Synthesis and biological evaluation of selective survivin inhibitors derived from the MX-106 hydroxyquinoline scaffold

European Journal of Medicinal Chemistry 224 (2021) 1 13719

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Synthesis and biological evaluation of selective survivin inhibitors

derived from the MX-106 hydroxyquinoline scaffold

,

Najah Albadari ^{a 1}, Shanshan Deng ^{a 1}, Hao Chen ^a, Guannan Zhao ^b, Junming Yue ^b,

Sicheng Zhang ^a, Duane D. Miller ^a, Zhongzhi Wu ^{a **}, Wei Li ^a

,

, *

^aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, 38163, USA

^bDepartment of Pathology and Laboratory Medicine, Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN, 38163, USA

a r t i c l e i n f o

a b s t r a c t

Article history:

The survivin (BIRC5) expression is very low in normal differentiated adult tissues, but it is one of the

most widely upregulated genes in tumor cells. The overexpression of survivin in many cancer types has

been positively correlated with resistance to chemotherapy, tumor metastasis, and poor patient survival.

Survivin is considered to be a cancer speciﬁc biomarker and serves as a potential cancer drug target. In

this report, we describe the design and syntheses of a series of novel selective survivin inhibitors based

on the hydroxyquinoline scaffold from our previously reported lead compound MX-106. The best

compound identiﬁed in this study is compound 12b. In vitro, 12b inhibited cancer cell proliferation with

Received 27 May 2021

Received in revised form

20 July 2021

Accepted 22 July 2021

Available online 30 July 2021

Keywords:

BIRC5 (survivin)

an average IC₅₀value of 1.4 mM, using a panel of melanoma, breast, and ovarian cancer cell lines. The

metabolic stability of 12b improved over MX-106 by 1.7-fold (88 vs 51 min in human microsomes).

Western blot analyses demonstrated that treatments with 12b selectively decreased survivin protein

levels, but negligibly affected other closely related members in the IAP family proteins, and strongly

induced cancer cell apoptosis. In vivo, compound 12b effectively inhibited melanoma tumor growth

when tested using a human A375 melanoma xenograft model. Further evaluation using an aggressive,

orthotopic ovarian cancer mouse model showed that 12b was highly efﬁcacious in suppressing both

primary tumor growth in ovaries and tumor metastasis to multiple peritoneal organs. Collectively, results

in this study strongly suggest that the hydroxyquinoline scaffold, represented by 12b and our earlier lead

compound MX-106, has abilities to selectively target survivin and is promising for further preclinical

development.

Selective survivin inhibitors

Structure-activity relationships

Antiproliferative activities

P-glycoprotein overexpression

Ovarian tumor metastasis

Orthotopic ovarian cancer mouse model

1. Introduction

differentiated cells while it is highly overexpressed in many types

of cancer cells, where it supports dysregulated cellular replication

The apoptotic process is essential for development and tissue

homeostasis and provides some measure of protection against er-

rors in DNA replication and other related processes. Cancer cells can

hijack and dysregulate this apoptotic process to protect their sur-

vival and growth [1]. Apoptosis is regulated in part by a family of

proteins called inhibitors of apoptosis proteins (IAPs), contain a

conserved 3-dimensional fold BIR domain that binds zinc. Survivin

(BIRC5) is unique among the members of the IAP family in that its

expression is low or undetectable in normal terminally

[2]. Clinically, the expression of survivin is positively correlated to

cancer cell metastasis, tumor invasiveness, chemoresistance, and

poor prognosis [3,4]. The pivotal role played by survivin in multiple

signaling pathways in cancer cells including apoptosis inhibition,

mitotic control, and cell cycle promotion make it an attractive

therapeutic target against cancer [5]. Several compounds have been

reported to inhibit survivin expression, and some of them have

been proposed as speciﬁc inhibitors of survivin [6], with YM155 as

the most known survivin inhibitor reported [7]. YM155 inhibits

transcription of the survivin gene, but recent reports have sug-

gested that it may actually be an DNA-damaging agent instead of a

bona-ﬁde survivin inhibitor [8]_.Thus, the exact mechanism of ac-

tion of YM155 is still debatable [9]. It made to clinical trials but

were suspended in phase II due to its high systemic toxicity and

* Corresponding author.

** Corresponding author.

E-mail address: wli@uthsc.edu (W. Li).

equal contributions to this work.

1

https://doi.org/10.1016/j.ejmech.2021.113719

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

relative low ability to degrade survivin [7,9]. Additional studies

indicate that YM155 is a substrate of the drug efﬂux pump P-

glycoprotein (P-gp), consistent with its limited efﬁcacy in drug

resistant cells [10].

They were synthesized by reacting 8-hydroxyquinoline with

formaldehyde and catalytic zinc chloride in concentrated hydro-

chloric acid to generate the salt of compound 2 [12]. Compounds 3a

and 3b were synthesized using step b, where phenyl-substituted

triazole and imidazole rings reacted with salt 2 in the presence of

K₂CO₃in DMF to form the desired products. Compounds 3c and 3d

were synthesized using steps c and d. In step c, substituted alcohols

were reacted directly with salt 2 with heating to form different

salts. In step d, these were converted into free bases by adjusting

the pH with NaHCO₃solution. Compounds 3a-3d were used in

Mannich reactions with paraformaldehyde and pyrrolidine in

anhydrous ethanol to form the ﬁnal compounds 4a-4d.

The general synthesis of MX-106 analogs that were substituted

in the C ring (compounds 7a-7k and 10) is outlined in Scheme 2.

Various terminal alkynes were reacted with azidotrimethylsilane

with catalytic amounts of CuI in a mixture of DMF and methanol to

prepare different 4-substituted-1-H-triazoles (compounds 5a-5k

and 8) [13]. Compounds 5a-5k and 8 were reacted with salt 2 to

form intermediate compounds 6a-6k and 9, which were submitted

to Mannich reaction with paraformaldehyde and pyrrolidine to

form compounds 7a-7k and 10 (Scheme 2).

MX-106 analogs that were substituted in the D ring (compounds

12a-12e and 14) are shown in Scheme 3. The intermediate com-

pounds 6a and 6b were reacted with different secondary amines via

Mannich reaction to form ﬁnal compounds 12a-12e and 13. The

ﬁnal compound 14 was obtained by removing the tert-

butyloxycarbonyl protecting group (BOC group) from compound

13 using triﬂuoroacetic acid (TFA) in dichloromethane.

Scheme 4 shows the general synthesis of compounds 19a-19h.

7-Bromo-8-hydroxyquinoline was reacted with formaldehyde and

catalytic zinc chloride in concentrated hydrochloric acid at 60 ^ꢀC for

16 h to produce compound 15. After introducing a chloromethyl

group to the 5-position of the hydroxyquinoline ring, compound 15

was reacted directly with 4-isopropylbenzyl alcohol with heating to

form a salt that was converted to a free base by adjusting the pH

with NaHCO₃solution to form intermediate compound 16. Com-

pound 16 was reacted with 2-(trimethylsilyl)ethoxymethyl chlo-

ride (SEMCl) and NaH in THF at RT to produce compound 17. The

Suzuki coupling reaction of compound 17 with appropriate aryl

boronic acid under Pd (PPh₃)₄condition generated the

We have previously reported the identiﬁcation of compound

UC-112 (Fig. 1), which selectively decreases survivin protein levels

and activates caspases 3/7 and 9 [1 1]. UC-112 also retains its po-

tency in multidrug-resistant cancer cell lines that overexpress P-gp.

A subsequent structure-activity relationship (SAR) study of UC-112

led to discovery of its derivative MX-106 [5-(((4-isopropylbenzyl)

oxy)methyl)-7-(pyrrolidin-1-ylmethyl)quinolin-8-ol] (Fig. 1). MX-

106 suppresses the in vivo growth of melanoma tumors [12].

Mechanistic studies indicated that MX-106 functions by selectively

suppressing expression of survivin and induces apoptosis of cancer

cells. The results of the SAR showed that the 8-hydroxyquinoline

moiety is important for its antiproliferative activities (A and B

rings, shown in Fig. 1). However, the benzyloxy ether linkage that

connects the C ring to the 8-hydroxyquinoline moiety (linker

moiety, Fig. 1) is metabolically labile group and we hypothesized

that we could improve the metabolic stability while maintaining

the potency of this scaffold by replacing the ether linker with more

stable moieties such as imidazole and triazole rings. We therefore

sought to further optimize the MX-106 hydroxyquinoline scaffold

by designing several focused sets of new MX-106 analogs (Fig. 1).

We introduced four major modiﬁcations to the MX-106 structure in

the present study. First, we modiﬁed the ﬂexible ether linker by

cyclization or replacing it with other ring systems. Second, we

modiﬁed the C ring by replacing the phenyl ring with other bio-

isosteres or by introducing additional substitutions. Third, we

modiﬁed the D ring with a variety of cyclic amines. Finally, we

removed the methylene linker between the B and D rings to obtain

a planar head scaffold. This combined approach led to the pro-

duction of thirty-one new MX-106 analogs, which we evaluated

in vitro and in vivo to identify compound 12b as a new lead com-

pound for future further preclinical studies.

2. Chemistry

Compounds 4a-4d contained different linkers between the B

and C rings relative to the parent compound MX-106 (Scheme 1).

Fig. 1. Targeted modiﬁcations used to produce new MX-106 analogs.

2

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

Scheme 1. Synthesis of Compounds 4a-4d

Reagents and conditions: (a) Conc. HCl, 37 % formaldehyde, ZnCl₂, r.t, 24 h; (b) K₂CO₃, DMF, r.t, 24 h; (c) substituted alcohol, 60 ^ꢀC; (d) hexane:ethylacetate (1:1), NaHCO₃, H₂O, pH

8e10; (e) pyrrolidine, paraformaldehyde, anhydrous EtOH, reﬂux, 3 h.

Scheme 2. Synthesis of Compounds 7a-7k and 10

Reagents and conditions: (a) azidotrimethylsilane (1.5 equiv), CuI(5 mol%), DMF/MeOH ¼ 5:1, reﬂux, 12 h; (b) K₂CO₃, DMF, r.t, overnight; (c) pyrrolidine, paraformaldehyde,

anhydrous EtOH, reﬂux, 3 h.

3

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

Scheme 3. Synthesis of Compounds 12a-12e and 14

Reagents and conditions: (a) amine, paraformaldehyde, anhydrous EtOH, reﬂux, 3 h; (b) triﬂuoroacetic acid (TFA), anhydrous CH₂Cl₂.

Scheme 4. Synthesis of Compounds 19a-19h

Reagents and conditions: (a) Conc. HCl, 37%formaldehyde, ZnCl₂, 60 ^ꢀC, 16 h; (b) 90 ^ꢀC; (c) hexane:ethylacetate (1:1), NaHCO₃, H₂O, pH 8e10; (d) SEMCl, NaH, THF; (e) Pd(PPh₃)₄,

Na₂CO₃, dioxane/H₂O; (f) triﬂuoroacetic acid (TFA), CH₂Cl₂.

intermediates 18a-18h. The ﬁnal compounds 19a-19h were ob-

tained by removing the SEMCl protecting group with triﬂuoroacetic

acid (TFA) in dichloromethane.

Table 1.

Compounds 4a-4d have less conformationally ﬂexible linkers

between rings B and C relative to the ﬂexible ether linker in

parental MX-106. Compounds 4a-4c showed less potency with an

average IC₅₀values of 4.2, 2.9, and 2.7

and 4c respectively as compared to 2.0

m

M for compounds 4a, 4b,

3. Results and discussion

mM of MX-106 (unless

speciﬁed, the IC₅₀value for each analogue is expressed as the

average of results obtained in all six cell lines). Compound 4d

showed activity comparable to that of MX-106 in the cell lines

3.1. In vitro antiproliferative assay in a panel of melanoma and

breast cancer cell lines

tested, with an IC₅₀value of 1.9 mM. Overall, converting the ﬂexible

All MX-106 analogs were evaluated for their cytotoxicity in a

panel of human cancer cell lines including melanoma cancer cell

lines A375, RPMI7951, MDA-MB-435 and its multidrug resistant

daughter line MDA-MB-435/LCC6MDR1 and breast cancer cell lines

MDA-MB-453 and SKBR3. MX-106 was included in the assays as a

positive control. These in vitro biological results are summarized in

ether linker in MX-106 to less conformationally ﬂexible linkers do

not seem to affect the potency signiﬁcantly in this scaffold. Next, we

kept the trizole ring as a linker and modiﬁed the C ring. We syn-

thesized compounds containing several substituted benzene rings

in the C ring position by introducing various electron donating

4

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

Table 1

In vitro growth inhibitory effects of MX-106 analogs in a panel of melanoma and breast cancer cell lines (IC₅₀standard error of the mean (SEM) [4], n ¼ 3).

Compound

A375

MDA-MB-435

RPMI7951

MDA-MB-453

SKBR3

MDA-MB-435/LCC6MDRI

Average IC₅₀

Resistance index (RI)^a

4a

4b

4c

4d

7a

7b

7c

7d

7e

7f

7g

7h

7i

7j

7k

10

2.2 0.2

2.0 0.3

1.9 0.3

1.0 0.2

0.7 0.1

0.8 0.1

1.1 0.2

0.7 0.1

0.9 0.1

1.0 0.1

0.9 0.1

0.7 0.1

0.6 0.1

1.1 0.2

1.0 0.1

0.7 0.1

0.9 0.1

1.0 0.1

1.0 0.2

0.8 0.1

1.0 0.2

0.5 0.1

0.6 0.1

0.8 0.1

0.7 0.1

0.8 0.1

0.9 0.1

0.6 0.1

nd^b

8.9 1.2

5.0 0.7

2.2 0.3

3.3 0.4

3.1 0.4

4.1 0.5

4.6 0.5

2.0 0.3

3.2 0.4

4.1 0.7

3.9 0.5

5.9 0.8

3.2 0.4

2.4 0.3

2.6 0.4

4.8 0.8

2.2 0.3

2.6 0.4

3.1 0.3

3.8 0.6

2.4 0.3

4.7 0.5

2.1 0.3

0.5 0.1

0.6 0.1

1.2 0.1

1.8 0.2

1.9 0.3

1.9 0.2

1.1 0.2

2.4 0.2

3.5 0.4

2.8 0.4

3.1 0.4

1.3 0.2

0.8 0.1

1.7 0.3

1.6 0.2

2.3 0.3

0.6 0.1

0.7 0.1

0.9 0.2

0.8 0.1

0.6 0.1

0.7 0.1

0.6 0.1

1.3 0.2

0.9 0.2

1.4 0.2

2.1 0.3

1.9 0.3

0.7 0.1

0.6 0.1

0.8 0.1

0.7 0.1

0.5 0.1

0.7 0.1

1.1 0.2

0.6 0.1

1.3 0.1

0.5 0.1

nd^b

3.1 0.3

2.9 0.4

2.4 0.3

1.3 0.2

3.1 0.4

3.5 0.5

5.9 0.7

2.9 0.5

3.3 0.5

4.1 0.6

2.2 0.4

4.1 0.7

1.8 0.2

3.1 0.4

2.8 0.5

2.2 0.4

1.5 0.3

2.5 0.3

6.3 0.7

6.2 0.7

2.4 0.3

2.0 0.3

1.3 0.3

0.5 0.1

1.3 0.1

1.7 0.1

0.9 0.1

1.7 0.2

0.8 0.1

4.4 0.6

0.6 0.1

nd^b

6.9 0.5

4.0 0.5

5.9 0.5

3.9 0.5

2.7 0.4

3.2 0.5

4.9 0.6

2.9 0.4

3.3 0.5

3.6 0.5

2.8 0.5

6.0 0.5

2.7 0.3

3.0 0.4

2.7 0.4

1.9 0.3

2.4 0.4

4.2 0.7

4.0 0.5

3.0 0.5

2.4 0.3

5.7 0.8

0.8 0.1

1.5 0.1

3.1 0.3

1.6 0.2

3.3 0.3

1.3 0.2

5.7 0.9

0.8 0.1

nd^b

1.3 0.2

0.6 0.1

2.2 0.2

1.3 0.1

0.6 0.1

1.1 0.1

1.7 0.1

0.7 0.1

1.0 0.1

1.1 0.2

1.4 0.1

1.8 0.2

2.2 0.2

0.5 0.1

0.9 0.1

1.6 0.2

1.2 0.1

0.7 0.1

1.6 0.1

1.7 0.1

0.9 0.1

5.1 0.6

0.2 0.0

1.4 0.1

1.0 0.1

2.3 0.2

1.0 0.1

3.0 0.4

1.2 0.2

0.9 0.1

4.2

2.9

2.7

1.9

2.0

2.4

3.4

1.6

2.0

2.4

2.0

3.3

1.9

1.7

2.3

1.5

1.7

3.0

3.1

1.7

2.6

1.9

0.7

0.9

1.4

1.6

1.4

2.6

0.8

nd^b

2.0

0.1

1.0

0.4

0.2

0.3

0.4

0.3

0.7

0.2

0.4

0.3

0.5

0.3

0.5

0.4

1.1

0.1

2.6

1.6

0.8

1.3

0.6

1.6

0.6

0.9

>425

0.6

12a

12b

12c

12d

12e

14

16

19a

19b

19c

19d

19e

19f

19g

19h

YM-155 (nM)

MX-106

>1

mM

1.0 0.1

1.1 0.1

1.6 0.2

2.3 0.2

2.1 0.1

a

Resistance Index (IR) is calculated by dividing IC₅₀values on multidrug-resistant cell line MDA-MB- 435/LCC6MDR1by IC₅₀values on the matching sensitive parental cell

line MDA-MB-435.

b

Not determined.

groups (methyl, ethyl, isopropyl, methoxy, and benzyloxy) or

electron withdrawing groups (bromo, triﬂuoromethyl, nitro, and

cyano) in different positions (para, ortho, and meta) on the phenyl C

ring.

