Design, synthesis and pharmacological evaluation of tricyclic derivatives as selective RXFP4 agonists

Bioorganic Chemistry 110 (2021) 104782

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Bioorganic Chemistry

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

Design, synthesis and pharmacological evaluation of tricyclic derivatives as

selective RXFP4 agonists

,

Lin Lin^a, Guangyao Lin^{b c}, Qingtong Zhou^d, Ross A.D. Bathgate^e, Grace Qun Gong^f,

,

f

,

,b,c,d,f,*

Dehua Yang^{c *}, Qing Liu^{f *}, Ming-Wei Wang^a

^aSchool of Pharmacy, Fudan University, Shanghai 201203, China

^bSchool of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China

^cUniversity of Chinese Academy of Sciences, Beijing 100049, China

^dSchool of Basic Medical Sciences, Fudan University, Shanghai 200032, China

^eFlorey Institute of Neuroscience and Mental Health, Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC 3052, Australia

^fThe National Center for Drug Screening and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS),

Shanghai 201203, China

A R T I C L E I N F O

A B S T R A C T

Keywords:

Relaxin family peptide receptors (RXFPs) are the potential therapeutic targets for neurological, cardiovascular,

and metabolic indications. Among them, RXFP3 and RXFP4 (formerly known as GPR100 or GPCR142) are ho-

mologous class A G protein-coupled receptors with short N-terminal domain. Ligands of RXFP3 or RXFP4 are

only limited to endogenous peptides and their analogues, and no natural product or synthetic agonists have been

reported to date except for a scaffold of indole-containing derivatives as dual agonists of RXFP3 and RXFP4. In

this study, a new scaffold of tricyclic derivatives represented by compound 7a was disclosed as a selective RXFP4

agonist after a high-throughput screening campaign against a diverse library of 52,000 synthetic and natural

compounds. Two rounds of structural modiﬁcation around this scaffold were performed focusing on three parts:

2-chlorophenyl group, 4-hydroxylphenyl group and its skeleton including cyclohexane-1,3-dione and 1,2,4-tria-

zole group. Compound 14b with a new skeleton of 7,9-dihydro-4H-thiopyrano[3,4-d][1,2,4]triazolo[1,5-a]

pyrimidin-8(5H)-one was thus obtained. The enantiomers of 7a and 14b were also resolved with their 9-(S)-

conformer favoring RXFP4 agonism. Compared with 7a, compound 9-(S)-14b exhibited 2.3-fold higher efﬁcacy

and better selectivity for RXFP4 (selective ratio of RXFP4 vs. RXFP3 for 9-(S)-14b and 7a were 26.9 and 13.9,

respectively).

Synthesis

Structure-activity relationship

Relaxin family peptide receptor 4

Selective agonist

Molecular docking

1. Introduction

[1,10]. The cognate ligands of RXFP3 and RXFP4 are relaxin-3 and

insulin-like peptide 5 (INSL5), respectively. Relaxin-3 also activates

Relaxin family is a group of peptide hormones that perform a variety

of biological functions after activation of the relaxin family peptide re-

ceptors 1-4 (RXFPs 1-4) [1,2], such as reproduction regulation, stress

responses, food intake and glucose homeostasis, etc. These activities

enable RXFPs to be the potential therapeutic targets for neurological,

cardiovascular and metabolic disorders [3–9]. Among them, RXFP3 and

RXFP4 (formerly known as GPR100 or GPCR142) are homologous class

A G protein-coupled receptors (GPCRs) with a short N-terminal domain.

RXFP4 is predominantly expressed in the colon and rectum with im-

plications of insulin secretion, appetite and regulation of colon motility

RXFP1 and RXFP4 in vitro [8,11]. Relaxin-3 interacts with both the LRR

domain and ECL2 of the TM domain of RXFP1 to produce the full

binding and cAMP signaling proﬁles [2]. R3/I5, a chimeric peptide,

contains the B chain of relaxin-3 and the A chain of INSL5 and activates

RXFP3 and RXFP4 at almost equal potency [2]. INSL5 is a two-chain,

three-disulﬁde-bonded peptide and mainly expressed in the color-

ectum and enteric nervous system together with glucagon-like peptide 1

(GLP-1) and peptide YY. Binding of INSL5 to RXFP4 increases GTPγS

activity, inhibits forskolin-stimulated cyclic adenosine monophosphate

(cAMP) accumulation and elevates phosphorylation of ERK1/2,

* Corresponding authors at: The National Center for Drug Screening and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences (CAS), Shanghai 201203, China (D. Yang, Q. Liu, M. W. Wang).

E-mail addresses: dhyang@simm.ac.cn (D. Yang), qliu@simm.ac.cn (Q. Liu), mwwang@simm.ac.cn (M.-W. Wang).

https://doi.org/10.1016/j.bioorg.2021.104782

Received 29 November 2020; Received in revised form 7 February 2021; Accepted 23 February 2021

Available online 2 March 2021

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

p38MAPK, Akt Ser⁴⁷³, Akt Thr³⁰⁸and S6 ribosomal protein in CHO-

RXFP4 cells [1,12]. INSL5 also suppresses glucose-stimulated insulin

secretion and Ca²⁺mobilization in MIN6 insulinoma cells and forskolin-

stimulated cAMP accumulation in NCI-H716 enteroendocrine cells [12].

Despite these attractive properties, ligands of RXFP3 or RXFP4 are only

limited to endogenous peptides and their analogues, and no natural

products or synthetic agonists have been reported to date except for a

scaffold of indole-containing derivatives disclosed by DeChristopher and

colleagues as dual agonists of RXFP3 and RXFP4 (shown as compound 1

in Fig. 1) [13].

group (7a-g and 7j-l), the effect of steric-hindrance (7f-g) or the

substituted position (7a-c and 7j-l) to improve their binding to RXFP4.

Subsequent SAR studies around 4-hydroxylphenyl and cyclohexane-

1,3-dione were carried out using a similar procedure, except that

different substituted benzaldehyde 8 and 1,3-cycloalkandione 10 were

employed to obtain derivatives 9 and 11, respectively. Compounds 9a-i

were thus designed and synthesized for the purpose of examining the

necessity of 9-aromatic nucleus (9b), restriction of the number and

position for 4-hydroxyl group (9a and 9c), effect of steric hindrance (9d

and 9h) and possibility of introduction of other heteroatom instead of

oxygen (9d-e and 9g-i). Compounds 11a-d were synthesized for the

purpose of increasing the steric hindrance (11a-b) by addition of di-

methyl group and inserting heteroatom such as oxygen (11c) and

sulphur (11d), aiming at ﬁnding other possible scaffolds except for

In an attempt to discover non-peptidic small molecules as selective

RXFP4 agonists, a high-throughput screening (HTS) campaign against a

diverse library of 52,000 synthetic and natural compounds was carried

out using a homogeneous time-resolved ﬂuorescence (HTRF) assay that

measures the inhibition of forskolin-stimulated cAMP accumulation in

human RXFP3- and RXFP4-overexpressing CHO cells. This led to the

discovery of a new scaffold, 5,6,7,9-tetrahydro-[1,2,4]triazolo[5,1-b]

quinazolin-8(4H)-one (represented by 7a), as selective RXFP4 agonist as

demonstrated by its activities in pCRE activation, ERK1/2 phosphory-

lation, intracellular calcium mobilization and β-arrestin recruitment

[14]. In this paper, we describe in detail the chemical synthesis,

structure-activity relationship (SAR) analysis, molecular docking and

subsequent bioactivity evaluation of 7a and its analogues.

5,6,7,9-tetrahydro-[1,2,4]triazolo[5,1-b]quinazolin-8(4H)-one,

compounds 7 and 9.

e.g.,

Further SAR analysis was made around the skeleton of 1,2,4-triazole

part with 4H-1,2,4-triazol-3-amine (12a) and tetrazole (12b) instead of

5-(2-chlorophenyl)-4H-1,2,4-triazol-3-amine 6a to examine if the 2-

chlorophenyl group is required for RXFP4 binding. Compounds 13a

and 13b were thus synthesized. Additionally, a derivative 13c was

synthesized with only two nitrogen atoms involved as compared with

compound 7a to see whether the number of nitrogen atom has any

impacts on its bioactivity (Scheme 2).

2. Results and discussion

2.2. Chiral resolution

2.1. SAR-based design and chemical synthesis

The enantiomers of compound 7a were resolved by CHIRALPAKIC

(IC00CD-NA012) column (0.46 cm × 15 cm) on Shimadzu LC-20AT

HPLC eluting with dichloromethane/ethanol = 90/10 (v/v) at a ﬂow

Based on SAR information, rational design around the scaffold of

5,6,7,9-tetrahydro-[1,2,4]triazolo[5,1-b]quinazolin-8(4H)-one (repre-

sented by 7a) was conducted focusing on ﬁve parts: 2-chlorophenyl

group, 4-hydroxylphenyl group, its skeleton including cyclohexane-

1,3-dione and 1,2,4-triazole group, and chiral resolution (Fig. 2).

The general synthetic route is described as follows. Substituted

methyl benzoate 2 or benzoyl chloride 3 was used as the starting ma-

terial, which underwent hydrazinolysis to obtain benzohydrazide de-

rivatives 4. The key intermediate 1,2,4-triazole 6 was synthesized by

reacting compound 4 with S-methylisothiourea sulfate which was

cyclized in the following step using p-toluenesulfonic acid as a catalyst.

Biginelli cyclocondensation [15–17] was then conducted under the

catalysis of acetic acid with the triazole derivative 6, cyclohexane-1,3-

dione and 4-hydroxybenzaldehyde to obtain the ﬁnal product 7

(Scheme 1). Compounds 7a-m were then designed and synthesized for

the purpose of ﬁnding the suitable substitutes on the phenyl group by

involving the electron-donating group (7h-i), electron-withdrawing

rate of 1.0 mL/min (35 ^◦C). Two peaks were separately collected at t_R

=

2.090 min (isomer 1) and t_R= 2.409 min (isomer 2) and the enantio-

meric excess (e.e.) value of each product was above 98% (Fig. 3A). The

white block-shaped single crystal of isomer 1 was acquired with ortho-

rhombic crystal system and its X-ray diffraction data were collected on a

Bruker D8 VENTURE single-crystal diffractometer. The absolute

conﬁguration of isomer 1 was determined as 9-(R)-7a. Its molecular

structure was made up of one aromatic heterocycle, two aromatic rings

and one aliphatic ring. The 2-chlorophenyl ring was almost coplanar

with the middle tricycle (Fig. 3B). The crystallographic data was shown

in Table S1-6 (Supplementary Information). CCDC2004638 contains the

supplementary crystallographic data for compound 9-(R)-7a and could

be obtained free of charge from The Cambridge Crystallographic Data

Centre via www.ccdc.cam.ac.uk/data_request/cif.

Fig. 1. Structures of compounds 1 and 7a.

2

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Fig. 2. Rational design of RXFP4 agonists.

Scheme 1. Synthetic route for compounds 7a-m. (i) 2 and NH₂NH₂⋅H₂O in EtOH, reﬂux or 3 and NH₂NH₂⋅H₂O in DCM, r.t. overnight; (ii) 4 and S-methylisothiourea

sulfate in dioxane and 2 N NaOH, reﬂux, two steps yield: 70–98%; (iii) 5 and p-TsOH in water and dioxane, reﬂux, yield: 75–97%; (iv) HOAc in EtOH, reﬂux,

yield: 30–61%.

3

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Scheme 2. Synthetic route for compounds 9a-i, 11a-d and 13a-c. (i) HOAc in EtOH, reﬂux, yields for 9: 36–59%; 11: 57–67%; 13: 69–76%.

2.3. cAMP accumulation and SAR analysis

while its potency was also elevated by nearly 4.9-fold compared to 7a

(EC₅₀= 1.9 ± 0.3 μM for 7f and EC₅₀= 9.3 ± 2.9 μM for 7a). Similar

RXFP4 agonist activities of compounds 7, 9, 11 and 13 were evalu-

ated with a HTRF assay that measures the inhibition of forskolin-

stimulated cAMP accumulation in human RXFP4-overexpressing CHO

cells. The speciﬁcity of these compounds for RXFP4 was also examined

in CHO-K1 cells stably expressing the human RXFP3, which is the most

closely related receptor to RXFP4, and RXFP1, which could be activated

by the same endogenous peptide relaxin-3. Agonist activity was

expressed as % INSL5 in hRXFP4-CHO-K1 cells, % R3/I5 in hRXFP3-

CHO-K1 cells or % relaxin-3 in hRXFP1-HEK293T cells (Table 1,

Figs. 4, S1 and S3).

phenomenon was observed for 7g (2-CF₃) that has a bigger steric hin-

drance than 7a (2-Cl) and exhibited almost an equal efﬁcacy (93.3 ±

11.2%) to 7f. Dual chloro-substituted analogues, such as 2,3-di-Cl (7j),

2,4-di-Cl (7k) and 2,5-di-Cl (7l) were designed for examining the

optimal substituted position of chloro group. The results showed that

compounds with 2,3-di-Cl (7j) or 2,4-di-Cl (7k) maintained about 63%-

78% efﬁcacy while 2,5-di-Cl (7l) displayed only 1/3 efﬁcacy compared

to 7a. Removal of this 2-chloro group led to nearly 60% decrease in

agonist activity (7m vs. 7a). The above observations indicate that 2-po-

sition on the phenyl group is essential.

Compound 7a was capable of inhibiting cAMP accumulation in

hRXFP4 overexpressing CHO-K1 cells, while exhibiting little or no

agonist activity in hRXFP3-overexpressing CHO-K1 cells and hRXFP1-

The inhibitory effects on forskolin-stimulated cAMP accumulation in

compounds with modiﬁcations around 4-hydroxyl phenyl part (strategy

2 in Fig. 2, 9a-i) suggest that this part is crucial for selective binding to

RXFP4. Change of 4-OH group to 3-OH and 9-aromatic nucleus to

cyclohexyl group led to 70% reduction and a total loss of efﬁcacy,

respectively (9a and 9b vs. 7a). 3,4-Dihydroxyl substituted derivative

(9c) only retained 65% efﬁcacy as 9a. Of note is that increase of steric

hindrance such as 4-morpholinophenyl (9d) and 4-(pyrrolidin-1-yl)

phenyl (9h) caused a weak agonist effect on RXFP3 (Efﬁcacy = 18.3 ±

6.2% for 9h), indicating that 4-phenyl position may be a key site that

deﬁnes receptor selectivity. Replacement of 4-OH group with 4-SH could

maintain its RXFP4 agonistic activity, however, the potency was nearly

decreased by 2-fold (9i vs. 7a).

overexpressing HEK293-T cells (EC₅₀= 9.3 ± 2.9 μM for RXFP4, Efﬁ-

cacy = 58.5 ± 14.7% for RXFP4 and 4.2 ± 2.8% for RXFP3, respec-

tively), indicating that this agonism was selective (Figs. 4A, 4B and S3).

Compound 7a also activated RXFP4-mediated signaling pathways

including ERK1/2 phosphorylation and β-arrestin 1/2 recruitment [14].

Chiral resolution of compound 7a resulted in a couple of enantiomers 9-

(S)-7a and 9-(R)-7a with its 9-(S)-conformer displaying full agonism as

INSL5 (Efﬁcacy = 106.6 ± 9.9% and 22.3 ± 9.3% for RXFP4 and RXFP3,

respectively), while its 9-(R)-conformer was inactive in both RXFP4- and

RXFP3-overexpressing cells.

