Structure-Based Design, Synthesis, and Biological Evaluation of New Triazolo[1,5- a]Pyrimidine Derivatives as Highly Potent and Orally Active ABCB1 Modulators

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Structure-Based Design, Synthesis, and Biological Evaluation of New

Triazolo[1,5‑a]Pyrimidine Derivatives as Highly Potent and Orally

Active ABCB1 Modulators

Shuai Wang,^⊥Sai-Qi Wang,^⊥Qiu-Xu Teng,^⊥Linlin Yang,^⊥Zi-Ning Lei, Xiao-Han Yuan, Jun-Feng Huo,

Xiao-Bing Chen, Mengru Wang, Bin Yu,* Zhe-Sheng Chen,* and Hong-Min Liu *

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Supporting Information

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ABSTRACT: ABCB1 is a promising therapeutic target for overcoming

multidrug resistance (MDR). In this work, we reported the structure-based

design of triazolo[1,5-a]pyrimidines as new ABCB1 modulators, of which

WS-691 signiﬁcantly increased sensitization of ABCB1-overexpressed

SW620/Ad300 cells to paclitaxel (PTX) (IC₅₀= 22.02 nM). Mechanistic

studies indicated that WS-691 signiﬁcantly increased the intracellular

concentration of PTX and [³H]-PTX while decreasing the eﬄux of [³H]-

PTX in SW620/Ad300 cells by inhibiting the eﬄux function of ABCB1. The

cellular thermal shift assay suggested that WS-691 could stabilize ABCB1 by

directly binding to ABCB1. WS-691 could stimulate the activity of ABCB1 ATPase but had almost no inhibitory activity against

CYP3A4. Importantly, WS-691 increased the sensitivity of SW620/Ad300 cells to PTX in vivo without observed toxicity.

Collectively, WS-691 is a highly potent and orally active ABCB1 modulator capable of overcoming MDR. The triazolo[1,5-

a]pyrimidine may be a promising scaﬀold for developing more potent ABCB1 modulators.

ABCB1 modulators. The third-generation ABCB1 modulators,

including tariquidar, zosuquidar, laniquidar, and elacridar in

the clinical trials (Figure 1C), could bind to ABCB1 with high

aﬃnity and exhibit almost no pharmacokinetic interaction with

other drugs.⁸Although some of the third-generation ABCB1

modulators (Figure 1C) have advanced into clinical trials,

none of them has been approved for clinical use.^12,26−29Thus,

the development of nontoxic and highly eﬃcacious ABCB1

modulators has been highly pursued for clinical application.

Our group has also been devoted to the development of

novel ABCB1 modulators, and some representative com-

pounds are shown in Figure 1D.³⁰⁻³²In this work, we reported

the discovery of the initial hit compound WS-36 (compound

5) from our in-house diverse compound library,³³⁻³⁹which

was cellularly engaged to ABCB1 and increased the sensitivity

of ABCB1-overexpressed SW620/Ad300 cells to PTX. Based

on the binding mode of compound WS-36, we performed the

structure-based design of triazolo[1,5-a]pyrimidine derivatives

as new ABCB1 modulators, leading to the discovery of WS-

691, which was well characterized as a nontoxic, highly potent,

INTRODUCTION

■

Multidrug resistance (MDR) is one of the major reasons for

the failure of successful cancer chemotherapy.¹Initially, cancer

patients can respond to drug treatment, while most of them

develop resistance to targeted chemotherapy during treat-

ment.²Although there are many underlying mechanisms of

MDR, the predominant one is the increased eﬄux of drugs

mediated by the upregulation of ATP-binding Cassette (ABC)

transporters.³⁻⁸The most well-studied and best-characterized

drug transporter is ABCB1 (also known as P-glycoprotein or

MDR1), as a member of ABC superfamily, which is

responsible for increasing the eﬄux of chemotherapeutic

drugs and decreasing the concentration of intracellular drugs.

ABCB1 is ubiquitously overexpressed in many MDR cancer

cells,⁹⁻¹²making ABCB1 a promising therapeutic target to

overcome MDR.¹³To date, numerous ABCB1 modulators

have been identiﬁed^5,8and can be generally divided into three

generations (Figure 1A−C). As shown in Figure 1A, the ﬁrst-

generation ABCB1 modulators, such as verapamil and

triﬂuoperazine,^14,15had deﬁciencies such as serious toxicity,

low therapeutic response, etc.¹⁶⁻¹⁹Relative to the ﬁrst-

generation ABCB1 modulators, the second-generation

ABCB1 modulators, including dexverapamil and biricodar

citrate (Figure 1B), showed improved potency, speciﬁcity, and

lower toxicity.²⁰⁻²²However, these ABCB1 modulators could

inhibit the activity of the drug-metabolizing enzyme CYP3A4

and thus cause unfavorable drug−drug interactions.²³⁻²⁵The

issues hampered further development of the second-generation

Received: October 5, 2020

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A

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Figure 1. Some representative ABCB1 modulators.

a

Scheme 1. Synthesis of Compounds 6−55

a

Reagents and Conditions: (a) Na₂CO₃, NaI, Acetone, 3−5 h, reﬂux; (b) acetic acid, 2−15 h, reﬂux; (c) POCl₃, 3−5 h, reﬂux; (d) EtOH, room

temperature (RT), 5 h.

and orally active ABCB1 modulator capable of overcoming

MDR.

RESULTS AND DISCUSSION

■

Structure-Based Design of Triazolo[1,5-a]Pyrimidine

Derivatives as New ABCB1 Inhibitors. To identify novel

and nontoxic ABCB1 inhibitors, we detected the IC₅₀values of

PTX against multidrug-resistant SW620/Ad300 cells in the

presence or absence of tested compounds. The cytotoxicity of

tested compounds against SW620/Ad300 cells was examined,

and the nontoxic concentrations (cell survival rate >90%,

Table S1 in the Supporting Information) were used for

examining their ability to reverse multidrug resistance. After

screening our in-house library, we successfully identiﬁed the hit

compound WS-36 (Figure 2A), which was nontoxic at 20 μM

(93.25% of cell survival rate) and increased the sensitivity of

CHEMISTRY

■

A novel series of triazolo[1,5-a]pyrimidine-based derivatives

targeting ABCB1 were designed and synthesized. As shown in

Scheme 1, compounds 2a−2v were obtained by reacting 5-

amino-4H-1,2,4-triazole-3-thiol (1) with various alkyl halides,

followed by cyclization with diﬀerent β-ketoesters, aﬀording

compounds 3a−3x. Treatment of compounds 3a−3x with

POCl₃under reﬂux gave compounds 4a−4x, which then

reacted with various amines in EtOH at room temperature to

give compounds 6−55.

B

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Figure 2. (A) Structure of WS-36. (B) In vitro antiproliferative

activity of PTX alone or in combination with WS-36 against SW620/

Ad300 cells. (C) Protein levels of ABCB1 in SW620 and SW620/

Ad300 cells, and β-actin was used as a loading control. (D) Eﬀect of

WS-36 on the thermostability of ABCB1.

SW620/Ad300 cells to PTX (Figure 2B). When combined

with WS-36 (20 μM), PTX inhibited the cell survival of

SW620/Ad300 cells with an IC₅₀value of 2.34 μM, lower than

that (4.23 μM) when PTX was used alone. As shown in Figure

2C, compared to parental SW620 cells, ABCB1 was up-

regulated in SW620/Ad300 cells. The cellular thermal shift

assay (CETSA) suggested that WS-36 could bind to ABCB1

and lead to the stabilization of ABCB1 (Figure 2D).

Given the binding property of WS-36 against ABCB1, we

docked it to a homology modeled human ABCB1, which was

established based on the apo form of mouse ABCB1 in the

inward-open conformation (PDB code: 4Q9H).⁴⁰The top-

scoring pose of WS-36 is bound to a central hydrophobic

cavity formed by aromatic and hydrophobic residues in TM5,

TM6, TM7, and TM10 (Figure 3A). A hydrogen bond was

predicted between the NH of WS-36 and the benzene

hydroxyl of Y310, and an edge-to-face π−π stacking interaction

was found between the phenyl ring of WS-36 and F983

(Figure 3B). Additionally, WS-36 was stabilized through

hydrophobic contacts with the side chains of F978, F732,

F336, L339, I340, and F343.

Figure 3. Initial evaluation of WS-36 as the ABCB1 inhibitor and

further structure-based design. (A) Three-dimensional (3D)-view of

interactions between WS-36 (cyan) and ABCB1 (green, PDB code:

4Q9H). The transporter is shown as a cartoon. Residues involved in

interactions with WS-36 are depicted in sticks. The hydrogen bond is

shown as red dashed lines. (B) Two-dimensional (2D)-view of

interactions between WS-36 and ABCB1. (C) WS-36 (cyan) binds

into a subpocket of a large hydrophobic cavity, the large space near

the triazole is marked with red dashed lines. The electrostatic surface

of this cavity is generated by PyMOL. The PyMOL charge-smoothed

potential bar is shown at the bottom of this panel. (D) Fluorophenyl

ring of WS-36 was located in a narrow cavity. (E) Structure-based

design of new triazolo[1,5-a]pyrimidine-based ABCB1 inhibitors from

WS-36.

Further analysis showed that WS-36 only occupied a

subpocket, leaving a large hydrophobic space (shown in a

dashed red ellipse) near the triazole ring (Figure 3C), while the

ﬂuorophenyl ring in WS-36 was located in a narrow cavity

(Figure 3D). The binding model of WS-36 not only

rationalized its inhibitory potency against ABCB1 but also

provided a structural basis for further structural optimization.

Based on this binding mode, we speculate that the attachment

of hydrophobic groups to the triazole ring could increase the

potency of WS-36 by forming extensive interactions with the

nonpolar residues in the pocket. Besides, structural mod-

iﬁcations at the methyl group and the phenyl ring may also

inﬂuence the inhibitory activity of the compounds against

ABCB1. Thus, we designed the triazolo[1,5-a]pyrimidine-

based ABCB1 inhibitors (Figure 3E).

resistant SW620/Ad300. The nontoxic concentrations of the

compounds (a survival rate above 90% toward SW620/Ad300,

provided in Table S1 in the Supporting Information) were

used for the following assays. Besides, RF, representative of the

reversal fold of MDR modulators based on the IC₅₀values of

PTX and PTX/modulator combination, was chosen as a

parameter for measuring the reversal ability of modulators. All

compounds at nontoxic concentrations in combination with

PTX were used to explore their eﬀects on the reversal activity

in PTX-resistant SW620/Ad300 cells, taking the well-

characterized MDR modulator verapamil (VPM) as a positive

control. The results are summarized in Tables 1 and 2. As

shown in Table 1, PTX exhibited cytotoxicity against SW620/

Ad300 cells with an IC₅₀value of 4.23 μM, while PTX,

together with VPM (4 μM), exhibited improved cytotoxicity

toward SW620/Ad300 cells with an IC₅₀value of 0.30 μM,

further conﬁrming that VPM could reverse PTX resistance

against SW620/Ad300 cells (RF = 13.90). Similarly,

compounds 5−55 were evaluated to examine their reversal

activity toward the SW620/Ad300 cells.

