Synthesis and anticancer evaluation of 6-azacyclonol-2,4,6-trimethylpyridin-3-ol derivatives: M3 muscarinic acetylcholine receptor-mediated anticancer activity of a cyclohexyl derivative in androgen-refractory prostate cancer

Article abstract of DOI:10.1016/j.bioorg.2021.104805

We recently reported 2,4,5-trimethylpyridin-3-ol with C(6)-azacyclonol, whose code name is BJ-1207, showing a promising anticancer activity by inhibiting NOX-derived ROS in A549 human lung cancer cells. The present study was focused on structural modification of the azacyclonol moiety of BJ-1207 to find a compound with better anticancer activity. Ten new compounds (3A–3J) were prepared and evaluated their inhibitory actions against proliferation of eighteen cancer cell lines as a primary screening. Among the ten derivatives of BJ-1207, the effects of compounds 3A and 3J on DU145 and PC-3, androgen-refractory cancer cell lines (ARPC), were greater than the parent compound, and compound 3A showed better activity than 3J. Antitumor activity of compound 3A was also observed in DU145-xenografted chorioallantoic membrane (CAM) tumor model. In addition, the ligand-based target prediction and molecular docking study using DeepZema server showed compound 3A was a ligand to M3 muscarinic acetylcholine receptor (M3R) which is overexpressed in ARPC. Carbachol, a muscarinic receptor agonist, concentration dependently increased proliferation of DU145 in the absence of serum, and it also activated NADPH oxidase (NOX). The carbachol-induced proliferation and NOX activity was significantly blocked by compounds 3A in a concentration-dependent manner. This finding might become a new milestone in the development of pyridinol-based anti-cancer agents against ARPC.

Full text of DOI:10.1016/j.bioorg.2021.104805

Bioorganic Chemistry 110 (2021) 104805
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and anticancer evaluation of 6-azacyclonol-2,4,6-trimethylpyri-
din-3-ol derivatives: M3 muscarinic acetylcholine receptor-mediated
anticancer activity of a cyclohexyl derivative in androgen-refractory
prostate cancer
a
,
1
a
,
1
b
a
Ujjwala Karmacharya , Prakash Chaudhary , Dongchul Lim , Sadan Dahal ,
a
b
a,
*
a,*
Bhuwan Prasad Awasthi , Hee Dong Park , Jung-Ae Kim , Byeong-Seon Jeong
a
College of Pharmacy, Yeungnam University, 280 Daehak-ro, Gyeongsan 38541, Republic of Korea
b
Innovo Therapeutics Inc., Daeduck Biz Center C-313, 17 Techno 4-ro, Yuseong-gu, Daejeon 34013, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
We recently reported 2,4,5-trimethylpyridin-3-ol with C(6)-azacyclonol, whose code name is BJ-1207, showing a
promising anticancer activity by inhibiting NOX-derived ROS in A549 human lung cancer cells. The present
study was focused on structural modification of the azacyclonol moiety of BJ-1207 to find a compound with
better anticancer activity. Ten new compounds (3A–3J) were prepared and evaluated their inhibitory actions
against proliferation of eighteen cancer cell lines as a primary screening. Among the ten derivatives of BJ-1207,
the effects of compounds 3A and 3J on DU145 and PC-3, androgen-refractory cancer cell lines (ARPC), were
greater than the parent compound, and compound 3A showed better activity than 3J. Antitumor activity of
compound 3A was also observed in DU145-xenografted chorioallantoic membrane (CAM) tumor model. In
addition, the ligand-based target prediction and molecular docking study using DeepZema® server showed
compound 3A was a ligand to M3 muscarinic acetylcholine receptor (M3R) which is overexpressed in ARPC.
Carbachol, a muscarinic receptor agonist, concentration dependently increased proliferation of DU145 in the
absence of serum, and it also activated NADPH oxidase (NOX). The carbachol-induced proliferation and NOX
activity was significantly blocked by compounds 3A in a concentration-dependent manner. This finding might
become a new milestone in the development of pyridinol-based anti-cancer agents against ARPC.
Chemical modification
Androgen-refractory prostate cancer (ARPC)
Antitumor
DeepZema
M3 muscarinic acetylcholine receptor (M3R)
1
. Introduction
evidence shows muscarinic receptors, G-protein coupled receptors
(
GPCRs), and their ligand, acetylcholine, are involved in the growth and
Prostate cancer is the most frequently diagnosed cancer and the
progression of prostate cancer [3,4]. Among five subtypes (M1R–M5R)
of muscarinic acetylcholine receptors, M1R and M3R are highly upre-
gulated in ARPC compared with hormone-sensitive prostate cancer in
cell lines and clinical specimens [3,5]. Also, the abnormal expression of
M1R and M3R corresponds to the poor prognosis of progression-free
survival in ARPC patients [6]. Because both M1R and M3R receptors
share common G protein involvement of coupling to Gq/11 G proteins,
phospholipase C and consequent activation of protein kinase C may be
involved in the progression. Several different signaling pathways of M1R
and M3R have been reported to induce ARPC progression; hedgehog [4]
and FAK-YAP (Focal adhesion kinase-Yes-associated protein) signaling
second leading cause of cancer mortality in men worldwide [1]. At
initial diagnosis, prostate cancer is sensitive to chemical or surgical
castration therapies that inhibit or deplete androgen action for prostate
cancer growth. However, the cancer eventually turns out to be
androgen-independent and undergo metastasis, which is referred to as
castration-resistant or androgen-refractory prostate cancer (ARPC).
The underlying mechanisms involved in the development of
androgen adaptation and therapeutic resistance of ARPC are related to
the progression of various mutations in the cancer genome and positive
regulatory signals from the tumor microenvironment [2]. Accumulating
*
Corresponding authors.
E-mail addresses: jakim@yu.ac.kr (J.-A. Kim), jeongb@ynu.ac.kr (B.-S. Jeong).
Both authors contributed equally to this work.
1
https://doi.org/10.1016/j.bioorg.2021.104805
Received 10 January 2021; Received in revised form 20 February 2021; Accepted 2 March 2021
Available online 6 March 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
[
6].