It follows that shifting the position of 2-chloro group (strategy 1 in

Fig. 2) from ortho- to that of meta- or para- resulted in a total loss of

activity (7a vs. 7b and 7c). The size of atomic radius (7d: R₁= F; 7a: R₁

= Cl; 7e: R₁= Br; 7f: R₁= I) also affected bioactivity with the tendency

that the bigger the atomic radius, the higher the agonist effect, i.e., 2-

iodine substituted analogue 7f showed 96.2 ± 9.1% efﬁcacy of INSL5,

Subsequent studies on the cyclohexane-1,3-dione skeleton was per-

formed through inserting a dimethyl group thereby increasing the steric

hindrance, i.e., 11a and 11b synthesized by Biginelli cyclocondensation

of 5,5-dimethylcyclohexane-1,3-dione (10a)/4,4-dimethylcyclohexane-

1,3-dione (10b) with 3-amine-5-(2-chlorophenyl)-4H-1,2,4-triazol (6a)

and 4-hydroxybenzaldehyde. A sharp reduction in agonism was

4

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Fig. 3. Chiral resolution of compound 7a. A, The condition of chiral resolution and its chromatography; B, Crystal structure of 9-(R)-7a with the atom labelling.

observed (Efﬁcacy = 9.9 ± 1.2% for 11a and 18.3 ± 3.5% for 11b,

respectively), suggesting that cyclohexane-1,3-dione skeleton is not

tolerant to structural modiﬁcation. Next, we tried to insert heteroatom

to the cyclohexane-1,3-dione skeleton, such as oxygen for 11c and

sulphur for 11d. In comparison with cyclohexane-1,3-dione skeleton

(7a), 2H-thiopyran-3,5(4H,6H)-dione skeleton (11d) still retained the

agonist effect while 2H-pyran-3,5(4H,6H)-dione skeleton (11c) signiﬁ-

cantly decreased its agonist activity (Efﬁcacy = 10.5 ± 2.4% for 11c and

60.8 ± 7.6% for 11d, respectively).

molinspiration.com/). Other parameters like molecular water solubil-

ity (MolLogS), molecular volume (MolVol) and drug-likeness score were

also assessed in silico (Table 2). A positive compound is determined

when (i) nHBD is ≤ 5; (ii) nHBA is ≤ 10; (iii) MW is ≤ 500; (iv) MolLogP

is < 5; (v) nRotB is ≤ 10; and (vi) PSA is ≤ 140 Å²or nHBD + nHBA ≤

12. Our analyses revealed that most of these synthesized compounds

conform to the two rules, indicative of optimal membrane permeability,

sound bioavailability and acceptable druggability (Table 2).

For the 1,2,4-triazole skeleton, replacing its triazole group (7a) with

pyrazole (13c) and keeping other substitutes unchanged resulted in

complete loss of activity. Removal of 2-chlorophenyl from this skeleton

(13a vs. 7a) or substitution of 1,2,4-triazole with tetrazole (13b vs. 7a)

caused a marked decline in agonism, indicating that 3-phenyl-1,2,4-tri-

azole skeleton is an important functional group.

2.5. Further optimization

Based on the above SAR studies, several conclusions could be drawn:

2-Br, 2-I and 2-CF₃substitutes are superior to 2-Cl (7e, 7f and 7g vs. 7a,

strategy 1 in Fig. 2); 4-OH phenyl is an essential group for receptor

selectivity (strategy 2 in Fig. 2); the skeleton of 5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b]quinazolin-8(4H)-one (7a) could be changed to

7,9-dihydro-4H-thiopyrano[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-8

(5H)-one (11d) without affecting the agonist effect on RXFP4 (strategy 3

in Fig. 2); and 3-phenyl-1,2,4-triazole skeleton is an key functional

group for RXFP4 binding (strategy 4 in Fig. 2). We then conducted the

2nd round structural optimization for the purpose of combining these

proponent functional groups. Compounds 14a-c were thus made by

Biginelli cyclocondensation of 5-(2-bromophenyl)-4H-1,2,4-triazol-3-

amine (6e), or 5-(2-iodophenyl)-4H-1,2,4-triazol-3-amine (6f), or 5-(2-

(triﬂuoromethyl)phenyl)-4H-1,2,4-triazol-3-amine (6 g), 2H-thiopyran-

3,5(4H, 6H)-dione and 4-hydroxybenzaldehyde under the catalysis of

2.4. Physicochemical properties

Physicochemical properties of synthesized compounds were calcu-

lated according to both Lipinski’s rule of ﬁve and Veber’s rule through

selecting appropriate molecules based on size, molecular weight (MW),

number of hydrogen bond donors (nHBD) and acceptors (nHBA), mo-

lecular octanol/water partition coefﬁcient (MolLogP), number of

rotatable bonds (nRotB) and molecular polar surface area (MolPSA).

This was carried out using Molsoft online software (http://molsoft.com/

mprop/) and Molinspiration cheminformatics software (https://www.

5

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Table 1

Inhibition of forskolin-stimulated cAMP accumulation by synthetic compounds in CHO-K1 cells stably overexpressing hRXFP4 or hRXFP3.^a,f

Cpd

EC₅₀

(μ

M)^b

Efﬁcacy (%)^c

Cpd

EC₅₀

(

μ

M)^b

Efﬁcacy (%)^c

RXFP4

RXFP3

RXFP4

RXFP3

RXFP4

RXFP3

RXFP4

RXFP3

7a

9-R-7a

9-S-7a

7b

9.3±2.9

N.D.

58.5±14.7

N.A.

4.2±2.8

N.A.

9d

9e

50.2±10.5

N.D.

33.9±8.1

7.0±1.6

N.A.

6.93^e

N.A.

6.8±1.3

N.D.

230.2±148.0

N.D.

106.6±9.9

N.A.

22.3±9.3

N.A.

9f

N.D.

8.2^e

9g

N.D.

N.A.

10.4^e

18.3±6.2

N.A.

7c

N.D.

6.5±0.4

17.6±4.7

78.2±5.3

96.2±9.1

93.3±11.2

30.4±6.0

12.4±4.6

36.9±3.5

45.7±6.5

21.3±3.4

24.6±9.2

17.1±3.2

N.A.

9h^d

N.D.

97.2±11.0

N.D.

N.A

7d

11.8±6.2

7.1±3.4

1.9±0.3

26.5±9.6

216±41.0

N.D.

N.A.

9i

18.7±3.5

19.3±12.3

6.5±2.3

35.2±12.3

21.6±0.9

N.D.

52.3±12.3

9.9±1.2

18.3±3.5

10.5±2.4

60.8±7.6

9.2±1.5

13.1±7.3

N.A.

7e

N.D.

5.34^e

13.6 ^e

N.A.

11a

11b

11c

N.D.

N.A.

7f

30.7 ^e

141.9 ^e

N.D.

N.A.

7g

N.D.

N.A.

7h

11d

13a

13b

13c

26.4

8.17^e

N.A.

7i

N.D.

N.A.

N.D.

7j

40.6±15.1

N.D.

N.A.

99.8±5.6

N.D.

N.A.

7k

N.D.

2.54^e

N.A.

N.D.

N.A.

7l

22.8±14.8

N.D.

14a

14b

9-R-14b

9-S-14b

14c

6.0±1.1

13.8±5.4

3.9±2.2

8.9±2.0

N.D.

76.0±12.3

113.9±13.9

27.8±8.9

134.4±5.2

>100.0

N.A.

7m

9a

N.D.

N.A.

N.D.

6.2±1.2

2.7±0.6

5.0±2.3

>100.0

100

N.D.

N.A.

N.D.

9b

N.D.

N.A.

N.D.

9c

21.8±9.9

N.D.

38.1±9.0

100

N.A.

N.D.

INSL5

0.011±0.009

N.D.

N.A.

R3/I5

0.045±0.005

0.2±0.1

84.5±3.5

a

Each compound was tested in duplicate and each experiment was repeated at least for three times.

EC₅₀values are presented as means ± SEM.

b

c

d

e

f

Agonist activity is expressed as % INSL5 in hRXFP4-CHO-K1 cells or % R3/I5 in hRXFP3-CHO-K1 cells.

The HCl salt of compound 9h was used in the test.

Compound was tested only once.

Cpd, compound; N.D., not detectable; N.A., not active.

acetic acid (Scheme 3). They were subsequently examined with cAMP

2.7. Docking studies

accumulation assay (Table 1, Figs. 4 and S1). Similar phenomenon was

observed for strategy 1 (Fig. 2) that bigger steric hindrance was bene-

ﬁcial to RXFP4 agonism as compared with compounds 14a and 14b

(Efﬁcacy = 76.0 ± 12.3 for 14a and 113.9 ± 13.9 for 14b, respectively).

Of these three compounds, 14b exhibited the highest efﬁcacy, close to

that of the isomer 9-(S)-7a with better selective ratio for RXFP4 than

RXFP3 (18.4 and 4.8 for 14b and 9-(S)-7a, respectively; Table 1). Also,

compound 14b showed no agonistic effect on RXFP1 (Fig. S3). In

addition, the solubility of 14b was ameliorated compared to 7a (MolLog

S = ꢀ 4.02 and ꢀ 5.69 for 14b and 7a, respectively; Table 2). Next, the

enantiomers of compound 14b were resolved by CHIRALPAK IC column

(0.46 cm × 15 cm) on Shimadzu LC-2010 HPLC eluting with ethanol at a

ﬂow rate of 1.0 mL/min at 25 ^◦C. Two peaks were separately collected at

the t_R= 4.568 min (9-R) and t_R= 5.981 min (9-S) and the enantiomeric

excess (e.e.) value of each product was above 99% (Fig. 5). Like com-

pound 7a, the conformer 9-(S)-14b presented a superior RXFP4 agonism

over that of 9-R (Efﬁcacy = 134.4 ± 5.2% and 27.8 ± 8.9% for 9-S and 9-

R, respectively), accompanied by an improved RXFP4 selective ratio vs.

RXFP3 (from 4.8 for 9-(S)-7a to 26.9 for 9-(S)-14b). Compound 14c

exhibited agonist effects on both RXFP4 and RXFP3, which may result

from the cytotoxicity (Fig. S2).

Molecular docking was conducted using the Dock Ligands module of

LibDock genetic algorithm program in BIOVIA Discovery Studio 2016

(Accelrys Software, San Diego, USA). Homology models of hRXFP4 and

hRXFP3 (SWISS-MODEL: Q8TDU9 and Q9NSD7, respectively), modeled

on the template of agonist-bound apelin receptor (PDB code: 5VBL),

were used because the apelin receptor is also determined with agonist.

The sequence identity between RXFP3/RXFP4 and apelin receptor is

32.1% and 28.2%, respectively. The proteins were prepared before

docking and then cavity searching was performed to ﬁnd the orthosteric

binding site, which showed the best pocket score. Next, ligands were

prepared using energy minimization by CHARMm forceﬁeld until RMS

gradient of 0.01 was reached. Compounds 7a and 14b were docked into

the hRXFP4 orthosteric binding site constructed by residues L118^3.29

T176^4.60, R208^5.42, F291^7.35, Q205^5.39, T266^6.55, G269^6.58, V265^6.54

Q287^7.31, K273^6.62, Y284^7.28, T288^7.32, L201^5.35, P196^5.30, L193^ECL2

,

L192^ECL2, L190^ECL2and Y204^5.38, as well as the hRXFP3 orthosteric

binding site constructed by residues T346^6.55, Y369^7.33, L345^6.54

L365^7.29, C366^7.30, S349^6.58, Y267^5.38, L264^5.35, I350^6.59, K353^6.62

,

F262^ECL2, W263^5.34, R250^ECL2and F251^ECL2(superscripts indicate

Ballesteros-Weinstein numbering for GPCRs, [Ballesteros and Weinstein,

1995]) with the top 10 poses presented and scored while keeping other

options in their default values (Figs. 7 and S4). LibDock ﬁtness scores of

114.064 and 111.977 with RXFP4, and 111.545 and 111.749 with

RXFP3 for 7a and 14b were then obtained respectively. After binding to

RXFP4, the phenolic hydroxyl group and nitrogen atom on the 1,2,4-tri-

azole interacted via hydrogen bonds with the carbonyl group of L193^ECL2

and the terminal amide of Q205^5.39, separately. Two aromatic rings (2-

Cl/I-Ph and 1,2,4-triazole) formed Pi-Pi stack with F291^7.35. The aro-

matic rings (2-Cl/I-Ph) also formed Pi-cation function with the terminal

amino group of K273^6.62. In addition, the sulfur atom on the thiopyrano

2.6. Receptor binding

Representative analogues 9-(S)-7a, 7e, 7g, 14a, 14b and 9-(S)-14b

that exhibited better agonistic effects in cAMP accumulation assay were

selected for competitive ligand binding assay in CHO-K1 cells stably

expressing human RXFP4 with europium-labeled Eu(A)-R3/I5 as con-

trol. The 9-(R) conformers of 7a and 14b were also tested in the binding

assay as comparison. The results showed that 9-(S)-7a and 9-(S)-14b

displayed superior binding afﬁnity to their corresponding 9-(R) con-

formers. However, their displacement curves were not paralleled with

that of peptide R3/I5, indicating that only parts of the binding site for

R3/I5 were competitively bound by the ligands. It was noted that 9-(R)-

7a showed an increased binding effect which might be caused by the cell

ring of compound 14b interacted with the phenolic ring of Y204^5.38

,

which may explain the efﬁcacy difference between 14b and 7a. As

comparison, docking studies of 7a and 14b with RXFP3 indicated that

no hydrogen bonds were formed between the receptor and ligands

except for Pi-Pi stack, Pi-cation and Pi-sulfur functions. This may be

interpreted as the selectivity of ligands for RXFP4 vs. RXFP3.

toxicity at high concentration (100 μM)Fig. 6 .

6

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Fig. 4. Inhibition of forskolin-stimulated cAMP accumulation by test compounds in CHO-K1 cells overexpressing hRXFP4 or hRXFP3 (A–F). Each compound was

tested in duplicate and each experiment was repeated three times. Agonist activity was expressed as % INSL5 in hRXFP4-CHO-K1 cells or % R3/I5 in hRXFP3-CHO-K1

cells. For each concentration, the value of 665/615 was calculated, and normalized to the corresponding maximum value obtained for INSL5 in hRXFP4-CHO-K1 cells

and for R3/I5 in hRXFP3-CHO-K1 cells. Normalized values were plotted vs. ligand concentration using GraphPad PRISM 8 (GraphPad Inc., San Diego, CA, USA) and

are expressed as means ± SEM. Cpd, compound.

3. Conclusions

representative compounds were performed which further demonstrated

9-(S)-14b as the most potent RXFP4 agonist with a pKi value of 5.86 ±

0.15.