Structure−Activity Relationship Studies. Based on the

binding mode of WS-36 within human ABCB1, structural

modiﬁcations were mainly focused on variations of substituents

around the triazolo[1,5-a]pyrimidine scaﬀold. Initially, we

evaluated the cytotoxicity of compounds 5−55 against PTX-

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Table 1. Reversal Activity of Compounds 6−29 toward the PTX-Resistant SW620/Ad300 Cells

As shown in Table 1, compared to WS-36 (IC₅₀= 2.34 μM,

RF = 1.88), compound 6 bearing an additional benzylthioether

group showed improved reversal activity (IC₅₀= 1.04 μM, RF

= 4.05). The data suggest that the introduction of the

benzylthioether group is beneﬁcial for the reversal activity.⁴¹

Replacement of the methyl group with larger ethyl and

isopropyl groups led to compounds 7 and 8, which exhibited

slightly decreased reversal activity with the IC₅₀values of 1.61

and 1.44 μM, respectively, indicating that the methyl group

was favorable for the reversal activity. Therefore, subsequent

modiﬁcations focused on variations of substituents attached to

the phenyl ring of the benzylthioether group (R²), aﬀording

compounds 9−19, which showed comparable or decreased

reversal activity with the IC₅₀values around 2.0 μM. The

results suggest that the position and electronic nature of

substituents on the phenyl ring of R²have no remarkable

eﬀects on the reversal activity. Replacement of the phenyl with

the naphthyl group gave compound 20, which showed

comparable reversal activity against SW620/Ad300 cells

(IC₅₀= 1.74 μM, RF = 2.43). Compared with compound 6,

the insertion of an additional one or two methylene spacers

was proved to be detrimental for the reversal activity.

Compound 22, when combined with PTX, exhibited no

reversal activity against SW620/Ad300 cells (IC₅₀= 4.22 μM).

We also found that compounds 23−26 bearing diﬀerent alkyl

groups failed to reverse PTX resistance against SW620/Ad300

cells. The results may suggest that the π−π and/or hydro-

phobic interactions with surrounding key amino acid residues

within the binding pocket are crucial for the reversal activity.

To further explore the structure−activity relationships (SARs),

we also introduced two representative heteroaromatic groups,

benzimidazole and benzothiophene, to the triazolo[1,5-a]-

pyrimidine scaﬀold aﬀording compounds WS-691 (27) and

28. To our delight, WS-691 showed signiﬁcantly improved

reversal activity against the PTX-resistant SW620/Ad300 cells

(IC₅₀= 22.02 nM), being around 15-fold more potent than

VRP (IC₅₀= 0.30 μM). WS-691 could almost completely

reverse the activity toward PTX-resistant SW620/Ad300 cells

with an RF value of up to 192.25. The results suggest that the

benzimidazole ring (R²) is essential for the reversal activity.

Besides, compound 29 bearing the benzoyl group showed

comparable or slightly increased activity against PTX-resistant

SW620/Ad300 cells with compounds 6−20.

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Table 2. Reversal Activity of WS-691 and Compounds 30−55 toward the PTX-Resistant SW620/Ad300 Cells

Inspired by the potent reversal activity of WS-691 against

PTX-resistant SW620/Ad300 cells and associated with the

binding mode of WS-36 within modeled human ABCB1

(Figure 3A−C), we next modiﬁed the structures focusing on

variations of substituents on the phenyl ring (Ar) of WS-691.

As indicated in Figure 3C, the ﬂuorophenyl ring in WS-36 was

located in a narrow cavity, and this binding mode suggests that

the introduction of steric substituents or inappropriate

substituent position may signiﬁcantly aﬀect the reversal

activity. Initially, we explored the eﬀect of the ﬂuorine atom

on the reversal activity. Clearly, the ﬂuorine-containing

compounds 30−38 showed signiﬁcantly decreased reversal

activity compared to WS-691, regardless of the position and

the number of ﬂuorine atoms. The results demonstrate that

modiﬁcations on this phenyl ring are not well tolerated. Of

note, compound 39 without any substituents on the phenyl

ring also exerted promising reversal activity with an IC₅₀of

0.046 μM (RF = 92). Similarly, the incorporation of other

common substituents into the phenyl ring (compounds 40−

53) also caused a decrease of the reversal activity. These results

are consistent with those observed in the ﬂuorine-containing

compounds. Notably, compounds 42, 50, and 51 bearing polar

groups (e.g., −COOH, −OH, and amine) showed signiﬁcantly

decreased reversal activity against PTX-resistant SW620/

Ad300 cells. Besides, compounds 54 and 55 possessing the

coumarin and pyrazole ring, respectively, also exhibited

decreased reversal activity. The above results suggest that

steric and polar groups on the phenyl ring (Ar) may be less

preferred for the reversal activity against PTX-resistant

SW620/Ad300 cells.

Eﬀect of WS-691 on Reversing PTX Resistance. In view

of the potent reversal activity of WS-691, we also explored the

eﬀect of WS-691 on the reversal of PTX resistance at diﬀerent

concentrations (cell survival rates under the nontoxic

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Table 3. WS-691 Signiﬁcantly Sensitizes SW620/Ad300 Cells to PTX

IC₅₀of PTX (mean SD, nM) [reversal fold]

treatment

SW620

SW620/Ad300

PTX

7.69 2.78 [1.00]

7.35 2.40 [1.05]

5.14 0.95 [1.50]

7.11 2.88 [1.08]

4233.18 499.10 [1.00]

73.60 10.32 [57.52]

22.02 4.80 [192.25]

303.53 47.93 [13.95]

PTX + WS-691 (10 μM)

PTX + WS-691 (20 μM)

PTX + VPM (4 μM)

concentration of WS-691 are provided in Table S2 in the

Supporting Information). As shown in Table 3, the IC₅₀values

of PTX against SW620 and SW620/Ad300 cells were 7.69 and

4233.18 nM, respectively, suggesting that SW620/Ad300 cells

were highly resistant to PTX. WS-691 at 10 and 20 μM

signiﬁcantly enhanced the sensitivity of SW620/Ad300 cell

lines to PTX, achieving about 57- and 192-fold reversal of PTX

resistance, respectively. When combined with WS-691 (20

μM), PTX signiﬁcantly inhibited the cell survival of SW620/

Ad300 cells with an IC₅₀value of 22.02 nM but was less potent

against parental SW620 cells. The expression diﬀerence of

ABCB1 in both SW620 and SW620/Ad300 cells may be

responsible for the reversal eﬀect of WS-691. VPM (4 μM)

failed to increase the cytotoxicity of PTX to SW620 cells and

was less potent than WS-691 in reversing MDR.

ABCB1 (Figure 4). As shown in Table 5, the HEK293/ABCB1

cells displayed about 47-fold resistance to PTX, compared to

the parental HEK293/pcDNA3.1 cells. WS-691, whether at 10

or 20 μM, showed little eﬀect on the cytotoxicity of PTX

against HEK293/pcDNA3.1 cells. However, WS-691, at 10

μM, exhibited almost the same reversal activity as VPM toward

PTX-resistant HEK293/ABCB1 cells, while WS-691, at 20

μM, showed 2-fold improvement of the reversal activity,

compared to VPM. WS-691, whether at 10 or 20 μM, showed

almost no eﬀect on the eﬃcacy of PTX against the parental cell

lines but could signiﬁcantly reverse the resistance of ABCB1-

overexpressed SW620/Ad300, KB-C2, and HEK293/ABCB1

cells to PTX. WS-691, especially at 20 μM, showed much

better reversal ability than the well-known ABCB1 modulator

VPM. The results strongly suggest that WS-691 reversed MDR

of SW620/Ad300, KB-C2, and HEK293/ABCB1 cells to PTX

by modulating ABCB1.

Eﬀect of WS-691 on Accumulation and Eﬄux of PTX.

Given the potent reversal activity of WS-691, we next

measured the intracellular concentration of PTX in SW620

and SW620/Ad300 cells by ultra-performance liquid chroma-

tography (UPLC). As illustrated in Figure 5A, because of

ABCB1-mediated drug eﬄux, the concentration of PTX in

ABCB1-overexpressed SW620/Ad300 cells was signiﬁcantly

lower than that in SW620 cells without treatment. Compared

to the untreated group, WS-691 signiﬁcantly increased the

intracellular concentration of PTX in SW620/Ad300 cells in a

dose-dependent manner. After treatment with WS-691 (20

μM), the intracellular concentration of PTX in SW620/Ad300

cells was slightly higher than that in cells treated with VPM (4

μM).

To further investigate the reversal eﬀect of WS-691, KB-3−1

and its MDR cell line KB-C2 were used, in which KB-C2

overexpressed ABCB1 (Figure 4). As depicted in Table 4, the

Figure 4. Protein levels of ABCB1 in KB-3-1, KB-C2, HEK293/

pcDNA.31, and HEK293/ABCB1 cell lines, and β-actin was used as

the loading control.

Besides, we also detected the concentration of tritium-

labeled PTX ([³H]-PTX) in SW620 and SW620/Ad300 cells.

As illustrated in Figure 5B, the concentration of [³H]-PTX in

ABCB1-overexpressed SW620/Ad300 cells was also signiﬁ-

cantly lower than that in SW620 cells without treatment with

WS-691 or VPM. Upon treatment of SW620 cells with VPM at

4 μM, the concentration of [³H]-PTX in SW620 cells was not

changed obviously, while WS-691 at 20 μM also failed to

increase [³H]-PTX in SW620 cells. To our delight, the

concentrations of [³H]-PTX in SW620/Ad300 cells treated

with WS-691 (20 μM) or VPM (4 μM) signiﬁcantly increased,

compared to the untreated control. The results indicate that

WS-691 could increase the intracellular concentration of PTX.

IC₅₀values of PTX against KB-3−1 and KB-C2 cells were 4.54

and 1886.37 nM, respectively, and the data indicated that

compared to the parental KC-3−1 cells, KB-C2 cells exhibited

415.50-fold resistance to PTX. WS-691 at 10 μM eﬀectively

reversed the PTX resistance with an RF value of 86.89, and

WS-691 at 20 μM totally reversed the PTX resistance of KB-

C2 cells with an IC₅₀value of 4.75 nM. Intriguingly, WS-691,

in combination with PTX, showed little eﬀect on the parental

KB-3-1 cells. The results supported that the reversal eﬀect of

WS-691 may be closely related to ABCB1.

To further investigate whether the reversal eﬀect of WS-691

was related to ABCB1, we detected the reversal eﬀect of WS-

691 against ABCB1 gene transfectant cell line HEK293/

Table 4. WS-691 Signiﬁcantly Sensitizes KB-C2 Cells to PTX

IC₅₀of PTX (mean SD, nM) [reversal fold]

KB-C2

treatment

KB-3-1

4.54 1.59 [1.00]

PTX

1886.37 243.05 [1.00]

21.71 3.27 [86.89]

4.75 1.52 [397.13]

7.05 2.81 [267.57]

PTX + WS-691 (10 μM)

PTX + WS-691 (20 μM)

PTX + VPM (4 μM)

3.95 0.81 [1.15]

2.99 0.47 [1.52]

3.84 1.25 [1.18]

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Table 5. WS-691 Signiﬁcantly Sensitizes HEK293/ABCB1 Cells to PTX

IC₅₀of PTX (mean SD, nM) [reversal fold]

treatment

HEK293/pcDNA3.1

HEK293/ABCB1

PTX

1.93 0.06 [1.00]

1.48 0.17 [1.30]

1.50 0.04 [1.29]

1.62 0.16 [1.19]

91.40 23.32 [1.00]

10.35 4.52 [8.83]

5.03 3.27 [18.17]

10.37 5.47 [8.81]

PTX + WS-691 (10 μM)

PTX + WS-691 (20 μM)

PTX + VPM (4 μM)

Figure 5. Eﬀect of WS-691 on accumulation and eﬄux of PTX. (A) Eﬀect of WS-691 on the intracellular concentration of PTX in SW620 and

SW620/Ad300 cells. (B) Eﬀect of WS-691 on the accumulation of [³H]-PTX in SW620 and SW620/Ad300 cells. (C) Eﬀect of WS-691 on the

eﬄux of [³H]-PTX in SW620 cells. (D) Eﬀect of WS-691 on the eﬄux of [³H]-PTX in SW620/Ad300 cells. Error bars represent the standard

deviation (SD) value obtained from the average of three independent experiments. *, P < 0.05 and **, P < 0.01.

