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

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

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 , Tara Man Kadayat , Til Bahadur Thapa Magar , Ganesh Bist ,

b,

a

c

b,

⇑

a,

⇑

Aarajana Shrestha , Younghwa Na , Youngjoo Kwon , Eung-Seok Lee

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

College of Pharmacy, Cha University, Pochon 487-010, Republic of Korea

b

c

a r t i c l e i n f o

a b s t r a c t

Article history:

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 cytotoxi-

city 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.

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

Ó 2015 Published by Elsevier Ltd.

Anticancer agents

1

. Introduction

novel compounds with improved therapeutic efﬁcacy and less toxi-

3

,8,9

city.

In an attempt to discover novel topo inhibitors, our research

Several of the most effective anticancer drugs such as etoposide,

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

camptothecin, and doxorubicin have been reported to interrupt the

normal catalytic cycle of DNA topoisomerase.¹This enzyme solves

various DNA topological problems associated with DNA replication,

–3

1

0–12

cancer cell lines.

4

transcription, recombination, and other vital cellular processes. In

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,

general, DNA topoisomerases (topos) are classiﬁed into two types:

topoisomerase I (topo I), which breaks single strands of DNA, and

5

topoisomerase II (topo II), which breaks double strands of DNA.

Unlike topo I, topo II acts as a homodimeric enzyme and requires

Mg (II) and ATP hydrolysis for enzyme turnover and rapid kinetics.

anti-inﬂammatory, antioxidant, antibacterial and antiangiogenic

1

,6

13–17

activities.

In the course of our research on hydroxylated

Inhibitionof topos duringtheir vital processes leads toapoptosis and

sequential cell death. This has established topos as a prime molecu-

lar target for the development of anticanceragents. A series of drugs

that speciﬁcally target topos have been widely used clinically as

anticancer agents. However, due to their severe side effects, exten-

sive research is being conducted to develop new derivatives and

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.¹

Results indicated that in general, dihydroxylated 2,4,6-triphenyl

pyridines exhibited stronger topo II inhibitory activity and cytotox-

icity compared to those of monohydroxylated 2,4,6-triphenyl

7

8–21

⇑

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.).

19

pyridines. The structure–activity relationship studies demon-

strated that hydroxylation at meta or para position of 2-phenyl ring

of central pyridine in dihydroxylated 2,4,6-triphenyl pyridines is

+

E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), eslee@yu.ac.kr (E.-S. Lee).

Authors have equally contributed to this work.

http://dx.doi.org/10.1016/j.bmc.2015.04.002

968-0896/Ó 2015 Published by Elsevier Ltd.

0

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

OH

R²

N

R¹

1

N

3

R³

N

HO

2

R , R , R = aryl

2

,4,6-triaryl pyridines

2,4,6-triphenyl pyridine

monohydroxylated 2,4,6-triphenyl pyridines

OH

N

HO

OH

HO

OH

dihydroxylated 2,4,6-triphenyl pyridines

trihydroxylated 2,4,6-triphenyl pyridine

OH

_R2

R1, R2 = OH

3

R

= 3 -thienyl

2 -furyl

4

2/ 3/ 4 -pyridyl

2

6

_R3

O

N

R¹

HO

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

2,4-diphenyl-6-aryl pyridine

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

important for signiﬁcant topo II inhibitory activity as well as cyto-

signiﬁcant topo II inhibitory activity and cytotoxicity.¹⁹As an

extension to this work, we further synthesized compounds hav-

ing 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 condensa-

toxicity.¹

8,19

Trihydroxylated 2,4,6-triphenyl pyridines were

reported as topo II inhibitors with better selectivity than mono-

and dihydroxylated 2,4,6-triphenyl pyridines only in high micro-

2

0

molar concentration. Our research group has recently reported

the synthesis of dihydroxylated 2,4-diphenyl-6-aryl pyridines pos-

sessing various aryl groups on 6-phenyl ring of central pyridines

tion reaction, eighteen hydroxylated propenone intermediates

were synthesized via base (NaOH) catalyzed reaction²

6,27

with-

(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-posi-

tion of central pyridine displayed the most potent topo inhibitory

activity in a low micromolar concentration and functioned as a

potent topo II poison. Moreover, we have found in previous stud-

ies that introduction of an aryl group such as thienyl or furyl group

out the protection of hydroxyl groups. In this method, 4 M aque-

ous solution of NaOH was added to the solution of equimolar

1

amounts of aryl ketone

1

(R = a–c) and aryl aldehyde

2

1

2

1

2

(R = d–i) in ethanol to obtain compounds 3 (R = a–c, R = d–i)

with the yield of 47–98%. In the second step, three pyridinium

3

at 4-position of central pyridine is important for signiﬁcant topo II

iodide salts 5 (R = a–c) were synthesized in quantitative yield

inhibitory activity and cytotoxicity.²

2–25

As a continuation of devel-

by the treatment of aryl ketone

4

(R = a–c) with iodine

1

28,29

oping novel potent anticancer agents, these encouraging results

have prompted us to synthesize and evaluate the biological activ-

ities of dihydroxylated 2,6-diphenyl-4-aryl pyridines possessing 2/

(1.2 equiv) in pyridine. Using modiﬁed Kröhnke synthesis,

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.

3

-thienyl, 2/3-furyl, and 2/3-pyridyl moieties on 4-position of cen-

tral 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 pyr-

idines. These compounds were evaluated for topo I and II inhibi-

tory activity and cytotoxicity against several human cancer cell

lines. Our work represents the ﬁrst systematic study of dihydroxy-

lated 2,6-diphenyl-4-aryl pyridines to develop potential anticancer

agents.

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 pyri-

dinium iodide salts as shown in Fig. 3. All the compounds con-

tained two hydroxyl moieties substituted at various positions

(ortho, meta, or para) of phenyl ring at 2- and 6-position of cen-

tral 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 2,6-

diphenyl-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 pre-

vious data. Yield (%) and HPLC purity (%) of the compounds are

shown in Table 1.

2

. Chemistry

Previously our research group reported the synthesis of dihy-

droxylated 2,4,6-triphenyl pyridine compounds which possessed

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

3

S

O

_R2

4

O

2

6

N

_R1

_R3

HO

OH

N

2

,4,6-trisubstituted pyridine dihydroxylated 2,4,6-triphenyl pyridine

significant topo II inhibition

S

O

N

HO

OH

HO

OH

HO

OH

Series A

Series B

Series C

O

N

HO

OH

HO

OH

HO

OH

Series D

Series E

Series F

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

O

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

H

_R2

_R1

2

CH3

2

(R

= d-i)

R¹

O

i

(R¹= a-c)

O

(R¹= a-c, R²= d-i)

1

3

.1. Topoisomerase I inhibitory activity

3

_R2

Compounds 6–11, which possess 2-thienyl moiety, exhibited

strong topo I inhibitory activity at 100

lM (Fig. 4). Compounds 7,

iii

R¹

N

R³

9–11 showed stronger effect than the positive control, camp-

6

-41

tothecin. 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

I

N

R³

CH3

R³

O

ii

₍_R1 = a-c)

₅₍_R3 = a-c)

topo I inhibition at 100 lM (59.2% and 61.7%, respectively). All

the compounds (18–23), which contain 2-furyl moiety at 4-posi-

4

tion of central pyridine, showed stronger topo I inhibition than

OH

the camptothecin (80.7%) at 100

ited 90.1% and 97.8% inhibition respectively, while 22 and 23

possessed 100% inhibition at 100 M. Only one, compound 29 with

3-furyl moiety at 4-position of central pyridine showed consider-

able inhibitory activity (74.9%) at 100 M. Compounds 25–28 from

lM: compounds 19 and 21 exhib-

OH

HO

1

R , R³

=

l

a

b

c

l

R²

=

S

the same group possessed weaker inhibition compared to the pos-

itive control. Compound 31 containing 2-pyridyl moiety showed

weak inhibition (35.4%), and compound 36 with 3-pyridyl moiety

O

N

S

O

N

d

e

f

g

h

i

exhibited moderate inhibitory activity (66.4%) at 100

compounds with 2- and 3-pyridyl moieties had no signiﬁcant

inhibitory activity at both 100 and 20 M. Taken together, several

evaluated compounds possessed signiﬁcant topo I inhibitory

activity at 100 M but none of the compounds were active at the

concentration of 20 M.

lM. All other

Scheme 1. General synthetic method of dihydroxylated 2,6-diphenyl-4-aryl

pyridines. Reagents and conditions: (i) aryl aldehydes 2 (R = d–i) (1.0 equiv),

NaOH, EtOH, 2–8 h, 20 °C, 47–98% yield; (ii) pyridine, iodine, 3 h, 140 °C; (iii)

2

l

NH

4

OAc (10.0 equiv), glacial acetic acid/methanol, 12–16 h, 80–100 °C, 30–83%

yield.

l

3

. Results and discussion

All the synthesized compounds (6–41) contained two hydroxyl

3.2. Topoisomerase II inhibitory activity

All compounds (6–11) with 2-thienyl moiety possessed moder-

moieties, one in each phenyl ring at 2- and 6-position with 2/3-

thienyl, 2/3-furyl, or 2/3-pyridyl at 4-position of central pyridine.

ate to signiﬁcant topo II inhibitory activity at 100

Compound 10 showed 88.6% inhibition which was higher than

lM (Fig. 5).

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

S

OH

HO

OH HO

N

6

7

8

OH

9

10

OH HO

11

OH

S

OH

HO

OH HO

N

12

13

14

OH

15

16

OH HO

17

OH

O

OH

HO

OH HO

N

19

20

21

22

23

18

OH

OH HO

OH

O

OH

HO

OH HO

N

24

25

2

6

OH

27

28

OH HO

29

OH

N

OH

HO

OH HO

N

31

32

33

34

35

30

OH

OH HO

OH

N

OH

HO

OH HO

N

36

37

38

OH

39

40

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% inhibi-

tion at 100 M. Compound 8 showed 76.4% and 30.2% inhibition

at 100 and 20 M, respectively. Compound 6 showed moderate

inhibition (55.9%) at 100 M while compound 11 possessed

4.6% and 30.3% inhibition at 100 and 20 M, respectively. All

the compounds (12–17), which contain 3-thienyl moiety, showed

stronger inhibition than etoposide at 100 M. Compounds 15–17

showed 88.1%, 91.5%, and 81.2% inhibition at 100 M and 23.0%,

4.7%, and 57.9% inhibition at 20 M, respectively. All the com-

moderate inhibitory activity (65.5%, 62.5%, and 52.2%, respectively)

at 100 M. 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

l

7

l

compounds exhibited moderate to signiﬁcant topo II inhibitory

l

activity at 100

well.

lM, with several compounds active at 20 lM as

l

4

l

pounds (19–23), which possess 2-furyl moiety, showed stronger

inhibitory activity than etoposide, except compound 18.

3.3. Cytotoxicity

Compound 18 showed moderate inhibition (52.2%) at 100

while compound 23 exhibited 92.3% and 48.4% inhibition at 100

and 20 M, respectively. Similarly, compounds 19, 21, and 22

showed signiﬁcant inhibition at 20 M. Among compounds con-

taining 3-furyl moiety (24–29), compounds 25 and 26 showed

moderate activity. Compound 28 possessed signiﬁcant inhibitory

l

M

The cytotoxicity of compounds were evaluated with four differ-

ent 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 camp-

tothecin, widely used anticancer drugs, were used as positive con-

trols. Several compounds displayed signiﬁcant cytotoxicity against

the tested cell lines at low micromolar concentration compared to

l

activity of 95% and 19.7% at 100 and 20

lM, respectively.

Compounds 30, 31, and 33, possessing 2-pyridyl moiety, showed

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

5

Table 1

position of hydroxyl group of phenyl ring. Among compounds con-

taining 3-furyl, 2-pyridyl, and 3-pyridyl moiety, compounds 25, 26,

Prepared compounds with yield, and purity by HPLC

2

8, 30, 31, 33, and 37 showed considerable topo II inhibitory activ-

ity at 100 M. Compounds 8–11, 15–17, 21–23, and 41 possessed

signiﬁcant topo II inhibitory activity at both 100 and 20 M. It is

interesting to note that most of these compounds contain hydroxyl

moiety at meta or para position of 2- and/or 6-phenyl ring attached

to central pyridine (Fig. 6).

