 1H), 4.16 (q, J = 7.0 Hz, 2H), 1.50 (t, J = 7.0 Hz, 3H), 1.45 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 192.1, 161.3 (2C), 160.2, 149.7, 143.4 (2C), 131.0, 128.5 (2C), 126.5, 119.5, 116.3, 115.5, 115.1, 114.7 (2C), 108.7, 78.0, 65.3, 28.7 (2C), 15.0; MS (FAB) m/z: 369 (M + H)+. 4-[3-(5-Hydroxy-2, 2-dimethyl-2H-chromen-6-yl)-3-oxo-propenyl]-benzonitrile (15): S-6

ACCEPTED MANUSCRIPT

Yield 56%; FT-IR (KBr, cm-1) 3430, 1631; 1H NMR (CDCl3, 200 MHz) δ 13.62 (s, 1H), 7.82 (d,

RI PT

J = 15.4 Hz, 1H), 7.62 (m, 3H), 7.61 (d, J = 15.4 Hz, 1H), 7.27 (m, 2H), 6.74 (d, J = 10.0 Hz, 1H), 6.39 (d, J = 8.9 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 191.5, 161.5, 160.8, 141.9, 139.5, 133.1, 132.7, 131.1, 129.7, 129.1, 128.7, 124.1,

SC

118.7, 116.1, 114.3, 113.9, 109.9, 109.1, 78.5, 28.8 (2C); MS (FAB) m/z: 332 (M + H)+.

M AN U

1-(5-Hydroxy-2, 2-dimethyl-2H-chromen-6-yl)-3-(4-nitro-phenyl)-propenone (16):

Yield 57%; FT-IR (KBr, cm-1) 3425, 1625; 1H NMR (CDCl3, 200 MHz) δ 13.29 (s, 1H), 8.28 (d,

TE D

J = 8.7 Hz, 2H), 7.79 (d, J = 15.4 Hz, 1H), 7.78 (d, J = 8.7 Hz, 2H), 7.70 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 15.4 Hz, 1H), 6.74 (d, J = 10.0 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); MS (FAB) m/z: 352 (M + H)+.

AC C

(17):

EP

4-Hydroxy-3-[3-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-oxo-propenyl]-benzaldehyde

Yield 58%; FT-IR (KBr, cm-1) 3383, 1660, 1629; 1H NMR (300 MHz, CD3OD+CDCl3) δ 9.85 (s, 1H), 8.21 (d, J = 1.8 Hz, 1H), 8.16 (d, J = 15.6 Hz, 1H), 7.95 (d, J = 15.6 Hz, 1H), 7.87 (d, J = 8.9 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 6.74 (d, J = 10.1 Hz, 1H), 6.41 (d, J = 8.9 Hz, 1H), 5.66 (d, J = 10.1 Hz, 1H), 1.46 (s, 6H); MS (FAB) m/z: 351 (M + H)+. S-7

ACCEPTED MANUSCRIPT (E)-3-(benzofuran-5-yl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one (18).

RI PT

Yield 62%; mp: 116-118 °C; FT-IR (KBr, cm-1): 3446, 1633; 1H NMR (CDCl3, 300 MHz) δ 13.77 (s, 1H), 8.01 (d, J = 15.4Hz, 1H), 7.89 (d, J = 1.1 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 7.1 Hz, 1H), 7.63 (s, 1H), 7.58 (d, J = 15.4Hz, 1H), 7.55 (d, J = 8.5Hz, 1H), 6.83 (d, J =

(s, 6H);

13

SC

2.2 Hz, 1H), 6.78 (d, J = 10.1Hz, 1H), 6.42 (d, J = 8.8 Hz, 1H), 5.61 (d, J = 10.1 Hz, 1H), 1.49 C NMR (CDCl3, 50 MHz) δ 191.9, 161.0, 159.8, 156.3, 146.1, 144.8, 130.6, 129.9,

M AN U

128.1 (2C), 124.7, 122.3, 119.1, 115.9, 114.1, 112.0, 109.4, 108.3, 106.8, 77.8, 28.4 (2C); MS (FAB) m/z:347 (M + H)+.

