A new series of 2-phenol-4-aryl-6-chlorophenyl pyridine derivatives as dual topoisomerase I/II inhibitors: Synthesis, biological evaluation and 3D-QSAR study

Article abstract of DOI:10.1016/j.ejmech.2016.02.050

As a continuous effort to develop novel antitumor agents, a new series of forty-five 2-phenol-4-aryl-6-chlorophenyl pyridine compounds were synthesized and evaluated for cytotoxicity against four different human cancer cell lines (DU145, HCT15, T47D, and HeLa), and topoisomerase I and II inhibitory activity. Several compounds (10-15, 20, 22, 24, 28, 42, and 49) displayed strong to moderate dual topoisomerase I and II inhibitory activity at 100 μM. It was observed that hydroxyl and chlorine moiety at meta or para position of phenyl ring is favorable for dual topoisomerase inhibitory activity and cytotoxicity. Most of the compounds displayed stronger cytotoxicities than those of all positive controls against the HCT15 and T47D cell lines. For investigation of the structure-activity relationships, a 3D-QSAR analysis using the method of comparative molecular field analysis (CoMFA) was performed. The generated 3D contour maps can be used for further rational design of novel terpyridine derivatives as highly selective and potent cytotoxic agents.

Full text of DOI:10.1016/j.ejmech.2016.02.050

Accepted Manuscript
A new series of 2-phenol-4-aryl-6-chlorophenyl pyridine derivatives as dual
topoisomerase I/II inhibitors: Synthesis, biological evaluation and 3D-QSAR study
Radha Karki, Kyu-Yeon Jun, Tara Man Kadayat, Somin Shin, Til Bahadur Thapa
Magar, Ganesh Bist, Aarajana Shrestha, Younghwa Na, Youngjoo Kwon, Eung-Seok
Lee
PII:
S0223-5234(16)30140-4
10.1016/j.ejmech.2016.02.050
EJMECH 8401
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 October 2015
Revised Date: 18 February 2016
Accepted Date: 19 February 2016
Please cite this article as: R. Karki,, K.-Y. Jun,, T.M. Kadayat,, S. Shin,, T.B. Thapa Magar,, G. Bist,,
A. Shrestha,, Y. Na,, Y. Kwon,, E.-S. Lee, A new series of 2-phenol-4-aryl-6-chlorophenyl pyridine
derivatives as dual topoisomerase I/II inhibitors: Synthesis, biological evaluation and 3D-QSAR study,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.050.
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ACCEPTED MANUSCRIPT
Graphical Abstract
4
6
N
HO
2
-phenol-4,6-diphenyl pyridines
Modification
at 4 and 6 position
R = pheny l, 2/3-thieny l, 2/3-f ur yl
R
R
R
N
OH
HO
N
N
Cl
Cl
Cl
HO
No topo Inhibition
Low cytotoxicity
Strong cytotoxicity and dual topo I/II Inhibition
2
-phenol-4-aryl-6-chlorophenyl pyridines

ACCEPTED MANUSCRIPT
A new series of 2-phenol-4-aryl-6-chlorophenyl pyridine derivatives as dual topoisomerase
I/II inhibitors: Synthesis, biological evaluation and 3D-QSAR study
#
,[a]
#,[b]
#,[a]
[b]
Radha Karki, Kyu-Yeon Jun,
Tara Man Kadayat, Somin Shin, Til Bahadur Thapa
[
a]
[a]
[a]
[c]
,[b]
Magar, Ganesh Bist, Aarajana Shrestha, Younghwa Na, Youngjoo Kwon,* Eung-Seok
,[a]
Lee*
[
a]
b]
College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Global Top 5
[
program Ewha Womans University, Seoul 120-750, Republic of Korea
[c]
College of Pharmacy, Cha University, Pochon 487-010, Republic of Korea
#
Authors are equal contributors.
*
Corresponding Authors:
Tel: +82 53 810-2827, fax: +82 53 810-4654, email: eslee@yu.ac.kr (E.-S.L.)
Tel: +82 2 3277-4653, fax: +82 2 3277-2851, email: ykwon@ewha.ac.kr (Y.K.)

ACCEPTED MANUSCRIPT
A new series of 2-phenol-4-aryl-6-chlorophenyl pyridine derivatives as dual topoisomerase
I/II inhibitors: Synthesis, biological evaluation and 3D-QSAR study
#
,[a]
#,[b]
#,[a]
[b]
Radha Karki, Kyu-Yeon Jun,
Tara Man Kadayat, Somin Shin, Til Bahadur Thapa
[
a]
[a]
[a]
[c]
,[b]
Magar, Ganesh Bist, Aarajana Shrestha, Younghwa Na, Youngjoo Kwon,* Eung-Seok
,[a]
Lee*
Abstract
As a continuous effort to develop novel antitumor agents, a new series of forty-five 2-phenol-4-
aryl-6-chlorophenyl pyridine compounds were synthesized and evaluated for cytotoxicity against
four different human cancer cell lines (DU145, HCT15, T47D, and HeLa), and topoisomerase I
and II inhibitory activity. Several compounds (10–15, 20, 22, 24, 28, 42, and 49) displayed
strong to moderate dual topoisomerase I and II inhibitory activity at 100 µM. It was observed
that hydroxyl and chlorine moiety at meta or para position of phenyl ring is favorable for dual
topoisomerase inhibitory activity and cytotoxicity. Most of the compounds displayed stronger
cytotoxicities than those of all positive controls against the HCT15 and T47D cell lines. For
investigation of the structure-activity relationships, a 3D-QSAR analysis using the method of
comparative molecular field analysis (CoMFA) was performed. The generated 3D contour maps
can be used for further rational design of novel terpyridine derivatives as highly selective and
potent cytotoxic agents.
Keywords: Antitumor agents; CoMFA; Cytotoxicity; 3D-QSAR; Dual topoisomerase I and II
inhibition; 2-phenol-4-aryl-6-chlorophenyl pyridine

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1
. Introduction
For the past several years, our research group has been seeking to identify and optimize low
molecular weight molecules capable of inhibiting dual topoisomerase I and II activity and cancer
cell proliferation. A series of mono, di and tri-hydroxylated 2,4,6-triphenyl pyridine derivatives
have recently been reported with significant topoisomerase inhibitory activity and cytotoxicity
against several human cancer cell lines [1, 2]. The importance of hydroxyl moieties has also been
reported in natural polyphenolic compounds such as resveratrol, curcumin, and epigallocatechin
for several biological activities including cytotoxicity and dual topoisomerase I and II inhibition
[3-6]. Similarly, formation of halogen bond with protein plays important role to increase the
protein-ligand binding affinity, and therefore insertion of halogen atoms is widely practiced in
drug design and discovery area to improve potency, blood brain barrier permeability, and
metabolic and chemical stability. Moreover, for the lead optimization of drug, many researchers
in medicinal chemistry are attracted to develop chlorinated compounds as chlorine can
accommodate in tight and deep cavities, as well as in hydrophobic pockets of the biological
targets that contribute to the stability and specificity of the binding site [7, 8].
Human DNA topoisomerases (topo I and topo II) are expressed at different level in different
cancer types [9-12]. For instance, topo I is overexpressed in colon cancer cell lines, while topo II
is overexpressed in breast and ovarian cancer cell lines [11, 13]. Camptothecin and etoposide,
topo I and topo II inhibitor, respectively, are clinically approved anticancer drugs [14, 15].
Recent studies have reported that use of single topo I enzyme inhibitor can develop its resistance
by inducing concomitant increase in expression level of topo II enzyme, and vice versa [16, 17].
Though this concept is still not very clear, some new class of compounds exhibited strong dual

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inhibitory activity in preclinical studies [18]. Furthermore treatment of dual topo I and II
inhibitors may exhibit synergistic effect to result in better efficacy, lower side effects and lower
resistance. Therefore, dual inhibition of topo I and II, which are involved in solving the
topological problems associated with nuclear processes, may be a promising strategy for the
design of anticancer agents [19, 20]. Previously reported 2,4,6-triaryl pyridines containing
chlorophenyl and phenolic moiety at 2- and 4-position of central pyridine, respectively, have
shown dual topo I and II inhibitory activity [21]. In addition, several reports on importance of
meta or para hydroxyl groups on 2-phenyl ring and thienyl or furyl moiety on 4-position of
central pyridine [22, 23] motivated us to synthesize a new series of 2-phenol-4-aryl-6-
chlorophenyl pyridines as potential antitumor agents.
The aim of the present work was to incorporate the chlorine moiety at 6-phenyl position and aryl
group at 4-position of central pyridine and to examine their biological effects. Herein, we report
the synthesis and three-dimensional quantitative structure-activity relationship (3D-QSAR) study
using the method of comparative molecular field analysis (CoMFA) for a series of novel 2-
phenol-4-aryl-6-chlorophenyl pyridines which inhibit both topo I and II activity and show
significant cytotoxicity. The substitution of hydroxyl and chlorine group allows us to investigate
their combined effect on cytotoxicity and dual topo inhibition with respect to the position (ortho,
meta, para) on phenyl ring.
2
. Design and synthesis
Previously, we reported 2-phenol-4,6-diphenyl pyridines, A, B and C (Figure 1) for the topo
inhibitory activity and cytotoxicity against several human cancer cell lines [1]. We were

