Design, Synthesis, and Evaluation of Small Molecule Gαq/11 Protein Inhibitors for the Treatment of Uveal Melanoma

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Design, Synthesis, and Evaluation of Small Molecule Gαq/11 Protein

Inhibitors for the Treatment of Uveal Melanoma

Yang Ge,^†Shuo Shi,^†Jun-Jie Deng, Xue-Ping Chen, Zhendong Song, Lu Liu, Linlin Lou, Xiaolei Zhang,*

and Xiao-Feng Xiong *

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ABSTRACT: Uveal melanoma is the ocular malignancy and

mainly driven by oncogenic mutations of Gαq/11 proteins.

Previous targeted therapy for melanoma treatment was limited to

speciﬁc downstream signaling pathway, and inhibiting the

“molecular switches” G proteins for melanoma treatment therapy

was rarely described. We herein report the discovery of

imidazopiperazine derivatives as Gαq/11 protein inhibitors. The

most promising compound GQ127 showed good eﬃcacy and

safety in inositol monophosphate (IP₁) assay by directly inhibiting

Gαq/11 proteins. GQ127 induced uveal melanoma cells apoptosis

and displayed potent antitumor activities in uveal melanoma cells

viability, migration, and invasion. The eﬀects of GQ127 on Gαq/

11 signaling pathway were conﬁrmed by analyzing the downstream

eﬀectors yes-associated protein (YAP) and extracellular signal-regulated kinase (ERK). More importantly, GQ127 signiﬁcantly

suppressed UM xenograft growth in mouse model without severe toxicity at the testing dose. These ﬁndings provide a lead

compound that directly targets the Gαq/11 proteins for uveal melanoma treatment.

conventionally considered as “molecular switches” that trans-

duce extracellular signals perceived by transmembrane G-

protein-coupled receptors (GPCRs) to intracellular signaling

networks.^11,12When agonists bind to and activate GPCRs, the

intracellular G proteins are activated by the conformational

changes of the GPCRs, followed by the accelerated exchange

of guanosine diphosphate (GDP) for guanosine triphosphate

(GTP). Subsequently, the Gα subunit is transformed from the

GDP-bound inactive state to the GTP-bound active state. As a

consequence, Gα-GTP and Gβγ dissociate and transmit

diﬀerent signals to their downstream eﬀectors separately.

The signaling cycle is terminated by hydrolysis of the GTP to

GDP, and the Gα returns to the inactive GDP-bound state

(Figure S1, Supporting Information).¹³In the case that the Gα

subunit is mutated or covalently modiﬁed, the signaling cycle is

disrupted, which ultimately converts the Gα into a

constitutively active form incapable of hydrolyzing GTP to

GDP. Constitutively activated Gαq or Gα11, as mentioned

earlier, is proposed to be the oncogenic drivers in ∼90% of

INTRODUCTION

■

Uveal melanoma (UM), a kind of melanoma that originates

from ocular melanocytes, is an aggressive malignancy in adults

and accounts for approximately 5% of all melanomas.^1,2More

than 90% of UM occurs in the choroid (a soft, smooth, elastic,

and vascular brown ﬁlm located between the retina and sclera),

with only 6% limited to the ciliary body and 4% to the iris.³

Although radiotherapy and surgery have been successfully used

for treating primary intraocular tumors, nearly 50% of UM

patients will eventually develop metastatic cancer to liver,

which leads to an average survival time of less than 1 year.^4,5

UM is insensitive to many therapeutic approaches, including

immunotherapy that shows eﬃcacy in treating cutaneous

melanoma (CM).^6,7Since almost 90% of UM patients carry

mutations in GNAQ (encoding Gαq subunit) and GNA11

(encoding Gα11 subunit), a reasonable explanation is that the

oncogenic mutations in these G proteins drive the UM

development, which is in contrast to cutaneous melanomas

dominated by v-raf murine sarcoma viral oncogene homolog

B1 (BRAF) or neuroblastoma rat sarcoma viral oncogene

(NRAS) mutations.⁸⁻¹⁰Thus, there is an urgent need to

develop novel and eﬀective therapeutic strategies for treating

UM.

Received: November 15, 2020

Published: March 15, 2021

The Gαq and Gα11 proteins are two subfamilies of the Gq

family, which belongs to the heterotrimeric G proteins. G

proteins are composed of Gα, Gβ, and Gγ subunits and are

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Figure 1. Structures of reported Gαq/11 protein inhibitors.

UM cases.^9,14It is worth noting that residues Q209 and R183

account for more than 99% of cancer-associated mutation sites

in Gαq and Gα11, indicating that these mutations may

decrease intrinsic guanosine triphosphatase (GTPase) activity

and contribute to cancer promotion.^13,15

While interesting leads, a lack of access to these materials

resulted in a worldwide completion to complete the successful

synthesis of 1 mg of 1 (https://www.innocentive.com/ar/

challenge/9933017). Fortunately, the ﬁrst total synthesis of 1

and 2 was achieved by our team in 2016 (Figure S2,

Supporting Information).²⁹This was the development of a

To date, enormous eﬀort has been focused on inhibiting the

downstream signaling of the activated Gαq/11 oncoproteins.¹⁶

For instance, one of the canonical downstream signaling is

mitogen-activated protein kinases (MAPKs)/extracellular

signal-regulated kinase (ERK) pathway (MAPKs/ERK)

which can be inhibited via phospholipase Cβ (PLCβ)/Ca²⁺/

diacylglycerol (DAG)/protein kinase C (PKC) pathway

(PLCβ/Ca²⁺/DAG/PKC).¹⁷⁻¹⁹Unfortunately, clinical trials

showed that patients with metastatic UM are insensitive to

MAPK or ERK inhibitors.^20,21Similarly, targeting other

downstream signaling pathways, including the phosphatidyli-

nositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B

(AKT)/mammalian target of rapamycin (mTOR) pathway

(PI3K/AKT/mTOR), or Trio rho guanine nucleotide

exchange factor (TRIO)/Ras homolog family member

(Rho)/Rac family small GTPase 1 (RAC)/Yes-associated

protein (YAP) pathway (TRIO/Rho/RAC/YAP), did not

show eﬃcacy in UM clinical trials either.^20,22One potential

explanation for those unsuccessful therapies is that the Gαq/11

oncoproteins activate multiple, individually dispensable down-

stream signaling networks²³so that disrupting a speciﬁc node

cannot achieve the desired treatment eﬃcacy. Therefore,

targeting the constitutively active Gαq/11 oncoproteins

directly may provide a promising strategy for UM treatment.

Thus far, few Gαq/11 protein inhibitors have been

described (Figure 1), including YM-254890 (1),²⁴FR900359

(2),²⁵and BIM-46187 (3).²⁶Depsipeptides 1 and 2 share a

common structural motif able to inhibit Gαq/11 proteins with

30

̈

biosynthesis approach to 2 by the Konig laboratory in 2018.

The established synthetic strategy provides the possibility of

performing structure−activity relationship (SAR) studies

within 1 and 2, which demonstrates that the intact structures

are critical for the activity and subtle changes to the molecules

would lead to a dramatic loss of Gαq/11 protein inhibition

potency.^31,32BIM-46187 (3), derived from BIM-46174 (4) by

oxidation, is a synthetic small molecular inhibitor of the Gαq/

11 proteins.³³The Gαq/11 protein inhibition mechanism

studies indicate that 3 permits the GDP exit but prevents GTP

entry, which is diﬀerent from 1 and 2.^33,34

Recently, Kuppers et al. synthesized analogs of 3 and found

̈

that imidazopiperazine fragment is essential for maintaining

the G protein inhibitory eﬃcacy.^35,36Compounds 3 and 4 are

small molecules with synthetic feasibility and perspectives;

however, some issues need to be addressed for this class of

compounds. For instance, the sulfhydryl group within 4 makes

the structure unstable in air and could be easily oxidized to

form 3 with large molecular weight, and the electron-rich

primary amine group is also lacking stability under oxidative

conditions.³³This issue is further complicated by high toxicity

to cell models along with a poorly deﬁned eﬃcacy in G protein

inhibition. In addition to these small molecules, two linear

peptides GP-2A (Arg-Pro-Lys-Pro-Gln-Gln-^D-Trp-Phe-D-Trp-

D-Trp-Met-NH₂)³⁷and 27mer (HQDYAEALINPIKHVSL-

MDQRARQLAA),³⁸could suppress the activity of the Gαq/

11 proteins. However, the mechanism of G protein inhibition,

cell permeability, and stability of the two peptides need to be

further investigated.³⁹Thus, the identiﬁcation of small

molecule Gαq/11 protein inhibitors with synthetic feasibility,

potency, and safety is still in high demand.

the IC₅₀values of 100

3 nM and 32

1 nM,

respectively.^24,25The crystal structure of 1 with chimeric

Gαq/i protein reveals that 1 binds to the hydrophobic cleft

between two interdomain linkers connecting the GTPase and

helical domains of the Gαq protein, thus disrupting the GDP/

GTP exchange by inhibiting the GDP release.²⁷Depsipeptide

2 is suggested to share the same mode of action (MOA) with

1, based on the molecular docking (MD) studies.²⁸Owing to

the unique bioactivity, 1 and 2 are precious research tools for

Gαq/11 proteins.

Herein, we report the discovery of a potent, stable, and safe

small molecule Gαq/11 protein inhibitor GQ127, which is

derived from 4 and demonstrates good antitumor activity and

low toxicity. Biological studies show that GQ127 exhibits good

Gαq/11 protein inhibitory activity and antitumor potency in

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Figure 2. Design and structural optimization strategies of GQ111-GQ166. Replacing the sulfhydryl group (red) and the primary amine (red) by

amino acids (blue) is to optimize the drug-like properties. Modifying the cyclohexyl fragment (green) is to discuss the signiﬁcance to molecular

activity.

vitro. More importantly, GQ127 signiﬁcantly inhibits the

growth of tumor in vivo.

RESULTS

■

Structure−Activity Relationship (SAR) Studies. The

Gαq/11 protein inhibition potency was characterized by

inositol monophosphate (IP₁) accumulation assay based on

homogeneous time-resolved ﬂuorescence (HTRF) technology.

BIM-46187 (3) was used as a reference compound, and the

data were summarized in Table 1 and Table 2. Compounds

GQ111-GQ113 with diﬀerent substituents on the primary

amine, and compounds GQ114-GQ116 with the methyl,

hydrogen, or hydroxyl group replacing the primary amine, were

designed and synthesized with the purpose to improve the

stability of the primary amine group in air. Compared to 3,

GQ111 displayed better inhibition potency; other compounds

GQ112-GQ116 showed similar or decreased inhibition

eﬃcacy toward Gαq/11 proteins, suggesting that the primary

amine or monosubstituted amine may be crucial for the Gαq/

11 protein inhibition potency.