(E)-3-(4-(2-(diethylamino)ethoxy)phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-

TE D

yl)prop-2-en-1-one (19):

EP

Yield 50%; FT-IR (KBr, cm-1) 3438, 1636; 1H NMR (CDCl3, 200 MHz) δ 13.41(s, 1H), 7.84 (d,

AC C

J = 15.5 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.6 Hz, 1H), 7.43 (d,= 15.5 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.37 (d, J = 8.8 Hz, 2H), 5.58 (d, J = 10.1 Hz, 1H), 4.10 (t, J = 6.2 Hz, 2H), 2.89 (t, J = 6.2 Hz, 2H), 2.67 (q, J = 7.1 Hz, 4H), 1.46 (s, 6H), 1.08 (q, J = 67.1Hz, 6H); MS (FAB) m/z: 422 (M + H)+. (E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-(2-(pyrrolidin-1yl)ethoxy)phenyl)prop-2-en-1-one(20):

S-8

ACCEPTED MANUSCRIPT

RI PT

Yield 35%; FT-IR (Neat, cm-1) 3438, 1636; 1H NMR (CDCl3, 200 MHz) δ 13.41(s, 1H), 7.84 (d, J = 15.5 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.6 Hz, 1H), 7.43 (d,= 15.5 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.75(d, J = 10.0 Hz, 1H), 6.37 (d, J = 8.8 Hz, 2H), 5.58 (d, J = 10.1 Hz, 1H),

= 67.1Hz, 6H);

13

SC

4.10 (t, J = 6.2 Hz, 2H), 2.89 (t, J = 6.2 Hz, 2H), 2.67 (q, J = 7.1 Hz, 4H), 1.46 (s, 6H), 1.08 (q, J C NMR (CDCl3, 50 MHz) δ 192.4, 161.8, 161.3, 160.0, 144.4, 130.9,

M AN U

130.7(2C), 128.4, 128.0, 118.3, 116.3, 115.4(2C), 114.5, 109.8, 108.6, 78.0, 67.3, 65.2, 55.0(2C), 28.4(2C), 23.8(2C); MS (FAB) m/z: 420 (M + H)+.

(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-(2-(piperidin-1-

EP

TE D

yl)ethoxy)phenyl)prop-2-en-1-one (21):

Yield 45%; FT-IR (Neat, cm-1) 3435, 1635; 1H NMR (CDCl3, 200 MHz) δ 13.80 (s, 1H), 7.86(d,

AC C

J = 15.5 Hz, 1H), 7.74(d, J = 8.9 Hz, 1H), 7.61 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 15.5 Hz, 1H), 6.95 (d, J = 8.3 Hz, 2H), 6.77 (d, J = 10.1Hz, 1H), 6.39 (d, J = 8.9 Hz, 1H), 5.61 (d, J = 10.1 Hz, 1H), 4.22 (t, J = 5.4 Hz, 2H), 2.28 (t, J = 5.4 Hz, 2H), 2.63 (brs, 4H), 2.13 (brs, 4H), 1.68 (t, J = 5.1Hz, 2H), 1.48 (s, 6H); MS (FAB) m/z: 434 (M + H)+. (E)-1-(5-(2-(diethylamino) ethoxy)-2, 2-dimethyl-2H-chromen-6-yl)-3-(4-(2-(diethylamino) ethoxy) phenyl)prop-2-en-1-one (22)

S-9

RI PT

ACCEPTED MANUSCRIPT

Yield: 47%; FT-IR (neat, cm−1) 1665; 1H NMR (CDCl3, 200 MHz) δ 7.71 (d, J = 14.9 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 14.9 Hz, 1H), 6.90 (d, J = 8.6 Hz,

SC

2H), 6.77 (d, J = 10.1 Hz, 1H), 6.63 (d, J = 8.8 Hz, 1H), 5.64 (d, J = 10.1 Hz, 1H), 5.10 (t, J = 6.0 Hz, 2H), 4.07 (t, J = 6.0 Hz, 2H), 3.93 (t, J = 6.0 Hz, 2H), 2.83 (t, J = 5.8 Hz, 2H), 2.55-

M AN U

2.61(m, 4H), 2.08(q, J = 8.1 Hz, 2H), 1.77 (t, J = 4.3 Hz, 2H), 1.71(t, J = 8.1 Hz, 3H), 1.67 (s, 3H), 1.46(s, 6H), 0.98(t, J = 9.0 Hz, 6H) ; MS (FAB) m/z: 521 (M + H)+. (E)-1-(2,2-dimethyl-5-(2-(pyrrolidin-1-yl)ethoxy)-2H-chromen-6-yl)-3-(4-(2-(pyrrolidin-1-

