Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

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Topoisomerase I and II inhibitory activity, cytotoxicity, and structure–activity relationship study of dihydroxylated 2,6-diphenyl-4-aryl pyridines Radha Karki a, , Chanju Song b, , Tara Man Kadayat a, Til Bahadur Thapa Magar a, Ganesh Bist a, Aarajana Shrestha a, Younghwa Na c, Youngjoo Kwon b,⇑, Eung-Seok Lee a,⇑ a

College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top 5 Program, Ewha Womans University, Seoul 120-750, Republic of Korea c College of Pharmacy, Cha University, Pochon 487-010, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 23 February 2015 Revised 2 April 2015 Accepted 3 April 2015 Available online xxxx Keywords: Dihydroxylated 2,6-diphenyl-4-aryl pyridines Topoisomerase I Topoisomerase II Cytotoxicity Anticancer agents

a b s t r a c t A new series of thirty-six dihydroxylated 2,6-diphenyl-4-aryl pyridines containing hydroxyl groups at the ortho, meta, or para position of 2- and 6-phenyl rings attached to the central pyridine were designed and synthesized. They were evaluated for topoisomerase I and II inhibitory activity and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Most of the compounds with hydroxyl moiety either at the meta or para position of 2- or 6-phenyl ring in combination with thienyl or furyl group at 4-position of central pyridine displayed significant topoisomerase II inhibitory activity and cytotoxicity. Positive correlation between topoisomerase II inhibitory activity and cytotoxicity was observed for the compounds 9–11, 15–17, 19, 21–23, 28, and 41. Among all the synthesized compounds, compound 17 emerged as the most promising topoisomerase II inhibitor with significant cytotoxicity. Ó 2015 Published by Elsevier Ltd.

1. Introduction Several of the most effective anticancer drugs such as etoposide, camptothecin, and doxorubicin have been reported to interrupt the normal catalytic cycle of DNA topoisomerase.1–3 This enzyme solves various DNA topological problems associated with DNA replication, transcription, recombination, and other vital cellular processes.4 In general, DNA topoisomerases (topos) are classified into two types: topoisomerase I (topo I), which breaks single strands of DNA, and topoisomerase II (topo II), which breaks double strands of DNA.5 Unlike topo I, topo II acts as a homodimeric enzyme and requires Mg (II) and ATP hydrolysis for enzyme turnover and rapid kinetics.1,6 Inhibition of topos during their vital processes leads to apoptosis and sequential cell death. This has established topos as a prime molecular target for the development of anticancer agents.7 A series of drugs that specifically target topos have been widely used clinically as anticancer agents. However, due to their severe side effects, extensive research is being conducted to develop new derivatives and ⇑ Corresponding authors. Tel.: +82 2 3277 4653; fax: +82 2 3277 2851 (Y.K.); tel.: +82 53 810 2827; fax: +82 53 810 4654 (E.-S.L.). E-mail addresses: [email protected] (Y. Kwon), [email protected] (E.-S. Lee).   Authors have equally contributed to this work.

novel compounds with improved therapeutic efficacy and less toxicity.3,8,9 In an attempt to discover novel topo inhibitors, our research group has previously designed and synthesized various 2,4,6-triaryl pyridines as terpyridine bioisosteres and evaluated them for topo I and/or II inhibitory activity and cytotoxicity against several human cancer cell lines.10–12 Several naturally available polyphenolic compounds such as curcumin, epigallocatechin gallate, resveratrol, and flavonoids bearing the hydroxyl or phenol group are reported to exhibit a wide range of pharmacological activities including antitumor, anti-inflammatory, antioxidant, antibacterial and antiangiogenic activities.13–17 In the course of our research on hydroxylated 2,4,6-triphenyl pyridines, we have obtained several compounds with the hydroxyl group at the ortho, meta, or para position of 2- or/and, 4- or/and, 6-phenyl ring attached to the central pyridine (Fig. 1) with moderate to significant topo I and II inhibitory activity and cytotoxicity against several human cancer cell lines.18–21 Results indicated that in general, dihydroxylated 2,4,6-triphenyl pyridines exhibited stronger topo II inhibitory activity and cytotoxicity compared to those of monohydroxylated 2,4,6-triphenyl pyridines.19 The structure–activity relationship studies demonstrated that hydroxylation at meta or para position of 2-phenyl ring of central pyridine in dihydroxylated 2,4,6-triphenyl pyridines is

http://dx.doi.org/10.1016/j.bmc.2015.04.002 0968-0896/Ó 2015 Published by Elsevier Ltd.

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OH R2

R1 1

2

N

R3

N

N

N

HO

3

R , R , R = aryl 2,4,6-triaryl pyridines

2,4,6-triphenyl pyridine

monohydroxylated 2,4,6-triphenyl pyridines

OH

OH

N

HO

N

HO

dihydroxylated 2,4,6-triphenyl pyridines

OH

N

HO

OH

trihydroxylated 2,4,6-triphenyl pyridine OH

R

1

4 2 R1

N

2

R , R = OH R3 = 3 -thienyl 2 -furyl 2/ 3/ 4 -pyridyl

2

6 R3

2,4-diphenyl-6-aryl pyridine

N

O

HO 2,4-Di-p-Phenolyl-6-2-Furanyl-Pyridine

Figure 1. Structures of previously synthesized 2,4,6-trisubstituted pyridines.

important for significant topo II inhibitory activity as well as cytotoxicity.18,19 Trihydroxylated 2,4,6-triphenyl pyridines were reported as topo II inhibitors with better selectivity than monoand dihydroxylated 2,4,6-triphenyl pyridines only in high micromolar concentration.20 Our research group has recently reported the synthesis of dihydroxylated 2,4-diphenyl-6-aryl pyridines possessing various aryl groups on 6-phenyl ring of central pyridines (Fig. 1). In this series, compounds containing a hydroxyl group at para position of 2- and 4-phenyl ring and 2-furyl group at 6-position of central pyridine displayed the most potent topo inhibitory activity in a low micromolar concentration and functioned as a potent topo II poison.21 Moreover, we have found in previous studies that introduction of an aryl group such as thienyl or furyl group at 4-position of central pyridine is important for significant topo II inhibitory activity and cytotoxicity.22–25 As a continuation of developing novel potent anticancer agents, these encouraging results have prompted us to synthesize and evaluate the biological activities of dihydroxylated 2,6-diphenyl-4-aryl pyridines possessing 2/ 3-thienyl, 2/3-furyl, and 2/3-pyridyl moieties on 4-position of central pyridine (Fig. 2). In addition, it would be very interesting to obtain the information about difference of biological activities and structure–activity relationships according to small change of position of dihydroxylated 2,6-diphenyl moiety on central pyridine ring such as of dihydroxylated 2,4-diphenyl-6-aryl pyridines to dihydroxylated 2,6-diphenyl-4-aryl pyridines. In this paper we describe the design, synthesis, and biological studies of thirty-six novel dihydroxylated 2,6-diphenyl-4-aryl pyridines. These compounds were evaluated for topo I and II inhibitory activity and cytotoxicity against several human cancer cell lines. Our work represents the first systematic study of dihydroxylated 2,6-diphenyl-4-aryl pyridines to develop potential anticancer agents. 2. Chemistry Previously our research group reported the synthesis of dihydroxylated 2,4,6-triphenyl pyridine compounds which possessed

significant topo II inhibitory activity and cytotoxicity.19 As an extension to this work, we further synthesized compounds having various aryl moieties at the 4-position of central pyridine, exchanging the phenyl ring as shown in Fig. 2. These compounds were synthesized by the method we previously reported, which is illustrated in Scheme 1. First, using Claisen–Schmidt condensation reaction, eighteen hydroxylated propenone intermediates were synthesized via base (NaOH) catalyzed reaction26,27 without the protection of hydroxyl groups. In this method, 4 M aqueous solution of NaOH was added to the solution of equimolar amounts of aryl ketone 1 (R1 = a–c) and aryl aldehyde 2 (R2 = d–i) in ethanol to obtain compounds 3 (R1 = a–c, R2 = d–i) with the yield of 47–98%. In the second step, three pyridinium iodide salts 5 (R3 = a–c) were synthesized in quantitative yield by the treatment of aryl ketone 4 (R1 = a–c) with iodine (1.2 equiv) in pyridine. Using modified Kröhnke synthesis,28,29 the final compounds 6–41 were synthesized by the reaction of appropriate hydroxylated chalcones 3 with pyridinium iodide salt 5 in the presence of ammonium acetate in glacial acetic acid or methanol in 30–83% yield. Total thirty-six dihydroxylated 2,6-diphenyl-4-aryl pyridine compounds (6–41) were systematically designed and synthesized from the reaction of eighteen intermediates and three pyridinium iodide salts as shown in Fig. 3. All the compounds contained two hydroxyl moieties substituted at various positions (ortho, meta, or para) of phenyl ring at 2- and 6-position of central pyridine. Compounds were designed and synthesized in six different series, with various aryl moiety at 4-position of central pyridine as shown in Fig. 2. Synthesis of dihydroxylated 2,6diphenyl-4-aryl pyridine compounds allowed us to investigate whether the alteration in the aryl moiety at 4-position of central pyridine has any effect on topo I and II inhibitory activity and cytotoxicity. Subsequently, ortho, meta, and para substitution of hydroxyl moiety would lead us to further confirm the effect of hydroxyl position on biological activity with respect to the previous data. Yield (%) and HPLC purity (%) of the compounds are shown in Table 1.

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S S 2 1

R

O

4

R2

N

R

HO

3

2,4,6-trisubstituted pyridine

O

6

N

N

N

OH

dihydroxylated 2,4,6-triphenyl pyridine significant topo II inhibition

S S

O

N

HO

OH

N

HO

OH

HO

OH

Series C

Series B

Series A

N

O

N N

N

HO

OH

N

HO

OH

Series E

Series D

HO

N

OH

Series F

Figure 2. Strategy for the design of dihydroxylated 2,6-diphenyl-4-aryl pyridines.

O 2

R R1

CH3

H

All these synthesized compounds were evaluated for topo I and II inhibitory activity and cytotoxicity against four different human cancer cell lines as illustrated in Figures 4 and 5 and Table 2.

R2

2 (R 2 = d-i) R1

O

i

1 (R 1 = a-c)

O

3.1. Topoisomerase I inhibitory activity

3 (R 1 = a-c, R 2 = d-i)

R2

iii

R

1

N

R

3

6-41 R3

R3

CH3 O

O 5 (R 3 = a-c)

ii

4 (R 1 = a-c)

I N

OH OH

HO

R 1, R 3 = a

R2 =

b

c

S

O

S d

N

O e

f

g

h

N i

Scheme 1. General synthetic method of dihydroxylated 2,6-diphenyl-4-aryl pyridines. Reagents and conditions: (i) aryl aldehydes 2 (R2 = d–i) (1.0 equiv), NaOH, EtOH, 2–8 h, 20 °C, 47–98% yield; (ii) pyridine, iodine, 3 h, 140 °C; (iii) NH4OAc (10.0 equiv), glacial acetic acid/methanol, 12–16 h, 80–100 °C, 30–83% yield.

Compounds 6–11, which possess 2-thienyl moiety, exhibited strong topo I inhibitory activity at 100 lM (Fig. 4). Compounds 7, 9–11 showed stronger effect than the positive control, camptothecin. Compound 9 showed the highest inhibition of 91.7%. Among compounds 12–17 with 3-thienyl moiety at 4-position of central pyridine, only compounds 15 and 17 possessed moderate topo I inhibition at 100 lM (59.2% and 61.7%, respectively). All the compounds (18–23), which contain 2-furyl moiety at 4-position of central pyridine, showed stronger topo I inhibition than the camptothecin (80.7%) at 100 lM: compounds 19 and 21 exhibited 90.1% and 97.8% inhibition respectively, while 22 and 23 possessed 100% inhibition at 100 lM. Only one, compound 29 with 3-furyl moiety at 4-position of central pyridine showed considerable inhibitory activity (74.9%) at 100 lM. Compounds 25–28 from the same group possessed weaker inhibition compared to the positive control. Compound 31 containing 2-pyridyl moiety showed weak inhibition (35.4%), and compound 36 with 3-pyridyl moiety exhibited moderate inhibitory activity (66.4%) at 100 lM. All other compounds with 2- and 3-pyridyl moieties had no significant inhibitory activity at both 100 and 20 lM. Taken together, several evaluated compounds possessed significant topo I inhibitory activity at 100 lM but none of the compounds were active at the concentration of 20 lM.

3. Results and discussion

3.2. Topoisomerase II inhibitory activity

All the synthesized compounds (6–41) contained two hydroxyl moieties, one in each phenyl ring at 2- and 6-position with 2/3thienyl, 2/3-furyl, or 2/3-pyridyl at 4-position of central pyridine.