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interested to observe the combined effect on biological results by incorporating hydroxyphenyl
moiety on 2-positon, phenyl, thienyl or furyl moieties on 4-position and chlorophenyl moiety on
6
-positon. Design of 2-phenol-4-aryl-6-chlorophenyl pyridines allowed us to investigate whether
the change in the aryl moiety at 4-position and chlorophenyl moiety at 6-position of central
pyridine has any effect on the dual topo I and II inhibitory activity and cytotoxicity.
Subsequently, the ortho, meta and para substitution of chlorine moiety will help us to further
confirm the effect of chlorine position on biological activity with respect to the previous data.
Total forty-five 2-phenol-4-aryl-6-chlorophenyl pyridine compounds (7–51) were systematically
designed in five different series as shown in Figure 2. All the designed compounds contain one
hydroxyl and one chlorine moiety at ortho, meta or para position of 2- and 6- phenyl ring,
respectively, attached to the central pyridine. The 4-position of central pyridine was substituted
with various aryl groups (phenyl, 2/3-thienyl, 2/3-furyl).
Figure 1
Figure 2
The synthetic route with three different steps for the target compounds 7–51 is outlined in
Scheme 1. In the first step, fifteen different hydroxylated chalcones were synthesized as
intermediates using Clasien-Schmidt condensation reaction [24]. In this method, 4 M aqueous
1
solution of NaOH was added to the solution of equimolar amounts of aryl ketone 1 (R = a-c)
2
1
2
and aryl aldehyde 2 (R = d-h) in ethanol to obtain compounds 3 (R = a-c, R = d-h) in 47–98%
3
yield. In the second step, three pyridinium iodide salts 5 (R = i-k) were synthesized in a
3
quantitative yield by the treatment of aryl ketone 4 (R = i-k) with iodine in pyridine. Finally

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using previously reported synthetic methods [25-29], final compounds (7-51) were synthesized
1
2
by the reaction of appropriate hydroxylated chalcones 3 (R = a-c, R = d-h) with pyridinium
3
iodide salt 5 (R = i-k) in the presence of ammonium acetate and glacial acetic acid in 36–95%
yield (Figure 3). The yields (%), purities (%) measured by HPLC, retention time, and melting
points of the synthesized compounds are listed in Table S1 (Supplementary Data).
Scheme 1
Figure 3
3
. Results and Discussion
All the synthesized 2-phenol-4-aryl-6-chlorophenyl pyridine compounds (7–51) were evaluated
for topo I and II inhibitory activity, and cytotoxicity against several human cancer cell lines. The
conversion of supercoiled plasmid DNA to relaxed DNA by topo I and II was examined in the
presence of synthesized 2-phenol-4-aryl-6-chlorophenyl pyridine compounds (7–51).
Camptothecin and etoposide, well known topo I and II inhibitors, respectively, were used as
positive controls. Figure 4 and 5 and Table 1 illustrate the topo I and II inhibitory activity and
cytotoxicity of synthesized compounds 7–51.
Figure 4
Figure 5
Table 1
3
.1. In vitro Cytotoxicity of the compounds

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We performed cytotoxicity assay using four different human cancer cell lines: 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 (topo II inhibitor) and camptothecin (topo I inhibitor), widely used anticancer drugs,
were used as positive controls. The inhibitory activity (IC ) is expressed as micromolar
5
0
concentration as illustrated in Table 1. Several compounds displayed significant cytotoxicity
against the tested cell lines at low micromolar concentration compared to the positive controls.
Compounds 10–15 (from Series A) containing phenyl moiety at 4-position of central pyridine
showed significant cytotoxicity (<2 µM) against the cell lines DU145, HCT15 and T47D, and
considerable cytotoxicity (<8 µM) against HeLa. Compound 14 possessed stronger cytotoxicity
(0.83 µM) than that of all the positive controls against HCT15. Compound 7–9 belonging to the
same series exhibited moderate to weak cytotoxicity against the tested cell lines. Similar type of
cytotoxicity pattern was observed with compounds containing 2-thienyl moiety at 4-position of
central pyridine (16–24, Series B). Compounds 19–24 exhibited significant cytotoxicity (<2 µM)
against the cell lines DU145, HCT15 and T47D. These compounds also showed considerable
cytotoxicity (<5 µM) against HeLa. Compound 16 exhibited significant cytotoxicity against
HCT15. Compounds 17 and 18 were less cytotoxic (>50 µM) to most of the cell lines.
Compounds 25–33 (from Series C), containing 3-thienyl moiety at 4-position of central pyridine,
displayed significant cytotoxicity to all the tested cell lines that were comparable to positive
controls with the exception of compound 26. Compound 28 possessed strong cytotoxicity against
DU145 and HCT15 (0.74 µM and 0.80 µM respectively) and compound 29 against DU145 (0.88
µM). Compounds 37–41 (from Series D), containing 2-furyl moiety at 4-position of central

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pyridine, displayed strong cytotoxicity against DU145, HCT15 and T47D while compounds 34–
3
6 and 42 exhibited moderate cytotoxicity. Among compounds 43–51 (from Series E) having 3-
furyl moiety at 4-position of central pyridine, compounds 46–50 possessed significant
cytotoxicity against DU145 and T47D (<2 µM) and HCT15 (<5 µM). Compounds 43–45, 51
exhibited moderate to weak activity. The cytotoxicity results indicate that most of the
compounds were active against the cell lines DU145, HCT15 and T47D with IC values less
5
0
than 2 µM. Interestingly, it was observed that combined effect of meta or para-hydroxyl group
and chlorine moiety on phenyl ring is favorable for significant cytotoxicity.
3
.2. Evaluation of topoisomerase I and II inhibition and structure-activity relationship study
Among 45 tested compounds as shown in Figure 4, several compounds 7, 9, 11, 13, 15, 16, 19,
2, 24, 28, 35, 42, and 49 exhibited stronger topo I inhibitory activity than positive control,
2
camptothecin (64.7%) at 100 µM concentration. Compounds 10, 12, 14, 20, and 36 exhibited
moderate topo I inhibition. However, compound 36 possessed significant topo I inhibitory
activity (44.6%) at 20 µM. All the other synthesized compounds exhibited weaker topo I
inhibition.
Figure 5 and Table 1 summarizes the topo II inhibitory activity of the synthesized compounds.
Most of the compounds showed significant to moderate topo II inhibitory activity at 100 µM as
compared to that of positive control, etoposide. Among compounds 7–15 from Series A, which
contain phenyl moiety at 4-position of central pyridine, compounds 13 and 14 exhibited strong
inhibition (89.1% and 79.9%, respectively) at 100 µM as compared to etoposide (76.4%
inhibition). Compounds 10, 11, 12, and 15 possessed moderate topo II inhibitory activities.