It is observed that with the exception of compound 37, all the

compounds which possessed an ortho hydroxyl moiety at 2-phenyl

position of central pyridine were less cytotoxic (IC50 >50 lM) in

R²

l

R¹

N

R³

Entry

R¹

R²

R³

Yield (%)

Purity (%)

0

6

7

8

9

2-OH phenyl

3-OH phenyl

4-OH phenyl

2-OH phenyl

3-OH phenyl

4-OH phenyl

2-OH phenyl

3-OH phenyl

4-OH phenyl

2-OH phenyl

3-OH phenyl

4-OH phenyl

2-OH phenyl

3-OH phenyl

4-OH phenyl

2-OH phenyl

3-OH phenyl

4-OH phenyl

2-Thienyl

3-Thienyl

2-Furyl

3-Furyl

2 -OH phenyl

3 -OH phenyl

4 -OH phenyl

3 -OH phenyl

4 -OH phenyl

2 -OH phenyl

3 -OH phenyl

4 -OH phenyl

3 -OH phenyl

4 -OH phenyl

2 -OH phenyl

3 -OH phenyl

4 -OH phenyl

3 -OH phenyl

4 -OH phenyl

2 -OH phenyl

3 -OH phenyl

4 -OH phenyl

3 -OH phenyl

4 -OH phenyl

2 -OH phenyl

3 -OH phenyl

4 -OH phenyl

3 -OH phenyl

4 -OH phenyl

2 -OH phenyl

3 -OH phenyl

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

98.6

98.4

99.2

95.1

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

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

1

2

3

4

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

22–25,30–33

activity. These results go in line with previous studies

and support the importance of 2-furyl, 2-thienyl, or 3-thienyl moi-

ety at the 4-position on central pyridine for signiﬁcant topo II inhi-

bitory 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 (<2 M)

l

against most of the cancer cell lines. Structure–activity relationship

study revealed that hydroxyl moiety at para position is more favor-

able for topo inhibitory activity and cytotoxicity with 2-furyl, 2-

thienyl, or 3-thienyl moiety at 4-position of central pyridine, as

indicated in Fig. 7. Among all the synthesized compounds, com-

pound 17 was found to be the most potent with signiﬁcant topo

II inhibition as well as cytotoxicity.

3-Furyl

2-Pyridyl

3-Pyridyl

4

. Conclusion

A total of thirty-six dihydroxylated 2,6-diphenyl-4-aryl pyri-

dine derivatives were systematically designed and synthesized

by efﬁcient synthetic routes and evaluated for topo I and II inhibi-

tory activity and cytotoxicity against various human cancer cell

lines. The topo inhibitory activities and cytotoxicity of 2,6-diphe-

nyl-4-aryl pyridines were greatly inﬂuenced by the position of

hydroxyl group in 2- and 6-phenyl rings, and types of aryl moieties

attached to central pyridine. Structure–activity relationship study

revealed that introduction of hydroxyl moiety either at meta or

para position of 2- or 6-phenyl rings in combination with 2-furyl,

0

4 -OH phenyl

3 -OH phenyl

4 -OH phenyl

the positive controls (Table 2). Among compounds 6–17, which pos-

sessed 2- or 3-thienyl moiety at 4-position of central pyridine, com-

pounds 6–8 and 12–14 showed relatively lower cytotoxicity (IC50

2

-thienyl, or 3-thienyl moiety at 4-position of central pyridine is

more important for signiﬁcant topo II inhibitory activity and cyto-

toxicity. Compounds 9, 10, 15–17, 19, 22, and 33–35 exhibited sig-

niﬁcant cytotoxicity (IC50 <2 lM) against T47D. Among all the

synthesized compounds, compound 41 possessed strong cytotoxi-

city against all four cancer cell lines. In general, it was observed

that several compounds were more cytotoxic against T47D than

other evaluated cell lines. Positive correlation between topo II inhi-

bition and cytotoxicity was observed for the compounds 9–11, 15–

>

50

and 16 possessed considerable cytotoxicity against DU145, HCT15,

and HeLa, but were much stronger (<0.78 M) than the positive con-

lM) in all four cancer cell lines (Table 2). Compounds 9, 10,

l

trols against T47D. Compound 11 also showed considerable cytotox-

icity against DU145. Compounds 15–17 had signiﬁcant activity

(

<2

or 3-furyl (18–29), compounds 18, 20 and 24–26 were less cytotoxic

>50 M) in most of the cell lines. Compounds 19, 21–23, and 27–29

exhibited signiﬁcant cytotoxicity (<2 M) in most of the cell lines.

Compounds with 2- or 3-pyridyl moiety (30–40) showed weak cyto-

toxicity (>50 M) in most of the cell lines. Compounds 33–35 and 41

lM) in most of the cell lines. Among compounds containing 2-

(

l

1

7, 19, 21–23, 28, and 41. Compound 17 emerged as the most

l

promising topo II inhibitor with signiﬁcant cytotoxicity.

In summary, for the ﬁrst time to our knowledge we investigated

dihydroxylated 2,6-diphenyl-4-aryl pyridines for the development

of potent anticancer agents. The present results revealed signiﬁ-

cant topo II inhibitory activity and cytotoxicity of some com-

pounds, which encourages further investigations to identify

modes of action involved in topo II inhibition and antitumor activ-

ity of these compounds.

l

exhibited signiﬁcant cytotoxicity against T47D. Compound 41 pos-

sessed strong cytotoxicity against all four cell lines. It was observed

that several compounds were more cytotoxic to T47D in comparison

to other used cell lines.

3

.4. Structure–activity relationship (SAR) study

A structure–activity relationship study was performed accord-

5. Experimental

ing to the results of topo II inhibitory activity and cytotoxicity of

the evaluated compounds. As shown in Table 2, most of the com-

pounds with 2-thienyl, 3-thienyl, and 2-furyl moiety exhibited sig-

Compounds used as starting materials and reagents were

obtained from Aldrich Chemical Co., Junsei or other chemical com-

panies, and used without further puriﬁcation. HPLC grade

niﬁcant topo II inhibitory activity at 100 lM irrelevant to the

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6

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

Figure 4. Human DNA topo I inhibitory activity of compounds 6–41.

Figure 5. Human DNA topo II inhibitory activity of compounds 6–41.

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

7

Table 2

Topo I and II inhibitory activities, and cytotoxicity of the prepared compounds 6–41

Compounds

% inhibition

IC50^a

(

lM)

Topo I

Topo II

DU145

HCT15

T47D

HeLa

100

lM

20

lM

100

l

M

20 lM

Camptothecin

Etoposide

Adriamycin

6

7

8

9

80.7

40.8

0.02 ± 0.00

2.09 ± 0.11

0.52 ± 0.28

>50

9.3 ± 1.05

>50

1.63 ± 0.14

1.89 ± 0.03

2.19 ± 0.03

>50

2.82 ± 0.24

1.84 ± 0.44

13.7 ± 0.81

1.34 ± 0.03

>50

0.77 ± 0.04

0.78 ± 0.02

11.68 ± 1.06

>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

0.18 ± 0.02

7.32 ± 0.15

0.88 ± 0.08

>50

1.69 ± 0.16

4.14 ± 0.20

>50

79.3

22.6

18.87 ± 0.34

1.23 ± 0.00

>50

3.48 ± 0.25

1.77 ± 0.04

>50

77.2

83.2

72.2

91.7

85.7

86.3

0.0

10.2

13.8

59.2

17.1

61.7

89.1

90.1

83.0

97.8

100.0

1.0

29.5

30.1

44.2

37.4

74.9

0.8

0.2

1.5

2.0

0.0

1.1

0.9

ND

4.5

ND

7.5

0.2

0.0

1.5

0.0

ND

3.9

2.0

1.3

0.0

ND

0.1

ND

0.0

1.9

2.8

5.0

55.9

78.1

76.4

78.3

88.6

74.6

84.5

78.2

80.6

88.1

91.5

81.2

52.2

81.2

84.8

88.2

92.8

92.3

12.2

62.1

69.3

7.3

95.0

12.0

65.5

62.5

7.8

52.2

11.9

27.4

5.2

10.8

12.1

30.2

22.3

21.5

30.3

4.7

1

2

3

4

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

2

3

4

5

6

7

8

9

0

1

11.0

9.7

>50

23.0

44.7

57.9

13.3

21.5

13.6

20.6

21.8

48.4

ND

7.0

12.8

ND

19.7

ND

4.3

1.5

ND

4.3

ND

8.1

ND

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

1.63 ± 0.08

1.18 ± 0.03

1.04 ± 0.01

>50

1.89 ± 0.8

0.84 ± 0.04

13.99 ± 0.68

>50

1.02 ± 0.13

0.93 ± 0.02

1.53 ± 0.36

>50

3.51 ± 0.23

>50

8.28 ± 1.69

>50

16.56 ± 1.08

2.58 ± 0.16

4.37 ± 0.3

>50

5.99 ± 0.37

5.17 ± 0.23

>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

5.58 ± 0.14

>50

6.39 ± 0.81

0.24 ± 0

>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

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

7.53 ± 0.58

>50

35.4

0.2

8.3

2.4

1.4

66.4

1.9

1.5

2.1

5.9

55.2

8.3

11.2

14.8

65.8

ND

25.7

>50

0.84 ± 0

>50

2.03 ± 0.14

8.7

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 chro-

matography (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

ESI LC/MS analyses were performed with a Finnigan LCQ

Advantage^ÒLC/MS/MS spectrometry utilizing Xcalibur program.

Ò

For ESI LC/MS, LC was performed with a 5

lL injection volume on

a Waters X Terra^Ò3.5

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, cap-

illary temperature: 215 °C, column temperature 40 °C, capillary

voltage: 21 V, tube lens offset: 10 V.

1

AMX 250 (250 MHz or 300 MHz, FT) for H NMR and 62.5 MHz

1

3

or 75.5 MHz for C 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 deter-

mined 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

5.1. General method for the preparation of 3

1

2

Compounds 3 (R = a–c, R = d–i) were synthesized by base

catalyzed Claisen–Schmidt condensation reaction. A solution of

(

SPD-M10A VP) using Shimadzu Class VP program. Sample volume

Ò

1

of 10

l

L was injected in Waters X- Terra

5

lM reverse-phase

equimolar amounts of aryl ketone 1 (R = a–c) and aryl aldehyde

2

C18 column (4.6 ꢀ 250 mm) with a gradient elution of 70% to

2 (R = d–i) in ethanol was added to 4 M aqueous solution of

1

00% of B in A for 10 min followed by 100% to 70% of B in A for

0 min at a ﬂow rate of 1.0 mL/min at 254 nm UV detection, where

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.

mobile phase A was 20 mM ammonium formate (AF) in doubly dis-

tilled water and B was 100% ACN. Purity of compound is described

as percent (%), and retention time is given in minutes.

Please cite this article in press as: Karki, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.002

8

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

S

HO

OH

HO

N

OH

HO

OH

11

9

10

(78.3%, 22.3%)

(88.6%, 21.5%)

(74.6%, 30.3%)

S

HO

OH

HO

N

OH

HO

OH

1

5

16

17

(

88.1%, 23.0%)

(91.5%, 44.7%)

(

81.2%, 57.9%)

O

HO

OH

HO

N

OH

HO

OH

2

1

22

23

(

88.3%, 20.6%)

(92.8%, 21.8%)

(92.3%, 48.4%)

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

l

M, respectively.

R²

N

R²

N

R²

N

>

OH

HO

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 chro-

matography to yield pure compound. Total eighteen hydroxylated

chalcone intermediates were synthesized.

3-thienyl H-3, H-5, CO–CH@CH), 7.15–7.07 (m, 2H, 1-phenyl H-4,

3-thienyl H-4), 6.57 (s, 1H, 1-phenyl 3-OH).