We also found that WS-691 exhibited better potency than

VPM in increasing the accumulation of PTX in SW620/Ad300

cells.

expression in SW620/Ad300 cells was investigated. As shown

in Figure 6A, after treatment of SW620/Ad300 cells with WS-

691 at 20 μM for 48, 72, or 96 h, respectively, the protein level

of ABCB1 was not changed. As an eﬄux pump, localization on

cytomembrane ensures its eﬄux function of ABCB1. In Figure

6B, treatment with WS-691 at 20 μM for 72 h did not change

the subcellular localization of ABCB1 (Green) in SW620/

Ad300 cells. It is believed that the inhibition of the eﬄux

function of ABCB1 or downregulation of ABCB1 has been

regarded as a potential therapeutic strategy to overcome

MDR.⁴²Therefore, these data suggest that WS-691 may

reverse the ABCB1-mediated MDR by inhibiting the eﬄux

function of ABCB1 rather than changing the expression and

subcellular localization of ABCB1.

Eﬀect of WS-691 on the ABCB1 ATP Hydrolysis. VPM,

a simulator of ABCB1-mediated ATP hydrolysis, has been

reported to stimulate the ABCB1-ATPase activity.⁴³In view of

the fact that ATP consumption could indirectly reﬂect the

activity of ATPase as drug eﬄux needs the energy derived from

ATP hydrolysis, we therefore assessed the eﬀect of WS-691 on

the basal activity of ABCB1 ATPase by detecting ATP

Next, we examined whether WS-691 could inhibit the eﬄux

function of ABCB1. The eﬀect of WS-691 on the eﬄux of

[³H]-PTX was also explored in SW620 and SW620/Ad300

cells by monitoring the concentration of [³H]-PTX at the four

diﬀerent time points. As depicted in Figure 5C, the treatment

of SW620 cells with WS-691 (20 μM) or VPM (4 μM) slightly

decreased eﬄux of [³H]-PTX, compared to the untreated cells.

Intriguingly, compared to the untreated group, after treatment

with WS-691 (20 μM) or VPM (4 μM), the eﬄux of [³H]-

PTX was signiﬁcantly inhibited in SW620/Ad300 cells, leading

to 64 and 62% loss of [³H]-PTX, respectively, in SW620/

Ad300 cells after 120 min, which was obviously lower than that

(97% loss) in the untreated group (Figure 5D). All of these

data suggest that WS-691 could inhibit the eﬄux function of

ABCB1, thus reducing PTX eﬄux and increasing the

intracellular concentration of PTX.

Eﬀect of WS-691 on ABCB1 Expression and Sub-

cellular Localization. The eﬀect of WS-691 on ABCB1

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performed to explore the interaction between CYP3A4 and

WS-691, a well-known CYP3A4 inhibitor ketoconazole and

VPM were used as the positive controls. As shown in Figure

8A, compared to the solvent control, at 30 μM, ketoconazole

and VPM sharply decreased the activity of CYP3A4 in a time-

dependent manner, while WS-691 displayed mild activation of

CYP3A4. Besides, as shown in Figure 8B, ketoconazole and

VPM showed strong and moderate inhibitory eﬀects on

CYP3A4 activity, respectively, while WS-691 displayed a very

slight activation eﬀect on CYP3A4. At 30 min, WS-691 (20

μM) changed the activity of CYP3A4 to 1.06-fold of that at 0

min. At 37 °C, the IC₅₀values of ketoconazole and VPM were

0.011 and 21.33 μM, respectively. These data suggested that

WS-691 had almost no eﬀect on the activity of CYP3A43,

indicating possible low toxicity of WS-691 as an ABCB1

modulator.

Cellular Thermal Shift Assay (CETSA). ABCC1 has been

reported to be the second most important ABC transporter in

MDR, and most ABCB1 modulators generally exhibited

inhibitory activity toward ABCC1.⁴⁵Therefore, to investigate

whether WS-691 could selectively bind to ABCB1 in SW620/

Ad300 cells, the cellular thermal shift assay was carried out.⁴⁶

As shown in Figure 9, pretreatment with WS-691 signiﬁcantly

inhibited the degradation of ABCB1 protein caused by high

temperature, compared to the solvent control. On the contrary,

WS-691 showed no obvious eﬀect on ABCC1 degradation.

The data suggest that WS-691 could selectively stabilize

ABCB1 by directly binding to ABCB1.

Molecular Modeling Analysis. Compound WS-691 was

docked into the human ABCB1 model. WS-691 had a similar

binding mode compared with WS-36 but formed more

hydrophobic interactions with ABCB1. The ﬂuorophenyl ring

of WS-691 almost overlapped with that of WS-36, and a π−π

stacking interaction was also formed with F983. The

triazolo[1,5-a]pyrimidine scaﬀold shifted a little from the

space occupied by WS-36 and, thus, lost the hydrogen bond

interaction with Y310 (Figure 10A,B). As expected, the

benzimidazole ring of WS-691 extended to TM7 and TM10,

forming two π−π stacking interactions with F983 and F728,

respectively, as well as many nonspeciﬁc contacts with Y307,

I306, and F303 (Figure 10C).

Figure 6. Eﬀect of WS-691 on ABCB1 expression and subcellular

localization. (A) Protein level of ABCB1 in SW620/Ad300 cells, and

β-actin was used as a loading control. (B) Eﬀect of WS-691 on the

subcellular localization of ABCB1 (green) after treatment for 72 h.

Hoechst 33342 (blue) counterstains the nuclei. The scale bar is 10

μm.

hydrolysis in the presence of WS-691 at diﬀerent concen-

trations (0−40 μM). As shown in Figure 7, WS-691 stimulated

ATP hydrolysis of ABCB1 to about 2.5-fold of basal activity.

The ABCB1-stimulation eﬀect of WS-691 reached maximal at

a concentration of about 19.1 μM, indicating that WS-691

could stimulate the activity of ABCB1 ATPase with a maximal

value being around 2.5-fold of basal activity.

Eﬀect of WS-691 on Cytochrome P3A4 (CYP3A4). The

second-generation ABCB1 modulators showed better potency

and speciﬁcity but with lower cell toxicity than the ﬁrst-

generation ABCB1 modulators. Unfortunately, these ABCB1

modulators could simultaneously inhibit the activity of the

drug-metabolizing enzyme CYP3A4, which may lead to

unexpected pharmacokinetic parameters and side ef-

fects.^{21−23,25,44}Therefore, the CYP3A4 activity assay was

Figure 7. Eﬀect of WS-691 on the ABCB1 ATP hydrolysis. The graph plotted the change of average ATPase activity of ABCB1 with SD as the

concentration of WS-691 changed. The inset shows the stimulation of ATP hydrolysis at a lower concentration of WS-691.

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Figure 8. Eﬀect of WS-691 on the activity of CYP3A4. (A) Reaction kinetics of ﬂuorogenic substrate metabolism in human liver microsomes

containing CYP3A4 at 37 °C. (B) Dose−response curve of WS-691, VPM, and ketoconazole on CYP3A4.

than VPM. No mortality or signiﬁcant change in body weight

was observed during the treatment with the tested doses

(Figure 11C). The HE and TUNEL staining of tumor tissues

(Figure 11E) showed that the combination of WS-691 and

PTX led to more death (nuclear pyknosis, dark purple in HE

staining) and apoptosis (bright green in TUNEL staining) of

tumor cells compared to vehicle and single-drug treatment.

The HE staining of liver tissues presented in Figure 11F

showed that all treatments did not cause obvious liver injury,

suggesting that the combination regimen did not result in

increased toxicity. These data indicated that WS-691 increased

the sensitivity of SW620/Ad300 cells to PTX in vivo without

increasing toxicity.

Figure 9. Binding of WS-691 with ABCB1. The aliquots of SW620/

Ad300 cell lysate in the presence or absence of WS-691 were heated,

and ABCB1 and ABCC1 were assessed by Western blotting.

CONCLUSIONS

■

In this work, we reported the discovery of the hit compound

WS-36 after screening our diverse small-molecule compound

library, and subsequent extensive medicinal chemistry eﬀorts

generated a novel series of triazolo[1,5-a]pyrimidine-based

ABCB1 modulators 6−55. In particular, PTX, in combination

with WS-691 at 20 μM, signiﬁcantly inhibited the growth of

PTX-resistant SW620/Ad300 cells with an IC₅₀value of 22.02

nM, being around 14-fold more potent than VPM (IC₅₀= 303

nM), and the results indicated that WS-691 almost completely

reversed the PTX resistance activity toward SW620/Ad300

cells with an RF value of up to 192.25. Further mechanistic

studies revealed that WS-691 reversed the ABCB1-mediated

MDR by inhibiting the eﬄux function of ABCB1 rather than

downregulating ABCB1. Besides, WS-691 signiﬁcantly in-

creased the concentration of PTX and [³H]-PTX while

decreasing the eﬄux of [³H]-PTX in SW620/Ad300 cells by

Xenograft Study. Inspired by the encouraging in vitro

potency of WS-691, we examined the antitumor eﬀect of WS-

691 and PTX in vivo using an established xenograft model

bearing SW620/Ad300 cells in nude mice. Animals were

treated with vehicle, PTX, WS-691, VPM, PTX plus WS-691,

or PTX plus VPM, respectively. During the therapy, the tumor

weight and volume were measured every 2 days. As shown in

Figure 11A,B, the combination of PTX with WS-691 showed

stronger antitumor activity than PTX alone. The tumor weight

and volume in the group treated with PTX/WS-691

combination were smaller than that treated with PTX alone,

while WS-691 showed little eﬀect on the tumor growth

compared to the vehicle group. It is remarkable that WS-691,

in combination with PTX, showed superior antitumor potency

Figure 10. Predicted binding mode of WS-691 (PDB code: 4Q9H). (A) 3D-view of interactions between WS-691 (magenta) and ABCB1 (green).

The transporter is shown as a cartoon. Residues involved in interactions with both WS-691 and WS-36 are depicted in green sticks. Residues that

only interact with WS-691 are depicted in orange sticks. (B) WS-691 (magenta) binds into the same hydrophobic cavity as WS-36 (cyan) and

occupies more space in the binding pocket. The electrostatic surface of this cavity is generated by PyMOL. The PyMOL Charge-smoothed

potential bar is shown in the picture. (C) 2D-view of interactions between WS-691 and ABCB1.

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Figure 11. Synergistic antitumor eﬀect of WS-691 and PTX in vivo. (A) Tumor volume during the in vivo study. (B) Tumor weight at the end of

the experiment. (C) Body weight of animals during the in vivo study. (D) Photographs of tumors at the end of the in vivo study. (E) HE and

TUNEL staining of tumor tissues. (E) HE staining of liver tissues. The scale bar is 100 μm. *, P < 0.05.

inhibition of the eﬄux function of ABCB1. WS-691 could

stimulate the activity of ABCB1 ATPase but had almost no

eﬀect on the inhibitory activity against CYP3A4, an important

enzyme of Cytochrome P450 responsible for drug metabolism

in the body, and the data suggest that WS-691 may be a safer

ABCB1 modulator than VPM and Ketoconazole in vivo and

could be synergistically used with other drugs without aﬀecting

drug−drug interactions. The cellular thermal shift assay

suggested that WS-691 could directly bind to ABCB1 and

then stabilize ABCB1. More importantly, WS-691 increased

the sensitivity of SW620/Ad300 cells to PTX in a xenograft

model bearing SW620/Ad300 cells without obvious toxicity.

Collectively, WS-691 is a highly eﬀective and nontoxic ABCB1

modulator capable of overcoming multidrug resistance in vitro

and in vivo and may deserve further preclinical development.

The success of WS-691 highlights the importance of the

triazolo[1,5-a]pyrimidine scaﬀold for the development of new

ABCB1 inhibitors.