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However, compounds 7–9 were devoid of inhibitory activity. The similar type of topo II
inhibitory pattern was observed for compounds (16–24) from Series B, which contain 2-thienyl
moiety at 4-position of central pyridine. Compounds 19–24 exhibited moderate to strong topo II
inhibition. Among compounds 25–33 from Series C, which contains 3-thienyl moiety at 4-
position of central pyridine, compounds 31 and 33 possessed strong inhibition (81.8% and 90.5%
respectively). Compounds 28-30 and 32 exhibited considerable inhibitory activities at 100 µM.
With the similar previous pattern compounds 25–27 displayed no activity. Series D containing 2-
furyl moiety at 4-position of central pyridine (34–42), compound 40 displayed stronger activity
(77%) than etoposide at 100 µM. Compounds 39, 41 and 42 exhibited moderate topo II
inhibitory activity at both 100 µM (56.5%, 56.1%, 63.9%) and 20 µM (25.5%, 22.3%, 29%),
respectively. Compounds 37 and 38 showed considerable activity at 100 µM and compounds 34–
3
6 possessed no activity. Among compounds 43–51 from Series E having 3-furyl moiety at 4-
position of central pyridine, compounds 48–51 possessed significant activity (78.15%, 81.9%,
4.1% and 86.6%, respectively) at 100 µM. Similarly, compounds 46 and 47 displayed moderate
8
inhibition while compound 43-45 possessed weak topo II inhibitory activity at 100 µM. It is
observed that most of the compounds exhibited significant to moderate topo II inhibitory activity
at 100 µM, with a few compounds active at 20 µM. It is interesting to note that compounds from
Series D containing 2-furyl moiety at 4-position of central pyridine displayed stronger or
moderate topo II inhibitory activity at both 100 µM and 20 µM.
Compounds (7–9, 16–18, 25–27, 34–36, 43–45) with ortho-hydroxyphenyl at 2-position of
central pyridine ring nevertheless substitution of aryl group at 4-position of central pyridine and
position of chloride on phenyl group at 6-position of central pyridine, showed weak to no topo II

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inhibition at 100 µM, and were less cytotoxic in most of the tested cell lines. However, all the
other compounds with hydroxyl moiety at meta or para position of 2-phenyl ring on central
pyridine (10–15, 19–24, 28–33, 37–42, 46–51) possessed significant topo II inhibition and
cytotoxicity (<2 µM) in most of the cell lines. In general, the results indicated that meta or para-
hydroxyphenyl substituents on 2-position of central pyridine showed the better topo II inhibitory
activity and cytotoxicity regardless of the substitution at 4, and 6 positions on central pyridine
(
Figure 6). Four compounds (13, 22, 24 and 49) were proved to possess both topo I and II
inhibitory activity stronger than positive control at 100 µM. Furthermore, compounds 10-12, 14,
5, 20, 28, 42 exhibited moderate topo I and II inhibitory activity. Structure-activity relationship
1
study revealed that hydroxyl moiety at meta or para position is more favorable for dual topo I
and II inhibitory activity and cytotoxicity with chlorine moiety on 6-phenyl ring, as indicated in
Figure 7.
Figure 6
Figure 7
3
.3. Analysis of the 3D-quantitative structure-activity relationship
The 3D-quantitative structure activity relationships for the compounds synthesized were
evaluated. In this study, compounds synthesized all share the common core of 2,6-
diphenylpyridine ring. For 3D-QSAR analysis, alignment of the compounds is necessary.
Compound 10 was chosen as the template since it had good topo I and topo II activities as well
as low IC50 for all four human cancer cell lines. Using compound 10 as a template the remaining
compounds were manually aligned using the common core 2,6-diphenylpyridine ring (Figure 8).

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The hydroxylphenyl ring was placed at the 2-position of pyridine and the chlorophenyl ring at 6-
position of pyridine ring. Comparative molecular field analysis (CoMFA) can be used to quantify
specific contributions of steric and electrostatic factors that influence the cytotoxicity of small
2
2
molecules [30]. According to CoMFA model, the cross-validated r (q ) of the training set was
2
0
.82 with 3 components, while the non-cross-validated r was 0.915 with a standard error of
estimate of 0.135 and F values of 86.404. The steric and electrostatic descriptors had 26.8% and
3.2% of relative contributions. Figure 9 shows the contour maps for the CoMFA model based
7
on one of the most active compound 38. In the steric contribution contour map (Figure 9A),
green represents regions that favor steric bulk groups and yellow the opposite. The
electropositive substitutions were favored meta and para site of 2-hydroxylphenyl ring as it is
represented in red and electropositive substitutions in blue (Figure 9B). The 6-chlorophenyl ring
did not have any significant effect on the inhibitory activity of the compound depending on the
different location of the chloro group substitution. The model was validated by a test set that
included eight compounds, indicated by asterisks in Table 2. The 3D-QSAR model gave good
statistical results and good prediction using the test set. In summary, hydroxyl group which is
able to form H-bonding was the most important factor for the activity of the compounds
according to the CoMFA model.
Figure 8
Figure 9
Table 2
4
. Conclusion

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In this study, we designed and synthesized forty-five 2-phenol-4-aryl-6-chlorophenyl pyridines
by efficient synthetic route. With an aim to develop novel antitumor agent, synthesized
compounds were evaluated for dual topo I and II inhibitory activity and cytotoxicity against
several human cancer cell lines. Compound 10–15, 20, 22, 24, 28, 42, and 49 showed strong to
moderate dual topoisomerase I and II inhibitory activity at 100 µM. Several compounds
exhibited strong cytotoxicity compared to all the positive controls against human colorectal
adenocarcinoma cell line (HCT15), and human ductal breast epithelial tumor cell line (T47D).
We also analyzed 3D-QSAR for the synthesized compounds using CoMFA. The CoMFA model
generated using the pIC50 data from T47D cells was validated by predicting a test set consisting
of eight compounds. Based on CoMFA analysis, substitution of chloro group at 6-phenyl ring did
not have any significant effect on the inhibitory activity of the compound. However, hydroxyl
group which is able to form H-bonding was the most important factor for the activity. Further
exploration by introducing other important electron withdrawing or donating substituents
including hydroxyl and chlorine group on central pyridine could lead the development of novel
and potent anticancer agents. Importantly, these results prompt us to investigate the possible
cytotoxic mechanisms of these compounds against HCT15 and T47D.
5
. Experimental
Compounds used as starting materials and reagents were obtained from Aldrich Chemical Co.,
Junsei or other chemical companies, and used without further purification. HPLC grade
acetonitrile (ACN) and methanol were purchased from Burdick and Jackson, USA. Thin-layer
chromatography (TLC) and column chromatography (CC) were performed with Kieselgel 60
F254 (Merck) and silica gel (Kieselgel 60, 230-400 mesh, Merck) respectively. Since all the

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compounds prepared contain aromatic ring, they were visualized and detected on TLC plates
with UV light (short wave, long wave or both). NMR spectra were recorded on a Bruker AMX
2
50 (250 MHz, FT) for 1H NMR and 62.5 MHz for 13C NMR, and chemical shifts were
calibrated according to TMS. Chemical shifts (δ) were recorded in ppm and coupling constants
J) in hertz (Hz). Melting points were determined in open capillary tubes on electrothermal 1A
100 digital melting point apparatus and were uncorrected.
(
9
HPLC analyses were performed using two Shimadzu LC-10AT pumps gradient-controlled
HPLC system equipped with Shimadzu system controller (SCL-10A VP) and photo diode array
detector (SPD-M10A VP) using Shimadzu Class VP program. Sample volume of 10 ꢀL was
injected in Waters X- Terra® 5 ꢀM reverse-phase C18 column (4.6ⅹ250 mm) with a gradient
elution of 70% to 100% of B in A for 10 min followed by 100% to 70% of B in A for 10 min at a
flow rate of 1.0 mL/min at 254 nm UV detection, where mobile phase A was solution (20 mM)
of ammonium formate (AF) in double distilled water and B was 100% ACN. Purity of compound
is described as percent (%), and retention time is given in minutes.
®
ESI LC/MS analyses were performed with a Finnigan LCQ Advantage LC/MS/MS
®
spectrometry utilizing Xcalibur program. For ESI LC/MS, LC was performed with a 5 ꢀL
®
injection volume on a Waters X Terra 3.5 ꢀm reverse-phase C column (2.1ⅹ100 mm) with a
1
8
gradient elution from 10% to 90% of B in A for 5 min followed by 90% to 10% of B in A for 7
min and 10% B in A for 6 min, at a flow rate of 250 ꢀL/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,

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capillary temperature: 215°C, column temperature 40 °C, capillary voltage: 21 V, tube lens
offset: 10 V.
5
.1. General method for the preparation of 3
1
2
Compounds 3 (R = a-c, R = d-h) were synthesized using Claisen-Schmidt condensation
1
2
reaction. To a solution of equimolar amounts of aryl ketone 1 (R = a-c) and aryl aldehyde 2 (R
d-h) in ethanol were added aqueous solution of 4 M sodium hydroxide and stirred for 2 - 8 h at
0 °C. The reaction mixture was neutralized with 6 M HCl and pH adjusted to 2. The mixture
=
2
was extracted with ethyl acetate, washed with water and brine. It was further purified by either
recrystallization or column chromatography to yield pure compound. Total fifteen hydroxylated
chalcone intermediates were synthesized which were reported earlier [1, 31].
5
.2. General method for the preparation of 5
3
A mixture of aryl methyl ketone 4 (R = i-k), iodine (1.2 equiv) and pyridine (15 equiv) was
refluxed at 140 °C for 3 h. After cooling the reaction mixture to room temperature, the
precipitate was filtered and washed with cold pyridine followed by drying overnight to yield 66-
3
8
7% of 5 (R = i-k). Three different pyridinium iodide salts (5i, 5j, and 5k) synthesized by this
method were used without further purification.
5
.2.1. Synthesis of 1-[2-(2-Chloro-phenyl)-2-oxo-ethyl]-pyridinium iodide (5i)
The same procedure described above for S-1 was followed with 2'-chloro acetophenone (4.01
mL, 30.0 mmol), iodine (7.61 g, 30.0 mmol), and pyridine (35 mL) to yield 7.13 g (19.8 mmol,
6
6.0%) as an off-white solid.