1

3

C NMR (62.5 MHz, CDCl ) d 190.13, 156.43, 140.22, 139.32,

1

37.84, 132.53, 129.91, 129.19, 128.42, 120.87, 120.47 (2C),

5

.1.1. 1-(2-Hydroxyphenyl)-3-(thiophen-2-yl)propenone (3a)

115.11.

The procedure described in Section 5.1 was employed with aryl

1

2

ketone 1 (R = a) (2.40 mL, 20.00 mmol), aryl aldehyde 2 (R = d)

5.1.3. 1-(4-Hydroxyphenyl)-3-(thiophen-2-yl)propenone (3c)

(

1.86 mL, 20.00 mmol), ethanol (10 mL) and sodium hydroxide

The procedure described in Section 5.1 was employed with aryl

ketone 1 (R = c) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = d)

(0.93 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide

1

2

4 M) to yield a yellow solid (3.10 g, 67.4%, 13.46 mmol). TLC (ethyl

1

acetate/n-hexane = 1:3) R

f

= 0.47. H NMR (250 MHz, CDCl

3

) d

12.85 (s, 1H, 1-phenyl 2-OH), 8.05 (d, J = 14.6 Hz, 1H, CO–

CH@CH), 7.89 (d, J = 7.9 Hz, 1H, 1-phenyl H-6), 7.53–7.41 (m, 4H,

(4 M) to yield yellow solid (2.10 g, 91.3%, 9.10 mmol). TLC (ethyl

1

acetate/n-hexane = 1:2) R

f

= 0.20. H NMR (250 MHz, DMSO-d

6

) d

1

0

6

-phenyl H-4, 3-thienyl H-3, H-5, CO–CH@CH), 7.11 (td, J = 4.4,

.4 Hz, 1H, 3-thienyl H-4), 7.03 (d, J = 8.4 Hz, 1H, 1-phenyl H-3),

.95 (t, J = 7.9 Hz, 1H, 1-phenyl H-5).

10.46 (s, 1H, 1-phenyl 4-OH), 7.99 (d, J = 7.7 Hz, 2H, 1-phenyl H-

2, H-6), 7.86 (d, J = 15.3 Hz, 1H, CO–CH@CH), 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–CH@CH), 7.16 (t, J = 5.0 Hz, 1H, 3-thienyl H-

4), 6.87 (d, J = 8.7 Hz, 2H, 1-phenyl H-3, H-5).

1

3

C NMR (62.5 MHz, CDCl ) d 193.46, 162.13, 144.33, 139.56,

1

37.17, 135.92, 129.53, 128.57, 123.19, 120.48, 119.82, 118.21,

15.66.

1

3

C NMR (62.5 MHz, DMSO-d

6

) d 187.24, 163.45, 150.49, 145.14,

1

32.14 (2C), 129.12, 129.26, 118.81, 116.59, 115.42 (2C), 112.24.

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 (R = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = d)

0.93 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide

5.1.4. 1-(2-Hydroxyphenyl)-3-(thiophen-3-yl)propenone (3d)

1

2

The procedure described in Section 5.1 was employed with aryl

1

2

(

ketone 1 (R = a) (1.20 mL, 10.00 mmol), aryl aldehyde 2 (R = e)

4 M) to yield a yellow solid (2.26 g, 98.2%, 9.81 mmol). TLC (ethyl

(0.91 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide

1

acetate/n-hexane = 1:3) R

f

= 0.25. H NMR (250 MHz, CDCl

3

) d 7.97

(4 M) to yield a yellow solid (2.15 g, 93.4%, 9.33 mmol). TLC (ethyl

1

(

d, J = 15.3 Hz, 1H, CO–CH@CH), 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,

acetate/n-hexane = 1:5) R

f

= 0.46. H NMR (250 MHz, CDCl

3

) d

12.87 (s, 1H, 1-phenyl 2-OH), 7.92 (d, J = 15.5 Hz, 1H, CO–CH@CH),

Please cite this article in press as: Karki, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.002

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

9

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–CH@CH),

7.02 (dd, J = 8.4, 0.9 Hz, 1H, 1-phenyl H-3), (t, J = 8.1 Hz, 1H, 1-phe-

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 (R = c) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = f)

1

2

nyl H-5).

¹³C NMR (62.5 MHz, CDCl

36.32, 130.05, 129.53, 127.26, 125.15, 119.96, 119.67, 118.80,

(0.82 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide

3

) d 193.88, 163.49, 138.81, 137.92,

(4 M) to yield yellow solid (1.90 g, 90.5%, 8.86 mmol). TLC (ethyl

1

acetate/n-hexane = 1:2) R

f

= 0.34. H NMR (250 MHz, DMSO-d

6

) d

18.59.

10.41 (s, 1H, 1-phenyl 4-OH), 7.96 (d, J = 8.7 Hz, 2H, 1-phenyl H-

, H-6), 7.87 (br, 1H, 3-furyl H-5), 7.53 (d, J = 15.6 Hz, 1H, CO–

2

5

.1.5. 1-(3-Hydroxyphenyl)-3-(thiophen-3-yl)propenone (3e)

CH@CH), 7.47 (d, J = 15.6 Hz, 1H, CO–CH@CH), 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).

C NMR (62.5 MHz, DMSO-d ) d 186.74, 162.37, 151.49, 146.04,

6

131.14 (2C), 129.61, 129.20, 119.01, 116.49, 115.66 (2C), 113.20.

The procedure described in Section 5.1 was employed with aryl

1

2

ketone 1 (R = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = e)

1

3

(

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)

1

R

f

= 0.32.

H NMR (250 MHz,

CDCl

3

) d 7.81 (d, J = 15.5 Hz, 1H, CO–CH@CH), 7.63–7.52 (m, 3H,

5.1.10. 3-(Furan-3-yl)-1-(2-hydroxyphenyl)propenone (3j)

1

4

-phenyl H-2, H-6, 3-thienyl H-2), 7.40–7.32 (m, 3H, 3-thienyl H-

, H-5, 1-phenyl H-5), 7.30 (d, J = 15.3 Hz, 1H, CO–CH@CH), 7.11

The procedure described in Section 5.1 was employed with aryl

ketone 1 (R = a) (1.80 mL, 15.00 mmol), aryl aldehyde 2 (R = g)

1

2

(

d, J = 8.1 Hz, 1H, 1-phenyl H-4), 6.52 (s, 1H, 1-phenyl 3-OH).

(1.25 mL, 15.00 mmol), ethanol (10 mL) and sodium hydroxide

1

3

C NMR (62.5 MHz, CDCl

3

) d 190.97, 156.41, 139.62, 138.79,

(4 M) to yield a yellow solid (1.50 g, 46.7%, 7.00 mmol). TLC (ethyl

1

38.11, 129.88, 129.44, 127.07, 125.26, 121.75, 120.92, 120.32,

15.18.

acetate/n-hexane = 1:5) R

f

= 0.47. H NMR (250 MHz, CDCl

3

) 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–CH@CH), 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–CH@CH), 7.02 (dd, J = 8.4, 0.9 Hz, 1H, 1-

phenyl H-3), 6.93 (td, J = 8.1, 1.1 Hz, 1H, 1-phenyl H-5), 6.73

(br, 1H, 3-furyl H-4).

5

.1.6. 1-(4-Hydroxyphenyl)-3-(thiophen-3-yl)propenone (3f)

The procedure described in Section 5.1 was employed with aryl

1

2

ketone 1 (R = c) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = e)

(

acetate/n-hexane = 1:2) R

1

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

1

3

C NMR (62.5 MHz, CDCl ) d 193.57, 163.60, 146.02, 144.70,

= 0.30. ¹H NMR (250 MHz, DMSO-d

136.27, 135.46, 129.53, 123.19, 120.01, 119.92, 118.77, 118.63,

107.43.

f

6

) d

0.42 (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–CH@CH, CO–CH@CH), 7.66–7.62 (m,

5.1.11. 3-(Furan-3-yl)-1-(3-hydroxyphenyl)propenone (3k)

1

5

H, 3-thienyl H-5), 6.87 (d, J = 8.7 Hz, 2H, 1-phenyl H-3, H-5).

The procedure described in Section 5.1 was employed with aryl

ketone 1 (R = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = g)

(1.00 mL, 12.00 mmol), ethanol (10 mL) and sodium hydroxide

1

3

1

2

C NMR (62.5 MHz, DMSO-d

6

) d 187.54, 162.36, 138.63, 136.84,

31.32 (2C), 130.24, 129.37, 127.85, 126.38, 121.75, 115.57 (2C).

(

4 M) to yield light yellow solid (1.95 g, 91.1%, 9.10 mmol). TLC

1

.1.7. 3-(Furan-2-yl)-1-(2-hydroxyphenyl)propenone (3g)

(ethyl acetate/n-hexane = 1:2)

R

f

= 0.34.

H NMR (250 MHz,

The procedure described in Section 5.1 was employed with aryl

ketone 1 (R = a) (1.80 mL, 15.00 mmol), aryl aldehyde 2 (R = f)

DMSO-d ) d 9.76 (s, 1H, 1-phenyl 3-OH), 8.18 (s, 1H, 3-furyl H-2),

6

1

2

7.76 (br, 1H, 3-furyl H-5), 7.64 (d, J = 15.50 Hz, 1H, CO–CH@CH),

7.52 (d, J = 15.4 Hz, 1H, CO–CH@CH), 7.51 (d, J = 7.2 Hz, 1H, 1-phe-

nyl H-6), 7.40 (br, 1H, 1-phenyl H-2), 7.34 (t, J = 7.8 Hz, 1H, 1-phe-

nyl 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).

(

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) R

CDCl ) d 12.85 (s, 1H, 1-phenyl 2-OH), 7.91 (dd, J = 8.1, 1.4 Hz,

H, 1-phenyl H-6), 7.68 (d, J = 15.2 Hz, 1H, CO–CH@CH), 7.89 (d,

= 0.44. ¹H NMR (250 MHz,

f

3

1

3

1

6

C NMR (62.5 MHz, DMSO-d ) d 189.20, 157.84, 146.84, 145.10,

J = 15.2 Hz, 1H, CO–CH@CH), 7.55–7.45 (m, 2H, 1-phenyl H-4, 3-

furyl H-5), 7.01 (d, J = 8.4 Hz, 1H, 1-phenyl H-3), 6.93 (td, J = 8.1,

139.17, 134.56, 129.90, 123.32, 122.09, 120.24, 119.50, 114.71,

108.17.

1

6

.0 Hz, 1H, 1-phenyl H-5), 6.76 (d, J = 3.4 Hz, 1H, 3-furyl H-3),

.53 (br, 1H, 3-furyl H-4).

5.1.12. 3-(Furan-3-yl)-1-(4-hydroxyphenyl)propenone (3l)

1

3

C NMR (62.5 MHz, CDCl ) d 190.19, 156.51, 151.50, 145.16,

The procedure described in Section 5.1 was employed with aryl

1

2

1

39.30, 131.20, 129.87, 120.89, 120.52, 119.01, 116.84, 115.12,

12.77.

ketone 1 (R = c) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = 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

1

5

.1.8. 3-(Furan-2-yl)-1-(3-hydroxyphenyl)propenone (3h)

f 6

acetate/n-hexane = 1:2) R = 0.24. H NMR (250 MHz, DMSO-d ) d

The procedure described in Section 5.1 was employed with aryl

ketone 1 (R = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = f)

0.82 mL, 10.00 mmol), ethanol (10 mL) and sodium hydroxide

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–CH@CH), 7.58 (d, J = 15.2 Hz, 1H, CO–

CH@CH), 7.12 (br, 1H, 3-furyl H-4), 6.88 (d, J = 8.7 Hz, 2H, 1-phenyl

H-3, H-5).

1

2

(

4 M) to yield light yellow solid (2.15 g, 93.4%, 9.33 mmol). TLC

1

ethyl acetate/n-hexane = 1:2)

R

f

= 0.37.