Epidemiological and clinical studies have demonstrated oxidative
activity among the fifty compounds 1 used [11]. Since the 2,4,5-trime-
thylpyridin-3-ol part is common to all fifty compounds, it can be said
that the variation in anticancer activity between each compound may be
due to the difference in the structure of amino moiety connected to C(6)-
position of the pyridine ring. The 6-amino part in BJ-1207 compound is
4-(hydroxydiphenylmethyl)piperidine, also known as azacyclonol,
which is the parent structure of fexofenadine, an antihistamine drug
used in the treatment of allergy symptoms.
stress plays a critical role in the development and progression of prostate
cancer [7]. ARPC cells produce a higher level of reactive oxygen species
(
ROS) than androgen-dependent prostate cancer cells. The major ROS
source in cancer is NADPH oxidase (NOX), and prostate cancer cells
express various NOX isoforms, including NOX1, NOX2, NOX4, and
NOX5 [8]. NOX activation is induced by stimuli such as growth factors,
cytokines, and calcium influx [9]. Upon stimulation, translocation and
unification of cytosolic subunits to membrane components require
phosphorylation by various protein kinases, including protein kinase B
and protein kinase C [10].
Therefore, this study was mainly focused on exploring the structur-
e–activity relationship on the azacyclonol moiety of BJ-1207, and its ten
representative analogs 3A–3J were proposed for this purpose as shown
in Fig. 2. Palladium-catalyzed amination was used as an assembly re-
action between the key intermediate bromopyridine 4 and ten different
cyclic amines 5.
We recently reported a 2,4,5-trimethylpyridin-3-ol compound con-
taining azacyclonol moiety at C(6)-position of the pyridine ring, whose
code name is BJ-1207 (2) (Fig. 1), showed a promising anticancer ac-
tivity by inhibiting NOX-derived ROS in A549 human lung cancer cell
line [11]. The present study began with a focus on structural modifi-
cation of BJ-1207 to find a compound showing better anticancer activity
by fine-tuning the azacyclonol moiety using its similar substructures. We
found that compound 3A, one of the BJ-1207 derivatives, showed more
potent antiproliferative activity than BJ-1207 against human prostate
cancer cell lines from this study. In particular, the antiproliferative effect
of compound 3A was better in ARPC cell lines like DU145 and PC-3 than
in androgen-sensitive cells such as LNCaP. We also found that the
anticancer effect of compound 3A is mediated through inhibition of
M3R, which was revealed by molecular docking analysis, receptor
binding assay, and carbachol-induced NOX activation and DU145 cell
proliferation assays.
The synthesis began with the preparation of the common key inter-
mediate (4) from commercially available pyridoxine hydrochloride (6)
following the well-established method developed by us (Scheme 1) [12].
Chlorination of two hydroxyl moieties of 6 was done using excess thi-
onyl chloride and a catalytic amount of N,N-dimethylformamide. The
product thus obtained was refluxed in acetic acid using zinc to cut off the
two chlorine groups affording 3-hydroxy-2,4,5-trimethylpyridine (7) in
a two-step reaction with an overall yield 85% from 6. Next, bromination
and benzylation were done consecutively using 1,3-dibromo-5,5-dime-
thylhydantoin in the first reaction to introduce bromine for the
palladium-catalyzed amination and using benzyl bromide for hydroxyl
protection. Commercially available 4-benzoylpiperdine hydrochloride
(8) was used for the synthesis of the compound 3A–3C. First, the free
amino group of 8 was protected from strong basic conditions of the
upcoming step by either Cbz or Boc. The addition of the carbonyl of 9-1
with cyclohexylmagnesium bromide gave 10A in 99% yield [13]. While
the commercially available Grignard reagent was used for the synthesis
of 10A, Grignard reagents for 10B and 10C were prepared by a halogen-
metal exchange reaction using 3-bromothiophene and n-butyllithium for
10B and 2-bromopyridine and isopropylmagnesium chloride for 10C,
2
. Results and discussion
2
.1. Synthesis
In the previous study, BJ-1207 showed the most potent anticancer
Fig. 1. The goal of this study: Structural modification of BJ-1207 by fine-tuning of the azacyclonol moiety.
2

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
Fig. 2. General synthetic strategy for compounds 3 and structures of the derivatives.
respectively. After the Grignard reactions, the protecting group was
removed for the next Buchwald-Hartwig amination reaction. For the Cbz
protective group in 10A, deprotection was readily carried out by cata-
lytic hydrogenolysis. However, in the case of 10B, the reaction did not
proceed well under the same reaction conditions, which might be due to
deactivation of the palladium catalyst on activated carbon by sulfur
atom in the structure of 10B [13]. Thus, an alternative approach for the
removal of Cbz in 10B was applied. The free piperidine compound 11B
was obtained in a high yield by treatment of 10B with an excess amount
of base in sealed-tube for a long time at high temperature. Typical re-
action conditions for the deprotection of Boc group afforded 11C. All
obtained piperidine substrates 11A–11C were then coupled with the
bromo-compound 4 under Buchwald-Hartwig amination conditions.
Finally, the benzyl protective group was cleaved by catalytic hydro-
genolysis using hydrogen gas to get 3A and 3B. 3C was obtained by a
milder reaction condition using boron trichloride and pentam-
ethylbenzene. Unlike the case of the deprotection of Cbz of 10B by
catalytic hydrogenolysis, there was no such a problem observed in the
transformation of 11B to 3B.
25, respectively. Then, the final target compounds 3G–3J were obtained
by debenzylation either with molecular hydrogen or boron trichloride.
On treating the intermediate 18 with sodium borohydride, the corre-
sponding alcohol 19 was formed which was then treated with molecular
hydrogen to obtain 3F [17].