Compounds GQ117-GQ135 were designed and synthesized

omitting the sulfhydryl group to improve the stability of the

molecules. Compared with 3, compounds GQ119,³⁶GQ127,

GQ130, and GQ134 displayed higher Gαq/11 protein

inhibitory eﬃcacy at 10 μM (Table 1), indicating that phenolic

hydroxyl or alkyl groups are better than sulfhydryl group for

Gαq/11 protein binding aﬃnity. More importantly, the newly

designed compounds were much more stable and no

dimerization side products were detected even after 2 weeks

of storage (Figure S3). Other compounds with diﬀerent amino

acids side chains, including imidazole, indole, carboxylic acid,

amide and aryl group, were less potent than 3.

SYNTHETIC DESIGN

■

Our design starts from 4, a redox precursor to 3 (Figure 2). To

avoid the instability caused by the sulfhydryl group and the

primary amine, the cysteine fragment was replaced to improve

the stability in our design. First, considering amino acids are

abundant building blocks with side chain diversity, GQ111-

GQ166 (Figure 2) was synthesized by introducing diﬀerent

amino acids or their derivatives to improve the stability under

air, as well as improving the Gαq/11 protein inhibition

potency. Second, the primary amine in 4 was either removed

or swapped to hydroxyl group or methyl group to investigate

its necessity. Third, the cyclohexyl fragment was also replaced

by an aliphatic chain or aromatic rings to further explore SARs.

In addition, the stereochemistry of the cyclohexyl fragment was

also investigated. Finally, we combined all the privileged

fragments and examined the synergistic eﬀects on Gαq/11

proteins inhibition. In total, 56 imidazopiperazine derivatives

were evaluated.

Imidazopiperazine derivatives GQ111-GQ166 were pre-

pared using the route depicted in Scheme 1.^36,40In addition to

the several known compounds, 52 new analogs GQ111-

GQ116, GQ118, GQ120-GQ137, GQ139, and GQ141-

GQ166 were prepared. Analogs were prepared to start from

the commercially available N-carbobenzoxy-protected amino

acids 5a−5i. 5a−5i were treated with 2-bromoacetophenone

and cesium carbonate in N,N-dimethylformamide (DMF),

followed by treating with NH₄OAc to obtain intermediates

7a−7i with the yields in the range of 60−85%.^36,40

Nucleophilic substitutions between 7a−7i and ethyl bromoa-

cetate were performed to deliver 8a−8i.^36,40Treating 8a−8i

with Pd/C under H₂atmosphere in CH₃OH led to the

deprotection of carbobenzoxy (Cbz) and spontaneous intra-

molecular lactamization to aﬀord 9a−9i. Reduction of the

amide groups within 9a−9i by borane gave the key building

blocks 10a−10i in good yields (80 4%). Finally, compounds

GQ136, GQ137, GQ151, GQ152, GQ158, GQ159, GQ165,

and GQ166 were furnished by the condensation of amine

intermediates with carboxylic acids. Compounds GQ111-

GQ135, GQ138-GQ150, GQ153-GQ157, and GQ160-

GQ164 were obtained after the condensation with amino

acids and deprotection of the protecting groups.

The importance of the primary amine and sulfhydryl group

was further investigated by GQ136 and GQ137, which had

both the primary amine and sulfhydryl groups removed.

Interestingly, GQ136 and GQ137 also showed obviously

improved eﬃcacy at 10 μM compared to 3, conﬁrming that the

sulfhydryl group is not crucial for the Gαq/11 protein

inhibition potency, which is in accordance with GQ119,³⁶

GQ127, GQ130, and GQ134.

To understand the eﬀect of cyclohexyl moiety on Gαq/11

protein inhibition more clearly, we only modiﬁed the

cyclohexyl group of 4 with the sulfhydryl group (Table 2).

Subsequently, compounds GQ138-GQ147 with diﬀerent alkyl

or aryl groups were designed and synthesized to investigate the

SAR of the cyclohexyl moiety. Besides, the impact of

stereochemistry for R¹was also examined. Previously, the

stereochemistry of the cyclohexylmethyl group has been

discussed by Ku

̈

ppers et al.³⁶Interestingly, compounds

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a

Scheme 1. Synthetic Route of the Imidazopiperazine Derivatives GQ111-GQ166

a

Reagents and conditions: (i) Cs₂CO₃, N,N-dimethylformamide (DMF), rt; (ii) NH₄OAc, toluene, reﬂux; (iii) K₂CO₃, DMF, rt; (iv) Pd/C, H₂(1

atm), methanol, rt; (v) BH₃·THF, THF, reﬂux; (vi) 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexaﬂuorophosphate (HATU),

N,N-diisopropylethylamine (DIPEA), DMF, rt; (vii) triﬂuoroacetic acid (TFA), (i-Pr)₃SiH, rt, Ar; (viii) TFA, CH₂Cl₂, rt; (ix) TFA, thioanisole, rt.

GQ141-GQ143 with (R)-isobutyl, (S)-isopropyl, or (R)-

isopropyl group showed much better potency than 3 at the

concentration of 10 μM, while GQ138-GQ140, GQ144, and

GQ145 displayed decreased potency, indicating that the

proper combination of the substituents and stereochemistry

of R¹are critical for the activity.

With the above SAR information in hand, we chose to

combine the three privileged R¹substituents (R)-isobutyl, (S)-

isopropyl, and (R)-isopropyl with seven diﬀerent R²sub-

stituents and three R³substituents which were beneﬁcial for

Gαq/11 protein inhibition activity. In total, 21 new analogs

GQ146-GQ166 were synthesized in searching for more potent

Gαq/11 protein inhibitors. To our surprise, none of the

compounds showed better inhibition potency than GQ127,

GQ130, GQ134, GQ136, GQ137, and GQ141-GQ143,

suggesting that the proper combination of alkyl substituents

R¹, alkyl substituents R², and amino or alkyl substituents R³

may be necessary to acquire the Gαq/11 protein inhibition

potency.

Thus, we summarize the SAR information as the following

(Figure 3): the primary amine or the monomethylated amine

group is important for keeping the Gαq/11 protein inhibition

potency for the compounds; the substitution of sulfhydryl

group by phenolic hydroxyl or alkyl groups can increase the

eﬃcacy and stability of the compounds; replacing the

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a

Table 1. Inhibition on Gαq/11-Mediated Signaling

Table 2. Inhibition on Gαq/11-Mediated Signaling

a

Gαq/11 protein inhibition activity was characterized using IP₁

a

b

Gαq/11 protein inhibition activity was characterized using IP₁

accumulation assay. Percentage of inhibition on IP₁accumulation

b

accumulation assay. Percentage of inhibition on IP₁accumulation

in CHO cells stably expressing M₁receptor by 10 μM compounds.

IC₅₀values were determined for compounds. Data represent the

mean SD of three independent experiments.

c

in CHO cells stably expressing M₁receptor by 10 μM compounds.

Data represent the mean SD of three independent experiments.

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Figure 3. Structure−activity relationship (SAR) studies of imidazopiperazine Gαq/11 protein inhibitors.

a

Table 3. Antiproliferative Activities of GQ111-GQ166

antiproliferative activity (IC₅₀, μM)

compd

92.1

MP41

compd

92.1

MP41

GQ111

GQ112

GQ113

GQ114

GQ115

GQ116

GQ117³⁶

GQ118

GQ119³⁶

GQ120

GQ121

GQ122

GQ123

GQ124

GQ125

GQ126

GQ127

GQ128

GQ129

GQ130

GQ131

GQ132

GQ133

GQ134

GQ135

GQ136

GQ137

GQ138³⁶

GQ139

24.0 1.3

>100

33.1 1.0

29.1 1.2

>100

42.0 3.6

35.5 2.8

78.4 4.2

>100

91.9 6.6

30.1 3.1

55.4 4.9

40.3 2.1

>100

19.3 1.3

10.1 0.6

>100

27.8 1.3

>100

56.3 1.4

>100

95.9 5.2

>100

25.5 1.6

33.5 2.8

83.0 3.7

>100

24.8 0.9

>100

GQ140³⁶

GQ141

GQ142

GQ143

GQ144

GQ145

GQ146

GQ147

GQ148

GQ149

GQ150

GQ151

GQ152

GQ153

GQ154

GQ155

GQ156

GQ157

GQ158

GQ159

GQ160

GQ161

GQ162

GQ163

GQ164

GQ165

GQ166

3

35.2 0.3

64.1 3.0

65.7 3.0

62.7 1.9

>100

84.4 5.6

45.0 3.2

>100

6.8 0.8

4.8 0.4

91.9 0.3

47.7 1.8

>100

59.3 4.7

>100

85.8 4.4

>100

97.2 5.1

97.6 5.3

>100

10.7 0.8

27.4 1.5

20.6 1.5

87.2 4.4

>100

22.6 0.6

63.4 3.6

15.6 1.1

>100

42.3 2.6

27.4 1.2

45.2 2.3

25.8 1.7

>100

79.0 4.2

19.8 1.3

20.0 1.0

10.5 0.5

2.3 0.3

4

a

The antiproliferative activities of the compounds were measured using CCK-8 assay. Data represent the mean

SD of three independent

experiments.

cyclohexyl group with an isopropyl group maintains the

potency of the compounds.

activities were characterized via cell counting kit-8 (CCK-8)

assay, and the results were summarized in Table 3 with the

IC₅₀values.

As shown in Table 3, compounds GQ111, GQ118, GQ122-

GQ124, GQ127, GQ130-GQ132, and GQ138-GQ140

displayed inhibitory potency on 92.1 and MP41 cells with

the IC₅₀values in micromolar range, which were comparable to

Evaluation of Antiproliferative Activities and Tox-

icities of Imidazopiperazines. Antiproliferative activities of

synthesized compounds GQ111-GQ166 were evaluated on

uveal melanoma cell lines 92.1 with GNAQ^Q209Lmutation and

MP41 with GNA11^Q209Lmutation. The cell growth inhibitory

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Figure 4. Cytotoxicity of 3 and GQ127 on CHO-M₁cells. (A) The percentage of cell viability in indicated concentrations of 3 and GQ127 was

assessed for 2 h using a CCK-8 assay. (B) The percentage of cell viability in indicated concentrations of 3 and GQ127 was assessed for 24 h using a

CCK-8 assay. The data are shown as the mean SD of three independent experiments.

Figure 5. Eﬀects of 3, 4, and GQ127 on agonist-mediated change of BRET signal. (A−C) HEK293 cells were transiently transfected to express

AT₁R, Gαq-RLuc8, Gγ2-Venus, and unlabeled Gβ1. The cells were pretreated with the indicated concentrations of 3, 4, and GQ127 for 1 h at 37

°C. BRET signal was measured after the addition of agonist AngII. (D) Dose−response of compounds on AT₁R-promoted BRET changes between

Gαq-RLuc8 and Gγ2-Venus after AngII stimulation. The data are shown as the mean SD of three independent experiments: ****p < 0.0001

versus control.