H NMR (250 MHz,

CDCl

3

) d 7.81 (d, J = 15.2 Hz, 1H, CO–CH@CH), 7.59 (d, J = 8.2 Hz,

¹³C NMR (62.5 MHz, DMSO-d

6

) d 187.20, 162.30, 146.56, 145.11,

1

7

H, 1-phenyl H-6), 7.56–7.52 (m, 2H, 1-phenyl H-2, 3-furyl H-5),

.42 (d, J = 15.3 Hz, 1H, CO–CH@CH), 7.37 (t, J = 7.7 Hz, 1H, 1-phe-

133.47, 131.25 (2C), 129.27, 123.52, 121.93, 115.56 (2C), 108.37.

nyl H-5), 7.01 (dd, J = 8.0, 2.1 Hz, 1H, 1-phenyl H-4), 6.73 (d,

5.1.13. 1-(2-Hydroxyphenyl)-3-(pyridin-2-yl)-propenone (3m)

3

1

.40 Hz, 1H, 3-furyl H-3), 6.52 (br, 1H, 3-furyl H-4), 5.66 (s, 1H,

-phenyl 3-OH).

The procedure described in Section 5.1 was employed with aryl

ketone 1 (R = a) (2.40 mL, 20.00 mmol), aryl aldehyde 2 (R = h)

(1.91 mL, 20.00 mmol), ethanol (10 mL) and sodium hydroxide

1

2

1

3

C NMR (62.5 MHz, CDCl ) d 190.43, 157.11, 141.32, 140.39,

1

37.80, 131.52, 129.88, 129.09, 128.31, 120.67, 119.89 (2C),

15.32.

(4 M) to yield a yellow solid (2.75 g, 61.0%, 12.20 mmol). TLC (ethyl

1

acetate/n-hexane = 1:2) R

f

= 0.33. H NMR (300 MHz, DMSO-d

6

) d

Please cite this article in press as: Karki, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.002

1

0

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

1

8

2.01 (s, 1H, 1-phenyl 2-OH), 8.68 (d, J = 4.8 Hz, 1H, 3-pyridyl H-6),

.21 (d, J = 15.3 Hz, 1H, CO–CH@CH), 8.03 (dd, J = 8.4, 1.5 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).

C NMR (75.5 MHz, DMSO-d ) d 189.45, 158.78, 144.02, 143.81,

6

143.57, 139.08, 137.98, 134.65, 130.76, 128.07, 127.70, 121.81,

120.73, 115.69.

1

3

phenyl H-6), 7.90–7.88 (m, 2H, 3-pyridyl H-3, H-4), 7.75

d, J = 15.3 Hz, 1H, CO–CH@CH), 7.55 (td, J = 7.8, 1.5 Hz, 1H, 1-phe-

nyl H-4), 7.43 (t, J = 8.7 Hz, 1H, 3-pyridyl H-5), 7.01 (d, J = 8.1 Hz,

(

1

H, 1-phenyl H-3), 7.43 (t, J = 8.1 Hz, 1H, 1-phenyl H-5).

1

3

5.1.18. 1-(4-Hydroxyphenyl)-3-(pyridin-3-yl)-propenone (3r)

6

C NMR (75.5 MHz, DMSO-d ) d 194.19, 162.06, 153.40, 150.94,

44.01, 138.08, 137.09, 131.55, 126.32, 126.13, 125.83, 122.24,

20.22, 118.56.

The procedure described in Section 5.1 was employed with aryl

1

2

ketone 1 (R = c) (0.68 g, 5.00 mmol), aryl aldehyde 2 (R = 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

(

5

.1.14. 1-(3-Hydroxyphenyl)-3-(pyridin-2-yl)-propenone (3n)

1

The procedure described in Section 5.1 was employed with aryl

(ethyl acetate/n-hexane = 3:1)

R

f

= 0.26.

H NMR (300 MHz,

1

2

ketone 1 (R = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = h)

DMSO-d ) d 10.71 (s, 1H, 1-phenyl 4-OH), 9.38 (s, 1H, 3-pyridyl

6

(

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

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–CH@CH), 8.12

(d, J = 8.7 Hz, 2H, 1-phenyl H-2, H-6), 8.02 (t, J = 7.8 Hz, 1H, 3-pyr-

idyl H-5), 7.78 (d, J = 15.6 Hz, 1H, CO–CH@CH), 6.94 (d, J = 8.4 Hz,

2H, 1-phenyl H-3, H-5).

C NMR (75.5 MHz, DMSO-d ) d 186.78, 163.19, 143.50, 142.89,

6

142.67, 136.29, 134.39, 131.83 (2C), 128.68, 127.67, 127.16, 115.84

(2C).

acetate/n-hexane = 1:1) R

8

f

= 0.40. ¹H NMR (300 MHz, DMSO-d

6

) d

.77 (br, 1H, 3-pyridyl H-6), 8.28 (d, J = 15.3 Hz, 1H, CO–CH@CH),

.19 (br, 2H, 3-pyridyl H-3, H-4), 7.75 (d, J = 15.9 Hz, 1H, CO–

1

3

CH@CH), 7.68–7.59 (m, 2H, 3-pyridyl H-5, 1-phenyl H-6), 7.47 (s,

H, 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).

1

3

6

C NMR (75.5 MHz, DMSO-d ) d 188.92, 158.21, 149.93, 146.17,

5

.2. General method for the preparation of 5

1

42.43, 138.44, 137.63, 130.34, 129.01, 126.51, 126.26, 121.34,

20.12, 114.96.

3

A mixture of aryl ketone 4 (R = a–c), iodine (1.2 equiv) and pyr-

5

.1.15. 1-(4-Hydroxyphenyl)-3-(pyridin-2-yl)-propenone (3o)

idine (15 equiv) was reﬂuxed at 140 °C for 3 h. Precipitate occurred

The procedure described in Section 5.1 was employed with aryl

during reaction which was cooled to room temperature. Then it

1

2

3

ketone 1 (R = c) (0.68 g, 5.00 mmol), aryl aldehyde 2 (R = h)

was ﬁltered and washed with cold pyridine to afford 5 (R = a–c)

(

0.47 mL, 5.00 mmol), ethanol (10 mL) and sodium hydroxide

in quantitative yield. Three different pyridinium iodide salts were

synthesized by this method.

4 M) to yield light yellow solid (0.80 g, 71.4%, 3.55 mmol). TLC

1

ethyl acetate/n-hexane = 1:1)

R

f

= 0.39.

H NMR (300 MHz,

5

.3. General method for the preparation of 6–41

DMSO-d

6

) d 10.74 (s, 1H, 1-phenyl 4-OH), 8.84 (d, J = 5.4 Hz, 1H,

3

7

-pyridyl H-6), 8.55 (d, J = 15.9 Hz, 1H, CO–CH@CH), 8.41 (br, 2H,

-pyridyl H-3, H-4), 8.11 (d, J = 9.0 Hz, 2H, 1-phenyl H-2, H-6),

.84 (t, J = 9.0 Hz, 1H, 3-pyridyl H-5), 7.78 (d, J = 15.6 Hz, 1H, CO–

1

A mixture of hydroxylated chalcone intermediate 3 (R = a–c,

2

3

R = d–i), pyridinium iodide salt 5 (R = a–c) and anhydrous ammo-

nium 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 evapo-

rated at reduced pressure, which was then puriﬁed by silica gel col-

umn chromatography with the gradient elution of ethyl acetate/n-

hexane 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.

CH@CH), 6.94 (d, J = 9.1 Hz, 2H, 1-phenyl H-3, H-5).

1

3

C NMR (75.5 MHz, DMSO-d

33.74, 131.68 (2C), 130.00, 128.35, 127.49, 126.44, 126.16, 115.74

2C).

6

) d 186.37, 163.12, 161.93, 144.77,

1

(

5

.1.16. 1-(2-Hydroxyphenyl)-3-(pyridin-3-yl)-propenone (3p)

The procedure described in Section 5.1 was employed with aryl

1

2

ketone 1 (R = a) (2.40 mL, 20.00 mmol), aryl aldehyde 2 (R = 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

= 0.30. ¹H NMR (300 MHz, DMSO-d

) d

0

acetate/n-hexane = 2:1) R

2.39 (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 H-

), 8.26 (d, J = 8.1 Hz, 1H, 1-phenyl H-6), 8.17 (d, J = 15.6 Hz, 1H,

CO–CH@CH), 7.84 (d, J = 15.9 Hz, 1H, CO–CH@CH), 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).

f

6

5.3.1. 2,2 -(4-(Thiophen-2-yl)pyridine-2,6-diyl)diphenol (6)

The same procedure described in Section 5.3 was employed

with 3 (R = a, R = d) (0.34 g, 1.50 mmol), dry ammonium acetate

(1.15 g, 15.00 mmol), 5 (R = 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

f

R = 0.27, mp: 217–218 °C, HPLC:

Retention time: 10.23 min, purity: 98.9%; ESI LC/MS: m/z calcd

1

2

3

4

(

acetate/n-hexane = 1:3)

1

3

+

1

C NMR (75.5 MHz, DMSO-d

6

) d 193.56, 162.09, 151.50, 150.83,

for C21

DMSO-d

2H, pyridine H-3, H-5), 8.08 (d, J = 3.6 Hz, 1H, 4-thiophene H-3),

.94 (d, J = 7.8 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6), 7.81 (d,

H15NO

2

S [MH] 346.09; found 346.35. H NMR (300 MHz,

1

41.49, 136.78, 135.59, 131.24, 130.54, 124.22, 123.94, 120.92,

19.48, 118.00.

6

) d 12.33 (s, 2H, 2-phenyl 2-OH, 6-phenyl 2-OH), 8.18 (s,

7

5

.1.17. 1-(3-Hydroxyphenyl)-3-(pyridin-3-yl)-propenone (3q)

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).

C NMR (62.5 MHz, DMSO-d ) d 157.55 (2C), 155.76 (2C),

6

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).

The procedure described in Section 5.1 was employed with aryl

1

2

ketone 1 (R = b) (1.36 g, 10.00 mmol), aryl aldehyde 2 (R = i)

1

3

(

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)

1

R

f

= 0.27.

H NMR (300 MHz,

6

DMSO-d ) d 9.39 (br, 1H, 3-pyridyl H-2), 8.97 (d, J = 8.1 Hz, 1H, 3-

pyridyl H-4), 8.87 (d, J = 5.7 Hz, 1H, 3-pyridyl H-6), 8.23 (d,

J = 15.9 Hz, 1H, CO–CH@CH), 8.03 (t, J = 8.1 Hz, 1H, 3-pyridyl H-

5.3.2. 2-(6-(3-Hydroxyphenyl)-4-(thiophen-2-yl)pyridin-2-

yl)phenol (7)

5

), 7.79 (d, J = 15.6 Hz, 1H, CO–CH@CH), 7.68 (d, J = 7.8 Hz, 1H, 1-

The same procedure described in Section 5.3 was employed

1

2

phenyl H-6), 7.51 (s, 1H, 1-phenyl H-2), 7.38 (t, J = 7.8 Hz, 1H, 1-

with 3 (R = a, R = d) (0.34 g, 1.50 mmol), dry ammonium acetate

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

11

3

(

1.15 g, 15.00 mmol), 5 (R = b) (0.51 g, 1.50 mmol) and acetic acid

(ethyl acetate/n-hexane = 1:2) R

f

= 0.20, mp: 240–241 °C, HPLC:

2 mL) to yield a yellow solid (0.33 g, 63.7%, 0.95 mmol). TLC (ethyl

retention time: 7.63 min, purity: 98.9%; ESI LC/MS: m/z calcd for

+

acetate/n-hexane = 1:3)

R

f

= 0.27, mp: 225–226 °C, HPLC:

C

21

H

15NO

2

S [MH] 346.09; found 346.35.

1

Retention time: 10.50 min, purity: 98.7%; ESI LC/MS: m/z calcd

H NMR (250 MHz, DMSO-d ) d 9.79 (br, 1H, 2-phenyl 3-OH),

6

+

for C21

1

H15NO

2

S [MH] 346.09; found 346.35.