2
.2. Anticancer activity evaluation
The new compounds 3A–3J along with BJ-1207 (2) at one fixed
concentration (30
μ
M) were tested for antiproliferative activity in
eighteen different human cancer cell lines: breast cancer cell lines (MCF-
, MCF-7/ADR, MCF-7/TAMR, and MDA-MB-231), colon cancer cell
lines (HT-29, SW620, Caco-2, and HCT-116), pancreatic cancer cell lines
PANC-1 and MiaPaCa-2), a liver cancer cell line (Hep3B), lung cancer
cell lines (A549 and H1299), prostate cancer cell lines (DU145 and PC-
), monoblastic leukemia cell lines (U937 and THP-1) and a glioblas-
7
(
3
toma cell line (A172) (Fig. 3). Antiproliferative activities of the new
compounds were less effective than the parent compound 2 except
compounds 3A and 3J. Looking more specifically, replacing one of the
phenyl rings of the azacyclonol group in the parent compound 2 with a
cyclic alkane (cyclohexyl (3A)) or two heteroaromatic rings (thiophen-
Commercially available 4-(hydroxydiphenylmethyl)piperidine (13)
was employed for the synthesis of two derivatives 3D and 3E as shown in
Scheme 2. Reductive removal of the hydroxyl group in 13 was done
under Gribble reduction conditions, sodium borohydride in trifluoro-
acetic acid, affording 4-diphenylmethylpiperidine (14) in good yield
3
-yl (3B) and pyridin-2-yl (3C)) produced opposite results. The cyclo-
hexyl analogue 3A showed comparable inhibitory activity in most can-
cer cell lines to the parent compound 2 at a fixed concentration. In
particular, the proliferation of DU145, a prostate cancer cell line, was
highly suppressed by compound 3A (71.3% inhibition) compared to
compound 2 (49.0% inhibition). On the other hand, compounds 3B and
[
14,15]. Dehydration reaction of 13 under acidic conditions with tri-
fluoroacetic acid gave 4-(diphenylmethylene)piperidine (16) [16]. Both
intermediates 14 and 16 were then coupled with the key intermediate 4
by Buchwald-Hartwig amination reaction to get 15 and 17, respectively.
Both were then debenzylated by hydrogenolysis to obtain 3D and 3E
3
C, replaced by heteroaromatic rings, significantly lost the inhibitory
activity. Removal of the hydroxyl group of azacyclonol as in analogues
D and 3E was detrimental to the inhibitory activity. Another group of
(
see Scheme 3).
3
On following common sequence of Buchwald-Hartwig reaction of 4
analogues, 3F, 3G, and 3I, in which one of the two phenyl rings of the
parent compound was removed, generally showed a decrease in the
inhibitory activity regardless of the structural change of the hydroxyl
group. Interestingly, the two piperazine analogues with no hydroxyl
group, 3I and 3J, showed significantly different activity. The inhibitory
with commercially available reagents, 4-benzoylpiperidine hydrochlo-
ride (8) and 4-benylpiperidine (20), respectively resulted in 18 and 21
while coupling with commercially available 1-benzhydrylpiperazine
(
22) and 1-(bis(4-fluorophenyl)-methyl)piperazine (24) gave 23 and
3

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
Scheme 1. Synthesis of compounds 3A–C. Reagents and Conditions: (a) SOCl
2
, DMF, reflux, 1 h; (b) Zn, AcOH, reflux, 12 h, 85% (2 steps); (c) DBDMH, THF, r.t., 3 h,
8
2
0%; (d) PhCH Br, K
11MgBr, Et
2
CO
3
, DMF, r.t., 12 h, 87%; (e) for 9-1: ClCO
2
Bn, K
2
CO
3
, THF, r.t., 2 h, 90%, for 9-2: Boc
2
O, Et
3
N, DMAP, r.t., 3 h, 66%; (f) for 10A: c-
O, ꢀ 78 ◦C, 2 h, 99%, for 10B, 3-bromothiophene, n-BuLi, THF, ꢀ 78 C, 3 h, 24%, for 10C, 2-bromopyridine, i-PrMgCl, THF, ꢀ 40 ◦C, 12 h, 30%; (g)
◦
C
6
H
2
◦
for 11A: H
2
, Pd/C, MeOH-THF, r.t., 18 h, 60%, for 11B: K
2
CO
3
, MeOH, 80 C, sealed tube, 3 d, 98%, for 11C: TFA, DCM, r.t., 12 h, 99%; (h) 4, Pd
2
(dba)
3
, BINAP,
t
◦
NaO Bu, toluene, 130 C, 12A (48 h, 33%), 12B (36 h, 35%), 12C (12 h, 36%); (i) for 3A and 3B: H
2
, Pd/C, MeOH, r.t., 3A (2 h, 90%), 3B (12 h, 98%), for 3C: BCl
3
,
◦
C
6
HMe
5
, DCM, 0 C,15 min, 99%.
activities of 3I with simple phenyl rings were almost as much as those of
the piperidine analogue 3D. However, the introduction of fluoro atoms
in the two phenyl rings as in 3J showed quite different results compared
to non-fluoro compound 3I. Suppression of most cancer cell prolifera-
tion with 3J was retained to the level of the parent compound 2. As can
be seen in Fig. 3, among ten new derivatives, compounds 3A and 3J
generally showed superior inhibition of proliferation in most cancer cell
lines to other analogs. The growth inhibitory effects of 3A and 3J were
better than compound 2 particularly in ARPC cell lines such as DU145
and PC-3.
so much different. In the case of sunitinib, the CC50 values were
observed 20.9, 7.3, 9.5
respectively [18].
μ
M against CHO-K1, HEK-293, and CCD841,
To confirm an anticancer activity of the derivatives in vivo, we
examined antitumor effects of 3A and 3J in chorioallantoic membrane
(CAM) tumor model, in which DU145 cells were xenografted, and the
effects of 3A and 3J were compared to that of the parent compound 2.
Compared to vehicle-treated controls, compounds 2, 3A and 3J sup-
pressed tumor growth (Fig. 4Aa, 4B, 4C) and angiogenesis (Fig. 4Ab and
4C) in a dose-dependent manner.