3. Other compounds, like GQ112 or GQ115, either did not

show antiproliferative activity on UM cells or could not inhibit

both of the UM cell lines. To our surprise, GQ119,³⁶GQ134,

GQ136, GQ137, GQ141-GQ143, and GQ160 displayed

discrepant antiproliferation activities in IP₁accumulation

assay and UM cell antiproliferation assay. One presumable

explanation for this phenomenon is that these compounds may

have distinct cell membrane permeability in CHO-M₁and UM

cell lines. The other reasonable explanation is that a compound

may exhibit diﬀerent inhibition activity in distinct test methods

and systems. To better understand the correlations of Gαq/11

protein inhibitory potency and antiproliferative activity, we

chose the most potent compounds GQ127 and GQ130 to

further characterize their dose response in IP₁accumulation

assay, together with 3. Compounds GQ127 and GQ130

eﬃciently inhibited Gαq/11 protein signaling with the IC₅₀

partially due to its cytotoxicity. To verify the hypothesis, we

chose the most eﬀective GQ127 to study its eﬀect on

morphological change and cytotoxicity of CHO-M₁cells, in

comparison with 3. As shown in Figure S5 (see Supporting

Information), 3 resulted in signiﬁcant morphological changes

in CHO-M₁cells at the concentration of 10 μM in 2 h, and the

changes were more obvious at the concentration of 20 μM,

indicating that 3 might have severe cytotoxicity and the Gαq/

11 protein inhibition of 3 might be partially resulted from its

toxicity. Unexpectedly, CHO-M₁cells did not show obvious

morphological changes when treated with GQ127, even at

concentrations up to 20 μM, suggesting that GQ127 is less

cytotoxic than 3. To further verify our hypothesis, we treated

the CHO-M₁cells with diﬀerent concentrations of 3 and

GQ127 for 2 and 24 h to measure the cell viability (Figure 4).

The data clearly showed that 3 had severe cytotoxicity toward

CHO-M₁cells, and most of the cells were dead even under the

concentration of 5 μM in 2 h, let alone treating cells with

higher concentration or longer time. To our delight, GQ127

showed satisfactory safety toward CHO-M₁cells; the cell

viability was not aﬀected even at concentrations up to 20 μM

for 24 h. These data clearly demonstrated that the

antiproliferative ability of 3 on UM cells may be partially

due to its cytotoxicity, whereas GQ127 has better safety and

inhibits UM cells through Gαq/11 protein signaling pathway.

values of 22.6

1.4 μM and 48.6

5.3 μM (Table 1),

respectively; however, we did not obtain the exact IC₅₀value of

3 as it did not show an inhibition trend at the indicated

concentrations (Figure S4, Supporting Information).

In the IP₁accumulation assay, the M₁receptor was

overexpressed in CHO cells. Surprisingly, we found that 3

obviously resulted in the morphological changes of CHO-M₁

cells in the IP₁accumulation assay, although it also showed

inhibitory potency on UM cell lines. The observation indicated

that the inhibition of 3 on CHO-M₁cells and UM cells may be

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Figure 6. Eﬀects of GQ127 on proliferative inhibition of UM cells with GNAQ knockdown and agonist-induced Ca²⁺release of HEK293-AT₁R

cells. (A) The percentage of cell viability in the indicated concentrations of compounds was assessed for 72 h by a CCK-8 assay. (B) The

concentration−response curves of Ca²⁺release for AngII in the absence and presence of 100 μM 3, 4, and GQ127 were measured. The data are

shown as the mean SD of three independent experiments: *p < 0.05 versus control; ***p < 0.001.

Figure 7. Apoptotic eﬀects of GQ127 in UM cells. (A, C) 92.1 and MP41 cells were exposed to the indicated concentrations of GQ127 for 48 h.

The apoptosis was measured via ﬂow cytometer. (B, D) The percentage of apoptosis cells at indicated concentrations of compound. The data are

shown as the mean SD of three independent experiments: **p < 0.01 versus control; ****p < 0.0001.

In addition, we further investigated the antiproliferative

potency of GQ127 on UM cells 92.1 and MP41 at diﬀerent

times and concentrations (Figure S6, Supporting Information).

The results showed that with increasing the concentration or

extending the action time, the inhibitory eﬀects of GQ127 on

UM cells gradually improved, indicating that GQ127 inhibits

UM cell proliferation in a time- and dose-dependent manner.

In brief, 3 exhibited antiproliferative activities in 92.1 and

MP41 cells; however, this eﬀect was mainly due to the high

cytotoxicity of 3, which limited its further application. The

newly discovered GQ127 eﬃciently suppressed the prolifer-

ation of UM cells and showed much higher safety to cells.

Eﬀects of GQ127 on Inhibiting BRET Signal. To

understand how GQ127 inhibits Gαq/11 protein signaling, we

employed bioluminescence resonance energy transfer (BRET)

technology to study the direct interactions between GQ127

and Gαq/11 proteins. The human embryonic kidney (HEK)

293 cells that were transiently co-transfected angiotensin type

1 receptor (AT₁R), Gαq-RLuc8 and Gγ2-Venus, were utilized

to assess the eﬀect of GQ127 on BRET alteration caused by

the activated AT₁R. The agonist-induced bioluminescence

resonance energy transfer (BRET) signal between Gα and Gβγ

was measured in HEK293-AT₁R cells pretreated with GQ127

at diﬀerent concentrations. As shown in Figure 5, GQ127

altered the BRET signal in a dose-dependent manner with the

IC₅₀value of 55.2 2.6 μM for Gαq/11 proteins, while 3 and

4 started to alter the BRET signal at the concentration of 100

μM. In addition, the statistical signiﬁcance of the data was

determined by Student’s test. The p value of GQ127 was less

than 0.0001, in comparison with 3 and 4 (p > 0.05 versus

control). The results suggested that GQ127 directly binds to

Gαq/11 proteins and disrupts the Gαq/11 protein signal

transduction.

Antiproliferative Activity of GQ127 in UM Cells

Depends on GNAQ. To further verify whether the inhibition

of GQ127 on UM cells proliferation depends on targeting

GNAQ, 92.1 cells were transfected with control or siRNA

targeting GNAQ to study the eﬀect of GQ127 on cancer cell

proliferation. As shown in Figure 6A, GQ127 eﬀectively

inhibited the proliferation of 92.1 cells with si#control but did

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Figure 8. Eﬀects of GQ127 on the migration of UM cells. (A, C) 92.1 and MP41 cells were exposed to GQ127 at indicated concentrations for 48

h. The migration of UM cells was observed using a light microscope. Photographs were obtained under magniﬁcation, ×40. (B, D) Quantitative

analysis of the relative migration. The results are shown as the mean SD of three independent experiments: **p < 0.01 versus control; ***p <

0.001; ****p < 0.0001.

Figure 9. Eﬀects of GQ127 on the invasion of UM cells. (A, C) 92.1 and MP41 cells were exposed to GQ127 at the indicated concentrations for

48 h. The invasion of UM cells was observed using a light microscope. Photographs were obtained under magniﬁcation, ×100. (B, D) Quantitative

analysis of the number of invading cells. The results are shown as the mean SD of three independent experiments: ***p < 0.001 versus control;

****p < 0.0001.

not suppress the proliferation of 92.1 cells with si#GNAQ,

indicating that inhibition of GQ127 on UM cell proliferation

depends on disrupting the Gαq/11 proteins directly.

Eﬀects of GQ127 on Apoptosis in UM Cells. To

understand the eﬀects of GQ127 on inducing cancerous cell

death, apoptosis assay was carried out for UM cell lines by ﬂow

cytometry analysis (Figure 7). 92.1 and MP41 cells were

treated with GQ127 for 48 h, stained with annexin V-FITC

and propidium iodide (PI), then analyzed by ﬂow cytometer.

As shown in Figure 7, GQ127 eﬀectively induced the apoptosis

of 92.1 cells with the apoptotic values of 9.48% at 5 μM and

20.1% at 15 μM and induced the apoptosis of MP41 cells with

apoptotic values of 9.74% at 15 μM and 24.1% at 30 μM.

Compounds 3 and 4 also induced apoptosis of UM cells,

similar to GQ127 (Figure S7, Supporting Information). Taken

together, GQ127 promoted the apoptosis of UM cells in a

dose-dependent manner.

Eﬀect of GQ127 on Agonist-Induced Ca²⁺Release. To

further understand how GQ127 inhibits Gαq/11 protein

signaling, we performed an extensive analysis to evaluate the

eﬀects of GQ127 on agonist-induced Ca²⁺release. HEK293

cells overexpressing AT₁R along with Gαq/11 were applied to

assess the eﬀects of GQ127 on agonist-induced Ca²⁺release

caused by the activated AT₁R. When the AngII binds to AT₁R,

the Gαq/11 proteins are activated by AT₁R, followed by PLC

activation. Consequently, Ca²⁺release is accelerated. HEK293-

AT₁R cells were pretreated with 3, 4, or GQ127 at the

concentration of 100 μM, and then the agonist-induced Ca²⁺

release was varied by the diﬀerent concentrations of AngII. As

shown in Figure 6B, GQ127 signiﬁcantly altered agonist-

induced Ca²⁺release, while 3 and 4 only had minimum eﬀects

on Ca²⁺release, suggesting that GQ127 targets Gαq/11

proteins directly.

Eﬀects of GQ127 on Inhibiting UM Cell Colony

Formation. Next, the eﬀects of GQ127 on colony formation

of 92.1 and MP41 cells were further studied by colony survival

assay (Figure S8, Supporting Information). In agreement with

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Figure 10. Eﬀects of GQ127 on tumor growth in MP41 xenograft model. GQ127 was administered via intraperitoneal injection (ip) for 21 days.

(A) The tumor volumes of the male mice were measured and calculated once every 3 days. (B) Tumor weight of the male mice on day 22. (C) The

male mice body weight was measured every 3 days. (D) Hematoxylin and eosin (H&E) staining showed that GQ127 has no obvious side eﬀects on

mice heart, liver, spleen, lung, and kidney. Photographs were obtained under magniﬁcation, ×100. ***p < 0.001 versus vehicle; ****p < 0.0001.

Figure 11. Eﬀects of 3 and GQ127 on tumor growth in MP41 xenograft model. Compounds 3 and GQ127 were administered via intraperitoneal

injection (ip) for 21 days. (A) The tumor volumes of the female mice were measured and calculated once every 3 days. (B) Tumor weight of the

female mice on day 22. (C) The female mice body weight was measured every 3 days. *p < 0.05 versus vehicle; ****p < 0.0001.

the antiproliferative activities, GQ127 eﬀectively suppressed

the colony formation of 92.1 and MP41 cells.

Eﬀects of GQ127 on UM Cell Xenograft Mice Model.

Encouraged by its promising anticancer eﬃcacy on the cell

model, MP41 xenograft model on severe combined nude (nu/

nu) BALB/c mice was employed to further evaluate the

antitumor potency of GQ127 in vivo. The tumor-bearing mice

were administrated with GQ127 at the dose of 10 mg/kg and

30 mg/kg per day for 21 consecutive days. Tumor volume and

body weight were measured every 3 days. Compound 4 with

the sulfhydryl group could easily be oxidized to form a disulﬁde

bond in air, making 4 exist as a mixture of dimerized 3 and 4

with an imprecise ratio. Therefore, 3 was used as positive

control in in vivo assay. As shown in Figure 10, GQ127

displayed robust antitumor eﬃcacy with the tumor growth

inhibition (TGI) achieving 99.0% in the high-dosage group.