) d 14.25 (s, 1H, 2-phenyl 2-OH),

9.65 (br, 1H, 6-phenyl 4-OH), 8.14 (d, J = 8.6 Hz, 2H, 6-phenyl H-

2, H-6), 8.04 (d, J = 3.6 Hz, 1H, 4-thiophene H-3), 7.97 (s, 1H, pyri-

dine H-3), 7.91 (s, 1H, pyridine H-5), 7.76 (d, J = 5.0 Hz, 4-thio-

phene H-5), 7.68 (s, 1H, 2-phenyl H-2), 7.66 (d, J = 7.8 Hz, 1H, 2-

phenyl 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-phe-

nyl H-3, H-5), 6.88 (dd, J = 8.1, 2.1 Hz, 1H, 2-phenyl H-4).

H NMR (250 MHz, DMSO-d

6

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,

-phenyl H-4), 7.31 (ddd, J = 5.0, 3.6, 0.5 Hz, 1H, 4-thiophene H-

), 7.00–6.92 (m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4).

1

3

2

4

6

C NMR (62.5 MHz, DMSO-d ) 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.

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 159.29, 158.23, 157.61, 155.04,

1

43.98, 140.32, 138.90, 131.76, 130.37, 129.28, 129.11, 128.08,

27.78, 119.14, 119.08, 117.95, 117.68, 117.10, 115.17, 114.27,

13.54.

0

5

.3.6. 4,4 -(4-(Thiophen-2-yl)pyridine-2,6-diyl)diphenol (11)

The same procedure described in Section 5.3 was employed

1

2

with 3 (R = c, R = d) (0.34 g, 1.50 mmol), dry ammonium acetate

5

.3.3. 2-(6-(4-Hydroxyphenyl)-4-(thiophen-2-yl)pyridin-2-

3

(

1.15 g, 15.00 mmol), 5 (R = 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)

Retention time: 7.50 min, purity: 97.8%; ESI LC/MS: m/z calcd for

yl)phenol (8)

The same procedure described in Section 5.3 was employed

f

R = 0.20, mp: 245–246 °C, HPLC:

1

2

with 3 (R = a, R = d) (0.34 g, 1.50 mmol), dry ammonium acetate

3

(

1.15 g, 15.00 mmol), 5 (R = c) (0.51 g, 1.50 mmol) and acetic acid

+

C

21

H

15NO

2

S [MH] 346.09; found 346.35.

(

2 mL) to yield a yellow solid (0.32 g, 62.0%, 0.92 mmol). TLC (ethyl

1

H NMR (250 MHz, DMSO-d ) d 9.81 (s, 2H, 2-phenyl 4-OH, 6-

6

acetate/n-hexane = 1:2)

Retention time: 10.50 min, purity: 98.5%; ESI LC/MS: m/z calcd

for C21

1

f

R = 0.31, mp: 293–294 °C, HPLC:

phenyl 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

15NO

2

S [MH] 346.09; found 346.35.

) d 14.57 (s, 1H, 2-phenyl 2-OH),

H NMR (250 MHz, DMSO-d

6

1

0.03 (br, 1H, 6-phenyl 4-OH), 8.23 (s, 1H, pyridine H-3), 8.22 (d,

H-3, H-5, 6-phenyl H-3, H-5).

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-thio-

phene H-4), 6.97 (d, J = 8.6 Hz, 2H, 6-phenyl H-3, H-5), 6.95–6.92

13

6

C NMR (62.5 MHz, DMSO-d ) d 158.96 (3C), 156.68 (2C),

42.73, 141.44, 129.81 (3C), 129.02, 128.48 (3C), 128.13, 126.88,

15.72 (3C), 112.66 (2C).

1

0

5

.3.7. 2,2 -(4-(Thiophen-3-yl)pyridine-2,6-diyl)diphenol (12)

(

m, 2H, 2-phenyl H-3, H-5).

1

3

The same procedure described in Section 5.3 was employed

6

C NMR (62.5 MHz, DMSO-d ) d 159.56, 159.53, 157.56, 155.01,

1

2

with 3 (R = a, R = e) (0.34 g, 1.50 mmol), dry ammonium acetate

1

44.01, 140.59, 131.84, 129.30, 129.21, 128.47 (2C), 128.26,

28.07, 127.75, 119.14, 119.12, 118.07, 116.23 (2C), 114.19,

13.20.

3

(

1.15 g, 15.00 mmol), 5 (R = 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)

f

R = 0.28, mp: 219–220 °C, HPLC:

Retention time: 9.59 min, purity: 99.7%; ESI LC/MS: m/z calcd for

0

5

.3.4. 3,3 -(4-(Thiophen-2-yl)pyridine-2,6-diyl)diphenol (9)

+

C

21

H

15NO

2

S [MH] 346.09; found 346.35.

H NMR (300 MHz, DMSO-d ) d 12.34 (s, 2H, 2-phenyl 2-OH, 6-

phenyl 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, 2-

The same procedure described in Section 5.3 was employed

1

2

6

with 3 (R = b, R = d) (0.27 g, 1.20 mmol), dry ammonium acetate

3

(

0.92 g, 12.00 mmol), 5 (R = 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) R = 0.28, mp: 249–250 °C, HPLC:

f

Retention time: 7.78 min, purity: 96.1%; ESI LC/MS: m/z calcd for

(

+

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).

C

21

H

15NO

2

S [MH] 346.09; found 346.36.

H NMR (250 MHz, DMSO-d ) d 9.63 (s, 2H, 2-phenyl 3-OH, 6-

phenyl 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),

.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,

1

6

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 157.55 (2C), 155.75 (2C),

1

44.44, 139.23, 131.19 (2C), 128.92 (2C), 128.06, 126.69, 125.67,

22.31 (2C), 119.34 (2C), 117.51 (2C), 116.97 (2C).

(

7

5.3.8. 2-(6-(3-Hydroxyphenyl)-4-(thiophen-3-yl)pyridin-2-

yl)phenol (13)

2

-phenyl H-4, 6-phenyl H-4).

1

3

C NMR (62.5 MHz, DMSO-d

42.82, 140.77, 139.96 (2C), 129.81 (2C), 128.95, 128.29, 127.09,

17.66 (2C), 116.44 (2C), 114.65 (2C), 113.68 (2C).

6

) d 157.81 (2C), 156.66 (2C),

The same procedure described in Section 5.3 was employed

1

2

1

with 3 (R = a, R = e) (0.34 g, 1.50 mmol), dry ammonium acetate

3

(1.15 g, 15.00 mmol), 5 (R = 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) = 0.28, mp: 230–231 °C, HPLC:

Retention time: 9.94 min, purity: 99.1%; ESI LC/MS: m/z calcd for

R

f

5

.3.5. 3-(6-(4-Hydroxyphenyl)-4-(thiophen-2-yl)pyridin-2-

yl)phenol (10)

+

C

21

H

15NO

2

S [MH] 346.09; found 346.35.

H NMR (250 MHz, DMSO-d ) d 14.62 (s, 1H, 2-phenyl 2-OH),

.81 (br, 1H, 6-phenyl 3-OH), 8.58 (dd, J = 2.5, 0.8 Hz, 1H, 4-thio-

The same procedure described in Section 5.3 was employed

1

2

with 3 (R = b, R = d) (0.34 g, 1.50 mmol), dry ammonium acetate

6

3

9

(

1.15 g, 15.00 mmol), 5 (R = c) (0.51 g, 1.50 mmol) and acetic acid

phene H-2), 8.44 (s, 1H, pyridine H-3), 8.31 (d, J = 7.8 Hz, 1H,

(

2 mL) to yield a light yellow solid (0.30 g, 57.4%, 0.86 mmol). TLC

Please cite this article in press as: Karki, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.002

1

2

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

2

-phenyl H-6), 8.18 (s, 1H, pyridine H-5), 8.03 (d, J = 5.0 Hz, 4-thio-

¹³C NMR (62.5 MHz, DMSO-d

) d 158.85, 157.89, 156.67, 156.46,

6

phene 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 H-

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.

4

), 6.99–6.93 (m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4).

1

3

0

C NMR (62.5 MHz, DMSO-d

6

) d 159.58, 158.32, 157.75, 154.89,

5.3.12. 4,4 -(4-(Thiophen-3-yl)pyridine-2,6-diyl)diphenol (17)

1

45.29, 139.21, 139.03, 131.84, 130.47, 127.97, 127.86, 126.93,

26.16, 119.16, 119.10, 118.09, 117.89, 117.09, 116.41, 115.26,

13.69.

The same procedure described in Section 5.3 was employed

1

2

with 3 (R = c, R = e) (0.34 g, 1.50 mmol), dry ammonium acetate

3

(1.15 g, 15.00 mmol), 5 (R = 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) = 0.50, mp: 236–237 °C, HPLC:

Retention time: 6.88 min, purity: 97.0%; ESI LC/MS: m/z calcd for

R

f

5

.3.9. 2-(6-(4-Hydroxyphenyl)-4-(thiophen-3-yl)pyridin-2-

yl)phenol (14)

+

C

21

H

15NO

2

S [MH] 346.09; found 346.35.

The same procedure described in Section 5.3 was employed

1

2

H NMR (250 MHz, DMSO-d ) d 9.70 (s, 2H, 2-phenyl 4-OH, 6-

with 3 (R = a, R = e) (0.34 g, 1.50 mmol), dry ammonium acetate

6

3

phenyl 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),

(

1.15 g, 15.00 mmol), 5 (R = 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)

f

R = 0.31, mp: 292–293 °C, HPLC:

7

.73 (dd, J = 5.0, 2.9 Hz, 1H, 4-thiophene H-5), 6.91 (d, J = 8.6 Hz,

Retention time: 9.75 min, purity: 99.4%; ESI LC/MS: m/z calcd for

+

4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5).

C

21

H

15NO

2

S [MH] 346.09; found 346.34.

H NMR (250 MHz, DMSO-d ) d 14.85 (s, 1H, 2-phenyl 2-OH),

.99 (br, 1H, 6-phenyl 4-OH), 8.56 (d, J = 2.8 Hz, 1H, 4-thiophene

1

3

1

6

C NMR (62.5 MHz, DMSO-d ) d 158.69 (2C), 156.42 (2C),

6

1

43.75, 139.84, 130.07 (2C), 128.32 (4C), 127.43, 126.63, 124.37,

15.51 (4C), 113.58 (2C).

9

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,

0

5

.3.13. 2,2 -(4-(Furan-2-yl)pyridine-2,6-diyl)diphenol (18)

The same procedure described in Section 5.3 was employed

2

4

2

.9 Hz, 1H, 4-thiophene H-5), 7.33 (t, J = 7.8 Hz, 1H, 2-phenyl H-

), 6.97 (d, J = 8.4 Hz, 2H, 6-phenyl H-3, H-5), 6.95–6.92 (m, 2H,

-phenyl H-3, H-5).

1

2

with 3 (R = a, R = f) (0.32 g, 1.50 mmol), dry ammonium acetate

3

(

1.15 g, 15.00 mmol), 5 (R = 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) = 0.37, mp: 217–218 °C, HPLC:

Retention time: 8.97 min, purity: 98.8%; ESI LC/MS: m/z calcd for

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 159.67, 159.46, 157.58, 154.78,

R

f

1

45.16, 139.19, 131.72, 128.47 (3C), 127.89, 127.73, 126.92,

25.96, 119.14, 119.01, 118.07, 116.18, 115.28, 114.14 (2C).

+

C

21

H

15NO

3

[MH] 330.11; found 330.35.