A new scaffold of tricyclic derivatives represented by 7a as non-

peptidic selective RXFP4 agonist was disclosed after HTS, capable of

suppressing forskolin-stimulated cAMP production in hRXFP4-

overexpressing CHO-K1 cells as opposed to hRXFP3 and hRXFP1. A

pair of enantiomers (9-R and 9-S) were resolved and their structures

were conﬁrmed by X-ray crystallography. Medicinal chemistry efforts in

modiﬁcation of 7a was then performed focusing on three parts: 2-chlor-

ophenyl group, 4-hydroxyphenyl group and its skeleton including

cyclohexane-1,3-dione and 1,2,4-triazole group. Initial optimization

revealed that 2-bromophenyl, 2-iodophenyl and 2-triﬂuoromethyl-

phenyl substitutes are superior to 2-chlorophenyl, 4-hydroxyphenyl is

an essential group for receptor selectivity, the skeleton of 5,6,7,9-tet-

rahydro-[1,2,4]triazolo[5,1-b]quinazolin-8(4H)-one could be changed

to 7,9-dihydro-4H-thiopyrano[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-8

(5H)-one without loss of agonist activity on RXFP4 and 3-phenyl-1,2,4-

triazole skeleton is a key functional group for RXFP4 binding. Based on

this, our follow-up optimization resulted in 9-(S)-14b with 2.3-fold

higher efﬁcacy and better selectivity (selective ratio of RXFP4 vs.

RXFP3 for 9-(S)-14b and 7a were 26.9 and 13.9, respectively). Subse-

quent molecular docking was carried out to elucidate a possible reason

of this selectivity for RXFP4 vs. RXFP3. Competitive binding assay of

4. Experimental protocols

4.1. Chemistry

Reagents are of commercial grade and were used as received unless

otherwise noted. The structures of all new compounds are consistent

with their ¹H, ¹³C NMR and mass spectra, and are judged to be ≥ 95%

pure by HPLC. NMR spectra were recorded on Bruker AN-400, AVANCE

III 500 and Varian Inova 600 spectrometers. Chemical shifts were re-

ported in parts per million (ppm), with the solvent resonance as the

internal standard (CD₃OD 3.31 ppm, CDCl₃7.26 ppm and DMSO‑d₆

2.50 ppm for ¹H NMR; CD₃OD 49.15 ppm, CDCl₃77.23 ppm and

DMSO‑d₆39.52 ppm for ¹³C NMR). Low resolution mass spectral data

(electrospray ionization) were acquired on a Finnigan LCQ-DECA mass

spectrometer. High resolution mass spectral data were collected on

Agilent G6520 Q-TOF mass spectrometer. Samples were analyzed for

purity on a HP1100 series equipped with a Zorbax SB-C18 column (5

μ

m, 4.6 mm × 250 mm). Purities of ﬁnal compounds were determined

using a 5 L injection with quantitation by AUC at 210 and 254 nm

μ

7

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Table 2

Calculated physicochemical properties of synthesized compounds.^a

Cpd

MolLogS

MolLogP

MW

nHBD

nHBA

nSC

nRotB

MolVol (A³)

MolPSA (A²)

Drug-likeness model score

7a

7b

ꢀ 5.69

ꢀ 6.05

ꢀ 6.07

ꢀ 5.73

ꢀ 6.00

ꢀ 6.20

ꢀ 5.91

ꢀ 5.30

ꢀ 5.37

ꢀ 6.60

ꢀ 6.62

ꢀ 6.56

ꢀ 5.11

ꢀ 5.71

ꢀ 5.70

ꢀ 3.89

ꢀ 4.71

ꢀ 4.41

ꢀ 5.67

ꢀ 5.42

ꢀ 5.70

ꢀ 5.02

ꢀ 7.34

ꢀ 6.75

ꢀ 5.33

ꢀ 6.85

ꢀ 3.27

ꢀ 3.15

ꢀ 5.72

ꢀ 3.98

ꢀ 4.02

ꢀ 4.15

4.08

4.2

392.1

2

1

3

1

2

1

2

4

5

4

3

5

4

3

4

3

4

5

4

5

3

5

1

2

3

2

3

2

1

2

3

372.53

374.56

374.48

362.08

378.22

385.73

394.3

67.96

75.51

67.96

50.54

83.44

61.71

73.61

50.34

54.16

50.34

67.96

68.12

76.87

67.96

69.17

83.65

57.33

67.96

1.21

0.7

7c

4.2

392.1

0.74

1.01

0.88

1.15

0.67

0.83

0.67

1.06

1.19

0.95

0.66

0.95

0.48

1.16

0.50

1.18

0.94

0.72

0.95

1.03

1.43

1.01

1.07

0.74

0.41

0.86

0.50

0.80

0.36

7d

7e

3.63

4.21

4.16

4.6

376.13

436.05

484.04

426.13

388.15

426.07

358.14

392.1

7f

7g

7h

7i

3.57

4.67

4.79

3.48

4.08

4.61

3.53

4.22

3.68

5.45 (>5)

5.13 (>5)

5.31 (>5)

4.81

4.8

389.21

389.13

388.2

7j

7k

389.8

7l

389.8

7m

9a

357.29

372.6

9b

382.16

408.10

461.16

433.13

404.14

422.10

445.17

408.08

420.14

394.08

410.06

282.11

283.11

391.11

454.01

502.00

444.09

393.03

385.25

447.99

422.94

401.06

399.73

440.74

378.40

418.86

415.37

358.12

374.53

283.32

277.09

374.22

380.21

387.73

396.29

9c

9d

9e

9f

9g

9h

9i

11a

11b

11c

11d

13a

13b

13c

14a

14b

14c

4.86

2.77

3.08

1.46

1.34

4.11

3.88

4.10

3.97

a

All the calculations were carried out online. MolLogS, molecular water solubility in Log (moles/L); MolLogP, molecular octanol/water partition coefﬁcient; nHBD,

number of hydrogen bond donors; nHBA, number of hydrogen bond acceptors; nSC, number of stereo centers; nRotB, number of rotatable bonds; MolVol, molecular

volume; MolPSA, molecular polar surface area. Drug-likeness score predicts an overall drug-likeness (druggability) using Molsoft’s chemical ﬁngerprints. The training

set for this mode consists of 5000 known drugs from WDI (positives) and 100,000 carefully selected non-drug-like compounds (negatives).

Scheme 3. Synthetic route for compounds 14a-c. (i) HOAc in EtOH, reﬂux, yield: 41–51%.

(Agilent diode array detector). X-ray diffraction was recorded on a

Bruker D8 VENTURE single-crystal diffractometer. Speciﬁc optical

rotation was determined on Autopol VI-Rudolph polarimeter. All the

melting points of synthesized compounds were measured on WRS-1B

digital melting point apparatus. The procedures for compounds 4–5

were included in Supplementary Information.

(t, J = 7.6 Hz, 1H, phenyl C₄-H), 7.41 (dt, J = 1.6 Hz, J = 7.2 Hz, 1H,

phenyl C₅-H), 7.50 (dd, J = 1.2 Hz, J = 7.6 Hz, 1H, phenyl C₃-H), 7.64

(dd, J = 2.0 Hz, J = 7.6 Hz, 1H, phenyl C₆-H). ¹³C NMR (CD₃OD, 125

MHz) 128.1 (phenyl C₅), 131.4 (phenyl C₆), 131.7 (phenyl C₂), 132.6

(phenyl C_3,4), 134.2 (phenyl C₁), 155.9 (CNH₂), 157.2 (CN₁N₃).

The procedures for compounds 6b-m are the same as 6a.

4.1.1. 3-Amine-5-(2-chlorophenyl)-4H-1,2,4-triazol (6a)

4.1.2. 5-(3-Chlorophenyl)-4H-1,2,4-triazol-3-amine (6b)

N-(2-Chlorobenzamido)-guanidine (5a, 997.7 mg, 4.71 mmol, 1 eq)

and p-toluenesulfonic acid monohydrate (116.2 mg, 0.61 mmol, 0.13

eq) were dissolved in a mixture of water (28 mL) and dioxane (14 mL).

The resulted solution was reﬂuxed overnight. After cooling, it was

ﬁltered and the solution was evaporated in vacuo into a small amount,

which was placed at room temperature (RT) for 1 h. The product was

precipitated and then ﬁltered as white powder (1.33 g, yield: 95.0%). m.

p. 222–223 ^◦C LR-ESI: 195.1 [M+H]⁺. HR-ESI m/z calcd for C₈H₈ClN₄

[M+H]⁺195.0437, found 195.0439. ¹H NMR (CD₃OD, 400 MHz) 7.37

Yield: 76.2%, white powder, m.p. 200–202 ^◦C. LR-ESI: 195.1

[M+H]⁺. HR-ESI m/z calcd for C₈H₈ClN₄[M+H]⁺195.0437, found

195.0436. ¹H NMR (CD₃OD, 400 MHz) 7.40 (m, 2H, phenyl C_4,5-H),

7.82 (d, J = 6.0 Hz, 1H, phenyl C₆-H), 7.91 (s, 1H, phenyl C₂-H). ¹³C

NMR (CD₃OD, 100 MHz) 125.5 (phenyl C₆), 127.1 (phenyl C₂), 130.2

(phenyl C₄), 131.3 (phenyl C₅), 134.2 (phenyl C₁), 135.7 (phenyl C₃),

159.1 (CNH₂), 159.7 (CN₁N₃).

8

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Fig. 5. Chiral resolution of compound 14b. The condition of chiral resolution and its chromatography.

Fig. 6. Competitive binding assay of selected compounds with hRXFP4 performed in CHO-K1 cells stably expressing the receptor RXFP4. Europium-labeled Eu(A)-

R3/I5 was used in the presence of increasing amounts of compounds. Each compound was measured in triplicate, and each experiment was repeated independently

three times. Data were analyzed using GraphPad PRISM 8 (GraphPad Inc., San Diego, CA) and expressed as means ± SEM.

4.1.3. 5-(4-Chlorophenyl)-4H-1,2,4-triazol-3-amine (6c)

4.1.6. 5-(2-Iodophenyl)-4H-1,2,4-triazol-3-amine (6f)

Yield: 74.5%, white powder, m.p. 227–229 ^◦C. LR-ESI: 195.1

[M+H]⁺. HR-ESI m/z calcd for C₈H₈ClN₄[M+H]⁺195.0437, found

195.0436. ¹H NMR (CD₃OD, 400 MHz) 7.42 (d, J = 8.4 Hz, 2H, phenyl

C_3,5-H), 7.88 (d, J = 8.4 Hz, 2H, phenyl C_2,6-H). ¹³C NMR (CD₃OD, 100

MHz) 128.7 (phenyl C_2,6), 129.9 (phenyl C_3,5), 132.1 (phenyl C₁), 136.2

(phenyl C₄), 158.8 (CNH₂), 159.3 (CN₁N₃).

Yield: 97.0%, m.p. 240–242 ^◦C. LR-ESI: 287.0 [M+H]⁺. HR-ESI m/z

calcd for C₈H₈IN₄[M+H]⁺286.9794, found 286.9795. ¹H NMR

(CD₃OD, 400 MHz) 7.10 (t, J = 7.2 Hz, 1H, phenyl C₅-H), 7.28 (d, J =

8.4 Hz, 1H, phenyl C₆-H), 7.47 (d, J = 7.6 Hz, 1H, phenyl C₄-H), 7.74 (d,

J = 8.0 Hz, 1H, phenyl C₃-H).

4.1.7. 5-(2-Triﬂuoromethylphenyl)-4H-1,2,4-triazol-3-amine (6g)

Yield: 81.6%, m.p. 165–167 ^◦C. LR-ESI: 228.9 [M+H]⁺. HR-ESI m/z

calcd for C₉H₈F₃N₄[M+H]⁺229.0701, found 229.0702. ¹H NMR

(CD₃OD, 400 MHz) 7.63 (m, 2H, phenyl C_4,5-H), 7.68 (d, J = 6.8 Hz, 1H,

phenyl C₃-H), 7.80 (d, J = 7.6 Hz, 1H, phenyl C₆-H). ¹³C NMR (CD₃OD,

125 MHz) 127.5 (CF₃), 130.3 (phenyl C₂), 130.5 (phenyl C₁), 133.0

(phenyl C_3,4), 133.1 (phenyl C_5,6), 157.8 (CNH₂), 158.8 (CN₁N₃).

4.1.4. 5-(2-Fluorophenyl)-4H-1,2,4-triazol-3-amine (6d)

Yield: 90.0%, white powder, m.p. 188–189 ^◦C. LR-ESI: 179.0

[M+H]⁺. HR-ESI m/z calcd for C₈H₈FN₄[M+H]⁺179.0733, found

179.0732. ¹H NMR (CD₃OD, 400 MHz) 7.23 (m, 2H, phenyl C_4,5-H),

7.44 (m, 1H, phenyl C₆-H), 7.87 (t, J = 7.6 Hz, 1H, phenyl C₃-H). ¹³

C

NMR (CD₃OD, 125 MHz) 117.3 (phenyl C₃), 119.6 (phenyl C₁), 125.5

(phenyl C₅), 131.0 (phenyl C₄), 132.3 (phenyl C₆), 160.6 (CNH₂), 162.6

(phenyl C₂, CN₁N₃).

4.1.8. 5-(3-Methoxyphenyl)-4H-1,2,4-triazol-3-amine (6h)

Yield: 79.1%, white powder, m.p. 223–225 ^◦C. LR-ESI: 191.1

[M+H]⁺. HR-ESI m/z calcd for C₉H₁₁N₄O [M+H]⁺191.0933, found

191.0930. ¹H NMR (CD₃OD, 400 MHz) 3.84 (s, 3H, OCH₃), 6.96 (d, J =

8.4 Hz, 1H, phenyl C₄-H), 7.32 (t, J = 8.4 Hz, 1H, phenyl C₅-H), 7.48 (m,

2H, phenyl C_2,6-H). ¹³C NMR (CD₃OD, 125 MHz) 55.9 (OCH₃), 112.4

(phenyl C₂), 116.5 (phenyl C₄), 119.6 (phenyl C₆), 130.8 (phenyl C₅),

133.1 (phenyl C₁), 159.9 (CNH₂), 161.5 (2C, OCH₃, CN₁N₃).

4.1.5. 5-(2-Bromophenyl)-4H-1,2,4-triazol-3-amine (6e)

Yield: 87.5%, m.p. 227–228 ^◦C. LR-ESI: 238.8 [M+H]⁺. HR-ESI m/z

calcd for C₈H₈BrN₄[M+H]⁺238.9932, found 238.9932. ¹H NMR

(CD₃OD, 400 MHz) 7.33 (t, J = 7.6 Hz, 1H, phenyl C₅-H), 7.42 (t, J = 7.6

Hz, 1H, phenyl C₄-H), 7.57 (d, J = 7.6 Hz, 1H, phenyl C₃-H), 7.69 (d, J =

8.0 Hz, 1H, phenyl C₃-H). ¹³C NMR (CD₃OD, 125 MHz) 123.5 (phenyl

C₂), 128.6 (phenyl C₅), 132.8 (phenyl C_4,6), 134.6 (phenyl C₃), 137.8

(phenyl C₁), 157.2 (CNH₂), 158.5 (CN₁N₃).

4.1.9. 5-(4-Methoxyphenyl)-4H-1,2,4-triazol-3-amine (6i)

Yield: 87.4%, white powder, m.p. 224–226 ^◦C. LR-ESI: 191.2

[M+H]⁺. HR-ESI m/z calcd for C₉H₁₁N₄O [M+H]⁺191.0933, found

9

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Fig. 7. A, Molecular docking of compounds 7a and 14b with hRXFP4 (SWISS-MODEL: Q8TDU9) at the orthosteric binding site. Compounds 7a and 14b were

displayed as cyan and pink sticks, respectively, while the amino acids of RXFP4 are shown as grey cartoon or sticks; B, Sequence alignment among RXFP3 (SWISS-

MODEL: Q9NSD7), RXFP4 and apelin receptor (PDB code: 5VBL). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web

version of this article.)