Meanwhile, the low-dosage group also showed eﬀective

antitumor activity with TGI at 65.4% by comparison to 3

with TGI at 35.9% (Figure 11). Even though GQ127 showed

the low micromolar potency in vitro, it exhibited the robust

antitumor activity in vivo. A rational explanation is that GQ127

targets the upstream “molecular switches” Gαq/11 proteins

which mediate complex downstream signals, therefore

disrupting a wide range of signaling pathways, which cannot

be achieved by inhibiting a single node. It is worth noting that

no obvious side eﬀects were observed on mice body weight,

heart, kidney, and spleen during and after the treatment

Eﬀects of GQ127 on UM Cell Migration and Invasion.

The metastatic feature of UM cells permits them to invade the

distant organs. It has been proved that Gαq/11 protein

signaling participates in the migration of UM cells, and

inhibition of Gαq/11 protein signaling can suppress the

migration and invasion of UM cells.²⁸Thus, we investigated

the eﬀects of GQ127 on the migration and invasion of UM

cells by conducting Boyden chamber transwell experiments. As

shown in Figure 8, the migration of UM cells was aggressive in

the absence of Gαq/11 protein inhibitors. To our delight, the

migration of UM cells 92.1 and MP41 was signiﬁcantly

suppressed by treating with GQ127 at the concentration of 15

μM. The migration of MP41 cells was also severely suppressed

by GQ127 at the concentration of 30 μM. In accordance with

the migration inhibitory eﬀect, the invasion capability of 92.1

and MP41 cells was signiﬁcantly suppressed by GQ127 at the

concentrations of 5 μM and 15 μM (Figure 9). Compounds 3

and 4 showed similar inhibitory eﬀects on the migration and

invasion of UM cells. (Figures S9 and S10, Supporting

Information). These results suggested that GQ127 may aﬀect

UM metastasis by suppressing the cell migration and invasion

through the Gαq/11 protein signaling pathway.

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Figure 12. Eﬀects of GQ127 on suppressing the downstream signaling eﬀectors of Gαq/11 proteins in UM cells and MP41 xenografts. (A) 92.1

and MP41 cells were treated with the indicated concentrations of GQ127 for 48 h: Western blot analysis of proteins (p-YAP, YAP, p-ERK, and

ERK) expression in 92.1 and MP41 cells. (B) MP41 xenografts were treated with the indicated doses of GQ127 for 21 consecutive days: Western

blot analysis of proteins (p-YAP, YAP, p-ERK, and ERK) expression in MP41 xenografts. GAPDH was used as a loading control. The results are

representative of experiments in triplicate. Full images of the blots can be found within the Supporting Information.

(Figure 10C, Figure 11C, and Figure S11, Supporting

Information). Histological analysis of heart, liver, spleen,

lung, and kidney with hematoxylin and eosin (H&E) further

conﬁrmed the safety of GQ127. As shown in Figure 10D, there

were no obvious morphological changes in these tissues after

the treatment. The results indicated that GQ127 is eﬀective in

MP41 xenograft without severe toxicity at the testing dose.

Eﬀects of GQ127 on Downstream Signaling Eﬀectors

ERK and YAP of Gαq/11 Proteins. ERK is the primary

eﬀector of canonical PLCβ signaling pathway and can mediate

mitogenic signals in the physiological and pathophysiological

process.¹⁷⁻¹⁹Suppression of Gαq/11 oncoproteins by GQ127

may potentially disrupt the phosphorylation of ERK, followed

by promoting of the apoptosis of UM cells. Thereby, we

evaluated the ERK phosphorylation via Western blot analysis

in vitro. As shown in Figure 12A, GQ127 clearly inhibited ERK

phosphorylation in both 92.1 and MP41 cells at the

concentrations of 15 μM and 30 μM.

YAP is also known to play important roles in UM

tumorigenesis and acts as a key eﬀector to transduce signals

in the Hippo pathway.²²When Gαq/11 oncoproteins are

activated, the dephosphorylation of YAP is triggered to

promote UM cell growth. Hence, we assessed the accumu-

lation of phosphorylated YAP through Western blot (Figure

12A). Unsurprisingly, treatment of 92.1 and MP41 cells with

GQ127 evidently enhanced the level of YAP phosphorylation,

suggesting GQ127 reduces UM tumorigenesis.

Table 4. Pharmacokinetic Parameters of GQ127 in Mice (n

= 6)

route of dosing

parameter

iv

po

dose (mg/kg)

t_1/2(h)

T_max(h)

C_max(ng/mL)

AUC_0−t(h·ng/mL)

MRT_0−t(h)

V_ss(L/kg)

CL ((mL/min)/kg)

F (%)

10

30

1.17 0.08

0.08 0.00

2608 255

2136 148

0.93 0.07

4.53 0.40

77.9 5.14

1.41 0.11

0.42 0.14

2888 505

5936 2210

1.88 0.19

92.6

0.42 h, the elimination half-life (t_1/2) of 1.41 h, and the mean

residence times (MRTs) of 1.88 h after the oral administration.

In addition, GQ127 showed an apparent volume of

distribution at steady state (V_ss) of 4.53 L/kg and a plasma

clearance (CL) of 77.9 (mL/min)/kg after the intravenous

injection. These results indicated that GQ127 exhibits

favorable pharmacokinetic properties.

DISCUSSION AND CONCLUSIONS

■

G proteins are conventionally considered as critical “molecular

switches” in GPCR signaling; however, directly targeting G

proteins with therapeutic purposes are rarely reported. The

mutated Gαq/11 proteins are witnessed to be the major

driving force for UM and may provide the insights into the

development of new therapeutic strategies against UM. Thus,

we performed SAR studies based on the structures of 3 and 4

in the search for easily prepared, potent, stable, and safe small

molecule Gαq/11 protein inhibitors. Consequently, we studied

the inhibitory mechanism and further explored the in vitro and

in vivo antitumor eﬀects for the most potent compound

GQ127.

In addition, we analyzed the expression and the levels of

ERK and YAP in MP41 xenografts tumor tissues. As shown in

Figure 12B, GQ127 eﬃciently promoted the phosphorylation

YAP and disrupted the phosphorylation of ERK in tumor

tissue with the doses of 10 mg/kg and 30 mg/kg, respectively.

Taken together, these data revealed that GQ127 suppresses

Gαq/11-driven tumorigenesis signaling both in vitro and in

vivo by promoting the phosphorylation YAP and disrupting the

phosphorylation of ERK.

Pharmacokinetic (PK) Study of GQ127. Given the

outstanding antitumor activities of GQ127 in vitro and in vivo,

single-dose PK studies were then performed with male

Institute of Cancer Research (ICR) mice at 10 mg/kg by

intravenous injection (iv) and 30 mg/kg by oral administration

(po), and the results were summarized in Table 4 and Figure

S12 (see Supporting Information). It was exciting that GQ127

displayed outstanding oral bioavailability (F) of 92.6% with an

area under the curve (AUC_0−t) of 5936 h·ng/mL. GQ127

achieved the maximum plasma concentration (C_max) of 2888

ng/mL, the time at maximum plasma concentration (T_max) of

The design of new analogs was based on using diverse and

commercially available amino acids derivatives in searching for

stable, potent, and safe Gαq/11 protein inhibitors. In total, 56

new analogs were designed and synthesized. The data obtained

from the SAR studies suggested that the cysteine fragment,

which is related to the air instability, is not critical for

maintaining the Gαq/11 inhibitory potency, and several new

compounds displayed improved Gαq/11 inhibitory potency

compared with 3 in IP₁accumulation assay. GQ127 was

witnessed to be the most eﬀective analog among them. We also

noticed that 3 can cause the morphological changes of CHO-

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synthesized compound was conﬁrmed to be over 95% by reverse-

phase analytical high-performance liquid chromatography (HPLC).

HPLC conditions: Agilent 1260 with a Poroshell 120 SB-C18 column

(3 mm × 100 mm, 2.7 μm); acetonitrile/water (gradient 0% to

100%); T = 25 °C; ﬂow rate = 0.6 mL/min.

M₁cells and eventually caused CHO-M₁cells death under the

concentration of 5 μM in 2 h, indicating that the Gαq/11

protein inhibition potency and UM cells suppression by 3 may

be partially due to its toxicity. Compared with 3, GQ127

displayed much lower cytotoxicity to CHO-M₁cells even

under the concentration of 20 μM for 24 h.

GQ127 showed potent antiproliferative activity on 92.1 and

MP41 cells and induced the apoptosis of the two cell lines in a

dose-dependent manner. Further studies on inhibiting UM

cells colony formation, migration, and invasion proved that

GQ127 indeed has antitumor activity. To verify the

mechanism of how GQ127 inhibits Gαq/11 proteins, BRET

experiment, rescue assay, and calcium release assay were

employed and conﬁrmed that GQ127 directly binds to and

inhibits the Gαq/11 proteins in a dose-dependent manner.

The eﬀects of GQ127 on Gαq/11 downstream signaling

eﬀectors ERK and YAP were also characterized, which revealed

that GQ127 suppresses the Gαq/11-driven tumorigenesis by

promoting the phosphorylation YAP and inhibiting the

phosphorylation of ERK in vitro and in vivo.

The antitumor eﬀects of GQ127 were also evaluated in

MP41 xenograft mouse model. GQ127 showed robust

antitumor activity with the TGI of 99.0% when administrating

the mice with 30 mg/kg per day, and with the TGI of 65.4% in

10 mg/kg group by comparison to 3 with the TGI of 35.9% at

dose of 10 mg/kg. Although GQ127 showed the low

micromolar potency in vitro, it exhibited the robust antitumor

activity in vivo. A reasonable explanation is that GQ127 targets

the upstream “molecular switches” Gαq/11 proteins which

mediate complex downstream signals, therefore disrupting a

wide range of signaling pathways which cannot be achieved by

inhibiting a single node. It is worth noting that no obvious side

eﬀects were observed on mice body weight, heart, kidney, and

spleen after the treatment, indicating that GQ127 is eﬀective in

MP41 xenograft without severe toxicity at the testing dose.

Signiﬁcantly, GQ127 also showed favorable pharmacokinetic

properties.

In conclusion, we discovered a novel Gαq/11 protein

inhibitor GQ127 with high stability, potency, and safety.

GQ127 showed promising antitumor activity both in vitro and

in vivo, and the mechanism of how GQ127 targets Gαq/11

proteins was also explored. The easily synthesized small

molecule GQ127 appears to have a great potential to become a

lead structure for designing anti-UM drugs by targeting Gαq/

11 proteins directly, which may provide a new and

straightforward approach for UM therapeutics.

General Procedure for the Synthesis of Intermediates 9a−

9i. Commercially available N-Cbz-protected amino acids 5a−5i (1.00

equiv) were added to a mixture of 2-bromoacetophenone (1.05

equiv) and cesium carbonate (1.50 equiv) in DMF. The reaction was

stirred at room temperature (rt) for 30 min and monitored by TLC.