All compounds (6–11) with 2-thienyl moiety possessed moderate to significant topo II inhibitory activity at 100 lM (Fig. 5). Compound 10 showed 88.6% inhibition which was higher than

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S

S

OH

OH

S

OH N

6

7

OH

OH

8

12

13

OH

OH

20

O

O

O

OH N

24

25

N

S

OH

N

HO

OH

S

N

15

OH

HO

N

OH

N

16

OH

17

HO

O

OH

N

HO

21

OH

OH

O

N

N

22

O

HO

N 26

N

OH

HO

O

OH

N

HO

27

OH

N

OH

23

OH

O

N

N

28

N

OH

HO

29

N

OH

N

OH

N

N

30

31

N

OH

HO

N 32

N

OH

11

HO

OH

N

OH

N OH

OH OH

19

OH

N 10

O

18

OH

HO

O

N

OH

HO

S

14

N

OH

OH

N

O OH

S

OH N

OH

OH

N 9

S

OH

N

O

HO

N

S

OH

S

OH

N

S

S

OH

N

OH

OH

N

HO

33

N 34

N

N OH

HO

35

N

OH

N

OH

N

N

36

37

OH

HO

N 38

OH

N

OH

HO

39

N 40

N OH

HO

41

OH

Figure 3. Structures of synthesized dihydroxylated 2,6-diphenyl-4-aryl pyridines.

etoposide (79.3%), and compounds 7 and 9 exhibited 78% inhibition at 100 lM. Compound 8 showed 76.4% and 30.2% inhibition at 100 and 20 lM, respectively. Compound 6 showed moderate inhibition (55.9%) at 100 lM while compound 11 possessed 74.6% and 30.3% inhibition at 100 and 20 lM, respectively. All the compounds (12–17), which contain 3-thienyl moiety, showed stronger inhibition than etoposide at 100 lM. Compounds 15–17 showed 88.1%, 91.5%, and 81.2% inhibition at 100 lM and 23.0%, 44.7%, and 57.9% inhibition at 20 lM, respectively. All the compounds (19–23), which possess 2-furyl moiety, showed stronger inhibitory activity than etoposide, except compound 18. Compound 18 showed moderate inhibition (52.2%) at 100 lM while compound 23 exhibited 92.3% and 48.4% inhibition at 100 and 20 lM, respectively. Similarly, compounds 19, 21, and 22 showed significant inhibition at 20 lM. Among compounds containing 3-furyl moiety (24–29), compounds 25 and 26 showed moderate activity. Compound 28 possessed significant inhibitory activity of 95% and 19.7% at 100 and 20 lM, respectively. Compounds 30, 31, and 33, possessing 2-pyridyl moiety, showed

moderate inhibitory activity (65.5%, 62.5%, and 52.2%, respectively) at 100 lM. Similarly, compounds 37 and 41, containing 3-pyridyl moiety, showed 55.2% and 65.8% inhibition at 100 lM. Compound 41 also showed stronger inhibition (25.7%) at 20 lM which is higher than etoposide (22.6%). To sum up, most of the compounds exhibited moderate to significant topo II inhibitory activity at 100 lM, with several compounds active at 20 lM as well. 3.3. Cytotoxicity The cytotoxicity of compounds were evaluated with four different human cancer cell lines: the human prostate tumor cell line (DU145), human colorectal adenocarcinoma cell line (HCT15), human ductal breast epithelial tumor cell line (T47D), and human cervix tumor cell line (HeLa). Adriamycin, etoposide, and camptothecin, widely used anticancer drugs, were used as positive controls. Several compounds displayed significant cytotoxicity against the tested cell lines at low micromolar concentration compared to

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R. Karki et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx Table 1 Prepared compounds with yield, and purity by HPLC R2

R1

Entry

R1

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

2-OH 2-OH 2-OH 3-OH 3-OH 4-OH 2-OH 2-OH 2-OH 3-OH 3-OH 4-OH 2-OH 2-OH 2-OH 3-OH 3-OH 4-OH 2-OH 2-OH 2-OH 3-OH 3-OH 4-OH 2-OH 2-OH 2-OH 3-OH 3-OH 4-OH 2-OH 2-OH 2-OH 3-OH 3-OH 4-OH

phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl

N

R3

R2

R3

2-Thienyl 2-Thienyl 2-Thienyl 2-Thienyl 2-Thienyl 2-Thienyl 3-Thienyl 3-Thienyl 3-Thienyl 3-Thienyl 3-Thienyl 3-Thienyl 2-Furyl 2-Furyl 2-Furyl 2-Furyl 2-Furyl 2-Furyl 3-Furyl 3-Furyl 3-Furyl 3-Furyl 3-Furyl 3-Furyl 2-Pyridyl 2-Pyridyl 2-Pyridyl 2-Pyridyl 2-Pyridyl 2-Pyridyl 3-Pyridyl 3-Pyridyl 3-Pyridyl 3-Pyridyl 3-Pyridyl 3-Pyridyl

20 -OH 30 -OH 40 -OH 30 -OH 40 -OH 40 -OH 20 -OH 30 -OH 40 -OH 30 -OH 40 -OH 40 -OH 20 -OH 30 -OH 40 -OH 30 -OH 40 -OH 40 -OH 20 -OH 30 -OH 40 -OH 30 -OH 40 -OH 40 -OH 20 -OH 30 -OH 40 -OH 30 -OH 40 -OH 40 -OH 20 -OH 30 -OH 40 -OH 30 -OH 40 -OH 40 -OH

phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl

Yield (%)

Purity (%)

60.8 63.7 62.0 77.1 57.4 52.6 83.7 56.2 80.8 77.3 76.5 77.6 65.6 56.0 55.0 57.3 56.6 70.8 75.8 55.0 60.1 60.3 60.0 59.1 30.0 33.7 58.0 59.3 62.2 56.0 41.2 61.2 51.0 40.8 53.0 45.1

98.9 98.7 98.5 96.1 98.9 97.8 99.7 99.1 99.4 98.2 99.3 97.0 98.8 97.5 97.4 99.3 99.3 98.6 98.4 99.2 95.1 98.8 98.8 96.3 97.6 97.0 99.2 95.7 95.9 95.0 99.5 99.4 97.5 97.0 95.5 97.9

the positive controls (Table 2). Among compounds 6–17, which possessed 2- or 3-thienyl moiety at 4-position of central pyridine, compounds 6–8 and 12–14 showed relatively lower cytotoxicity (IC50 >50 lM) in all four cancer cell lines (Table 2). Compounds 9, 10, and 16 possessed considerable cytotoxicity against DU145, HCT15, and HeLa, but were much stronger (50 lM) in most of the cell lines. In contrast, with the exception of compound 28 and 41, all the compounds (24–41) having 3-furyl or pyridyl moiety at 4-position did not show any significant topo inhibitory activity. These results go in line with previous studies22–25,30–33 and support the importance of 2-furyl, 2-thienyl, or 3-thienyl moiety at the 4-position on central pyridine for significant topo II inhibitory activity. Compounds containing either meta or para hydroxyl moiety at 2- or 6-phenyl position of central pyridine (9, 10, 15–17, 21, 22, 27–29, 33, 34, 41) exhibited significant cytotoxicity (50 1.63 ± 0.14 1.89 ± 0.03 2.19 ± 0.03 >50 >50 >50 1.31 ± 0.3 2.23 ± 0.01 2.06 ± 0.03 >50 1.79 ± 0.02 >50 2.15 ± 0.03 2.07 ± 0.41 >50 >50 2.79 ± 0.19 28 ± 0.26 1.65 ± 0.15 2.7 ± 0.77 3.44 ± 0.66 >50 17.05 ± 4.83 25.40 ± 0.73 3.61 ± 0.03 1.98 ± 0.04 >50 >50 5.58 ± 0.14 >50 >50 6.39 ± 0.81 0.24 ± 0

2.82 ± 0.24 18.87 ± 0.34 1.23 ± 0.00 >50 >50 >50 3.48 ± 0.25 1.77 ± 0.04 >50 >50 >50 >50 1.63 ± 0.08 1.18 ± 0.03 1.04 ± 0.01 >50 >50 >50 1.89 ± 0.8 0.84 ± 0.04 13.99 ± 0.68 >50 >50 >50 1.02 ± 0.13 0.93 ± 0.02 1.53 ± 0.36 >50 >50 >50 3.51 ± 0.23 >50 >50 >50 8.28 ± 1.69 >50 >50 >50 0.84 ± 0

1.84 ± 0.44 13.7 ± 0.81 1.34 ± 0.03 >50 >50 >50 0.77 ± 0.04 0.78 ± 0.02 11.68 ± 1.06 >50 >50 >50 1.51 ± 0.07 0.76 ± 0.05 1.77 ± 0.04 >50 0.82 ± 0.03 >50 3.52 ± 0.49 1.28 ± 0.15 21.14 ± 0.93 >50 >50 >50 2.59 ± 0.13 2.61 ± 0.29 3.46 ± 0.07 >50 2.56 ± 0.13 >50 1.41 ± 0.02 0.98 ± 0.1 1.04 ± 0.16 >50 6.23 ± 0.73 >50 >50 >50 2.03 ± 0.14

0.18 ± 0.02 7.32 ± 0.15 0.88 ± 0.08 >50 >50 >50 1.69 ± 0.16 4.14 ± 0.20 >50 >50 >50 >50 16.56 ± 1.08 2.58 ± 0.16 4.37 ± 0.3 >50 >50 >50 5.99 ± 0.37 5.17 ± 0.23 >50 >50 >50 >50 4.63 ± 0.08 3.31 ± 0.03 3.42 ± 0.33 >50 2.02 ± 0.03 >50 6.5 ± 0.12 1.93 ± 0.19 >50 >50 7.53 ± 0.58 >50 >50 >50 0.6 ± 0.01

a Each data represents mean ± SD from three different experiments performed in triplicate; ND: Not determined; DU145: human prostate tumor; HCT15: human colorectal adenocarcinoma; T47D: Human breast ductal carcinoma; HeLa: human cervix adenocarcinoma cell line; Camptothecin: positive control for topo I and cytotoxicity; Etoposide: positive control for topo II and cytotoxicity; Adriamycin: positive control for cytotoxicity.

acetonitrile (ACN) and methanol were purchased from Burdick and Jackson, USA. Thin-layer chromatography (TLC) and column chromatography (CC) were performed with Kieselgel 60 F254 (Merck) and silica gel (Kieselgel 60, 230–400 mesh, Merck), respectively. Since all the compounds prepared contain aromatic ring, they were visualized and detected on TLC plates with UV light (short wave, long wave or both). NMR spectra were recorded on a Bruker AMX 250 (250 MHz or 300 MHz, FT) for 1H NMR and 62.5 MHz or 75.5 MHz for 13C NMR, and chemical shifts were calibrated according to TMS. Chemical shifts (d) were recorded in ppm and coupling constants (J) in hertz (Hz). Melting points were determined in open capillary tubes on electrothermal 1A 9100 digital melting point apparatus and were uncorrected. HPLC analyses were performed using two Shimadzu LC-10AT pumps gradient-controlled HPLC system equipped with Shimadzu system controller (SCL-10A VP) and photodiode array detector (SPD-M10A VP) using Shimadzu Class VP program. Sample volume of 10 lL was injected in Waters X- TerraÒ 5 lM reverse-phase C18 column (4.6  250 mm) with a gradient elution of 70% to 100% of B in A for 10 min followed by 100% to 70% of B in A for 10 min at a flow rate of 1.0 mL/min at 254 nm UV detection, where mobile phase A was 20 mM ammonium formate (AF) in doubly distilled water and B was 100% ACN. Purity of compound is described as percent (%), and retention time is given in minutes.

ESI LC/MS analyses were performed with a Finnigan LCQ AdvantageÒ LC/MS/MS spectrometry utilizing XcaliburÒ program. For ESI LC/MS, LC was performed with a 5 lL injection volume on a Waters X TerraÒ 3.5 lm reverse-phase C18 column (2.1  100 mm) with a gradient elution from 10% to 90% of B in A for 5 min followed by 90% to 10% of B in A for 10 min and 10% B in A for 5 min, at a flow rate of 250 lL/min, where mobile phase A was 100% distilled water with 0.1% formic acid and mobile phase B was 100% ACN. MS ionization conditions were: Sheath gas flow rate: 70 arb, aux gas flow rate: 20 arb, I spray voltage: 4.5 KV, capillary temperature: 215 °C, column temperature 40 °C, capillary voltage: 21 V, tube lens offset: 10 V. 5.1. General method for the preparation of 3 Compounds 3 (R1 = a–c, R2 = d–i) were synthesized by base catalyzed Claisen–Schmidt condensation reaction. A solution of equimolar amounts of aryl ketone 1 (R1 = a–c) and aryl aldehyde 2 (R2 = d–i) in ethanol was added to 4 M aqueous solution of sodium hydroxide and stirred for 2–8 h at 20 °C. The reaction mixture was neutralized with 6 M HCl until pH 7 for compounds containing 2/3-pyridyl. 6 M HCl was added until pH to 2 for compounds containing 2/3-thienyl and 2/3-furyl. The mixture was extracted with ethyl acetate, washed with water and brine.

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HO

S

S

S

OH

N

HO

N

N OH

11

10

9

(78.3%, 22.3%)

(88.6%, 21.5%)

(74.6%, 30.3%)

S

S

S

HO

HO

OH

N

N

N

OH

HO

OH

17

15

16

(88.1%, 23.0%)

(91.5%, 44.7%)

(81.2%, 57.9%)

O

O

O

HO

OH

HO

HO

OH

N

N

N OH

21

OH

HO

22

23

(92.3%, 48.4%)

(92.8%, 21.8%)

(88.3%, 20.6%)

Figure 6. Structures of compounds having significant topo II inhibitory activity at 100 and 20 lM, respectively.