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1
Mp: 205.3-206.8 °C, H NMR (250 MHz, DMSO-d ) δ 9.00 (d, J = 5.6 Hz, 2H, pyridinium H-2,
6
H-6), 8.74 (t, J = 7.6 Hz, 1H, pyridinium H-4), 8.28 (t, J = 7.2 Hz, 2H, pyridinium H-3, H-5),
8
.10 (d, J = 7.2 Hz, 1H, phenyl H-6), 7.77 - 7.59 (m, 3H, phenyl H-3, H-4, H-5), 6.39 (s, 2H, -
CO-CH -).
2
5
.2.2. Synthesis of 1-[2-(3-Chloro-phenyl)-2-oxo-ethyl]-pyridinium iodide (5j)
The same procedure described above for S-1 was followed with 3'-chloro acetophenone (6.45
mL, 50.0 mmol), iodine (12.69 g, 50.0 mmol), and pyridine (60 mL) to yield 16.90 g (47.0
mmol, 94.0%) as an off-white solid.
1
Mp: 197.5-198.6 °C, H NMR (250 MHz, DMSO-d ) δ 8.96 (d, J = 5.9 Hz, 2H, pyridinium H-2,
6
H-6), 8.73 (t, J = 7.7 Hz, 1H, pyridinium H-4), 8.27 (t, J = 6.9 Hz, 2H, pyridinium H-3, H-5),
8
.08 (br, 1H, phenyl H-2), 7.99 (d, J = 7.7 Hz, 1H, phenyl H-6), 7.80 (d, J = 8.2 Hz, 1H, phenyl
H-4), 7.74 (t, J = 7.8 Hz, 1H, phenyl H-5), 6.45 (s, 2H, -CO-CH -).
2
5
.2.3. Synthesis of 1-[2-(4-Chloro-phenyl)-2-oxo-ethyl]-pyridinium iodide (5k)
The same procedure described above for S-1 was followed with 4'-chloro acetophenone (7.78
mL, 60.0 mmol), iodine (15.22 g, 60.0 mmol), and pyridine (70 mL) to yield 18.76 g (52.1
mmol, 86.9%) as a creamy white solid.
1
Mp: 198.3-199.5 °C, H NMR (250 MHz, DMSO-d
6
) δ 8.97 (d, J = 5.9 Hz, 2H, pyridinium H-2,
H-6), 8.73 (t, J = 7.7 Hz, 1H, pyridinium H-4), 8.27 (t, J = 7.0 Hz, 2H, pyridinium H-3, H-5),
.09 (d, J = 8.4 Hz, 2H, phenyl H-2, H-6), 7.77 (d, J = 8.4 Hz, 2H, phenyl H-3, H-5), 6.44 (s, 2H,
CO-CH -).
8
-
2
5
.3. General method for the preparation of 7–51
1
2
A mixture of hydroxylated chalcone intermediate 3 (R = a-c, R = d-h), pyridinium iodide salt 5
3
(
R = i-k) and anhydrous ammonium acetate in glacial acetic acid were heated at 80-100 °C for
1
2–24 h. The reaction mixture was then extracted with ethyl acetate, washed with water and

ACCEPTED MANUSCRIPT
brine. The organic layer was dried over magnesium sulfate and filtered. The filtrate was
evaporated at reduced pressure, which was then purified by silica gel column chromatography
with the gradient elution of ethyl acetate/n-hexane to afford solid compounds 7-51 in 36–95%
yield. Total forty-five 2-phenol-4-aryl-6-chlorophenyl pyridine compounds were synthesized by
this method.
5
.3.1. Synthesis of 2-(6-(2-Chlorophenyl)-4-phenylpyridin-2-yl)phenol (7)
1
2
The same procedure described in section 5.3 was employed with 3 (R = a, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = i) (0.72 g, 2.00 mmol) and acetic
acid (2 mL) to yield a yellow solid (0.37 g, 51.4%, 1.02 mmol). TLC (ethyl acetate / n-hexane =
1
:5) R = 0.46, Mp: 165-166 °C, HPLC: Retention time: 11.78 min, purity: 100%; ESI LC/MS:
f
+
+
m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.37 (100%), [MH+2]: 360.38 (30%).
2
3
16
1
H NMR (250 MHz, CDCl ) δ 14.32 (s, 1H, 2-phenyl 2-OH), 8.11 (s, 1H, pyridine H-3), 7.97 (d,
3
J = 7.9 Hz, 1H, 2-phenyl H-6), 7.79 (s, 1H, pyridine H-5), 7.78-7.73 (m, 2H, 4-phenyl H-2, H-6),
7
7
1
1
1
.69-7.66 (m, 1H, 6-phenyl H-6), 7.56-7.50 (m, 4H, 4-phenyl H-3, H-4, H-5, 6-phenyl H-3),
.42-7.39 (m, 2H, 6-phenyl H-4, H-5), 7.34 (t, J = 8.1 Hz, 2-phenyl H-4), 7.05 (d, J = 8.2 Hz,
1
3
H, 2-phenyl H-3), 6.96 (t, J = 7.6 Hz, 1H, 2-phenyl H-5). C NMR (62.5 MHz, CDCl ) δ
3
59.99, 157.92, 154.09, 150.37, 138.21, 137.82, 132.22, 131.63, 131.22, 130.46, 130.15 (2C),
29.47, 129.25, 127.30 (3C), 126.34, 121.17, 118.94, 118.87, 118.65, 115.93.
5
.3.2. Synthesis of 2-(6-(3-Chlorophenyl)-4-phenylpyridin-2-yl)phenol (8)
1
2
The same procedure described in section 5.3 was employed with 3 (R = a, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = j) (0.72 g, 2.00 mmol) and acetic

ACCEPTED MANUSCRIPT
acid (2 mL) to yield a yellow solid (0.58 g, 81.4%, 1.62 mmol). TLC (ethyl acetate / n-hexane =
1
:3 v/v) R = 0.48, Mp: 109-110 °C; HPLC: Retention time: 13.28 min, purity: 100%; ESI
f
+
+
LC/MS: m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.36 (100%), [MH+2]:
2
3
16
3
60.37 (30%)
1
H NMR (250 MHz, CDCl ) δ 14.51 (s, 1H, 2-phenyl 2-OH), 8.07 (d, J = 1.1 Hz, 1H, pyridine
3
H-3), 7.95-7.92 (m, 2H, 6-phenyl H-2, 2-phenyl H-6), 7.90-7.7.86 (m, 1H, 6-phenyl H-6), 7.80
(d, J = 1.2 Hz, 1H, pyridine H-5), 7.76-7.72 (m, 2H, 4-phenyl H-2, H-6), 7.56-7.51 (m, 3H, 4-
phenyl H-3, H-4, H-5), 7.47-7.45 (m, 2H, 6-phenyl H-4, H-5), 7.35 (td, J = 8.4, 1.5 Hz, 2-phenyl
H-4), 7.09 (dd, J = 8.2, 1.1 Hz, 1H, 2-phenyl H-3), 6.96 (td, J = 8.2, 1.2 Hz, 1H, 2-phenyl H-5).
1
3
C NMR (62.5 MHz, CDCl ) δ 159.92, 158.26, 153.95, 151.43, 139.97, 138.21, 135.07, 131.79,
3
1
1
30.41, 129.62, 129.57, 129.28 (2C), 127.23 (2C), 127.08, 126.46, 125.16, 118.97, 118.88,
18.56, 117.39, 116.36.
5
.3.3. Synthesis of 2-(6-(4-Chlorophenyl)-4-phenylpyridin-2-yl)phenol (9)
1
2
The same procedure described in section 5.3 was employed with 3 (R = a, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = k) (0.72 g, 2.00 mmol) and acetic
acid (2 mL) to yield a yellow solid (0.65 g, 91.8%, 1.83 mmol). TLC (ethyl acetate / n-hexane =
1
:5 v/v) R = 0.50; Mp: 159-160 °C; HPLC: Retention time: 13.45 min, purity: 100%; ESI
f
+
+
LC/MS: m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.38 (100%), [MH+2]:
2
3
16
3
60.38 (30%)
1
H NMR (250 MHz, CDCl ) δ 14.62 (s, 1H, 2-phenyl 2-OH), 8.05 (d, J = 1.1 Hz, 1H, pyridine
3
H-3), 7.94 (d, J = 8.6 Hz, 2H, 6-phenyl H-2, H-6), 7.94-7.91 (m, 1H, 2-phenyl H-6), 7.79 (d, J =
1
.1 Hz, 1H, pyridine H-5), 7.75-7.71 (m, 2H, 4-phenyl H-2, H-6), 7.56-7.51 (m, 3H, 4-phenyl H-