EXPERIMENTAL SECTION

■

Chemistry. Reagents and solvents were commercially available for

direct use without further puriﬁcation. A Bruker DPX 400 MHz

spectrometer was used to characterize all of the NMR data and TMS

in DMSO-d₆or CDCl₃selected as the internal standard. Relative to

TMS, δ ppm values were used to represent Chemical shifts. A Waters

Micromass Q-T of Micromass spectrometer by electrospray ionization

(ESI) was used to give high-resolution mass spectra (HRMS). Waters

e2695 HPLC was used to measure the purity of compounds 5−55, all

of which were >95% pure (Phenomenex column, C-18, 5.0 μm, 4.6

mm × 250 mm). Compounds 5−26 were detected by the acetonitrile

in H₂O (40:60, v/v), and the ﬂow rate is 1.0 mL/min, while

compounds 27−41 and 43−55 were measured by acetonitrile in H₂O

(55:45, v/v). Besides, compound 42 was subjected to acetonitrile in

H₂O (70:30, v/v)

General Method for the Synthesis of 4a−4x. Compounds

4a−4x were prepared according to the previously reported method.³⁷

General Method for the Synthesis of Compounds 6−55.

Compound 4a (290.7 mg, 1 mmol) was dissolved in ethanol (2 mL),

followed by the addition of commercially available 4-ﬂuoroaniline

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(111.1 mg, 1 mmol). The reaction mixture was stirred at room

temperature for 5 h. Upon completion of the reaction, the solvent was

evaporated, the resulting residue was then puriﬁed by column

chromatography to give compound 6. Other compounds were

synthesized following a similar route as described for the synthesis

of compound 6.

2-(Benzylthio)-N-(4-ﬂuorophenyl)-5-methyl-[1,2,4]triazolo[1,5-

a]pyrimidin-7-amine (6). Yellow solid, yield: 92%. HPLC purity:

99.02%. m.p.: 227−231 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.52−

7.45 (m, 4H), 7.36 (m, 4H), 7.29−7.23 (m, 1H), 6.37 (s, 1H), 4.57

(s, 2H), 2.45 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.20,

161.90, 160.23, 159.47, 152.44, 146.64, 137.60, 132.00, 128.95,

128.48, 127.54, 127.46, 127.36, 116.66, 116.43, 90.38, 34.58, 22.00.

HRMS (ESI): m/z calcd for C₁₉H₁₅FN₅S (M − H)⁺, 364.1032;

found, 364.1040.

HRMS (ESI): m/z calcd for C₁₉H₁₆BrFN₅S (M + H)⁺, 444.0294;

found, 444.0281.

2-((3-Bromobenzyl)thio)-N-(4-ﬂuorophenyl)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (13). White solid, yield: 84%.

HPLC purity: 99.85%. m.p.: 208−213 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.77 (s, 1H), 7.57−7.43 (m, 4H), 7.38 (t, J = 8.8 Hz,

2H), 7.29 (t, J = 7.8 Hz, 1H), 6.37 (s, 1H), 4.56 (s, 2H), 2.45 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆) δ 164.84, 161.89, 160.39, 159.46,

152.57, 146.59, 140.80, 131.99, 131.66, 130.57, 130.16, 128.08,

127.55, 127.46, 121.50, 116.66, 116.44, 90.36, 33.69, 22.10. HRMS

(ESI): m/z calcd for C₁₉H₁₆BrFN₅S (M + H)⁺, 444.0294; found,

444.0281.

N-(4-Fluorophenyl)-5-methyl-2-((4-(triﬂuoromethyl)benzyl)thio)-

[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (14). White solid, yield:

89%. HPLC purity: 98.26%. m.p.:180−188 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 7.73 (dd, J = 28.3, 8.1 Hz, 4H), 7.49 (dd, J = 8.8,

4.9 Hz, 2H), 7.37 (t, J = 8.7 Hz, 2H), 6.36 (s, 1H), 4.65 (s, 2H), 2.45

(s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.71, 161.86, 160.63,

159.43, 152.79, 146.53, 143.02, 132.06, 129.76, 127.98, 127.67,

127.52, 127.44, 125.54, 125.28, 125.24, 122.84, 116.63, 116.40, 90.31,

33.81, 22.23. HRMS (ESI): m/z calcd for C₂₀H₁₆F₄N₅S (M + H)⁺,

434.1063; found, 434.1062.

N-(4-Fluorophenyl)-5-methyl-2-((4-nitrobenzyl)thio)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (15). White solid, yield: 92%.

HPLC purity: 99.28%. m.p.: 171−175 °C. ¹H NMR (400 MHz,

CDCl₃) δ 8.13 (d, J = 8.7 Hz, 2H), 7.73−7.53 (m, 3H), 7.32 (dd, J =

8.8, 4.6 Hz, 2H), 7.18 (t, J = 8.4 Hz, 2H), 6.16 (s, 1H), 4.59 (s, 2H),

2.51 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.48, 165.10, 162.60,

160.13, 155.79, 147.16, 145.82, 144.86, 131.34, 129.92, 126.59,

126.51, 123.70, 117.19, 116.96, 89.01, 34.77, 25.38. HRMS (ESI): m/

z calcd for C₁₉H₁₆FN₆O₂S (M + H)⁺, 411.1039; found, 411.1024.

N-(4-Fluorophenyl)-5-methyl-2-((3-nitrobenzyl)thio)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (16). White solid, yield: 88%.

HPLC purity: 99.28%. m.p.: 162−167 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.69 (s, 1H), 7.54−7.41 (m, 2H), 7.30 (d, J = 7.8 Hz,

1H), 6.84 (t, J = 8.7 Hz, 2H), 6.55 (dd, J = 7.9, 4.9 Hz, 2H), 5.82 (s,

1H), 4.44 (s, 2H), 2.30 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ

162.16, 155.85, 155.31, 153.56, 151.73, 151.29, 145.54, 141.09,

132.00, 131.04, 130.61, 128.42, 122.00, 115.65, 115.43, 115.14,

115.07, 99.04, 34.22, 19.03. HRMS (ESI): m/z calcd for

C₁₉H₁₆FN₆O₂S (M + H)⁺, 411.1039; found, 411.1024.

2-((2,6-Dichlorobenzyl)thio)-N-(4-ﬂuorophenyl)-5-methyl-

[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (17). White solid, yield:

77%. HPLC purity: 98.85%. m.p.: 200−206 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 7.56 (d, J = 8.1 Hz, 2H), 7.52−7.32 (m, 5H), 6.38

(s, 1H), 4.86 (s, 2H), 2.46 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

δ 164.42, 161.75, 160.05, 159.33, 146.41, 135.12, 132.21, 131.83,

130.66, 128.86, 127.36, 127.27, 116.60, 116.38, 90.22, 31.58, 22.64.

HRMS (ESI): m/z calcd for C₁₉H₁₅Cl₂FN₅S (M + H)⁺, 434.0409;

found, 434.0396.

N-(4-Fluorophenyl)-5-methyl-2-((4-methylbenzyl)thio)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (18). White solid, yield: 86%.

HPLC purity: 98.99%. m.p.: 171−174 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.49 (dd, J = 8.9, 4.9 Hz, 2H), 7.38 (dd, J = 12.1, 5.3

Hz, 4H), 7.13 (d, J = 7.9 Hz, 2H), 6.38 (s, 1H), 4.53 (s, 2H), 2.46 (s,

3H), 2.27 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.28,

161.92, 160.05, 159.49, 152.26, 146.68, 136.60, 134.37, 131.96,

131.93, 129.03, 128.87, 127.56, 127.47, 116.66, 116.43, 90.41, 34.43,

21.87, 20.66. HRMS (ESI): m/z calcd for C₂₀H₁₉FN₅S (M + H)⁺,

380.1345; found, 380.1332.

2-(Benzylthio)-5-ethyl-N-(4-ﬂuorophenyl)-[1,2,4]triazolo[1,5-a]-

pyrimidin-7-amine (7). White solid, yield: 79%. HPLC purity:

99.56%. m.p.: 214−223 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.56−

7.46 (m, 4H), 7.32 (m, 4H), 7.28−7.22 (m, 1H), 6.27 (s, 1H), 4.57

(s, 2H), 2.66 (q, J = 7.5 Hz, 2H), 1.19 (t, J = 7.5 Hz, 3H). ¹³C NMR

(100 MHz, DMSO-d₆) δ 168.59, 164.65, 161.22, 158.80, 155.90,

145.35, 137.99, 133.01, 132.98, 128.86, 128.41, 127.19, 126.84,

126.76, 116.36, 116.14, 88.23, 34.51, 30.90, 12.85. HRMS (ESI): m/z

calcd for C₂₀H₁₉FN₅S (M + H)⁺, 380.1345; found, 380.1331.

2-(Benzylthio)-N-(4-ﬂuorophenyl)-5-isopropyl-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-amine (8). White solid, yield: 84%. HPLC purity:

1

96.35%. m.p.: 192−198 °C. H NMR (400 MHz, DMSO-d₆) δ 7.46

(d, J = 7.1 Hz, 2H), 7.33 (t, J = 7.1 Hz, 2H), 7.28 (d, J = 7.0 Hz, 1H),

6.84 (t, J = 8.8 Hz, 2H), 6.55 (dd, J = 7.7, 5.2 Hz, 2H), 5.82 (s, 1H),

4.46 (s, 2H), 2.86 (m, 1H), 1.24 (d, J = 6.8 Hz, 6H). ¹³C NMR (100

MHz, DMSO-d₆) δ 162.63, 160.21, 155.86, 155.73, 153.58, 151.88,

145.52, 137.93, 129.31, 128.95, 127.78, 115.64, 115.42, 115.16,

115.08, 95.98, 35.06, 32.12, 21.33. HRMS (ESI): m/z calcd for

C₂₁H₂₁FN₅S (M + H)⁺, 394.1502; found, 394.1487.

2-((4-Fluorobenzyl)thio)-N-(4-ﬂuorophenyl)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (9). White solid, yield: 92%. HPLC

purity: 98.94%. m.p.: 184−191 °C. ¹H NMR (400 MHz, DMSO-d₆) δ

7.56 (dd, J = 8.6, 5.6 Hz, 2H), 7.52−7.46 (m, 2H), 7.37 (dd, J = 12.2,

5.4 Hz, 2H), 7.15 (t, J = 8.9 Hz, 2H), 6.37 (s, 1H), 4.57 (s, 2H), 2.45

(s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.04, 162.61, 161.89,

160.19, 159.46, 152.55, 146.60, 134.07, 134.04, 132.01, 131.03,

130.95, 127.55, 127.46, 116.65, 116.42, 115.32, 115.10, 90.35, 33.69,

22.07. HRMS (ESI): m/z calcd for C₁₉H₁₆F₂N₅S (M + H)⁺,

384.1094; found, 384.1093.

2-((4-Chlorobenzyl)thio)-N-(4-ﬂuorophenyl)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (10). White solid, yield: 93%.

HPLC purity: 97.76%. m.p.: 183−189 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.55 (d, J = 8.4 Hz, 2H), 7.49 (dd, J = 9.0, 4.9 Hz, 2H),

7.37 (t, J = 8.8 Hz, 4H), 6.36 (s, 1H), 4.56 (s, 2H), 2.45 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 164.89, 161.87, 160.46, 159.44,

152.64, 146.56, 137.03, 132.03, 131.92, 130.84, 128.37, 127.53,

127.45, 116.64, 116.41, 90.31, 33.71, 22.14. HRMS (ESI): m/z calcd

for C₁₉H₁₆ClFN₅S (M + H)⁺, 400.0799; found, 400.0799.

2-((4-Bromobenzyl)thio)-N-(4-ﬂuorophenyl)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (11). White solid, yield: 89%.

HPLC purity: 99.94%. m.p.: 180−186 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.50 (m, 6H), 7.37 (t, J = 8.8 Hz, 2H), 6.37 (s, 1H),

4.54 (s, 2H), 2.45 (s, 3H). ¹³C NMR (100 MHz, DMSO-d_6′) δ

164.91, 161.90, 160.28, 159.47, 152.42, 146.63, 137.43, 131.97,

131.29, 131.20, 127.56, 127.48, 120.45, 116.65, 116.42, 90.39, 33.77,

22.00. HRMS (ESI): m/z calcd for C₁₉H₁₆BrFN₅S (M + H)⁺,

444.0294; found, 444.0276.