To compare the growth inhibitory power of the compounds, 2, 3A,
and 3J, the concentration for 50% of maximal inhibition of cell prolif-
eration (IC50) was measured (Table 1). IC50 values of 3A and 3J were a
little higher in A549 and H1299 human lung cancer cell lines, but the
values were lower in DU145 and PC-3 than the parent compound 2. In
case of LNCaP cell line, compounds 3A and 3J were a little bit better or
equivalent to compound 2. In the case of anticancer drug sunitinib, a
multi-receptor tyrosine kinase inhibitor, IC50 values against prolifera-
2
.3. Mechanism of action study
In order to search the possible molcular targets of compound 3A
showing a selective inhibitory effect on ARPC, we used the DeepZema®
web server which is an interactive and integrated software system for
accelerating drug discovery projects [19]. Firstly we predicted targets of
compound 3A by conformal prediction methods [20] which can be
regarded as a new type of quantitavie structure activity relationship
tion of DU145, PC-3, and LNCaP cell lines were 13.1, 15.5, and 20.8 M,
μ
respectively. The results indicate that compounds 3A and 3J selectively
inhibits ARPC proliferation.
(
QSAR) with information on the certainty of a prediction. Predicted
targets of compound 3A were M3R and M4R. The targets of compound 2
were also predicted to be M3R and M4R. Then compounds 2 and 3A
were docked into the binding site of rat M3R (PDB 5ZHP) using auto-
dock vina [21] in order to understand protein–ligand binding in-
teractions. Compound 3A had a better docking score (ꢀ 9.0 kCal/mol)
than compound 2 (ꢀ 8.1 kCal/mol). A proposed binding mode of com-
pound 3A to the rat M3R is shown in Fig. 5. Positively charged nitrogen
We then checked how selective the inhibitory activities of the com-
pounds against cancer cells compared to normal cells were by measuring
the concentration for 50% of maximal cytotoxicity (CC50) in three
normal cell lines, CHO-KI, HEK-293, and CCD841 (Table 2). The CC50
values of the three compounds, 2, 3A, and 3J, were much higher than
IC50 values, and the CC50 values between the three compounds were not
4

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
◦
t
◦
4 2 3
Scheme 2. Synthesis of compounds 3D and 3E. Reagents and Conditions: (a) NaBH , TFA, 0 C, 50 min, 88%; (b) 4, Pd (dba) , BINAP, NaO Bu, toluene, 130 C, 15
(
2 3
20 h, 46%), 17 (12 h, 48%); (c) H , Pd/C, MeOH-CHCl , r.t., 3D (6 h, 86%), 3E (7 h, 14%); (d) TFA, DCM, r.t., 9 h, 95%.
atom of the pyridine ring forms a hydrogen bond to Tyr148, while the
methyl groups contribute to the binding energy via hydrophobic in-
teractions with residues with alkyl side chains such as Ile225, Leu225
and Val 510. The cyclohexyl and phenyl rings also make hydrophobic
interactions.
induced NOX activity. The inhibitory activity of compound 3A was the
best and much stronger than that of VAS2870, a NOX2/4 inhibitor,
which also showed concentration-dependent inhibitory effect on
carbachol-induced NOX activity. Compound 3J which showed the
lowest inhibitory activity was almost similar to that of VAS2870. The
results implicate that compounds 2 and 3A have similar target, M3R,
which might not be the case for compound 3J.
In conjunction with the reports that ARPC expresses high levels of
M1R and M3R [22], we performed a competitive M3R binding assay for
compound 2 and 3A, in which each compound competed with the
3
binding of [ H]-labeled 1,1-dimethyl-4-diphenylacetoxypiperidinium
3
. Conclusion
iodide (4-DAMP), a selective M3R antagonist [23], to M3R. In the
competitive binding assay, compound 3A showed K
.4 M (Fig. 6). However, IC50 of compound 2 was greater than >10
and K
i
= 1
μ
M and IC50
=
In the current study, we found that anti-proliferative activity of
1
μ
μ
M
compound 3A was better than the parent compound, 6-azacyclonol-
,4,6-trimethylpyridin-3-ol, particularly in ARPC cell line. The results
i
was not calculable because higher concentrations of compound 2
2
could not be prepared for the assay due to solubility problem. The results
from molecular docking, competitive receptor binding assay, and
carbachol-induced cell viability and NOX activation indicate that ARPC-
selective activity of compound 3A was associated with its antagonistic
action to M3R which is highly expressed in ARPC and serves as a pros-
tate cancer progression marker. Based on the structure of compound 3A,
it may be possible to design and develop anticancer compounds with
high affinity and selectivity to M3R for the treatment of ARPC which
overexpresses the receptor. However, M1R is also upregulated in patient
tissues and cell lines of ARPC [4,6], and plays a role in the proliferation,
migration, and invasion of prostate cancers [4]. A previous study has
focused on targeting the common signaling pathways of M1R and M3R
which are involved in ARPC progression [6]. From the current study, we
propose that it may be possible to develop an ARPC therapeutic agent
that simultaneously inhibits both M1R and M3R through rational
chemical modification of compound 3A.
indicate that compound 3A was more than seven fold potent than
3
compound 2 in inhibiting the binding of [ H]4-DAMP, although binding
affinity of compound 3A to M3R is relatively low.
We, then, compared the inhibitory effects of compounds 2 and 3A on
carbachol-induced proliferation of DU145 cells. Treatment of DU145
cells with carbachol in the presence of low level serum (1%) induced a
minimal level increase in cell proliferation (Fig. 7A). This might be
because the proliferative action of serum counteracts the effect of
carbachol. The carbachol and serum (1%)-induced proliferation was
concentration dependently inhibited by compounds 2 (Fig. 7B) and 3A
(
Fig. 7C), and the inhibitory effect of compound 3A was much stronger
than that of compound 2. In the absence of serum, carbachol increased
cell survival in a concentration- and time-dependent manner (Fig. 7D).
The carbachol (300 M) alone-induced viability of DU145 cells was
μ
inhibited by compounds 2 and 3A, and compound 3A was more potent
than compound 2 (Fig. 7E). These results support that action of com-
pound 3A in enhancing viable number of DU145 cells was mediated
through M3R.