R2

R2

R2

> N

> HO

OH

N

OH

OH N

OH

HO

R2 = 2- thienyl, 3- thienyl, 2- furyl Figure 7. Favorable order of substitution for topo II inhibitory activity and cytotoxicity in dihydroxylated 2,6-diphenyl-4-aryl pyridine compounds.

It was further purified by either recrystallization or column chromatography to yield pure compound. Total eighteen hydroxylated chalcone intermediates were synthesized. 5.1.1. 1-(2-Hydroxyphenyl)-3-(thiophen-2-yl)propenone (3a) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = a) (2.40 mL, 20.00 mmol), aryl aldehyde 2 (R2 = d) (1.86 mL, 20.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield a yellow solid (3.10 g, 67.4%, 13.46 mmol). TLC (ethyl acetate/n-hexane = 1:3) Rf = 0.47. 1H NMR (250 MHz, CDCl3) d 12.85 (s, 1H, 1-phenyl 2-OH), 8.05 (d, J = 14.6 Hz, 1H, CO– [email protected]), 7.89 (d, J = 7.9 Hz, 1H, 1-phenyl H-6), 7.53–7.41 (m, 4H, 1-phenyl H-4, 3-thienyl H-3, H-5, CO–[email protected]), 7.11 (td, J = 4.4, 0.4 Hz, 1H, 3-thienyl H-4), 7.03 (d, J = 8.4 Hz, 1H, 1-phenyl H-3), 6.95 (t, J = 7.9 Hz, 1H, 1-phenyl H-5). 13 C NMR (62.5 MHz, CDCl3) d 193.46, 162.13, 144.33, 139.56, 137.17, 135.92, 129.53, 128.57, 123.19, 120.48, 119.82, 118.21, 115.66. 5.1.2. 1-(3-Hydroxyphenyl)-3-(thiophen-2-yl)propenone (3b) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = d) (0.93 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield a yellow solid (2.26 g, 98.2%, 9.81 mmol). TLC (ethyl acetate/n-hexane = 1:3) Rf = 0.25. 1H NMR (250 MHz, CDCl3) d 7.97 (d, J = 15.3 Hz, 1H, CO–[email protected]), 7.67 (br, 1H, 1-phenyl H-2), 7.56 (d, J = 7.7 Hz, 1H, 1-phenyl H-6), 7.44–7.26 (m, 4H, 1-phenyl H-5,

3-thienyl H-3, H-5, CO–[email protected]), 7.15–7.07 (m, 2H, 1-phenyl H-4, 3-thienyl H-4), 6.57 (s, 1H, 1-phenyl 3-OH). 13 C NMR (62.5 MHz, CDCl3) d 190.13, 156.43, 140.22, 139.32, 137.84, 132.53, 129.91, 129.19, 128.42, 120.87, 120.47 (2C), 115.11. 5.1.3. 1-(4-Hydroxyphenyl)-3-(thiophen-2-yl)propenone (3c) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = c) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = d) (0.93 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield yellow solid (2.10 g, 91.3%, 9.10 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.20. 1H NMR (250 MHz, DMSO-d6) d 10.46 (s, 1H, 1-phenyl 4-OH), 7.99 (d, J = 7.7 Hz, 2H, 1-phenyl H2, H-6), 7.86 (d, J = 15.3 Hz, 1H, CO–[email protected]), 7.74 (d, J = 5.0 Hz, 1H, 3-thienyl H-3), 7.64 (d, J = 3.3 Hz, 1H, 3-thienyl H-5), 7.55 (d, J = 15.3 Hz, 1H, CO–[email protected]), 7.16 (t, J = 5.0 Hz, 1H, 3-thienyl H4), 6.87 (d, J = 8.7 Hz, 2H, 1-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 187.24, 163.45, 150.49, 145.14, 132.14 (2C), 129.12, 129.26, 118.81, 116.59, 115.42 (2C), 112.24. 5.1.4. 1-(2-Hydroxyphenyl)-3-(thiophen-3-yl)propenone (3d) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = a) (1.20 mL, 10.00 mmol), aryl aldehyde 2 (R2 = e) (0.91 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield a yellow solid (2.15 g, 93.4%, 9.33 mmol). TLC (ethyl acetate/n-hexane = 1:5) Rf = 0.46. 1H NMR (250 MHz, CDCl3) d 12.87 (s, 1H, 1-phenyl 2-OH), 7.92 (d, J = 15.5 Hz, 1H, CO–[email protected]),

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7.89 (d, J = 7.9 Hz, 1H, 1-phenyl H-6), 7.67 (br, 1H, 3-thienyl H-2), 7.53–7.38 (m, 4H, 1-phenyl H-4, 3-thienyl H-4, H-5, CO–[email protected]), 7.02 (dd, J = 8.4, 0.9 Hz, 1H, 1-phenyl H-3), (t, J = 8.1 Hz, 1H, 1-phenyl H-5). 13 C NMR (62.5 MHz, CDCl3) d 193.88, 163.49, 138.81, 137.92, 136.32, 130.05, 129.53, 127.26, 125.15, 119.96, 119.67, 118.80, 118.59. 5.1.5. 1-(3-Hydroxyphenyl)-3-(thiophen-3-yl)propenone (3e) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = e) (0.91 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield light yellow solid (2.20 g, 95.6%, 9.55 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.32. 1H NMR (250 MHz, CDCl3) d 7.81 (d, J = 15.5 Hz, 1H, CO–[email protected]), 7.63–7.52 (m, 3H, 1-phenyl H-2, H-6, 3-thienyl H-2), 7.40–7.32 (m, 3H, 3-thienyl H4, H-5, 1-phenyl H-5), 7.30 (d, J = 15.3 Hz, 1H, CO–[email protected]), 7.11 (d, J = 8.1 Hz, 1H, 1-phenyl H-4), 6.52 (s, 1H, 1-phenyl 3-OH). 13 C NMR (62.5 MHz, CDCl3) d 190.97, 156.41, 139.62, 138.79, 138.11, 129.88, 129.44, 127.07, 125.26, 121.75, 120.92, 120.32, 115.18. 5.1.6. 1-(4-Hydroxyphenyl)-3-(thiophen-3-yl)propenone (3f) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = c) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = e) (0.91 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield yellow solid (2.18 g, 94.7%, 9.46 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.30. 1H NMR (250 MHz, DMSO-d6) d 10.42 (s, 1H, 1-phenyl 4-OH), 8.04 (s, 1H, 3-thienyl H-2), 8.03 (d, J = 8.7 Hz, 2H, 1-phenyl H-2, H-6), 7.77 (d, J = 3.8 Hz, 1H, 3-thienyl H-4), 7.75–7.70 (m, 2H, CO–[email protected], CO–[email protected]), 7.66–7.62 (m, 1H, 3-thienyl H-5), 6.87 (d, J = 8.7 Hz, 2H, 1-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 187.54, 162.36, 138.63, 136.84, 131.32 (2C), 130.24, 129.37, 127.85, 126.38, 121.75, 115.57 (2C). 5.1.7. 3-(Furan-2-yl)-1-(2-hydroxyphenyl)propenone (3g) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = a) (1.80 mL, 15.00 mmol), aryl aldehyde 2 (R2 = f) (1.24 mL, 15.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield a greenish yellow solid (2.93 g, 91.3%, 13.67 mmol). TLC (ethyl acetate/n-hexane = 1:5) Rf = 0.44. 1H NMR (250 MHz, CDCl3) d 12.85 (s, 1H, 1-phenyl 2-OH), 7.91 (dd, J = 8.1, 1.4 Hz, 1H, 1-phenyl H-6), 7.68 (d, J = 15.2 Hz, 1H, CO–[email protected]), 7.89 (d, J = 15.2 Hz, 1H, CO–[email protected]), 7.55–7.45 (m, 2H, 1-phenyl H-4, 3furyl H-5), 7.01 (d, J = 8.4 Hz, 1H, 1-phenyl H-3), 6.93 (td, J = 8.1, 1.0 Hz, 1H, 1-phenyl H-5), 6.76 (d, J = 3.4 Hz, 1H, 3-furyl H-3), 6.53 (br, 1H, 3-furyl H-4). 13 C NMR (62.5 MHz, CDCl3) d 190.19, 156.51, 151.50, 145.16, 139.30, 131.20, 129.87, 120.89, 120.52, 119.01, 116.84, 115.12, 112.77. 5.1.8. 3-(Furan-2-yl)-1-(3-hydroxyphenyl)propenone (3h) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = f) (0.82 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield light yellow solid (2.15 g, 93.4%, 9.33 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.37. 1H NMR (250 MHz, CDCl3) d 7.81 (d, J = 15.2 Hz, 1H, CO–[email protected]), 7.59 (d, J = 8.2 Hz, 1H, 1-phenyl H-6), 7.56–7.52 (m, 2H, 1-phenyl H-2, 3-furyl H-5), 7.42 (d, J = 15.3 Hz, 1H, CO–[email protected]), 7.37 (t, J = 7.7 Hz, 1H, 1-phenyl H-5), 7.01 (dd, J = 8.0, 2.1 Hz, 1H, 1-phenyl H-4), 6.73 (d, 3.40 Hz, 1H, 3-furyl H-3), 6.52 (br, 1H, 3-furyl H-4), 5.66 (s, 1H, 1-phenyl 3-OH). 13 C NMR (62.5 MHz, CDCl3) d 190.43, 157.11, 141.32, 140.39, 137.80, 131.52, 129.88, 129.09, 128.31, 120.67, 119.89 (2C), 115.32.

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5.1.9. 3-(Furan-2-yl)-1-(4-hydroxyphenyl)propenone (3i) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = c) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = f) (0.82 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield yellow solid (1.90 g, 90.5%, 8.86 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.34. 1H NMR (250 MHz, DMSO-d6) d 10.41 (s, 1H, 1-phenyl 4-OH), 7.96 (d, J = 8.7 Hz, 2H, 1-phenyl H2, H-6), 7.87 (br, 1H, 3-furyl H-5), 7.53 (d, J = 15.6 Hz, 1H, CO– [email protected]), 7.47 (d, J = 15.6 Hz, 1H, CO–[email protected]), 7.04 (d, J = 3.4 Hz, 1H, 3-furyl H-3), 6.87 (d, J = 8.7 Hz, 2H, 1-phenyl H-3, H-5), 6.66 (br, 1H, 3-furyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 186.74, 162.37, 151.49, 146.04, 131.14 (2C), 129.61, 129.20, 119.01, 116.49, 115.66 (2C), 113.20. 5.1.10. 3-(Furan-3-yl)-1-(2-hydroxyphenyl)propenone (3j) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = a) (1.80 mL, 15.00 mmol), aryl aldehyde 2 (R2 = g) (1.25 mL, 15.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield a yellow solid (1.50 g, 46.7%, 7.00 mmol). TLC (ethyl acetate/n-hexane = 1:5) Rf = 0.47. 1H NMR (250 MHz, CDCl3) d 12.80 (s, 1H, 1-phenyl 2-OH), 7.87 (dd, J = 7.7, 1.5 Hz, 1H, 1-phenyl H-6), 7.84 (d, J = 15.2 Hz, 1H, CO–[email protected]), 7.78 (s, 1H, 3-furyl H-2), 7.49 (td, J = 8.1, 1.5 Hz, 1H, 1-phenyl H-4), 7.49 (s, 1H, 3-furyl H-5), 7.30 (d, J = 15.2 Hz, 1H, CO–[email protected]), 7.02 (dd, J = 8.4, 0.9 Hz, 1H, 1phenyl H-3), 6.93 (td, J = 8.1, 1.1 Hz, 1H, 1-phenyl H-5), 6.73 (br, 1H, 3-furyl H-4). 13 C NMR (62.5 MHz, CDCl3) d 193.57, 163.60, 146.02, 144.70, 136.27, 135.46, 129.53, 123.19, 120.01, 119.92, 118.77, 118.63, 107.43. 5.1.11. 3-(Furan-3-yl)-1-(3-hydroxyphenyl)propenone (3k) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = g) (1.00 mL, 12.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield light yellow solid (1.95 g, 91.1%, 9.10 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.34. 1H NMR (250 MHz, DMSO-d6) d 9.76 (s, 1H, 1-phenyl 3-OH), 8.18 (s, 1H, 3-furyl H-2), 7.76 (br, 1H, 3-furyl H-5), 7.64 (d, J = 15.50 Hz, 1H, CO–[email protected]), 7.52 (d, J = 15.4 Hz, 1H, CO–[email protected]), 7.51 (d, J = 7.2 Hz, 1H, 1-phenyl H-6), 7.40 (br, 1H, 1-phenyl H-2), 7.34 (t, J = 7.8 Hz, 1H, 1-phenyl H-5), 7.12 (br, 1H, 3-furyl H-4), 7.03 (ddd, J = 8.0, 2.5, 0.8 Hz, 1H, 1-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 189.20, 157.84, 146.84, 145.10, 139.17, 134.56, 129.90, 123.32, 122.09, 120.24, 119.50, 114.71, 108.17. 5.1.12. 3-(Furan-3-yl)-1-(4-hydroxyphenyl)propenone (3l) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = c) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = g) (1.00 mL, 12.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield a yellow solid (1.73 g, 80.8%, 8.07 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.24. 1H NMR (250 MHz, DMSO-d6) d 10.40 (s, 1H, 1-phenyl 4-OH), 8.16 (s, 1H, 3-furyl H-2), 8.01 (d, J = 8.7 Hz, 2H, 1-phenyl H-2, H-6), 7.76 (br, 1H, 3-furyl H-5), 7.64 (d, J = 15.1 Hz, 1H, CO–[email protected]), 7.58 (d, J = 15.2 Hz, 1H, CO– [email protected]), 7.12 (br, 1H, 3-furyl H-4), 6.88 (d, J = 8.7 Hz, 2H, 1-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 187.20, 162.30, 146.56, 145.11, 133.47, 131.25 (2C), 129.27, 123.52, 121.93, 115.56 (2C), 108.37. 5.1.13. 1-(2-Hydroxyphenyl)-3-(pyridin-2-yl)-propenone (3m) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = a) (2.40 mL, 20.00 mmol), aryl aldehyde 2 (R2 = h) (1.91 mL, 20.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield a yellow solid (2.75 g, 61.0%, 12.20 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.33. 1H NMR (300 MHz, DMSO-d6) d