EP

TE D

yl)ethoxy)phenyl)prop-2-en-1-one(23):

AC C

Yield: 39%; FT-IR (neat, cm−1) 1652; 1H NMR (CDCl3, 200 MHz) δ 7.66 (d, J = 15.7 Hz, 1H), 7.56 (d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 15.7 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 6.73 (d, J = 10.0 Hz, 1H), 6.63 (d, J = 8.5 Hz, 1H), 5.68 (d, J = 10.0 Hz, 1H), 4.17 (t, J = 5.7 Hz, 2H), 3.96 (t, J = 5.8 Hz, 2H), 2.95 (t, J = 5.6 Hz, 2H), 2.83 (t, J = 5.7 Hz, 2H), 2.69 (m, 4H),2.56(m, 4H), 1.83(m, 4H), 1.73(m, 4H), 1.45(s, 6H); MS (FAB) m/z: 517 (M + H)+.

S-10

ACCEPTED MANUSCRIPT 1-[2,2-Dimethyl-5-(2-piperidin-1-yl-ethoxy)-2H-chromen-6-yl]-3-[4-(2-piperidin-1-ylethoxy)-phenyl]-propenone (24): O

O N

O

RI PT

O

N

Yield: 47%; FT-IR (neat, cm−1) 1652; 1H NMR (CDCl3, 200 MHz) δ 7.67 (d, J = 14.7 Hz, 1H),

SC

7.55 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 14.7 Hz, 1H), 6.91 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 10.0 Hz, 1H), 6.63 (d, J = 8.7 Hz, 1H), 5.67 (d, J = 10.0 Hz, 1H), 4.13 (t, J =

M AN U

5.9 Hz, 2H), 3.93 (t, J = 5.7 Hz, 2H), 2.78 (t, J = 5.9 Hz, 2H), 2.62 (t, J = 5.7 Hz, 2H), 2.53–2.34 (m, 8H), 1.61–1.25 (m, 18H); 13C NMR (CDCl3, 50 MHz) δ 191.3, 161.1, 157.6, 155.6, 143.2, 131.5, 130.7, 130.5, 128.2 (2C), 126.6, 124.5, 117.3, 115.4, 115.3 (2C), 113.0, 77.1, 73.9, 66.4, 58.9, 58.1, 55.4 (2C), 55.2 (2C), 28.4 (2C), 26.2 (4C), 24.6, 24.5; MS (FAB) m/z: 545 (M + H)+.

EP

TE D

1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-pyridin-2-yl-propenone (25):

AC C

Yield 49%; FT-IR (neat, cm−1) 1642; 1H NMR (CDCl3, 200 MHz) δ 13.60 (s, 1H), 8.69 (d, J = 4.3 Hz, 1H), 8.12 (d, J = 15.1 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 15.1 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.31(m, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.38 (d, J = 8.9 Hz, 1H), 5.59 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 192.5, 161.4, 160.6, 153.5, 150.6, 142.5, 137.3, 131.8, 128.5, 126.0, 124.8 (2C), 116.2, 114.6, 109.7, 108.9, 78.3, 28.8 (2C); MS (FAB) m/z: 308 (M + H)+. 1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-pyridin-3-yl-propenone (26): S-11

ACCEPTED MANUSCRIPT

Yield 55%; FT-IR (neat, cm−1) 3442, 1641; 1H NMR (CDCl3, 200 MHz) δ 13.62 (s, 1H), 8.91

RI PT

(br s, 1H), 8.73 (br s, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 15.5 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.62 (d, J = 15.5 Hz, 1H), 7.32 (m, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.40 (d, J = 8.9 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 191.6, 161.5, 160.6,

SC

151.5, 150.4, 140.7, 135.0, 131.1 (2C), 128.7, 124.2, 122.7, 116.1, 114.2, 109.9, 109.0, 78.1,

M AN U

28.8 (2C); MS (FAB) m/z: 308 (M + H)+.