4
. Experimental section
4
4
.1. Synthesis [26]
We also compared the inhibitory effects of compounds 2, 3A, and 3J
on carbachol-induced NOX activity, based on the reports that GPCR is
linked to NOX activation [24], and carbachol, a muscarinic receptor
agonist, increases NOX-dependent ROS (Fig. 8) [25]. Compounds 2, 3A,
.1.1. General
Unless noted otherwise, materials were purchased from commercial
suppliers and used without further purification. Air or moisture-
sensitive reactions were carried out under an inert gas atmosphere.
and 3J concentration-dependently inhibited carbachol (300
μM)-
5

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
t
◦
Scheme 3. Synthesis of compounds 3F–3J. Reagents and Conditions: (a) 4, Pd
2
(dba)
3
, BINAP, NaO Bu, toluene, 130 C, 18 (24 h, 27%), 21 (3 h, 72%), 23 (3 h, 56%),
3
, r.t., 3F (2 h, 77%), 3G (1.5 h, 76%), 3H (3 h, 95%), for 3I: BCl ,
2
5 (5 h, 41%); (b) NaBH
4
, MeOH-H
2
O, r.t., 30 min, 99%; (c) for 3F–3H: H
2
, Pd/C, MeOH-CHCl
3
◦
C
6
HMe
5
, DCM, 0 C, 30 min, 87%, for 3J: H
2
, Pd/C, MeOH, r.t., 3 h, 95%.
Fig. 3. Bar graph showing the mean percentage inhibition of each compound at 30 M concentration against eighteen cancer cell proliferation.
μ
The reaction progress was monitored by thin layer-chromatography
TLC) using silica gel F254 plates. The products were purified by flash
not corrected. Low-resolution mass spectra (LRMS) were obtained using
a Jeol ColdSpray-LC-TOF-MS and recorded in a positive ion mode with
an electrospray (ESI) source. NMR spectra were obtained using a Bruker-
(
column chromatography using silica gel 60 (70–230 mesh) or by using
the Biotage ‘Isolera One’ system with indicated solvents. Melting points
were determined using a Fisher–Johns melting point apparatus and were
1
13
250 spectrometer (250 MHz for H NMR and 62.5 MHz for C NMR)
1
and a Bruker Avance Neo 400 spectrometer (400 MHz for H NMR).
6

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
Table 1
mmol). The mixture was stirred for 2 h maintaining anhydrous condition
at ꢀ 78 ◦C. After completion of the reaction, it was quenched with
IC50 of the selected compounds against proliferation of human cancer cell lines.
a
aqueous NH
over MgSO
solid. R
4
Cl and extracted with EtOAc. The EtOAc layer was dried
Cancer cell line
IC50
(μM)
4
and concentrated to get 10A (727 mg, 99% yield) as a white
2
3A
3J
◦
f
0.50 (DCM:MeOH = 20:1); m.p. 132 C; MS m/z 408.28 [M +
MCF-7
10.9 ± 1.0
33.2 ± 3.2
29.3 ± 2.7
14.7 ± 1.8
18.6 ± 0.8
24.3 ± 2.0
28.6 ± 1.2
29.1 ± 1.7
21.9 ± 3.5
18.2 ± 2.9
31.6 ± 2.8
8.2 ± 0.7
11.9 ± 1.5
29.0 ± 3.4
22.2 ± 2.4
18.6 ± 2.6
17.8 ± 1.3
27.9 ± 1.8
29.6 ± 3.9
30.6 ± 2.0
16.0 ± 1.3
18.8 ± 2.0
64.1 ± 3.6
15.0 ± 1.9
14.8 ± 2.2
1.7 ± 0.1
17.0 ± 2.7
31.9 ± 3.7
18.4 ± 1.5
21.7 ± 2.9
20.6 ± 2.3
25.0 ± 1.7
25.4 ± 1.4
29.2 ± 1.9
14.2 ± 1.3
24.8 ± 2.7
35.5 ± 1.0
12.4 ± 1.5
14.3 ± 1.8
5.0 ± 0.3
+
1
H] ; H NMR (CDCl
3
) δ 7.42–7.26 (m, 10H), 5.07 (s, 2H), 4.21 (s, 2H),
MCF-7/ADR
MCF-7/TMAR
MDA-MB-231
HT-29
2
1
.73 (q, J = 12.7 Hz, 2H), 2.06 (d, J = 11.9 Hz, 1H), 1.96–1.65 (m, 8H),
1
3
.53–1.37 (m, 2H), 1.32–1.12 (m, 4H), 1.08–0.82 (m, 4H); C NMR
) δ 155.09, 142.82, 136.89, 128.41(2C), 127.85, 127.73(2C),
27.64(2C), 126.46, 126.26(2C), 79.97, 66.89, 44.34(2C), 42.45, 35.54,
7.33, 26.71, 26.57, 26.51, 26.45(2C), 24.13.
(CDCl
3
SW620
1
2
Caco-2
HCT116
PANC-1
MIA PaCa-2
Hep3B
4.1.4. Cyclohexyl(phenyl)(piperidin-4-yl)methanol (11A) [CAS RN
64061-56-9]
A549
To a solution of 10A (145 mg, 0.35 mmol) in MeOH-THF (1:1, 10
mL) was added 10% palladium on carbon (37 mg, 0.035 mmol). The
mixture was stirred under hydrogen atmosphere at room temperature
for 18 h. The mixture was filtered through Celite pad and the filtrate was
H1299
11.0 ± 1.4
18.3 ± 2.2
26.5 ± 4.4
51.6 ± 2.4
31.8 ± 2.3
41.0 ± 3.0
25.4 ± 1.7
DU145
PC-3
11.3 ± 3.8
45.5 ± 1.4
28.8 ± 2.6
33.1 ± 2.6
29.2 ± 1.9
13.3 ± 2.4
50.8 ± 2.8
33.0 ± 2.2
39.1 ± 3.3
23.8 ± 1.3
LNCaP
U937
concentrated to give 11A (58 mg, 60% yield) as a white solid. R
f
0.20
THP-1
1
(
DCM:MeOH = 9:1); H NMR (CDCl
3
) δ 7.39–7.17 (m, 5H), 3.17 (dd, J
A172
=
=
24.8, 12.1 Hz, 2H), 2.63 (ddd, J = 17.9, 12.3, 8.1 Hz, 2H), 2.11 (dd, J
8.7, 5.7 Hz, 1H), 1.92–1.66 (m, 6H), 1.25 (ddd, J = 12.9, 11.2, 3.9 Hz,
a
The values are mean ± SEM of three independent experiments performed in
triplicate.