We introduced different para-substitutions with electron-

withdrawing functional groups, such as ꢁBr (compound 7a), eCF₃

(compound 7b), eNO₂(compound 7f), and eCN (compound 7h) to

the C phenyl ring. Compound 7a (with a bromo substitution in the

para position) exhibited same cytotoxic activity to that of MX-106

the pyrrolidine ring and replaced it with different cyclic and acyclic

amines. Compounds 12a, 12b, and 12e possess larger D rings (12a:

para-bromophenyl C ring and piperidine D ring, 12b: para-tri-

ﬂuorophenyl C ring and piperidine D ring,12e: para-bromophenyl C

ring and morpholine D ring). These three analogs exhibited slightly

higher cytotoxic activity than the parent compound in all cancer

cell lines tested (IC₅₀values:12a, 1.4

MX-106 2.0 M). Conversely, compound 14 (with a para-bromo-

phenyl C ring and piperazine D ring) exhibited less cytotoxic ac-

tivity (IC₅₀2.6 M) than did MX-106. The D ring of analog 14 was a

mM; 12b, 1.7 mM; 12e, 1.7 mM;

m

(IC₅₀2.0

mM for 7a vs 2.0

mM for MX-106). But stronger electron

m

withdrawing substitutions resulted in decreased activity (IC₅₀

piperzine ring rather than the morpholine ring found in 12e, sug-

gesting that perhaps a hydrogen acceptor functional group in D ring

is favorable for cytotoxic activity. Compound 12d with diethyl

amine, an open ring analog of pyrrolidine, exhibited lower activity

values are 2.4, 2.4, and 3.3

respectively).

mM for compounds 7b, 7f, and 7h,

Next, we shifted the bromo substitution position in this C ring in

order to probe its best location. While the para-Br compound 7a

was same as MX-106 in potency, 7j (ortho-Br) and 7k (meta-Br)

(IC₅₀3.1

the pyrrolidine ring was replaced by an azepane ring, resulting in

decreased activity (IC₅₀3.0 M) relative to MX-106. On the whole, a

mM) than MX-106 in all cell lines tested. In compound 12c,

were slightly more active, with IC₅₀value of 1.7

mM, suggesting that

m

the ortho- and meta-bromine substitutions in the C phenyl ring

were slightly favorable for antiproliferative activity. Interestingly,

compound 7d, in which an electron donating methyl substitution is

introduced in the para position, exhibited improved potency (IC₅₀

5-membered or 6-membered D ring is favorable in this hydrox-

yquinoline scaffold of MX-106.

The parent compound MX-106 contains a methylene linker

between the B and D rings. To test the signiﬁcance of this methy-

lene group for the cytotoxic activity of MX-106, we synthesized

several analogs that lacked it. Most of these compounds were more

active than the MX-106 parent, suggesting that the methylene

linker may not be important for its cytotoxic activity. Analogs 19a

(with a phenyl D-ring) and 19h (with thiophene D-ring) demon-

strated the best cytotoxic activity in all cancer cell lines tested, with

1.6 mM vs 2.0 mM for MX-106). But analogs with a larger substitu-

tion, 7e (para-CH₂CH₃), 7g (para-OCH₃), 7i (para-benzyloxy), or 7c

(para-isopropyl) did not provide further enhancement in potency.

Bioisosterically replacing the phenyl ring with a thiophene ring to

generate compound 10 resulted in slightly decreased activity (IC₅₀

of 2.3 mM) compared to MX-106.

We continued our modiﬁcation of MX-106 with the pyrrolidine

IC₅₀values of 0.7 and 0.8

mM, respectively.

D ring. We compared the effects of different ring sizes or removed

Compounds 19d (para-methylphenyl

D ring), 19e (meta-

5

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

methylphenyl D ring), and 19f (para-methoxyphenyl D ring)

Therefore, the smaller the RI value, the better the ability of the drug

to overcome resistance. Twenty-ﬁve of the thirty-one new MX-106

analogs tested exhibited more potent inhibitory effects against the

resistant MDA-MB-435/LCCMDR1 cell line that overexpressed P-gp

than those observed in the parental drug sensitive MDA-MB-

435 cell line. These compounds exhibited RI values that were less

than 1, suggesting that they could circumvent drug resistance

mediated by P-gp. In contrast, small molecule inhibitor YM155

displayed signiﬁcantly reduced activity against P-gp-over-

expressing MDA-MB-435/LCCMDR1 cells, where it exhibited an

exhibited higher cytotoxic activity (IC₅₀values: 19d, 1.4

mM; 19e,

1.6 M; 19f, 1.4 M) than MX-106 in the cancer cell lines tested,

m

although all were lower than that of compound 19a. Interestingly,

substituting the phenyl D ring with an electron with drawing group

such as para-triﬂuoromethyl (compound 19g, IC₅₀2.6

mM)

decreased the activity of the compound more than did substituting

it with electron donating groups such as in compounds 19d and 19f.

Finally, compounds 19b (with 4-isoquinoline D ring) and 19c (with

3-quinoline D ring) exhibited higher potency (IC₅₀values: 19b,

0.9

mM; 19c, 1.4

m

M) than MX-106 in all cancer cell lines tested but

IC₅₀of more than 1 mM comparing to 2.4 nM in the sensitive cell. It

they were less cytotoxic than 19a.

is worth noting that compounds 19a, 19b, 19d, and 19f lacking the

ﬂexible methylene linker between the B and D rings exhibit poor RI

values (RI 2.6, 1.6, 1.3, and 1.6, respectively). In contrast MX-106 (RI

0.6) and the remaining analogs, which retained the methylene

linker between the B and D rings, exhibited RI values less than 1

(Table 1). These data suggest that the ﬂexibility of the methylene

linker might be beneﬁcial for the ability to overcome P-gp over-

expression. Collectively, more than 80 % of the new MX-106 analogs

we describe here were able to overcome drug-resistance mediated

by P-gp overexpression in MDA-MB-435/LCCMDR1 cells.

3.2. In vitro antiproliferative assay in ovarian cancer cell lines

Survivin is highly expressed in ovarian cancer, where it is

negatively correlated with overall patient survival [14]. Knockout

(KO) of survivin using CRISPR/Cas9 nickase or pharmacological

treatment of ovarian cancer cells with small molecule survivin in-

hibitor YM155 inhibits the epithelial to mesenchymal transition

(EMT), which contributes to ovarian tumor metastasis and

chemotherapy drug resistance [15e1 7]. MX106 effectively over-

comes chemoresistance in vitro by inhibiting EMT in ovarian cancer

cells. MX106 also suppresses primary tumor growth in mouse

ovaries and metastases in multiple peritoneal organs as compared

to vehicle-treated control mice in an orthotopic ovarian cancer

mouse model [14]. Therefore, we selected analogs 7a, 7k, 12a, 12b,

and 19a described in this study to be evaluated for their cytotoxicity

in the cultured human ovarian cell lines OVCAR3 and OVCAR8. The

results of this experimental procedure are summarized in Table 2.

Compounds 7a, 7k, 12a, and 12b exhibited average IC₅₀values of

3.4. MX-106 analogs retain selective inhibition for survivin among

IAPs

We showed previously that MX-106 selectively downregulated

the expression of survivin in cancer cells as compared with other

members of the IAP family [12]. To determine whether the new

MX-106 analogs maintained the same selective degradation of

survivin, we treated A375, a melanoma cell line with compounds

7a, 12b, and 19a and three ovarian cancer cell lines (SKOV3,

OVCAR3, and OVCAR8) with compounds 7k and 12b and performed

western blotting assay to monitor survivin protein levels (Figs. 2

and 3). MX-106 served as positive control. Analogs 7a and 12b

selectively suppressed survivin levels in A375 cancer cells in a dose-

dependent manner, while the levels of other IAPs were minimally

affected (Fig. 2A and B). Also, compound 19a selectively down-

regulated survivin in A375 cells, although to a lesser extent than we

observed for compounds 7a and 12b (Fig. 2C). The Western blot

1.3

m

M (7a),1.1

mM (7k, 12a, and 12b) in OVCAR3 and OVCAR8 cells,

while that of compound 19a was 2

mM.

3.3. Inhibitory effect of MX-106 analogs against cell lines that

overexpress P-gp

P-glycoprotein (P-gp) is a clinically relevant drug transporter

that acts as a unidirectional efﬂux pump. It limits the cellular up-

take and intracellular concentration of anticancer drugs, leading to

ineffective treatment outcomes in multidrug resistant (MDR) cells

[18]. Therefore, drug candidates that possess the ability to over-

come or circumvent overexpression of P-gp are expected to retain

their therapeutic efﬁcacy even in MDR cancer cells. MDR melanoma

cell line MDA-MB-435/LCCMDR1 that overexpresses P-gp and its

parental sensitive cancer cell line MDA-MB-435 were used to

evaluate the abilities of the newly described analogs to surmount P-

gp overexpression. The results are shown in Table 1, where they are

reported as a function of the cell line tested. The small molecule

survivin inhibitor YM155 was also tested on both the multidrug

resistant melanoma cells and their parental cells. The resistance

index (RI) was calculated by dividing the IC₅₀value of the tested

compound in resistant MDA-MB-435/LCCMDR1 cells by the IC₅₀

value obtained for the same compound in MDA-MB-435 cells.

result for 19a is not conclusive since 10 mM treatment did not show

survivin downregulation unlike 7a and 12b treatments. Consistent

with downregulation of survivin, compounds 7a, 12b, and 19a

effectively induced apoptosis in cancer cells, as indicated by our

detection of elevated levels of an apoptosis, cleaved poly (ADP-

ribose) polymerase (cleaved-PARP) (Fig. 2A-C). Similarly, treatment

of ovarian cancer cell lines OVCAR8 (Fig. 3A), SKOV3 (Fig. 3B), and

OVCAR3 (Fig. 3C) with compounds 7k and 12b reduced the accu-

mulation of survivin protein.

3.5. Metabolic stability of MX-106 analogs in vitro in liver

microsomes

MX-106 has a half-life of 51 min with a clearance rate at

136.6

mL/min/mg in human liver microsomes [12]. MX-106 can

potentially be deactivated metabolically by the hydrolysis of the

ether linker. To improve the metabolic stability of MX-106, we

blocked its metabolic labile site and designed new MX-106 analogs

described above. We examined the metabolic stabilities of analogs

7a, 7k, 12a, 12b, 19a, and 19b in vitro by measuring their half-lives

on incubation with mouse, rat, and human liver microsomes in the

presence of an NADPH regenerating system. The results are sum-

marized in Table 3. All tested compounds possessed longer half-

lives and lower clearance in comparison to MX-106 in human

liver microsomes.

Table 2

In vitro growth inhibitory effects of MX-106 analogs in ovarian cancer cell lines

(IC₅₀standard error of the mean (SEM) [1], n ¼ 3).

Compound

OVCAR3

OVCAR8

Average IC₅₀

7a

7k

12a

12b

19a

1.0 0.2

0.8 0.6

0.5 0.0

0.6 0.0

1.3 0.2

1.7 0.2

1.3 0.1

1.7 0.1

1.5 0.1

2.6 1.3

1.3

1.1

2.0

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N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

Fig. 2. Compounds 7a, 12b, and 19a selectively downregulate survivin in melanoma cancer cells. Western blot analysis of A375 cells treated for 24 h with increasing doses of (A)

MX-106 and compound 7a, (B) MX-106 and compound 12b, or (C) compound 19a. Primary antibodies were speciﬁc for cleaved apoptosis marker poly(ADP-ribose) polymerase

(cleaved-PARP); IAP family members cellular inhibitor of apoptosis protein-1 (CIAP1), X-linked inhibitor of apoptosis protein (XIAP), melanoma inhibitor of apoptosis protein (Livin),

and baculoviral inhibitor of apoptosis repeat-containing protein 5 (survivin or BIRC5); and for the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Fig. 3. Compounds 7k and 12b downregulate survivin in ovarian cancer cells. Western blot analysis of ovarian cancer cells treated for 24 h with gradient increasing doses of

compounds 7k, 12b, and MX-106: (A) OVCAR8 cells, (B) SKOV3 cells and (C) OVCAR3 cells were treated, respectively. Primary antibodies were speciﬁc for apoptosis marker cleaved-

PARP, IAP survivin, or for the internal control GAPDH as described in the legend to Fig. 2.

Table 3

In vitro microsomal stabilities of compounds 7a, 7k, 12a, 12b, 19a, and 19b in liver microsomes from mice, rats, or humans.

Metabolic stability in mouse

Metabolic stability in rat

Metabolic stability in human

Compounds

t_1/2(h)

Cl_int

(m

L/min/mg)

t_1/2(h)

Cl_int

(

m

L/min/mg)

t_1/2(h)

Cl_int(mL/min/mg)

7a

7k

12a

12b

19a

19b

Verapamil

0.8

1.0

0.7

1.1

>4

1.2

0.1

27.8

24.3

35

21.6

5.5

1.0

0.9

1.3

1.6

2.5

0.6

0.2

22.6

24.8

18.0

14.2

9.2

1.9

2.3

1.3

1.5

>4

1.4

0.3

12.0

10.0

17.5

15.7

3.2

19.9

192.5

36.7

100.4

16.3

85.6

3.6. Binding kinetics of compound 12b to survivin

condition in order to obtain the crystal structures of survivin pro-

tein in complexes with this scaffold and will report the results in

the future.

To determine if 12b suppresses survivin expression by directly

interacting with the survivin protein, we used a PlexArray HT sys-

tem (Plexera Bioscience, Woodinville, WA) that employs surface

plasmon resonance (SPR) technology to evaluate the interaction

and measure its afﬁnity. Biotinylated survivin protein was

expressed in E.coli BL21 (DE3) strains (Invitrogen; Thermo Fisher

Scientiﬁc) and immobilized to a gold-coated sensor chip via avidin-

biotin conjugation and compound 12b added at different concen-

trations for detection of binding in relative resonance units (RU)

and determination of afﬁnity of 12b for survivin. Resonance values

for 12b increased in a concentration-dependent manner (Fig. 4).