1-(5-Hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-quinolin-3-yl-propenone (27):

TE D

Yield 51%; FT-IR (KBr, cm-1) 3435, 1646; 1H NMR (CDCl3, 200 MHz) δ 13.59 (s, 1H), 9.20 (d, J = 2.1 Hz, 1H), 8.35 (d, J = 2.1 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 8.01 (d, J = 15.7 Hz, 1H),

EP

7.80 (d, J = 8.8 Hz, 1H), 7.78 (m, 2H), 7.73 (d, J = 15.7 Hz, 1H), 7.64 (m, 1H), 6.76 (d, J = 10.0 Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 5.61 (d, J = 10.0 Hz, 1H), 1.48 (s, 6H); 13C NMR (CDCl3, 50

AC C

MHz) δ 191.6, 161.5, 160.6, 149.8, 149.0, 141.0, 136.4, 131.2 (2C), 129.8, 128.8, 128.6, 128.2, 128.1, 127.9, 122.4, 116.2, 114.3, 109.8, 109.0, 78.1, 28.8 (2C); MS (FAB) m/z: 358 (M + H)+. 3-(2-Chloro-quinolin-3-yl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (28):

O N OH

O

S-12

Cl

ACCEPTED MANUSCRIPT Yield 57%; FT-IR (neat, cm−1) 3392, 1639; 1H NMR (CDCl3, 200 MHz) δ 13.49 (s, 1H), 8.48 (s, 1H), 8.27 (d, J = 15.5 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 15.5 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 6.9 Hz, 1H), 7.63 (d, J = 6.9 Hz, 1H), 6.75 (d,

RI PT

J = 10.0 Hz, 1H), 6.41 (d, J = 8.8 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.48 (s, 6H); MS (FAB) m/z: 392 (M+H)+.

3-(2-Chloro-6-methyl-quinolin-3-yl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-

SC

propenone (29):

O

M AN U

CH3

N

OH

O

Cl

Yield 41%; FT-IR (neat, cm−1) 3442, 1629; 1H NMR (CDCl3, 200 MHz) δ 13.62 (s, 1H), 8.37 (s, 1H), 8.24 (d, J = 15.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.64 (d, J =

TE D

15.4 Hz, 1H), 7.62 (m, 1H), 7.60 (d, J = 8.9 Hz, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 2.55 (s, 3H), 1.48 (s, 6H); 13C NMR (CDCl3, 50 MHz) δ 191.4, 161.5, 160.7, 149.9, 146.9, 139.3, 138.3, 136.0, 134.4, 131.2, 128.6, 128.5, 128.2, 127.4,

EP

127.2, 124.8, 116.1, 114.3, 109.9, 109.0, 78.5, 28.8 (2C), 22.0; MS (FAB) m/z: 406 (M + H)+.

AC C

(E)-3-(4-(dimethylamino) phenyl)-1-(4-(3-methylbut-2-enyloxy)phenyl)prop-2-en-1-one(35)

N O

O

Yield: 59%; IR (KBr) 1637 cm-1; 1H NMR (CDCl3, 300 MHz) δ 8.02 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 15.4 Hz, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 15.4 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), S-13

ACCEPTED MANUSCRIPT 6.69 (d, J = 8.8 Hz, 2H), 5.51 (t, J = 6.1 Hz, 1H), 4.58 (d, J = 6.1 Hz, 2H), 3.03 (s, 6H), 1.81 (s, 3H), 1.76 (s, 3H); 13C NMR (CDCl3, 50 MHz) δ 189.3, 162.7, 152.3, 145.3, 139.2, 132.1, 130.9 (2C), 130.7 (2C), 123.3, 119.5, 117.1, 114.7 (2C), 112.2 (2C), 65.4, 40.5 (2C), 26.2, 18.7; MS

RI PT

(FAB) m/z 336 (M+H)+. (E)-1-(4-((E)-3,7-dimethylocta-2,6-dienyloxy)phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (36) OH

O

SC

O

M AN U

Yield: 35%; mp 72-74 oC; FT-IR (neat, cm−1) 3437, 1642; 1H NMR (CDCl3, 200 MHz) δ 8.02 (d, J = 15.5 Hz, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 15.5 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 5.49(m, 1H), 5.09(m, 1H), 4.62 (d, J = 6.7 Hz, 2H), 1.73 (t, J = 9.1 Hz, 2H), 1.69 (q, J = 7.1 Hz, 2H), 1.67 (s, 6H), 1.59 (s, 3H); MS (FAB) m/z

TE D

377 (M+H)+.