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R. Karki et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx

12.01 (s, 1H, 1-phenyl 2-OH), 8.68 (d, J = 4.8 Hz, 1H, 3-pyridyl H-6), 8.21 (d, J = 15.3 Hz, 1H, CO–[email protected]), 8.03 (dd, J = 8.4, 1.5 Hz, 1H, 1phenyl H-6), 7.90–7.88 (m, 2H, 3-pyridyl H-3, H-4), 7.75 (d, J = 15.3 Hz, 1H, CO–[email protected]), 7.55 (td, J = 7.8, 1.5 Hz, 1H, 1-phenyl H-4), 7.43 (t, J = 8.7 Hz, 1H, 3-pyridyl H-5), 7.01 (d, J = 8.1 Hz, 1H, 1-phenyl H-3), 7.43 (t, J = 8.1 Hz, 1H, 1-phenyl H-5). 13 C NMR (75.5 MHz, DMSO-d6) d 194.19, 162.06, 153.40, 150.94, 144.01, 138.08, 137.09, 131.55, 126.32, 126.13, 125.83, 122.24, 120.22, 118.56. 5.1.14. 1-(3-Hydroxyphenyl)-3-(pyridin-2-yl)-propenone (3n) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = h) (0.95 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield a yellow solid (1.10 g, 48.8%, 4.88 mmol). TLC (ethyl acetate/n-hexane = 1:1) Rf = 0.40. 1H NMR (300 MHz, DMSO-d6) d 8.77 (br, 1H, 3-pyridyl H-6), 8.28 (d, J = 15.3 Hz, 1H, CO–[email protected]), 8.19 (br, 2H, 3-pyridyl H-3, H-4), 7.75 (d, J = 15.9 Hz, 1H, CO– [email protected]), 7.68–7.59 (m, 2H, 3-pyridyl H-5, 1-phenyl H-6), 7.47 (s, 1H, 1-phenyl H-2), 7.39 (t, J = 7.8 Hz, 1H, 1-phenyl H-5), 7.11 (d, J = 6.6 Hz, 1H, 1-phenyl H-4), 5.18 (br, 1H, 1-phenyl 3-OH). 13 C NMR (75.5 MHz, DMSO-d6) d 188.92, 158.21, 149.93, 146.17, 142.43, 138.44, 137.63, 130.34, 129.01, 126.51, 126.26, 121.34, 120.12, 114.96. 5.1.15. 1-(4-Hydroxyphenyl)-3-(pyridin-2-yl)-propenone (3o) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = c) (0.68 g, 5.00 mmol), aryl aldehyde 2 (R2 = h) (0.47 mL, 5.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield light yellow solid (0.80 g, 71.4%, 3.55 mmol). TLC (ethyl acetate/n-hexane = 1:1) Rf = 0.39. 1H NMR (300 MHz, DMSO-d6) d 10.74 (s, 1H, 1-phenyl 4-OH), 8.84 (d, J = 5.4 Hz, 1H, 3-pyridyl H-6), 8.55 (d, J = 15.9 Hz, 1H, CO–[email protected]), 8.41 (br, 2H, 3-pyridyl H-3, H-4), 8.11 (d, J = 9.0 Hz, 2H, 1-phenyl H-2, H-6), 7.84 (t, J = 9.0 Hz, 1H, 3-pyridyl H-5), 7.78 (d, J = 15.6 Hz, 1H, CO– [email protected]), 6.94 (d, J = 9.1 Hz, 2H, 1-phenyl H-3, H-5). 13 C NMR (75.5 MHz, DMSO-d6) d 186.37, 163.12, 161.93, 144.77, 133.74, 131.68 (2C), 130.00, 128.35, 127.49, 126.44, 126.16, 115.74 (2C). 5.1.16. 1-(2-Hydroxyphenyl)-3-(pyridin-3-yl)-propenone (3p) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = a) (2.40 mL, 20.00 mmol), aryl aldehyde 2 (R2 = i) (1.88 mL, 20.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield yellow solid (3.30 g, 73.3%, 14.65 mmol). TLC (ethyl acetate/n-hexane = 2:1) Rf = 0.30. 1H NMR (300 MHz, DMSO-d6) d 12.39 (s, 1H, 1-phenyl 2-OH), 9.03 (s, 1H, 3-pyridyl H-2), 8.62 (d, J = 4.8 Hz, 1H, 3-pyridyl H-6), 8.36 (d, J = 7.8 Hz, 1H, 3-pyridyl H4), 8.26 (d, J = 8.1 Hz, 1H, 1-phenyl H-6), 8.17 (d, J = 15.6 Hz, 1H, CO–[email protected]), 7.84 (d, J = 15.9 Hz, 1H, CO–[email protected]), 7.57 (t, J = 7.5 Hz, 1H, 1-phenyl H-4), 7.49 (t, J = 7.8 Hz, 1H, 3-pyridyl H-5), 7.03–6.99 (m, 2H, 1-phenyl H-3, H-5). 13 C NMR (75.5 MHz, DMSO-d6) d 193.56, 162.09, 151.50, 150.83, 141.49, 136.78, 135.59, 131.24, 130.54, 124.22, 123.94, 120.92, 119.48, 118.00. 5.1.17. 1-(3-Hydroxyphenyl)-3-(pyridin-3-yl)-propenone (3q) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R2 = i) (0.95 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield light yellow solid (1.50 g, 66.7%, 6.65 mmol). TLC (ethyl acetate/n-hexane = 3:1) Rf = 0.27. 1H NMR (300 MHz, DMSO-d6) d 9.39 (br, 1H, 3-pyridyl H-2), 8.97 (d, J = 8.1 Hz, 1H, 3pyridyl H-4), 8.87 (d, J = 5.7 Hz, 1H, 3-pyridyl H-6), 8.23 (d, J = 15.9 Hz, 1H, CO–[email protected]), 8.03 (t, J = 8.1 Hz, 1H, 3-pyridyl H5), 7.79 (d, J = 15.6 Hz, 1H, CO–[email protected]), 7.68 (d, J = 7.8 Hz, 1H, 1phenyl H-6), 7.51 (s, 1H, 1-phenyl H-2), 7.38 (t, J = 7.8 Hz, 1H, 1-

phenyl H-5), 7.11 (dd, J = 8.1, 1.8 Hz, 1H, 1-phenyl H-4), 4.90 (br, 1H, 1-phenyl 3-OH). 13 C NMR (75.5 MHz, DMSO-d6) d 189.45, 158.78, 144.02, 143.81, 143.57, 139.08, 137.98, 134.65, 130.76, 128.07, 127.70, 121.81, 120.73, 115.69. 5.1.18. 1-(4-Hydroxyphenyl)-3-(pyridin-3-yl)-propenone (3r) The procedure described in Section 5.1 was employed with aryl ketone 1 (R1 = c) (0.68 g, 5.00 mmol), aryl aldehyde 2 (R2 = i) (0.47 mL, 5.00 mmol), ethanol (10 mL) and sodium hydroxide (4 M) to yield light yellow solid (0.76 g, 67.8%, 3.37 mmol). TLC (ethyl acetate/n-hexane = 3:1) Rf = 0.26. 1H NMR (300 MHz, DMSO-d6) d 10.71 (s, 1H, 1-phenyl 4-OH), 9.38 (s, 1H, 3-pyridyl H-2), 8.96 (d, J = 8.1 Hz, 1H, 3-pyridyl H-4), 8.86 (d, J = 5.4 Hz, 1H, 3-pyridyl H-6), 8.26 (d, J = 15.9 Hz, 1H, CO–[email protected]), 8.12 (d, J = 8.7 Hz, 2H, 1-phenyl H-2, H-6), 8.02 (t, J = 7.8 Hz, 1H, 3-pyridyl H-5), 7.78 (d, J = 15.6 Hz, 1H, CO–[email protected]), 6.94 (d, J = 8.4 Hz, 2H, 1-phenyl H-3, H-5). 13 C NMR (75.5 MHz, DMSO-d6) d 186.78, 163.19, 143.50, 142.89, 142.67, 136.29, 134.39, 131.83 (2C), 128.68, 127.67, 127.16, 115.84 (2C). 5.2. General method for the preparation of 5 A mixture of aryl ketone 4 (R3 = a–c), iodine (1.2 equiv) and pyridine (15 equiv) was refluxed at 140 °C for 3 h. Precipitate occurred during reaction which was cooled to room temperature. Then it was filtered and washed with cold pyridine to afford 5 (R3 = a–c) in quantitative yield. Three different pyridinium iodide salts were synthesized by this method. 5.3. General method for the preparation of 6–41 A mixture of hydroxylated chalcone intermediate 3 (R1 = a–c, R2 = d–i), pyridinium iodide salt 5 (R3 = a–c) and anhydrous ammonium acetate in glacial acetic acid/methanol were heated at 80– 100 °C for 12–16 h. The reaction mixture was then extracted with ethyl acetate, washed with water and brine. The organic layer was dried with magnesium sulfate and filtered. The filtrate was evaporated at reduced pressure, which was then purified by silica gel column chromatography with the gradient elution of ethyl acetate/nhexane to afford solid compounds 6–41 in 30–83% yield. Total thirty-six dihydroxylated 2,6-diphenyl-4-aryl pyridine compounds were synthesized by this method. 5.3.1. 2,20 -(4-(Thiophen-2-yl)pyridine-2,6-diyl)diphenol (6) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = d) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = a) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.31 g, 60.8%, 0.91 mmol). TLC (ethyl acetate/n-hexane = 1:3) Rf = 0.27, mp: 217–218 °C, HPLC: Retention time: 10.23 min, purity: 98.9%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1H NMR (300 MHz, DMSO-d6) d 12.33 (s, 2H, 2-phenyl 2-OH, 6-phenyl 2-OH), 8.18 (s, 2H, pyridine H-3, H-5), 8.08 (d, J = 3.6 Hz, 1H, 4-thiophene H-3), 7.94 (d, J = 7.8 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.81 (d, J = 5.1 Hz, 1H, 4-thiophene H-5), 7.35–7.27 (m, 3H, 2-phenyl H-4, 6-phenyl H-4, 4-thiophene H-4), 6.98–6.94 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 157.55 (2C), 155.76 (2C), 143.20, 140.62, 131.37 (2C), 129.28, 129.17, 128.79 (2C), 127.82, 121.98 (2C), 119.46 (2C), 117.56 (2C), 115.94 (2C). 5.3.2. 2-(6-(3-Hydroxyphenyl)-4-(thiophen-2-yl)pyridin-2yl)phenol (7) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = d) (0.34 g, 1.50 mmol), dry ammonium acetate