We used a steady-state ﬁtting model to calculate the equilibrium

dissociation constant (KD) of compound 12b binding to survivin at

3.7. Molecular modeling of the interaction between survivin and

compound 12b

To explain the observed potency of compound 12b we per-

formed a molecular modeling study using the known complex of

human survivin-SMAC AVPI, determined by solving the crystal

structure for human survivin and the 1e15 peptide from Smac/

DIABLO (PDB entry: 3UIH), as a scaffold. The results are shown in

Fig. 5A and B. Compound 12b was predicted to form salt bridge and

p-p stacking interactions with the survivin protein BIR domain. The

key contacts predicted are as follows: (1) a possible salt bridge

interaction between the 8-hydroxyquinoline of 12b and residue

Lys79 of survivin (Fig. 5B, purple arrow at right); (2) two possible

salt bridge interactions between the piperidine of 12b and survivin

residues Asp71 and Glu 65 (Fig. 5B, purple arrows at top and left);

4.27 mM. Although preliminary and the exact binding site on sur-

vivin protein is yet to be determined, this result demonstrates that

12b directly interact with survivin. We are currently working with a

CRO (Creative Biostructures, Inc) to optimize the crystallization

7

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

immunodeﬁcient NOD scid gamma (NSG) mice and allowed to

grow for 2 weeks until the average tumor volume reached 100 mm³

prior to treatment. Groups of mice (n ¼ 7/group) were treated with

vehicle alone or with 20 mg/kg or 40 mg/kg doses of 12b by

intraperitoneal (IP) administration 3 days/week for 15 days. Tumor

size (volume) and body weight were measured every 2 days.

As shown in Fig. 6A, Compound 12b signiﬁcantly inhibited tu-

mor growth in a dose-dependent manner. All of the mice were

stable and there was no signiﬁcant body weight loss observed,

suggesting that doses of 20 mg/kg and 40 mg/kg were equally well

tolerated (Fig. 6B). At the end of the experiment, the mice were

sacriﬁced, and tumors were removed, weighed, and measured.

Compared to the vehicle control, both doses of compound 12b

signiﬁcantly inhibited growth of the tumors, with inhibition of

tumor volume by approximately 41 % (20 mg/kg) and 66 % (40 mg/

kg) and reduction of tumor weight by approximately 32 % (20 mg/

kg) and 55 % (40 mg/kg) (Fig. 6C-E). These results demonstrated

that treatment of mice with compound 12b attenuated the pro-

gression of melanoma tumors at a safe dosage.

Fig. 4. Kinetics of binding survivin protein by compound 12b at 25, 50, 100, 200,

400 nM. Binding of compound 12b to survivin protein was measured using SPR

technology. Resonance was measured in arbitrary units at each concentration and

plotted as a function of incubation time.

3.8.2. Treatment with compound 12b reduced the growth rate and

metastasis of ovarian tumors in an orthotopic OVCAR8 ovarian

cancer mouse model

(3)

hydroxyquinoline and triazole ring of 12b and residue His80 of

survivin (Fig. 5B, green lines at right); and (4) a -cation interaction

between the piperidine of 12b and residue Trp67 of survivin

(Fig. 5B, red lines at left).

a possible p- p stacking interactions between the 8-

Survivin is overexpressed in ovarian cancer and our lead com-

pound MX106 suppresses the growth of primary ovarian tumors

and subsequent metastasis [14]. We utilized an established ortho-

topic ovarian cancer mouse model to further evaluate the thera-

peutic potential of compound 12b for treatment of survivin-

overexpressing ovarian tumors. This was done by surgically

injecting OVCAR8 tumor cells expressing luciferase into the left side

of the ovarian bursa (left ovary) of female mice. One week following

orthotopic implantation, the mice were separated into 2 groups

that were treated with either vehicle or 20 mg/kg compound 12b (3

times/week by IP injection). We began treatment with 4 iterations

of the lower dose in case the implantation surgery had weakened

the mice, then increased the dose to 40 mg/kg for an additional 4

treatments. The mice were monitored for body weight and biolu-

minescence imaging after administration of D-luciferin. Mice were

sacriﬁced after 9 days of treatment and all the major organs/tissues

were harvested and bioimaged to visualize metastases.

p

3.8. In vivo assessment of antitumor efﬁcacy

3.8.1. Antitumor activity of compound 12b in A375 subcutaneous

and OVCAR8 orthotopic xenograft models in mice

As one of the most potent MX-106 analogs we describe here,

compound 12b signiﬁcantly inhibited the accumulation of survivin

protein at a low micromolar concentration in a dose dependent

manner and was stable in mouse and human liver microsomes.

Therefore, we selected compound 12b for further evaluation of its

antitumor efﬁcacy in two xenograft mouse models for cancer. We

investigated the effect of treatment with 12b on inhibition of solid

tumors in a subcutaneous human melanoma A375 xenograft model

in mice. A375 melanoma tumors were implanted in the ﬂank of

After 2.5 weeks of treatment, mice that had received treatment

Fig. 5. Potential binding of 12b to survivin based on modeling of 12b onto the crystal structure of survivin bound to the Smac/DIABLO AVPI peptide. (A) The best docking pose of 12b

binding to survivin (survivin PDB: 3UIH), shown with the surface of the protein color-coded by electron potential (red: electron negative potential; blue: electron positive potential).

(B) Types of interactions predicted between 12b and residues in survivin protein are shown in a 2D interaction map. Predicted contacts include: 1) a salt bridge interaction between

8-hydroxyquinoline of 12b and residue Lys79 of survivin (purple arrow at right); (2) two salt bridge interactions between the piperidine of 12b and survivin residues Asp71 and Glu

65 (purple arrows at top and left); (3) a

p- p stacking interactions between the 8-hydroxyquinoline and triazole ring of 12b and residue His80 of survivin (green lines at right); and

(4) a -cation interaction between the piperidine of 12b and residue Trp67 of survivin (red lines at left).

p

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N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

Fig. 6. The antitumor efﬁcacy of compound 12b against growth of A375 xenograft tumors in NSG mice. A375 cells were subcutaneously injected into the dorsal right ﬂank of NSG

mice. When average tumor volume reached 100 mm³, mice were randomized into 3 groups (n ¼ 7/group) and treated with vehicle control or with 20 mg/kg or 40 mg/kg doses of

compound 12b. (A) Tumor growth curves with 20 mg/kg and 40 mg/kg compound 12b versus vehicle. Tumor volumes were monitored 3 times/week. Statistical signiﬁcance was

assessed using two-way ANOVA with Dunnett's multiple comparisons test (****p < 0.0001). (B) Mouse body weight was recorded thrice a week throughout the 15 days treatment

period. (C-D) The ﬁnal volume (C) (one-way ANOVA, *p ¼ 0.0165, ***p ¼ 0.0004) and weight (D) (one-way ANOVA, *p ¼ 0.0450, **p ¼ 0.0015) of inoculated tumors were measured

at the end of the experiment. (E) Images of tumors isolated from each treatment group.

with compound 12b exhibited a signiﬁcant reduced total photon

ﬂux in tumors relative to that in mice treated with only the vehicle

control, (Fig. 7A and B). Examination of body weight during the

treatment period showed no severe weight loss during the course

of the 12b treatment (Fig. 7C). Bioimaging of the excised uterus and

ovaries from each mouse ex vivo is shown in Fig. 7D. In each case,

the right ovary was normal whereas the left ovary bore the tumor.

In the vehicle control group, the tumor-bearing left ovaries

exhibited higher luciferase activity than did the normal right

ovaries due to increased tumor burden. However, treatment with

compound 12b resulted in a remarkable decrease in luciferase ac-

tivity (photon ﬂux) of the excised ovaries, presumably due to

delayed tumor progression (Fig. 7D). We conﬁrmed the results of

the bioluminescence measurements by visual inspection of the

representative uterus images and by measurement of tumor weight

(Fig. 7E). The tumor masses of the animals treated with compound

12b displayed a very strong decrease in tumor weight compared to

the control group (12b: 0.11 0.02; control: 0.18 0.01), as shown

in the right side panel of Fig. 7E.

4. Conclusions

We designed and synthesized thirty-one novel MX-106 analogs

that optimized the linker, C ring, and D ring moieties of MX-106.

Several analogs showed comparable potency to that of MX-106 in

killing tumor cells and improved metabolic stability in human liver

microsomes. The new MX-106 analogs selectively downregulate

the accumulation of survivin protein in a dose-dependent manner.

They also exhibited signiﬁcant abilities to overcome P-gp-mediated

drug resistance not observed for small molecule survivin inhibitor

YM-155. The in vivo efﬁcacy of compound 12b was determined in

melanoma and ovarian cancer xenograft models, where it exhibited

signiﬁcant inhibitory effect of tumor growth. Further optimization

of the 8-hydroxyquinoline MX-106 scaffold may generate survivin

inhibitors that selective for survivin and even more potent in their

anti-tumor activity.

5. Experimental section

Since distant metastases often occur as a late complication in

ovarian cancer patients, we resected nearby major organs (liver,

kidney, spleen, stomach, and intestine) from both groups of mice

and imaged them ex vivo to look for bioluminescence signals that

represented metastases. This allowed us to determine the effect of

12b in suppressing peritoneal multi-organ metastasis in the

orthotopic OVCAR8 ovarian cancer model. The area and total

photon ﬂux of metastatic tumors in liver (Fig. 8A), kidney (Fig. 8B),

spleen (Fig. 8C) and stomach (Fig. 8D) were signiﬁcantly less in

number and smaller in mice treated with compound 12b than in

control animals. However, there was no signiﬁcant inhibition of

metastatic intestinal tumors in mice treated with compound 12b

(Fig. 8E). Taken together, the results of our in vivo studies using the

orthotopic ovarian cancer model demonstrate that treatment of

mice with compound 12b led to inhibition of tumor growth and

visceral metastases in ovarian tumors that overexpressed survivin.

5.1. Chemistry

Chemical reagents and solvents were purchased from AK Sci-

entiﬁc (Mountain View, CA), Alfa Aesar (Ward Hill, MA), or Sigma-

Aldrich Chemical Co. (St. Louis, MO) and were used without further

puriﬁcation. Aluminum-backed uniplates (Analtech, Newark, DE)

were used for routine thin layer chromatography (TLC), which was

monitored using ultraviolet (UV) light. Silica gel (230e400 mesh;

Fisher Scientiﬁc, Pittsburgh, PA) was used for ﬂash chromatog-

raphy. A Bruker Ascend 400 (Billerica, MA) spectrometer was used

to obtain NMR spectra. Coupling constants (J) are provided in Hertz

(Hz). Chemical shifts are reported as parts per million (ppm) rela-

tive to DMSO‑d₆or tetramethylsilane (TMS) in CDCl₃. Melting

points were recorded on a Fisher-Johns melting point apparatus

(uncorrected). High resolution mass spectra (HRMS) were collected

in positive detection mode on a Waters Xevo G2-S time-of-ﬂight

9

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

Fig. 7. Compound 12b reduced growth of primary tumors in an orthotopic ovarian cancer mouse model. Luciferase-expressing OVCAR8 cells were injected into the left side of the

ovarian bursa of 7-week old NSG mice. After one week of tumor progression, mice were randomly separated into 2 groups (vehicle and 12b treatment groups) based on the photon

ﬂux in the tumors and mice body weight and drug treatment was initiated. (A) Representative pseudocolor images tracking OVCAR8 xenografts in mice at the experimental

endpoint. The tumor photon ﬂux (B) (two-way ANOVA, **p ¼ 0.0068) and mouse body weight (C) were monitored during the treatment. (D) Bioluminescence images of excised

ovarian tumor mass ex vivo and scatter plot with a bar showing the ﬁnal tumor photon ﬂux SEM of excised tumors (Student t-test, **p ¼ 0.0031). (E) Pathological images of uterus

and ovaries from each mouse and scatter plot with a bar representing the wet weight of ovarian tumor SEM (Student t-test, **p ¼ 0.0027).

(Tof) instrument equipped with an electron-spray ionization (ESI)

source (Milford, MA).

Method B was used to synthesize compounds 3c and 3d. Com-

pound 2 was suspended in substituted alcohol 3 (5 equivalents) and

the mixture was heated at 70 ^ꢀC for 1 h. The solution was poured

into a 200 mL solution of hexane: ethyl acetate (1:1) and ﬁltered,

then NaHCO₃solution was added to the ﬁltrate. The mixture was

extracted 3 times with dichloromethane (30 mL, 3 times), washed

with brine, dried over anhydrous Na₂SO₄and evaporated to dryness

to yield the desired compounds.

5.1.1. Preparation of 5-(chloromethyl)quinolin-8-ol (2)

A mixture of 5.84 g (40.0 mmol) of 8-hydroxyquinoline, 50 mL of

concentrated hydrochloric acid, and 6.4 mL of 37 % formaldehyde

was treated with 0.6 g of zinc chloride and stirred at room tem-

perature (RT) for 48 h. The mixture was ﬁltered, washed with

copious acetone, and dried to produce compound 2 as a yellow

solid (7.2g, 88 %). ¹H NMR (400 MHz, DMSO‑d₆)

d 11.77 (s, 1H),

5.1.2.1. 5-((4-phenyl-1H-imidazole-1-yl)methyl)quinolin-8-ol (3a).

Pale brown solid (Yield: 97 %). ¹H NMR (400 MHz, Chloroform-d)

9.11e9.07 (m, 2H), 8.03 (dd, J ¼ 8.5, 5.1 Hz, 1H), 7.82 (d, J ¼ 8.0 Hz,

d

8.83 (dd, J ¼ 4.2, 1.4 Hz, 1H), 8.17 (d, J ¼ 8.5 Hz, 1H), 7.73e7.69

1H), 7.36 (d, J ¼ 8.0 Hz, 1H), 5.32 (s, 2H).

(m, 2H), 7.59 (s, 1H), 7.49 (dd, J ¼ 8.6, 4.2 Hz, 1H), 7.42 (d, J ¼ 7.8 Hz,

1H), 7.33 (t, J ¼ 7.6 Hz, 2H), 7.23e7.16 (m, 2H), 7.13 (d, J ¼ 1.2 Hz,1H),

5.49 (s, 2H).

5.1.2. General procedure for the preparation of compounds 3a-3d

Method A was used to synthesize compounds 3a and 3b. To a

solution of compound 3 in DMF (12 mL) was added potassium

carbonate (3 equivalents). The suspension was stirred at RT for

30 min. Salt of 5-chloromethyl-8-quinolinol hydrochloride (com-

pound 2; 1 equivalent) was added to the suspension. The mixture

was stirred overnight at RT. Water was added to the suspension and

the mixture was extracted with ethyl acetate (30 mL, 3 times). The

combined organic phase was washed with brine, dried over anhy-

drous Na₂SO₄and concentrated to obtain the crude compound,

which was puriﬁed by ﬂash chromatography (dichloromethane:

methanol; 10:1).

5.1.2.2. 5-((4-phenyl-2H-1,2,3-triazol-2-yl)methyl)quinolin-8-ol

(3b). Pale yellow solid (Yield: 90 %). ¹H NMR (400 MHz,

Chloroform-d)

d

8.80e8.77 (m, 1H), 8.63 (dd, J ¼ 8.7, 1.5 Hz, 1H),

7.81 (s, 1H), 7.77e7.73 (m, 2H), 7.62 (d, J ¼ 7.8 Hz, 1H), 7.51 (dd,

J ¼ 8.6, 4.2 Hz, 1H), 7.41 (t, J ¼ 7.6 Hz, 2H), 7.36e7.32 (m, 1H), 7.17 (d,

J ¼ 7.8 Hz, 1H), 5.95 (s, 2H).

5.1.2.3. 5-(((2,3-dihydro-1H-inden-2-yl)oxy)methyl)quinolin-8-ol

(3c). White solid (Yield: 70 %). ¹H NMR (400 MHz, Chloroform-d)

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N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

Fig. 8. Anti-metastatic effect of compound 12b in the orthotopic OVCAR8 ovarian cancer mouse model. At the endpoint of the study described in Fig. 7, all the major organs were

harvested from the mice and bioimaged ex vivo to visualize metastases. Images of metastases in (A) liver (Student t-test, **p ¼ 0.0021); (B) kidney (Student t-test, *p ¼ 0.0154); (C)

spleen (Student t-test, ****p < 0.0001); (D) stomach (Student t-test, *p ¼ 0.0408); and (E) intestine of each mouse in the vehicle group (top panel) and 12b treatment group (bottom

panel) are shown on the left and scatter plots with the bar representing the mean SEM of total photon ﬂux for each tissue are shown on the right.

d

8.79 (dd, J ¼ 4.2,1.6 Hz,1H), 8.49 (dd, J ¼ 8.5,1.5 Hz,1H), 7.49e7.42

5.1.3. General procedure for the preparation of compounds 4a-4d

An equimolar mixture of compound 3, paraformaldehyde, and

pyrrolidine in anhydrous ethanol (20 mL) was reﬂuxed for 3 h

under argon. After cooling, the solvent was evaporated under

reduced pressure. The crude compound was puriﬁed by ﬂash

chromatography (dichloromethane: methanol; 20:1).