ACCEPTED MANUSCRIPT
3
4
, H-4, H-5), 7.51 (d, J = 8.5 Hz, 2H, 6-phenyl H-3, H-5), 7.35 (td, J = 8.3, 1.4 Hz, 2-phenyl H-
1
3
), 7.08 (d, J = 8.3, 1.0 Hz, 1H, 2-phenyl H-3), 6.96 (td, J = 8.0, 1.0 Hz, 1H, 2-phenyl H-5). C
NMR (62.5 MHz, CDCl ) δ 159.95, 158.19, 154.08, 151.41, 138.31, 136.53, 135.82, 131.75,
3
1
1
29.53, 129.31 (2C), 129.27 (2C), 128.21 (2C), 127.22 (2C), 126.44, 118.95, 118.90, 118.54,
17.08, 116.08.
5
.3.4. Synthesis of 3-(6-(2-chlorophenyl)-4-phenylpyridin-2-yl)phenol (10)
1
2
The same procedure described in section 5.3 was employed with 3 (R = b, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = i) (0.72 g, 2.00 mmol) and acetic
acid (2 mL) to yield a light yellow solid (0.29 g, 40.7%, 0.81 mmol). TLC (ethyl acetate / n-
hexane = 1:3 v/v) R = 0.38; Mp: 104-105 °C; HPLC: Retention time: 9.42 min, purity:
f
+
+
1
00%; ESI LC/MS: m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.40 (100%),
23 16
[MH+2]: 360.39 (30%)
1
H NMR (250 MHz, CDCl ) δ 7.90 (d, J = 1.4 Hz, 1H, pyridine H-3), 7.81 (d, J = 1.4 Hz, 1H,
3
pyridine H-5), 7.76-7.71 (m, 3H, 4-phenyl H-2, H-6, 6-phenyl H-6), 7.66-7.61 (m, 2H, 2-phenyl
H-2, H-6), 7.56-7.46 (m, 4H, 4-phenyl H-3, H-4, H-5, 6-phenyl H-3), 7.39-7.34 (m, 2H, 6-phenyl
H-4, H-5), 7.33 (t, J = 7.5 Hz, 2-phenyl H-5), 6.90 (ddd, J = 8.0, 2.5, 1.0 Hz, 1H, 2-phenyl H-4),
1
3
5
1
.46 (s, 1H, 2-phenyl 3-OH). C NMR (62.5 MHz, CDCl ) δ 157.29, 157.10, 156.09, 149.38,
3
40.96, 139.28, 138.49, 132.37, 131.79, 130.20, 129.96, 129.66, 129.14, 129.11 (2C), 127.22
(2C), 127.00, 121.66, 119.46, 117.59, 116.18, 114.23.
5
.3.5. Synthesis of 3-(6-(3-Chlorophenyl)-4-phenylpyridin-2-yl)phenol (11)

ACCEPTED MANUSCRIPT
1
2
The same procedure described in section 5.3 was employed with 3 (R = b, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = j) (0.72 g, 2.00 mmol) and acetic
acid (2 mL) to yield a white solid (0.54 g, 76.5%, 1.53 mmol). TLC (ethyl acetate / n-hexane =
1
:5 v/v) R = 0.18; Mp: 179-180 °C; HPLC: Retention time: 11.62 min, purity: 100%; ESI
f
+
+
LC/MS: m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.40 (100%), [MH+2]:
2
3
16
3
60.40 (30%)
1
H NMR (250 MHz, CDCl
3
) δ 8.19-8.17 (m, 1H, 6-phenyl H-2), 8.05-8.02 (m, 1H, 6-phenyl H-
6
4
7
), 7.88 (d, J = 1.3 Hz, 1H, pyridine H-3), 7.85 (d, J = 1.3 Hz, 1H, pyridine H-5), 7.75-7.68 (m,
H, 4-phenyl H-2, H-6, 2-phenyl H-2, H-6), 7.54-7.48 (m, 3H, 4-phenyl H-3, H-4, H-5), 7.43-
.41 (m, 2H, 6-phenyl H-4, H-5), 7.38 (t, J = 7.8 Hz, 2-phenyl H-5), 6.95 (ddd, J = 8.0, 2.5, 0.8
1
3
Hz, 1H, 2-phenyl H-4), 5.13 (s, 1H, 2-phenyl 3-OH). C NMR (62.5 MHz, CDCl ) δ 157.05,
3
1
1
55.99, 155.97, 150.43, 141.23, 140.88, 138.60, 134.77, 130.01, 129.93, 129.16 (3C), 129.04,
27.25 (2C), 127.14, 125.17, 119.49, 117.75, 117.43, 116.23, 114.02.
5
.3.6. Synthesis of 3-(6-(4-Chlorophenyl)-4-phenylpyridin-2-yl)phenol (12)
1
2
The same procedure described in section 5.3 was employed with 3 (R = b, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = k) (0.72 g, 2.00 mmol) and acetic
acid (2 mL) to yield a white solid (0.55 g, 77.9%, 1.55 mmol). TLC (ethyl acetate / n-hexane =
1
:3 v/v) R = 0.34; Mp: 148-149 °C; HPLC: Retention time: 11.73 min, purity: 100%; ESI
f
+
+
LC/MS: m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.40 (100%), [MH+2]:
2
3
16
3
60.40 (30%)
1
H NMR (250 MHz, CDCl
3
) δ 8.12 (dd, J = 8.6, 1.8 Hz, 2H, 6-phenyl H-2, H-6), 7.85 (d, J = 1.2
Hz, 1H, pyridine H-3), 7.83 (d, J = 1.2 Hz, 1H, pyridine H-5), 7.73-7.65 (m, 4H, 4-phenyl H-2,

ACCEPTED MANUSCRIPT
H-6, 2-phenyl H-2, H-6), 7.55-7.47 (m, 3H, 4-phenyl H-3, H-4, H-5), 7.46 (d, J = 8.6 Hz, 2H, 6-
phenyl H-3, H-5), 7.35 (t, J = 7.9 Hz, 2-phenyl H-5), 6.92 (ddd, J = 8.0, 2.5, 0.7 Hz, 1H, 2-
1
3
phenyl H-4), 5.18 (br, 1H, 2-phenyl 3-OH). C NMR (62.5 MHz, CDCl ) δ 157.02, 156.25,
3
1
1
55.99, 150.41, 140.97, 138.71, 137.84, 135.17, 129.98, 129.15, 129.13 (2C), 128.86 (2C),
28.38 (2C), 127.14 (2C), 119.48, 117.49, 117.16, 116.19, 114.04.
5
.3.7. Synthesis of 4-(6-(2-Chlorophenyl)-4-phenylpyridin-2-yl)phenol (13)
1
2
The same procedure described in section 5.3 was employed with 3 (R = c, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = i) (0.72 g, 2.00 mmol) and acetic
acid (2 mL) to yield a white solid (0.30 g, 41.7%, 0.83 mmol). TLC (ethyl acetate / n-hexane =
1
:2 v/v) R = 0.40; Mp: 179-180 °C; HPLC: Retention time: 9.47 min, purity: 100%; ESI LC/MS:
f
+
+
m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.40 (100%), [MH+2]: 360.40 (30%)
2
3
16
1
H NMR (250 MHz, CDCl ) δ 8.00 (d, J = 8.6 Hz, 2H, 2-phenyl H-2, H-6), 7.84 (d, J = 1.3 Hz,
3
1
6
H, pyridine H-3), 7.75 (s, 1H, pyridine H-5), 7.75-7.72 (m, 3H, 4-phenyl H-2, H-6, 6-phenyl H-
), 7.55-7.45 (m, 4H, 6-phenyl H-3, 4-phenyl H-3, H-4, H-5), 7.39-7.30 (m, 2H, 6-phenyl H-4,
1
3
H-5), 6.89 (d, J = 8.6 Hz, 1H, 2-phenyl H-3, H-5), 5.60 (s, 1H, 2-phenyl 4-OH). C NMR (62.5
MHz, CDCl ) δ 157.49, 156.96, 156.68, 149.27, 139.33, 138.65, 132.36, 132.04, 131.78, 130.16,
3
1
29.59, 129.10 (2C), 129.02, 128.71 (2C), 127.20 (2C), 126.95, 120.88, 116.84, 115.64 (2C).
5
.3.8. Synthesis of 4-(6-(3-Chlorophenyl)-4-phenylpyridin-2-yl)phenol (14)
1
2
The same procedure described in section 5.3 was employed with 3 (R = c, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = j) (0.72 g, 2.00 mmol) and acetic
acid (2 mL) to yield a white solid (0.60 g, 83.1%, 1.66 mmol). TLC (ethyl acetate / n-hexane =