Subsequently, the solution was evaporated under reduced pressure,

and toluene was added to resuspend the crude product. To the

suspension was added NH₄OAc (9.00 equiv), and the mixture was

heated to reﬂux for 3 h. After completion of the reaction, the mixture

was concentrated under reduced pressure. The residue was diluted

with ethyl acetate (EtOAc) and washed with a saturated solution of

sodium bicarbonate (NaHCO₃) and with a saturated solution of

sodium chloride (NaCl). The EtOAc phase was dried over anhydrous

sodium sulfate (Na₂SO₄), ﬁltered, and concentrated under vacuum.

The crude products were puriﬁed by silica gel column chromatog-

raphy to obtain 7a−7i as a reddish brown oil.

To a solution of 7a−7i (1.00 equiv) in DMF were added ethyl

bromoacetate (1.10 equiv) and K₂CO₃(1.50 equiv). The mixture was

stirred at rt for 2 h. Upon completion, the mixture was diluted with

EtOAc and washed with water and with a saturated solution of NaCl.

The EtOAc phase was dried over anhydrous Na₂SO₄, ﬁltered, and

evaporated under reduced pressure. 8a−8i were obtained without

additional puriﬁcation.

To a solution of 8a−8i (1.00 equiv) in methanol were added Pd/C

(10%). The suspension was stirred under a hydrogen atmosphere at rt

for 24 h. The Pd/C and formed precipitate were ﬁltered oﬀ with

diatomite. The ﬁltrate was concentrated under vacuum and puriﬁed

by ﬂash chromatography to give light yellow products 9a−9i.

General Procedure for the Synthesis of Intermediates 10a−

10i. To a solution of 9a−9i (1.00 equiv) in THF was added 1 M

solution of BH₃in THF, and the mixture was heated to reﬂux for 6 h.

After completion, the mixture was concentrated under reduced

pressure, followed by addition of methanol to resuspend. The

suspension was heated to reﬂux for 2 h. Next, the solution was

evaporated under vacuum. The crude product was puriﬁed by silica

gel column chromatography to obtain 10a−10i as a bright yellow oil.

( S ) - 8 - ( C y c l o h e x y l m e t h y l ) - 2 - p h e n y l - 5 , 6 , 7 , 8 -

tetrahydroimidazo[1,2-a]pyrazine (10a). Bright yellow oil, yield

1

81%. H NMR (400 MHz, CDCl₃) δ 7.73 (dd, J = 8.4, 1.2 Hz, 2H),

7.34 (t, J = 7.6 Hz, 2H), 7.20 (t, J = 7.6 Hz, 1H), 7.06 (s, 1H), 4.15

(dd, J = 10.0, 3.6 Hz, 1H), 4.04−3.88 (m, 2H), 3.37 (dt, J = 13.2, 4.0

Hz, 1H), 3.16 (ddd, J = 18.0, 8.8, 4.8 Hz, 1H), 2.19 (ddd, J = 25.6,

10.4, 2.8 Hz, 1H), 1.90 (d, J = 12.8 Hz, 1H), 1.79 (s, 1H), 1.75−1.65

(m, 4H), 1.63−1.55 (m, 2H), 1.33−1.18 (m, 3H), 1.12−0.98 (m,

2H). ¹³C NMR (100 MHz, CDCl₃) δ 147.4, 140.6, 134.4, 128.5 (2C),

126.5, 124.9 (2C), 113.9, 51.7, 45.5, 41.7, 41.5, 34.5, 33.9, 32.0, 26.6,

26.5, 26.3. MS (ESI) m/z: calcd for C₁₉H₂₅N₃[M + H]⁺, 296.2;

found, 296.1.

EXPERIMENTAL SECTION

■

(S)-8-Benzyl-2-phenyl-5,6,7,8-tetrahydroimidazo[1,2-a]-

1

pyrazine (10b). Bright yellow oil, yield 80%. H NMR (400 MHz,

Chemistry. All reagents and solvents were purchased from

commercial sources and used without further puriﬁcation. All

CDCl₃) δ 7.82−7.69 (m, 2H), 7.36 (t, J = 7.6 Hz, 2H), 7.33−7.26

(m, 4H), 7.26−7.20 (m, 2H), 7.08 (s, 1H), 4.28 (dd, J = 10.0, 3.2 Hz,

1H), 4.03−3.81 (m, 2H), 3.74 (dd, J = 14.0, 3.6 Hz, 1H), 3.27 (ddd, J

= 12.8, 4.4, 3.2 Hz, 1H), 3.05 (ddd, J = 12.8, 9.8, 4.4 Hz, 1H), 2.97

(dd, J = 14.0, 10.0 Hz, 1H), 1.98 (s, 1H). ¹³C NMR (100 MHz,

CDCl₃) δ 146.2, 140.7, 138.1, 134.4, 129.5 (2C), 128.7 (2C), 128.6

(2C), 126.7, 126.7, 124.9 (2C), 114.0, 55.8, 45.4, 42.0, 40.1. MS

(ESI) m/z: calcd for C₁₉H₁₉N₃[M + H]⁺, 290.2; found, 290.1.

(R)-8-Benzyl-2-phenyl-5,6,7,8-tetrahydroimidazo[1,2-a]-

chemical structures were conﬁrmed by H and ¹³C nuclear magnetic

1

resonance (NMR) spectra and high-resolution mass spectrometry

(HRMS). ¹H and ¹³C NMR spectra were recorded using

tetramethylsilane (TMS) as the internal standard, CD₃OD, CDCl₃,

or DMSO-d₆with a Bruker BioSpin GmbH spectrometer at 400 and

100 MHz, respectively. Chemical shifts (δ) were reported in parts per

million (ppm). Multiplicity of ¹H NMR signals was reported as singlet

(s), doublet (d), triplet (t), quartert (q), and multiplet (m). Coupling

constants (J) are reported in hertz (Hz). HRMS was recorded on a

Shimadzu LCMS-IT-TOF mass spectrometer. Chemical HSGF254

silica gel plates (0.2 mm, Huanghai) were used for thin-layer

chromatography (TLC) analysis. Flash column chromatography was

performed with silica gel (200−300 mesh) purchased from Yantai

Jiangyou Silica Gel Development Co., Ltd. The purity of the

1

pyrazine (10c). Bright yellow oil, yield 85%. H NMR (400 MHz,

CDCl₃) δ 7.82−7.69 (m, 2H), 7.36 (t, J = 7.6 Hz, 2H), 7.33−7.26

(m, 4H), 7.26−7.20 (m, 2H), 7.08 (s, 1H), 4.28 (dd, J = 10.0, 3.2 Hz,

1H), 4.03−3.81 (m, 2H), 3.74 (dd, J = 14.0, 3.6 Hz, 1H), 3.27 (ddd, J

= 12.8, 4.4, 3.2 Hz, 1H), 3.05 (ddd, J = 12.8, 9.8, 4.4 Hz, 1H), 2.97

(dd, J = 14.0, 10.0 Hz, 1H), 1.98 (s, 1H). ¹³C NMR (100 MHz,

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CDCl₃) δ 146.2, 140.7, 138.1, 134.4, 129.5 (2C), 128.7 (2C), 128.6

(2C), 126.7, 126.7, 124.9 (2C), 114.0, 55.8, 45.4, 42.0, 40.1. MS

(ESI) m/z: calcd for C₁₉H₁₉N₃[M + H]⁺, 290.2; found, 290.1.

(S)-8-Isobutyl-2-phenyl-5,6,7,8-tetrahydroimidazo[1,2-a]-

Next, to a solution of protected intermediates (1.00 equiv) in

triﬂuoroacetic acid (TFA) were added (i-Pr)₃SiH (2.00 equiv). The

mixture was stirred under Ar at rt for 6 h. After completion of the

reaction, the formed precipitate was ﬁltered oﬀ. The ﬁltrate was

concentrated under vacuum and puriﬁed by reverse-phase HPLC

(column, Amethyst C18-H, 250 mm × 10 mm × 5 μm) using a

gradient of 0−50% of CH₃CN in an aqueous solution of TFA at 0.1%

for 40 min. The products GQ111-GQ115, GQ121, GQ129, GQ132,

GQ133, GQ138-GQ146, GQ153, and GQ160 were obtained by

lyophilizing.

The building blocks with protective groups (1.00 equiv) were

added to CH₂Cl₂containing 30% TFA. The mixture was stirred at rt

for 0.5 h. After completion of the reaction, the mixture was

concentrated under vacuum and puriﬁed by reverse-phase HPLC

using a gradient of 0−50% of CH₃CN in an aqueous solution of TFA

at 0.1% for 40 min. The products GQ116-GQ120, GQ122-GQ128,

GQ130, GQ131, GQ134, GQ147-GQ150, GQ154-GQ157 and

GQ161-GQ164 were obtained by lyophilizing.

The N-Mtr-protected intermediate (1.00 equiv) was added to a

solution containing 95% TFA and 5% thioanisole. The mixture was

stirred at rt for 1 h. Upon completion, the mixture was concentrated

under reduced pressure and puriﬁed by reverse-phase HPLC using a

gradient of 0−60% of CH₃CN in an aqueous solution of TFA at 0.1%

for 30 min. The product GQ135 were obtained by lyophilizing.

Besides, to a solution of coupling partners 10a and 10e (1.00

equiv) in DMF were added carboxylic acid fragments (1.10 equiv),

HATU (1.50 equiv), and DIPEA (2.00 equiv). The mixture was

stirred at rt for 4 h and monitored by TLC. The mixture was

concentrated under reduced pressure, diluted with EtOAc, and

washed with water and with a saturated solution of NaCl. The EtOAc

phase was dried over anhydrous Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The crude products were puriﬁed by silica gel

column chromatography to obtain the solid products GQ136,

GQ137, GQ151, GQ152, GQ158, GQ159, GQ165, and GQ166.

(S)-1-((S)-8-(Cyclohexylmethyl)-2-phenyl-5, 6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-mercapto-2-

(methylamino)propan-1-one (GQ111). White solid, yield 67%.

¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H), 7.76−7.69 (m, 2H),

7.54−7.44 (m, 3H), 6.20 (dd, J = 11.2, 3.2 Hz, 1H), 4.83 (t, J = 5.6

Hz, 1H), 4.50 (dd, J = 15.2, 4.0 Hz, 1H), 4.38 (ddd, J = 17.6, 10.4, 4.4

Hz, 2H), 4.09−3.97 (m, 1H), 3.13 (dd, J = 15.2, 6.0 Hz, 1H), 3.07

(dd, J = 14.8, 5.2 Hz, 1H), 2.77 (s, 3H), 2.30 (d, J = 12.0 Hz, 1H),

2.16−2.05 (m, 1H), 1.86−1.64 (m, 5H), 1.34−1.18 (m, 4H), 1.12−

1.01 (m, 2H). ¹³C NMR (100 MHz, CD₃OD): δ 167.0, 145.3, 135.1,

130.7, 130.3 (2C), 127.7, 126.5 (2C), 117.8, 60.8, 48.1, 46.7, 41.4,

38.8, 34.9, 34.9, 33.0, 32.3, 27.2 (2C), 26.9, 24.6. HRMS: m/z calcd

for C₂₃H₃₂N₄OS [M + H]⁺, 413.2370; found, 413.2379. Purity:

98.1%.