(m, 2H), 7.21e7.18 (m, 2H), 7.17e7.13 (m, 2H), 7.10 (d, J ¼ 7.7 Hz, 1H),

4.90 (s, 2H), 4.46 (tt, J ¼ 6.5, 4.9 Hz, 1H), 3.18 (d, J ¼ 6.5 Hz, 1H), 3.14

(d, J ¼ 6.5 Hz, 1H), 3.05 (d, J ¼ 5.0 Hz, 1H), 3.01 (d, J ¼ 5.0 Hz, 1H).

5.1.2.4. 5-(((1,2,3,4-tetrahydronaphthalen-2-yl)oxy)methyl)quinolin-

8-ol (3d). Yellow solid (Yield: 66 %). ¹H NMR (400 MHz,

Chloroform-d)

d

9.01 (s, 1H), 8.87 (dd, J ¼ 4.4, 1.5 Hz, 1H), 8.56 (dd,

5.1.3.1. 5-((4-phenyl-1H-imidazole-1-yl)methyl)-7-(pyrrolidin-1-

J ¼ 8.6, 1.5 Hz, 1H), 7.51 (dd, J ¼ 8.5, 4.4 Hz, 1H), 7.46 (d, J ¼ 7.8 Hz,

1H), 7.18 (d, J ¼ 7.8 Hz, 1H), 7.09 (dddd, J ¼ 12.0, 9.2, 5.8, 3.2 Hz, 4H),

4.94 (d, J ¼ 2.0 Hz, 2H), 3.90 (dddd, J ¼ 8.9, 7.7, 4.9, 3.0 Hz, 1H), 3.71

(d, J ¼ 10.9 Hz, 2H), 2.87 (ddt, J ¼ 31.5, 15.3, 7.7 Hz, 2H), 1.88 (dtd,

J ¼ 12.7, 8.9, 5.7 Hz, 1H), 1.42e1.22 (m, 1H).

ylmethyl)quinolin-8-ol (4a). Yellow solid (Yield: 81 %). Mp:

123e125 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

1.6 Hz, 1H), 8.12 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.74e7.70 (m, 2H), 7.57 (d,

J ¼ 1.4 Hz, 1H), 7.42 (dd, J ¼ 8.6, 4.2 Hz, 1H), 7.34 (t, J ¼ 7.7 Hz, 2H),

7.25e7.19 (m, 1H), 7.15e7.12 (m, 2H), 5.47 (s, 2H), 4.00 (s, 2H), 2.72

d

8.92 (dd, J ¼ 4.1,

11

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European Journal of Medicinal Chemistry 224 (2021) 113719

(q, J ¼ 5.2, 4.1 Hz, 4H), 1.89 (p, J ¼ 3.1 Hz, 4H). ¹³C NMR (101 MHz,

(s, 1H), 7.76 (d, J ¼ 7.9 Hz, 2H), 7.32 (d, J ¼ 7.8 Hz, 2H), 2.95 (h,

J ¼ 6.9 Hz, 1H), 1.28 (dd, J ¼ 6.9, 1.3 Hz, 6H).

Chloroform-d)

d 154.29, 149.04, 142.46, 139.67, 137.31, 133.97,

130.84, 128.60, 128.48, 126.86, 126.22, 124.70, 121.91, 120.06, 118.19,

114.87, 57.55, 53.77, 48.24, 23.66. HRMS: calculated for C₂₄H₂₄N₄O

[2]^þ385.2023, found 385.2029 (mass error ¼ 1.6 ppm).

5.1.4.4. 4-(p-tolyl)-2H-1,2,3-triazole (5d). Pale yellow solid (Yield:

90 %). ¹H NMR (400 MHz, Chloroform-d)

d

7.95 (s, 1H), 7.75e7.68

(m, 2H), 7.28 (d, J ¼ 0.9 Hz, 2H), 2.40 (s, 3H).

5.1.3.2. 5-((4-phenyl-2H-1,2,3-triazol-2-yl)methyl)-7-(pyrrolidin-1-

ylmethyl)quinolin-8-ol (4b). Pale orange solid (Yield: 88 %). Mp:

5.1.4.5. 4-(4-ethylphenyl)-2H-1,2,3-triazole (5e). White solid (Yield:

93 %) ¹H NMR (400 MHz, Chloroform-d)

7.96 (s, 1H), 7.76e7.72 (m,

102e103 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d

8.87 (dd, J ¼ 4.2,

d

1.6 Hz, 1H), 8.57 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.81 (s, 1H), 7.77e7.73 (m,

2H), 7.50 (s,1H), 7.46 (dd, J ¼ 8.6, 4.2 Hz,1H), 7.43e7.40 (m,1H), 7.39

(d, J ¼ 1.6 Hz, 1H), 7.37e7.31 (m, 1H), 5.93 (s, 2H), 4.11 (s, 2H), 2.84

(br s, 4H), 1.95e1.90 (m, 4H). ¹³C NMR (101 MHz, Chloroform-d)

2H), 7.31e7.28 (m, 2H), 2.70 (q, J ¼ 7.6 Hz, 2H), 1.27 (t, J ¼ 7.6 Hz,

3H).

5.1.4.6. 4-(4-nitrophenyl)-2H-1,2,3-triazole (5f). Pale brown solid

(Yield: 88 %) ¹H NMR (400 MHz, Chloroform-d)

d

8.37e8.29 (m,

d

148.71, 147.80, 139.42, 132.19, 131.27, 130.24, 130.14, 128.85,

128.47, 126.99, 125.90, 121.88, 120.45, 56.80, 56.12, 53.61, 23.58.

HRMS: calculated for C₂₃H₂₃N₅O [9]^þ386.1975, found 386.1976

(mass error ¼ 0.3 ppm).

2H), 8.09 (s, 1H), 8.03 (d, J ¼ 1.8 Hz, 2H).

5.1.4.7. 4-(4-methoxyphenyl)-2H-1,2,3-triazole (5g). White solid

(Yield: 93 %) ¹H NMR (400 MHz, CDCl₃)

d 7.92 (s, 1H), 7.78e7.73 (m,

5.1.3.3. 5-(((2,3-dihydro-1H-inden-2-yl)oxy)methyl)-7-(pyrrolidin-

2H), 7.01e6.96 (m, 2H), 3.86 (s, 3H).

1-ylmethyl)quinolin-8-ol (4c). Red orange semisolid (Yield: 86 %).

¹H NMR (400 MHz, Chloroform-d)

d 8.87e8.82 (m, 1H), 8.46 (d,

5.1.4.8. 4-(2H-1,2,3-triazol-4-yl)benzonitrile (5h). Pale orange solid

J ¼ 8.5 Hz, 1H), 7.59 (s, 1H), 7.46 (dd, J ¼ 8.4, 4.2 Hz, 1H), 7.23e7.11

(m, 4H), 4.89 (s, 2H), 4.53e4.46 (m, 1H), 4.22 (s, 2H), 3.60 (d,

J ¼ 10.7 Hz, 1H), 3.22 (d, J ¼ 6.5 Hz, 1H), 3.18 (d, J ¼ 6.5 Hz, 1H), 3.07

(d, J ¼ 4.8 Hz, 1H), 3.04e2.98 (m, 4H), 2.01e1.95 (m, 4H). ¹³C NMR

(Yield: 73 %) ¹H NMR (400 MHz, CDCl₃)

d 11.88 (s, 1H), 8.05 (s, 1H),

7.98e7.94 (m, 2H), 7.77e7.73 (m, 2H).

5.1.4.9. 4-(4-(benzyloxy)phenyl)-2H-1,2,3-triazole (5i). White solid

(Yield: 91 %) ¹H NMR (400 MHz, DMSO‑d₆)

8.18 (s, 1H), 7.78 (d,

(101 MHz, Chloroform-d)

d 152.73, 148.47, 140.76, 138.99, 133.43,

d

129.73, 127.67, 126.56, 124.70, 124.41, 122.06, 79.78, 69.12, 53.23,

44.80, 39.30, 24.46, 23.38. HRMS: calculated for C₂₄H₂₆N₂O₂[9]^þ

375.2067, found 375.2065 (mass error ¼ ꢁ0.5 ppm).

J ¼ 8.3 Hz, 2H), 7.49e7.45 (m, 2H), 7.43e7.38 (m, 2H), 7.36e7.31 (m,

1H), 7.10 (d, J ¼ 8.4 Hz, 2H), 5.15 (s, 2H).

5.1.4.10. 4-(2-bromophenyl)-2H-1,2,3-triazole (5j). White solid

5 . 1. 3 . 4 . 7 - ( p y r r o l i d i n - 1 - y l m e t h y l ) - 5 - ( ( ( 1, 2 , 3 , 4 -

tetrahydronaphthalen-2-yl)oxy)methyl)quinolin-8-ol (4d). Pale or-

ange solid (Yield: 72 %). Mp: 72e73 ^ꢀC. ¹H NMR (400 MHz,

(Yield: 95 %) ¹H NMR (400 MHz, DMSO‑d₆)

d 8.32 (s, 1H), 7.76 (dd,

J ¼ 8.0, 1.2 Hz, 3H), 7.49 (td, J ¼ 7.5, 1.3 Hz, 2H), 7.34 (td, J ¼ 7.7,

1.8 Hz, 2H).

Chloroform-d)

d

9.88 (s, 1H), 8.86 (d, J ¼ 4.2 Hz, 1H), 8.38 (d,

J ¼ 8.5 Hz, 1H), 7.38 (dd, J ¼ 8.7, 4.1 Hz, 1H), 7.30 (s, 1H), 7.09 (th,

J ¼ 7.3, 3.6 Hz, 4H), 4.91 (s, 2H), 4.01 (s, 2H), 3.96e3.87 (m, 1H), 3.11

(dd, J ¼ 16.3, 4.9 Hz,1H), 3.00e2.78 (m, 3H), 2.77e2.72 (m, 4H), 2.13

(dt, J ¼ 13.0, 4.8 Hz, 1H), 1.89 (q, J ¼ 3.1 Hz, 4H), 1.87e1.81 (m, 1H).

5.1.4.11. 4-(3-bromophenyl)-2H-1,2,3-triazole (5k). White solid

(Yield: 94 %) ¹H NMR (400 MHz, Chloroform-d)

d

8.00 (t, J ¼ 1.8 Hz,

1H), 7.98 (s, 1H), 7.78e7.75 (m, 1H), 7.52 (ddd, J ¼ 8.0, 2.0, 1.0 Hz,

¹³C NMR (101 MHz, Chloroform-d)

d 153.34, 148.57, 139.60, 136.02,

1H), 7.33 (t, J ¼ 7.9 Hz, 1H).

134.46, 132.87, 129.42, 128.56, 127.25, 125.91, 125.79, 123.83, 121.32,

117.11, 73.76, 68.32, 57.10, 53.66, 35.48, 28.43, 27.24, 23.59. HRMS:

calculated for C₂₅H₂₈N₂O₂[MþH]^þ389.2224, found 389.2226

(mass error ¼ 0.5 ppm).

5.1.4.12. 4-(thiophen-3-yl)-1H-1,2,3-triazole (8). White solid (Yield:

95 %) ¹H NMR (400 MHz, Chloroform-d)

d 7.88 (s, 1H), 7.69 (dd,

J ¼ 2.9, 1.3 Hz, 1H), 7.50 (dd, J ¼ 5.1, 1.3 Hz, 1H), 7.43 (dd, J ¼ 5.0,

2.9 Hz, 1H).

5.1.4. General procedure for the preparation of compounds 5a-5k

and 8

5.1.5. General procedure for the preparation of compounds 6a-6k

and 9

The terminal alkyne and azidotrimethylsilane (1.5 equivalent)

were added to a solution of DMF and MeOH (5:1) under argon at-

mosphere. After adding CuI (5 mol%), the reaction mixture was

reﬂuxed for 12 h and quenched with water (10 mL) [13,19]. The

mixture was extracted with dichloromethane (30 mL.3 times),

dried with anhydrous Na₂SO₄and concentrated to obtain the crude

compounds, which were puriﬁed by ﬂash chromatography (hex-

ane: ethyl acetate; 2:1).

These compounds were derived from compounds 5a-5k and 8,

respectively, and were synthesized individually in parallel. To a

solution of compound 5(a-k) or 8 (5 mmol) in DMF (12 mL) was

added potassium carbonate (15 mmol). The suspension was stirred

at RT for 30 min. Salt of compound 2 (5 mmol) was added to the

suspension and the mixture was stirred overnight at RT. Water was

added to the suspension and the mixture was extracted 3 times

with ethyl acetate (30 mL). The combined organic phase was

washed with brine, dried over anhydrous Na₂SO₄and concentrated

to obtain the crude compounds. The crude compounds were puri-

ﬁed by ﬂash chromatography (dichloromethane: ethyl acetate;

20:1).

5.1.4.1. 4-(4-bromophenyl)-2H-1,2,3-triazole

(5a). White

solid

(Yield: 96 %). ¹H NMR (400 MHz, CDCl₃)

d

11.78 (s, 1H), 7.97 (s, 1H),

7.71 (d, J ¼ 8.2 Hz, 2H), 7.59 (d, J ¼ 8.1 Hz, 2H).

5.1.4.2. 4-(4-(triﬂuoromethyl)phenyl)-2H-1,2,3-triazole

(5b).

11.80 (s, 1H),

White solid (Yield: 93 %). ¹H NMR (400 MHz, CDCl₃)

d

5.1.5.1. 5-((4-(4-bromophenyl)-2H-1,2,3-triazol-2-yl)methyl)quino-

8.04 (s, 1H), 7.96 (d, J ¼ 8.0 Hz, 2H), 7.72 (d, J ¼ 8.0 Hz, 2H).

lin-8-ol (6a). White solid (Yield: 90 %). ¹H NMR (400 MHz,

Chloroform-d)

d

8.80 (d, J ¼ 4.2 Hz, 1H), 8.63 (d, J ¼ 8.6 Hz, 1H), 8.40

5.1.4.3. 4-(4-isopropylphenyl)-2H-1,2,3-triazole (5c). White solid

(s, 1H), 7.79 (s, 1H), 7.62 (d, J ¼ 8.2 Hz, 3H), 7.56e7.48 (m, 3H), 7.17

(Yield: 99 %). ¹H NMR (400 MHz, Chloroform-d)

d 13.41 (s, 1H), 8.00

(d, J ¼ 7.8 Hz, 1H), 5.94 (s, 2H).

12

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

5.1.5.2. 5-((4-(4-(triﬂuoromethyl)phenyl)-2H-1,2,3-triazol-2-yl)

methyl)quinolin-8-ol (6b). Pale yellow solid (Yield: 90 %). ¹H NMR

1.2 Hz, 1H), 7.63 (d, J ¼ 7.9 Hz, 1H), 7.53 (dd, J ¼ 8.6, 4.2 Hz, 1H), 7.46

(ddd, J ¼ 8.0, 2.0, 1.0 Hz, 1H), 7.28 (d, J ¼ 7.9 Hz, 1H), 7.18 (d,

J ¼ 7.8 Hz, 1H), 5.95 (s, 2H).

(400 MHz, CDCl₃)

7.86 (d, J ¼ 6.3 Hz, 3H), 7.65 (t, J ¼ 7.9 Hz, 3H), 7.53 (dd, J ¼ 8.7,

d

8.80 (d, J ¼ 4.2 Hz, 1H), 8.64 (d, J ¼ 8.6 Hz, 1H),

4.2 Hz, 1H), 7.18 (d, J ¼ 7.8 Hz, 1H), 5.97 (s, 2H).

5.1.5.12. 5-((4-(thiophen-3-yl)-2H-1,2,3-triazol-2-yl)methyl)quino-

lin-8-ol (9). Pale pink solid (Yield: 78 %). ¹H NMR (400 MHz,

5.1.5.3. 5-((4-(4-isopropylphenyl)-2H-1,2,3-triazol-2-yl)methyl)qui-

Chloroform-d)

d

8.79 (dd, J ¼ 4.2,1.5 Hz,1H), 8.61 (dd, J ¼ 8.6,1.5 Hz,

nolin-8-ol (6c). White solid (Yield: 86 %). ¹H NMR (400 MHz,

1H), 7.70 (s,1H), 7.62e7.58 (m, 2H), 7.51 (dd, J ¼ 8.6, 4.2 Hz,1H), 7.42

(dd, J ¼ 5.0, 1.3 Hz, 1H), 7.37 (dd, J ¼ 5.1, 2.9 Hz, 1H), 7.17 (d,

J ¼ 7.8 Hz, 1H), 5.93 (s, 2H).