10

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

191.0930. ¹H NMR (CD₃OD, 500 MHz) 3.83 (s, 3H, OCH₃), 6.97 (d, J =

4.1.15. 2-(3-Chlorophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b]quinazolin- 8(4H)-one (7b)

8.0 Hz, 2H, phenyl C_3,5-H), 7.82 (d, J = 8.5 Hz, 2H, phenyl C_2,6-H). ¹³

C

NMR (CD₃OD, 125 MHz) 56.0 (OCH₃), 115.2 (2C, phenyl C_3,5), 124.6

(phenyl C₁), 128.8 (2C, phenyl C_2,6), 149.1 (CNH₂), 152.3 (CN₁N₃),

159.4 (phenyl C₄).

Yield: 43.3%, yellowish powder. m.p. 297–299 ^◦C. LR-ESI: 391.1 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₆ClN₄O₂[M-H]^-391.0962, found

391.0962. ¹H NMR (CD₃OD, 500 MHz) 2.03 (m, 1H, C₆-H), 2.11 (m, 1H,

C₆-H), 2.40 (m, 2H, C₅-H), 2.78 (m, 2H, C₇-H), 6.30 (s, 1H, C₉-H), 6.71

(d, J = 8.5 Hz, 2H, 4-OH phenyl C_3,5-H), 7.14 (d, J = 8.5 Hz, 2H, 4-OH

phenyl C_2,6-H), 7.37 (m, 2H, 3-Cl phenyl C_4,5-H), 7.86 (m, 1H, 3-Cl

phenyl C₆-H), 7.93 (s, 1H, 3-Cl phenyl C₂-H). ¹³C NMR (CD₃OD, 125

MHz) 22.3 (C₆), 28.0 (C₅), 37.7 (C₇), 56.0 (C₉), 109.8 (C_8a), 116.3 (2C,

4-OH phenyl C_3,5), 125.6 (3-Cl phenyl C₆), 127.2 (3-Cl phenyl C₂), 129.6

(2C, 4-OH phenyl C_2,6), 130.4 (3-Cl phenyl C₄), 131.3 (3-Cl phenyl C₅),

133.7 (4-OH phenyl C₁), 134.1 (3-Cl phenyl C₁), 135.7 (3-Cl phenyl C₃),

151.4 (C_5a), 154.5 (CN₂N₄), 159.2 (4-OH phenyl C₄), 160.4 (CN₁N₃),

197.3 (CO).

4.1.10. 5-(2, 3-Dichlorophenyl)-4H-1,2,4-triazol-3-amine (6j)

Yield: 82.1%, white powder, m.p. 243–245 ^◦C. LR-ESI: 229.0

[M+H]⁺. HR-ESI m/z calcd for C₈H₇C_l2N₄[M+H]⁺229.0048, found

229.0046. ¹H NMR (CD₃OD, 400 MHz) 7.36 (t, J = 7.6 Hz, 1H, phenyl

C₅-H), 7.57 (d, J = 7.6 Hz, 1H, phenyl C₆-H), 7.61 (d, J = 8.0 Hz, 1H,

phenyl C₄-H). ¹³C NMR (CD₃OD, 125 MHz) 128.8 (phenyl C₆), 131.1

(2C, phenyl C_4,5), 132.4 (phenyl C₂), 132.7 (phenyl C₃), 135.0 (phenyl

C₁), 158.6 (CNH₂), 158.9 (CN₁N₃).

4.1.11. 5-(2, 4-Dichlorophenyl)-4H-1, 2, 4-triazol-3-amine (6k)

Yield: 89.8%, white powder, m.p. 242–243 ^◦C. LR-ESI: 229.0

[M+H]⁺. HR-ESI m/z calcd for C₈H₇C_l2N₄[M+H]⁺229.0048, found

229.0046. ¹H NMR (CD₃OD, 400 MHz) 7.41 (dd, J = 1.6 Hz, J = 8.4 Hz,

1H, phenyl C₅-H), 7.58 (s, 1H, phenyl C₃-H), 7.66 (d, J = 8.4 Hz, 1H,

phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 128.4 (phenyl C₅), 131.2

(phenyl C₆), 133.6 (phenyl C₃), 135.1(2C, phenyl C_2,4), 136.7 (phenyl

C₁), 158.4 (CNH₂), 159.3 (CN₁N₃).

4.1.16. 2-(4-Chlorophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b]quinazolin- 8(4H)-one (7c)

Yield: 33.4%, yellowish powder. m.p. 297–298 ^◦C. LR-ESI: 391.2 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₆ClN₄O₂[M-H]^-391.0962, found

391.0954. ¹H NMR (CD₃OD, 400 MHz) 2.03 (m, 1H, C₆-H), 2.10 (m, 1H,

C₆-H), 2.40 (m, 2H, C₅-H), 2.76 (m, 2H, C₇-H), 6.29 (s, 1H, C₉-H), 6.70

(d, J = 8.4 Hz, 2H, 4-OH phenyl C_3,5-H), 7.12 (d, J = 8.4 Hz, 2H, 4-OH

phenyl C_2,6-H), 7.39 (d, J = 8.4 Hz, 2H, 4-Cl phenyl C_3,5-H), 7.91 (d, J =

8.8 Hz, 2H, 4-Cl phenyl C_2,6-H). ¹³C NMR (CD₃OD, 125 MHz) 22.2 (C₆),

28.0 (C₅), 37.7 (C₇), 59.5 (C₉), 109.8 (C_8a), 116.3 (2C, 4-OH phenyl

4.1.12. 5-(2, 5-Dichlorophenyl)-4H-1, 2, 4-triazol-3-amine (6l)

Yield: 87.0%, white powder, m.p. 268–269 ^◦C. LR-ESI: 229.1

[M+H]⁺. HR-ESI m/z calcd for C₈H₇C_l2N₄[M+H]⁺229.0048, found

229.0047. ¹H NMR (CD₃OD, 400 MHz) 7.42 (d, J = 8.8 Hz, 1H, phenyl

C₃-H), 7.49 (d, J = 8.8 Hz, 1H, phenyl C₄-H), 7.70 (s, 1H, phenyl C₆-H).

¹³C NMR (CD₃OD, 125 MHz) 130.3 (phenyl C₂), 131.4 (phenyl C₆),

132.1 (phenyl C₄), 132.6 (phenyl C₅), 133.0 (phenyl C₃), 133.8 (phenyl

C₁), 157.1 (CNH₂), 158.3 (CN₁N₃).

C

_3,5), 128.9 (2C, 4-Cl phenyl C_2,6), 129.6 (2C, 4-Cl phenyl C_3,5), 129.9

(2C, 4-OH phenyl C_2,6), 130.9 (4-OH phenyl C₁), 133.8 (4-Cl phenyl C₁),

136.4 (4-Cl phenyl C₄), 149.3 (C_5a), 154.5 (CN₂N₄), 158.6 (4-OH phenyl

C₄), 160.8 (CN₁N₃), 197.3 (CO).

4.1.17. 2-(2-Fluorophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b]quinazolin- 8(4H)-one (7d)

4.1.13. 5-Phenyl-4H-1,2,4-triazol-3-amine (6m)

Yield: 52.8%, yellowish powder, m.p. 263–264 ^◦C. LR-ESI: 375.2 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₆FN₄O₂[M-H]^-375.1257, found

375.1256. ¹H NMR (CD₃OD, 400 MHz) 2.09 (m, 2H, C₆-H), 2.40 (m, 2H,

C₅-H), 2.77 (m, 2H, C₇-H), 6.34 (s, 1H, C₉-H), 6.70 (d, J = 8.0 Hz, 2H, 4-

OH phenyl C_3,5-H), 7.14 (d, J = 8.4 Hz, 2H, 4-OH phenyl C_2,6-H), 7.20

(m, 2H, 2-F phenyl C_5,6-H), 7.41 (m, 1H, 2-F phenyl C₄-H), 7.88 (t, J =

7.6 Hz, 1H, 2-F phenyl C₃-H). ¹³C NMR (CD₃OD, 125 MHz) 22.3 (C₆),

28.0 (C₅), 37.7 (C₇), 59.4 (C₉), 110.0 (C_8a), 116.3 (2C, 4-OH phenyl

Yield: 90.0%, white powder, m.p. 187–188 ^◦C. LR-ESI: 161.1

[M+H]⁺. HR-ESI m/z calcd for C₈H₉N₄[M+H]⁺161.0827, found

161.0827. ¹H NMR (CD₃OD, 400 MHz) 7.40 (m, 3H, phenyl C_3,4,5-H),

7.89 (d, J = 6.8 Hz, 2H, phenyl C_2,6-H). ¹³C NMR (CD₃OD, 125 MHz)

127.3 (2C, phenyl C_2,6), 129.8 (2C, phenyl C_3,5), 130.5 (phenyl C₄),

131.9 (phenyl C₁), 159.9 (CNH₂), 160.2 (CN₁N₃).

4.1.14. 2-(2-Chlorophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

C_3,5), 117.4 (2-F phenyl C₃), 120.1 (2-F phenyl C₁), 125.4 (2-F phenyl

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (7a)

C₅), 129.6 (2C, 4-OH phenyl C_2,6), 131.4 (2-F phenyl C₆), 132.3 (2-F

phenyl C₄), 133.7 (4-OH phenyl C₁), 149.0 (C_5a), 154.4 (CN₂N₄), 158.6

(4-OH phenyl C₄), 160.7 (2-F phenyl C₂), 162.7 (CN₁N₃), 197.2 (CO).

Acetic acid (600 μL) was added to the solution of 5-(2-chloro)phenyl-

4H-1,2,4-triazol-3-amine (6a, 4 g, 20.6 mmol, 1 eq), cyclohexane-1,3-

dione (2.3 g, 20.6 mmol, 1 eq) and 4-hydroxybenzaldehyde (2.5 g,

20.6 mmol, 1 eq) in EtOH (130 mL). The reaction was reﬂuxed over-

night. After cooling, the solvent was removed in vacuo and the residue

was separated on the Biotage® SNAP Cartridge Sil-100 g column eluting

with 0–10% methanol/dichloromethane to obtain the product as

yellowish powder (1.8 g, yield: 32.3%). m.p. 297–298 ^◦C. LR-ESI: 391.2

[M-H]^-. HR-ESI m/z calcd for C₂₁H₁₆ClN₄O₂[M-H]^-391.0962, found

391.0968. ¹H NMR (CD₃OD, 500 MHz) 2.05 (m, 1H, C₆-H), 2.10 (m, 1H,

C₆-H), 2.40 (m, 2H, C₅-H), 2.75 (m, 2H, C₇-H), 6.34 (s, 1H, C₉-H), 6.70

(d, J = 8.5 Hz, 2H, 4-OH phenyl C_3,5-H), 7.14 (d, J = 9.0 Hz, 2H, 4-OH

phenyl C_2,6-H), 7.33 (dt, J = 1.0 Hz, J = 7.5 Hz, 1H, 2-Cl phenyl C₅-H),

7.38 (dt, J = 1.0 Hz, J = 7.5 Hz, 1H, 2-Cl phenyl C₄-H), 7.47 (dd, J = 1.0

Hz, J = 7.5 Hz, 1H, 2-Cl phenyl C₃-H), 7.63 (dd, J = 2.0 Hz, J = 8.0 Hz,

1H, 2-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 22.3 (C₆), 28.0 (C₇),

37.7 (C₅), 59.4 (C₉), 109.8 (C_8a), 116.3 (2C, 4-OH phenyl C_3,5), 128.0 (2-

Cl phenyl C₅), 129.6 (2C, 4-OH phenyl C_2,6), 131.5 (2C, 2-Cl phenyl

4.1.18. 2-(2-Bromophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b]quinazolin- 8(4H)-one (7e)

Yield: 55.3%, yellowish powder, m.p. > 300 ^◦C. LR-ESI: 435.1 [M-

H]^-, 437.1. HR-ESI m/z calcd for C₂₁H₁₆BrN₄O₂[M-H]^-435.0457, found

435.0463. ¹H NMR (CD₃OD, 400 MHz) 2.03 (m, 2H, C₆-H), 2.37 (m, 2H,

C₅-H), 2.69 (m, 2H, C₇-H), 6.33 (s, 1H, C₉-H), 6.71 (d, J = 8.0 Hz, 2H, 4-

OH phenyl C_3,5-H), 7.14 (d, J = 8.0 Hz, 2H, 4-OH phenyl C_2,6-H), 7.28 (t,

J = 7.6 Hz, 1H, 2-Br phenyl C₄-H), 7.36 (t, J = 7.2 Hz, 1H, 2-Br phenyl

C₅-H), 7.55 (d, J = 7.6 Hz, 1H, 2-Br phenyl C₃-H), 7.64 (d, J = 7.6 Hz,

1H, 2-Br phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 22.2 (C₆), 28.0 (C₅),

37.7 (C₇), 59.3 (C₉), 109.7 (C_8a), 116.3 (2C, 4-OH phenyl C_3,5), 123.4 (2-

Br phenyl C₂), 128.5 (2-Br phenyl C₅), 129.6 (2C, 4-OH phenyl C_2,6),

132.0 (2-Br phenyl C₆), 132.8 (2-Br phenyl C₃), 133.5 (4-OH phenyl C₁),

133.7 (2-Br phenyl C₁), 134.7 (2-Br phenyl C₃), 148.8 (C_5a), 154.7

(CN₂N₄), 158.6 (4-OH phenyl C₄), 161.2 (CN₁N₃), 197.2 (CO).

C

_1,3), 131.8 (2-Cl phenyl C₄), 132.7 (2-Cl phenyl C₆), 133.6 (4-OH

phenyl C₁), 134.3 (2-Cl phenyl C₂), 148.8 (C_5a), 154.5 (CN₂N₄), 158.6

(4-OH phenyl C₄), 160.2 (CN₁N₃), 197.2 (CO).

4.1.19. 2-(2-Iodophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-[1,2,4]

triazolo[5,1-b]quinazolin- 8(4H)-one (7f)

Compounds 7b-m, 9a-i, 11a-d, 13a-c and 14a-c were prepared using

similar procedures as compound 7a.

Yield: 50.0%, yellowish powder, m.p. > 300 ^◦C. LR-ESI: 483.0 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₆IN₄O₂[M-H]^-483.0318, found

11

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

483.0321. ¹H NMR (CD₃OD, 400 MHz) 2.05 (m, 2H, C₆-H), 2.38 (m, 2H,

C₅-H), 2.69 (m, 2H, C₇-H), 6.32 (s, 1H, C₉-H), 6.70 (d, J = 8.0 Hz, 2H, 4-

OH phenyl C_3,5-H), 7.14 (d, J = 7.6 Hz, 2H, 4-OH phenyl C_2,6-H), 7.39

(m, 2H, 2-I phenyl C_5,6-H), 7.47 (d, J = 7.6 Hz, 1H, 2-I phenyl C₄-H),

7.93 (d, J = 8.0 Hz, 1H, 2-I phenyl C₃-H). ¹³C NMR (CD₃OD, 125 MHz)

22.2 (C₆), 28.0 (C₅), 37.7 (C₇), 59.4 (C₉), 109.9 (C_8a), 116.9 (2C, 4-OH

phenyl C_3,5), 128.2 (2-I phenyl C₅), 129.2 (2-I phenyl C₆), 130.6 (2C, 4-

OH phenyl C_2,6), 130.9 (2-I phenyl C₄), 132.0 (4-OH phenyl C₁), 132.3

(2-I phenyl C₃), 133.6 (2-I phenyl C₁), 133.8 (2-I phenyl C₂), 149.2 (C_5a),

154.5 (CN₂N₄), 158.5 (4-OH phenyl C₄), 161.8 (CN₁N₃), 197.3 (CO).

phenyl C₄), 135.0 (2,3-di-Cl phenyl C₃), 135.1 (2,3-di-Cl phenyl C₁),

148.8 (C_5a), 154.6 (CN₂N₄), 158.6 (4-OH phenyl C₄), 159.7 (CN₁N₃),

197.3 (CO).