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(1.15 g, 15.00 mmol), 5 (R3 = b) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.33 g, 63.7%, 0.95 mmol). TLC (ethyl acetate/n-hexane = 1:3) Rf = 0.27, mp: 225–226 °C, HPLC: Retention time: 10.50 min, purity: 98.7%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1 H NMR (250 MHz, DMSO-d6) d 14.25 (s, 1H, 2-phenyl 2-OH), 9.75 (s, 1H, 6-phenyl 3-OH), 8.32 (s, 1H, pyridine H-3), 8.23 (d, J = 7.3 Hz, 1H, 2-phenyl H-6), 8.15 (d, J = 3.6 Hz, 1H, 4-thiophene H-3), 8.00 (s, 1H, pyridine H-5), 7.83 (d, J = 5.0 Hz, 4-thiophene H-5), 7.50 (d, J = 7.8 Hz, 1H, 6-phenyl H-6), 7.44 (s, 1H, 6-phenyl H-2), 7.41 (t, J = 7.8 Hz, 1H, 6-phenyl H-5), 7.38 (t, J = 7.7 Hz, 1H, 2-phenyl H-4), 7.31 (ddd, J = 5.0, 3.6, 0.5 Hz, 1H, 4-thiophene H4), 7.00–6.92 (m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.29, 158.23, 157.61, 155.04, 143.98, 140.32, 138.90, 131.76, 130.37, 129.28, 129.11, 128.08, 127.78, 119.14, 119.08, 117.95, 117.68, 117.10, 115.17, 114.27, 113.54. 5.3.3. 2-(6-(4-Hydroxyphenyl)-4-(thiophen-2-yl)pyridin-2yl)phenol (8) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = d) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.32 g, 62.0%, 0.92 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.31, mp: 293–294 °C, HPLC: Retention time: 10.50 min, purity: 98.5%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1 H NMR (250 MHz, DMSO-d6) d 14.57 (s, 1H, 2-phenyl 2-OH), 10.03 (br, 1H, 6-phenyl 4-OH), 8.23 (s, 1H, pyridine H-3), 8.22 (d, J = 7.3 Hz, 1H, 2-phenyl H-6), 8.14 (d, J = 3.7 Hz, 1H, 4-thiophene H-3), 7.98 (s, 1H, pyridine H-5), 7.93 (d, J = 8.6 Hz, 2H, 6-phenyl H-2, H-6), 7.83 (d, J = 5.0 Hz, 1H, 4-thiophene H-5), 7.37 (t, J = 7.8 Hz, 1H, 2-phenyl H-4), 7.30 (dd, J = 4.9, 4.0 Hz, 1H, 4-thiophene H-4), 6.97 (d, J = 8.6 Hz, 2H, 6-phenyl H-3, H-5), 6.95–6.92 (m, 2H, 2-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.56, 159.53, 157.56, 155.01, 144.01, 140.59, 131.84, 129.30, 129.21, 128.47 (2C), 128.26, 128.07, 127.75, 119.14, 119.12, 118.07, 116.23 (2C), 114.19, 113.20. 5.3.4. 3,30 -(4-(Thiophen-2-yl)pyridine-2,6-diyl)diphenol (9) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = d) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = b) (0.40 g, 1.20 mmol) and acetic acid (2 mL) to yield a light yellow solid (0.31 g, 77.1%, 0.92 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.28, mp: 249–250 °C, HPLC: Retention time: 7.78 min, purity: 96.1%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.36. 1 H NMR (250 MHz, DMSO-d6) d 9.63 (s, 2H, 2-phenyl 3-OH, 6phenyl 3-OH), 8.07 (dd, J = 3.6, 0.9 Hz, 1H, 4-thiophene H-3), 8.00 (s, 2H, pyridine H-3, H-5), 7.77 (dd, J = 5.3, 1.0 Hz, 1H, 4-thiophene H-5), 7.69–7.64 (m, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.36 (t, J = 7.8 Hz, 2H, 2-phenyl H-5, 6-phenyl H-5), 7.27 (dd, J = 5.0, 3.7 Hz, 1H, 4-thiophene H-4), 6.89 (dd, J = 7.5, 1.6 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 157.81 (2C), 156.66 (2C), 142.82, 140.77, 139.96 (2C), 129.81 (2C), 128.95, 128.29, 127.09, 117.66 (2C), 116.44 (2C), 114.65 (2C), 113.68 (2C). 5.3.5. 3-(6-(4-Hydroxyphenyl)-4-(thiophen-2-yl)pyridin-2yl)phenol (10) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = d) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a light yellow solid (0.30 g, 57.4%, 0.86 mmol). TLC

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(ethyl acetate/n-hexane = 1:2) Rf = 0.20, mp: 240–241 °C, HPLC: retention time: 7.63 min, purity: 98.9%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1 H NMR (250 MHz, DMSO-d6) d 9.79 (br, 1H, 2-phenyl 3-OH), 9.65 (br, 1H, 6-phenyl 4-OH), 8.14 (d, J = 8.6 Hz, 2H, 6-phenyl H2, H-6), 8.04 (d, J = 3.6 Hz, 1H, 4-thiophene H-3), 7.97 (s, 1H, pyridine H-3), 7.91 (s, 1H, pyridine H-5), 7.76 (d, J = 5.0 Hz, 4-thiophene H-5), 7.68 (s, 1H, 2-phenyl H-2), 7.66 (d, J = 7.8 Hz, 1H, 2phenyl H-6), 7.35 (t, J = 7.8 Hz, 1H, 2-phenyl H-5), 7.27 (dd, J = 4.8, 3.9 Hz, 1H, 4-thiophene H-4), 6.93 (d, J = 8.6 Hz, 2H, 6-phenyl H-3, H-5), 6.88 (dd, J = 8.1, 2.1 Hz, 1H, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.05, 157.97, 156.84, 156.63, 142.86, 141.19, 140.27, 129.97, 129.64, 129.07, 128.50 (2C), 128.29, 127.06, 117.78, 116.51, 115.74 (2C), 113.80 (2C), 113.67. 5.3.6. 4,40 -(4-(Thiophen-2-yl)pyridine-2,6-diyl)diphenol (11) The same procedure described in Section 5.3 was employed with 3 (R1 = c, R2 = d) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield orange solid (0.27 g, 52.6%, 0.78 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.20, mp: 245–246 °C, HPLC: Retention time: 7.50 min, purity: 97.8%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1 H NMR (250 MHz, DMSO-d6) d 9.81 (s, 2H, 2-phenyl 4-OH, 6phenyl 4-OH), 8.12 (d, J = 8.3 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 8.01 (d, J = 3.6 Hz, 4-thiophene H-3), 7.88 (s, 2H, pyridine H-3, H-5), 7.74 (d, J = 5.0 Hz, 1H, 4-thiophene H-5), 7.24 (t, J = 4.8 Hz, 1H, 4-thiophene H-4), 6.91 (d, J = 8.2 Hz, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 158.96 (3C), 156.68 (2C), 142.73, 141.44, 129.81 (3C), 129.02, 128.48 (3C), 128.13, 126.88, 115.72 (3C), 112.66 (2C). 5.3.7. 2,20 -(4-(Thiophen-3-yl)pyridine-2,6-diyl)diphenol (12) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = e) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = a) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.43 g, 83.7%, 1.25 mmol). TLC (ethyl acetate/n-hexane = 1:3) Rf = 0.28, mp: 219–220 °C, HPLC: Retention time: 9.59 min, purity: 99.7%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1 H NMR (300 MHz, DMSO-d6) d 12.34 (s, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 8.45 (dd, J = 2.8, 1.2 Hz, 1H, 4-thiophene H-2), 8.23 (s, 2H, pyridine H-3, H-5), 7.96 (dd, J = 8.3, 1.3 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.90 (d, J = 5.0 Hz, 1H, 4-thiophene H-4), 7.77 (dd, J = 5.0, 2.9 Hz, 1H, 4-thiophene H-5), 7.31 (t, J = 7.6 Hz, 2H, 2phenyl H-4, 6-phenyl H-4), 6.99–6.93 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 157.55 (2C), 155.75 (2C), 144.44, 139.23, 131.19 (2C), 128.92 (2C), 128.06, 126.69, 125.67, 122.31 (2C), 119.34 (2C), 117.51 (2C), 116.97 (2C). 5.3.8. 2-(6-(3-Hydroxyphenyl)-4-(thiophen-3-yl)pyridin-2yl)phenol (13) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = e) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = b) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.29 g, 56.2%, 0.84 mmol). TLC (ethyl acetate/n-hexane = 1:3) Rf = 0.28, mp: 230–231 °C, HPLC: Retention time: 9.94 min, purity: 99.1%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1 H NMR (250 MHz, DMSO-d6) d 14.62 (s, 1H, 2-phenyl 2-OH), 9.81 (br, 1H, 6-phenyl 3-OH), 8.58 (dd, J = 2.5, 0.8 Hz, 1H, 4-thiophene H-2), 8.44 (s, 1H, pyridine H-3), 8.31 (d, J = 7.8 Hz, 1H,

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2-phenyl H-6), 8.18 (s, 1H, pyridine H-5), 8.03 (d, J = 5.0 Hz, 4-thiophene H-4), 7.78 (dd, J = 5.0, 2.9 Hz, 1H, 4-thiophene H-5), 7.53 (d, J = 7.7 Hz, 1H, 6-phenyl H-6), 7.46 (s, 1H, 6-phenyl H-2), 7.41 (t, J = 8.0 Hz, 1H, 6-phenyl H-5), 7.35 (t, J = 8.0 Hz, 1H, 2-phenyl H4), 6.99–6.93 (m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.58, 158.32, 157.75, 154.89, 145.29, 139.21, 139.03, 131.84, 130.47, 127.97, 127.86, 126.93, 126.16, 119.16, 119.10, 118.09, 117.89, 117.09, 116.41, 115.26, 113.69. 5.3.9. 2-(6-(4-Hydroxyphenyl)-4-(thiophen-3-yl)pyridin-2yl)phenol (14) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = e) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.42 g, 80.8%, 1.21 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.31, mp: 292–293 °C, HPLC: Retention time: 9.75 min, purity: 99.4%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.34. 1 H NMR (250 MHz, DMSO-d6) d 14.85 (s, 1H, 2-phenyl 2-OH), 9.99 (br, 1H, 6-phenyl 4-OH), 8.56 (d, J = 2.8 Hz, 1H, 4-thiophene H-2), 8.36 (s, 1H, pyridine H-3), 8.29 (d, J = 7.8 Hz, 1H, 2-phenyl H-6), 8.14 (s, 1H, pyridine H-5), 8.02 (d, J = 5.1 Hz, 1H, 4-thiophene H-4), 7.96 (d, J = 8.3 Hz, 2H, 6-phenyl H-2, H-6), 7.77 (dd, J = 4.2, 2.9 Hz, 1H, 4-thiophene H-5), 7.33 (t, J = 7.8 Hz, 1H, 2-phenyl H4), 6.97 (d, J = 8.4 Hz, 2H, 6-phenyl H-3, H-5), 6.95–6.92 (m, 2H, 2-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.67, 159.46, 157.58, 154.78, 145.16, 139.19, 131.72, 128.47 (3C), 127.89, 127.73, 126.92, 125.96, 119.14, 119.01, 118.07, 116.18, 115.28, 114.14 (2C). 5.3.10. 3,30 -(4-(Thiophen-3-yl)pyridine-2,6-diyl)diphenol (15) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = e) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = b) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.40 g, 77.3%, 1.15 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.28, mp: 248–249 °C, HPLC: Retention time: 7.17 min, purity: 98.2%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.36. 1 H NMR (250 MHz, DMSO-d6) d 9.62 (s, 2H, 2-phenyl 3-OH, 6phenyl 3-OH), 8.46 (dd, J = 2.8, 1.0 Hz, 1H, 4-thiophene H-2), 8.13 (s, 2H, pyridine H-3, H-5), 7.95 (d, J = 5.0 Hz, 1H, 4-thiophene H4), 7.75–7.68 (m, 5H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6, 4-thiophene H-5), 7.35 (t, J = 7.9 Hz, 2H, 2-phenyl H-5, 6-phenyl H-5), 6.89 (dd, J = 7.9, 2.0 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 157.98 (2C), 156.70 (2C), 144.18, 140.50 (2C), 139.56, 129.90 (2C), 127.78, 126.79, 125.01, 117.92 (2C), 116.46 (2C), 115.93 (2C), 113.96 (2C). 5.3.11. 3-(6-(4-Hydroxyphenyl)-4-(thiophen-3-yl)pyridin-2yl)phenol (16) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = e) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.39 g, 76.5%, 1.14 mmol). TLC (ethyl acetate/n-hexane = 1:1) Rf = 0.52, mp: 229–230 °C, HPLC: Retention time: 7.00 min, purity: 99.3%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1 H NMR (250 MHz, DMSO-d6) d 9.64 (br, 2H, 2-phenyl 3-OH, 6phenyl 4-OH), 8.43 (d, J = 2.8, 1.0 Hz, 1H, 4-thiophene H-2), 8.18 (d, J = 8.6 Hz, 2H, 6-phenyl H-2, H-6), 8.09 (s, 1H, pyridine H-3), 8.04 (s, 1H, pyridine H-5), 7.93 (dd, J = 5.2, 1.0 Hz, 4-thiophene H-4), 7.74–7.67 (m, 3H, 2-phenyl H-2, H-6, 4-thiophene H-5), 7.34 (t, J = 7.8 Hz, 1H, 2-phenyl H-5), 6.92 (d, J = 8.6 Hz, 2H, 6-phenyl H3, H-5), 6.87 (dd, J = 8.2, 2.1 Hz, 1H, 2-phenyl H-4).