6
H), 1.06–0.80 (m, 2H), 0.75–0.58 (m, 1H).
4
.1.5. (1-(5-(Benzyloxy)-3,4,6-trimethylpyridin-2-yl)piperidin-4-yl)
Table 2
(
cyclohexyl)(phenyl)methanol (12A)
-(Benzyloxy)-6-bromo-2,4,5-trimethylpyridine (4, 50 mg, 0.17
CC50 of the selected compounds against normal cell lines.
3
a
Normal cell line
CC50
( M)
μ
mmol) was taken in a round-bottomed flask and to it were added tris
′
2
3A
3J
(dibenzylideneacetone)dipalladium(0) (4 mg, 0.003 mmol), 2,2 -bis
′
(
diphenylphosphino)-1,1 -binaphthyl (4 mg, 0.006 mmol), sodium tert-
CHO-K1
HEK 293
CCD 841
67.6 ± 1.3
61.7 ± 1.2
69.2 ± 3.8
69.1 ± 2.1
76.9 ± 2.4
75.8 ± 3.1
63.1 ± 4.3
72.4 ± 3.0
70.2 ± 2.8
butoxide (13 mg, 0.13 mmol), 11A (55 mg, 0.20 mmol), and anhydrous
toluene (5 mL). The reaction mixture was refluxed for 48 h. After
cooling, brine and EtOAc were added to the mixture and was then
washed several times with brine. The EtOAc layer was dried with
a
The values are mean ± SEM of three independent experiments performed in
triplicate.
4
MgSO , filtered, and concentrated. The residue was purified by silica gel
column chromatography (EA:HX = 1:8) to give 12A (28 mg, 33% yield)
Chemical shifts (δ) were expressed in ppm using a solvent as an internal
standard and the coupling constant (J) in hertz. HPLC analyses were
performed on a system consisted of an LC-20AD pump, a CBM-20A
communication bus module, an SPD-20A UV–visible detector, and a
DGU-20A5 degasser from Shimadzu Corporation (Kyoto, Japan). A
◦
as a white solid. R
f
0.30 (EA:HX = 1:5); m.p. 130 C; MS m/z 499.38 [M
+
1
+
=
H] ; H NMR (CDCl
3
) δ 7.53–7.29 (m, 10H), 4.73 (s, 2H), 3.28 (dd, J
22.5, 12.3 Hz, 2H), 2.77 (d, J = 9.3 Hz, 2H), 2.42 (s, 3H), 2.12 (d, J =
1
2
1
1
2
1
9.3 Hz, 6H), 1.90 (d, J = 9.1 Hz, 2H), 1.78–1.57 (m, 6H), 1.52–1.42 (m,
13
H), 1.02–0.68 (m, 6H); C NMR (CDCl ) δ 157.82, 147.78, 146.05,
3
Phenomenex Luna C18 column (250 × 4.6 mm, 5.0
μm) (Torrance, CA,
43.27, 140.24, 137.36, 128.53(2C), 128.05, 127.85 (2C), 127.51(2C),
26.41(2C), 126.24, 122.69, 80.14, 74.71, 51.33, 51.22, 44.39, 42.32,
9.69, 27.42, 26.95, 26.80, 26.74, 26.63, 26.49, 26.29, 19.26, 14.43,
3.02.
USA) was used with a gradient solvent system consisted of acetonitrile
and water (from 30% to 100% of acetonitrile over 20 min, then 100% of
acetonitrile for 10–20 min) at a flow rate of 1.0 mL/min at 254 nm UV
detection. Purity of compound was recorded as a percentage (%) and
retention time was given in minutes.
4
.1.6. 6-(4-(Cyclohexyl(hydroxy)(phenyl)methyl)piperidin-1-yl)-2,4,5-
trimethylpyridin-3-ol (3A)
4
2
.1.2. Benzyl 4-benzoylpiperidine-1-carboxylate (9–1) [CAS RN 922504-
7-6]
To a solution of 12A (25 mg, 0.05 mmol) in MeOH (4 mL) was added
1
0% palladium on carbon (5 mg, 0.005 mmol). The mixture was stirred
To a mixture of 4-benzoylpiperidine hydrochloride (8, 3.0 g, 13.29
under hydrogen atmosphere at room temperature for 2 h, and the
mixture was filtered through Celite pad. The filtrate was concentrated to
mmol) in THF (40 mL) were successively added potassium carbonate
(
5.5 g, 39.80 mmol), THF (40 mL), and benzyl chloroformate (2.1 mL,
◦
get 3A (18 mg, 90% yield) as a white solid. R 0.30 (DCM:MeOH = 20:1);
1
5.28 mmol) at 0 C. The mixture was brought to room temperature
f
◦
+
1
m.p. 108 C; MS m/z 409.3 [M + H] ; H NMR (CD
3
OD) δ 7.90 (s, 1H),
slowly and was stirred for 2 h. DCM was added to it and was washed
thrice with brine. It was dried over MgSO , filtered and concentrated to
get 9-1 (3.8 g, 90% yield) as a colorless oil. R
0.50 (DCM:MeOH = 20:1);
) δ 7.98–7.90 (m, 2H), 7.62–7.43 (m, 3H), 7.41–7.28 (m,
H), 5.15 (s, 2H), 4.24 (d, J = 12.9 Hz, 2H), 3.50–3.37 (m, 1H), 3.00 (t,
J = 12.4 Hz, 2H), 1.97–1.63 (m, 4H).