2-((2-Bromobenzyl)thio)-N-(4-ﬂuorophenyl)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (12). White solid, yield: 85%.

HPLC purity: 99.08%. m.p.: 174−181 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.76−7.63 (m, 2H), 7.48 (dd, J = 8.9, 5.0 Hz, 2H),

7.40−7.30 (m, 3H), 7.24 (m, 1H), 6.26 (s, 1H), 4.64 (s, 2H), 2.39 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.16, 163.81, 158.92,

155.57, 145.38, 136.85, 132.88, 132.73, 131.41, 129.61, 127.92,

127.08, 126.99, 124.50, 124.02, 116.42, 116.19, 89.33, 35.27, 24.48.

N-(4-Fluorophenyl)-2-((4-methoxybenzyl)thio)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (19). White solid, yield: 91%.

HPLC purity: 96.78%. m.p.: 168−172 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.37 (d, J = 8.2 Hz, 2H), 6.93−6.79 (m, 4H), 6.55 (dd, J

= 7.8, 5.1 Hz, 2H), 5.82 (s, 1H), 4.39 (s, 2H), 3.73 (s, 3H), 2.30 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.61, 159.02, 155.85,

155.34, 153.56, 151.66, 151.22, 145.55, 130.57, 129.59, 115.65,

115.43, 115.14, 115.07, 114.35, 98.99, 55.53, 34.63, 19.02. HRMS

(ESI): m/z calcd for C₂₀H₁₉FN₅OS (M + H)⁺, 396.1294; found,

396.1280.

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N-(4-Fluorophenyl)-5-methyl-2-((naphthalen-1-ylmethyl)thio)-

[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (20). White solid, yield:

81%. HPLC purity: 99.22%. m.p.:183−187 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 8.21 (d, J = 8.2 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H),

7.89 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 6.9 Hz, 1H), 7.66−7.34 (m,

7H), 6.38 (s, 1H), 5.09 (s, 2H), 2.47 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 165.18, 161.87, 160.54, 159.44, 152.70, 146.57, 133.49,

132.61, 132.03, 130.89, 128.73, 128.39, 127.77, 127.51, 127.42,

126.49, 126.01, 125.48, 123.71, 116.65, 116.42, 90.33, 32.65, 22.20.

HRMS (ESI): m/z calcd for C₂₃H₁₉FN₅S (M + H)⁺, 416.1345; found,

416.1330.

N-(4-Fluorophenyl)-5-methyl-2-(phenethylthio)-[1,2,4]triazolo-

[1,5-a]pyrimidin-7-amine (21). White solid, yield: 79%. HPLC

purity: 97.05%. m.p.: 201−203 °C. ¹H NMR (400 MHz, DMSO-d₆) δ

7.38−7.20 (m, 5H), 6.84 (t, J = 8.9 Hz, 2H), 6.55 (m, 2H), 5.82 (s,

1H), 3.41 (t, J = 7.5 Hz, 2H), 3.04 (t, J = 7.5 Hz, 2H), 2.31 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆) δ 162.65, 155.85, 155.36, 153.57,

151.70, 151.15, 145.56, 140.39, 129.03, 128.83, 126.83, 115.64,

115.43, 115.14, 115.07, 98.97, 35.59, 32.40, 19.01. HRMS (ESI): m/z

calcd for C₂₀H₁₉FN₅S (M + H)⁺, 380.1345; found, 380.1332.

N-(4-Fluorophenyl)-5-methyl-2-((3-phenylpropyl)thio)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (22). White solid, yield: 81%.

HPLC purity: 99.82%. m.p.: 172−175 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.52−7.45 (m, 2H), 7.37 (m, 2H), 7.23 (m, 5H), 6.37

(s, 1H), 3.29 (t, J = 7.1 Hz, 2H), 2.80−2.70 (m, 2H), 2.46 (s, 3H),

2.14−2.00 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.53,

161.95, 159.75, 159.53, 152.09, 146.76, 141.03, 131.92, 128.34,

128.29, 127.62, 127.54, 125.89, 116.67, 116.44, 90.43, 33.91, 30.91,

30.42, 21.71. HRMS (ESI): m/z calcd for C₂₁H₂₁FN₅S (M + H)⁺,

394.1502; found, 394.1489.

6.27 (s, 1H), 4.88 (s, 2H), 2.37 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 164.13, 163.35, 161.32, 158.90, 155.72, 150.82, 145.29,

135.49, 132.71, 127.09, 127.00, 123.35, 116.41, 116.19, 114.48, 89.29,

27.21, 24.54. HRMS (ESI): m/z calcd for C₂₀H₁₅FN₇S (M − H)⁺,

404.1094; found, 404.1102.

2-(((5-Chlorobenzo[b]thiophen-3-yl)methyl)thio)-N-(4-ﬂuoro-

phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (28).

White solid, yield: 76%. HPLC purity: 99.48%. m.p.: 191−196 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 8.10 (d, J = 1.9 Hz, 1H), 8.03 (d, J

= 8.6 Hz, 1H), 7.94 (s, 1H), 7.54−7.39 (m, 3H), 7.37−7.28 (m, 2H),

6.26 (s, 1H), 4.83 (s, 2H), 2.39 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 164.30, 164.01, 161.32, 158.90, 155.76, 145.31, 139.13,

138.23, 132.92, 131.49, 129.50, 128.43, 127.09, 127.00, 124.71,

124.54, 121.64, 116.40, 116.18, 89.20, 27.77, 24.62. HRMS (ESI): m/

z calcd for C₂₁H₁₆ClFN₅S₂(M + H)⁺, 456.0520; found, 456.0503.

2-((7-((4-Fluorophenyl)amino)-5-methyl-[1,2,4]triazolo[1,5-a]-

pyrimidin-2-yl)thio)-1-phenylethan-1-one (29). White solid, yield:

81%. HPLC purity: 98.18%. m.p.: 201−207 °C. ¹H NMR (400 MHz,

CDCl₃) δ 8.10−8.03 (m, 2H), 7.66 (s, 1H), 7.58 (t, J = 7.4 Hz, 1H),

7.47 (t, J = 7.6 Hz, 2H), 7.36−7.30 (m, 2H), 7.21−7.13 (m, 2H),

6.12 (s, 1H), 4.89 (s, 2H), 2.46 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃) δ 193.60, 165.78, 164.88, 162.54, 160.07, 155.83, 144.87,

135.68, 133.66, 131.54, 131.51, 128.76, 128.56, 126.65, 126.57,

117.10, 116.87, 88.89, 39.60, 25.32. HRMS (ESI): m/z calcd for

C₂₀H₁₇FN₅OS (M + H)⁺, 394.1138; found, 394.1137.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(2-ﬂuorophenyl)-

5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (30). White solid,

yield: 91%. HPLC purity: 96.76%. m.p.: 181−186 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 7.85−7.77 (m, 2H), 7.63−7.45 (m, 5H), 7.43−

7.36 (m, 1H), 6.09 (d, J = 1.8 Hz, 1H), 5.09 (s, 2H), 2.40 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 163.26, 162.28, 158.18, 155.70,

154.04, 150.46, 146.06, 130.67, 129.89, 129.81, 129.24, 125.93,

125.44, 123.08, 122.96, 117.02, 116.83, 113.95, 90.25, 25.21, 22.95.

HRMS (ESI): m/z calcd for C₂₀H₁₇FN₇S (M + H)⁺, 406.1250; found,

406.1249.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(3-ﬂuorophenyl)-

5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (31). White solid,

yield: 93%. HPLC purity: 99.92%. m.p.: 198−202 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 7.81 (m, 2H), 7.55 (m, 4H), 7.39 (m, 2H), 6.34

(s, 1H), 5.08 (s, 2H), 2.39 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

δ 162.98, 162.13, 161.75, 159.32, 154.19, 150.57, 146.08, 132.10,

130.68, 127.60, 127.52, 125.94, 116.52, 116.29, 113.97, 89.99, 25.19,

23.06. HRMS (ESI): m/z calcd for C₂₀H₁₇FN₇S (M + H)⁺, 406.1250;

found, 406.1250.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(2,6-diﬂuorophen-

yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (32). White

solid, yield: 86%. HPLC purity: 98.74%. m.p.: 195−201 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 7.84−7.77 (m, 2H), 7.66−7.52 (m,

3H), 7.39 (t, J = 8.2 Hz, 2H), 6.06 (s, 1H), 5.08 (s, 2H), 2.38 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆) δ 163.57, 163.27, 159.70, 157.20,

N-(4-Fluorophenyl)-5-methyl-2-(propylthio)-[1,2,4]triazolo[1,5-

a]pyrimidin-7-amine (23). White solid, yield: 83%. HPLC purity:

1

99.57%. m.p.: 187−192 °C. H NMR (400 MHz, CDCl₃) δ 7.66 (s,

1H), 7.39−7.30 (m, 2H), 7.23−7.14 (m, 2H), 6.14 (s, 1H), 3.31−

3.20 (m, 2H), 2.49 (s, 3H), 1.91−1.77 (m, 2H), 1.05 (t, J = 7.4 Hz,

3H). ¹³C NMR (100 MHz, CDCl₃) δ 167.26, 164.67, 162.46, 160.01,

155.92, 144.67, 131.60, 126.54, 126.46, 117.09, 116.86, 88.62, 33.44,

25.33, 23.28, 13.34. HRMS (ESI): m/z calcd for C₁₅H₁₇FN₅S (M +

H)⁺, 318.1189; found, 318.1178.

2-(Allylthio)-N-(4-ﬂuorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]-

pyrimidin-7-amine (24). White solid, yield: 81%. HPLC purity:

1

99.17%. m.p.: 203−208 °C. H NMR (400 MHz, DMSO-d₆) δ 7.43

(m, 2H), 7.28 (m, 2H), 6.20 (s, 1H), 5.99 (m, 1H), 5.31 (dd, J =

17.0, 1.2 Hz, 1H), 5.08 (d, J = 10.0 Hz, 1H), 3.90 (d, J = 6.9 Hz, 2H),

2.33 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.32, 163.95,

155.78, 145.25, 133.96, 132.97, 127.02, 126.93, 118.06, 116.38,

116.15, 89.12, 33.27, 24.64. HRMS (ESI): m/z calcd for C₁₅H₁₅FN₅S

(M + H)⁺, 316.1032; found, 316.1031.

N-(4-Fluorophenyl)-5-methyl-2-(prop-2-yn-1-ylthio)-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-amine (25). White solid, yield: 74%.

HPLC purity: 99.86%. m.p.: 201−205 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.57−7.45 (m, 2H), 7.37 (m, 2H), 6.38 (s, 1H), 4.17 (d,

J = 2.5 Hz, 2H), 3.28 (t, J = 2.5 Hz, 1H), 2.45 (s, 3H). ¹³C NMR

(100 MHz, DMSO-d₆) δ 164.05, 162.20, 161.15, 159.39, 152.95,

146.56, 127.42, 127.33, 116.64, 116.41, 90.33, 79.95, 74.17, 22.37,

19.26. HRMS (ESI): m/z calcd for C₁₅H₁₃FN₅S (M + H)⁺, 314.0876;

found, 314.0877.