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C NMR (62.5 MHz, DMSO-d6) d 158.85, 157.89, 156.67, 156.46, 143.98, 140.59, 139.69, 129.98, 129.78, 128.44 (2C), 127.60, 126.69, 124.66, 117.81, 116.29, 115.63 (2C), 114.80, 114.70, 113.87. 5.3.12. 4,40 -(4-(Thiophen-3-yl)pyridine-2,6-diyl)diphenol (17) The same procedure described in Section 5.3 was employed with 3 (R1 = c, R2 = e) (0.34 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.40 g, 77.6%, 1.16 mmol). TLC (ethyl acetate/n-hexane = 1:1) Rf = 0.50, mp: 236–237 °C, HPLC: Retention time: 6.88 min, purity: 97.0%; ESI LC/MS: m/z calcd for C21H15NO2S [MH]+ 346.09; found 346.35. 1 H NMR (250 MHz, DMSO-d6) d 9.70 (s, 2H, 2-phenyl 4-OH, 6phenyl 4-OH), 8.39 (d, J = 2.9, 1.2 Hz, 4-thiophene H-2), 8.16 (d, J = 8.6 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 8.00 (s, 2H, pyridine H-3, H-5), 7.91 (dd, J = 5.0, 1.0 Hz, 1H, 4-thiophene H-4), 7.73 (dd, J = 5.0, 2.9 Hz, 1H, 4-thiophene H-5), 6.91 (d, J = 8.6 Hz, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 158.69 (2C), 156.42 (2C), 143.75, 139.84, 130.07 (2C), 128.32 (4C), 127.43, 126.63, 124.37, 115.51 (4C), 113.58 (2C). 5.3.13. 2,20 -(4-(Furan-2-yl)pyridine-2,6-diyl)diphenol (18) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = f) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = a) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.32 g, 65.6%, 0.98 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.37, mp: 217–218 °C, HPLC: Retention time: 8.97 min, purity: 98.8%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.35. 1 H NMR (250 MHz, DMSO-d6) d 12.27 (s, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 8.20 (d, J = 0.9 Hz, 2H, pyridine H-3, H-5), 7.94 (s, 1H, 4-furan H-5), 7.91 (d, J = 7.9 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.55 (d, J = 3.4 Hz, 1H, 4-furan H-3), 7.32 (t, J = 7.9 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4), 6.99–6.93 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5), 6.75 (dd, J = 3.4, 1.9 Hz, 1H, 4-furan H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 157.45 (2C), 155.60 (2C), 150.75, 145.36, 139.13, 131.19 (2C), 128.59 (2C), 121.99 (2C), 119.31 (2C), 117.48 (2C), 113.71 (2C), 112.88, 111.19. 5.3.14. 2-(4-(Furan-2-yl)-6-(3-hydroxyphenyl)pyridin-2yl)phenol (19) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = f) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = b) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.28 g, 56.0%, 0.83 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.37, mp: 199–200 °C, HPLC: Retention time: 9.46 min, purity: 97.5%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.36. 1 H NMR (250 MHz, DMSO-d6) d 14.34 (s, 1H, 2-phenyl 2-OH), 9.74 (s, 1H, 6-phenyl 3-OH), 8.35 (s, 1H, pyridine H-3), 8.19 (d, J = 7.3 Hz, 1H, 2-phenyl H-6), 8.06 (s, 1H, pyridine H-5), 7.97 (d, J = 0.8 Hz, 1H, 4-furan H-5), 7.64 (d, J = 3.3 Hz, 4-furan H-3), 7.48 (d, J = 7.7 Hz, 1H, 6-phenyl H-6), 7.43 (s, 1H, 6-phenyl H-2), 7.41 (t, J = 7.8 Hz, 1H, 6-phenyl H-5), 7.35 (t, J = 7.5 Hz, 1H, 2-phenyl H-4), 6.99–6.92 (m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4), 6.77 (dd, J = 3.3, 1.7 Hz, 1H, 4-furan H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.33, 158.24, 157.56, 154.88, 150.60, 145.52, 139.94, 138.91, 131.76, 130.39, 127.60, 119.09 (2C), 117.99, 117.58, 117.10, 113.45, 112.95 (2C), 112.09, 111.64. 5.3.15. 2-(4-(Furan-2-yl)-6-(4-hydroxyphenyl)pyridin-2yl)phenol (20) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = f) (0.32 g, 1.50 mmol), dry ammonium acetate

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(1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.27 g, 55.0%, 0.82 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.30, mp: 278–279 °C, HPLC: Retention time: 9.36 min, purity: 97.4%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.35. 1 H NMR (250 MHz, DMSO-d6) d 14.64 (s, 1H, 2-phenyl 2-OH), 9.99 (s, 1H, 6-phenyl 4-OH), 8.27 (s, 1H, pyridine H-3), 8.18 (d, J = 7.9 Hz, 1H, 2-phenyl H-6), 8.03 (s, 1H, pyridine H-5), 7.96 (s, 1H, 4-furan H-5), 7.91(d, J = 8.6 Hz, 2H, 6-phenyl H-2, H-6), 7.63 (d, J = 3.0 Hz, 4-furan H-3), 7.36 (t, J = 7.8 Hz, 1H, 2-phenyl H-5), 6.97 (d, J = 8.4 Hz, 2H, 6-phenyl H-3, H-5), 6.95–6.92 (m, 2H, 2-phenyl H-3, H-5), 6.77 (dd, J = 3.2, 1.7 Hz, 1H, 4-furan H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.53, 159.51, 157.47, 154.83, 150.80, 145.51, 139.93, 131.78, 128.33, 128.25 (2C), 127.55, 119.08, 119.06, 118.07, 116.19 (2C), 113.03, 111.92, 111.56, 111.01. 5.3.16. 3,30 -(4-(Furan-2-yl)pyridine-2,6-diyl)diphenol (21) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = f) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = b) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a light yellow solid (0.28 g, 57.3%, 0.86 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.35, mp: 241–242 °C, HPLC: Retention time: 6.77 min, purity: 99.3%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.36. 1 H NMR (250 MHz, DMSO-d6) d 9.56 (s, 2H, 2-phenyl 3-OH, 6phenyl 3-OH), 8.05 (s, 2H, pyridine H-3, H-5), 7.92 (d, J = 1.5 Hz, 1H, 4-furan H-5), 7.70 (t, J = 1.6 Hz, 2H, 2-phenyl H-2, 6-phenyl H-2), 7.66 (d, J = 7.8 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.53 (d, J = 3.3 Hz, 1H, 4-furan H-3), 7.36 (t, J = 7.9 Hz, 2H, 2-phenyl H-5, 6-phenyl H-5), 6.89 (dd, J = 8.0, 1.9 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4), 6.73 (dd, J = 3.4, 1.7 Hz, 1H, 4-furan H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 157.93 (2C), 156.59 (2C), 151.10, 144.85, 140.12 (2C), 139.03, 129.84 (2C), 117.63 (2C), 116.49 (2C), 113.73 (2C), 112.73 (2C), 112.54, 110.38. 5.3.17. 3-(4-(Furan-2-yl)-6-(4-hydroxyphenyl)pyridin-2yl)phenol (22) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = f) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.28 g, 56.6%, 0.85 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.32, mp: 213–214 °C, HPLC: Retention time: 6.68 min, purity: 99.3%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.36. 1 H NMR (250 MHz, DMSO-d6) d 9.79 (s, 1H, 2-phenyl 3-OH), 9.57 (s, 1H, 6-phenyl 4-OH), 8.13 (d, J = 8.7 Hz, 2H, 6-phenyl H-2, H-6), 8.02 (d, J = 0.8 Hz, 1H, pyridine H-3), 7.97 (d, J = 1.0 Hz, 1H, pyridine H-5), 7.91 (d, J = 1.2 Hz, 1H, 4-furan H-5), 7.70 (t, J = 2.0 Hz, 1H, 2-phenyl H-2), 7.65 (d, J = 7.8 Hz, 1H, 2-phenyl H6), 7.51 (d, J = 3.2 Hz, 1H, 4-furan H-3), 7.31 (t, J = 7.9 Hz, 1H, 2phenyl H-5), 6.93 (d, J = 8.7 Hz, 2H, 6-phenyl H-3, H-5), 6.88 (dd, J = 8.6, 2.4 Hz, 1H, 2-phenyl H-4), 6.73 (dd, J = 3.4, 1.7 Hz, 1H, 4-furan H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.99, 157.96, 156.61, 156.39, 151.33, 144.81, 140.29, 138.97, 129.91, 129.67, 128.36 (2C), 117.63, 116.44, 115.69 (2C), 113.69, 112.77, 111.54, 111.46, 110.25. 5.3.18. 4,40 -(4-(Furan-2-yl)pyridine-2,6-diyl)diphenol (23) The same procedure described in Section 5.3 was employed with 3 (R1 = c, R2 = f) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.51 g, 1.50 mmol) and acetic acid (2 mL) to yield a yellow solid (0.35 g, 70.8%, 1.06 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.17, mp: 253–254 °C, HPLC:

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Retention time: 6.55 min, purity: 98.6%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.36. 1 H NMR (250 MHz, DMSO-d6) d 9.77 (s, 2H, 2-phenyl 4-OH, 6phenyl 4-OH), 8.12 (d, J = 8.4 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.93 (s, 2H, pyridine H-3, H-5), 7.90 (dd, J = 1.5, 0.7 Hz, 4-furan H-5), 7.47 (d, J = 3.4 Hz, 1H, 4-furan H-3), 6.91 (d, J = 8.2 Hz, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5), 6.72 (dd, J = 3.4, 1.7 Hz, 1H, 4-furan H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.82, 156.41 (3C), 151.48 (2C), 144.57, 138.79, 129.81 (3C), 128.24 (4C), 115.60 (4C), 112.61, 110.38, 109.88. 5.3.19. 2,20 -(4-(Furan-3-yl)pyridine-2,6-diyl)diphenol (24) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = g) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = a) (0.61 g, 1.80 mmol) and acetic acid (2 mL) to yield a yellow solid (0.37 g, 75.8%, 1.13 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.46, mp: 209–210 °C, HPLC: Retention time: 8.29 min, purity: 98.4%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.35. 1 H NMR (250 MHz, DMSO-d6) d 12.27 (s, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 8.63 (s, 1H, 4-furan H-2), 8.13 (s, 2H, pyridine H-3, H-5), 7.93 (dd, J = 8.3, 1.7 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.86 (t, J = 1.6 Hz, 1H, 4-furan H-5), 7.34–7.30 (m, 2H, 2-phenyl H-4, 6-phenyl H-4), 7.29 (dd, J = 4.8, 1.5 Hz, 1H, 4-furan H-4), 6.98–6.92 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 157.46 (2C), 155.58 (2C), 145.09, 142.76, 142.03, 131.04 (2C), 128.75 (2C), 124.23, 122.20 (2C), 119.16 (2C), 117.41 (2C), 116.31 (2C), 108.84. 5.3.20. 2-(4-(Furan-3-yl)-6-(3-hydroxyphenyl)pyridin-2yl)phenol (25) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = g) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = b) (0.61 g, 1.80 mmol) and acetic acid (2 mL) to yield a yellow solid (0.27 g, 55.0%, 0.82 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.44, mp: 208–209 °C, HPLC: Retention time: 8.67 min, purity: 99.2%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.35. 1 H NMR (250 MHz, DMSO-d6) d 14.61 (s, 1H, 2-phenyl 2-OH), 9.74 (s, 1H, 6-phenyl 3-OH), 8.74 (s, 1H, 4-furan H-2), 8.35 (s, 1H, pyridine H-3), 8.26 (d, J = 7.7 Hz, 1H, 2-phenyl H-6), 8.09 (s, 1H, pyridine H-5), 7.87 (t, J = 1.4 Hz, 1H, 4-furan H-5), 7.51 (d, J = 7.8 Hz, 1H, 6-phenyl H-6), 7.44 (s, 1H, 6-phenyl H-2), 7.42– 7.31 (m, 3H, 2-phenyl H-4, 6-phenyl H-5, 4-furan H-4), 6.99–6.91 (m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.52, 158.21, 157.55, 154.66, 145.05, 143.19, 142.98, 139.03, 131.68, 130.28, 127.62, 124.12, 118.97, 118.90, 117.99, 117.70, 116.98, 115.75, 114.56, 113.58, 108.93. 5.3.21. 2-(4-(Furan-3-yl)-6-(4-hydroxyphenyl)pyridin-2yl)phenol (26) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = g) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.61 g, 1.80 mmol) and acetic acid (2 mL) to yield a yellow solid (0.30 g, 60.1%, 0.90 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.39, mp: 261–262 °C, HPLC: Retention time: 8.57 min, purity: 95.1%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.35. 1 H NMR (250 MHz, DMSO-d6) d 14.83 (s, 1H, 2-phenyl 2-OH), 9.92 (br, 1H, 6-phenyl 4-OH), 8.71 (s, 1H, 4-furan H-2), 8.26 (s, 1H, pyridine H-3), 8.24 (d, J = 7.8 Hz, 1H, 2-phenyl H-6), 8.05 (s, 1H, pyridine H-5), 7.93 (d, J = 8.6 Hz, 2H, 6-phenyl H-2, H-6), 7.86 (t, J = 1.6 Hz, 1H, 4-furan H-5), 7.40 (d, J = 1.1 Hz, 1H, 4-furan H-