4.1.24. 2-(2,4-Dichlorophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (7k)

Yield: 55.9%, yellowish powder, m.p. 281–283 ^◦C. LR-ESI: 425.2 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₅Cl₂N₄O₂[M-H]^-425.0572, found

425.0580. ¹H NMR (CD₃OD, 500 MHz) 2.03 (m, 1H, C₆-H), 2.09 (m, 1H,

C₆-H), 2.39 (m, 2H, C₅-H), 2.75 (m, 2H, C₇-H), 6.33 (s, 1H, C₉-H), 6.70

(d, J = 8.5 Hz, 2H, 4-OH phenyl C_3,5-H), 7.13 (d, J = 8.5 Hz, 2H, 4-OH

phenyl C_2,6-H), 7.36 (dd, J = 2.5 Hz, J = 8.5 Hz, 1H, 2,4-di-Cl phenyl C₅-

H), 7.52 (d, J = 2.0 Hz, 1H, 2,4-di-Cl phenyl C₃-H), 7.67 (d, J = 8.5 Hz,

1H, 2,4-di-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 22.2 (C₆), 28.0

(C₅), 37.7 (C₇), 59.4 (C₉), 109.7 (C_8a), 116.2 (2C, 4-OH phenyl C_3,5),

128.3 (2,4-di-Cl phenyl C₅), 129.6 (2C, 4-OH phenyl C_2,6), 130.2 (2,4-di-

Cl phenyl C₂), 131.3 (2,4-di-Cl phenyl C₆), 133.6 (2,4-di-Cl phenyl C₄),

135.0 (4-OH phenyl C₁), 136.8 (2,4-di-Cl phenyl C₁), 148.8 (C_5a), 154.6

(CN₂N₄), 158.6 (4-OH phenyl C₄), 159.3 (CN₁N₃), 197.2 (CO).

4.1.20. 2-(2-Triﬂuoromethylphenyl)-9-(4-hydroxyphenyl)-5,6,7,9-

tetrahydro-[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (7g)

Yield: 57.1%, yellowish powder, m.p. > 300 ^◦C. LR-ESI: 425.2 [M-

H]^-. HR-ESI m/z calcd for C₂₂H₁₆F₃N₄O₂[M-H]^-425.1225, found

425.1221. ¹H NMR (CD₃OD, 400 MHz) 2.10 (m, 2H, C₆-H), 2.41 (m, 2H,

C₅-H), 2.77 (m, 2H, C₇-H), 6.33 (s, 1H, C₉-H), 6.71 (d, J = 8.0 Hz, 2H, 4-

OH phenyl C_3,5-H), 7.12 (d, J = 7.6 Hz, 2H, 4-OH phenyl C_2,6-H), 7.63

(m, 3H, 2-CF₃phenyl C_3,4,5-H), 7.77 (d, J = 7.2 Hz, 1H, 2-CF₃phenyl C₆-

H). ¹³C NMR (CD₃OD, 125 MHz) 22.2 (C₆), 28.0 (C₅), 37.7 (C₇), 59.3

(C₉), 109.8 (C_8a), 116.2 (2C, 4-OH phenyl C_3,5), 124.4 (CF₃), 127.6 (2-

CF₃phenyl C₃), 129.5 (2C, 4-OH phenyl C_2,6), 130.3 (2-CF₃phenyl C₂),

130.5 (4-OH phenyl C₁), 130.9 (2-CF₃phenyl C₄), 133.0 (2-CF₃phenyl

C₆), 133.1 (2-CF₃phenyl C₅), 133.5 (2-CF₃phenyl C₁), 148.9 (C_5a),

154.6 (CN₂N₄), 158.5 (4-OH phenyl C₄), 160.4 (CN₁N₃), 197.3 (CO).

4.1.25. 2-(2,5-Dichlorophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (7l)

Yield: 61.4%, yellowish powder, 283–284 ^◦C. LR-ESI: 425.1 [M-H]^-.

HR-ESI m/z calcd for C₂₁H₁₅Cl₂N₄O₂[M-H]^-425.0572, found 425.0578.

¹H NMR (DMSO‑d₆, 400 MHz) 1.96 (m, 2H, C₆-H), 2.28 (m, 2H, C₅-H),

2.68 (m, 2H, C₇-H), 6.21 (s, 1H, C₉-H), 6.66 (d, J = 7.6 Hz, 2H, 4-OH

phenyl C_3,5-H), 7.07 (d, J = 8.0 Hz, 2H, 4-OH phenyl C_2,6-H), 7.49 (d,

J = 8.8 Hz, 1H, 2,5-di-Cl phenyl C₄-H), 7.57 (d, J = 8.8 Hz , 1H, 2,5-di-Cl

phenyl C₃-H), 7.76 (brs, 1H, 2,5-di-Cl phenyl C₆-H), 9.40 (s, OH), 11.26

(brs, NH). ¹³C NMR (DMSO‑d₆, 125 MHz) 20.6 (C₆), 26.4 (C₅), 36.3 (C₇),

57.3 (C₉), 107.0 (C_8a), 115.0 (2C, 4-OH phenyl C_3,5), 128.2 (2C, 4-OH

phenyl C_2,6), 129.9 (2,5-di-Cl phenyl C₆), 130.0 (4-OH phenyl C₁),

130.1 (2,5-di-Cl phenyl C₄), 131.2 (2,5-di-Cl phenyl C₂), 131.6 (2,5-di-Cl

phenyl C₅), 131.7 (C_5a), 132.4 (2,5-di-Cl phenyl C₃), 147.1 (2,5-di-Cl

phenyl C₁), 152.1 (CN₂N₄), 156.5 (4-OH phenyl C₁), 157.0 (CN₁N₃),

193.2 (CO).

4.1.21. 2-(3-Methoxyphenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (7h)

Yield: 52.8%, white powder, m.p. 291–292 ^◦C. LR-ESI: 387.3 [M-H]^-.

HR-ESI m/z calcd for C₂₂H₁₉N₄O₃[M-H]^-387.1457, found 387.1455. ¹H

NMR (CD₃OD, 400 MHz) 2.09 (m, 2H, C₆-H), 2.40 (m, 2H, C₅-H), 2.75

(m, 2H, C₇-H), 3.82 (s, 3H, OCH₃), 6.30 (s, 1H, C₉-H), 6.70 (d, J = 8.4

Hz, 2H, 4-OH phenyl C_3,5-H), 6.94 (dd, J = 2.0 Hz, J = 8.8 Hz, 1H, 3-

OCH₃phenyl C₄-H), 7.13 (d, J = 8.4 Hz, 2H, 4-OH phenyl C_2,6-H), 7.29

(t, J = 8.0 Hz, 1H, 3-OCH₃phenyl C₅-H), 7.50 (d, J = 2.8 Hz, 1H, 3-OCH₃

phenyl C₂-H), 7.52 (d, J = 8.8 Hz, 1H, 3-OCH₃phenyl C₆-H). ¹³C NMR

(CD₃OD, 125 MHz) 22.3 (C₆), 28.0 (C₅), 37.7 (C₇), 55.9 (OCH₃), 59.4

(C₉), 110.0 (C_8a), 112.4 (3-OCH₃phenyl C₂), 116.3 (2C, 4-OH phenyl

4.1.26. 2-(Phenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-[1,2,4]

triazolo[5,1-b]quinazolin-8(4H)- one (7m)

C

_3,5), 116.7 (3-OCH₃phenyl C₄), 119.8 (3-OCH₃phenyl C₆), 129.6 (2C,

4-OH phenyl C_2,6), 130.8 (3-OCH₃phenyl C₅), 133.3 (4-OH phenyl C₁),

133.8 (3-OCH₃phenyl C₁), 149.2 (C_5a), 154.4 (CN₂N₄), 158.6 (4-OH

phenyl C₄), 161.5 (CN₁N₃), 161.7 (3-OCH₃phenyl C₃), 197.2 (CO).

Yield: 60.4%, yellowish powder, m.p. 260–262 ^◦C. LR-ESI: 357.2 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₇N₄O₂[M-H]^-357.1352, found

357.1354. ¹H NMR (CD₃OD, 400 MHz) 2.07 (m, 2H, C₆-H) , 2.39 (m, 2H,

C₅-H), 2.77 (m, 2H, C₇-H), 6.29 (s, 1H, C₉-H), 6.71 (d, J = 8.4 Hz, 2H, 4-

OH phenyl C_3,5-H), 7.14 (d, J = 8.4 Hz, 2H, 4-OH phenyl C_2,6-H), 7.38

(m, 3H, phenyl C_3,4,5-H), 7.92 (m, 2H, phenyl C_2,6-H). ¹³C NMR (CD₃OD,

125 MHz) 22.2 (C₆), 30.8 (C₅), 37.7 (C₇), 55.8 (C₉), 109.8 (C_8a), 116.2

(2C, 4-OH phenyl C_3,5), 127.3 (2C, phenyl C_2,6), 129.5 (4-OH phenyl C₁),

129.6 (2C, phenyl C_3,5), 129.7 (2C, 4-OH phenyl C_2,6), 130.1 (phenyl

C₄), 133.8 (C_5a), 134.4 (phenyl C₁), 153.2 (CN₂N₄), 154.6 (4-OH phenyl

C₄), 158.5 (CN₁N₃), 197.3 (CO).

4.1.22. 9-(4-Hydroxyphenyl)-2-(4-methoxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (7i)

Yield: 30.1%, yellowish powder. LR-ESI: 387.2 [M-H]^-. HR-ESI m/z

calcd for C₂₂H₁₉N₄O₃[M-H]^-387.1457, found 387.1457. ¹H NMR

(CD₃OD, 400 MHz) 2.09 (m, 2H, C₆-H), 2.39 (m, 2H, C₅-H), 2.77 (m, 2H,

C₇-H), 3.81 (s, 3H, OCH₃), 6.28 (s, 1H, C₉-H), 6.70 (d, J = 8.4 Hz, 2H, 4-

OH phenyl C_3,5-H), 6.94 (d, J = 8.8 Hz, 2H, 4-OCH₃phenyl C_3,5-H), 7.12

(d, J = 8.4 Hz, 2H, 4-OH phenyl C_2,6-H), 7.85 (d, J = 9.2 Hz, 2H, 4-OCH₃

phenyl C_2,6-H).

4.1.27. 2-(2-Chlorophenyl)-9-(3-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b]quinazolin- 8(4H)-one (9a)

4.1.23. 2-(2,3-Dichlorophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (7j)

Yield: 43.6%, yellowish powder, m.p. 280–282 ^◦C. LR-ESI: 391.3 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₆ClN₄O₂[M-H]^-391.0962, found

391.0960. ¹H NMR (CD₃OD, 400 MHz) 2.11 (m, 1H, C₆-H), 2.20 (t, J =

7.2 Hz, 1H, C₆-H), 2.42 (m, 2H, C₅-H), 2.78 (m, 2H, C₇-H), 6.36 (s, 1H,

C₉-H), 6.67 (d, J = 8.4 Hz, 1H, 3-OH phenyl C₄-H), 6.75 (s, 1H, 3-OH

phenyl C₂-H), 6.78 (d, J = 7.6 Hz, 1H, 3-OH phenyl C₆-H), 7.11 (t, J

= 8.0 Hz, 1H, 3-OH phenyl C₅-H), 7.34 (t, J = 7.6 Hz, 1H, 2-Cl phenyl C₄-

H), 7.39 (t, J = 7.6 Hz, 1H, 2-Cl phenyl C₅-H), 7.47 (d, J = 7.6 Hz, 1H, 2-

Cl phenyl C₃-H) , 7.65 (d, J = 6.8 Hz, 1H, 2-Cl phenyl C₆-H). ¹³C NMR

(CD₃OD, 125 MHz) 22.1 (C₆), 28.0 (C₅), 37.7 (C₇), 59.6 (C₉), 109.6

(C_8a), 115.2 (3-OH phenyl C₄), 116.2 (3-OH phenyl C₂), 119.5 (3-OH

phenyl C₆), 128.0 (2-Cl phenyl C₅), 130.6 (2-Cl phenyl C₆), 131.5 (2-Cl

phenyl C₃), 131.8 (3-OH phenyl C₅), 132.7 (2-Cl phenyl C₄), 134.3 (2-Cl

Yield: 50.6%, yellowish powder, m.p. 280–281 ^◦C. LR-ESI: 425.1 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₅Cl₂N₄O₂[M-H]^-425.0572, found

425.0574. ¹H NMR (CD₃OD, 400 MHz) 2.06 (m, 1H, C₆-H), 2.12 (m, 1H,

C₆-H), 2.41 (m, 2H, C₅-H), 2.77 (m, 2H, C₇-H), 6.34 (s, 1H, C₉-H), 6.71

(d, J = 8.8 Hz, 2H, 4-OH phenyl C_3,5-H), 7.14 (d, J = 8.8 Hz, 2H, 4-OH

phenyl C_2,6-H), 7.33 (t, J = 8.0 Hz, 1H, 2,3-di-Cl phenyl C₅-H), 7.57 (d, J

= 8.0 Hz, 1H, 2,3-di-Cl phenyl C₆-H), 7.59 (d, J = 8.0 Hz, 1H, 2,3-di-Cl

phenyl C₄-H). ¹³C NMR (CD₃OD, 125 MHz) 22.3 (C₆), 28.0 (C₅), 37.7

(C₇), 59.4 (C₉), 109.8 (C_8a), 116.3 (2C, 4-OH phenyl C_3,5), 128.8 (2,3-di-

Cl phenyl C₆), 129.6 (2C, 4-OH phenyl C_2,6), 131.2 (2,3-di-Cl phenyl C₅),

132.5 (2,3-di-Cl phenyl C₄), 133.5 (2,3-di-Cl phenyl C₂), 133.9 (4-OH

12

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

phenyl C₂), 138.6 (2-Cl phenyl C₁), 139.0 (C_5a), 143.8 (3-OH phenyl C₁),

148.9 (CN₂N₄), 154.8 (3-OH phenyl C₃), 158.8 (CN₁N₃), 197.2 (CO).

1H, 2-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 100 MHz) 22.2 (C₆), 24.1

(AcNH), 28.0 (C₅), 37.7 (C₇), 59.5 (C₉), 109.4 (C_8a), 125.9 (2C, 4-AcNH

phenyl C_3,5), 128.5 (2-Cl phenyl C₅), 128.9 (2C, 4-AcNH phenyl C_2,6),

129.8 (2-Cl phenyl C₆), 130.8 (2-Cl phenyl C₃), 131.3 (2-Cl phenyl C₄),

132.8 (2-Cl phenyl C₂), 133.0 (4-AcNH phenyl C₁), 134.3 (4-AcNH

phenyl C₄), 139.9 (2-Cl phenyl C₁), 151.0 (C_5a), 154.9 (CN₂N₄), 160.4

(CN₁N₃), 168.6 (CH₃CO), 197.2 (CO).