H NMR (250 MHz, DMSO-d ) d 12.27 (s, 2H, 2-phenyl 2-OH, 6-

phenyl 2-OH), 8.20 (d, J = 0.9 Hz, 2H, pyridine H-3, H-5), 7.94 (s, 1H,

1

0

6

5

.3.10. 3,3 -(4-(Thiophen-3-yl)pyridine-2,6-diyl)diphenol (15)

The same procedure described in Section 5.3 was employed

4

7

-furan H-5), 7.91 (d, J = 7.9 Hz, 2H, 2-phenyl H-6, 6-phenyl H-6),

.55 (d, J = 3.4 Hz, 1H, 4-furan H-3), 7.32 (t, J = 7.9 Hz, 2H, 2-phenyl

1

2

with 3 (R = b, R = e) (0.34 g, 1.50 mmol), dry ammonium acetate

3

(

1.15 g, 15.00 mmol), 5 (R = b) (0.51 g, 1.50 mmol) and acetic acid

H-4, 6-phenyl H-4), 6.99–6.93 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl

2 mL) to yield a yellow solid (0.40 g, 77.3%, 1.15 mmol). TLC (ethyl

H-3, H-5), 6.75 (dd, J = 3.4, 1.9 Hz, 1H, 4-furan H-4).

acetate/n-hexane = 1:2)

R

f

= 0.28, mp: 248–249 °C, HPLC:

13

6

C NMR (62.5 MHz, DMSO-d ) d 157.45 (2C), 155.60 (2C),

Retention time: 7.17 min, purity: 98.2%; ESI LC/MS: m/z calcd for

1

50.75, 145.36, 139.13, 131.19 (2C), 128.59 (2C), 121.99 (2C),

19.31 (2C), 117.48 (2C), 113.71 (2C), 112.88, 111.19.

+

C

21

H

15NO

2

S [MH] 346.09; found 346.36.

H NMR (250 MHz, DMSO-d ) d 9.62 (s, 2H, 2-phenyl 3-OH, 6-

phenyl 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 H-

), 7.75–7.68 (m, 5H, 2-phenyl H-2, H-6, 6-phenyl H-2, H-6, 4-thio-

phene H-5), 7.35 (t, J = 7.9 Hz, 2H, 2-phenyl H-5, 6-phenyl H-5),

1

6

5

.3.14. 2-(4-(Furan-2-yl)-6-(3-hydroxyphenyl)pyridin-2-

(

4

yl)phenol (19)

The same procedure described in Section 5.3 was employed

1

2

with 3 (R = a, R = f) (0.32 g, 1.50 mmol), dry ammonium acetate

6

.89 (dd, J = 7.9, 2.0 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4).

3

(

1.15 g, 15.00 mmol), 5 (R = b) (0.51 g, 1.50 mmol) and acetic acid

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 157.98 (2C), 156.70 (2C),

2 mL) to yield a yellow solid (0.28 g, 56.0%, 0.83 mmol). TLC (ethyl

1

44.18, 140.50 (2C), 139.56, 129.90 (2C), 127.78, 126.79, 125.01,

17.92 (2C), 116.46 (2C), 115.93 (2C), 113.96 (2C).

acetate/n-hexane = 1:2)

f

R = 0.37, mp: 199–200 °C, HPLC:

Retention time: 9.46 min, purity: 97.5%; ESI LC/MS: m/z calcd for

+

C

21

H

15NO

3

[MH] 330.11; found 330.36.

H NMR (250 MHz, DMSO-d ) d 14.34 (s, 1H, 2-phenyl 2-OH),

.74 (s, 1H, 6-phenyl 3-OH), 8.35 (s, 1H, pyridine H-3), 8.19 (d,

1

5

.3.11. 3-(6-(4-Hydroxyphenyl)-4-(thiophen-3-yl)pyridin-2-

6

yl)phenol (16)

9

The same procedure described in Section 5.3 was employed

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

1

2

with 3 (R = b, R = e) (0.34 g, 1.50 mmol), dry ammonium acetate

3

(

1.15 g, 15.00 mmol), 5 (R = c) (0.51 g, 1.50 mmol) and acetic acid

(

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

2 mL) to yield a yellow solid (0.39 g, 76.5%, 1.14 mmol). TLC (ethyl

acetate/n-hexane = 1:1)

f

R = 0.52, mp: 229–230 °C, HPLC:

H-4), 6.99–6.92 (m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4), 6.77

Retention time: 7.00 min, purity: 99.3%; ESI LC/MS: m/z calcd for

(

dd, J = 3.3, 1.7 Hz, 1H, 4-furan H-4).

+

1

3

C

21

H

15NO

2

S [MH] 346.09; found 346.35.

C NMR (62.5 MHz, DMSO-d

6

) d 159.33, 158.24, 157.56, 154.88,

1

H NMR (250 MHz, DMSO-d ) d 9.64 (br, 2H, 2-phenyl 3-OH, 6-

6

1

50.60, 145.52, 139.94, 138.91, 131.76, 130.39, 127.60, 119.09

phenyl 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

(

2C), 117.99, 117.58, 117.10, 113.45, 112.95 (2C), 112.09, 111.64.

(

7

s, 1H, pyridine H-5), 7.93 (dd, J = 5.2, 1.0 Hz, 4-thiophene H-4),

.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 H-

, H-5), 6.87 (dd, J = 8.2, 2.1 Hz, 1H, 2-phenyl H-4).

5

.3.15. 2-(4-(Furan-2-yl)-6-(4-hydroxyphenyl)pyridin-2-

yl)phenol (20)

The same procedure described in Section 5.3 was employed

1

2

3

with 3 (R = a, R = f) (0.32 g, 1.50 mmol), dry ammonium acetate

Please cite this article in press as: Karki, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.002

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

13

3

(

1.15 g, 15.00 mmol), 5 (R = c) (0.51 g, 1.50 mmol) and acetic acid

Retention time: 6.55 min, purity: 98.6%; ESI LC/MS: m/z calcd for

+

2 mL) to yield a yellow solid (0.27 g, 55.0%, 0.82 mmol). TLC (ethyl

C

21

H

15NO

3

[MH] 330.11; found 330.36.

1

acetate/n-hexane = 1:2)

R

f

= 0.30, mp: 278–279 °C, HPLC:

H NMR (250 MHz, DMSO-d ) d 9.77 (s, 2H, 2-phenyl 4-OH, 6-

6

Retention time: 9.36 min, purity: 97.4%; ESI LC/MS: m/z calcd for

phenyl 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).

C NMR (62.5 MHz, DMSO-d ) d 158.82, 156.41 (3C), 151.48

6

(2C), 144.57, 138.79, 129.81 (3C), 128.24 (4C), 115.60 (4C),

112.61, 110.38, 109.88.

+

C

21

H

15NO

3

[MH] 330.11; found 330.35.

H NMR (250 MHz, DMSO-d ) d 14.64 (s, 1H, 2-phenyl 2-OH),

.99 (s, 1H, 6-phenyl 4-OH), 8.27 (s, 1H, pyridine H-3), 8.18 (d,

1

6

9

J = 7.9 Hz, 1H, 2-phenyl H-6), 8.03 (s, 1H, pyridine H-5), 7.96 (s,

H, 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),

.97 (d, J = 8.4 Hz, 2H, 6-phenyl H-3, H-5), 6.95–6.92 (m, 2H, 2-phe-

nyl H-3, H-5), 6.77 (dd, J = 3.2, 1.7 Hz, 1H, 4-furan H-4).

1

3

1

(

6

1

3

0

C NMR (62.5 MHz, DMSO-d

6

) d 159.53, 159.51, 157.47, 154.83,

5.3.19. 2,2 -(4-(Furan-3-yl)pyridine-2,6-diyl)diphenol (24)

1

50.80, 145.51, 139.93, 131.78, 128.33, 128.25 (2C), 127.55,

19.08, 119.06, 118.07, 116.19 (2C), 113.03, 111.92, 111.56,

11.01.

The same procedure described in Section 5.3 was employed

1

2

with 3 (R = a, R = g) (0.32 g, 1.50 mmol), dry ammonium acetate

3

(1.15 g, 15.00 mmol), 5 (R = 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) = 0.46, mp: 209–210 °C, HPLC:

Retention time: 8.29 min, purity: 98.4%; ESI LC/MS: m/z calcd for

0

5

.3.16. 3,3 -(4-(Furan-2-yl)pyridine-2,6-diyl)diphenol (21)

R

f

The same procedure described in Section 5.3 was employed

with 3 (R = b, R = f) (0.32 g, 1.50 mmol), dry ammonium acetate

1.15 g, 15.00 mmol), 5 (R = 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) R = 0.35, mp: 241–242 °C, HPLC:

f

Retention time: 6.77 min, purity: 99.3%; ESI LC/MS: m/z calcd for

1

2

+

C

21

1

H

15NO

3

[MH] 330.11; found 330.35.

3

(

H NMR (250 MHz, DMSO-d ) d 12.27 (s, 2H, 2-phenyl 2-OH, 6-

6

phenyl 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).

+

C

21

H

15NO

3

[MH] 330.11; found 330.36.

H NMR (250 MHz, DMSO-d ) d 9.56 (s, 2H, 2-phenyl 3-OH, 6-

phenyl 3-OH), 8.05 (s, 2H, pyridine H-3, H-5), 7.92 (d, J = 1.5 Hz,

H, 4-furan H-5), 7.70 (t, J = 1.6 Hz, 2H, 2-phenyl H-2, 6-phenyl

1

6

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 157.46 (2C), 155.58 (2C),

1

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.

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

5

.3.20. 2-(4-(Furan-3-yl)-6-(3-hydroxyphenyl)pyridin-2-

H-4), 6.73 (dd, J = 3.4, 1.7 Hz, 1H, 4-furan H-4).

yl)phenol (25)

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 157.93 (2C), 156.59 (2C),

The same procedure described in Section 5.3 was employed

1

51.10, 144.85, 140.12 (2C), 139.03, 129.84 (2C), 117.63 (2C),

16.49 (2C), 113.73 (2C), 112.73 (2C), 112.54, 110.38.

1

2

with 3 (R = a, R = g) (0.32 g, 1.50 mmol), dry ammonium acetate

3

(

1.15 g, 15.00 mmol), 5 (R = 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

5

.3.17. 3-(4-(Furan-2-yl)-6-(4-hydroxyphenyl)pyridin-2-

acetate/n-hexane = 1:2)

Retention time: 8.67 min, purity: 99.2%; ESI LC/MS: m/z calcd for

C

f

R = 0.44, mp: 208–209 °C, HPLC:

yl)phenol (22)

The same procedure described in Section 5.3 was employed

+

21

H

15NO

3

[MH] 330.11; found 330.35.

H NMR (250 MHz, DMSO-d ) d 14.61 (s, 1H, 2-phenyl 2-OH),

.74 (s, 1H, 6-phenyl 3-OH), 8.74 (s, 1H, 4-furan H-2), 8.35 (s, 1H,

1

2

with 3 (R = b, R = f) (0.32 g, 1.50 mmol), dry ammonium acetate

1

6

3

(

1.15 g, 15.00 mmol), 5 (R = c) (0.51 g, 1.50 mmol) and acetic acid

9

(

2 mL) to yield a yellow solid (0.28 g, 56.6%, 0.85 mmol). TLC (ethyl

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

(

acetate/n-hexane = 1:2)

Retention time: 6.68 min, purity: 99.3%; ESI LC/MS: m/z calcd for

f

R = 0.32, mp: 213–214 °C, HPLC:

+

C

21

H

15NO

3

[MH] 330.11; found 330.36.

H NMR (250 MHz, DMSO-d ) d 9.79 (s, 1H, 2-phenyl 3-OH),

.57 (s, 1H, 6-phenyl 4-OH), 8.13 (d, J = 8.7 Hz, 2H, 6-phenyl H-2,

.31 (m, 3H, 2-phenyl H-4, 6-phenyl H-5, 4-furan H-4), 6.99–6.91

1

6

m, 3H, 2-phenyl H-3, H-5, 6-phenyl H-4).

9

13

6

C NMR (62.5 MHz, DMSO-d ) d 159.52, 158.21, 157.55, 154.66,

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 H-

1

45.05, 143.19, 142.98, 139.03, 131.68, 130.28, 127.62, 124.12,

18.97, 118.90, 117.99, 117.70, 116.98, 115.75, 114.56, 113.58,

08.93.