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4), 7.33 (td, J = 7.8, 1.3 Hz, 1H, 2-phenyl H-4), 6.98 (d, J = 8.5 Hz, 2H, 6-phenyl H-3, H-5), 6.98–6.92 (m, 2H, 2-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 159.61, 159.34, 157.37, 154.53, 144.99, 143.04, 142.82, 131.55, 128.28 (2C), 128.26, 127.49, 124.23, 118.96, 118.80, 117.96, 116.04 (2C), 114.60, 113.46, 108.92. 5.3.22. 3,30 -(4-(Furan-3-yl)pyridine-2,6-diyl)diphenol (27) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = g) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = b) (0.61 g, 1.50 mmol) and acetic acid (2 mL) to yield a light yellow solid (0.30 g, 60.3%, 0.90 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.27, mp: 224–225 °C, HPLC: Retention time: 6.07 min, purity: 98.8%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.36. 1 H NMR (250 MHz, DMSO-d6) d 9.60 (s, 2H, 2-phenyl 3-OH, 6-phenyl 3-OH), 8.68 (s, 1H, 4-furan H-2), 8.06 (s, 2H, pyridine H-3, H-5), 7.85 (t, J = 1.8 Hz, 1H, 4-furan H-5), 7.73 (t, J = 1.8 Hz, 2H, 2-phenyl H-2, 6-phenyl H-2), 7.70 (d, J = 7.8 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.37 (d, J = 1.2 Hz, 1H, 4-furan H-4), 7.32 (t, J = 7.8 Hz, 2H, 2-phenyl H-5, 6-phenyl H-5), 6.89 (dd, J = 7.5, 1.8 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 157.98 (2C), 156.51 (2C), 144.99, 142.56, 141.71, 140.43 (2C), 129.86 (2C), 124.52, 117.84 (2C), 116.43 (2C), 115.38 (2C), 113.92 (2C), 109.03. 5.3.23. 3-(4-(Furan-3-yl)-6-(4-hydroxyphenyl)pyridin-2yl)phenol (28) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = g) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.61 g, 1.80 mmol) and acetic acid (2 mL) to yield a light orange solid (0.30 g, 60.0%, 0.90 mmol). TLC (ethyl acetate/n-hexane = 1:1) Rf = 0.51, mp: 226–227 °C, HPLC: Retention time: 5.92 min, purity: 98.8%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.36. 1 H NMR (250 MHz, DMSO-d6) d 9.69 (br, 1H, 2-phenyl 3-OH), 9.51 (br, 1H, 6-phenyl 4-OH), 8.62 (s, 1H, 4-furan H-2), 8.15 (d, J = 8.6 Hz, 2H, 6-phenyl H-2, H-6), 8.00 (s, 1H, pyridine H-3), 7.96 (s, 1H, pyridine H-5), 7.82 (t, J = 1.6 Hz, 4-furan H-5), 7.71 (t, J = 1.8 Hz, 1H, 2-phenyl H-2), 7.67 (d, J = 7.9 Hz, 1H, 2-phenyl H6), 7.33 (t, J = 7.8 Hz, 1H, 2-phenyl H-5), 7.32 (d, J = 1.6 Hz, 1H, 4-furan H-4), 6.92 (d, J = 8.6 Hz, 2H, 6-phenyl H-3, H-5), 6.87 (dd, J = 7.8, 2.2 Hz, 1H, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.80, 157.84, 156.43, 156.25, 144.77, 142.20, 141.41, 140.48, 129.85, 129.66, 128.30 (2C), 124.54, 117.68, 116.20, 115.54 (2C), 114.21, 114.09, 113.79, 108.89. 5.3.24. 4,40 -(4-(Furan-3-yl)pyridine-2,6-diyl)diphenol (29) The same procedure described in Section 5.3 was employed with 3 (R1 = c, R2 = g) (0.32 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = c) (0.61 g, 1.80 mmol) and acetic acid (2 mL) to yield a light yellow solid (0.29 g, 59.1%, 0.88 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.22, mp: 259–260 °C, HPLC: Retention time: 5.81 min, purity: 96.3%; ESI LC/MS: m/z calcd for C21H15NO3 [MH]+ 330.11; found 330.35. 1 H NMR (300 MHz, DMSO-d6) d 9.75 (s, 2H, 2-phenyl 4-OH, 6phenyl 4-OH), 8.61 (s, 1H, 4-furan H-2), 8.15 (d, J = 8.7 Hz, 4H, 2phenyl H-2, H-6, 6-phenyl H-2, H-6), 7.93 (s, 2H, pyridine H-3, H-5), 7.83 (t, J = 1.8 Hz, 1H, 4-furan H-5), 7.32 (d, J = 1.2 Hz, 1H, 4-furan H-4), 6.91 (d, J = 8.4 Hz, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (67.5 MHz, DMSO-d6) d 158.60 (2C), 156.10 (2C), 144.67, 142.04, 141.13, 129.83 (2C), 128.18 (4C), 124.57, 115.40 (4C), 112.91 (2C), 108.81.

5.3.25. 2,20 -([2,40 -Bipyridine]-20 ,60 -diyl)diphenol (30) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = h) (0.33 g, 1.50 mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R3 = a) (0.61 g, 1.80 mmol) and methanol (10 mL) to yield a brown solid (0.15 g, 30.0%, 0.45 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.31, mp: 213–214 °C, HPLC: Retention time: 7.71 min, purity: 97.6%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.36. 1 H NMR (250 MHz, DMSO-d6) d 12.30 (s, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 8.80 (d, J = 3.9 Hz, 1H, 4-pyridine H-6), 8.60 (s, 2H, pyridine H-3, H-5), 8.39 (d, J = 7.9 Hz, 1H, 4-pyridine H-3), 8.05 (td, J = 7.7, 1.6 Hz, 1H, 4-pyridine H-4), 7.96 (dd, J = 7.8, 1.0 Hz, 2phenyl H-6, 6-phenyl H-6), 7.56 (dd, J = 7.0, 4.8 Hz, 1H, 4-pyridine H-5), 7.36 (td, J = 7.7, 1.3 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4), 7.01–6.95 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 157.50 (2C), 155.72 (2C), 153.62, 150.18, 148.15, 137.90, 131.29 (2C), 128.81 (2C), 124.89, 122.23 (2C), 122.08, 119.47 (2C), 117.55 (2C), 117.33 (2C). 5.3.26. 2-(60 -(3-Hydroxyphenyl)-[2,40 -bipyridin]-20 -yl)phenol (31) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = h) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = b) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield a yellow solid (0.13 g, 33.7%, 0.40 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.26, mp: 219–220 °C, HPLC: Retention time: 8.03 min, purity: 97.0%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.37. 1 H NMR (250 MHz, DMSO-d6) d 14.32 (s, 1H, 2-phenyl 2-OH), 9.80 (s, 1H, 6-phenyl 3-OH), 8.82 (d, J = 3.9 Hz, 1H, 4-pyridine H6), 8.76 (s, 1H, pyridine H-3), 8.49 (s, 1H, pyridine H-5), 8.48 (d, J = 7.9 Hz, 1H, 4-pyridine H-3), 8.26 (dd, J = 7.3, 1.3 Hz, 1H, 2-phenyl H-6), 8.07 (td, J = 7.8, 1.7 Hz, 1H, 4-pyridine H-4), 7.57 (dd, J = 7.4, 4.8 Hz, 1H, 4-pyridine H-5), 7.52 (d, J = 7.6 Hz, 1H, 6-phenyl H-6), 7.47 (s, 1H, 6-phenyl H-2), 7.42 (t, J = 7.9 Hz, 1H, 2-phenyl H-4), 7.36 (t, J = 7.6 Hz, 1H, 6-phenyl H-5), 7.01–6.92 (m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.31, 158.34, 157.68, 154.96, 153.28, 150.16, 148.94, 139.10, 137.89, 131.85, 130.57, 127.81, 125.04, 122.23, 119.35, 119.26, 118.06, 117.69, 117.14, 116.46, 115.82, 113.52. 5.3.27. 2-(60 -(4-Hydroxyphenyl)-[2,40 -bipyridin]-20 -yl)phenol (32) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = h) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = c) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield a yellow solid (0.23 g, 58.0%, 0.69 mmol). TLC (ethyl acetate/n-hexane = 1:2) Rf = 0.25, mp: 291–292 °C, HPLC: Retention time: 7.99 min, purity: 99.2%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.37. 1 H NMR (300 MHz, DMSO-d6) d 14.58 (s, 1H, 2-phenyl 2-OH), 9.29 (s, 1H, 6-phenyl 4-OH), 8.81 (d, J = 3.9 Hz, 1H, 4-pyridine H6), 8.67 (s, 1H, pyridine H-3), 8.45–8.43 (m, 2H, 4-pyridine H-3, pyridine H-5), 8.23 (d, J = 7.5 Hz, 1H, 2-phenyl H-6), 8.06 (td, J = 7.5, 1.5 Hz, 1H, 4-pyridine H-4), 7.95 (d, J = 8.7 Hz, 2H, 6-phenyl H-2, H-6), 7.56 (dd, J = 7.2, 4.8 Hz, 1H, 4-pyridine H-5), 7.37 (td, J = 8.4, 1.2 Hz, 1H, 2-phenyl H-4), 6.99 (d, J = 8.7 Hz, 2H, 6-phenyl H-3, H-5), 6.97–6.94 (m, 2H, 2-phenyl H-3, H-5). 13 C NMR (75.5 MHz, DMSO-d6) d 159.34, 159.30, 157.34, 154.75, 153.29, 149.96, 148.69, 137.69, 131.60, 128.26 (3C), 127.47, 124.80, 122.02, 119.10, 119.02, 117.91, 116.09 (2C), 115.27, 114.42. 5.3.28. 3,30 -([2,40 -Bipyridine]-20 ,60 -diyl)diphenol (33) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = h) (0.27 g, 1.20 mmol), dry ammonium acetate

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(0.92 g, 12.00 mmol), 5 (R3 = b) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield an off-white solid (0.24 g, 59.3%, 0.71 mmol). TLC (ethyl acetate/n-hexane = 1:1) Rf = 0.37, mp: 249–250 °C, HPLC: Retention time: 5.32 min, purity: 95.7%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.37. 1 H NMR (250 MHz, DMSO-d6) d 9.62 (s, 2H, 2-phenyl 3-OH, 6phenyl 3-OH), 8.79 (d, J = 4.3 Hz, 1H, 4-pyridine H-6), 8.47 (s, 2H, pyridine H-3, H-5), 8.41 (d, J = 7.9 Hz, 1H, 4-pyridine H-3), 8.04 (td, J = 7.7, 1.5 Hz, 1H, 4-pyridine H-4), 7.74 (s, 2H, 2-phenyl H-2, 6-phenyl H-2), 7.72 (d, J = 8.0 Hz, 2-phenyl H-6, 6-phenyl H-6), 7.54 (dd, J = 7.2, 4.8 Hz, 1H, 4-pyridine H-5), 7.34 (t, J = 7.8 Hz, 2H, 2-phenyl H-5, 6-phenyl H-5), 6.90 (dd, J = 7.9, 2.0 Hz, 2H, 2phenyl H-4, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.04 (2C), 156.73 (2C), 153.90, 150.08, 148.00, 140.31 (2C), 137.83, 130.02 (2C), 124.68, 121.83, 117.78 (2C), 116.55 (2C), 116.02 (2C), 113.81 (2C). 5.3.29. 3-(60 -(4-Hydroxyphenyl)-[2,40 -bipyridin]-20 -yl)phenol (34) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = h) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = c) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield a yellow solid (0.25 g, 62.2%, 0.74 mmol). TLC (ethyl acetate/n-hexane = 1:1) Rf = 0.35, mp: 234–235 °C, HPLC: Retention time: 5.33 min, purity: 95.9%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.36. 1 H NMR (250 MHz, DMSO-d6) d 9.80 (s, 1H, 2-phenyl 3-OH), 9.58 (s, 1H, 6-phenyl 4-OH), 8.79 (d, J = 4.0 Hz, 1H, 4-pyridine H6), 8.41 (s, 1H, pyridine H-3), 8.38–8.35 (m, 2H, 4-pyridine H-3, pyridine H-5), 8.17 (d, J = 8.5 Hz, 6-phenyl H-2, H-6), 8.03 (t, J = 7.8 Hz, 1H, 4-pyridine H-4), 7.72 (s, 1H, 2-phenyl H-2), 7.70 (d, J = 7.7 Hz, 2-phenyl H-6), 7.53 (dd, J = 7.4, 4.9 Hz, 1H, 4-pyridine H-5), 7.36 (t, J = 7.9 Hz, 1H, 2-phenyl H-5), 6.94 (d, J = 8.5 Hz, 2H, 6phenyl H-3, H-5), 6.88 (d, J = 8.0 Hz, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.98, 158.01 (2C), 156.79, 156.50, 154.12, 150.04, 147.88, 140.45, 137.77, 130.00, 129.86, 128.46 (2C), 124.58, 121.76, 117.72, 116.43, 115.76 (2C), 114.91 (2C), 113.73. 5.3.30. 4,40 -([2,40 -Bipyridine]-20 ,60 -diyl)diphenol (35) The same procedure described in Section 5.3 was employed with 3 (R1 = c, R2 = h) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = c) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield a light yellow solid (0.22 g, 56.0%, 0.67 mmol). TLC (ethyl acetate/n-hexane = 1:1) Rf = 0.29, mp: 268–269 °C, HPLC: Retention time: 5.24 min, purity: 95.0%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.36. 1 H NMR (300 MHz, DMSO-d6) d 9.80 (s, 2H, 2-phenyl 4-OH, 6phenyl 4-OH), 8.78 (d, J = 4.5 Hz, 1H, 4-pyridine H-6), 8.36–8.33 (m, 3H, 4-pyridine H-3, pyridine H-3, H-5), 8.16 (d, J = 8.7 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 8.02 (td, J = 7.8, 1.8 Hz, 1H, 4-pyridine H-4), 7.52 (dd, J = 7.5, 4.8 Hz, 1H, 4-pyridine H-5), 6.92 (d, J = 8.7 Hz, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (75.5 MHz, DMSO-d6) d 158.71 (2C), 156.41 (2C), 154.16, 149.85, 147.58, 137.58, 129.84 (2C), 128.25 (4C), 124.34, 121.54, 115.56 (4C), 113.65 (2C). 5.3.31. 2,20 -([3,40 -Bipyridine]-20 ,60 -diyl)diphenol (36) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = i) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = a) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield a yellow solid (0.16 g, 41.2%, 0.49 mmol). TLC (ethyl acetate/n-hexane = 2:1) Rf = 0.23, mp: 269–270 °C, HPLC: Retention time: 5.73 min, purity: 99.5%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.37.