Chloroform-d)

d

8.79 (d, J ¼ 4.2 Hz, 1H), 8.62 (d, J ¼ 8.6 Hz, 1H), 8.39

(s, 1H), 7.77 (d, J ¼ 1.3 Hz, 1H), 7.67 (d, J ¼ 7.8 Hz, 2H), 7.60 (d,

J ¼ 7.7 Hz, 1H), 7.50 (dd, J ¼ 9.0, 4.2 Hz, 1H), 7.28 (s, 2H), 7.17 (d,

J ¼ 7.8 Hz, 1H), 5.94 (s, 2H), 2.92 (p, J ¼ 6.8 Hz, 1H), 1.26 (dd, J ¼ 6.9,

1.2 Hz, 6H).

5.1.6. General procedure for the preparation of compounds 7a-7k

and 10

These compounds were derived from compounds 6a-6k and 9,

respectively, and were synthesized individually in parallel. An

equimolar mixture of the substrates 6(a-k) or 9, paraformaldehyde,

and pyrrolidine in anhydrous ethanol (15 mL) was reﬂuxed for 3 h

under argon. After cooling, the solvent was evaporated under

reduced pressure. The crude compound was puriﬁed by ﬂash

chromatography (dichloromethane: methanol 20: 1) to produce

the pure products.

5.1.5.4. 5-((4-(p-tolyl)-2H-1,2,3-triazol-2-yl)methyl)quinolin-8-ol

(6d). White solid (Yield: 66 %) ¹H NMR (400 MHz, Chloroform-d)

d

8.78 (dd, J ¼ 4.2, 1.5 Hz, 1H), 8.63 (dd, J ¼ 8.6, 1.5 Hz, 1H), 8.42

(s, 1H), 7.77 (s, 1H), 7.67e7.57 (m, 3H), 7.50 (dd, J ¼ 8.6, 4.2 Hz, 1H),

7.23e7.15 (m, 3H), 5.94 (s, 2H), 2.37 (s, 3H).

5.1.5.5. 5-((4-(4-ethylphenyl)-2H-1,2,3-triazol-2-yl)methyl)quinolin-

8-ol (6e). Pale brown solid (Yield: 73 %) ¹H NMR (400 MHz, CDCl₃)

d

8.79 (dd, J ¼ 4.2, 1.5 Hz, 1H), 8.63 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.78 (s,

5.1.6.1. 5-((4-(4-bromophenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-(pyr-

1H), 7.68e7.65 (m, 2H), 7.61 (d, J ¼ 7.8 Hz, 1H), 7.50 (dd, J ¼ 8.6,

4.2 Hz, 1H), 7.24 (d, J ¼ 8.1 Hz, 2H), 7.17 (d, J ¼ 7.8 Hz, 1H), 5.94 (s,

2H), 2.67 (q, J ¼ 7.6 Hz, 2H), 1.24 (t, J ¼ 7.6 Hz, 3H).

rolidin-1-ylmethyl)quinolin-8-ol (7a). Pink solid (Yield: 87 %). Mp:

146e148 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d

8.87 (d, J ¼ 4.1 Hz,

1H), 8.54 (d, J ¼ 8.5 Hz, 1H), 7.79 (s, 1H), 7.61 (d, J ¼ 8.2 Hz, 2H), 7.51

(d, J ¼ 8.2 Hz, 2H), 7.42 (dd, J ¼ 8.6, 4.1 Hz, 1H), 7.37 (s, 1H), 5.90 (s,

2H), 4.01 (s, 2H), 2.70 (d, J ¼ 5.9 Hz, 4H), 1.87 (q, J ¼ 3.9, 3.4 Hz, 4H).

5.1.5.6. 5-((4-(4-nitrophenyl)-2H-1,2,3-triazol-2-yl)methyl)quinolin-

8-ol (6f). Pale orange solid (Yield: 59 %). ¹H NMR (400 MHz, CDCl₃)

¹³C NMR (101 MHz, Chloroform-d)

d 154.43, 148.78, 146.77, 139.63,

d

8.81 (dd, J ¼ 4.2, 1.5 Hz, 1H), 8.64 (dd, J ¼ 8.6, 1.5 Hz, 1H),

131.98, 131.19, 129.77, 129.24,127.43, 126.80, 122.37, 121.63, 119.99,

118.17, 57.65, 56.18, 53.76, 23.65. HRMS: calculated for C₂₃H₂₂BrN₅O

[MþH]^þ464.1080, found 464.1068 (mass error ¼ ꢁ2.6 ppm).

8.29e8.24 (m, 2H), 7.94e7.89 (m, 3H), 7.65 (d, J ¼ 7.8 Hz, 1H), 7.54

(dd, J ¼ 8.6, 4.2 Hz, 1H), 7.19 (d, J ¼ 7.8 Hz, 1H), 5.98 (s, 2H).

5.1.5.7. 5-((4-(4-methoxyphenyl)-2H-1,2,3-triazol-2-yl)methyl)qui-

5.1.6.2. 7-(pyrrolidin-1-ylmethyl)-5-((4-(4-(triﬂuoromethyl)phenyl)-

2H-1,2,3-triazol-2-yl)methyl)quinolin-8-ol (7b). Pale brown solid

(Yield: 66 %). Mp: 144e145 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

nolin-8-ol (6g). White solid (Yield: 76 %). ¹H NMR (400 MHz,

Chloroform-d)

d

8.79 (dd, J ¼ 4.2,1.5 Hz,1H), 8.63 (dd, J ¼ 8.6,1.5 Hz,

1H), 7.73 (s, 1H), 7.69e7.65 (m, 2H), 7.61 (d, J ¼ 7.8 Hz, 1H), 7.51 (dd,

J ¼ 8.6, 4.2 Hz,1H), 7.17 (d, J ¼ 7.8 Hz,1H), 6.96e6.91 (m, 2H), 5.93 (s,

2H), 3.83 (s, 3H).

d

8.88 (d, J ¼ 4.1 Hz, 1H), 8.56 (d, J ¼ 8.6 Hz, 1H), 7.86 (d,

J ¼ 7.6 Hz, 3H), 7.65 (d, J ¼ 8.0 Hz, 2H), 7.47e7.38 (m, 2H), 5.93 (s,

2H), 4.03 (s, 2H), 2.73 (d, J ¼ 5.8 Hz, 4H), 1.89 (d, J ¼ 4.9 Hz, 4H). ¹³

C

NMR (101 MHz, Chloroform-d)

d 154.46, 148.78, 146.40, 139.61,

5.1.5.8. 4-(2-((8-hydroxyquinolin-5-yl)methyl)-2H-1,2,3-triazol-4-

133.71, 131.92, 131.62, 130.20 (q, J ¼ 33.3 Hz), 129.84, 126.80, 126.04,

125.81 (q, J ¼ 3.0 Hz), 124.03 (q, J ¼ 273.7 Hz), 121.64, 119.86, 118.12,

57.59, 56.27, 53.73, 23.63. ¹⁹F NMR (376 MHz, Chloroform-d)

yl)benzonitrile (6h). Pale brown solid (Yield: 77 %). ¹H NMR

(400 MHz, CDCl₃)

d

8.81 (dd, J ¼ 4.2, 1.5 Hz, 1H), 8.63 (dd, J ¼ 8.6,

1.5 Hz, 1H), 7.88e7.83 (m, 3H), 7.71e7.67 (m, 2H), 7.64 (d, J ¼ 7.8 Hz,

1H), 7.53 (dd, J ¼ 8.6, 4.2 Hz, 1H), 7.18 (d, J ¼ 7.8 Hz, 1H), 5.97 (s, 2H).

d

ꢁ62.64. HRMS: calculated for C₂₄H₂₂F₃N₅O [MþH]^þ454.1849,

found 454.1839 (mass error ¼ ꢁ2.2 ppm).

5.1.5.9. 5-((4-(4-(benzyloxy)phenyl)-2H-1,2,3-triazol-2-yl)methyl)

5.1.6.3. 5-((4-(4-isopropylphenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-

quinolin-8-ol (6i). White solid (Yield: 86 %). ¹H NMR (400 MHz,

(pyrrolidin-1-ylmethyl)quinolin-8-ol (7c). Pale yellow solid (Yield:

Chloroform-d)

d

8.79 (dd, J ¼ 4.2,1.5 Hz,1H), 8.63 (dd, J ¼ 8.6,1.5 Hz,

78 %). Mp: 106e107 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 8.87 (d,

1H), 7.73 (s, 1H), 7.69e7.65 (m, 2H), 7.61 (d, J ¼ 7.8 Hz, 1H), 7.51 (dd,

J ¼ 8.6, 4.2 Hz,1H), 7.44 (d, J ¼ 7.4 Hz, 2H), 7.41e7.36 (m, 2H), 7.33 (t,

J ¼ 7.1 Hz, 1H), 7.17 (d, J ¼ 7.8 Hz, 1H), 7.03e6.98 (m, 2H), 5.93 (s,

2H), 5.09 (s, 2H).

J ¼ 3.9 Hz, 1H), 8.55 (d, J ¼ 8.6 Hz, 1H), 7.78 (d, J ¼ 1.9 Hz, 1H),

7.71e7.64 (m, 2H), 7.44 (s, 2H), 7.27 (d, J ¼ 7.4 Hz, 2H), 5.92 (s, 2H),

4.08 (s, 2H), 2.92 (p, J ¼ 6.9 Hz, 1H), 2.81 (br s, 4H), 1.91 (br s, 4H),

1.26 (dd, J ¼ 7.0, 1.9 Hz, 6H).¹³C NMR (101 MHz, Chloroform-d)

d

149.39, 147.85, 132.22, 131.14, 127.82, 126.93, 125.92, 56.07,

5.1.5.10. 5-((4-(2-bromophenyl)-2H-1,2,3-triazol-2-yl)methyl)quino-

53.58, 33.96, 23.93, 23.56. HRMS: calculated for C₂₆H₂₉N₅O [MþH]^þ

428.2445, found 428.2438 (mass error ¼ ꢁ1.6 ppm).

lin-8-ol (6j). White solid (Yield: 54 %). ¹H NMR (400 MHz,

Chloroform-d)

d

8.80 (dd, J ¼ 4.2,1.5 Hz,1H), 8.66 (dd, J ¼ 8.6,1.5 Hz,

1H), 8.07 (s, 1H), 7.69e7.62 (m, 3H), 7.54e7.50 (m, 1H), 7.35 (td,

J ¼ 7.6, 1.3 Hz, 1H), 7.22 (dd, J ¼ 7.6, 1.8 Hz, 1H), 7.18 (d, J ¼ 7.8 Hz,

1H), 5.97 (s, 2H).

5.1.6.4. 7-(pyrrolidin-1-ylmethyl)-5-((4-(p-tolyl)-2H-1,2,3-triazol-2-

yl)methyl)quinolin-8-ol (7d). Pale orange solid (Yield: 74 %). Mp:

102e103 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 10.31 (s, 1H), 8.86

(dd, J ¼ 4.3, 1.6 Hz, 1H), 8.54 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.78 (s, 1H),

7.66e7.61 (m, 2H), 7.39 (dd, J ¼ 8.6, 4.1 Hz, 1H), 7.35 (s, 1H), 7.19 (d,

J ¼ 7.9 Hz, 2H), 5.89 (s, 2H), 3.98 (s, 2H), 2.68 (dq, J ¼ 7.6, 4.2, 3.7 Hz,

4H), 2.34 (s, 3H), 1.89e1.78 (m, 4H). ¹³C NMR (101 MHz,

5.1.5.11. 5-((4-(3-bromophenyl)-2H-1,2,3-triazol-2-yl)methyl)quino-

lin-8-ol (6k). White solid (Yield: 62 %). ¹H NMR (400 MHz,

Chloroform-d)

d

8.80 (dd, J ¼ 4.2,1.5 Hz,1H), 8.62 (dd, J ¼ 8.6,1.5 Hz,

1H), 8.43 (s, 1H), 7.91 (t, J ¼ 1.8 Hz, 1H), 7.80 (s, 1H), 7.66 (dt, J ¼ 7.7,

Chloroform-d)

d 154.26, 148.69, 147.81, 139.59, 138.30, 132.04,

13

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

131.03, 129.61, 129.51, 127.42, 126.78, 125.78, 121.55, 120.26, 118.17,

57.60, 56.01, 53.71, 23.62, 21.30. HRMS: calculated for C₂₄H₂₅N₅O

[MþH]^þ400.2132, found 400.2141 (mass error ¼ 2.2 ppm).

Mp: 57e59 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 8.91e8.85 (m,

1H), 8.57 (dd, J ¼ 8.7, 1.6 Hz, 1H), 8.07 (s, 1H), 7.65 (ddd, J ¼ 16.9, 7.9,

1.5 Hz, 2H), 7.43 (dd, J ¼ 8.6, 4.1 Hz, 1H), 7.38 (s, 1H), 7.33 (td, J ¼ 7.5,

1.3 Hz, 1H), 7.18 (td, J ¼ 7.7, 1.8 Hz, 1H), 5.94 (s, 2H), 4.01 (s, 2H),

2.75e2.67 (m, 4H), 1.94e1.79 (m, 4H). ¹³C NMR (101 MHz,

5.1.6.5. 5-((4-(4-ethylphenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-(pyr-

rolidin-1-ylmethyl)quinolin-8-ol (7e). Pale orange solid (Yield:

Chloroform-d) d 154.42, 148.78, 146.29, 139.64, 134.27, 133.65,

82 %). Mp: 90e92 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d

8.86 (dd,

132.07, 131.23, 130.95, 129.76, 129.72, 127.54, 126.83, 121.86, 121.63,

120.06, 118.21, 57.68, 56.15, 53.76, 23.65. HRMS: calculated for

J ¼ 4.2, 1.6 Hz, 1H), 8.54 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.78 (s, 1H),

7.71e7.63 (m, 2H), 7.41 (dd, J ¼ 8.6, 4.1 Hz, 1H), 7.38 (s, 1H),

7.26e7.20 (m, 2H), 5.90 (s, 2H), 4.01 (s, 2H), 2.75e2.69 (m, 4H), 2.65

(q, J ¼ 7.6 Hz, 2H),1.93e1.82 (m, 4H),1.23 (t, J ¼ 7.6 Hz, 3H). ¹³C NMR

C

₂₃H₂₂BrN₅O [MþH]^þ464.1080, found 464.1082 (mass

error ¼ 0.4 ppm).

(101 MHz, Chloroform-d)

d

154.22, 148.70, 147.85, 144.73, 139.56,

5.1.6.11. 5-((4-(3-bromophenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-

132.07, 131.09, 129.71, 128.35, 127.67, 126.83, 125.90, 121.63, 120.32,

117.93, 57.43, 56.05, 53.69, 28.68, 23.62, 15.57. HRMS: calculated for

(pyrrolidin-1-ylmethyl)quinolin-8-ol (7k). Solid (Yield: 64 %). Mp:

70e72 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 8.91e8.85 (m, 1H),

C

₂₅H₂₇N₅O

[MþH]^þ

414.2288,

found

414.2285

(mass

8.54 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.92 (t, J ¼ 1.8 Hz, 1H), 7.80 (s, 1H), 7.65

(dt, J ¼ 7.7, 1.4 Hz, 1H), 7.44 (dt, J ¼ 8.7, 3.0 Hz, 2H), 7.39 (s, 1H), 7.25

(t, J ¼ 7.9 Hz, 1H), 5.91 (s, 2H), 4.03 (s, 2H), 2.72 (q, J ¼ 4.6, 3.4 Hz,

error ¼ ꢁ0.7 ppm).