6

), 7.51 (d, J = 3.2 Hz, 1H, 4-furan H-3), 7.31 (t, J = 7.9 Hz, 1H, 2-

phenyl 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-fu-

ran H-4).

5

.3.21. 2-(4-(Furan-3-yl)-6-(4-hydroxyphenyl)pyridin-2-

yl)phenol (26)

The same procedure described in Section 5.3 was employed

¹³C NMR (62.5 MHz, DMSO-d

) d 158.99, 157.96, 156.61, 156.39,

6

1

2

with 3 (R = a, R = g) (0.32 g, 1.50 mmol), dry ammonium acetate

1

51.33, 144.81, 140.29, 138.97, 129.91, 129.67, 128.36 (2C),

17.63, 116.44, 115.69 (2C), 113.69, 112.77, 111.54, 111.46,

10.25.

3

(

1.15 g, 15.00 mmol), 5 (R = 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)

f

R = 0.39, mp: 261–262 °C, HPLC:

Retention time: 8.57 min, purity: 95.1%; ESI LC/MS: m/z calcd for

+

0

C₂₁H₁₅NO [MH] 330.11; found 330.35.

5

.3.18. 4,4 -(4-(Furan-2-yl)pyridine-2,6-diyl)diphenol (23)

3

1

The same procedure described in Section 5.3 was employed

6

H NMR (250 MHz, DMSO-d ) d 14.83 (s, 1H, 2-phenyl 2-OH),

1

2

with 3 (R = c, R = f) (0.32 g, 1.50 mmol), dry ammonium acetate

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-

3

(

1.15 g, 15.00 mmol), 5 (R = 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) R = 0.17, mp: 253–254 °C, HPLC:

f

Please cite this article in press as: Karki, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.002

1

4

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

0

4

6

), 7.33 (td, J = 7.8, 1.3 Hz, 1H, 2-phenyl H-4), 6.98 (d, J = 8.5 Hz, 2H,

5.3.25. 2,2 -([2,4 -Bipyridine]-2 ,6 -diyl)diphenol (30)

-phenyl H-3, H-5), 6.98–6.92 (m, 2H, 2-phenyl H-3, H-5).

The same procedure described in Section 5.3 was employed

with 3 (R = a, R = h) (0.33 g, 1.50 mmol), dry ammonium acetate

(1.15 g, 15.00 mmol), 5 (R = 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

1

3

1

2

C NMR (62.5 MHz, DMSO-d

6

) d 159.61, 159.34, 157.37, 154.53,

3

1

44.99, 143.04, 142.82, 131.55, 128.28 (2C), 128.26, 127.49,

24.23, 118.96, 118.80, 117.96, 116.04 (2C), 114.60, 113.46,

08.92.

(ethyl acetate/n-hexane = 1:2) R

f

= 0.31, mp: 213–214 °C, HPLC:

Retention time: 7.71 min, purity: 97.6%; ESI LC/MS: m/z calcd for

0

+

5

.3.22. 3,3 -(4-(Furan-3-yl)pyridine-2,6-diyl)diphenol (27)

C

22

H

16

N

2

O

2

[MH] 341.13; found 341.36.

1

The same procedure described in Section 5.3 was employed

with 3 (R = b, R = g) (0.32 g, 1.50 mmol), dry ammonium acetate

1.15 g, 15.00 mmol), 5 (R = 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) R = 0.27, mp: 224–225 °C, HPLC:

f

Retention time: 6.07 min, purity: 98.8%; ESI LC/MS: m/z calcd for

H NMR (250 MHz, DMSO-d ) d 12.30 (s, 2H, 2-phenyl 2-OH, 6-

6

1

2

phenyl 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, 2-

phenyl 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),

3

(

+

C

21

H

15NO

3

[MH] 330.11; found 330.36.

H NMR (250 MHz, DMSO-d ) d 9.60 (s, 2H, 2-phenyl 3-OH,

-phenyl 3-OH), 8.68 (s, 1H, 4-furan H-2), 8.06 (s, 2H, pyridine

7.01–6.95 (m, 4H, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5).

1

13

6

C NMR (62.5 MHz, DMSO-d ) d 157.50 (2C), 155.72 (2C),

6

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).

H-3, H-5), 7.85 (t, J = 1.8 Hz, 1H, 4-furan H-5), 7.73 (t, J = 1.8 Hz,

H, 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,

.8 Hz, 2H, 2-phenyl H-4, 6-phenyl H-4).

C NMR (62.5 MHz, DMSO-d

44.99, 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.

.3.23. 3-(4-(Furan-3-yl)-6-(4-hydroxyphenyl)pyridin-2-

2

0

5

(

.3.26. 2-(6 -(3-Hydroxyphenyl)-[2,4 -bipyridin]-2 -yl)phenol

31)

The same procedure described in Section 5.3 was employed

(

1

2

1

3

with 3 (R = a, R = h) (0.27 g, 1.20 mmol), dry ammonium acetate

0.92 g, 12.00 mmol), 5 (R = 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

f

ethyl acetate/n-hexane = 1:2) R = 0.26, mp: 219–220 °C, HPLC:

6

) d 157.98 (2C), 156.51 (2C),

3

(

1

(

Retention time: 8.03 min, purity: 97.0%; ESI LC/MS: m/z calcd for

5

+

C

22

H

16

N

2

O

2

[MH] 341.13; found 341.37.

yl)phenol (28)

1

H NMR (250 MHz, DMSO-d ) d 14.32 (s, 1H, 2-phenyl 2-OH),

6

The same procedure described in Section 5.3 was employed

with 3 (R = b, R = g) (0.32 g, 1.50 mmol), dry ammonium acetate

1.15 g, 15.00 mmol), 5 (R = 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) R = 0.51, mp: 226–227 °C, HPLC:

f

Retention time: 5.92 min, purity: 98.8%; ESI LC/MS: m/z calcd for

1

2

9.80 (s, 1H, 6-phenyl 3-OH), 8.82 (d, J = 3.9 Hz, 1H, 4-pyridine H-

6), 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-phe-

nyl 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,

3

(

+

C

21

H

15NO

3

[MH] 330.11; found 330.36.

H NMR (250 MHz, DMSO-d ) d 9.69 (br, 1H, 2-phenyl 3-OH),

.51 (br, 1H, 6-phenyl 4-OH), 8.62 (s, 1H, 4-furan H-2), 8.15 (d,

1

6

2

-phenyl H-3, H-5, 6-phenyl H-4).

9

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 159.31, 158.34, 157.68, 154.96,

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 H-

), 7.33 (t, J = 7.8 Hz, 1H, 2-phenyl H-5), 7.32 (d, J = 1.6 Hz, 1H, 4-fu-

1

53.28, 150.16, 148.94, 139.10, 137.89, 131.85, 130.57, 127.81,

25.04, 122.23, 119.35, 119.26, 118.06, 117.69, 117.14, 116.46,

15.82, 113.52.

(

6

ran 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).

0

5

(

.3.27. 2-(6 -(4-Hydroxyphenyl)-[2,4 -bipyridin]-2 -yl)phenol

32)

The same procedure described in Section 5.3 was employed

with 3 (R = a, R = h) (0.27 g, 1.20 mmol), dry ammonium acetate

0.92 g, 12.00 mmol), 5 (R = 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

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 158.80, 157.84, 156.43, 156.25,

1

44.77, 142.20, 141.41, 140.48, 129.85, 129.66, 128.30 (2C),

24.54, 117.68, 116.20, 115.54 (2C), 114.21, 114.09, 113.79,

08.89.

1

2

3

(

f

ethyl acetate/n-hexane = 1:2) R = 0.25, mp: 291–292 °C, HPLC:

Retention time: 7.99 min, purity: 99.2%; ESI LC/MS: m/z calcd for

0

5

.3.24. 4,4 -(4-(Furan-3-yl)pyridine-2,6-diyl)diphenol (29)

The same procedure described in Section 5.3 was employed

+

C

22

H

16

N

2

O

2

[MH] 341.13; found 341.37.

1

2

with 3 (R = c, R = g) (0.32 g, 1.50 mmol), dry ammonium acetate

1

H NMR (300 MHz, DMSO-d ) d 14.58 (s, 1H, 2-phenyl 2-OH),

.29 (s, 1H, 6-phenyl 4-OH), 8.81 (d, J = 3.9 Hz, 1H, 4-pyridine H-

), 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

6

3

(

1.15 g, 15.00 mmol), 5 (R = c) (0.61 g, 1.80 mmol) and acetic acid

9

6

2 mL) to yield a light yellow solid (0.29 g, 59.1%, 0.88 mmol). TLC

ethyl acetate/n-hexane = 1:2) R = 0.22, mp: 259–260 °C, HPLC:

f

Retention time: 5.81 min, purity: 96.3%; ESI LC/MS: m/z calcd for

+

C

21

H

15NO

3

[MH] 330.11; found 330.35.

1

H NMR (300 MHz, DMSO-d ) d 9.75 (s, 2H, 2-phenyl 4-OH, 6-

6

phenyl 4-OH), 8.61 (s, 1H, 4-furan H-2), 8.15 (d, J = 8.7 Hz, 4H, 2-

phenyl 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,

H-3, H-5), 6.97–6.94 (m, 2H, 2-phenyl H-3, H-5).

13

6

C NMR (75.5 MHz, DMSO-d ) d 159.34, 159.30, 157.34, 154.75,

53.29, 149.96, 148.69, 137.69, 131.60, 128.26 (3C), 127.47, 124.80,

22.02, 119.10, 119.02, 117.91, 116.09 (2C), 115.27, 114.42.

1

4

-furan H-4), 6.91 (d, J = 8.4 Hz, 4H, 2-phenyl H-3, H-5, 6-phenyl

H-3, H-5).

¹³C NMR (67.5 MHz, DMSO-d

44.67, 142.04, 141.13, 129.83 (2C), 128.18 (4C), 124.57, 115.40

4C), 112.91 (2C), 108.81.

6

) d 158.60 (2C), 156.10 (2C),

0

5

.3.28. 3,3 -([2,4 -Bipyridine]-2 ,6 -diyl)diphenol (33)

The same procedure described in Section 5.3 was employed

with 3 (R = b, R = h) (0.27 g, 1.20 mmol), dry ammonium acetate

1

(

1

2

Please cite this article in press as: Karki, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.002

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

15

3

¹H NMR (300 MHz, DMSO-d

(

0.92 g, 12.00 mmol), 5 (R = b) (0.40 g, 1.20 mmol) and methanol

6

) d 12.25 (s, 2H, 2-phenyl 2-OH, 6-

(

10 mL) to yield an off-white solid (0.24 g, 59.3%, 0.71 mmol).

phenyl 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, 2-

phenyl H-3, H-5, 6-phenyl H-3, H-5).

TLC (ethyl acetate/n-hexane = 1:1)

HPLC: Retention time: 5.32 min, purity: 95.7%; ESI LC/MS: m/z

calcd for C22

f

R = 0.37, mp: 249–250 °C,

+

H

16

N

2

O

2

[MH] 341.13; found 341.37.

1

H NMR (250 MHz, DMSO-d ) d 9.62 (s, 2H, 2-phenyl 3-OH, 6-

6

phenyl 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

1

3

6

C NMR (75.5 MHz, DMSO-d ) d 157.35 (2C), 155.62 (2C),

(

td, J = 7.7, 1.5 Hz, 1H, 4-pyridine H-4), 7.74 (s, 2H, 2-phenyl H-2,

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).

6

7

2

-phenyl H-2), 7.72 (d, J = 8.0 Hz, 2-phenyl H-6, 6-phenyl H-6),

.54 (dd, J = 7.2, 4.8 Hz, 1H, 4-pyridine H-5), 7.34 (t, J = 7.8 Hz,

H, 2-phenyl H-5, 6-phenyl H-5), 6.90 (dd, J = 7.9, 2.0 Hz, 2H, 2-

0

5.3.32. 2-(6 -(3-Hydroxyphenyl)-[3,4 -bipyridin]-2 -yl)phenol

(37)

phenyl H-4, 2-phenyl H-4).