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H NMR (300 MHz, DMSO-d6) d 12.25 (s, 2H, 2-phenyl 2-OH, 6phenyl 2-OH), 9.19 (s, 1H, 4-pyridine H-2), 8.72 (d, J = 3.9 Hz, 1H, 4-pyridine H-6), 8.41 (d, J = 8.1 Hz, 1H, 4-pyridine H-4), 8.28 (s, 2H, pyridine H-3, H-5), 8.00 (d, J = 7.2 Hz, 2-phenyl H-6, 6-phenyl H-6), 7.62 (dd, J = 7.8, 4.8 Hz, 1H, 4-pyridine H-5), 7.35 (t, J = 7.2 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4), 6.99–6.94 (m, 4H, 2phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (75.5 MHz, DMSO-d6) d 157.35 (2C), 155.62 (2C), 150.42, 148.39, 146.95, 135.12, 133.24, 131.18 (2C), 128.93 (2C), 124.11, 121.99 (2C), 119.26 (2C), 117.84 (2C), 117.34 (2C). 5.3.32. 2-(60 -(3-Hydroxyphenyl)-[3,40 -bipyridin]-20 -yl)phenol (37) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = i) (0.27 g, 1.50 mmol), dry ammonium acetate (0.92 g, 15.00 mmol), 5 (R3 = b) (0.40 g, 1.50 mmol) and MeOH (10 mL) to yield a yellow solid (0.25 g, 61.2%, 0.73 mmol). TLC (ethyl acetate/n-hexane = 3:1) Rf = 0.34, mp: 239–240 °C, HPLC: Retention time: 5.96 min, purity: 99.4%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.37. 1 H NMR (250 MHz, DMSO-d6) d 14.32 (s, 1H, 2-phenyl 2-OH), 9.74 (br, 1H, 6-phenyl 3-OH), 9.26 (d, J = 1.9 Hz, 1H, 4-pyridine H-2), 8.73 (dd, J = 4.7, 1.5 Hz, 1H, 4-pyridine H-6), 8.49 (s, 1H, pyridine H-3), 8.46–8.45 (m, 1H, 2-phenyl H-6), 8.33 (dd, J = 8.4, 1.6 Hz, 1H, 4-pyridine H-4), 8.20 (d, J = 1.1 Hz, 1H, pyridine H-5), 7.62 (dd, J = 7.5, 4.8 Hz, 1H, 4-pyridine H-5), 7.57 (d, J = 7.5 Hz, 1H, 6-phenyl H-6), 7.49 (t, J = 2.0 Hz, 1H, 6-phenyl H-2), 7.38 (t, J = 7.8 Hz, 1H, 6-phenyl H-5), 7.35 (dt, J = 7.8, 1.4 Hz, 1H, 2-phenyl H-4), 6.99–6.95 (m, 2H, 2-phenyl H-3, H-5), 6.94 (dd, J = 7.9, 2.4 Hz, 1H, 6-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.29, 158.24, 157.62, 154.95, 150.56, 148.61, 147.89, 138.99, 135.28, 133.02, 131.78, 130.33, 128.01, 124.04, 119.20, 119.07, 117.92, 117.88, 117.25, 117.08, 116.49, 113.67. 5.3.33. 2-(60 -(4-Hydroxyphenyl)-[3,40 -bipyridin]-20 -yl)phenol (38) The same procedure described in Section 5.3 was employed with 3 (R1 = a, R2 = i) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = c) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield an off-white solid (0.20 g, 51.0%, 0.61 mmol). TLC (ethyl acetate/n-hexane = 3:1) Rf = 0.25, mp: 308–309 °C, HPLC: Retention time: 5.98 min, purity: 97.5%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.37. 1 H NMR (300 MHz, DMSO-d6) d 14.64 (s, 1H, 2-phenyl 2-OH), 10.00 (s, 1H, 6-phenyl 4-OH), 9.26 (d, J = 1.8 Hz, 1H, 4-pyridine H-2), 8.72 (dd, J = 4.5, 1.2 Hz, 1H, 4-pyridine H-6), 8.48 (dt, J = 8.1, 1.8 Hz, 1H, 4-pyridine H-4), 8.40 (s,1H, pyridine H-3), 8.31 (d, J = 6.9 Hz, 1H, 2-phenyl H-6), 8.17 (s, 1H, pyridine H-5), 7.99 (d, J = 8.7 Hz, 2H, 6-phenyl H-2, H-6), 7.61 (dd, J = 7.8, 4.8 Hz, 1H, 4-pyridine H-5), 7.37 (t, J = 8.4 Hz, 1H, 2-phenyl H-4), 6.97 (d, J = 8.7 Hz, 2H, 6-phenyl H-3, H-5), 6.96–6.93 (m, 2H, 2-phenyl H3, H-5). 13 C NMR (75.5 MHz, DMSO-d6) d 159.54 (2C), 157.58, 154.92, 150.64, 148.71, 147.86, 135.39, 133.23, 131.83, 128.62 (2C), 128.37, 127.99, 124.18, 119.18, 119.11, 118.04, 116.26, 116.19 (2C), 115.35. 5.3.34. 3,30 -([3,40 -Bipyridine]-20 ,60 -diyl)diphenol (39) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = i) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = b) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield an off-white solid (0.16 g, 40.8%, 0.48 mmol). TLC (ethyl acetate/n-hexane = 3:1) Rf = 0.25, mp: 329-330 °C, HPLC: Retention time: 4.66 min, purity: 97.0%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.37.

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H NMR (250 MHz, DMSO-d6) d 9.64 (s, 2H, 2-phenyl 3-OH, 6phenyl 3-OH), 9.21 (s, 1H, 4-pyridine H-2), 8.70 (d, J = 4.0 Hz, 1H, 4-pyridine H-6), 8.45 (d, J = 8.0 Hz, 1H, 4-pyridine H-4), 8.17 (s, 2H, pyridine H-3, H-5), 7.79-7.72 (m, 4H, 2-phenyl H-2, 6-phenyl H-2, 2-phenyl H-6, 6-phenyl H-6), 7.60 (dd, J = 7.9, 4.8 Hz, 1H, 4pyridine H-5), 7.36 (t, J = 7.8 Hz, 2H, 2-phenyl H-5, 6-phenyl H5), 6.90 (dd, J = 8.0, 1.7 Hz, 2H, 2-phenyl H-4, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 158.02 (2C), 156.79 (2C), 150.32, 148.52, 146.81, 140.30 (2C), 135.18, 133.64, 129.96 (2C), 124.20, 118.03 (2C), 116.91 (2C), 116.59 (2C), 114.03 (2C). 5.3.35. 3-(60 -(4-Hydroxyphenyl)-[3,40 -bipyridin]-20 -yl)phenol (40) The same procedure described in Section 5.3 was employed with 3 (R1 = b, R2 = i) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = c) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield an off-white solid (0.21 g, 53.0%, 0.63 mmol). TLC (ethyl acetate/n-hexane = 3:1) Rf = 0.25, mp: 277-278 °C, HPLC: Retention time: 4.33 min, purity: 95.5%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.36. 1 H NMR (250 MHz, DMSO-d6) d 9.76 (br, 1H, 2-phenyl 3-OH), 9.65 (br, 1H, 6-phenyl 4-OH), 9.21 (d, J = 2.0 Hz, 1H, 4-pyridine H-2), 8.70 (dd, J = 4.7, 1.2 Hz, 1H, 4-pyridine H-6), 8.43 (dt, J = 8.0, 1.7 Hz, 1H, 4-pyridine H-4), 8.21 (d, J = 8.6 Hz, 6-phenyl H-2, H-6), 8.13 (s, 1H, pyridine H-3), 8.08 (s, 1H, pyridine H-5), 7.75 (s, 1H, 2-phenyl H-2), 7.73 (d, J = 8.0 Hz, 2-phenyl H-6), 7.59 (dd, J = 7.9, 4.8 Hz, 1H, 4-pyridine H-5), 7.34 (t, J = 7.8 Hz, 1H, 2phenyl H-5), 6.93 (d, J = 8.6 Hz, 2H, 6-phenyl H-3, H-5), 6.88 (dd, J = 7.5, 2.0 Hz, 2-phenyl H-4). 13 C NMR (62.5 MHz, DMSO-d6) d 159.00, 157.95 (2C), 156.78, 156.55, 150.21, 148.43, 146.59, 140.39, 135.05, 133.74, 129.86, 129.77, 128.60 (2C), 124.12, 117.92, 116.42, 115.66 (3C), 113.93. 5.3.36. 4,40 -([3,40 -Bipyridine]-20 ,60 -diyl)diphenol (41) The same procedure described in Section 5.3 was employed with 3 (R1 = c, R2 = i) (0.27 g, 1.20 mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R3 = c) (0.40 g, 1.20 mmol) and methanol (10 mL) to yield a light orange solid (0.18 g, 45.1%, 0.54 mmol). TLC (ethyl acetate/n-hexane = 3:1) Rf = 0.23, mp: 316-317 °C, HPLC: Retention time: 4.27 min, purity: 97.9%; ESI LC/MS: m/z calcd for C22H16N2O2 [MH]+ 341.13; found 341.37. 1 H NMR (250 MHz, DMSO-d6) d 9.79 (s, 2H, 2-phenyl 4-OH, 6phenyl 4-OH), 9.19 (d, J = 1.4 Hz, 1H, 4-pyridine H-2), 8.69 (dd, J = 4.7, 1.2 Hz, 1H, 4-pyridine H-6), 8.42 (dt, J = 8.3, 2.0 Hz, 1H, 4pyridine H-4), 8.19 (d, J = 8.7 Hz, 4H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6), 8.03 (s, 2H, pyridine H-3, H-5), 7.59 (dd, J = 7.8, 4.8 Hz, 1H, 4-pyridine H-5), 6.92 (d, J = 8.7 Hz, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5). 13 C NMR (62.5 MHz, DMSO-d6) d 158.90 (2C), 156.61 (2C), 150.14, 148.39, 146.42, 134.98, 133.90, 129.92 (2C), 128.56 (4C), 124.11, 115.63 (4C), 114.51 (2C).

volume of 10 lL was terminated by adding 2.5 lL of the stop solution containing 5% sarcosyl, 0.0025% bromophenol blue and 25% glycerol. DNA samples were then electrophoresed on a 1% agarose gel at 15 V for 7 h with a running buffer of TAE. Gels were stained for 30 min in an aqueous solution of ethidium bromide (0.5 lg/ mL). DNA bands were visualized by transillumination with UV light and were quantitated using AlphaImager™ (Alpha Innotech Corporation). 5.4.2. Assay for DNA Topoisomerase II Inhibition in vitro DNA topo II inhibitory activity of compounds were measured as follows. 35 The mixture of 200 ng of supercoiled pBR322 plasmid DNA and 1 unit of human DNA topoisomerase IIa (Usb Corp., USA) was incubated without and with the prepared compounds in the assay buffer (10 mM Tris–HCl (pH 7.9) containing 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15 lg/mL bovine serum albumin) for 30 min at 30 °C. The reaction in a final volume of 20 lL was terminated by the addition of 3 lL of 7 mM EDTA. Reaction products were analyzed on 1% agarose gel at 25 V for 4 h with a running buffer of TAE. Gels were stained for 30 min in an aqueous solution of ethidium bromide (0.5 lg/mL). DNA bands were visualized by transillumination with UV light and supercoiled DNA was quantitated using AlphaImager™ (Alpha Innotech Corporation). 5.4.3. Cytotoxicity assay Cancer cells were cultured according to the supplier’s instructions. Cells were seeded in 96-well plates at a density of 2– 4  104 cells per well and incubated for overnight in 0.1 mL of media supplied with 10% Fetal Bovine Serum (Hyclone, USA) in 5% CO2 incubator at 37 °C. On day 2, after FBS starvation for 4 h, culture medium in each well was exchanged with 0.1 mL aliquots of medium containing graded concentrations of compounds. On day 4, each well was added with 5 lL of the cell counting kit-8 solution (Dojindo, Japan) then incubated for additional 4 h under the same condition. The absorbance of each well was determined by an Automatic Elisa Reader System (Bio-Rad 3550) at 450 nm wavelength. For determination of the IC50 values, the absorbance readings at 450 nm were fitted to the four-parameter logistic equation. The compounds like adriamycin, etoposide, and camptothecin were purchased from Sigma and used as positive controls. Acknowledgement This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A2A2A01046188). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.04.002.

5.4. Pharmacology References and notes 5.4.1. Assay for DNA topoisomerase I inhibition in vitro DNA topo I inhibition assay was determined following the method reported by Fukuda et al. 34 with minor modifications. The test compounds were dissolved in DMSO at 20 mM as stock solution. The activity of DNA topo I was determined by assessing the relaxation of supercoiled DNA pBR322. The mixture of 100 ng of plasmid pBR322 DNA and 1 unit of recombinant human DNA topoisomerase I (TopoGEN INC., USA) was incubated without and with the prepared compounds at 37 °C for 30 min in the relaxation buffer (10 mM Tris–HCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5% glycerol). The reaction in the final

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

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Topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship study of dihydroxylated 2,6-diphenyl-4-aryl pyridines.

A new series of thirty-six dihydroxylated 2,6-diphenyl-4-aryl pyridines containing hydroxyl groups at the ortho, meta, or para position of 2- and 6-ph...
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