1-[4-(3,7-Dimethyl-octa-2,6-dienyloxy)-phenyl]-3-(4-methoxy-phenyl)-propenone (37): OMe

EP

O

AC C

O

Yield: 70%; mp 68‒69 oC; IR (KBr, cm-1) 1627, .1H NMR (CDCl3, 200 MHz) δ 8.02 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 15.5 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 15.5 Hz, 1H), 6.98 (d, J = 9.1 Hz, 2H), 6.93 (d, J = 9.1 Hz, 2H), 5.09 (t, J = 6.2 Hz, 1H), 5.47 (t, J = 6.7 Hz, 1H), 4.62 (t, J = 6.2 Hz, 2H), 3.85 (s, 3H), 2.16‒2.06 (m, 4H), 1.75 (s, 3H), 1.68 (s, 3H), 1.60 (s, 3H); 13C NMR (CDCl3, 50 MHz) δ 189.1, 163.0, 161.8, 144.1, 142.2, 131.5, 131.0 (2C), 130.5 (2C), 128.2, 124.3, 124.1, 119.9, 119.2, 114.8 (2C), 114.7 (2C), 65.5, 55.7, 39.9, 26.6, 26.0, 18.1, 17.1; MS (FAB) m/z 391 (M+H)+.

S-14

ACCEPTED MANUSCRIPT (E)-3-(4-(dimethylamino)phenyl)-1-(4-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-

RI PT

trienyloxy)phenyl)prop-2-en-1-one (38):

Yield: 65%; mp 52‒54 oC; IR (KBr) 1630 cm-1;1H NMR (CDCl3, 200 MHz) δ 8.01 (d, J = 8.7

SC

Hz, 2H), 7.78 (d, J = 15.4 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 15.4 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 5.47 (t, J = 5.9 Hz, 1H), 5.08 (t, J = 5.2 Hz, 2H), 4.61 (d,

M AN U

J = 6.5 Hz, 2H), 3.04 (s, 6H), 2.12‒2.04 (m, 8H), 1.76 (s, 3H), 1.67 (s, 3H), 1.59 (s, 6H); MS (FAB) m/z 472 (M+H)+.

1-[4-(3,7-Dimethyl-octa-2,6-dienyloxy)-phenyl]-3-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-

TE D

propenone (39): O

O

N

O

EP

Yield: 66%; semisolid; IR (neat) 1627 cm-1; 1H NMR (CDCl3, 200 MHz) δ 8.02 (d, J = 8.8 Hz,

AC C

2H), 7.77 (d, J = 15.5 Hz, 1H), 7.59 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 15.5 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 5.49 (t, J = 6.4 Hz, 1H), 5.12 (t, J = 6.2 Hz, 1H), 4.62 (t, J = 6.4 Hz, 2H), 4.20 (t, J = 5.8 Hz, 2H), 2.99 (t, J = 5.8 Hz, 2H), 2.59 (m, 4H), 2.17 (m, 2H), 1.86 (m, 2H), 1.76 (s, 3H), 1.67 (s, 3H), 1.60 (s, 3H), 1.25 (m, 4H); MS (FAB) m/z 474 (M+H)+; (E)-3-(4-ethoxyphenyl)-1-(4-(2-(piperidin-1-yl)ethoxy)phenyl)prop-2-en-1-one (40):

S-15

ACCEPTED MANUSCRIPT

RI PT

Yield: 66%; mp 78‒80 oC; IR (KBr) 1650 cm-1;1H NMR (CDCl3, 200 MHz) δ 8.02 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 15.5 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 15.5 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 4.20 (q, J = 6.0 Hz, 2H), 4.10 (t, J = 7.0 Hz, 2H), 3.58

1.40‒1.47 (t, J = 6.9 Hz, 3H); MS (FAB) m/z 380 (M+H)+.

SC

(q, J = 7.0 Hz, 2H), 2.82 (t, J = 6.0 Hz, 4H), 2.68 (t, J = 7.0 Hz, 4H), 1.59‒1.60 (m, 2H),

M AN U

(E)-3-(4-(dimethylamino)phenyl)-1-(4-(2-(piperidin-1-yl)ethoxy)phenyl)prop-2-en-1-one (41):

NMe2

O

N

TE D

O

Yield: 66%; mp 102‒104 oC; IR (KBr) 1650 cm-1; 1H NMR (CDCl3, 200 MHz) δ 8.01 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 15.4 Hz, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 15.4 Hz, 1H), 6.97 (d, J

EP

= 8.7 Hz, 2H), 6.69 (d, J = 8.7 Hz, 2H), 4.18 (t, J = 6.0 Hz, 2H), 3.03 (s, 6H), 2.80 (t, J = 5.9 Hz,