4.1.28. 2-(2-Chlorophenyl)-9-cyclohexyl-5,6,7,9-tetrahydro-[1,2,4]

triazolo[5,1-b]quinazolin-8(4H)- one (9b)

Yield: 59.2%, white powder. LR-ESI: 381.2 [M-H]^-. HR-ESI m/z calcd

for C₂₁H₂₂ClN₄O [M-H]^-381.1482, found 381.1485. ¹H NMR (CD₃OD,

400 MHz) 1.02 (m, 1H, cyclohexyl C₁-H), 1.17 (m, 2H, cyclohexyl C_2,6

-

H), 1.66 (m, 8H, cyclohexyl), 2.07 (m, 2H, C₆-H), 2.45 (m, 2H, C₅-H),

2.66 (brs, 2H, C₇-H), 5.30 (s, 1H, C₉-H), 7.39 (m, 2H, 2-Cl phenyl C_4,5

4.1.32. 2-(2-Chlorophenyl)-9-(4-ethylphenyl)-5,6,7,9-tetrahydro-[1,2,4]

triazolo[5,1-b]quinazolin- 8(4H)-one (9f)

-

H), 7.50 (d, J = 7.2 Hz, 1H, 2-Cl phenyl C₃-H), 7.72 (d, J = 6.8 Hz, 1H, 2-

Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 22.2 (C₆), 27.4 (cyclo-

hexyl C₃), 27.5 (cyclohexyl C₅), 27.6 (cyclohexyl C₄), 27.8 (cyclohexyl

C₂), 27.9 (cyclohexyl C₆), 32.4 (C₅), 37.8 (C₇), 46.7 (cyclohexyl C₁), 60.6

(C₉), 108.1 (C_8a), 128.0 (2-Cl phenyl C₅), 131.5 (2-Cl phenyl C₆), 131.6

(2-Cl phenyl C₂), 131.7 (2-Cl phenyl C₃), 132.6 (2-Cl phenyl C₄), 134.3

(2-Cl phenyl C₁), 149.9 (C_5a), 156.4 (CN₂N₄), 159.3 (CN₁N₃), 197.6

(CO).

Yield: 45.7%, yellowish powder. LR-ESI: 403.3 [M-H]^-. HR-ESI m/z

calcd for C₂₃H₂₀ClN₄O [M-H]^-403.1326, found 403.1324. ¹H NMR

(CD₃OD, 400 MHz) 1.17 (t, J = 7.6 Hz, 3H, CH₂CH₃), 2.04 (m, 2H, C₆-

H), 2.39 (m, 2H, C₅-H), 2.58 (q, J = 7.6 Hz, 2H, CH₂CH₃), 2.76 (m, 2H,

C₇-H), 6.39 (s, 1H, C₉-H), 7.12 (d, J = 8.0 Hz, 2H, 4-Et phenyl C_3,5-H),

7.21 (d, J = 8.0 Hz, 2H, 4-Et phenyl C_2,6-H), 7.32 (dt, J = 7.6 Hz, J = 1.6

Hz, 1H, 2-Cl phenyl C₄-H), 7.37 (dt, J = 8.0 Hz, J = 2.0 Hz, 1H, 2-Cl

phenyl C₅-H), 7.46 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H, 2-Cl phenyl C₃-H),

7.63 (dd, J = 7.6 Hz, J = 2.0 Hz, 1H, 2-Cl phenyl C₆-H). ¹³C NMR

(CD₃OD, 125 MHz) 16.2 (CH₂CH₃), 22.2 (C₆), 28.0 (C₅), 29.6 (CH₂CH₃),

37.7 (C₇), 59.6 (C₉), 109.6 (C_8a), 128.0 (2-Cl phenyl C₅), 128.4 (2C, 4-Et

phenyl C_3,5), 129.1 (2C, 4-Et phenyl C_2,6), 131.4 (2-Cl phenyl C₂), 131.5

(2-Cl phenyl C₆), 131.8 (2-Cl phenyl C₃), 132.6 (2-Cl phenyl C₄), 134.2

(4-Et phenyl C₁), 139.9 (2-Cl phenyl C₁), 145.7 (4-Et phenyl C₄), 148.9

(C_5a), 154.8 (CN₂N₄), 160.3 (CN₁N₃), 197.2 (CO).

4.1.29. 2-(2-Chlorophenyl)-9-(3,4-dihydroxyphenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (9c)

Yield: 61.3%, yellowish powder, m.p. 298–300 ^◦C. LR-ESI: 407.1 [M-

H]^-. HR-ESI m/z calcd for C₂₁H₁₆ClN₄O₃[M-H]^-407.0911, found

407.0916. ¹H NMR (CD₃OD, 400 MHz) 2.04 (m, 2H, C₆-H), 2.40 (brs,

2H, C₅-H), 2.74 (brs, 2H, C₇-H), 6.28 (s, 1H, C₉-H), 6.66 (d, J = 8.4 Hz,

1H, 3,4-di-OH phenyl C₆-H), 6.70 (d, J = 8.4 Hz, 1H, 3,4-di-OH phenyl

C₅-H), 6.76 (s, 1H, 3,4-di-OH phenyl C₂-H), 7.33 (t, J = 7.2 Hz, 1H, 2-Cl

phenyl C₄-H), 7.38 (t, J = 7.2 Hz, 1H, 2-Cl phenyl C₅-H), 7.46 (d, J = 8.0

Hz, 1H, 2-Cl phenyl C₃-H), 7.64 (d, J = 7.2 Hz, 1H, 2-Cl phenyl C₆-H).

¹³C NMR (CD₃OD, 125 MHz) 22.2 (C₆), 28.0 (C₅), 37.7 (C₇), 59.3 (C₉),

109.8 (C_8a), 115.5 (3,4-di-OH phenyl C₅), 116.2 (3,4-di-OH phenyl C₂),

120.0 (3,4-di-OH phenyl C₆), 128.0 (2-Cl phenyl C₅), 131.5 (2-Cl phenyl

C₆), 131.8 (2-Cl phenyl C₃), 132.7 (2-Cl phenyl C₄), 134.2 (3,4-di-OH

phenyl C₁), 134.3 (2-Cl phenyl C₂), 146.3 (3,4-di-OH phenyl C₄), 146.4

(3,4-di-OH phenyl C₃), 148.7 (C_5a), 154.5 (CN₂N₄), 160.1 (CN₁N₃),

197.3 (CO).

4.1.33. 2-(2-Chlorophenyl)-9-(4-(methylthio)phenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (9g)

Yield: 49.7%, yellowish powder, m.p. 255–256 ^◦C. LR-ESI: 421.1 [M-

H]^-. HR-ESI m/z calcd for C₂₂H₁₈ClN₄OS [M-H]^-421.0890, found

421.0897. ¹H NMR (CD₃OD, 400 MHz) 2.06 (m, 2H, C₆-H), 2.41 (m, 2H,

C₅-H), 2.43 (s, 3H, CH₃S), 2.76 (m, 2H, C₇-H), 6.39 (s, 1H, C₉-H), 7.19

(d, J = 8.8 Hz, 2H, 4-CH₃S phenyl C_2,6-H), 7.24 (d, J = 8.4 Hz, 2H, 4-

CH₃S phenyl C_3,5-H), 7.33 (dt, J = 7.6 Hz, J = 1.6 Hz, 1H, 2-Cl phenyl C₄-

H), 7.38 (dt, J = 8.0 Hz, J = 2.0 Hz, 1H, 2-Cl phenyl C₅-H), 7.46 (dd, J =

8.0 Hz, J = 1.6 Hz, 1H, 2-Cl phenyl C₃-H), 7.64 (dd, J = 7.6 Hz, J = 2.0

Hz, 1H, 2-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 15.6 (CH₃S),

22.2 (C₆), 28.0 (C₅), 37.7 (C₇), 59.5 (C₉), 109.3 (C_8a), 127.5 (2C, 4-CH₃S

phenyl C_3,5), 128.0 (2-Cl phenyl C₅), 128.9 (2C, 4-CH₃S phenyl C_2,6),

131.5 (2-Cl phenyl C₂), 131.6 (2-Cl phenyl C₆), 131.8 (2-Cl phenyl C₃),

132.6 (2-Cl phenyl C₄), 134.3 (4-CH₃S phenyl C₁), 139.3 (2-Cl phenyl

C₁), 140.3 (4-CH₃S phenyl C₄), 148.9 (C_5a), 155.0 (CN₂N₄), 160.4

(CN₁N₃), 197.1 (CO).

4.1.30. 2-(2-Chlorophenyl)-9-(4-morpholinophenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (9d)

Yield: 35.8%, yellowish powder, m.p. 284–286 ^◦C. LR-ESI: 460.2 [M-

H]^-. HR-ESI m/z calcd for C₂₅H₂₃ClN₅O₂[M-H]^-460.1540, found

460.1543. ¹H NMR (CD₃OD, 400 MHz) 2.07 (m, 2H, C₆-H), 2.41 (m, 2H,

C₅-H), 2.75 (m, 2H, C₇-H), 3.09 (t, J = 5.2 Hz, 4H, morpholino NCH₂),

3.78 (t, J = 4.8 Hz, 4H, morpholino OCH₂), 6.36 (s, 1H, C₉-H), 6.88 (d, J

= 8.8 Hz, 2H, phenyl C_3,5-H), 7.20 (d, J = 8.8 Hz, 2H, phenyl C_2,6-H),

7.33 (dt, J = 7.2 Hz, J = 1.6 Hz, 1H, 2-Cl phenyl C₄-H), 7.40 (dt, J = 8.0

Hz, J = 2.0 Hz, 1H, 2-Cl phenyl C₅-H), 7.46 (dd, J = 7.6 Hz, J = 1.2 Hz,

1H, 2-Cl phenyl C₃-H), 7.64 (dd, J = 7.6 Hz, J = 2.0 Hz, 1H, 2-Cl phenyl

C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 22.2 (C₆), 28.0 (C₅), 37.7 (C₇), 50.7

(2C, morpholino NCH₂), 59.3 (C₉), 68.1 (2C, morpholino OCH₂), 109.7

(C_8a), 116.8 (2C, phenyl C_3,5), 128.0 (2-Cl phenyl C₅), 129.2 (2C, phenyl

C_2,6), 131.5 (phenyl C₁), 131.6 (2-Cl phenyl C₆), 131.8 (2-Cl phenyl C₃),

132.6 (2-Cl phenyl C₄), 134.0 (2-Cl phenyl C₂), 134.3 (2-Cl phenyl C₁),

148.8 (C_5a), 152.8 (phenyl C₄), 154.5 (CN₂N₄), 160.2 (CN₁N₃), 197.2

(CO).

4.1.34. 2-(2-Chlorophenyl)-9-(4-(pyrrolidin-1-yl)phenyl)-5,6,7,9-

tetrahydro-[1,2,4]triazolo[5,1-b] quinazolin-8(4H)-one (9h)

Yield: 39.0%, yellowish powder, m.p. 280–282 ^◦C. LR-ESI: 444.2 [M-

H]^-. HR-ESI m/z calcd for C₂₅H₂₃ClN₅O [M-H]^-444.1591, found

444.1593. ¹H NMR (CD₃OD, 400 MHz) 1.98 (m, 4H, pyrrolidinyl C_3,4

-

H), 2.03 (m, 1H, C₆-H), 2.20 (m, 1H, C₆-H), 2.40 (m, 2H, C₅-H), 2.77 (m,

2H, C₇-H), 3.22 (m, 4H, pyrrolidinyl C_2,5-H), 6.31 (s, 1H, C₉-H), 6.48 (d,

J = 8.4 Hz, 2H, 4-pyrrolidinyl phenyl C_3,5-H), 7.12 (d, J = 8.8 Hz, 2H, 4-

pyrrolidinyl phenyl C_2,6-H), 7.33 (dt, J = 7.6 Hz, J = 1.6 Hz, 1H, 2-Cl

phenyl C₄-H), 7.38 (dt, J = 7.2 Hz, J = 1.6 Hz, 1H, 2-Cl phenyl C₅-H),

7.47 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H, 2-Cl phenyl C₃-H), 7.63 (dd, J = 7.6

Hz, J = 2.0 Hz, 1H, 2-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 22.3

(C₆), 26.5 (2C, pyrrolidinyl C_3,4), 28.0 (C₅), 37.7 (C₇), 58.5 (2C, pyrro-

lidinyl C_2,5), 59.5 (C₉), 110.1 (C_8a), 112.7 (2C, 4-pyrrolidinyl phenyl

4.1.31. N-(4-(2-(2-Chlorophenyl)-8-oxo-4,5,6,7,8,9-hexahydro-[1,2,4]

triazolo[5,1-b]quinazolin-9-yl) phenyl)acetamide (9e)

Yield: 55.8%, yellowish powder, m.p. 290–291 ^◦C. LR-ESI: 432.2 [M-

H]^-. HR-ESI m/z calcd for C₂₃H₁₉ClN₅O₂[M-H]^-432.1233, found

432.1237. ¹H NMR (CD₃OD, 400 MHz) 2.08 (m, 2H, C₆-H), 2.09 (s, 3H,

AcNH), 2.40 (m, 2H, C₅-H), 2.75 (m, 2H, C₇-H), 6.39 (s, 1H, C₉-H), 7.26

(d, J = 8.8 Hz, 2H, 4-AcNH phenyl C_2,6-H), 7.33 (dt, J = 7.6 Hz, J = 1.6

Hz, 1H, 2-Cl phenyl C₄-H), 7.38 (dt, J = 7.2 Hz, J = 2.0 Hz, 1H, 2-Cl

phenyl C₅-H), 7.48 (d, J = 8.8 Hz, 2H, 4-AcNH phenyl C_3,5-H), 7.64

(dd, J = 7.6 Hz, J = 2.0 Hz, 1H, 2-Cl phenyl C₃-H), 7.78 (d, J = 8.4 Hz,

C_3,5), 128.0 (2-Cl phenyl C₅), 128.1 (2-Cl phenyl C₆), 129.1 (2C, 4-pyr-

rolidinyl phenyl C_2,6), 129.3 (4-pyrrolidinyl phenyl C₁), 131.5 (2-Cl

phenyl C₂), 131.9 (2-Cl phenyl C₃), 132.7 (2-Cl phenyl C₄), 140.0 (2-Cl

phenyl C₁), 148.8 (C_5a), 149.6 (4-pyrrolidinyl phenyl C₄), 154.9

(CN₂N₄), 160.1 (CN₁N₃), 197.4 (CO).