ACCEPTED MANUSCRIPT
1
:2 v/v) R
f
= 0.45; Mp: 181-182 °C; HPLC: Retention time: 11.60 min, purity: 100%; ESI
+
+
LC/MS: m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.40 (100%), [MH+2]:
2
3
16
3
60.40 (30%)
1
H NMR (250 MHz, CDCl ) δ 8.18 (s, 1H, 6-phenyl H-2), 8.11 (d, J = 8.7 Hz, 2H, 2-phenyl H-2,
3
H-6), 8.06-8.02 (m, 1H, 6-phenyl H-6), 7.83 (s, 1H, pyridine H-3), 7.80 (d, J = 1.1 Hz, 1H,
pyridine H-5), 7.75-7.71 (m, 2H, 4-phenyl H-2, H-6), 7.57-7.47 (m, 3H, 4-phenyl H-3, H-4, H-
5
1
1
1
), 7.43-7.38 (m, 2H, 6-phenyl H-4, H-5), 6.97 (d, J = 8.7 Hz, 2H, 2-phenyl H-3, H-5), 5.15 (s,
1
3
3
H, 2-phenyl 4-OH). C NMR (62.5 MHz, CDCl ) δ 157.23, 156.65, 155.85, 150.34, 141.39,
38.82, 134.73, 132.09, 129.90, 129.12 (2C), 129.07, 128.94, 128.66 (2C), 127.25, 127.14 (2C),
25.14, 116.91, 116.60, 115.61 (2C).
5
.3.9. Synthesis of 4-(6-(4-Chlorophenyl)-4-phenylpyridin-2-yl)phenol (15)
1
2
The same procedure described in section 5.3 was employed with 3 (R = c, R = d) (0.44 g, 2.00
3
mmol), dry ammonium acetate (1.54 g, 20.00 mmol), 5 (R = k) (0.72 g, 2.00 mmol) and acetic
acid (2 mL) to yield a yellow solid (0.56 g, 78.5%, 1.57 mmol). TLC (ethyl acetate / n-hexane =
1
:2 v/v) R
f
= 0.45; Mp: 240-241 °C; HPLC: Retention time: 11.64 min, purity: 100%; ESI
+
+
LC/MS: m/z calcd for C H ClNO [MH] 358.10; found [MH] : 358.39 (100%), [MH+2]:
2
3
16
3
60.40 (30%)
1
H NMR (250 MHz, DMSO-d ) δ 9.92 (br, 1H, 2-phenyl 4-OH), 8.37 (d, J = 8.5 Hz, 2H, 2-
6
phenyl H-2, H-6), 8.19 (d, J = 8.6 Hz, 2H, 6-phenyl H-2, H-6), 8.12 (s, 1H, pyridine H-3), 8.08
(s, 1H, pyridine H-5), 8.03-8.00 (m, 2H, 4-phenyl H-2, H-6), 7.60 (d, J = 8.6 Hz, 2H, 6-phenyl
H-3, H-5), 7.57-7.53 (m, 3H, 4-phenyl H-3, H-4, H-5), 6.93 (d, J = 8.5 Hz, 2H, 2-phenyl H-3, H-
1
3
5
6
). C NMR (62.5 MHz, DMSO-d ) δ 159.10, 156.90, 155.11, 149.70, 138.01, 137.97, 134.19,

ACCEPTED MANUSCRIPT
1
1
29.73, 129.51, 129.31 (2C), 128.94 (2C), 128.90 (2C), 128.64 (2C), 127.56 (2C), 115.91,
15.75 (2C), 115.69.
5
.3.10. Synthesis of 2-(6-(2-Chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (16)
1
2
The same procedure described in section 5.3 was employed with 3 (R = a, R = e) (0.27 g, 1.20
3
mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R = i) (0.43 g, 1.20 mmol) and acetic
acid (1 mL) to yield a light yellow solid (0.26 g, 59.5%, 0.71 mmol). TLC (ethyl acetate / n-
hexane = 1:3 v/v) R
00%; ESI LC/MS: m/z calcd for C21
MH+2]: 366.34 (35%)
f
= 0.47; Mp: 157-158 °C; HPLC: Retention time: 12.01 min, purity:
+
+
1
H14ClNOS [MH] 364.06; found [MH] : 364.34 (100%),
[
1
H NMR (250 MHz, CDCl ) δ 14.21 (s, 1H, 2-phenyl 2-OH), 8.07 (s, 1H, pyridine H-3), 7.94 (d,
3
J = 8.1 Hz, 1H, 2-phenyl H-6), 7.75 (d, J = 0.7 Hz, 1H, pyridine H-5), 7.67 (d, J = 4.4 Hz, 1H, 4-
thiophene H-3), 7.65-7.63 (m, 1H, 6-phenyl H-6), 7.56-7.52 (m, 1H, 6-phenyl H-3), 7.50 (d, J =
5
.0 Hz, 1H, 4-thiophene H-5), 7.42-7.37 (m, 2H, 6-phenyl H-4, H-5), 7.34 (td, J = 8.2, 1.0 Hz, 2-
phenyl H-4), 7.21 (t, J = 4.0 Hz, 1H, 4-thiophene H-4), 7.04 (d, J = 8.0 Hz, 1H, 2-phenyl H-3),
1
3
6
1
1
3
.97 (t, J = 7.3 Hz, 1H, 2-phenyl H-5). C NMR (62.5 MHz, CDCl ) δ 159.99, 158.09, 154.31,
43.21, 140.98, 137.64, 132.22, 131.72, 131.11, 130.45, 130.21, 128.62, 127.89, 127.28, 126.31,
26.09, 119.21, 118.88, 118.75, 118.66, 113.94.
5
.3.11. Synthesis of 2-(6-(3-chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (17)
1
2
The same procedure described in section 5.3 was employed with 3 (R = a, R = e) (0.27 g, 1.20
3
mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R = j) (0.43 g, 1.20 mmol) and acetic
acid (1 mL) to yield a yellow solid (0.41 g, 94.2%, 1.13 mmol). TLC (ethyl acetate / n-hexane =