(S)-1-((S)-8-(Cyclohexylmethyl)-2-phenyl-5, 6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-(dimethylamino)-3-

mercaptopropan-1-one (GQ112). White solid, yield 69%. ¹H

NMR (400 MHz, CD₃OD) δ 7.82 (s, 1H), 7.73−7.69 (m, 2H),

7.53−7.43 (m, 3H), 6.20 (dd, J = 11.6, 3.2 Hz, 1H), 4.89−4.86 (m,

1H), 4.55 (d, J = 15.6 Hz, 1H), 4.40 (dd, J = 11.6, 6.0 Hz, 2H), 4.11−

3.98 (m, 1H), 3.21−3.12 (m, 2H), 2.98 (s, 6H), 2.30 (d, J = 12.0 Hz,

1H), 2.17−2.05 (m, 1H), 1.86−1.67 (m, 5H), 1.31−1.18 (m, 4H),

1.12−1.03 (m, 2H). ¹³C NMR (100 MHz, CD₃OD): δ 167.2, 145.3,

135.9, 130.5, 130.3 (2C), 128.3, 126.4 (2C), 117.7, 66.3, 48.3, 46.5,

42.2, 42.2, 41.4, 39.1, 35.3, 34.9, 32.9, 27.3, 27.2, 27.0, 23.0. HRMS:

m/z calcd for C₂₄H₃₄N₄OS [M + H]⁺, 427.2526; found, 427.2516.

Purity: 96.3%.

(S)-1-((S)-8-(Cyclohexylmethyl)-2-phenyl-5, 6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-(diethylamino)-3-

mercaptopropan-1-one (GQ113). White solid, yield 73%. ¹H

NMR (400 MHz, CD₃OD) δ 7.83 (s, 1H), 7.75−7.68 (m, 2H),

7.54−7.44 (m, 3H), 6.24 (dd, J = 11.6, 2.8 Hz, 1H), 4.96−4.91 (m,

1H), 4.67 (dd, J = 15.2, 4.0 Hz, 1H), 4.44 (ddd, J = 17.2, 12.6, 4.2 Hz,

2H), 4.14−4.00 (m, 1H), 3.42 (td, J = 14.2, 7.0 Hz, 2H), 3.27 (dd, J =

14.6, 7.8 Hz, 2H), 3.20 (dd, J = 14.4, 3.6 Hz, 1H), 3.10 (dd, J = 14.8,

8.0 Hz, 1H), 2.32 (d, J = 12.4 Hz, 1H), 2.19−2.09 (m, 1H), 1.87−

1

pyrazine (10d). Bright yellow oil, yield 78%. H NMR (400 MHz,

CDCl₃) δ 7.81−7.65 (m, 2H), 7.40−7.30 (m, 2H), 7.24−7.17 (m,

1H), 7.06 (s, 1H), 4.11 (dd, J = 10.4, 3.6 Hz, 1H), 4.04−3.88 (m,

2H), 3.36 (dt, J = 13.2, 4.2 Hz, 1H), 3.15 (ddd, J = 18.0, 9.2, 5.2 Hz,

1H), 2.15 (ddd, J = 13.8, 10.0, 3.8 Hz, 1H), 1.98−1.89 (m, 2H), 1.65

(ddd, J = 14.2, 10.4, 4.2 Hz, 1H), 1.02 (t, J = 6.4 Hz, 6H). ¹³C NMR

(100 MHz, CDCl₃) δ 147.3, 140.5, 134.4, 128.5 (2C), 126.5, 124.9

(2C), 113.9, 52.4, 45.5, 42.9, 41.7, 24.4, 23.8, 21.3. MS (ESI) m/z:

calcd for C₁₆H₂₁N₃[M + H]⁺, 256.2; found, 256.1.

(R)-8-Isobutyl-2-phenyl-5,6,7,8-tetrahydroimidazo[1,2-a]-

1

pyrazine (10e). Bright yellow oil, yield 80%. H NMR (400 MHz,

CDCl₃) δ 7.81−7.65 (m, 2H), 7.40−7.30 (m, 2H), 7.24−7.17 (m,

1H), 7.06 (s, 1H), 4.11 (dd, J = 10.4, 3.6 Hz, 1H), 4.04−3.88 (m,

2H), 3.36 (dt, J = 13.2, 4.2 Hz, 1H), 3.15 (ddd, J = 18.0, 9.2, 5.2 Hz,

1H), 2.15 (ddd, J = 13.8, 10.0, 3.8 Hz, 1H), 1.98−1.89 (m, 2H), 1.65

(ddd, J = 14.2, 10.4, 4.2 Hz, 1H), 1.02 (t, J = 6.4 Hz, 6H). ¹³C NMR

(100 MHz, CDCl₃) δ 147.3, 140.5, 134.4, 128.5 (2C), 126.5, 124.9

(2C), 113.9, 52.4, 45.5, 42.9, 41.7, 24.4, 23.8, 21.3. MS (ESI) m/z:

calcd for C₁₆H₂₁N₃[M + H]⁺, 256.2; found, 256.1.

(S)-8-Isopropyl-2-phenyl-5,6,7,8-tetrahydroimidazo[1,2-a]-

1

pyrazine (10f). Bright yellow oil, yield 82%. H NMR (400 MHz,

CDCl₃) δ 7.85−7.66 (m, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.20 (t, J = 7.2

Hz, 1H), 7.07 (s, 1H), 4.07−3.87 (m, 3H), 3.38 (ddd, J = 12.8, 4.4,

2.4 Hz, 1H), 3.14 (ddd, J = 13.0, 10.4, 4.4 Hz, 1H), 2.71−2.61 (m,

1H), 1.80 (s, 1H), 1.13 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H).

¹³C NMR (100 MHz, CDCl₃) δ 146.3, 140.5, 134.5, 128.5 (2C),

126.5, 124.9 (2C), 113.8, 59.5, 45.5, 42.3, 31.2, 19.3, 16.7. MS (ESI)

m/z: calcd for C₁₅H₁₉N₃[M + H]⁺, 242.2; found, 242.1.

(R)-8-Isopropyl-2-phenyl-5,6,7,8-tetrahydroimidazo[1,2-a]-

1

pyrazine (10g). Bright yellow oil, yield 81%. H NMR (400 MHz,

CDCl₃) δ 7.85−7.66 (m, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.20 (t, J = 7.2

Hz, 1H), 7.07 (s, 1H), 4.07−3.87 (m, 3H), 3.38 (ddd, J = 12.8, 4.4,

2.4 Hz, 1H), 3.14 (ddd, J = 13.0, 10.4, 4.4 Hz, 1H), 2.71−2.61 (m,

1H), 1.80 (s, 1H), 1.13 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H).

¹³C NMR (100 MHz, CDCl₃) δ 146.3, 140.5, 134.5, 128.5 (2C),

126.5, 124.9 (2C), 113.8, 59.5, 45.5, 42.3, 31.2, 19.3, 16.7. MS (ESI)

m/z: calcd for C₁₅H₁₉N₃[M + H]⁺, 242.2; found, 242.1.

(S)-8-Methyl-2-phenyl-5,6,7,8-tetrahydroimidazo[1,2-a]-

1

pyrazine (10h). Bright yellow oil, yield 83%. H NMR (400 MHz,

CDCl₃) δ 7.72 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.26−

7.16 (m, 1H), 7.04 (s, 1H), 4.16 (q, J = 6.8 Hz, 1H), 4.04−3.84 (m,

2H), 3.33 (dt, J = 13.2, 3.4 Hz, 1H), 3.15 (ddd, J = 10.6, 9.8, 4.6 Hz,

1H), 2.55 (s, 1H), 1.59 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz,

CDCl₃) δ 147.5, 140.5, 134.3, 128.5 (2C), 126.6, 124.9 (2C), 113.9,

50.2, 45.5, 41.9, 20.2. MS (ESI) m/z: calcd for C₁₃H₁₅N₃[M + H]⁺,

214.1; found, 214.1.

(R)-8-Methyl-2-phenyl-5,6,7,8-tetrahydroimidazo[1,2-a]-

1

pyrazine (10i). Bright yellow oil, yield 80%. H NMR (400 MHz,

CDCl₃) δ 7.72 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.26−

7.16 (m, 1H), 7.04 (s, 1H), 4.16 (q, J = 6.8 Hz, 1H), 4.04−3.84 (m,

2H), 3.33 (dt, J = 13.2, 3.4 Hz, 1H), 3.15 (ddd, J = 10.6, 9.8, 4.6 Hz,

1H), 2.55 (s, 1H), 1.59 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz,

CDCl₃) δ 147.5, 140.5, 134.3, 128.5 (2C), 126.6, 124.9 (2C), 113.9,

50.2, 45.5, 41.9, 20.2. MS (ESI) m/z: calcd for C₁₃H₁₅N₃[M + H]⁺,

214.1; found, 214.1.

General Procedure for the Synthesis of GQ111-GQ166. To a

solution of 10a−10i (1.00 equiv) in DMF were added protected

amino acids (1.10 equiv), 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium hexaﬂuorophosphate (HATU, 1.50 equiv) and

N,N-diisopropylethylamine (DIPEA, 2.00 equiv). The mixture was

stirred at rt for 4 h. After completion of the reaction, the mixture was

diluted with EtOAc and washed with water and with a saturated

solution of NaCl. The EtOAc phase was dried over anhydrous

Na₂SO₄, ﬁltered, and concentrated under vacuum. The crude

products were puriﬁed by silica gel column chromatography to

obtain intermediates with protective groups as an oﬀ-white solid.

3143

https://dx.doi.org/10.1021/acs.jmedchem.0c01977

J. Med. Chem. 2021, 64, 3131−3152

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1.63 (m, 5H), 1.38 (t, J = 7.6 Hz, 6H), 1.30−1.17 (m, 4H), 1.13−

1.03 (m, 2H). ¹³C NMR (100 MHz, CD₃OD): δ 167.7, 145.2, 135.7,

130.6, 130.3 (2C), 128.1, 126.5 (2C), 117.7, 63.5, 48.2, 48.2 (2C),

46.5, 41.5, 39.1, 35.4, 34.9, 32.9, 27.5, 27.2, 27.1, 22.9, 9.2, 9.1.

HRMS: m/z calcd for C₂₆H₃₈N₄OS [M + H]⁺, 455.2839; found,

455.2830. Purity: 95.7%.

MHz, CD₃OD) δ 8.87 (d, J = 1.2 Hz, 1H), 7.86 (s, 1H), 7.75−7.71

(m, 2H), 7.55−7.44 (m, 4H), 6.14 (dd, J = 10.8, 3.2 Hz, 1H), 5.05−

5.00 (m, 1H), 4.65 (dd, J = 15.6, 4.0 Hz, 1H), 4.44 (dd, J = 13.2, 3.6

Hz, 1H), 4.38−4.29 (m, 1H), 4.14−3.99 (m, 1H), 3.47 (dd, J = 16.0,

4.4 Hz, 1H), 3.30−3.20 (m, 1H), 2.27 (d, J = 12.0 Hz, 1H), 2.09

(ddd, J = 14.2, 11.0, 3.4 Hz, 1H), 1.83−1.63 (m, 5H), 1.46−1.38 (m,

1H), 1.35−1.19 (m, 3H), 1.03 (dt, J = 12.6, 6.4 Hz, 2H). ¹³C NMR

(100 MHz, CD₃OD): δ 168.3, 145.6, 136.1, 135.4, 130.7, 130.3 (2C),

127.9, 127.3, 126.5 (2C), 120.2, 117.8, 50.3, 48.2, 46.6, 41.9, 39.0,

34.9, 34.2, 33.2, 27.3, 27.0, 26.9, 26.6. HRMS: m/z calcd for

C₂₅H₃₂N₆O [M + H]⁺, 433.2710; found, 433.2701. Purity: 97.4%.