1

3

C NMR (62.5 MHz, DMSO-d

6

) d 158.04 (2C), 156.73 (2C),

The same procedure described in Section 5.3 was employed

1

2

1

53.90, 150.08, 148.00, 140.31 (2C), 137.83, 130.02 (2C), 124.68,

21.83, 117.78 (2C), 116.55 (2C), 116.02 (2C), 113.81 (2C).

with 3 (R = a, R = i) (0.27 g, 1.50 mmol), dry ammonium acetate

3

(0.92 g, 15.00 mmol), 5 (R = 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) R = 0.34, mp: 239–240 °C, HPLC:

Retention time: 5.96 min, purity: 99.4%; ESI LC/MS: m/z calcd for

0

5

(

.3.29. 3-(6 -(4-Hydroxyphenyl)-[2,4 -bipyridin]-2 -yl)phenol

f

34)

The same procedure described in Section 5.3 was employed

with 3 (R = b, R = h) (0.27 g, 1.20 mmol), dry ammonium acetate

0.92 g, 12.00 mmol), 5 (R = 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

+

C

22

1

H

16

N

2

O

2

[MH] 341.13; found 341.37.

1

2

H NMR (250 MHz, DMSO-d ) d 14.32 (s, 1H, 2-phenyl 2-OH),

6

3

(

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, pyr-

idine 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).

f

ethyl acetate/n-hexane = 1:1) R = 0.35, mp: 234–235 °C, HPLC:

Retention time: 5.33 min, purity: 95.9%; ESI LC/MS: m/z calcd for

+

C

22

H

16

N

2

O

2

[MH] 341.13; found 341.36.

1

H NMR (250 MHz, DMSO-d ) d 9.80 (s, 1H, 2-phenyl 3-OH),

.58 (s, 1H, 6-phenyl 4-OH), 8.79 (d, J = 4.0 Hz, 1H, 4-pyridine H-

), 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

6

9

6

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 159.29, 158.24, 157.62, 154.95,

(

d, J = 7.7 Hz, 2-phenyl H-6), 7.53 (dd, J = 7.4, 4.9 Hz, 1H, 4-pyridine

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.

H-5), 7.36 (t, J = 7.9 Hz, 1H, 2-phenyl H-5), 6.94 (d, J = 8.5 Hz, 2H, 6-

phenyl H-3, H-5), 6.88 (d, J = 8.0 Hz, 2-phenyl H-4).

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 158.98, 158.01 (2C), 156.79,

0

5

(

.3.33. 2-(6 -(4-Hydroxyphenyl)-[3,4 -bipyridin]-2 -yl)phenol

38)

The same procedure described in Section 5.3 was employed

1

(

56.50, 154.12, 150.04, 147.88, 140.45, 137.77, 130.00, 129.86,

28.46 (2C), 124.58, 121.76, 117.72, 116.43, 115.76 (2C), 114.91

2C), 113.73.

1

2

with 3 (R = a, R = i) (0.27 g, 1.20 mmol), dry ammonium acetate

0.92 g, 12.00 mmol), 5 (R = 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

f

ethyl acetate/n-hexane = 3:1) R = 0.25, mp: 308–309 °C, HPLC:

3

(

0

5

.3.30. 4,4 -([2,4 -Bipyridine]-2 ,6 -diyl)diphenol (35)

The same procedure described in Section 5.3 was employed

1

2

with 3 (R = c, R = h) (0.27 g, 1.20 mmol), dry ammonium acetate

Retention time: 5.98 min, purity: 97.5%; ESI LC/MS: m/z calcd for

3

(

0.92 g, 12.00 mmol), 5 (R = 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).

+

C

22

H

16

N

2

O

2

[MH] 341.13; found 341.37.

H NMR (300 MHz, DMSO-d ) d 14.64 (s, 1H, 2-phenyl 2-OH),

0.00 (s, 1H, 6-phenyl 4-OH), 9.26 (d, J = 1.8 Hz, 1H, 4-pyridine

1

6

TLC (ethyl acetate/n-hexane = 1:1)

HPLC: Retention time: 5.24 min, purity: 95.0%; ESI LC/MS: m/z

f

R = 0.29, mp: 268–269 °C,

1

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

+

calcd for C22

H

16

N

2

O

2

[MH] 341.13; found 341.36.

H NMR (300 MHz, DMSO-d ) d 9.80 (s, 2H, 2-phenyl 4-OH, 6-

phenyl 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,

1

6

(

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,

2

1

6

-phenyl H-2, H-6, 6-phenyl H-2, H-6), 8.02 (td, J = 7.8, 1.8 Hz,

H, 4-pyridine H-4), 7.52 (dd, J = 7.5, 4.8 Hz, 1H, 4-pyridine H-5),

.92 (d, J = 8.7 Hz, 2-phenyl H-3, H-5, 6-phenyl H-3, H-5).

J = 8.7 Hz, 2H, 6-phenyl H-3, H-5), 6.96–6.93 (m, 2H, 2-phenyl H-

3

, H-5).

13_C_N_M_R₍₇₅_.₅_M_H_z_,_D_M_S_O_-_d

6

) d 159.54 (2C), 157.58, 154.92,

1

3

6

C NMR (75.5 MHz, DMSO-d ) d 158.71 (2C), 156.41 (2C),

1

50.64, 148.71, 147.86, 135.39, 133.23, 131.83, 128.62 (2C),

28.37, 127.99, 124.18, 119.18, 119.11, 118.04, 116.26, 116.19

1

54.16, 149.85, 147.58, 137.58, 129.84 (2C), 128.25 (4C), 124.34,

21.54, 115.56 (4C), 113.65 (2C).

(

2C), 115.35.

0

5

.3.31. 2,2 -([3,4 -Bipyridine]-2 ,6 -diyl)diphenol (36)

0

5

.3.34. 3,3 -([3,4 -Bipyridine]-2 ,6 -diyl)diphenol (39)

The same procedure described in Section 5.3 was employed

with 3 (R = a, R = i) (0.27 g, 1.20 mmol), dry ammonium acetate

0.92 g, 12.00 mmol), 5 (R = 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) R = 0.23, mp: 269–270 °C, HPLC:

f

Retention time: 5.73 min, purity: 99.5%; ESI LC/MS: m/z calcd for

The same procedure described in Section 5.3 was employed

with 3 (R = b, R = i) (0.27 g, 1.20 mmol), dry ammonium acetate

0.92 g, 12.00 mmol), 5 (R = 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)

HPLC: Retention time: 4.66 min, purity: 97.0%; ESI LC/MS: m/z

16 2 2

calcd for C22H N O [MH] 341.13; found 341.37.

1

2

1

2

3

(

f

R = 0.25, mp: 329-330 °C,

+

C

22

H

16

N

2

O

2

[MH] 341.13; found 341.37.

Please cite this article in press as: Karki, R.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.002

1

6

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

¹H NMR (250 MHz, DMSO-d

phenyl 3-OH), 9.21 (s, 1H, 4-pyridine H-2), 8.70 (d, J = 4.0 Hz, 1H,

) d 9.64 (s, 2H, 2-phenyl 3-OH, 6-

volume of 10 lL was terminated by adding 2.5 lL of the stop solu-

6

tion 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

4

2

-pyridine H-6), 8.45 (d, J = 8.0 Hz, 1H, 4-pyridine H-4), 8.17 (s,

H, 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, 4-

pyridine H-5), 7.36 (t, J = 7.8 Hz, 2H, 2-phenyl H-5, 6-phenyl H-

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

), 6.90 (dd, J = 8.0, 1.7 Hz, 2H, 2-phenyl H-4, 2-phenyl H-4).

1

3

6

C NMR (62.5 MHz, DMSO-d ) d 158.02 (2C), 156.79 (2C),

1

50.32, 148.52, 146.81, 140.30 (2C), 135.18, 133.64, 129.96 (2C),

24.20, 118.03 (2C), 116.91 (2C), 116.59 (2C), 114.03 (2C).

5.4.2. Assay for DNA Topoisomerase II Inhibition in vitro

DNA topo II inhibitory activity of compounds were measured as

3

5

follows.

The mixture of 200 ng of supercoiled pBR322 plasmid

(Usb Corp.,

0

5

(

.3.35. 3-(6 -(4-Hydroxyphenyl)-[3,4 -bipyridin]-2 -yl)phenol

DNA and 1 unit of human DNA topoisomerase II

USA) was incubated without and with the prepared compounds

in the assay buffer (10 mM Tris–HCl (pH 7.9) containing 50 mM

a

40)

The same procedure described in Section 5.3 was employed

with 3 (R = b, R = i) (0.27 g, 1.20 mmol), dry ammonium acetate

0.92 g, 12.00 mmol), 5 (R = 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).

1

2

NaCl, 5 mM MgCl

serum albumin) for 30 min at 30 °C. The reaction in a ﬁnal volume

of 20 L was terminated by the addition of 3 L of 7 mM EDTA.

Reaction products were analyzed on 1% agarose gel at 25 V for

h with a running buffer of TAE. Gels were stained for 30 min in

an aqueous solution of ethidium bromide (0.5 g/mL). DNA bands

were visualized by transillumination with UV light and supercoiled

DNA was quantitated using AlphaImager™ (Alpha Innotech

Corporation).

2

, 1 mM EDTA, 1 mM ATP, and 15 lg/mL bovine

3

(

l

TLC (ethyl acetate/n-hexane = 3:1)

HPLC: Retention time: 4.33 min, purity: 95.5%; ESI LC/MS: m/z

f

R = 0.25, mp: 277-278 °C,

4

+

calcd for C22

H

16

N

2

O

2

[MH] 341.13; found 341.36.

H NMR (250 MHz, DMSO-d ) d 9.76 (br, 1H, 2-phenyl 3-OH),

.65 (br, 1H, 6-phenyl 4-OH), 9.21 (d, J = 2.0 Hz, 1H, 4-pyridine

l

1

6

9

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),

.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, 2-

phenyl 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).

6

C NMR (62.5 MHz, DMSO-d ) d 159.00, 157.95 (2C), 156.78,

56.55, 150.21, 148.43, 146.59, 140.39, 135.05, 133.74, 129.86,

29.77, 128.60 (2C), 124.12, 117.92, 116.42, 115.66 (3C), 113.93.

5

.4.3. Cytotoxicity assay

Cancer cells were cultured according to the supplier’s instruc-

tions. Cells were seeded in 96-well plates at a density of 2–

ꢀ 10 cells per well and incubated for overnight in 0.1 mL of

media supplied with 10% Fetal Bovine Serum (Hyclone, USA) in

% CO 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

7

(

4

1

3

5

2

1

day 4, each well was added with 5 lL of the cell counting kit-8

0

5

.3.36. 4,4 -([3,4 -Bipyridine]-2 ,6 -diyl)diphenol (41)

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 equa-

tion. The compounds like adriamycin, etoposide, and camptothecin

were purchased from Sigma and used as positive controls.

The same procedure described in Section 5.3 was employed

1

2

with 3 (R = c, R = i) (0.27 g, 1.20 mmol), dry ammonium acetate

3

(

0.92 g, 12.00 mmol), 5 (R = 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)

HPLC: Retention time: 4.27 min, purity: 97.9%; ESI LC/MS: m/z

f

R = 0.23, mp: 316-317 °C,

+

calcd for C22

H

16

N

2

O

2

[MH] 341.13; found 341.37.

1

H NMR (250 MHz, DMSO-d ) d 9.79 (s, 2H, 2-phenyl 4-OH, 6-

6

Acknowledgement

phenyl 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, 4-

pyridine 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,

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

1

H, 4-pyridine H-5), 6.92 (d, J = 8.7 Hz, 2-phenyl H-3, H-5, 6-phenyl

H-3, H-5).

¹³C NMR (62.5 MHz, DMSO-d

50.14, 148.39, 146.42, 134.98, 133.90, 129.92 (2C), 128.56 (4C),

Technology (NRF-2012R1A2A2A01046188).

6

) d 158.90 (2C), 156.61 (2C),

Supplementary data

1

24.11, 115.63 (4C), 114.51 (2C).

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

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

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