ACCEPTED MANUSCRIPT
1
:3 v/v) R
f
= 0.47; Mp: 111-112 °C; HPLC: Retention time: 13.60 min, purity: 100%; ESI
+
+
LC/MS: m/z calcd for C H ClNOS [MH] 364.06; found [MH] : 364.33 (100%), [MH+2]:
2
1
14
3
66.34 (35%)
1
H NMR (250 MHz, CDCl ) δ 14.35 (br, 1H, 2-phenyl 2-OH), 8.04 (d, J = 1.1 Hz, 1H, pyridine
3
H-3), 7.92 (d, J = 7.7 Hz, 1H, 2-phenyl H-6), 7.91-7.89 (m, 1H, 6-phenyl H-2), 7.88-7.84 (m,
1
H, 6-phenyl H-6), 7.76 (d, J = 1.3 Hz, 1H, pyridine H-5), 7.67 (dd, J = 3.7, 1.0 Hz, 1H, 4-
thiophene H-3), 7.52 (dd, J = 5.0, 1.0 Hz, 1H, 4-thiophene H-5), 7.47-7.45 (m, 2H, 6-phenyl H-
4
7
, H-5), 7.35 (td, J = 8.5, 1.5 Hz, 2-phenyl H-4), 7.22 (dd, J = 5.0, 3.7 Hz, 1H, 4-thiophene H-4),
1
3
.08 (dd, J = 8.3, 1.0 Hz, 1H, 2-phenyl H-3), 6.98 (td, J = 8.2, 1.1 Hz, 1H, 2-phenyl H-5). C
NMR (62.5 MHz, CDCl ) δ 159.92, 158.45, 154.17, 144.11, 140.87, 139.80, 135.07, 131.88,
3
1
1
30.40, 129.70, 128.65, 127.97, 127.03, 126.42, 126.13, 125.14, 118.98, 118.71, 118.57, 115.46,
14.31.
5
.3.12. Synthesis of 2-(6-(4-Chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (18)
1
2
The same procedure described in section 5.3 was employed with 3 (R = a, R = e) (0.27 g, 1.20
3
mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R = k) (0.43 g, 1.20 mmol) and acetic
acid (1 mL) to yield a yellow solid (0.37 g, 85.8%, 1.03 mmol). TLC (ethyl acetate / n-hexane =
1
:3 v/v) R = 0.52; Mp: 181-182 °C; HPLC: Retention time: 13.68 min, purity: 100%; ESI
f
+
+
LC/MS: m/z calcd for C H ClNOS [MH] 364.06; found [MH] : 364.35 (100%), [MH+2]:
2
1
14
3
66.35 (35%)
1
H NMR (250 MHz, CDCl ) δ 14.52 (s, 1H, 2-phenyl 2-OH), 8.02 (d, J = 1.1 Hz, 1H, pyridine
3
H-3), 7.91 (dd, J = 8.6, 1.8 Hz, 2H, 6-phenyl H-2, H-6), 7.91-7.87 (m, 1H, 2-phenyl H-6), 7.76
(d, J = 1.3 Hz, 1H, pyridine H-5), 7.65 (dd, J = 3.7, 1.0 Hz, 1H, 4-thiophene H-3), 7.51 (d, J =

ACCEPTED MANUSCRIPT
8
.6 Hz, 2H, 6-phenyl H-3, H-5), 7.50-7.48 (m, 1H, 4-thiophene H-5), 7.35 (td, J = 8.5, 1.5 Hz, 2-
phenyl H-4), 7.22 (dd, J = 5.0, 3.7 Hz, 1H, 4-thiophene H-4), 7.07 (dd, J = 8.2, 1.0 Hz, 1H, 2-
1
3
phenyl H-3), 6.94 (td, J = 8.2, 1.2 Hz, 1H, 2-phenyl H-5). C NMR (62.5 MHz, CDCl ) δ
3
1
59.96, 158.37, 154.30, 144.08, 140.97, 136.34, 135.90, 131.84, 129.31 (2C), 128.63, 128.18
(2C), 127.91, 126.39, 126.06, 118.96, 118.72, 118.56, 115.15, 114.03.
5
.3.13. Synthesis of 3-(6-(2-Chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (19)
1
2
The same procedure described in section 5.3 was employed with 3 (R = b, R = e) (0.27 g, 1.20
3
mmol), dry ammonium acetate (0.92 g, 12.00 mmol), 5 (R = i) (0.43 g, 1.20 mmol) and acetic
acid (1 mL) to yield a light yellow solid (0.17 g, 40.2%, 0.48 mmol). TLC (ethyl acetate / n-
hexane = 1:2 v/v) R = 0.48; Mp: 151-152 °C; HPLC: Retention time: 9.65 min, purity:
f
+
+
1
00%; ESI LC/MS: m/z calcd for C H ClNOS [MH] 364.06; found [MH] : 364.37 (100%),
21 14
[MH+2]: 366.35 (35%)
1
H NMR (250 MHz, CDCl ) δ 7.85 (d, J = 1.4 Hz, 1H, pyridine H-3), 7.77 (d, J = 1.4 Hz, 1H,
3
pyridine H-5), 7.72-7.69 (m, 1H, 6-phenyl H-6), 7.61-7.58 (m, 3H, 2-phenyl H-2, H-6, 4-
thiophene H-3), 7.51-7.47 (m, 1H, 6-phenyl H-3), 7.45 (dd, J = 5.0, 1.0 Hz, 1H, 4-thiophene H-
5
3
), 7.40-7.33 (m, 2H, 6-phenyl H-4, H-5), 7.33 (t, J = 8.0 Hz, 2-phenyl H-5), 7.18 (dd, J = 5.0,
.7 Hz, 1H, 4-thiophene H-4), 6.89 (ddd, J = 7.2, 2.5, 0.7 Hz, 1H, 2-phenyl H-4), 5.68 (s, 1H, 2-
1
3
phenyl 3-OH). C NMR (62.5 MHz, CDCl ) δ 157.55, 157.32, 156.10, 142.36, 141.43, 140.68,
3
1
1
39.02, 132.37, 131.65, 130.19, 129.95, 129.74, 128.45, 127.25, 126.98, 125.56, 119.77, 119.42,
16.32, 115.87, 114.25.
5
.3.14. Synthesis of 3-(6-(3-Chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (20)

ACCEPTED MANUSCRIPT
1
2
The same procedure described in section 5.3 was employed with 3 (R = b, R = e) (0.34 g, 1.50
3
mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R = j) (0.54 g, 1.50 mmol) and acetic
acid (2 mL) to yield a light yellow solid (0.52 g, 95.6%, 1.43 mmol). TLC (ethyl acetate / n-
hexane = 1:2 v/v) R = 0.45; Mp: 147-148 °C; HPLC: Retention time: 11.86 min, purity:
f
+
+
1
00%; ESI LC/MS: m/z calcd for C H ClNOS [MH] 364.06; found [MH] : 364.37 (100%),
21 14
[MH+2]: 366.35 (35%)
1
H NMR (250 MHz, CDCl
3
) δ 8.15 (s, 1H, 6-phenyl H-2), 8.03-7.99 (m, 1H, 6-phenyl H-6), 7.85
(s, 1H, pyridine H-3), 7.82 (d, J = 1.1 Hz, 1H, pyridine H-5), 7.69-7.66 (m, 2H, 2-phenyl H-2, H-
6
5
3
), 7.62 (dd, J = 3.6, 0.9 Hz, 1H, 4-thiophene H-3), 7.47 (dd, J = 5.0, 0.8 Hz, 1H, 4-thiophene H-
), 7.44-7.41 (m, 2H, 6-phenyl H-4, H-5), 7.38 (t, J = 8.1 Hz, 2-phenyl H-5), 7.20 (dd, J = 5.0,
.7 Hz, 1H, 4-thiophene H-4), 6.95 (ddd, J = 8.0, 2.5, 0.8 Hz, 1H, 2-phenyl H-4), 5.13 (s, 1H, 2-
1
3
phenyl 3-OH). C NMR (62.5 MHz, CDCl ) δ 157.25, 156.19, 155.96, 143.22, 141.46, 141.03,
3
1
1
40.68, 134.77, 130.01, 129.93, 129.13, 128.46, 127.22 (2C), 125.48, 125.15, 119.48, 116.32,
15.89, 115.55, 113.99.
5
.3.15. Synthesis of 3-(6-(4-Chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (21)
1
2
The same procedure described in section 5.3 was employed with 3 (R = c, R = e) (0.34 g, 1.50
3
mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R = k) (0.54 g, 1.50 mmol) and acetic
acid (2 mL) to yield an off-white solid (0.46 g, 85.6%, 1.28 mmol). TLC (ethyl acetate / n-
hexane = 1:2 v/v) R = 0.43; Mp: 174-175 °C; HPLC: Retention time: 11.95 min, purity:
f
+
+
1
00%; ESI LC/MS: m/z calcd for C H ClNOS [MH] 364.06; found [MH] : 364.37 (100%),
21 14
[MH+2]: 366.345 (35%)