2-((Cyclopropylmethyl)thio)-N-(4-ﬂuorophenyl)-5-methyl-

[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (26). White solid, yield:

80%. HPLC purity: 98.10%. m.p.: 175−182 °C. ¹H NMR (400

MHz, CDCl₃) δ 7.68 (s, 1H), 7.35 (dd, J = 8.8, 4.7 Hz, 2H), 7.19 (t, J

= 8.5 Hz, 2H), 6.14 (s, 1H), 3.24 (d, J = 7.3 Hz, 2H), 2.49 (s, 3H),

1.29−1.18 (m, 1H), 0.65−0.55 (m, 2H), 0.40−0.30 (m, 2H). ¹³C

NMR (100 MHz, CDCl₃) δ 167.27, 164.70, 162.47, 160.01, 155.89,

144.68, 131.63, 131.60, 126.55, 126.47, 117.09, 116.86, 88.65, 37.39,

25.34, 11.19, 5.92. HRMS (ESI): m/z calcd for C₁₆H₁₇FN₅S (M +

H)⁺, 330.1189; found, 330.1177.

154.80, 150.53, 145.59, 130.68, 130.31, 125.91, 113.91, 112.89,

112.67, 89.95, 25.29, 23.58. HRMS (ESI): m/z calcd for C₂₀H₁₆F₂N₇S

(M + H)⁺, 424.1156; found, 424.1157.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(3-chloro-4-ﬂuo-

rophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (33).

White solid, yield: 90%. HPLC purity: 99.75%. m.p.: 183−189 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 7.85−7.75 (m, 3H), 7.63−7.50

(m, 4H), 6.46 (s, 1H), 5.09 (s, 2H), 2.41 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆) δ 163.08, 162.43, 156.98, 154.53, 154.22, 150.49,

145.85, 133.15, 130.64, 127.49, 126.22, 126.14, 125.93, 120.22,

120.03, 117.79, 117.57, 113.97, 90.40, 25.12, 23.10. HRMS (ESI): m/

z calcd for C₂₀H₁₆ClFN₇S (M + H)⁺, 440.0860; found, 440.0860.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-N-(2,4,5-

triﬂuorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (34).

White solid, yield: 89%. HPLC purity: 99.22%. m.p.: 210−216 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 7.94−7.75 (m, 4H), 7.60−7.49

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(4-ﬂuorophenyl)-

5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (27). White solid,

yield: 91%. HPLC purity: 99.33%. m.p.: 207−215 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 7.63 (m, 2H), 7.49 (m, 2H), 7.43−7.30 (m, 4H),

(m, 2H), 6.21 (d, J = 1.5 Hz, 1H), 5.07 (s, 2H), 2.38 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 163.49, 163.12, 156.05, 154.87,

150.56, 145.62, 140.20, 130.68, 130.54, 125.92, 117.85, 117.65,

L

https://dx.doi.org/10.1021/acs.jmedchem.0c01741

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

116.86, 113.96, 107.41, 107.19, 90.36, 25.23, 23.66. HRMS (ESI): m/

z calcd for C₂₀H₁₅F₃N₇S (M + H)⁺, 442.1062; found, 442.1062.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(4-ﬂuoro-3-meth-

ylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (35).

White solid, yield: 88%. HPLC purity: 99.17%. m.p.: 193−198 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 7.86−7.77 (m, 2H), 7.58−7.51

(m, 2H), 7.45−7.39 (m, 1H), 7.39−7.28 (m, 2H), 6.33 (s, 1H), 5.09

(s, 2H), 2.40 (s, 3H), 2.31 (d, J = 1.5 Hz, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 163.08, 161.61, 160.45, 158.03, 153.80, 150.50, 146.30,

131.52, 130.67, 128.67, 128.62, 125.94, 125.78, 125.60, 125.05,

124.97, 116.09, 115.86, 113.97, 90.12, 25.16, 22.74, 14.25, 14.22.

HRMS (ESI): m/z calcd for C₂₁H₁₉FN₇S (M + H)⁺, 420.1407; found,

420.1407.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(2-ﬂuoro-4-meth-

ylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (36).

White solid, yield: 83%. HPLC purity: 98.82%. m.p.: 202−207 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 7.84−7.77 (m, 2H), 7.58−7.51

(m, 2H), 7.44 (t, J = 8.2 Hz, 1H), 7.31 (d, J = 11.3 Hz, 1H), 7.20 (d, J

= 7.6 Hz, 1H), 6.06 (d, J = 1.7 Hz, 1H), 5.08 (s, 2H), 2.41 (s, 3H),

2.39 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.22, 162.25,

157.94, 155.47, 154.08, 150.44, 146.20, 140.29, 140.22, 130.65,

128.90, 125.93, 120.24, 120.11, 117.26, 117.07, 113.94, 90.14, 30.65,

25.21, 22.97, 20.62. HRMS (ESI): m/z calcd for C₂₁H₁₉FN₇S (M +

H)⁺, 420.1407; found, 420.1408.

MHz, DMSO-d₆) δ 8.07 (d, J = 8.5 Hz, 2H), 7.81 (m, 2H), 7.69 (d, J

= 8.5 Hz, 2H), 7.54 (dd, J = 6.2, 3.1 Hz,2H), 6.64 (s, 1H), 5.11 (s,

2H), 2.44 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.64,

163.04, 162.70, 154.38, 150.60, 145.01, 140.26, 130.67, 130.64,

128.25, 125.94, 123.94, 113.96, 90.88, 25.15, 23.25. HRMS (ESI): m/

z calcd for C₂₁H₁₆N₇O₂S (M − H)⁺, 430.1086; found, 430.1094.

4-((2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-yl)amino)benzonitrile (43). White solid,

yield: 89%. HPLC purity: 98.96%. m.p.: 236−239 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 7.96 (d, J = 8.6 Hz, 2H), 7.85−7.73 (m, 4H), 7.51

(m, 2H), 6.68 (s, 1H), 5.08 (s, 2H), 2.42 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆) δ 163.02, 154.48, 150.51, 144.49, 140.88, 133.59,

130.55, 125.86, 124.23, 118.54, 113.88, 107.87, 91.25, 25.04, 23.34.

HRMS (ESI): m/z calcd for C₂₁H₁₅N₈S (M − H)⁺, 411.1140; found,

411.1148.

1-(4-((2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-

[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)amino)phenyl)ethan-1-one

(44). White solid, yield: 82%. HPLC purity: 99.44%. m.p.: 183−186

°C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.05 (d, J = 8.4 Hz, 2H), 7.63

(d, J = 8.4 Hz, 2H), 7.56 (m, 2H), 7.22 (m, 2H), 6.65 (s, 1H), 4.84

(s, 2H), 2.60 (s, 3H), 2.44 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

δ 196.70, 163.90, 161.23, 155.90, 150.70, 143.95, 141.51, 133.52,

129.70, 122.80, 122.45, 114.74, 90.67, 27.75, 26.62, 24.69. HRMS

(ESI): m/z calcd for C₂₂H₂₀N₇OS (M + H)⁺, 430.1450; found,

430.1451.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(2,4-dichloro-

phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (45).

White solid, yield: 76%. HPLC purity: 96.90%. m.p.: 170−176 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (d, J = 1.5 Hz, 1H), 7.84−

7.76 (m, 2H), 7.66−7.60 (m, 2H), 7.58−7.51 (m, 2H), 6.00 (s, 1H),

5.08 (s, 2H), 2.36 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ

163.11, 162.90, 154.57, 150.54, 145.72, 133.32, 132.54, 132.21,

131.24, 130.67, 130.05, 128.81, 125.92, 113.95, 90.37, 25.30, 23.35.

HRMS (ESI): m/z calcd for C₂₀H₁₆Cl₂N₇S (M + H)⁺, 456.0565;

found, 456.0564.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-N-(4-

(triﬂuoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(37). White solid, yield: 89%. HPLC purity: 97.45%. m.p.: 185−189

1

°C. H NMR (400 MHz, DMSO-d₆) δ 7.93 (s, 1H), 7.86 (d, J = 7.8

Hz, 1H), 7.83−7.70 (m, 4H), 7.58−7.49 (m, 2H), 6.52 (s, 1H), 5.10

(s, 2H), 2.42 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.04,

162.81, 154.50, 150.57, 145.42, 137.07, 130.79, 130.65, 130.36,

130.05, 128.60, 125.93, 125.12, 123.08, 122.41, 121.55, 113.95, 90.39,

25.14, 23.35. HRMS (ESI): m/z calcd for C₂₁H₁₇F₃N₇S (M + H)⁺,

456.1218; found, 456.1219.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-N-(4-

(triﬂuoromethoxy)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(38). White solid, yield: 82%. HPLC purity: 99.07%. m.p.: 203−207

°C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.81 (m, 2H), 7.66 (d, J = 8.9

Hz, 2H), 7.54 (m, 4H), 6.48 (s, 1H), 5.09 (s, 2H), 2.41 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 162.94, 162.84, 154.59, 150.64,

146.20, 145.43, 135.23, 130.67, 126.69, 125.93, 124.50, 122.21,

121.34, 118.79, 113.96, 101.15, 90.12, 25.16, 23.41. HRMS (ESI): m/

z calcd for C₂₁H₁₇F₃N₇S (M + H)⁺, 456.1218; found, 456.1213.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-N-phenyl-

[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (39). White solid, yield:

79%. HPLC purity: 99.37%. m.p.: 180−191 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 7.50 (m, 6H), 7.33 (d, J = 7.0 Hz, 1H), 7.18 (m,

2H), 6.34 (s, 1H), 4.79 (s, 2H), 2.38 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 164.03, 163.80, 155.80, 150.66, 144.94, 136.53, 129.48,

126.12, 124.44, 122.01, 114.75, 89.29, 28.00, 24.60. HRMS (ESI): m/

z calcd for C₂₀H₁₆N₇S (M − H)⁺, 386.1188; found, 386.1196.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(4-chlorophenyl)-

5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (40). White solid,

yield: 81%. HPLC purity: 97.17%. m.p.: 213−217 °C. ¹H NMR (400

MHz, DMSO-d₆) δ 7.78 (m, 2H), 7.61−7.48 (m, 6H), 6.45 (s, 1H),

5.07 (s, 2H), 2.39 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ

163.11, 161.84, 153.87, 150.47, 145.79, 134.79, 131.02, 130.63,

129.53, 126.89, 125.94, 113.95, 90.39, 25.12, 22.77. HRMS (ESI): m/

z calcd for C₂₀H₁₅ClN₇S (M − H)⁺, 420.0798; found, 420.0807.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(4-bromophenyl)-

5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (41). White solid,

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-N-(p-

tolyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (46). White solid,

yield: 86%. HPLC purity: 99.06%. m.p.: 240−247 °C. ¹H NMR

(400 MHz, DMSO-d₆) δ 7.79 (s, 2H), 7.53 (s, 2H), 7.37 (t, J = 6.7

Hz, 4H), 6.37 (s, 1H), 5.09 (s, 2H), 2.41 (s, 3H), 2.37 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 163.37, 152.43, 150.23, 146.66,

136.98, 132.59, 130.62, 130.10, 125.96, 125.35, 125.17, 113.96, 90.51,

25.10, 21.51, 20.64. HRMS (ESI): m/z calcd for C₂₁H₁₈N₇S (M −

H)⁺, 400.1344; found, 400.1352.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(4-methoxyphen-

yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (47). White

solid, yield: 82%. HPLC purity: 99.14%. m.p.: 213−219 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 7.84−7.78 (m, 2H), 7.59−7.51 (m,

2H), 7.50−7.43 (m, 1H), 7.40 (dd, J = 7.8, 1.5 Hz, 1H), 7.27 (d, J =

7.7 Hz, 1H), 7.11 (dd, J = 11.8, 4.3 Hz, 1H), 5.93 (s, 1H), 5.08 (s,

2H), 3.80 (s, 3H), 2.37 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ

163.18, 161.22, 154.32, 153.69, 150.54, 146.35, 130.72, 129.46,

128.23, 125.93, 123.35, 120.96, 113.98, 112.95, 90.48, 55.72, 25.30,

22.65. HRMS (ESI): m/z calcd for C₂₁H₂₀N₇OS (M + H)⁺,

418.1450; found, 418.1451.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(3-methoxyphen-

yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (48). White

solid, yield: 80%. HPLC purity: 98.55%. m.p.: 203−208 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 7.81 (m, 2H), 7.54 (m, 2H), 7.45 (t, J

= 8.1 Hz, 1H), 7.11 (m, 2H), 6.98 (m, 1H), 6.48 (s, 1H), 5.10 (s,

2H), 3.82 (s, 3H), 2.43 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ

163.10, 161.54, 160.00, 153.72, 150.50, 145.92, 136.81, 130.65,

130.37, 125.93, 116.95, 113.96, 112.47, 110.99, 90.47, 55.98, 55.33,

25.14, 22.67, 18.52. HRMS (ESI): m/z calcd for C₂₁H₂₀N₇OS (M +

H)⁺, 418.1450; found, 418.1451.