(S)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-phenylpropan-1-

one (GQ122). White solid, yield 49%. ¹H NMR (400 MHz,

CD₃OD) δ 7.73−7.68 (m, 2H), 7.56 (s, 1H), 7.50 (t, J = 6.8 Hz, 2H),

7.44 (t, J = 7.2 Hz, 1H), 7.31−7.17 (m, 5H), 6.03 (dd, J = 10.8, 3.6

Hz, 1H), 4.83−4.79 (m, 1H), 4.16 (dd, J = 15.2, 4.8 Hz, 1H), 3.94−

3.76 (m, 2H), 3.18 (d, J = 7.2 Hz, 2H), 2.38 (td, J = 12.2, 5.0 Hz,

1H), 2.28 (d, J = 12.8 Hz, 1H), 2.04−1.93 (m, 1H), 1.78−1.64 (m,

5H), 1.42−1.34 (m, 1H), 1.29−1.20 (m, 3H), 1.07−0.98 (m, 2H).

¹³C NMR (100 MHz, CD₃OD): δ 169.7, 145.1, 135.2, 130.6 (2C),

(S)-1-((S)-8-(Cyclohexylmethyl)-2-phenyl-5, 6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-mercapto-2-meth-

1

ylpropan-1-one (GQ114). Yellow-white solid, yield 70%. H NMR

(400 MHz, CD₃OD) δ 7.84 (s, 1H), 7.70 (d, J = 7.2 Hz, 2H), 7.54−

7.44 (m, 3H), 6.27 (dd, J = 12.0, 2.8 Hz, 1H), 4.64−4.52 (m, 1H),

4.41−4.24 (m, 2H), 3.91 (ddd, J = 15.8, 10.2, 4.6 Hz, 1H), 3.26−3.17

(m, 1H), 2.82 (dd, J = 13.2, 9.2 Hz, 1H), 2.60 (ddd, J = 33.4, 13.2, 4.8

Hz, 1H), 2.22 (dd, J = 33.2, 11.6 Hz, 1H), 2.11−2.02 (m, 1H), 1.83−

1.60 (m, 6H), 1.24 (dt, J = 13.8, 6.6 Hz, 6H), 1.06 (dd, J = 24.6, 12.1

Hz, 2H). ¹³C NMR (100 MHz, CD₃OD) δ 175.9, 144.6, 133.7, 129.6,

129.1 (2C), 126.4, 125.3 (2C), 116.7, 46.3, 45.6, 40.6, 39.9, 37.5,

34.0, 33.8, 32.0, 26.7, 26.1, 26.0, 25.8, 17.2. HRMS: m/z calcd for

C₂₃H₃₁N₃OS [M + H]⁺, 398.2261; found, 398.2266. Purity: 95.5%.

(S)-1-(8-(Cyclohexylmethyl)-2-phenyl-5,6-dihydroimidazo-

[1,2-a]pyrazin-7(8H)-yl)-3-mercaptopropan-1-one (GQ115).

Yellow-white solid, yield 68%. ¹H NMR (400 MHz, CD₃OD) δ

7.83 (s, 1H), 7.72−7.68 (m, 2H), 7.55−7.44 (m, 3H), 6.21 (dd, J =

11.8, 3.0 Hz, 1H), 4.42 (dd, J = 15.2, 3.8 Hz, 1H), 4.36−4.22 (m,

2H), 3.92−3.80 (m, 1H), 3.00−2.91 (m, 1H), 2.83 (ddd, J = 20.4,

13.0, 6.2 Hz, 3H), 2.22 (d, J = 12.4 Hz, 1H), 2.08−1.99 (m, 1H),

1.78−1.62 (m, 5H), 1.36−1.20 (m, 4H), 1.11−0.98 (m, 2H). ¹³C

NMR (100 MHz, CD₃OD) δ 171.5, 144.8, 133.6, 129.6, 129.1 (2C),

126.4, 125.2 (2C), 116.7, 45.7, 45.5, 40.0, 37.3, 36.7, 33.8, 33.7, 31.9,

26.1 (2C), 25.7, 19.1. HRMS: m/z calcd for C₂₂H₂₉N₃OS [M + H]⁺,

384.2104; found, 384.2098. Purity: 95.8%.

(S)-1-((S)-8-(Cyclohexylmethyl)-2-phenyl-5, 6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-hydroxypropan-1-

one (GQ116). White solid, yield 60%. ¹H NMR (400 MHz,

CD₃OD) δ 7.82 (s, 1H), 7.74−7.67 (m, 2H), 7.55−7.44 (m, 3H),

6.12 (d, J = 9.6 Hz, 1H), 4.79−4.71 (m, 1H), 4.64 (dd, J = 12.8, 6.4

Hz, 1H), 4.29 (td, J = 13.0, 9.2 Hz, 2H), 3.91−3.78 (m, 1H), 2.24 (d,

J = 12.0 Hz, 1H), 2.11−2.01 (m, 1H), 1.80−1.63 (m, 5H), 1.42 (d, J

= 6.4 Hz, 3H), 1.36−1.18 (m, 4H), 1.10−0.99 (m, 2H). ¹³C NMR

(100 MHz, CD₃OD): δ 175.0, 146.0, 135.0, 130.7, 130.3 (2C), 127.9,

126.4 (2C), 117.8, 67.6, 47.3, 46.9, 41.5, 38.3, 35.0, 34.7, 33.3, 27.3,

27.2, 26.8, 20.3. HRMS: m/z calcd for C₂₂H₂₉N₃O₂[M + H]⁺,

368.2333; found, 368.2315. Purity: 96.9%.

130.2 (2C), 130.2 (2C), 130.0 (2C), 129.1 (2C), 126.3 (2C), 117.3,

51.9, 48.2, 45.3, 41.5, 39.0, 38.7, 34.9, 34.3, 33.3, 27.3, 27.0, 26.7.

HRMS: m/z calcd for C₂₈H₃₄N₄O [M + H]⁺, 443.2805; found,

443.2791. Purity: 98.8%.

(S)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-4-methylpentan-1-

one (GQ123). White solid, yield 55%. ¹H NMR (400 MHz,

CD₃OD) δ 7.82 (s, 1H), 7.74−7.69 (m, 2H), 7.53−7.43 (m, 3H),

6.09 (dd, J = 10.8, 3.2 Hz, 1H), 4.66 (dd, J = 10.4, 3.2 Hz, 1H), 4.49−

4.38 (m, 1H), 4.33−4.24 (m, 2H), 4.03 (dd, J = 20.0, 8.8 Hz, 1H),

2.25 (d, J = 12.0 Hz, 1H), 2.11−2.01 (m, 1H), 1.90−1.57 (m, 9H),

1.47−1.15 (m, 5H), 1.09 (d, J = 6.4 Hz, 3H), 1.02 (d, J = 6.0 Hz,

3H). ¹³C NMR (100 MHz, CD₃OD): δ 169.9, 145.6, 135.5, 130.6,

130.3 (2C), 128.1, 126.5 (2C), 117.8, 50.5, 48.1, 46.2, 41.9, 41.0,

38.8, 34.9, 34.2, 33.3, 27.3, 26.9, 26.6, 25.3, 23.4, 21.2. HRMS: m/z

calcd for C₂₅H₃₆N₄O [M + H]⁺, 409.2962; found, 409.2967. Purity:

96.6%.

(S)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-methylbutan-1-one

1

(GQ124). White solid, yield 57%. H NMR (400 MHz, CD₃OD) δ

7.83 (s, 1H), 7.74−7.70 (m, 2H), 7.53−7.44 (m, 3H), 6.11 (dd, J =

10.4, 3.6 Hz, 1H), 4.53 (d, J = 4.0 Hz, 1H), 4.42 (dd, J = 12.8, 4.0 Hz,

2H), 4.25 (td, J = 12.8, 4.6 Hz, 1H), 4.06−3.93 (m, 1H), 2.31−2.22

(m, 2H), 2.09−2.02 (m, 1H), 1.82−1.64 (m, 5H), 1.44−1.23 (m,

4H), 1.18 (d, J = 6.8 Hz, 3H), 1.10−1.02 (m, 2H), 1.00 (d, J = 6.8

Hz, 3H). ¹³C NMR (100 MHz, CD₃OD): δ 169.1, 145.6, 135.5,

130.6, 130.3 (2C), 128.0, 126.5 (2C), 117.8, 56.7, 48.1, 46.5, 41.9,

38.8, 34.9, 34.2, 33.3, 30.4, 27.3, 26.9, 26.6, 19.3, 16.0. HRMS: m/z

calcd for C₂₄H₃₄N₄O [M + H]⁺, 395.2805; found, 395.2792. Purity:

96.2%.

(S)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

1

(GQ118). White solid, yield 64%. H NMR (400 MHz, CD₃OD) δ

7.83 (s, 1H), 7.73−7.69 (m, 2H), 7.54−7.44 (m, 3H), 6.09 (dd, J =

10.8, 2.8 Hz, 1H), 4.64 (q, J = 6.8 Hz, 1H), 4.45−4.33 (m, 2H),

4.32−4.23 (m, 1H), 4.04−3.94 (m, 1H), 2.26 (d, J = 12.0 Hz, 1H),

2.08 (dd, J = 14.2, 3.0 Hz, 1H), 1.80−1.63 (m, 5H), 1.54 (d, J = 6.8

Hz, 3H), 1.40−1.20 (m, 4H), 1.10−0.98 (m, 2H). ¹³C NMR (100

MHz, CD₃OD): δ 170.1, 145.7, 135.5, 130.6, 130.3 (2C), 128.1,

126.5 (2C), 117.7, 48.3, 48.0, 46.5, 41.8, 38.5, 35.0, 34.3, 33.2, 27.3,

27.0, 26.6, 17.0. HRMS: m/z calcd for C₂₂H₃₀N₄O [M + H]⁺,

367.2492; found, 367.2480. Purity: 98.0%.