ACCEPTED MANUSCRIPT
1
H NMR (250 MHz, CDCl
3
) δ 8.11 (dd, J = 8.6, 1.8 Hz, 2H, 6-phenyl H-2, H-6), 7.84 (d, J = 1.3
Hz, 1H, pyridine H-3), 7.81 (d, J = 1.3 Hz, 1H, pyridine H-5), 7.69-7.64 (m, 2H, 2-phenyl H-2,
H-6), 7.61 (dd, J = 3.6, 1.0 Hz, 1H, 4-thiophene H-3), 7.48 (d, J = 8.6 Hz, 2H, 6-phenyl H-3, H-
5
), 7.47-7.46 (m, 1H, 4-thiophene H-5), 7.37 (t, J = 8.1 Hz, 2-phenyl H-5), 7.19 (dd, J = 5.0, 3.7
Hz, 1H, 4-thiophene H-4), 6.94 (ddd, J = 8.1, 2.7, 0.9 Hz, 1H, 2-phenyl H-4), 5.17 (br, 1H, 2-
1
3
phenyl 3-OH). C NMR (62.5 MHz, CDCl ) δ 157.21, 156.44, 155.95, 143.17, 141.54, 140.76,
3
1
1
37.62, 135.26, 129.99, 128.86 (2C), 128.45, 128.35 (2C), 127.16, 125.42, 119.46, 116.28,
15.63, 115.29, 113.99.
5
.3.16. Synthesis of 4-(6-(2-Chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (22)
1
2
The same procedure described in section 5.3 was employed with 3 (R = c, R = e) (0.34 g, 1.50
3
mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R = i) (0.54 g, 1.50 mmol) and acetic
acid (2 mL) to yield a white solid (0.30 g, 56.3%, 0.84 mmol). TLC (ethyl acetate / n-hexane =
1
:2 v/v) R = 0.36; Mp: 173-174 °C; HPLC: Retention time: 9.66 min, purity: 100%; ESI LC/MS:
f
+
+
m/z calcd for C21H14ClNOS [MH] 364.06; found [MH] : 364.36 (100%), [MH+2]: 366.35
(35%)
1
H NMR (250 MHz, CDCl ) δ 7.97 (d, J = 8.7 Hz, 2H, 2-phenyl H-2, H-6), 7.81 (d, J = 1.4 Hz,
3
1
H, pyridine H-3), 7.72 (d, J = 1.4 Hz, 1H, pyridine H-5), 7.70-7.69 (m, 1H, 6-phenyl H-6), 7.59
(dd, J = 3.6, 0.9 Hz, 1H, 4-thiophene H-3), 7.52-7.48 (m, 1H, 6-phenyl H-3), 7.44 (dd, J = 5.0,
0
1
.8 Hz, 1H, 4-thiophene H-5), 7.38-7.31 (m, 2H, 6-phenyl H-4, H-5), 7.18 (dd, J = 5.0, 3.7 Hz,
H, 4-thiophene H-4), 6.88 (d, J = 8.7 Hz, 1H, 2-phenyl H-3, H-5), 5.68 (s, 1H, 2-phenyl 4-OH).
1
3
3
C NMR (62.5 MHz, CDCl ) δ 157.69, 157.18, 156.77, 142.20, 141.60, 139.12, 132.37, 131.78,

ACCEPTED MANUSCRIPT
1
1
31.65, 130.16 , 129.66, 128.70 (2C), 128.41, 127.10, 126.95, 125.43, 119.00, 115.64 (2C),
15.07.
5
.3.17. Synthesis of 4-(6-(3-Chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (23)
1
2
The same procedure described in section 5.3 was employed with 3 (R = c, R = e) (0.34 g, 1.50
3
mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R = j) (0.54 g, 1.50 mmol) and acetic
acid (2 mL) to yield a light yellow solid (0.38 g, 69.5%, 1.04 mmol). TLC (ethyl acetate / n-
hexane = 1:2 v/v) R
00%; ESI LC/MS: m/z calcd for C21
MH+2]: 366.35 (35%)
f
= 0.39; Mp: 179-180 °C; HPLC: Retention time: 11.87 min, purity:
+
+
1
H14ClNOS [MH] 364.06; found [MH] : 364.37 (100%),
[
1
H NMR (250 MHz, CDCl ) δ 8.17-8.15 (m, 1H, 6-phenyl H-2), 8.09 (d, J = 8.7 Hz, 2H, 2-
3
phenyl H-2, H-6), 8.04-7.99 (m, 1H, 6-phenyl H-6), 7.81 (d, J = 1.3 Hz, 1H, pyridine H-3), 7.78
d, J = 1.3 Hz, 1H, pyridine H-5), 7.62 (dd, J = 3.6, 1.0 Hz, 1H, 4-thiophene H-3), 7.46 (dd, J =
.0, 1.0 Hz, 1H, 4-thiophene H-5), 7.44-7.41 (m, 2H, 6-phenyl H-4, H-5), 7.20 (dd, J = 5.0, 3.7
Hz, 1H, 4-thiophene H-4), 6.97 (d, J = 8.7 Hz, 2H, 2-phenyl H-3, H-5), 5.10 (s, 1H, 2-phenyl 4-
(
5
1
3
OH). C NMR (62.5 MHz, CDCl
3
) δ 157.40, 156.72, 156.04, 143.10, 141.68, 141.18, 134.73,
1
1
31.89, 129.90, 129.04, 128.65 (2C), 128.42, 127.22, 127.05, 125.35, 125.12, 115.60 (2C),
15.05, 114.77.
5
.3.18. Synthesis of 4-(6-(4-Chlorophenyl)-4-(thiophen-2-yl)pyridin-2-yl)phenol (24)
1
2
The same procedure described in section 5.3 was employed with 3 (R = c, R = e) (0.34 g, 1.50
3
mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R = k) (0.54 g, 1.50 mmol) and acetic
acid (2 mL) to yield an off-white solid (0.49 g, 90.8%, 1.36 mmol). TLC (ethyl acetate / n-

ACCEPTED MANUSCRIPT
hexane = 1:2 v/v) R
f
= 0.42; Mp: 244-245 °C; HPLC: Retention time: 11.97 min, purity:
+
+
1
00%; ESI LC/MS: m/z calcd for C H ClNOS [MH] 364.06; found [MH] : 364.37 (100%),
21 14
[MH+2]: 366.35 (35%)
1
H NMR (250 MHz, DMSO-d ) δ 9.86 (s, 1H, 2-phenyl 4-OH), 8.33 (d, J = 8.6 Hz, 2H, 2-phenyl
6
H-2, H-6), 8.15 (d, J = 8.7 Hz, 2H, 6-phenyl H-2, H-6), 8.08 (dd, J = 3.9, 1.1 Hz, 1H, 4-
thiophene H-3), 8.05 (d, J = 1.0 Hz, 1H, pyridine H-3), 8.00 (d, J = 0.9 Hz, 1H, pyridine H-5),
7
7
.78 (dd, J = 5.0, 0.8 Hz, 1H, 4-thiophene H-5), 7.60 (d, J = 8.6 Hz, 2H, 6-phenyl H-3, H-5),
1
3
.28 (dd, J = 5.0, 3.7 Hz, 1H, 4-thiophene H-4), 6.93 (d, J = 8.7 Hz, 2H, 2-phenyl H-3, H-5). C
) δ 159.13, 156.99, 155.24, 143.11, 141.03, 137.63, 134.30, 129.41,
29.04, 128.95 (2C), 128.81 (2C), 128.56 (2C), 128.50, 127.30, 115.74 (2C), 113.95, 113.79.
NMR (62.5 MHz, DMSO-d
6
1
5
.3.19. Synthesis of 2-(6-(2-Chlorophenyl)-4-(thiophen-3-yl)pyridin-2-yl)phenol (25)
1
2
The same procedure described in section 5.3 was employed with 3 (R = a, R = f) (0.34 g, 1.50
3
mmol), dry ammonium acetate (1.15 g, 15.00 mmol), 5 (R = i) (0.54 g, 1.50 mmol) and acetic
acid (2 mL) to yield a light yellow solid (0.35 g, 64.1%, 0.96 mmol). TLC (ethyl acetate / n-
hexane = 1:5 v/v) R
f
= 0.46; Mp: 152-153 °C; HPLC: Retention time: 11.36 min, purity:
+
+
1
00%; ESI LC/MS: m/z calcd for C H ClNOS [MH] 364.06; found [MH] : 364.34 (100%),
2
1
14
[MH+2]: 366.34 (35%)
1
H NMR (250 MHz, CDCl ) δ 14.18 (s, 1H, 2-phenyl 2-OH), 8.08 (d, J = 1.1 Hz, 1H, pyridine
3
H-3), 7.93 (dd, J = 8.0, 1.4 Hz, 1H, 2-phenyl H-6), 7.79 (dd, J = 2.8, 1.4 Hz, 1H, 4-thiophene H-
), 7.75 (d, J = 1.3 Hz, 1H, pyridine H-5), 7.67-7.63 (m, 1H, 6-phenyl H-6), 7.55-7.52 (m, 2H, 6-
phenyl H-3, 4-thiophene H-4), 7.51 (dd, J = 5.0, 2.9 Hz, 1H, 4-thiophene H-5), 7.41-7.36 (m,
H, 6-phenyl H-4, H-5), 7.32 (td, J = 8.4, 1.5 Hz, 2-phenyl H-4), 7.03 (dd, J = 8.3, 1.0 Hz, 1H,
2
2