5.1.6.6. 5-((4-(4-nitrophenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-(pyr-

4H), 1.95e1.82 (m, 4H). ¹³C NMR (101 MHz, Chloroform-d)

d 154.42,

rolidin-1-ylmethyl)quinolin-8-ol (7f). Orange solid (Yield: 57 %).

148.78, 146.39, 139.61, 132.31, 131.96, 131.38, 131.33, 130.39, 129.83,

128.84, 126.81, 124.43, 122.96, 121.69, 119.97, 118.10, 57.58, 56.22,

53.75, 23.65. HRMS: calculated for C₂₃H₂₂BrN₅O [MþH]^þ464.1080,

found 464.1083 (mass error ¼ 0.6 ppm).

Mp: 168e170 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 8.89 (dd,

J ¼ 4.2, 1.6 Hz, 1H), 8.56 (dd, J ¼ 8.6, 1.6 Hz, 1H), 8.31e8.23 (m, 2H),

7.91 (dd, J ¼ 6.9, 2.0 Hz, 3H), 7.49e7.42 (m, 2H), 5.95 (s, 2H), 4.04 (s,

2H), 2.73 (td, J ¼ 5.3, 4.3, 2.3 Hz, 4H), 1.95e1.83 (m, 4H). ¹³C NMR

(101 MHz, Chloroform-d)

d

154.56, 148.82, 147.45, 145.64, 139.61,

5.1.6.12. 7-(pyrrolidin-1-ylmethyl)-5-((4-(thiophen-3-yl)-2H-1,2,3-

136.52, 132.09, 131.88, 130.00, 126.78, 126.40, 124.26, 121.70, 119.60,

118.18, 57.60, 56.42, 53.76, 23.65. HRMS: calculated for C₂₃H₂₂N₆O₃

[MþH]þ 431.1826, found 431.1831 (mass error ¼ 1.2 ppm).

triazol-2-yl)methyl)quinolin-8-ol (10). Orange solid (Yield: 69 %).

Mp: 57e59 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 9.14 (s, 1H), 8.84

(d, J ¼ 4.2 Hz, 1H), 8.53 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.70 (s, 1H), 7.58 (dd,

J ¼ 3.0, 1.3 Hz, 1H), 7.49 (s, 1H), 7.46e7.37 (m, 2H), 7.35 (dd, J ¼ 5.0,

2.9 Hz, 1H), 5.90 (s, 2H), 4.07 (s, 2H), 2.85e2.77 (m, 4H), 1.90 (h,

5.1.6.7. 5-((4-(4-methoxyphenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-

(pyrrolidin-1-ylmethyl)quinolin-8-ol (7g). Pink solid (Yield: 77 %).

J ¼ 3.0 Hz, 4H). ¹³C NMR (101 MHz, Chloroform-d)

d 154.03, 148.64,

Mp: 142e144 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d

8.87 (dd,

143.96, 139.35, 132.18, 131.51, 131.37, 130.21, 126.95, 126.43, 125.87,

121.85, 121.61, 120.43, 116.93, 56.56, 55.96, 53.55, 23.54. HRMS:

calculated for C₂₁H₂₁N₅OS [MþH]^þ392.1540, found 392.1541 (mass

error ¼ 0.3 ppm).

J ¼ 4.2, 1.6 Hz, 1H), 8.55 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.74 (s, 1H),

7.69e7.65 (m, 2H), 7.41 (dd, J ¼ 8.6, 4.1 Hz, 1H), 7.35 (s, 1H),

6.95e6.90 (m, 2H), 5.89 (s, 2H), 4.00 (s, 2H), 3.81 (s, 3H), 2.70 (tt,

J ¼ 5.0, 2.4 Hz, 4H), 1.89e1.82 (m, 4H). ¹³C NMR (101 MHz,

Chloroform-d)

d

159.78, 154.29, 148.72, 147.64, 139.62, 132.04,

5.1.7. General procedure for the preparation of compounds 12a-12e

and 13

The method used to synthesize compounds 12a-12e and 13 was

the same used to synthesize compounds 7a-7k and 10.

130.70, 129.58, 127.21, 126.80, 122.95, 121.56, 120.28, 118.18, 114.22,

57.69, 56.00, 55.31, 53.74, 23.64. HRMS: calculated for C₂₄H₂₅N₅O₂

[MþH]^þ416.2081, found 416.2077 (mass error ¼ ꢁ1.0 ppm).

5.1.6.8. 4-(2-((8-hydroxy-7-(pyrrolidin-1-ylmethyl)quinolin-5-yl)

5.1.7.1. 5-((4-(4-bromophenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-

methyl)-2H-1,2,3-triazol-4-yl)benzonitrile (7h). White solid (Yield:

(piperidin-1-ylmethyl)quinolin-8-ol (12a). White solid (Yield: 74 %).

68 %). Mp: 164e166 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d

8.88

Mp: 131e132 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 9.24 (s, 1H),

(dd, J ¼ 4.2, 1.6 Hz, 1H), 8.55 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.90e7.81 (m,

3H), 7.72e7.64 (m, 2H), 7.49e7.40 (m, 2H), 5.94 (s, 2H), 4.04 (s, 2H),

2.74 (q, J ¼ 5.5, 4.7 Hz, 4H), 1.95e1.83 (m, 4H).¹³C NMR (101 MHz,

8.92e8.86 (m, 1H), 8.53 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.79 (s, 1H), 7.61 (d,

J ¼ 8.3 Hz, 2H), 7.52 (d, J ¼ 8.2 Hz, 2H), 7.42 (dd, J ¼ 8.6, 4.2 Hz, 1H),

7.32 (s, 1H), 5.90 (s, 2H), 3.87 (s, 2H), 2.60 (br s, 4H), 1.68 (p,

J ¼ 5.6 Hz, 4H), 1.57e1.45 (m, 2H). ¹³C NMR (101 MHz, Chloroform-

Chloroform-d)

d 154.49, 148.79, 145.97, 139.57, 134.64, 132.68,

131.93, 131.85, 130.02, 126.81, 126.29, 121.72, 119.73, 118.70, 118.02,

111.71, 57.46, 56.35, 53.73, 23.63. HRMS: calculated for C₂₄H₂₂N₆O

[MþH]^þ411.1928, found 411.1928 (mass error ¼ 0.0 ppm).

d) d 155.02, 148.91, 146.79, 139.76, 131.99, 131.91, 131.19, 129.86,

129.24, 127.44, 126.89, 122.38, 121.62, 119.99, 117.07, 61.49, 56.18,

54.10, 25.80, 23.91. HRMS: calculated for C₂₄H₂₄BrN₅O [MþH]^þ

478.1237, found 478.1243 (mass error ¼ 1.3 ppm).

5.1.6.9. 5-((4-(4-(benzyloxy)phenyl)-2H-1,2,3-triazol-2-yl)methyl)-

7-(pyrrolidin-1-ylmethyl)quinolin-8-ol (7i). Pale orange (Yield:

5.1.7.2. 7-(piperidin-1-ylmethyl)-5-((4-(4-(triﬂuoromethyl)phenyl)-

2H-1,2,3-triazol-2-yl)methyl)quinolin-8-ol (12b). Pale yellow solid

(Yield: 82 %). Mp: 120e121 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

68 %). Mp: 132e134 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 8.88

(dd, J ¼ 4.1, 1.6 Hz, 1H), 8.55 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.76e7.64 (m,

3H), 7.47e7.29 (m, 7H), 7.06e6.97 (m, 2H), 5.91 (s, 2H), 5.10 (s, 2H),

4.03 (s, 2H), 2.74 (br s, 4H), 1.89 (br s, 4H). ¹³C NMR (101 MHz,

d

11.56 (s, 1H), 8.90 (d, J ¼ 4.1 Hz, 1H), 8.55 (d, J ¼ 8.6 Hz, 1H),

7.85 (d, J ¼ 7.2 Hz, 3H), 7.65 (d, J ¼ 7.9 Hz, 2H), 7.43 (dd, J ¼ 8.6,

4.1 Hz, 1H), 7.33 (s, 1H), 5.92 (s, 2H), 3.87 (s, 2H), 2.60 (br s, 4H), 1.67

(q, J ¼ 5.7 Hz, 4H), 1.51 (br s, 2H). ¹³C NMR (101 MHz, Chloroform-d)

Chloroform-d)

d 158.93, 154.28, 148.74, 147.59, 139.59, 136.68,

132.04, 130.73, 129.62, 128.61, 128.04, 127.47, 127.22, 126.82, 123.19,

121.61, 120.29, 117.97, 115.16, 69.99, 57.59, 56.02, 53.71, 23.62.

d

155.12, 148.94, 146.42, 139.80, 133.74, 131.85, 131.64, 130.21 (q,

HRMS: calculated for

492.2394 (mass error ¼ 0.0 ppm).

C

₃₀H₂₉N₅O₂[MþH]^þ492.2394, found

J ¼ 32.3 Hz), 129.88, 126.88, 126.07, 125.82 (q, J ¼ 3.0 Hz), 124.04 (q,

J ¼ 272.7 Hz), 121.62, 119.85, 117.13, 61.55, 56.28, 54.10, 25.83, 23.92.

¹⁹F NMR (376 MHz, Chloroform-d)

C

error ¼ 0.9 ppm). Conclusive structural characterization was

d

ꢁ62.64. HRMS: calculated for

5.1.6.10. 5-((4-(2-bromophenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-

(pyrrolidin-1-ylmethyl)quinolin-8-ol (7j). Orange solid (Yield: 59 %).

₂₅H₂₄F₃N₅O [MþH]^þ468.2006, found 468.2010 (mass

14

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

obtained from X-ray analysis of single crystals of compound 12b

and summarized in Supplementary Tables S1 and S2, and Fig. S1.

J ¼ 8.2, 4.0 Hz, 4H), 6.07 (s, 2H), 3.75 (s, 2H), 3.05 (t, J ¼ 5.2 Hz, 4H),

2.63 (br s, 4H).¹³C NMR (101 MHz, Chloroform-d)

d 153.53, 148.79,

146.85, 139.35, 132.14, 132.00, 131.22, 130.47, 129.18, 127.41, 126.83,

122.43, 121.89, 120.60, 116.88, 66.86, 59.94, 56.09, 53.20. HRMS:

calculated for C₂₃H₂₃BrN₆O [MþH]^þ479.1189, found 479.1179

(mass error ¼ ꢁ2.1 ppm).

5.1.7.3. 7-(azepan-1-ylmethyl)-5-((4-(4-(triﬂuoromethyl)phenyl)-

2H-1,2,3-triazol-2-yl)methyl)quinolin-8-ol (12c). Pale yellow solid

(Yield: 69 %). Mp: 64e66 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d

8.91 (dd, J ¼ 4.1, 1.6 Hz, 1H), 8.56 (dd, J ¼ 8.6, 1.6 Hz, 1H),

7.90e7.83 (m, 3H), 7.66 (d, J ¼ 8.2 Hz, 2H), 7.44 (dd, J ¼ 8.6, 4.1 Hz,

1H), 7.32 (s,1H), 5.93 (s, 2H), 4.01 (s, 2H), 2.81 (t, J ¼ 5.7 Hz, 4H),1.76

(dd, J ¼ 7.6, 4.0 Hz, 4H), 1.67 (q, J ¼ 3.2 Hz, 4H). ¹³C NMR (101 MHz,

5.1.8. Preparation of 7-bromo-5-(chloromethyl)quinolin-8-ol (15)

A mixture of 3 g (13.38 mmol) of 7-bromo-8-hydroxyquinoline,

20 mL of concentrated hydrochloric acid, and 6 mL of 37 % form-

aldehyde was treated with 0.6 g of zinc chloride and stirred at 60 ^ꢀC

for 12 h. The mixture was ﬁltered, washed with copious acetone,

and dried to produce compound 15 as a white solid (1.67g, Yield:

Chloroform-d)

d 155.65, 148.97, 146.44, 139.90, 133.75, 131.86,

131.66, 130.24 (q, J ¼ 32.3 Hz), 129.71, 127.00, 126.09, 125.84 (q,

J ¼ 4.0 Hz), 124.04 (q, J ¼ 272.7 Hz), 121.65, 119.75, 117.72, 61.90,

56.30, 55.67, 27.65, 26.59. ¹⁹F NMR (376 MHz, Chloroform-d)

44.5 %). ¹H NMR (400 MHz, DMSO‑d₆)

8.63e8.59 (m, 1H), 7.73e7.70 (m, 1H), 7.69 (s, 1H), 4.86 (s, 2H).

d

8.94 (dd, J ¼ 4.3, 1.5 Hz, 1H),

d

ꢁ62.64. HRMS: calculated for C₂₆H₂₆F₃N₅O [MþH]^þ482.2162,

found 482.2159 (mass error ¼ ꢁ0.6 ppm).

5.1.9. Preparation of 7-bromo-5-(((4-isopropylbenzyl)oxy)methyl)

quinolin-8-ol (16)

5.1.7.4. 7-((diethylamino)methyl)-5-((4-(4-(triﬂuoromethyl)phenyl)-

2H-1,2,3-triazol-2-yl)methyl)quinolin-8-ol (12d). Pale orange solid

(Yield: 69 %). Mp: 116e117 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

7-Bromo-5-chloromethyl-8-quinolinol hydrochloride, com-

pound 15 (1.5 g, 5 mmol) was suspended in 4-isopropyl benzyl

alcohol (25 mmol) and the mixture was heated at 90 ^ꢀC for 2 h,

during which time the solution became completely homogeneous.

The solution was poured into 200 mL solution of hexane: ethyl

acetate (1:1) and ﬁltered, then NaHCO₃solution was added to the

ﬁltrate. The mixture was extracted by dichloromethane, washed

with brine, dried over anhydrous Na₂SO₄and evaporated to dryness

to yield the desired compound as white solid (Yield: 76 %). Mp:

d

8.90 (dd, J ¼ 4.2, 1.6 Hz, 1H), 8.55 (dd, J ¼ 8.7, 1.6 Hz, 1H), 7.86

(d, J ¼ 6.5 Hz, 3H), 7.66 (d, J ¼ 8.2 Hz, 2H), 7.43 (dd, J ¼ 8.5, 4.1 Hz,

1H), 7.35 (s,1H), 5.93 (s, 2H), 3.98 (s, 2H), 2.72 (q, J ¼ 7.2 Hz, 4H),1.17

(t, J ¼ 7.2 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d)

d 155.37,

148.91, 146.41, 139.82, 133.72, 131.85, 131.64, 130.21 (q, J ¼ 33.3 Hz),

129.76, 126.84, 126.06, 125.81 (q,

J

¼

4.0 Hz), 124.01 (q,

J ¼ 273.7 Hz), 121.61, 119.75, 117.49, 56.52, 56.28, 46.65, 11.33.¹⁹

F

NMR (376 MHz, Chloroform-d)

d

ꢁ62.64. HRMS: calculated for

167e168 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d

8.80 (dd, J ¼ 4.3,

C

₂₄H₂₄F₃N₅O [MþH]^þ456.2006, found 456.2010 (mass

1.5 Hz,1H), 8.45 (dd, J ¼ 8.5, 1.6 Hz,1H), 7.61 (s, 1H), 7.50 (dd, J ¼ 8.5,

4.2 Hz, 1H), 7.26e7.20 (m, 4H), 4.84 (s, 2H), 4.53 (s, 2H), 2.91 (h,

J ¼ 6.9 Hz, 1H), 1.25 (d, J ¼ 6.9 Hz, 6H). ¹³C NMR (101 MHz,

error ¼ 0.9 ppm).

5.1.7.5. 5-((4-(4-bromophenyl)-2H-1,2,3-triazol-2-yl)methyl)-7-

Chloroform-d) d 149.81, 148.69, 148.53, 138.73, 135.07, 133.84,

(morpholinomethyl)quinolin-8-ol (12e). White solid (Yield: 66 %).

131.88, 128.14, 126.58, 126.37, 125.62, 122.07, 102.84, 72.03, 69.36,

33.89, 24.02. HRMS: calculated for C₂₀H₂₀BrNO₂[MþH]þ 386.0750,

found 386.0754 (mass error ¼ 1.0 ppm).