7
.42 (d, J = 7.4 Hz, 2H), 7.33 (dd, J = 13.5, 6.2 Hz, 2H), 7.22 (t, J = 7.3
4
Hz, 1H), 3.28–3.15 (m, 2H), 2.93 (dd, J = 26.4, 13.1 Hz, 2H), 2.40 (s,
3H), 2.23 (d, J = 7.5 Hz, 3H), 2.14 (s, 3H), 1.99–1.90 (m, 3H), 1.74 (t, J
f
1
H NMR (CDCl
3
=
14.7 Hz, 2H), 1.67–1.49 (m, 4H), 1.40–1.20 (m, 4H), 1.11–0.98 (m,
5
13
2
H), 0.77 (dd, J = 12.6, 3.0 Hz, 1H); C NMR (CDCl
2C), 126.35(2C), 124.98, 80.14, 51.37(2C), 44.45(2C), 41.67, 27.40,
6.74 (2C), 26.59, 26.42, 26.27, 16.78, 14.43, 13.57; HPLC retention
3
) δ 142.73, 127.57
(
2
4
.1.3. Benzyl 4-(cyclohexyl(hydroxy)(phenyl)methyl)piperidine-1-
time 17.6 min, purity 97.7%.
carboxylate (10A)
In a flame-dried round bottomed flask, 9-1 (600 mg, 1.80 mmol) was
◦
4.1.7. 1-(5-(Benzyloxy)-3,4,6-trimethylpyridin-2-yl)-4-(bis(4-
fluorophenyl)methyl)piperazine (25)
added at ꢀ 78 C. Dry ethyl ether (18 mL) was added to it followed by the
dropwise addition of cyclohexylmagnesium bromide (2 M, 2.8 mL, 5.58
To a mixture of 3-(benzyloxy)-6-bromo-2,4,5-trimethylpyridine (4,
7

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
Fig. 4. Effect of compounds, 2, 3A, and 3J on
tumor growth in DU145-xenografted CAM tumor
model. (A, B) Comparison of inhibitory effects of
compounds, 2, 3A, and 3J on DU145-induced
angiogenesis (A, scale bars, 5 mm) and tumor
size (B). The images of angiogenesis are repre-
sentative of each group. The control CAM was
exposed to the vehicle. (C-E) Quantitation of
angiogenesis and tumor size. The quantitation of
new branches formed from existing blood vessels
and weighing of tumors grown on the CAM were
performed five days after cancer cell implanta-
tion. Data are presented as the mean ± standard
deviation from seven different samples. *P < 0.05
#
vs. vehicle-treated control. P < 0.05 vs. com-
pound 2 or 3J-treated group.
1
00 mg, 0.33 mmol), 1-(bis(4-fluorophenyl)methyl)piperazine (24, 94
2.52 (s, 4H), 2.43 (s, 3H), 2.17 (s, 3H), 2.12 (s, 3H); 13C NMR (CDCl
) δ
3
mg, 0.33 mmol) in toluene (5 mL) were added tris(dibenzylideneace-
163.75, 159.86, 157.10, 147.99, 146.31(2C), 140.42, 138.40, 137.30,
129.36, 129.24, 128.67, 128.55 (2C), 128.09, 127.98, 127.86 (2C),
122.49, 115.53 (2C), 115.19 (2C), 74.75, 74.52, 52.05 (2C), 50.46 (2C),
19.29, 14.41, 13.03.
′
tone)dipalladium(0) (7 mg, 0.007 mmol), 2,2 -bis(diphenylphosphino)-
′
1
0
,1 -binaphthyl (8 mg, 0.013 mmol), and sodium tert-butoxide (44 mg,
.44 mmol). The mixture was refluxed for 5 h and cooled to room
temperature. EtOAc and brine were added to the mixture and the
organic layer was washed with brine several times. The organic solution
4.1.8. 6-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)-2,4,5-
trimethylpyridin-3-ol (3J)
4
was dried over MgSO , filtered, and concentrated. The residue was pu-
rified by silica gel column chromatography (EA:HX = 1:10) to give 25
To a solution of 25 (40 mg, 0.07 mmol) in methanol (5 mL) was
added 10% palladium on carbon (7 mg, 0.007 mmol). The mixture was
stirred under hydrogen atmosphere at room temperature for 3 h then
filtered through Celite pad. The filtrate was concentrated to give 3J (28
(
68 mg, 41% yield) as a pale yellow solid. R
f
0.30 (Hx:EA = 5:1); m.p.
18 C; MS m/z 514.29 [M + H] ; H NMR (CDCl ) δ 7.50–7.33 (m, 9H),
.98 (t, J = 8.7 Hz, 4H), 4.74 (s, 2H), 4.31 (s, 1H), 3.11–3.02 (m, 4H),
◦
+
1
1
6
3
8

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
(
Chinese hamster ovary cell line) and HEK-293 (human embryonic
kidney cell line), were obtained from KCLB. The cells were cultured in
standard growth medium supplemented with 10% fetal bovine serum
◦
(
FBS), 1% penicillin/streptomycin and incubated at 37 C under a 5%
2
CO atmosphere.
4
.2.2. Cell proliferation assay
Cells were seeded at a density of 5000 cells/well in a 96-well plate.
After overnight incubation, the cells were serum-starved using 1% FBS.
The next day, the cells were pre-treated with the indicated concentra-
tions of drugs for 1 h prior to the treatment with serum (10% FBS). After
7
2
h
of incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) dye solution was added and incubated for 4 h.
Next, DMSO was added, and after 30 min, the color intensities were
measured using a microplate reader (Versamax, Molecular Devices, Inc.,
USA) at 490 nm.
4
.2.3. Cytotoxicity assay
The cytotoxicity of the compounds was assessed by measuring the
cell viability decrease using the MTT staining method. Briefly, cells were
4
seeded in a 96-well plate (Falcon, USA) at a density of 1 × 10 cells per
well. The next day, culture medium was changed into 1% serum con-
taining medium, and cells were incubated with different concentrations
of each compound for 24 h. Then, 10
μ
L of MTT solution was added to
Fig. 5. Binding mode of M3R-compound 3A complex.
◦
the well and further incubated for 4 h at 37 C. At the end of the incu-
bation, the media were removed, and 200
μ
L of dimethyl sulfoxide
◦
mg, 95% yield) as a white solid. R
f
0.25 (EA:Hx = 1:5); m.p. 89 C; MS
(
DMSO) was added into each well to solubilize the formazan crystals.
+
1
m/z 424.25 [M + H] ; H NMR (CDCl
3
) δ 7.38 (dd, J = 8.3, 5.6 Hz, 4H),
Finally, the absorbance was measured at 540 nm using a microplate
reader (Molecular Devices, Versa MAX Sunnyvale, CA, USA).