1

yield: 78%. HPLC purity: 100%. m.p.: 206−210 °C. H NMR (400

MHz, DMSO-d₆) δ 7.66 (d, J = 8.7 Hz, 2H), 7.56−7.47 (m, 2H), 7.42

(d, J = 8.7 Hz, 2H), 7.15 (s, 1H), 6.42 (s, 1H), 4.77 (s, 2H), 2.40 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.24, 164.06, 155.84,

150.61, 144.67, 136.19, 132.38, 126.38, 121.74, 118.26, 89.70, 28.34,

24.66. HRMS (ESI): m/z calcd for C₂₀H₁₇BrN₇S (M + H)⁺,

466.0450; found, 466.0277.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-N-(3,4,5-

trimethoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (49).

White solid, yield: 88%. HPLC purity: 99.64%. m.p.: 228−235 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 7.80 (m, 2H), 7.53 (m, 2H), 6.84

4-((2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-yl)amino)benzoic acid (42). White solid,

yield: 85%. HPLC purity: 98.45%. m.p.: 230−235 °C. ¹H NMR (400

(s, 2H), 6.52 (s, 1H), 5.08 (s, 2H), 3.81 (s, 6H), 3.72 (s, 3H), 2.41 (s,

M

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3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.05, 153.91, 153.24,

150.54, 145.99, 136.19, 131.32, 130.66, 125.93, 113.97, 103.01, 90.51,

60.09, 56.07, 25.16, 22.84. HRMS (ESI): m/z calcd for C₂₃H₂₂N₇O₃S

(M − H)⁺, 476.1505; found, 476.1513.

fecting the HEK293 cells with ABCB1 expression vector or empty

vector and were cultured in a medium with puromycin (1 μg/mL). All

cell lines were maintained in the Dulbecco’s modiﬁed Eagle’s medium

(DMEM), containing 10% fetal bovine serum and 1% penicillin and

streptomycin at 37 °C with 5% CO₂.

MTT Assay. The MTT assay was performed to detect the

sensitivity of cells to anticancer drugs in vitro.⁴⁹Brieﬂy, 3000 cells

were seeded in each well of a 96-well plate. After attachment to the

bottom of the well, cells were preincubated with or without the

reversal agents (100 μL/well) for 4 h, and then diﬀerent

concentrations of paclitaxel were added into designated wells. After

72 h of incubation, 20 μL of MTT solution (5 mg/mL) was added to

each well for cell staining. After 4 h, the medium was removed and

150 μL of DMSO was added to each well. Finally, the absorbance was

determined at 570 nm using an ELx 800 Universal microplate reader

(Bio-Tek, Inc.). IC₅₀was calculated using SPSS software. The data

4-((2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-yl)amino)phenol (50). White solid, yield:

1

76%. HPLC purity: 100%. m.p.: 206−212 °C. H NMR (400 MHz,

DMSO-d₆) δ 7.82 (m, 2H), 7.54 (m, 2H), 7.28 (d, J = 8.7 Hz, 2H),

6.97 (d, J = 8.7 Hz, 2H), 6.27 (s, 1H), 5.10 (s, 2H), 2.42 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 163.26, 159.57, 157.01, 152.51,

150.28, 147.00, 130.65, 127.13, 125.95, 116.18, 113.97, 90.31, 25.17,

21.60. HRMS (ESI): m/z calcd for C₂₀H₁₈N₇OS (M + H)⁺,

404.1294; found, 404.1293.

4-((2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-yl)amino)-2-chlorophenol (51). White

solid, yield: 83%. HPLC purity: 95.75%. m.p.: 218−225 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 7.82 (m, 2H), 7.54 (m, 2H), 7.46 (d,

J = 2.5 Hz, 1H), 7.26 (m, 1H), 7.20 (d, J = 8.7 Hz, 1H), 6.30 (s, 1H),

5.08 (s, 2H), 2.40 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ

163.08, 161.25, 154.07, 153.57, 152.61, 150.47, 146.59, 130.68,

127.35, 127.11, 125.95, 125.76, 119.95, 117.12, 113.98, 90.20, 25.21,

22.53. HRMS (ESI): m/z calcd for C₂₀H₁₇ClN₇OS (M + H)⁺,

438.0904; found, 438.0905.

were shown as the mean

experiments.

SD was obtained by three dependent

Docking Analysis. To investigate the interaction modes between

compounds and human ABCB1, molecular docking studies were

conducted using the AutoDock Tools package (version 1.5.6).⁵⁰The

model of human ABCB1 was obtained from homology modeling on

the SWISS-MODEL server.⁵¹The apo form of mouse ABCB1 in the

inward-open conformation (PDB code: 4Q9H), which could bind to

a series of cyclic peptides such as QZ-Ala (PDB code: 4Q9I), QZ-Val

(4Q9J), QZ-Leu (4Q9K), and QZ-Phe (4Q9L),⁴⁰was used as the

template for human ABCB1 modeling. In the docking simulation,

WS-36 and WS-691 were used as ligands, while the human ABCB1

model was used as the receptor. The structures of ABCB1 and

compounds were optimized and prepared to the pdbqt format ﬁles

used for docking by AutoDock tools. For the human ABCB1 model,

ﬁve grids ﬁles with varying xyz coordinates (70.95/−4.65/−9.74,

48.43/1.88/1.32, 76.87/13.42/5.17, 40.68/7.98/17.71, and 75.17/

29.6/27.6) were generated to probe the whole structure of ABCB1,

except for two ATPase domains. The dimensions of the docking grid

were 20 Å × 20 Å × 20 Å. All of the other parameters were set to the

default values. After this, WS-36 and WS-691 were docked to the

receptor human ABCB1 model with 20 complexes output per docking

run, and the results were ranked based on the value of their binding

energies. The most minimal energy complex for each compound was

then selected as the reliable binding mode. All results were analyzed

and visualized using PyMOL (http://www.pymol.org).

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-N-(3-ethynylphen-

yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (52). White

solid, yield: 78%. HPLC purity: 98.92%. m.p.: 226−231 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 7.86−7.79 (m, 2H), 7.63 (s, 1H),

7.61−7.46 (m, 5H), 6.45 (s, 1H), 5.10 (s, 2H), 4.37 (s, 1H), 2.42 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.07, 162.10, 154.06,

150.53, 145.76, 136.27, 130.65, 130.05, 128.01, 125.94, 125.73,

122.98, 113.96, 90.37, 82.66, 81.84, 25.13, 22.96. HRMS (ESI): m/z

calcd for C₂₂H₁₈N₇S (M + H)⁺, 412.1344; found, 412.1345.

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-N-(4-(4-

methylpiperazin-1-yl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-

amine (53). White solid, yield: 83%. HPLC purity: 97.52%. m.p.:

1

168−172 °C. H NMR (400 MHz, DMSO-d₆) δ 7.77 (m, 2H), 7.50

(m, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.17 (s,

1H), 5.03 (s, 2H), 3.87 (s, 2H), 3.51 (s, 2H), 3.19 (s, 4H), 2.83 (s,

3H), 2.34 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.80,

162.68, 155.85, 150.89, 148.12, 145.69, 131.74, 128.16, 126.26,

125.37, 116.58, 114.07, 89.18, 51.98, 45.33, 41.87, 25.57, 24.43.

HRMS (ESI): m/z calcd for C₂₅H₂₈N₉S (M + H)⁺, 486.2188; found,

486.2148.

Western Blotting. Cells were lysed on the ice for 30 min in RIPA

buﬀer (Beyotime Biotechnology, Shanghai, China) with complete

protease inhibitors (Biotool, Houston, TX). After centrifugation, the

samples were loaded onto sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE) and electrophoretically transferred onto

poly(vinylidene ﬂuoride) (PVDF) membranes. The membranes were

blocked by 5% bovine serum albumin (BSA) in TBST and then

incubated with the primary antibody at 4 °C overnight. The next day,

the membranes were incubated with secondary antibody for 2 h at

room temperature. The protein−antibody complex was detected by

enhanced chemiluminescence.

7-((2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-yl)amino)-4-methyl-2H-chromen-2-one

(54). White solid, yield: 87%. HPLC purity: 98.20%. m.p.: 202−211

°C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.88 (d, J = 9.2 Hz, 1H), 7.81

(m, 2H), 7.60 (m, 2H), 7.54 (m, 2H), 6.73 (s, 1H), 6.42 (d, J = 1.1

Hz, 1H), 5.11 (s, 2H), 2.47 (d, J = 0.8 Hz, 3H), 2.45 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆) δ 163.23, 163.00, 159.64, 154.70,

153.51, 152.89, 150.63, 144.64, 139.68, 130.64, 126.38, 125.93,

119.81, 117.33, 113.95, 113.59, 111.16, 91.15, 25.15, 23.56, 18.06.

HRMS (ESI): m/z calcd for C₂₄H₂₀N₇O₂S (M + H)⁺, 470.1399;

found, 470.1355.

Immunoﬂuorescence Assay. Around 5000 cells were planted on

the coverslip in the 24-well plate and incubated with WS-691 at 20

μM for 72 h. The cells were ﬁxed by 4% formaldehyde, permeabilized

by 0.1% Triton X-100, and blocked with 5% bull serum albumin

before being incubated with primary antibody at 4 °C overnight. After

rinsing, the cells were incubated with Alexa Fluor 488 rabbit anti-goat

IgG for 2 h and Hoechst 33342. A laser scanning confocal microscope

(Nikon A1R, Tokyo, Japan) was used to collect ﬂuorescence

images.⁵²

UPLC. The intracellular concentration of paclitaxel was detected by

ultra-performance liquid chromatography (UPLC).³²Then, 5 × 10⁶

SW620/Ad300 cells were cultured in the medium in the presence or

absence of WS-691 (10 and 20 μM) or verapamil (4 μM) for 72 h.

Then, the medium was replaced with fresh medium with 20 μM PTX

for another 3 h incubation. Subsequently, the cells were washed with

cold phosphate-buﬀered saline (PBS) and resuspended in 1 mL of

PBS. The cells were lysed by sonication and paclitaxel was extracted

2-(((1H-Benzo[d]imidazol-2-yl)methyl)thio)-5-methyl-N-(5-

methyl-1H-pyrazol-3-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine

(55). White solid, yield: 87%. HPLC purity: 97.87%. m.p.: 206−213

°C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.80 (m, 2H), 7.61−7.47 (m,

2H), 7.39 (s, 1H), 6.39 (s, 1H), 5.11 (s, 2H), 2.49 (s, 3H), 2.30 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 162.76, 161.77, 153.58,

150.63, 145.91, 143.80, 139.50, 130.63, 125.92, 124.50, 113.93, 97.33,

92.49, 55.98, 25.12, 23.09, 18.51, 10.60. HRMS (ESI): m/z calcd for

C₁₈H₁₈N₉S (M + H)⁺, 392.1406; found, 392.1402.