Mp: 142e144 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d 8.87 (dd,

J ¼ 4.2, 1.6 Hz, 1H), 8.56 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.79 (s, 1H),

7.63e7.58 (m, 2H), 7.51 (d, J ¼ 8.4 Hz, 2H), 7.45 (d, J ¼ 12.6 Hz, 2H),

5.91 (s, 2H), 3.88 (s, 2H), 3.77 (t, J ¼ 4.6 Hz, 4H), 2.63 (d, J ¼ 9.1 Hz,

5.1.10. Preparation of 7-bromo-5-(((4-isopropylbenzyl)oxy)

methyl)-8 ((2(trimethylsilyl)ethoxy)methoxy)quinoline (17)

4H). ¹³C NMR (101 MHz, Chloroform-d)

d

153.53, 148.79, 146.85,

139.35, 132.14, 132.00, 131.22, 130.47, 129.18, 127.41, 126.83, 122.43,

121.89, 120.60, 116.88, 66.86, 59.94, 56.09, 53.20. HRMS: calculated

To a solution of compound 16 in THF was added sodium hydride

(3 equivalents) at 0 ^ꢀC under inert conditions and stirred at RT for

30 min. SEMCl (2 equivalents) was added dropwise to the reaction

mixture and the solution was stirred for 3 h. The reaction was

quenched with water and extracted 3 times with ethyl acetate

(15 mL). The combined organic phase was washed by brine and

water and dried over Na₂SO_4,then concentrated under reduced

pressure to yield a crude product. The crude material was puriﬁed

by column chromatography (hexane: ethyl acetate; 2:1) to obtain

the pure compound as yellow oily liquid (Yield: 88 %). ¹H NMR

for

C

₂₃H₂₂BrN₅O₂[MþH]^þ480.1030, found 480.1039 (mass

error ¼ 1.9 ppm).

5.1.7.6. tert-butyl 4-((5-((4-(4-bromophenyl)-2H-1,2,3-triazol-2-yl)

methyl)-8 hydroxyquinolin-7-yl)methyl)piperazine-1-carboxylate

(13). Brown solid (Yield: 86 %).¹H NMR (400 MHz, Chloroform-d)

d

8.86 (dd, J ¼ 4.1, 1.5 Hz, 1H), 8.56 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.79

(s, 1H), 7.62e7.58 (m, 2H), 7.52e7.43 (m, 4H), 5.91 (s, 2H), 3.88 (s,

2H), 3.51 (t, J ¼ 5.0 Hz, 4H), 2.57 (t, J ¼ 5.1 Hz, 4H), 1.46 (d, J ¼ 1.5 Hz,

9H).

(400 MHz, Chloroform-d)

d

8.87 (dd, J ¼ 4.2, 1.6 Hz, 1H), 8.37 (dd,

J ¼ 8.6, 1.7 Hz, 1H), 7.66 (s, 1H), 7.39 (dd, J ¼ 8.5, 4.1 Hz, 1H),

7.26e7.18 (m, 4H), 5.67 (s, 2H), 4.83 (s, 2H), 4.52 (s, 2H), 4.07e4.01

(m, 2H), 2.88 (p, J ¼ 6.9 Hz, 1H), 1.22 (d, J ¼ 6.9 Hz, 6H), 1.00e0.90

(m, 2H), 0.00 (s, 9H).

5.1.7.7. Preparation of 5-((4-(4-bromophenyl)-2H-1,2,3-triazol-2-yl)

methyl)-7-(piperazin-1-ylmethyl)quinolin-8-ol (14). To a solution of

tert-butyl

4-((5-((4-(4-bromophenyl)-2H-1,2,3-triazol-2-yl)

methyl)-8 hydroxyquinolin-7-yl)methyl)piperazine-1-carboxylate

(compound 13) (100 mg, 0.2 mmol) in 10 mL of CH₂Cl₂was

added TFA (2 mL) at 0 ^ꢀC. The mixture was stirred at RT for 1 h.

Upon completion, the mixture was poured into NaHCO₃solution

and extracted three times with CH₂Cl₂(30 mL). The combined

organic layer was washed with brine, dried over Na₂SO₄, and

evaporated to dryness. The crude product was puriﬁed by column

chromatography (dichloromethane: methanol 20: 1) to yield target

compound 14 as a pale green solid (Yield: 90 %). Mp: 212e213 ^ꢀC. ¹H

5.1.11. General procedure for the preparation of compounds 18a-

18h

To a solution of compound 17 (1 g, 5.4 mmol) and different aryl

boronic acid or aryl boronic acid pinacol ester reagents (0.83 g,

5.4 mmol) in 1,4-dioxane/H₂O (45 mL, v/v ¼ 2/1) was added Pd

(PPh₃)₄and Na₂CO₃(1.1 g, 10.8 mmol). The reaction mixture was

reﬂuxed for 4 h under argon. The solvent was removed, 20 mL of

water was added, and the mixture was extracted with dichloro-

methane (15 mL.3 times).The combined organic layers were dried

over Na₂SO₄, ﬁltered, and concentrated under reduced pressure to

yield a crude product, which was used for the next step without

NMR (400 MHz, DMSO‑d₆)

d

8.87 (dd, J ¼ 4.2, 1.4 Hz, 1H), 8.64 (dd,

J ¼ 8.6, 1.5 Hz, 1H), 8.28 (s, 1H), 7.73 (d, J ¼ 8.3 Hz, 2H), 7.63 (dd,

15

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

puriﬁcation.

5.1.12.5. 5-(((4-isopropylbenzyl)oxy)methyl)-7-(m-tolyl)quinolin-8-

ol (19e). Pale yellow solid (Yield: 56 %). Mp: 72e74 ^ꢀC. ¹H NMR

(400 MHz, Chloroform-d)

d

8.82 (dd, J ¼ 4.3, 1.5 Hz, 1H), 8.49 (dd,

5.1.12. General procedure for the preparation of compounds 19a-

19h

J ¼ 8.4, 1.5 Hz, 1H), 7.62e7.56 (m, 3H), 7.47 (dd, J ¼ 8.5, 4.2 Hz, 1H),

7.39 (t, J ¼ 7.8 Hz, 1H), 7.27 (d, J ¼ 8.1 Hz, 2H), 7.20 (dd, J ¼ 8.4,

6.8 Hz, 3H), 4.92 (s, 2H), 4.54 (s, 2H), 2.90 (h, J ¼ 6.9 Hz, 1H), 2.45 (s,

3H), 1.24 (d, J ¼ 6.9 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d)

To a solution of 18(a-h) (100 mg) in 10 mL of CH₂Cl₂was added

TFA (2 mL) at 0 ^ꢀC. The mixtures were stirred at RT for 1 h. After

completion of the reaction, the mixture was poured into NaHCO₃

solution, and extracted 3 times with dichloromethane (10 mL). The

combined organic layer was washed with brine, dried over Na₂SO₄,

and concentrated to dryness. The crude products were puriﬁed by

column chromatography (hexane/ethyl acetate 2:1) to produce the

target compounds.

d

148.87, 148.55, 147.89, 139.02, 137.97, 137.40, 135.38, 133.55,

130.76, 129.96, 128.32, 128.15, 128.07, 126.53, 126.42, 124.14, 121.72,

121.65, 71.82, 70.19, 33.89, 24.04, 21.65. HRMS: calculated for

C

₂₇H₂₇NO₂

[MþH]^þ

398.2115,

found

398.2113

(mass

error ¼ ꢁ0.5 ppm).

5.1.12.6. 5-(((4-isopropylbenzyl)oxy)methyl)-7-(4-methoxyphenyl)

quinolin-8-ol (19f). Pale yellow solid (Yield: 58 %). Mp: 107e108 ^ꢀC.

5.1.12.1. 5-(((4-isopropylbenzyl)oxy)methyl)-7-phenylquinolin-8-ol

(19a). Yellow solid (Yield: 57 %). Mp: 112e113 ^ꢀC. ¹H NMR

¹H NMR (400 MHz, Chloroform-d)

d

9.43 (s, 1H), 8.92 (dd, J ¼ 4.9,

(400 MHz, Chloroform-d)

d

8.78 (dd, J ¼ 4.3, 1.6 Hz, 1H), 8.45 (dd,

1.5 Hz, 1H), 8.84 (dd, J ¼ 8.5, 1.5 Hz, 1H), 7.70 (d, J ¼ 8.9 Hz, 3H), 7.66

(dd, J ¼ 8.5, 4.8 Hz, 1H), 7.24 (q, J ¼ 8.2 Hz, 4H), 7.05e7.00 (m, 2H),

4.94 (s, 2H), 4.56 (s, 2H), 3.87 (s, 3H), 2.91 (p, J ¼ 6.9 Hz, 1H), 1.24 (d,

J ¼ 8.4, 1.6 Hz, 1H), 7.82e7.78 (m, 2H), 7.58 (s, 1H), 7.48 (t, J ¼ 7.7 Hz,

2H), 7.42 (dd, J ¼ 8.5, 4.2 Hz, 1H), 7.37e7.33 (m, 1H), 7.28e7.17 (m,

4H), 4.88 (s, 2H), 4.53 (s, 2H), 2.89 (h, J ¼ 6.9 Hz, 1H), 1.23 (d,

J ¼ 6.9 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d)

d 159.48, 148.84,

J ¼ 6.9 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d)

d 148.98, 148.59,

146.39, 144.75, 139.25, 135.40, 134.85, 132.49, 130.77, 128.74, 128.18,

127.41, 126.62, 125.71, 120.73, 114.05, 72.21, 69.72, 55.38, 33.87,

24.00. HRMS: calculated for C₂₇H₂₇NO₃[MþH]^þ414.2064, found

414.2060 (mass error ¼ ꢁ1.0 ppm).

147.97, 139.07, 137.55, 135.45, 133.58, 130.65, 129.38, 128.48, 128.21,

127.32, 126.62, 126.59, 124.31, 121.81, 121.55, 71.93, 70.22, 33.95,

24.12. HRMS: calculated for C₂₆H₂₅NO₂[MþH]^þ384.1958, found

384.1944 (mass error ¼ ꢁ3.6 ppm).

5.1.12.7. 5-(((4-isopropylbenzyl)oxy)methyl)-7-(4-(triﬂuoromethyl)

phenyl)quinolin-8-ol (19g). Pale brown solid (Yield: 56 %). Mp:

5.1.12.2. 5-(((4-isopropylbenzyl)oxy)methyl)-7-(isoquinolin-4-yl)

99e100 ^ꢀC. ¹H NMR (400 MHz, Chloroform-d)

d

8.83 (dd, J ¼ 4.3,

quinolin-8-ol (19b). Pale green solid (Yield: 55 %). Mp: 64e65 ^ꢀC. ¹H

1.5 Hz, 1H), 8.48 (dd, J ¼ 8.5, 1.6 Hz, 1H), 7.94e7.90 (m, 2H), 7.73 (d,

J ¼ 8.2 Hz, 2H), 7.57 (s, 1H), 7.50 (dd, J ¼ 8.5, 4.2 Hz, 1H), 7.27 (d,

J ¼ 8.1 Hz, 2H), 7.22 (d, J ¼ 8.2 Hz, 2H), 4.91 (s, 2H), 4.57 (s, 2H), 2.90

(h, J ¼ 6.9 Hz, 1H), 1.24 (d, J ¼ 6.9 Hz, 6H). ¹³C NMR (101 MHz,

NMR (400 MHz, Chloroform-d)

d

9.32 (s, 1H), 8.87 (dd, J ¼ 4.3,

1.5 Hz, 1H), 8.63 (s, 1H), 8.57 (dd, J ¼ 8.5, 1.5 Hz, 1H), 8.09e8.05 (m,

1H), 7.78e7.73 (m, 1H), 7.65 (qd, J ¼ 7.0, 3.4 Hz, 2H), 7.57 (dd, J ¼ 8.5,

4.2 Hz, 1H), 7.52 (s, 1H), 7.28 (d, J ¼ 7.9 Hz, 2H), 7.21 (d, J ¼ 8.0 Hz,

2H), 4.93 (s, 2H), 4.59 (s, 2H), 2.90 (h, J ¼ 6.8 Hz, 1H), 1.23 (d,

Chloroform-d) d 149.31,148.66,148.16,141.11,138.93,135.25,133.61,

J ¼ 7.0 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d)

d 152.39, 150.12,

129.83, 129.56, 129.09 (q, J ¼ 32.3 Hz), 128.15, 126.95, 126.58, 125.31

(q, J ¼ 4.0 Hz), 124.68, 124.33(q, J ¼ 272.7 Hz), 122.23, 119.89, 72.07,

148.65, 148.24, 143.90, 138.64, 135.21, 133.75, 131.57, 130.41, 128.17,

127.95, 127.24, 126.57, 125.44, 124.43, 122.28, 117.25, 72.12, 69.98,

33.88, 24.02. HRMS: calculated for C₂₉H₂₆N₂O₂[MþH]^þ435.2067,

found 435.2064 (mass error ¼ ꢁ0.7 ppm).

70.02, 33.90, 24.04. ¹⁹F NMR (376 MHz, Chloroform-d)

d

ꢁ62.45.

HRMS: calculated for

C

₂₇H₂₄F₃NO₂[MþH]^þ452.1832, found

452.1831 (mass error ¼ ꢁ0.2 ppm).

5.1.12.8. 5-(((4-isopropylbenzyl)oxy)methyl)-7-(thiophen-3-yl)qui-

5.1.12.3. 5'-(((4-isopropylbenzyl)oxy)methyl)-[4,7⁰-biquinolin]-8⁰-ol

nolin-8-ol (19h). Pale yellow solid (Yield: 55 %). Mp: 126e127 ^ꢀC. ¹H

(19c). Pale orange solid (Yield: 53 %). Mp: 59e61 ^ꢀC ¹H NMR

NMR (400 MHz, CDCl₃)

d

8.78 (dd, J ¼ 4.2, 1.5 Hz, 1H), 8.43 (dd,

(400 MHz, Chloroform-d)

d

9.02 (d, J ¼ 4.5 Hz, 1H), 8.86 (dd, J ¼ 4.2,

J ¼ 8.5, 1.6 Hz, 1H), 7.96 (dd, J ¼ 3.0, 1.3 Hz, 1H), 7.73e7.70 (m, 2H),

7.45e7.39 (m, 2H), 7.26 (d, J ¼ 8.2 Hz, 2H), 7.21 (d, J ¼ 8.2 Hz, 2H),

4.89 (s, 2H), 4.53 (s, 2H), 2.90 (h, J ¼ 6.9 Hz, 1H), 1.24 (d, J ¼ 6.9 Hz,

1.5 Hz, 1H), 8.55 (dd, J ¼ 8.5,1.6 Hz, 1H), 8.24e8.20 (m, 1H), 7.79 (dd,

J ¼ 8.4,1.4 Hz,1H), 7.73 (ddd, J ¼ 8.4, 6.8,1.4 Hz,1H), 7.56 (dd, J ¼ 8.5,

4.2 Hz, 1H), 7.53e7.50 (m, 1H), 7.49e7.46 (m, 2H), 7.28 (d, J ¼ 8.2 Hz,

2H), 7.22e7.18 (m, 2H), 4.91 (s, 2H), 4.59 (s, 2H), 2.89 (h, J ¼ 6.9 Hz,

1H), 1.23 (d, J ¼ 6.9 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d)

6H). ¹³C NMR (101 MHz, Chloroform-d)

d 148.52, 148.50, 147.86,

139.18, 137.44, 135.31, 133.42, 129.17, 128.12, 127.68, 126.50, 126.21,

125.03, 124.11, 123.70, 121.55, 116.24, 71.78, 70.09, 33.85, 24.01.

d

149.80, 149.68, 148.69, 148.39, 148.34, 144.75, 138.66, 135.17,

HRMS: calculated for

390.1528 (mass error ¼ 1.5 ppm).

C

₂₄H₂₃NO₂S [MþH]^þ390.1522, found

133.77, 130.67, 129.67, 129.52, 128.18, 127.40, 126.91, 126.61, 126.59,

126.38, 124.53, 122.52, 118.08, 72.21, 69.91, 33.89, 24.04. HRMS:

calculated for C₂₉H₂₆N₂O₂[MþH]þ 435.2067, found 435.2084

(mass error ¼ 3.9 ppm).