6
2
1
1
.98 (t, J = 8.5 Hz, 4H), 4.36 (s, 1H), 4.30 (s, 1H), 3.07–2.92 (m, 4H),
.50 (s, 4H), 2.39 (s, 3H), 2.14 (s, 6H); 13C NMR (CD
OD) δ 163.78,
3
59.90, 154.06, 145.68, 140.41, 138.64, 138.59, 135.53, 129.34(2C),
29.21(2C), 123.14, 114.93(2C), 114.59(2C), 74.60, 52.13(2C), 50.20
4
.2.4. NOX activity measurement
NOX activity was assayed with lucigenin method as previously re-
(
2C), 17.34, 12.90, 11.38; HPLC retention time 19.5 min, purity 98.2%.
ported [11] with some modification. In short, cells were washed thor-
oughly with ice cold 1x Phosphate-buffered saline (PBS) and collected in
Krebs-HEPES buffer of pH 7.4 (118 mM NaCl, 4 mM KCl, 2.5 mM CaCl2,
4
4
.2. Biological evaluation
1
4 2 4 3
.18 mM MgSO , 1.18 mM KH PO , 24.9 mM NaHCO , 11 mM glucose,
.2.1. Cell lines and culture
0.03 mM EDTA, and 20 mM HEPES) containing protease and phospha-
tase inhibitors (Thermo Scientific, Rockford, IL, USA). Cells were then
homogenized with Dounce homogenizer and protein concentration was
determined using BCA protein assay kit (Thermo Scientific). The
chemiluminescence was measured using a FLUOstar Optima microplate
Seventeen human cancer cell lines derived from breast (MCF-7,
MDA-MB-231), colon (HT-29, SW620, Caco-2, HCT-116), pancreas
PANC-1, MiaPaCa-2), liver (Hep3B), lung (A549, H1299), prostate (DU-
45, PC-3, LNCaP), blood (U937, TPH-1), and brain (A172) were ob-
(
1
tained from American Type Culture Collection (ATCC, USA) and Korean
Cell Line Bank (KCLB). Adriamycin-resistant and tamoxifen-resistant
MCF cell lines, MCF-7/ADR and MCF-7/TAMR, respectively, were
generously provided by Prof. Keon Wook Kang (Seoul National Uni-
versity, College of Pharmacy). Normal human colon epithelial cell line
CCD841 was obtained from ATCC, and two normal cell lines, CHO-K1
reader (BMG LABTECH) immediately after adding 50
well containing mixture of test compounds (3, 10, and 30
(5 M), NADPH (100 M) and inducer (10% FBS or 300
μ
g protein to each
M), lucigenin
M carbachol).
μ
μ
μ
μ
3
Fig. 6. Competition binding assay of compounds 2 and 3A for [ H]4-DAMP binding sites in human recombinant M3R which were expressed in CHO cells. K
i
and IC50
were calculated as described in Experimental Section.
9

U. Karmacharya et al.
Bioorganic Chemistry 110 (2021) 104805
Fig. 7. The inhibitory effects of compound 3A on carbachol-induced DU145 cell proliferation and survival. (A) DU145 cells were treated with different concen-
trations of carbachol in the presence of low level serum (1%). (B, C) DU145 cells were treated with increasing concentrations of compound 2 or 3A with different
concentrations of carbachol in the presence of 1% serum. (D) DU145 cells were treated with increasing concentrations of carbachol alone in the absence of serum for
4
8 or 72 h. (E) DU145 cells were co-treated with 300
μ
M carbachol and 10
μ
M of compound 2 or 3A for 48 or 72 h. *P < 0.05 versus the vehicle-treated control
#
group. P < 0.05 versus carbachol-treated group.
4
.2.5. CAM (chick chorioallantoic membrane) tumor model
4.2.6. Radioligand binding assay
Fertile chicken eggs were purchased from Byeolbichon Farm
Competitive M3R binding assay was performed in the laboratory of
Eurofins Discovery (France). Briefly, human recombinant M3R overex-
◦
(
Gyeongbuk, South Korea) and incubated at 37 C and under 55%
3
relative humidity. On the 9th day of egg incubation (post-fertilization),
false air sac was generated on the relatively flat side of eggs by a
pressed in CHO cells were incubated with 0.2 nM [ H]4-DAMP and se-
rial dilutions of compounds 2 and 3A for 1 hr at room temperature.
2
negative pressure technique. A small window (1 cm ) was created on the
Nonspecific binding was determined in the presence of 1 M atropine.
μ
false air sac surface of the eggs by separating the shell and membrane
beneath (technique) using a grinding wheel (Dremel, Racine, WI, USA).
The results were expressed as a percent inhibition of control specific
binding in the presence of the test compounds. Concentration causing a
half-maximal inhibition of control specific binding (IC50) and Hill co-
efficients (nH) were determined by non-linear analysis of the competi-
tion curves generated with mean replicate values using Hill equation
6
Next, DU145 cells were loaded at a density of 1.5 × 10 cells/CAM with
or without compound. After five days of drug treatment, the tumor
weight, number of vessel branch points within the tumor region were
analyzed.
i
curve fitting. The inhibition constant (K ) was calculated using the
The chick embryo experiments were approved beforehand by the
Institutional Animal Care and Use Committee of Yeungnam University
and were performed accordingly the guidelines issued by the Institute of
Laboratory Animal Resources (1996) and Yeungnam University (The
care and use of animals 2009).
Cheng Prusoff equation.
4.2.7. Carbachol-induced DU145 cell survival assay
DU145 cells were seeded in 96-well plates at a density 5000 cells/
well using complete (10% FBS containing) media and incubated for 24
h. Then, cells were washed three times with warm Hank’s balanced salt
1
0

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Bioorganic Chemistry 110 (2021) 104805
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.2.8. Statistical analysis
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introducing artificial intelligence to a traditional computational drug discovery
solution that can improve the accuracy of prediction throughout the entire process
of new drug discovery and development from hit discovery, lead optimization,
selectivity prediction, and ADME/Toxicity.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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26] Procedures for the synthesis of 3A and 3J are provided here. Synthetic procedures
for all compounds are described in Supplementary Material.
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