13

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Bioorganic Chemistry 110 (2021) 104782

4.1.35. 2-(2-Chlorophenyl)-9-(4-mercaptophenyl)-5,6,7,9-tetrahydro-

[1,2,4]triazolo [5,1-b] quinazolin-8(4H)-one (9i)

H]^-. HR-ESI m/z calcd for C₂₀H₁₄ClN₄O₂S [M-H]^-409.0526, found

409.0531. ¹H NMR (CD₃OD, 500 MHz) 3.18 (dd, J = 2.0 Hz, J = 16.0 Hz,

1H, C₅-H), 3.56 (dd, J = 2.0 Hz, J = 16.5 Hz, 1H, C₅-H), 3.62 (dd, J = 1.5

Hz, J = 17.0 Hz, 1H, C₇-H), 3.95 (d, J = 17.0 Hz, 1H, C₇-H), 6.39 (s, 1H,

C₉-H), 6.72 (d, J = 8.5 Hz, 2H, 4-OH phenyl C_3,5-H), 7.18 (d, J = 8.5 Hz,

2H, 4-OH phenyl C_2,6-H), 7.34 (dt, J = 1.5 Hz, J = 7.5 Hz, 1H, 2-Cl

phenyl C₄-H), 7.39 (dt, J = 1.5 Hz, J = 7.5 Hz, 1H, 2-Cl phenyl C₅-H),

7.47 (dd, J = 1.5 Hz, J = 8.0 Hz, 1H, 2-Cl phenyl C₃-H), 7.64 (dd, J = 1.5

Hz, J = 7.5 Hz, 1H, 2-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 27.6

(C₅), 35.4 (C₇), 59.3 (C₉), 108.8 (C_8a), 116.3 (2C, 4-OH phenyl C_3,5),

128.0 (2-Cl phenyl C₅), 129.6 (2C, 4-OH phenyl C_2,6), 131.4 (4-OH

phenyl C₁), 131.5 (2-Cl phenyl C₆), 131.8 (2-Cl phenyl C₃), 132.7 (2-Cl

phenyl C₄), 133.2 (2-Cl phenyl C₂), 134.3 (2-Cl phenyl C₁), 148.3 (C_5a),

152.1 (CN₂N₄), 158.7 (4-OH phenyl C₄), 160.3 (CN₁N₃), 191.6 (CO).

LR-ESI: 409.1 [M+H]⁺. HR-ESI m/z calcd for C₂₁H₁₈ClN₄OS

[M+H]⁺409.0890, found 409.0895. ¹H NMR (CD₃OD, 400 MHz) 2.09

(m, 2H, C₆-H), 2.40 (m, 2H, C₅-H), 2.76 (m, 2H, C₇-H), 6.34 (s, 1H, C₉-

H), 7.04 (d, J = 8.8 Hz, 2H, 4-SH phenyl C_2,6-H), 7.24 (d, J = 8.4 Hz, 2H,

4-SH phenyl C_3,5-H), 7.33 (dt, J = 1.2 Hz, J = 7.6 Hz, 1H, 2-Cl phenyl C₅-

H), 7.38 (dt, J = 1.6 Hz, J = 7.6 Hz, 1H, 2-Cl phenyl C₄-H), 7.47 (d, J =

8.0 Hz, 1H, 2-Cl phenyl C₃-H), 7.63 (dd, J = 2.0 Hz, J = 7.6 Hz, 1H, 2-Cl

phenyl C₆-H).

4.1.36. 2-(2-Chlorophenyl)-9-(4-hydroxyphenyl)-6,6-dimethyl-5,6,7,9-

tetrahydro-[1,2,4]triazolo [5,1-b]quinazolin-8(4H)-one (11a)

Yield: 57.2%, yellowish powder, m.p. 228–229 ^◦C. LR-ESI: 419.2 [M-

H]^-. HR-ESI m/z calcd for C₂₃H₂₀ClN₄O₂[M-H]^-419.1275, found

419.1278. ¹H NMR (CD₃OD, 400 MHz) 1.06 (s, 3H, C₆-CH₃), 1.14 (s, 3H,

C₆-CH₃), 2.21 (d, J = 16.4 Hz, 1H, C₅-H), 2.34 (d, J = 16.4 Hz, 1H, C₅-H),

2.59 (d, J = 17.2 Hz, 1H, C₇-H), 2.65 (d, J = 16.8 Hz, 1H, C₇-H), 6.31 (s,

1H, C₉-H), 6.71 (d, J = 8.0 Hz, 2H, 4-OH phenyl C_3,5-H), 7.13 (d, J = 8.0

Hz, 2H, 4-OH phenyl C_2,6-H), 7.34 (t, J = 7.6 Hz, 1H, 2-Cl phenyl C₄-H),

7.39 (t, J = 7.6 Hz, 1H, 2-Cl phenyl C₅-H), 7.47 (d, J = 8.0 Hz, 1H, 2-Cl

phenyl C₃-H), 7.63 (d, J = 7.2 Hz, 1H, 2-Cl phenyl C₆-H). ¹³C NMR

(CD₃OD, 125 MHz) 27.6 (C₆-CH₃), 29.3 (C₆-CH₃), 33.7 (C₆), 41.3 (C₅),

51.3 (C₇), 59.6 (C₉), 108.9 (C_8a), 116.3 (2C, 4-OH phenyl C_3,5), 128.0 (2-

Cl phenyl C₅), 129.6 (2C, 4-OH phenyl C_2,6), 131.5 (2-Cl phenyl C₆),

131.8 (2-Cl phenyl C₃), 132.7 (2-Cl phenyl C₄), 132.9 (C_5a), 133.6 (4-OH

phenyl C₁), 134.3 (2-Cl phenyl C₂), 148.9 (2-Cl phenyl C₁), 152.6

(CN₂N₄), 158.6 (4-OH phenyl C₄), 160.3 (CN₁N₃), 196.9 (CO).

4.1.40. 9-(4-Hydroxyphenyl)-5,6,7,9-tetrahydro-[1,2,4]triazolo[5,1-b]

quinazolin-8(4H)-one (13a)

Yield: 69.5%, white powder, m.p. 230–231 ^◦C. LR-ESI: 281.2 [M-1]^-.

HR-ESI m/z calcd for C₁₅H₁₃N₄O₂[M-H]^-281.1039, found 281.1042. ¹H

NMR (DMSO‑d₆, 400 MHz) 1.94 (m, 2H, C₆-H), 2.25 (m, 2H, C₅-H), 2.65

(m, 2H, C₇-H), 6.11 (s, 1H, C₉-H), 6.64 (d, J = 7.6 Hz, 2H, 4-OH phenyl

C

_3,5-H), 6.99 (d, J = 7.6 Hz, 2H, 4-OH phenyl C_2,6-H), 7.65 (s, 1H, C₂-H),

9.36 (s, OH), 11.0 (s, NH). ¹³C NMR (DMSO‑d₆, 125 MHz) 20.7 (C₆),

26.4 (C₅), 36.4 (C₇), 57.1 (C₉), 106.8 (C_8a), 114.9 (2C, 4-OH phenyl

C_3,5), 128.1 (2C, 4-OH phenyl C_2,6), 132.1 (4-OH phenyl C₁), 146.7

(C_5a), 149.8 (C₂), 152.3 (CN₂N₄), 156.9 (4-OH phenyl C₄), 193.2 (CO).

4.1.41. 9-(4-Hydroxyphenyl)-5,6,7,9-tetrahydrotetrazolo[5,1-b]

quinazolin-8(4H)-one (13b)

4.1.37. 2-(2-Chlorophenyl)-9-(4-hydroxyphenyl)-5,5-dimethyl-5,6,7,9-

tetrahydro-[1,2,4]triazolo [5,1-b]quinazolin-8(4H)-one (11b)

Yield: 75.8%, yellowish powder. LR-ESI: 282.1 [M-H]^-. HR-ESI m/z

calcd for C₁₄H₁₂N₅O₂[M-H]^-282.0991, found 282.0993. ¹H NMR

(CD₃OD, 400 MHz) 2.09 (m, 2H, C₆-H), 2.40 (m, 2H, C₅-H), 2.78 (m, 2H,

C₇-H), 6.58 (s, 1H, C₉-H), 6.72 (d, J = 8.0 Hz, 2H, 4-OH phenyl C_3,5-H),

7.13 (d, J = 8.0 Hz, 2H, 4-OH phenyl C_2,6-H). ¹³C NMR (CD₃OD, 125

MHz) 22.2 (C₆), 28.3 (C₅), 37.7 (C₇), 58.9 (C₉), 109.2 (C_8a), 116.5 (2C,

4-OH phenyl C_3,5), 129.7 (2C, 4-OH phenyl C_2,6), 132.7 (4-OH phenyl

C₁), 150.3 (C_5a), 155.5 (4-OH phenyl C₄), 159.0 (tetrazole), 196.9 (CO).

Yield: 67.2%, white powder, m.p. 227–229 ^◦C. LR-ESI: 419.1 [M-H]^-.

HR-ESI m/z calcd for C₂₃H₂₀ClN₄O₂[M-H]^-419.1275, found 419.1279.

¹H NMR (CD₃OD, 400 MHz) 1.02 (s, 3H, C₅-CH₃), 1.11 (s, 3H, C₅-CH₃),

1.91 (m, 2H, C₆-H), 2.74 (m, 2H, C₇-H), 6.29 (s, 1H, C₉-H), 6.70 (d, J =

8.4 Hz, 2H, 4-OH phenyl C_3,5-H), 7.12 (d, J = 8.4 Hz, 2H, 4-OH phenyl

C

_2,6-H), 7.32 (t, J = 7.6 Hz, 1H, 2-Cl phenyl C₃-H), 7.37 (t, J = 8.0 Hz,

1H, 2-Cl phenyl C₅-H), 7.46 (d, J = 8.0 Hz, 1H, 2-Cl phenyl C₃-H), 7.62

(d, J = 7.2 Hz, 1H, 2-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz) 24.8

(C₅-CH₃), 25.3 (C₅-CH₃), 35.7 (2C, C_6,7), 41.3 (C₅), 59.6 (C₉), 108.2

(C_8a), 116.3 (2C, 4-OH phenyl C_3,5), 128.0 (2-Cl phenyl C₅), 129.6 (2C,

4-OH phenyl C_2,6), 131.5 (2-Cl phenyl C₆), 131.8 (2-Cl phenyl C₃), 132.6

(2-Cl phenyl C₄), 133.6 (4-OH phenyl C₁), 134.3 (2-Cl phenyl C₂), 148.7

(2-Cl phenyl C₁), 152.6 (CN₂N₄), 158.5 (4-OH phenyl C₄), 160.2

(CN₁N₃), 172.4 (C_5a), 202.1 (CO).

4.1.42. 2-(2-Chlorophenyl)-9-(4-hydroxyphenyl)-5,6,7,9-

tetrahydropyrazolo[5,1-b]quinazolin-8(4H)- one (13c)

Yield: 75.0%, yellowish powder, m.p. 256–258 ^◦C. LR-ESI: 390.1 [M-

H]^-. HR-ESI m/z calcd for C₂₂H₁₇ClN₃O₂[M-H]^-390.1009, found

390.1012. ¹H NMR (CD₃OD, 400 MHz) 2.02 (m, 2H, C₆-H), 2.35 (m, 2H,

C₅-H), 2.70 (m, 2H, C₇-H), 6.21 (s, 1H, C₉-H), 6.32 (s, 1H, C₃-H), 6.66 (d,

J = 7.6 Hz, 2H, 4-OH phenyl C_3,5-H), 7.07 (d, J = 7.6 Hz, 2H, 4-OH

phenyl C_2,6-H), 7.28 (m, 2H, 2-Cl phenyl C_4,5-H), 7.43 (m, 1H, 2-Cl

phenyl C₃-H), 7.56 (m, 1H, 2-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125

MHz) 22.3 (C₆), 28.1 (C₅), 37.6 (C₇), 58.7 (C₉), 91.3 (C₃), 108.8 (C_8a),

116.0 (2C, 4-OH phenyl C_3,5), 128.1 (2-Cl phenyl C₆), 129.2 (2C, 4-OH

phenyl C_2,6), 130.6 (2-Cl phenyl C₄), 131.3 (2-Cl phenyl C₅), 131.8

(CN₂N₃), 132.0 (2-Cl phenyl C₃), 133.6 (2-Cl phenyl C₁), 135.1 (4-OH

phenyl C₁), 139.2 (2-Cl phenyl C₂), 150.9 (C_5a), 153.9 (C₂), 158.0 (4-OH

phenyl C₄), 196.9 (CO).

4.1.38. 2-(2-Chlorophenyl)-9-(4-hydroxyphenyl)-7,9-dihydro-4H-pyrano

[3,4-d][1,2,4]triazolo[1,5-a] pyrimidin-8(5H)-one (11c)

Yield: 58.8%, yellowish powder, m.p. 208–209 ^◦C. LR-ESI: 393.1 [M-

H]^-. HR-ESI m/z calcd for C₂₀H₁₄ClN₄O₃[M-H]^-393.0754, found

393.0757. ¹H NMR (CD₃OD, 400 MHz) 4.13 (s, 2H, C₅-H), 4.61 (d, J =

16.0 Hz, 1H, C₇-H), 4.69 (d, J = 16.4 Hz, 1H, C₇-H), 6.40 (s, 1H, C₉-H),

6.73 (d, J = 7.6 Hz, 2H, 4-OH phenyl C_3,5-H), 7.16 (d, J = 7.6 Hz, 2H, 4-

OH phenyl C_2,6-H), 7.33 (t, J = 7.2 Hz, 1H, 2-Cl phenyl C₄-H), 7.38 (t, J

= 7.6 Hz, 1H, 2-Cl phenyl C₅-H), 7.46 (d, J = 8.0 Hz, 1H, 2-Cl phenyl C₃-

H), 7.65 (d, J = 7.2 Hz, 1H, 2-Cl phenyl C₆-H). ¹³C NMR (CD₃OD, 125

MHz) 58.8 (C₉), 65.0 (C₇), 72.6 (C₅), 107.1 (C_8a), 116.4 (2C, 4-OH

phenyl C_3,5), 128.0 (2-Cl phenyl C₅), 129.6 (2C, 4-OH phenyl C_2,6),

131.4 (4-OH phenyl C₁), 131.5 (2-Cl phenyl C₆), 131.8 (2-Cl phenyl C₃),

132.6 (2-Cl phenyl C₄), 132.8 (2-Cl phenyl C₂), 134.2 (2-Cl phenyl C₁),

148.3 (CN₂N₄), 152.4 (C_5a), 158.8 (4-OH phenyl C₄), 160.3 (CN₁N₃),

192.6 (CO).

4.1.43. 2-(2-Bromophenyl)-9-(4-hydroxyphenyl)-7,9-dihydro-4H-

thiopyrano[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-8(5H)-one (14a)

Yield: 49.5%, yellowish powder, m.p. 295–296 ^◦C. LR-ESI: 455.0

[M+H]⁺. HR-ESI m/z calcd for C₂₀H₁₆BrN₄O₂S [M+H]⁺455.0172,

found 455.0162. ¹H NMR (CD₃OD, 400 MHz) 3.16 (d, J = 16.0 Hz, 1H,

C₅-H), 3.54 (d, J = 17.2 Hz, 2H, C_5,7-H), 3.89 (d, J = 17.2 Hz, 1H, C₇-H),

6.38 (s, 1H, C₉-H), 6.71 (d, J = 8.8 Hz, 2H, 4-OH phenyl C_3,5-H), 7.18 (d,

J = 8.8 Hz, 2H, 4-OH phenyl C_2,6-H), 7.30 (t, J = 8.0 Hz, 1H, 2-Br phenyl

C₄-H), 7.38 (t, J = 7.6 Hz, 1H, 2-Br phenyl C₅-H), 7.57 (dd, J = 1.6 Hz, J

= 7.6 Hz, 1H, 2-Br phenyl C₃-H), 7.66 (d, J = 8.4 Hz, 1H, 2-Br phenyl C₆-

H). ¹³C NMR (CD₃OD, 125 MHz) 27.5 (C₅), 35.4 (C₇), 59.3 (C₉), 108.9

(C_8a), 116.3 (2C, 4-OH phenyl C_3,5), 123.4 (2-Br phenyl C₂), 128.5 (2-Br

4.1.39. 2-(2-Chlorophenyl)-9-(4-hydroxyphenyl)-5,9-dihydro-4H-

thiopyrano[3,4-d][1,2,4]triazolo [1,5-a]pyrimidin-8(7H)-one (11d)

Yield: 58.6%, yellowish powder, m.p. 211–213 ^◦C. LR-ESI: 409.1 [M-

14

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

Isomer 2: 9-(S)-14b, [α ₂^D₀

]

+22^◦(c 0.1 in methanol).

phenyl C₅), 129.7 (2C, 4-OH phenyl C_2,6), 132.0 (2-Br phenyl C₆), 132.8

(2-Br phenyl C₄), 133.2 (4-OH phenyl C₁), 133.6 (C_5a), 134.8 (2-Br

phenyl C₃), 148.2 (2-Br phenyl C₁), 151.9 (CN₂N₄), 158.7 (4-OH phenyl

C₄), 161.3 (CN₁N₃), 191.5 (CO).