5.2. Cell culture and reagents

Two human melanoma (A375 and RPMI7951) and two human

breast (SKBR3 and MDA-MB-453) cell lines were purchased from

ATCC. The P-gp overexpressed MDA-MB-435/LCC6MDR1 cell line

and its parental MDA-MB-435 melanoma cell line were gifts from

Dr. Robert Clarke at Georgetown University [20]_.All these cell lines

were grown in DMEM (Mediatech, Inc) supplemented with 10 %

FBS (Atlanta Biologicals) and 1 % antibiotic-antimycotic solution

(Sigma-Aldrich) at 37 ^ꢀC in a humidiﬁed incubator containing 5 %

carbon dioxide (CO₂). Two ovarian cancer cell lines, OVCAR3 and

OVCAR8 were obtained from ATCC and cultured in RPMI 1640

supplemented with 10 % FBS (Hyclone; Logan, UT), 100 U/ml

5.1.12.4. 5-(((4-isopropylbenzyl)oxy)methyl)-7-(p-tolyl)quinolin-8-ol

(19d). White solid (Yield: 53 %). Mp: 85e87 ^ꢀC. ¹H NMR (400 MHz,

CDCl₃)

d

8.80 (dd, J ¼ 4.3, 1.6 Hz, 1H), 8.48 (dd, J ¼ 8.5, 1.6 Hz, 1H),

7.72e7.67 (m, 2H), 7.59 (s, 1H), 7.45 (dd, J ¼ 8.5, 4.2 Hz, 1H),

7.32e7.28 (m, 2H), 7.25 (s, 2H), 7.20 (d, J ¼ 8.2 Hz, 2H), 4.90 (s, 2H),

4.53 (s, 2H), 2.90 (hept, J ¼ 6.9 Hz, 1H), 2.41 (s, 3H), 1.24 (d,

J ¼ 7.0 Hz, 6H). ¹³C NMR (101 MHz, Chloroform-d)

d 148.68, 148.51,

147.68, 138.83, 137.03, 135.34, 134.45, 133.74, 130.68, 129.12, 128.11,

126.50, 126.45, 124.18, 121.81, 121.55, 71.80, 70.13, 33.86, 24.01,

21.26. HRMS: calculated for C₂₇H₂₇NO₂[MþH]^þ398.2115, found

398.2114 (mass error ¼ ꢁ0.3 ppm).

penicillin, and 100 mg/mL streptomycin (Invitrogen; Carlsbad, CA)

16

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

at 37 ^ꢀC in 5 % CO₂. Cell lines were authenticated using Short

Tandem Repeat (STR) analysis by ATCC and tested negative for

mycoplasma contamination using luciferase assay (Lonza, Allen-

dale, NJ).

initiate the reaction, the plate was incubated at 37 ^ꢀC for the indi-

cated time, and reactions were quenched with a 3-fold volume

excess of cold acetonitrile containing the internal standard. The

ﬁnal concentration of each component in this reaction was as fol-

lows: 1 mM EDTA, 0.5 mg/mL liver microsome, 10 mM test com-

5.3. Cytotoxicity assay

pound, 1.3 mM NADPH A, and 0.4 U/mL NADPH B (Glucose-6-

phosphate dehydrogeneous). Plates were sealed, mixed at

600 rpm for 10 min after quenching, and centrifuged at 4000 rpm

for 20 min to remove debris. The supernatants were transferred to

the appropriate plates for analysis by UPLCeMS. Microsomal half-

life was determined for each compound (and the control) and the

metabolic stability of each compound was calculated from the half-

life by least-squares ﬁt of the multiple time points, based on ﬁrst-

order kinetics.

5.3.1. Melanoma and breast cancer cell lines

The human melanoma (A375, RPMI7951, MDA-MB-435, and

MDA-MB-435/LCC6MDR1) and human breast (SKBR3 and MDA-

MB-453) cell lines were plated into 96-well plates at a density of

3500 to 5000 cells per well, depending on the proliferation rate of

each cell line. After overnight incubation, cells were treated with

the test compounds (concentration range: 3 nM-100 mM) for 72 h.

Each compound treatment was carried out in four replicates. At the

endpoint, the MTS reagent (Promega) was added to the cells, they

were incubated in the dark 37 ^ꢀC for 1.5 h, and cell viability was

determined by the absorbance a wavelength of 490 nm using a

plate reader (BioTek Instruments Inc., Winooski, VT). IC₅₀values

were normalized against untreated cells and calculated using

GraphPad Prism 7 software using nonlinear regression.

5.5. Western blotting

5.5.1. A375

A375 cells were seeded into 10-cm dishes at a density of

2 ꢂ 10⁶cells/plate and allowed to attach overnight. The medium

was replaced with either fresh complete DMEM medium or DMEM

containing the desired concentration (1, 5 and 10 mM) of MX-106, or

5.3.2. Ovarian cancer cell lines

compounds 7a, 12b, or 19a. After 24 h of incubation, cells were

washed with ice-cold PBS, lysed with RIPA buffer (Thermo Fisher

Scientiﬁc) containing Halt protease and phosphatase inhibitor

(Thermo Fisher Scientiﬁc), and centrifuged at 13,000 rpm at 4 ^ꢀC for

15 min. The Bradford assay method was used to quantify the pro-

OVCAR3 and OVCAR8 cells (3000 per well) were seeded into 96-

well plates. The culture medium was changed, and cells were

treated with compounds 7a, 7k, 12a, 12b, or 19a at 10 concentra-

tions ranging from 3 nM to 100 mM or with a DMSO vehicle control

in three replicates. Cells were incubated at 37 ^ꢀC for 72 h. After

treatment, MTS reagent (Promega, Madison, WI) was added and the

cells were incubated at 37 ^ꢀC for 1 h. We subsequently measured

the absorbance at a wavelength of 490 nm using a plate reader

(BioTek Instruments). IC₅₀values were calculated by nonlinear

regression analysis using OriginLab dats analysis and graphing

software.

tein in each sample. 50 mg of each samples were subjected to

Western blot analysis as described [25]. Brieﬂy, the protein samples

were separated via SDS-PAGE, transferred to PVDF membranes,

blocked with 5 % nonfat milk for 1 h at RT followed by overnight

incubation at 4 ^ꢀC with primary antibodies, which included

cleaved-PARP (#9185), CIAP1 (#7065), XIAP (#2045), Livin (#5471),

Survivin (#2808) and GAPDH (HRP Conjugate) (#3683), all pur-

chased from Cell Signaling Technology, Inc. Primary antibodies

were detected with HRP-conjugated secondary antibodies (#7074,

Cell Signaling Technology, Inc). and immunoreactive bands were

visualized on ﬁlm using enhanced chemiluminescent substrate

(ECL, Thermo Fisher Scientiﬁc).

5.4. Liver microsome stability assay [21,22]

This assay showed the metabolic liability of drug discovery

compounds in liver microsomes. Degradation of the compounds in

human, mouse, and rat liver microsomes was determined in mul-

tiple time points to monitor the rate of disappearance of the parent

compound during incubation. Pooled human liver microsomes

were obtained from XenoTech (Lenexa, KS), while pooled mouse

(CD-1) and rat (Sprague Dawley) liver microsomes were purchased

from Gibco (Ottawa, Ontario). NADPH regenerating agent solutions

A and B were purchased from BD Gentest (Woburn, MA). Ninety-

six-well analytical plates were obtained from Corning Incorpo-

rated (Acton, MA), while ninety-six-well deep well plates were

purchased from Midsci (St. Louis, MO).

Preparation of samples for determination of stability in micro-

somes was modiﬁed from the methods described by Di et al.

[23,24]. We used incubation times of 0, 15, 30, 60, 120, and 240 min.

Test compounds and system control (verapamil) were prepared as

10 mM stock solutions in DMSO. Concentrated mouse or human

liver microsomes (20 mg/mL protein concentration) and 0.5 M

EDTA were diluted into 0.1 M potassium phosphate buffer (pH7.4)

and mixed well prior to and after addition of each compound so-

5.5.2. Ovarian cancer cell lines

Ovarian cancer cells were treated with vehicle, or compounds

7k (2 and 4 mM), 12b (2, 4 mM), or MX-106 (2, 4 mM) for 24 h in

serum-free RPMI 1640 medium and collected in RIPA buffer

(Thermo Scientiﬁc; Rockford, IL) containing 1 % Halt Proteinase

Inhibitor Cocktail (Thermo Scientiﬁc; Rockford, IL). Protein was

quantitated using a Bradford assay as above. An equal amount of

protein (100

mg/lane) was loaded onto 10 % SDS-PAGE gels and

transferred onto PVDF membranes. The membranes were blocked

with 5 % nonfat milk for 1 h and incubated with primary antibodies

against GAPDH (Santa Cruz Biotechnology; St. Louis, MO), MCL1

and Survivin (both from Cell Signaling Technology, Inc.). Primary

antibodies were detected with HRP-conjugated secondary anti-

bodies (#7074, Cell Signaling Technology, Inc). and immunoreactive

bands were visualized on ﬁlm using enhanced chemiluminescent

substrate (ECL, Thermo Fisher Scientiﬁc).

lution. 90

m

L each of these solutions were transferred to triplicate

5.6. Surface plasmon resonance (SPR) for to measure binding

wells on each of six 96-well plates (one for each time point). For the

time 0 plate, a 3-fold (v/v) excess of cold acetonitrile containing

afﬁnity of 12b for survivin

4

m

g/mL warfarin as an internal standard was added to each well,

The binding afﬁnity of survivin was analyzed using SPR tech-

nology using a PlexAray HT system equipped with a bare gold-

coated (thickness 47 nm) PlexArray® Nanocapture® Sensor Chip

(Plexera Bioscience, Woodinville, WA). Biodo bioprinting was used

to manually print recombinant biotinylated survivin protein (in

along with NADPH regenerating solutions A and B, which were

mixed together in PBS, pH7.4. The samples were prepared for

analysis without further incubation. For the other ﬁve time-point

plates, NADPH regenerating agent was added to each well to

17

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

water) at various concentrations onto the sensor chip at 40 % hu-

midity via avidin-biotin conjugation. Each spot contained 0.2 mL of

consecutive days. During the treatment, tumor volume and body

weight were recorded 3 times/week; tumor volume was calculated

using the formula V ¼ (L ꢂ W²)/2, where L and W are the length and

width of the tumor. All mice were sacriﬁced at the conclusion of the

treatment and tumors were collected, weighed, and photographed.

12b solution and each concentration was printed in triplicate. The

chip was incubated overnight at 4 ^ꢀC in 80 % humidity, then rinsed

for 10 min with 10 ꢂ phosphate buffered saline with Tween 20

detergent (PBST), 10 min with 1 ꢂ PBST, and twice for 10 min each

in deionized water. The chip was blocked at RT overnight with 5 %

(w/v) non-fat milk in MilliQ water, and washed for 10 min in

10 ꢂ PBST, 10 min in 1 ꢂ PBST, and twice for 10 min each in

deionized water, then dried under a stream of nitrogen. SPR mea-

surements were performed using the PlexAray HT system (Plexera

Bioscience, Seattle, WA, US). Collimated light of wavelength 660 nm

passes through a coupling prism, reﬂects off the gold surface of the

chip, is received by an integrated CCD camera. A nonpulsatile piston

5.8.2. Ovarian cancer model

The therapeutic experiment was further conducted in an

orthotopic ovarian cancer mouse model. 5 ꢂ 10⁵OVCAR8 cells were

transduced with the lentiviral vector pEF1a-Luc2 to label them with

luciferase and surgically injected into left side of the ovarian bursa

of 7-week-old female (NSG) mice (Jackson Laboratory) under a

dissecting microscope. After one week, the mice were subjected to

bioluminescence imaging using the IVIS In Vivo Imaging System

(PerkinElmer XMRS) and Living Image software after IP adminis-

tration of the substrate D-luciferin. Based on the total ﬂux (pho-

tons/s), calculated by the Living Image software from luminescence

and body weight, mice were randomized into two groups (n ¼ 7/

group) and subjected to IP administration of vehicle or vehicle-

compound 12b. The 12b treatment was initiated at a dose of

20 mg/kg thrice per week. After 4 treatments, the dose was

increased to 40 mg/kg for an additional 4 treatments. Body weight

were recorded 3 times/week throughout the course of the experi-

ment. Mice were euthanized after 19 days of treatment and all the

major organs/tissues were harvested and bioimaged to visualize

metastases speciﬁc to that organ.

pump was used to inject samples and buffers into a 30 mL ﬂowcell

mounted on the coupling prim. All measurements were performed

at RT (25 ^ꢀC). Each measurement cycle involved 1) washing with

running buffer (PBST) at a constant rate (2

baseline, 2) injection of the 12b solution (5

washing of the surface with PBST for 300 s (2

eration with 0.5 % (v/v) H₃PO₄for 300 s (2 L/s). Changes in signal

m

L/s) to obtain a stable

L/s for binding), 3)

L/s), and 4) regen-

m

after binding and washing were recorded as the assay values (in

AU). We analyzed selected protein-grafted regions in the SPR im-

ages and the average reﬂectivity variations of the selected areas

were plotted as a function of time. Real-time binding signals were

collected and analyzed using the PlexArray® Analysis Software

Package (Plexera Bioscience). Kinetic analysis was performed using

BIAevaluation 4.1 software (Biacore, Inc., a division of Cytiva,

Uppsala, Sweden).

Authors' contribution

5.7. Molecular modeling

Najah Albadari and Shanshan Deng have equally contributed to

this work.

Molecular docking studies of 12b with survivin were conducted

in Schrodinger Molecular Modeling Suite 2014 (Schrodinger Inc.,

Portland, OR) following previously described procedures [1 1,12].

Compound 12b was prepared to generate various conformations,

then docked into the AVPI SMAC binding motif in a human survivin

crystal structure (Protein Data Bank entry: 3UIH). Docking to

minimize the energy of potential ligand binding poses was per-

formed prior to calculation of the molecular dynamics. Results were

visualized using the Maestro interface of the Schrodinger software.

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.

Acknowledgments

5.8. In vivo efﬁcacies of compound 12b on A375 subcutaneous

melanoma and OVCAR8 orthotopic ovary models in mice

This work was supported by NIH grant 1R01CA193609 to WL.

The content is solely the responsibility of the authors and does not

necessarily represent the ofﬁcial views of the National Institutes of

Health. We thank Dr. Lei Yang and Dr. Yong Li at St. Jude Children's

Research Hospital for the metabolic stability evaluation of com-

pounds 7a, 7k, 12a, 12b, 19a, and 19b.

All animal studies were performed under the guidelines of NIH

Principles of Laboratory Animal Care and protocols approved by the

UTHSC Institutional Animal Care and Use Committee (IACUC)

(protocol #17e056 for melanoma xenograft model and protocol

#19e0117 for ovary xenograft model). Animals were housed under

a 12:12 h light/dark cycle at 20e26 ^ꢀC with 30e70 % humidity.

Abbreviations

ESI

HPLC

IAP

ppm

Pgp

SAR

TLC

electron-spray ionization

high performance liquid chromatography

inhibitor of apoptosis proteins

parts per million

5.8.1. A375 melanoma model

6-7-week-old male humanized immunodeﬁcient NOD scid

gamma (NSG) mice (Jackson Laboratory) were used for implanta-

tion of A375 melanoma tumors. 2.5 ꢂ 10⁶logarithmically growing

P-glycoprotein

A375 cells in 50

mL of phenol red-free, FBS-free DMEM medium

structure-activity relationships

thin layer chromatography

tetramethylsilane

were mixed with 50

mL Matrigel and subcutaneously injected into

the dorsal right ﬂank of each NSG mouse. The tumors were allowed

to grow for 2 weeks until the average tumor volume reached

100 mm³. The mice were randomly assigned into 3 groups (n ¼ 7/

group): vehicle (5 % DMSO, 20 % PEG300, 5 % Tween 80, 70 % saline),

20 mg/kg compound 12b treatment group and 40 mg/kg com-

pound 12b treatment group. Vehicle or 12b treatments were

TMS

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113719.

administrated intraperitoneally (IP)

3

times/week for 15

18

N. Albadari, S. Deng, H. Chen et al.

European Journal of Medicinal Chemistry 224 (2021) 113719

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19

Article Doi

Synthesis and biological evaluation of selective survivin inhibitors derived from the MX-106 hydroxyquinoline scaffold

DOI: 10.1016/j.ejmech.2021.113719

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Full text of DOI:10.1016/j.ejmech.2021.113719

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