AC C

2H), 2.50‒2.52 (m, 4H), 1.59‒1.61 (m, 6H); MS (FAB) m/z 379 (M+H)+. 1,3-Bis-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-propenone (42): O N

N

O

O

Yield: 56%; mp 98‒100 oC; IR (KBr) 1602, 1668 cm-1; 1H NMR (CDCl3, 200 MHz) δ 8.01 (d, J = 8.8 Hz, 2H), 7.77 (d, J = 15.5 Hz, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 15.5 Hz, 1H), 6.98 S-16

ACCEPTED MANUSCRIPT (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 4.19 (t, J = 6.2 Hz, 2H), 4.12 (t, J = 6.1 Hz, 2H), 2.82 (t, J = 2.2 Hz, 2H), 2.78 (t, J = 2.2 Hz, 2H), 2.52 (t, J = 4.4 Hz, 8H), 1.59‒1.68 (m, 8H), 1.45‒1.47 (m, 4H); 13C NMR (CDCl3, 50 MHz) δ 189.1, 162.9, 161.1, 144.1, 131.7, 131.0 (2C),

RI PT

130.4 (2C), 128.2, 119.9, 115.3 (2C), 114.7 (2C), 66.5(2C), 58.2 (2C), 55.5 (4C), 26.3 (4C),

AC C

EP

TE D

M AN U

SC

24.5(2C); MS (FAB) m/z 463 (M+H)+.

S-17

ACCEPTED MANUSCRIPT

O

RI PT

NMR spectra of the selected chalcones

OEt

O

H-NMR spectrum of 4 (200 MHz, CDCl3)

AC C

EP

TE D

1

M AN U

SC

OH

O

OEt

OH

O

S-18

ACCEPTED MANUSCRIPT

C-NMR spectrum of 4 (50 MHz, CDCl3)

O

RI PT

13

OMe OMe O

H-NMR spectrum of 7 (200 MHz, CDCl3)

EP

TE D

1

M AN U

SC

OH

AC C

O

OMe OMe OH

O

S-19

ACCEPTED MANUSCRIPT

C-NMR spectrum of 7 (50 MHz, CDCl3)

O

Cl

O

H-NMR spectrum of 13 (200 MHz, CDCl3)

EP

TE D

1

M AN U

SC

OH

Cl

RI PT

13

O

Cl

O

AC C

OH

Cl

13

C-NMR spectrum of 13 (50 MHz, CDCl3) S-20

ACCEPTED MANUSCRIPT

O

O

H-NMR spectrum of 18 (300 MHz, CDCl3)

EP

TE D

1

M AN U

SC

OH

RI PT

O

AC C

O

13

OH

O

O

C-NMR spectrum of 18 (50 MHz, CDCl3) S-21

RI PT

ACCEPTED MANUSCRIPT

O N O

O

H-NMR spectrum of 21 (300 MHz, CDCl3)

TE D

1

M AN U

SC

OH

EP

O N

O

O

AC C

OH

13

C-NMR spectrum of 21 (50 MHz, CDCl3)

S-22

ACCEPTED MANUSCRIPT

O

RI PT

O

N

O

O

H-NMR spectrum of 24 (200 MHz, CDCl3)

TE D

1

M AN U

SC

N

O

EP

O

O

N

O

AC C

N

13

C-NMR spectrum of 24 (50 MHz, CDCl3) S-23

O N O

H-NMR spectrum of 25 (200 MHz, CDCl3)

EP

TE D

1

M AN U

SC

OH

RI PT

ACCEPTED MANUSCRIPT

AC C

O

13

OH

N

O

C-NMR spectrum of 25 (50 MHz, CDCl3) S-24

RI PT

ACCEPTED MANUSCRIPT

NMe 2 O

H-NMR spectrum of 35 (300 MHz, CDCl3)

TE D

1

M AN U

SC

O

NMe 2

O

AC C

EP

O

13

C-NMR spectrum of 35 (50 MHz, CDCl3)

S-25

ACCEPTED MANUSCRIPT

RI PT

OMe O

H-NMR spectrum of 37 (200 MHz, CDCl3)

EP

TE D

1

M AN U

SC

O

OMe

AC C

O

13

O

C-NMR spectrum of 37 (50 MHz, CDCl3) S-26