4.4. X-ray structure determination of 9-(R)-7a (isomer 1)

Diffraction data were collected on a Bruker D8 VENTURE single-

4.1.44. 9-(4-Hydroxyphenyl)-2-(2-iodophenyl)-7,9-dihydro-4H-

crystal diffractometer using a graphite-monochromated MoK

α radia-

thiopyrano[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-8(5H)-one (14b)

Yield: 50.5%, yellowish powder, m.p. > 300 ^◦C. LR-ESI: 501.1 [M-

1]^-. HR-ESI m/z calcd for C₂₀H₁₄IN₄O₂S [M-H]^-500.9882, found

500.9885. ¹H NMR (CD₃OD, 400 MHz) 3.15 (dd, J = 2.0 Hz, J = 16.0 Hz,

1H, C₅-H), 3.53 (m, 2H, C_5,7-H), 3.87 (dd, J = 2.0 Hz, J = 17.2 Hz, 1H,

C₇-H), 6.36 (s, 1H, C₉-H), 6.71 (d, J = 8.8 Hz, 2H, 4-OH phenyl C_3,5-H),

7.11 (dt, J = 1.6 Hz, J = 7.6 Hz, 1H, 2-I phenyl C₅-H), 7.18 (d, J = 8.8 Hz,

2H, 4-OH phenyl C_2,6-H), 7.39 (dt, J = 1.2 Hz, J = 7.6 Hz, 1H, 2-I phenyl

C₄-H), 7.48 (dd, J = 1.6 Hz, J = 7.6 Hz, 1H, 2-I phenyl C₆-H), 7.93 (d, J

= 8.0 Hz, 1H, 2-I phenyl C₃-H). ¹³C NMR (CD₃OD, 125 MHz) 27.6 (C₅),

35.4 (C₇), 59.3 (C₉), 97.4 (2-I phenyl C₂), 108.8 (C_8a), 116.3 (2C, 4-OH

phenyl C_3,5), 127.4 (2-I phenyl C₅), 129.2 (2-I phenyl C₆), 129.7 (2C, 4-

OH phenyl C_2,6), 132.0 (2-I phenyl C₄), 133.2 (4-OH phenyl C₁), 137.6

(C_5a), 141.3 (2-I phenyl C₃), 148.2 (2-I phenyl C₁), 152.0 (CN₂N₄), 158.6

(4-OH phenyl C₄), 163.0 (CN₁N₃), 191.5 (CO).

tion (0.71073 Å) at 193 K in the

ω-2θ scan mode. In this case, an

empirical absorption correction by SADABS was applied to the intensity

data. The structure was solved by direct methods and reﬁned by full-

matrix least-squares on F²methods using the SHELXTL crystallo-

graphic software package. All non-hydrogen atoms were reﬁned aniso-

tropically with hydrogen atoms included in calculated positions (riding

model). Crystallographic data for compound 9-(R)-7a is given in

Table S1-6. CCDC2004638 contains the supplementary crystallographic

data for compound 9-(R)-7a, which can be obtained free of charge from

The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.

uk/data_request/cif.

4.5. Bioassays

4.5.1. Reagents

4.1.45. 9-(4-Hydroxyphenyl)-2-(2-(triﬂuoromethyl)phenyl)-7,9-dihydro-

4H-thiopyrano[3,4-d][1,2,4] triazolo[1,5-a]pyrimidin-8(5H)-one (14c)

Yield: 40.9%, yellowish powder, m.p. 240–241 ^◦C. LR-ESI: 443.2 [M-

1]^-. HR-ESI m/z calcd for C₂₁H₁₄F₃N₄O₂S [M-H]^-443.0790, found

443.0797. ¹H NMR (CD₃OD, 400 MHz) 3.18 (dd, J = 16.4 Hz, J = 2.4 Hz,

1H, C₅-H), 3.57 (dd, J = 16.0 Hz, J = 2.0 Hz, 1H, C₅-H), 3.60 (dd, J =

16.8 Hz, J = 2.0 Hz, 1H, C₇-H), 3.94 (d, J = 16.8 Hz, 1H, C₇-H), 6.38 (s,

1H, C₉-H), 6.71 (d, J = 8.8 Hz, 2H, 4-OH phenyl C_3,5-H), 7.16 (d, J = 8.8

Hz, 2H, 4-OH phenyl C_2,6-H), 7.64 (m, 3H, 2-CF₃phenyl C_3,4,5-H), 7.78

(d, J = 7.6 Hz, 1H, 2-CF₃phenyl C₆-H). ¹³C NMR (CD₃OD, 125 MHz)

27.5 (C₅), 35.4 (C₇), 59.3 (C₉), 108.9 (C_8a), 116.3 (2C, 4-OH phenyl

Human INSL5 (hINSL5), R3/I5 and relaxin-3 were purchased from

Phoenix Pharmaceuticals (Burlingame, CA, USA). LANCE Ultra cAMP

and AlphaScreen SureFire p-ERK1/2 assay kits were obtained from

PerkinElmer (Waltham, MA, USA). Forskolin, 3-isobutyl-1-methylxan-

thine (IBMX), dimethyl sulfoxide (DMSO) and bovine serum albumin

were supplied by Sigma-Aldrich (St. Louis, MO, USA). The methods for

plasmid construction, cell culture and assay validation were described in

our previous paper [14].

4.5.2. cAMP accumulation assay

For off-target examination, inhibition of forskolin-induced cAMP

accumulation by test compounds was carried out in parental CHO cells.

For selective agonist effect evaluation, compounds were tested for their

ability to inhibit cAMP accumulation in CHO-K1 cells stably over-

expressing human RXFP4 or human RXFP3. Cells were seeded at a

density of 8 × 10⁵cells/mL and stimulated with different concentrations

of individual testing compounds (250, 100, 40, 16, 6.4, 2.56, 1.024 and

C

_3,5), 127.6 (CF₃), 127.7 (2-CF₃phenyl C₃), 129.6 (2C, 4-OH phenyl

_2,6), 130.5 (2-CF₃phenyl C₂), 131.0 (2-CF₃phenyl C₄), 131.5 (C_5a),

133.0 (2-CF₃phenyl C₆), 133.1 (2-CF₃phenyl C₅), 133.2 (4-OH phenyl

C₁), 148.4 (2-CF₃phenyl C₁), 152.1 (CN₂N₄), 158.7 (4-OH phenyl C₄),

160.6 (CN₁N₃), 191.5 (CO).

0.4096

500 M IBMX. Peptides INSL5 and R3/I5 were used as positive controls

at different concentrations M). For cAMP assay on hRXFP1-

μM) plus 500 nM forskolin for 40 min at RT in the presence of

4.2. Chiral resolution of compound 7a

μ

(

μ

The enantiomers of compound 7a (1.57 g) were resolved by CHIR-

ALPAK IC (IC00CD-NA012) column (0.46 cm × 15 cm) on Shimadzu LC-

20AT HPLC eluting with dichloromethane/ethanol [90/10 (v/v)] with a

ﬂow rate of 1.0 mL/min at 35 ^◦C under the wavelength of UV 254 nm..

Two peaks were separately collected at the t_R= 2.090 min (isomer 1)

and t_R= 2.409 min (isomer 2) and the enantiomeric excess (e.e.) value of

each product is determined by HPLC as > 98%. Two isomers were thus

obtained as light yellowish powder (isomer 1: 0.71 g; isomer 2: 0.71 g).

The absolute conﬁguration of isomer 1 was determined by X-ray

diffraction as 9-(R)-7a. Speciﬁc optical rotation was also detected for

overexpressing HEK293T cells, relaxin-3 was used as positive control

without forskolin stimulation. Eu-cAMP tracer and ULight-anti-cAMP

working solution were then applied followed by incubation for 40 min

at RT. Time-resolved ﬂuorescence resonance energy transfer (TR-FRET)

signals were read on an EnVision® multimode plate reader (Perki-

nElmer) with excitation at 320 or 340 nm and emission at 665 nm and

615 nm. Each compound was tested in duplicate and each experiment

was performed independently three times. Agonist activity was

expressed as % INSL5 in hRXFP4-CHO-K1 cells, % R3/I5 in hRXFP3-

CHO-K1 cells or % relaxin-3 in hRXFP1-HEK293T cells. For each

ligand-concentration, the value of 665/615 was calculated, and

normalized to the corresponding maximum value obtained for INSL5 in

hRXFP4-CHO-K1 cells, R3/I5 in hRXFP3-CHO-K1 cells and relaxin-3 in

hRXFP1-HEK293T cells. The normalized values were plotted vs. ligand

concentration using GraphPad PRISM 8 and are expressed as means ±

SEM.

these two isomers. Isomer 1: 9-(R)-7a, [α ^D₂₀-110^◦(c 0.1 in methanol);

]

Isomer 2: 9-(S)-7a, [α ₂^D₀

]

+107^◦(c 0.1 in methanol).

4.3. Chiral resolution of compound 14b

The enantiomers of compound 14b (0.0705 g) were resolved by

CHIRALPAK IC column (0.46 cm × 15 cm) on Shimadzu LC-2010 HPLC

◦

eluting with ethanol at a ﬂow rate of 1.0 mL/min at 25 C under the

4.5.3. Cytotoxicity assay

wavelength of UV 210 nm. Two peaks were separately collected at the t_R

= 4.568 min (isomer 1) and t_R= 5.981 min (isomer 2) and the enan-

tiomeric excess (e.e.) value of each product is determined by HPLC as >

99%. Two isomers were thus obtained as white powder (isomer 1:

0.0309 g; isomer 2: 0.0255 g). Speciﬁc optical rotation was detected for

Cytotoxicity was assessed in hRXFP4-CHO cells using the Cell

Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). Cells were seeded

into 96-well plates at a density of 30,000 cells/well and incubated

overnight, in which different concentrations of compounds were added

and incubated for 24 h. CCK-8 solution was then added and incubated

for another 1 h. Absorbance values at 450 nm were recorded on a

these two isomers. Isomer 1: 9-(R)-14b, [α ^D₂₀-22^◦(c 0.1 in methanol);

]

15

L. Lin et al.

Bioorganic Chemistry 110 (2021) 104782

SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA).

Data were normalized to the vehicle-treated samples (Supplementary

Information).

Author Contributions

QL, DHY and M-WW designed research. LL, GYL, QTZ, GQG and QL

performed research, LL, GYL, QTZ, QL, RADB, DHY and M-WW analyzed

data. LL, QL and M-WW wrote the manuscript.

4.5.4. Receptor binding assay

CHO-K1 cells stably expressing human RXFP4 were plated out at the

density of 50,000 cells per well per 200 μL in a 96-well ViewPlate with

Declaration of Competing Interest

clear bottom and white walls precoated with poly-^L-lysine. Competitive

binding assay was performed with 5 nM of europium-labeled Eu(A)-R3/

I5 in the presence of increasing amounts of test compounds dissolved in

DMSO following the protocol described previously [18]. Fluorescence

measurement was carried out at an excitation wavelength of 340 nm and

an emission wavelength of 614 nm on a Victor Plate Reader (Perki-

nElmer). Each concentration point was measured in triplicate, and each

experiment was performed independently three times. Data were

analyzed using GraphPad PRISM 8 and expressed as means ± SEM.

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.

Acknowledgements

We thank Sharon Layﬁeld and Tania Ferraro for technical assistance.

This work was partially supported by grants from the National Natural

Science Foundation of China 81872915 (M.-W.W.), 82073904 (M-WW),

21302202 (QL) 81973373 (DHY) and 81773792 (DHY), the National

Science & Technology Major Project “Key New Drug Creation and

Manufacturing Program” of China (2018ZX09735-001 to M-WW,

2018ZX09711002-002-005 to DHY and 2018ZX09711002-002-011 to

QL), the National Key R&D Program of China 2018YFA0507000 (M-

WW) and Novo Nordisk-CAS Research Fund (NNCAS-2017-1-CC to

DHY). The funders had no role in study design, data collection, and

analysis, decision to publish, or manuscript preparation.

4.5.5. Statistical analysis

Dose-response data were analyzed with Prism software (GraphPad

PRISM 8) using a sigmoidal model with variable slope. Statistical sig-

niﬁcance was determined using two tailed student’s t-test, and P < 0.05

was considered signiﬁcant.

4.6. Molecular modelling

Molecular docking for the binding of derivatives to hRXFP4 and

hRXFP3 was performed using the LibDock docking protocol in BIOVIA

Discovery Studio 2016 (Accelrys).

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.104782.

4.6.1. Preparation of target protein

Homology models of RXFP4 (SWISS-MODEL: Q8TDU9) and RXFP3

(SWISS-MODEL: Q9NSD7), which were modeled on the template of

agonist-bound apelin receptor (PDB code: 5VBL), were downloaded

from SWISS-MODEL at https://swissmodel.expasy.org/repository/

uniprot/. The energy of the system was minimized using CHARMm

forceﬁeld.

References

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4.6.2. Preparation of ligands

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default values using CHARMm forceﬁeld until RMS gradient of 0.01 was

reached.

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4.6.3. Molecular docking

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binding site constructed by residues L118^3.29, T176^4.60, R208^5.42

F291^7.35, Q205^5.39, T266^6.55, G269^6.58, V265^6.54, Q287^7.31, K273^6.62

,

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and Y204^5.38, and the hRXFP3 orthosteric binding site constructed by

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residues T346^6.55, Y369^7.33, L345^6.54, L365^7.29, C366^7.30, S349^6.58

,

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213–224, https://doi.org/10.1530/JME-17-0152.

Y267^5.38, L264^5.35, I350^6.59, K353^6.62, F262^ECL2, W263^5.34, R250^ECL2

and F251^ECL2. Then the prepared ligands 7a and 14b were docked into

the binding site using LibDock protocol, with the top 10 poses presented

and scored while keeping other options in their default values. LibDock

ﬁtness scores of 114.064 (7a) and 111.977 (14b) for hRXFP4, and

111.545 (7a) and 111.749 (14b) for hRXFP3 were thus obtained.

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17

Article Doi

Design, synthesis and pharmacological evaluation of tricyclic derivatives as selective RXFP4 agonists

DOI: 10.1016/j.bioorg.2021.104782

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