Cell Culture. The human colon cancer cell line SW620 and its

MDR cell line, SW620/Ad300, were kindly supplied by Dr. Susan

Bates’s lab (NIH, MD). The ABCB1-overexpressed KB-C2 cell line

was established by a step-wise exposure of KB-3-1 cells, a parental

human epidermoid carcinoma cell line, to increasing concentrations

(up to 2 μg/mL) of colchicine.^47,48The human primary embryonic

kidney cell line HEK293’s ABCB1 stable gene-transfected cell line

HEK293/ABCB1 and HEK293/pcDNA3.1 were obtained by trans-

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with ethyl acetate. The standard curve was established using paclitaxel

standard samples at a concentration range of 31−8000 ng/mL by

UPLC. The retention time was 1.56 min. Finally, the concentration of

intracellular PTX was calculated by a standard curve of UPLC.

[³H]-PTX Accumulation and Eﬄux Assay. The accumulation

assay was performed as previously described.⁵³The accumulation of

[³H]-PTX in SW620 and SW620/Ad300 was detected in the presence

or absence of verapamil or WS-691. The cells were incubated with

WS-691 at 20 μM at or VPM at 4 μM, respectively, for 4 h at 37 °C,

followed by the addition of 0.1 μM [³H]-PTX for another 2 h, which

was then lysed and placed in 5 mL of scintillation liquid after washing

with cold PBS three times. The Packard TRICARB 1900CA liquid

scintillation analyzer (Packard Instrument, Downers Grove, IL) was

used to detect the radioactivity. As for the [³H]-PTX eﬄux assay, after

the addition of 0.1 μM [³H]-PTX for 2 h, the cells were incubated in

the [³H]-free medium for another 2 h, during which each radioactivity

data were collected at 0, 30, 60, and 120 min, respectively.

ATPase Assay. The ATPase assay was performed as previously

described.⁵⁴ABCB1 crude membrane vesicles were purchased from

BD Biosciences (San Jose, CA), and 10 μg of membrane was

incubated with or without sodium orthovanadate (0.3 mM) in assay

buﬀer for 3 min. After that, 0−40 μM varying concentration of WS-

691 was added to the assay buﬀer, respectively. The reaction was

initiated upon adding 5 mM of Mg-ATP, followed by incubation at 37

°C for 20 min, and then was terminated by adding 100 μL 5% SDS

solution. The ATPase activity due to ABCB1 was determined from

the amount of inorganic phosphate (Pi) released detected at 880 nm

using a spectrophotometer.

Cellular Thermal Shift Assay (CETSA). Typically, SW620/

Ad300 cells (approximately 3.2 × 10⁷) were collected, washed, and

then resuspended with PBS. The cell suspensions were freeze-thawed

ﬁve times using liquid nitrogen. The lysate was separated by

centrifugation at 15 000g for 20 min at 4 °C. The cell lysates were

divided into two aliquots, with one aliquot being treated with WS-691

(200 μM) and the other aliquot with an equal amount of DMSO (1%,

v/v). After 30 min incubation at room temperature, the respective

lysates were divided into nine aliquots and heated individually at

diﬀerent temperatures for 3 min. The heated lysates were centrifuged

at 20 000g for 20 min at 4 °C. The supernatants were transferred to

new microtubes and analyzed by Western blotting analysis.

CYP3A4 Assay. The cytochrome P4503A4 (CYP3A4) activity

assay was performed to study the eﬀect of WS-691 and VPM on

CYP3A4, exactly according to the instruction of the kit (Catalog #

K701, Biovision). Ketoconazole was used as a positive CYP3A4

inhibitor control. Brieﬂy, the compound (Ketoconazole, WS-691, or

VPM), P450 Reaction Mix and CYP3A4 Assay Buﬀer were mixed and

incubated for 10 min. The reaction was started by adding the

CYP3A4 substrate/NADP⁺, and the ﬂuorescence was immediately

measured every 2 minutes at Ex/Em = 531/595 nm in kinetic mode at

37 °C.

chloric acid in alcohol (v/v). Then, the sections were stained in Eosin

solution. After being rinsed and paraﬃned, the slides were mounted in

neutral balsam.

TUNEL Assay. TUNEL staining was performed according to the

manufacturer’s instructions (Dalian Meilun Biotechnology, Dalian,

China). Brieﬂy, tumor tissue sections were incubated with proteinase

K (20 μg/mL) for 20 min at RT, then were rinsed with PBS. The

sections were subsequently incubated with a buﬀer containing TdT

enzyme and FITC-12-dUTP for 1 h at 37 °C in the dark conditions.

The sections were sealed with antiﬂuorescence attenuation sealant

and the ﬂuorescence of positive staining was examined using a

ﬂuorescence microscope.

Statistical Analysis. All experiments were repeated at least three

times, and the diﬀerences were determined using the two-tailed

Student’s t-test and statistical signiﬁcance was determined at P < 0.05.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01741.

■

sı

Cell survival rates under the nontoxic concentration of

compounds toward selected cancer cells NMR, HRMS,

and HPLC spectra of compounds 5−55 (PDF)

Coordinates of modeled structures in PDB format: WS-

36 in complex with ABCB1 (PDB code: 4Q9H) (PDB)

Coordinates of modeled structures in PDB format: WS-

691 in complex with ABCB1 (PDB code: 4Q9H)

(PDB)

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Bin Yu − School of Pharmaceutical Sciences & Key Laboratory

of Advanced Drug Preparation Technologies, Ministry of

Education, Zhengzhou University, Zhengzhou 450001,

China; orcid.org/0000-0002-7207-643X; Phone: +86-

0371-67781908; Email: yubin@zzu.edu.cn; Fax: +86-

0371-67781908

Zhe-Sheng Chen − College of Pharmacy and Health Sciences,

St. John’s University, Queens, New York 11439, United

States; orcid.org/0000-0002-8289-097X; Phone: +1-

718-990-1432; Email: chenz@stjohns.edu; Fax: +1-718-

990-1877

Hong-Min Liu − School of Pharmaceutical Sciences & Key

Laboratory of Advanced Drug Preparation Technologies,

Ministry of Education, Zhengzhou University, Zhengzhou

450001, China; orcid.org/0000-0001-6771-9421;

Phone: +86-0371-67781908; Email: liuhm@zzu.edu.cn;

Fax: +86-0371-67781908

Xenograft Studies. Animals were treated according to protocols

established by the ethics committee of Zhengzhou University, and the

in vivo experiments were carried out in accordance with the approved

guidelines and approved by the ethics committee of Zhengzhou

University. Brieﬂy, male BALB/c nude mice (15−17 g, aged 5−6

weeks, Hunan SJA Laboratory Animal Co., Ltd, Hunan, China) were

housed in individually ventilated cages and maintained with food and

sterile water. Approximately 8 × 10⁶SW620/Ad300 cells were

inoculated subcutaneously into the right forelimb of each mouse. The

mice were randomized into ﬁve groups (n = 6) and treated with one

of the following regimens: (i) vehicle (containing 90% corn oil, 5%

castor oil, and 5% DMSO, 10 mL/kg/day, i.g.); (ii) PTX (5 mg/kg/3

days, i.v.); (iii) WS-691 (20 mg/kg/day, i.g.); (iv) PTX plus VPM

hydrochloride (6 mg/kg/day, i.g.); (v) PTX plus WS-691. Following

21-day treatment, animals were euthanized by carbon dioxide, and

tumor and liver tissue were ﬁxed in 4% paraformaldehyde for

immunohistochemistry.

Authors

Shuai Wang − School of Pharmaceutical Sciences & Key

Laboratory of Advanced Drug Preparation Technologies,

Ministry of Education, Zhengzhou University, Zhengzhou

450001, China

Sai-Qi Wang − Department of Oncology, The Aﬃliated Cancer

Hospital of Zhengzhou University, Henan Cancer Hospital,

Henan Cancer Institute, Zhengzhou 450008, China

Qiu-Xu Teng − College of Pharmacy and Health Sciences, St.

John’s University, Queens, New York 11439, United States

Linlin Yang − Department of Pharmacology, School of Basic

Medical Sciences, Zhengzhou University, Zhengzhou 450001,

China

HE Staining. Histopathology analysis was performed using HE

staining. Brieﬂy, tissue sections were deparaﬃnized and stained by

Hematoxylin before being rinsed and diﬀerentiated by 1% hydro-

O

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Zi-Ning Lei − College of Pharmacy and Health Sciences, St.

John’s University, Queens, New York 11439, United States

Xiao-Han Yuan − School of Pharmaceutical Sciences & Key

Laboratory of Advanced Drug Preparation Technologies,

Ministry of Education, Zhengzhou University, Zhengzhou

450001, China

Jun-Feng Huo − School of Pharmaceutical Sciences & Key

Laboratory of Advanced Drug Preparation Technologies,

Ministry of Education, Zhengzhou University, Zhengzhou

450001, China

Xiao-Bing Chen − Department of Oncology, The Aﬃliated

Cancer Hospital of Zhengzhou University, Henan Cancer

Hospital, Henan Cancer Institute, Zhengzhou 450008, China

Mengru Wang − Department of Pharmacology, School of

Basic Medical Sciences, Zhengzhou University, Zhengzhou

450001, China

chromatography; PBS, phosphate-buﬀered saline; Pi, inorganic

phosphate; DMSO, dimethyl sulfoxide; i.v., intravenous

injection; i.g., intragastrical injection; RT, room temperature

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■

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Author Contributions

^⊥S.W., S.-Q.W., Q.-X.T., and L.Y. contributed equally to this

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Notes

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The authors declare no competing ﬁnancial interest.

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Xavier, C. P. R.; Vasconcelos, M. H. The multi-factorial nature of

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ACKNOWLEDGMENTS

■

We would like to acknowledge ﬁnancial support from the

National Natural Science Foundation of China (Nos.

81773562, 81973177, and 81703326), the Program for Science

& Technology Innovation Talents in Universities of Henan

Province (No. 21HASTIT045), the China Postdoctoral

Science Foundation (Nos. 2018M630840, and

2019T120641), Medical Science and Technique Foundation

of Henan Province (Nos. 2018020486 and SB201901101), the

Science and Technique Foundation of Henan Province (No.

202102310413), and 1000 Talents Program of Central plains

(No. 204200510023). We appreciate Drs. Susan E. Bates and

Robert Robey for the SW620 and SW620/Ad300 cell lines

used in the study (NIH, Bethesda, MD). We thank Dr. Shin-

Ichi Akiyama for KB-3-1 and KB-C2 cell lines (Kagoshima

University, Kagoshima, Japan). Besides, we are thankful to Dr.

Tanaji T. Talele (St. John’s University, New York, NY) for

providing the computing resources for docking analysis.

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ABBREVIATIONS USED

■

ABC, ATP-binding Cassette; ABCB1, MDR1, or P-glyco-

protein, ATP-binding cassette subfamily B member 1; ATP,

adenosine triphosphate; NBDs, nucleotide-binding domains;

TMDs, transmembrane domains; MDR, multidrug resistance;

ATP, adenosine triphosphate; CYP3A4, cytochrome P3A4;

PTX, paclitaxel; RF, reversal fold; VPM, verapamil; SARs,

structure−activity relationships; IFD, induce-ﬁt docking; XP,

the Glide extra precision; CETSA, cellular thermal shift assay;

Cpd., compound; TLC, thin-layer chromatography; ESI,

electrospray ionization; HPLC, high-performance liquid

chromatography; NIH, National Institutes of Health; MTT,

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

DMEM, Dulbecco’s modiﬁed Eagle’s medium; PDB, protein

data bank; SDS-PAGE, sodium dodecyl sulfate polyacrylamide

gel electrophoresis; BSA, bovine serum albumin; PVDF,

electrophoretically transferred onto polyvinylidene ﬂuoride;

HRP, horseradish peroxide; UPLC, ultra-performance liquid

P

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Article Doi

Structure-Based Design, Synthesis, and Biological Evaluation of New Triazolo[1,5- a]Pyrimidine Derivatives as Highly Potent and Orally Active ABCB1 Modulators

DOI: 10.1021/acs.jmedchem.0c01741

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