(2S,3R)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-hydroxybutan-1-

one (GQ120). White solid, yield 58%. ¹H NMR (400 MHz,

CD₃OD) δ 7.83 (s, 1H), 7.74−7.69 (m, 2H), 7.54−7.44 (m, 3H),

6.14 (d, J = 8.4 Hz, 1H), 4.64 (d, J = 3.2 Hz, 1H), 4.50 (d, J = 14.8

Hz, 1H), 4.42−4.31 (m, 2H), 4.24 (td, J = 9.8, 6.0 Hz, 1H), 4.07−

3.86 (m, 1H), 2.27 (d, J = 11.6 Hz, 1H), 2.07 (ddd, J = 14.2, 11.0, 3.4

Hz, 1H), 1.81−1.64 (m, 5H), 1.40−1.20 (m, 4H), 1.30 (d, J = 6.0 Hz,

3H), 1.03 (dt, J = 14.2, 7.0 Hz, 2H). ¹³C NMR (100 MHz, CD₃OD):

δ 167.9, 145.6, 135.5, 130.6, 130.3 (2C), 128.0, 126.5 (2C), 117.7,

66.6, 56.5, 48.0, 46.7, 41.8, 38.9, 35.0, 34.3, 33.3, 27.3, 27.0, 26.6,

19.0. HRMS: m/z calcd for C₂₃H₃₂N₄O₂[M + H]⁺, 397.2598; found,

397.2587. Purity: 99.1%.

(S)-3-Amino-4-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-4-oxobutanoic Acid

1

(GQ125). White solid, yield 68%. H NMR (400 MHz, CD₃OD) δ

7.81 (s, 1H), 7.73−7.67 (m, 2H), 7.52−7.43 (m, 3H), 6.08 (dd, J =

11.2, 3.2 Hz, 1H), 4.93−4.92 (m, 1H), 4.48 (d, J = 16.4 Hz, 1H),

4.41−4.28 (m, 2H), 4.01 (ddd, J = 15.4, 10.4, 5.6 Hz, 1H), 3.05 (dd, J

= 18.0, 6.0 Hz, 1H), 2.87 (dd, J = 18.0, 7.6 Hz, 1H), 2.25 (d, J = 12.4

Hz, 1H), 2.10−2.03 (m, 1H), 1.82−1.66 (m, 5H), 1.46−1.36 (m,

1H), 1.34−1.20 (m, 3H), 1.04 (dd, J = 22.4, 10.8 Hz, 2H). ¹³C NMR

(100 MHz, CD₃OD): δ 172.3, 168.9, 145.6, 136.0, 130.5, 130.2 (2C),

128.5, 126.4 (2C), 117.6, 49.4, 48.0, 46.1, 41.8, 39.0, 35.9, 34.9, 34.3,

33.2, 27.3, 27.0, 26.7. HRMS: m/z calcd for C₂₃H₃₀N₄O₃[M + H]⁺,

411.2391; found, 411.2379. Purity: 98.3%.

(S)-2-Amino-1-(8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethan-1-one (GQ126).

White solid, yield 61%. ¹H NMR (400 MHz, CD₃OD) δ 7.84 (s, 1H),

7.74−7.68 (m, 2H), 7.53−7.44 (m, 3H), 6.13 (dd, J = 11.6, 2.8 Hz,

1H), 4.33 (d, J = 5.6 Hz, 2H), 4.27−4.10 (m, 3H), 3.99−3.86 (m,

1H), 2.25 (d, J = 12.4 Hz, 1H), 2.12−2.02 (m, 1H), 1.81−1.62 (m,

5H), 1.41−1.32 (m, 1H), 1.31−1.19 (m, 3H), 1.11−1.02 (m, 2H).

(S)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-(1H-imidazol-4-yl)-

propan-1-one (GQ121). White solid, yield 65%. ¹H NMR (400

3144

https://dx.doi.org/10.1021/acs.jmedchem.0c01977

J. Med. Chem. 2021, 64, 3131−3152

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

¹³C NMR (100 MHz, CD₃OD): δ 167.1, 145.5, 135.4, 130.6, 130.3

(2C), 128.0, 126.4 (2C), 117.8, 47.7, 46.3, 41.3, 41.1, 38.0, 34.9, 34.7,

33.0, 27.3, 27.2, 26.8. HRMS: m/z calcd for C₂₁H₂₈N₄O [M + H]⁺,

353.2336; found, 353.2322. Purity: 98.9%.

propan-1-one (GQ132). White solid, yield 61%. H NMR (400

MHz, CD₃OD) δ 7.69−7.59 (m, 2H), 7.57−7.48 (m, 3H), 7.37 (d, J

= 7.2 Hz, 1H), 7.29 (s, 1H), 7.23 (s, 1H), 7.19 (dd, J = 7.2, 1.6 Hz,

1H), 6.78−6.71 (m, 2H), 5.82 (dd, J = 11.2, 3.2 Hz, 1H), 4.76 (dd, J

= 9.6, 6.0 Hz, 1H), 4.14 (dd, J = 14.8, 5.2 Hz, 1H), 3.72−3.58 (m,

2H), 3.46−3.33 (m, 2H), 2.33 (d, J = 12.0 Hz, 1H), 1.93−1.72 (m,

3H), 1.69 (d, J = 9.6 Hz, 2H), 1.62−1.52 (m, 2H), 1.39−1.21 (m,

4H), 1.04−0.95 (m, 2H). ¹³C NMR (100 MHz, CD₃OD): δ 171.1,

144.1, 137.3, 134.9, 130.7, 130.4 (2C), 128.0, 126.4 (3C), 125.9,

122.8, 120.0, 118.5, 117.8, 112.7, 108.2, 51.7, 48.1, 44.7, 41.3, 38.3,

34.9, 34.2, 33.2, 29.7, 27.3, 27.0, 26.7. HRMS: m/z calcd for

C₃₀H₃₅N₅O [M + H]⁺, 482.2914; found, 482.2890. Purity: 98.6%.

(S)-4-Amino-5-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-5-oxopentanamide

(2S,3R)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-methylpentan-1-

one (GQ127). White solid, yield 53%. ¹H NMR (400 MHz,

CD₃OD) δ 7.80 (s, 1H), 7.71 (d, J = 6.8 Hz, 2H), 7.52−7.43 (m,

3H), 6.09 (dd, J = 10.0, 3.6 Hz, 1H), 4.53 (d, J = 4.0 Hz, 1H), 4.42

(dt, J = 14.6, 5.6 Hz, 2H), 4.24−4.14 (m, 1H), 4.06−3.95 (m, 1H),

2.24 (d, J = 12.0 Hz, 1H), 2.07−1.95 (m, 2H), 1.82−1.65 (m, 5H),

1.57−1.51 (m, 1H), 1.46−1.38 (m, 1H), 1.36−1.19 (m, 4H), 1.16 (d,

J = 7.2 Hz, 3H), 1.08−1.00 (m, 2H), 0.94 (t, J = 7.6 Hz, 3H). ¹³C

NMR (100 MHz, CD₃OD): δ 169.0, 145.6, 135.9, 130.5, 130.2 (2C),

128.4, 126.5 (2C), 117.7, 56.7, 48.3, 46.6, 42.0, 39.0, 37.1, 34.9, 34.3,

33.4, 27.3, 26.9, 26.6, 23.6, 15.6, 11.7. HRMS: m/z calcd for

C₂₅H₃₆N₄O [M + H]⁺, 409.2962; found, 409.2954. Purity: 98.2%.

(S)-4-Amino-5-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-5-oxopentanoic Acid

1

(GQ133). White solid, yield 60%. H NMR (400 MHz, CD₃OD) δ

7.85 (d, J = 4.8 Hz, 1H), 7.74−7.68 (m, 2H), 7.54−7.45 (m, 3H),

6.19−6.04 (m, 1H), 4.88−4.84 (m, 1H), 4.72−4.55 (m, 1H), 4.49−

4.33 (m, 2H), 4.07−3.88 (m, 1H), 2.54 (dd, J = 9.8, 3.4 Hz, 1H),

2.44−2.32 (m, 1H), 2.31−2.15 (m, 2H), 2.15−1.99 (m, 2H), 1.80−

1.61 (m, 5H), 1.39−1.18 (m, 4H), 1.03 (dd, J = 22.0, 10.0 Hz, 2H).

¹³C NMR (100 MHz, CD₃OD): δ 176.3, 169.4, 145.7, 135.1, 130.8,

1

(GQ128). White solid, yield 61%. H NMR (400 MHz, CD₃OD) δ

7.85 (s, 1H), 7.76−7.67 (m, 2H), 7.56−7.43 (m, 3H), 6.06 (dd, J =

11.6, 3.2 Hz, 1H), 4.74 (dd, J = 7.2, 3.6 Hz, 1H), 4.64−4.46 (m, 2H),

4.40 (dd, J = 12.8, 4.0 Hz, 1H), 4.09−3.94 (m, 1H), 2.68−2.53 (m,

2H), 2.26 (d, J = 12.4 Hz, 1H), 2.20−2.00 (m, 3H), 1.81−1.61 (m,

5H), 1.42−1.19 (m, 4H), 1.04 (dd, J = 23.6, 12.0 Hz, 2H). ¹³C NMR

(100 MHz, CD₃OD): δ 175.6, 169.3, 145.7, 135.3, 130.7, 130.3 (2C),

128.0, 126.4 (2C), 117.8, 51.1, 48.0, 46.3, 41.7, 38.4, 35.0, 34.2, 33.2,

28.2, 27.3, 27.0, 26.6 (2C). HRMS: m/z calcd for C₂₄H₃₂N₄O₃[M +

H]⁺, 425.2547; found, 425.2526. Purity: 98.7%.

130.3 (2C), 127.7, 126.5 (2C), 117.9, 51.6, 48.0, 46.4, 41.8, 38.5,

35.0, 34.6, 34.2, 33.2, 29.7, 27.3, 27.0, 26.6. HRMS: m/z calcd for

C₂₄H₃₃N₅O₂[M + H]⁺, 424.2707; found, 424.2696. Purity: 97.8%.

(S)-2,6-Diamino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)hexan-1-one (GQ134).

White solid, yield 69%. ¹H NMR (400 MHz, CD₃OD) δ 7.82 (s, 1H),

7.75−7.69 (m, 2H), 7.55−7.42 (m, 3H), 6.09 (dd, J = 10.8, 3.2 Hz,

1H), 4.66 (dd, J = 7.6, 4.0 Hz, 1H), 4.45−4.38 (m, 2H), 4.30−4.20

(m, 1H), 4.09−3.94 (m, 1H), 2.93 (t, J = 8.0 Hz, 2H), 2.25 (d, J =

12.0 Hz, 1H), 2.06 (ddd, J = 14.2, 10.8, 3.6 Hz, 1H), 1.97−1.84 (m,

2H), 1.81−1.63 (m, 7H), 1.59−1.48 (m, 2H), 1.40−1.19 (m, 4H),

1.02 (dt, J = 12.4, 7.0 Hz, 2H). ¹³C NMR (100 MHz, CD₃OD): δ

169.2, 145.6, 135.7, 130.6, 130.3 (2C), 128.3, 126.5 (2C), 117.7, 51.7,

48.2, 46.6, 41.9, 40.0, 38.8, 34.9, 34.3, 33.3, 31.3, 28.0, 27.3, 27.0,

26.6, 22.2. HRMS: m/z calcd for C₂₅H₃₇N₅O [M + H]⁺, 424.3071;

found, 424.3057. Purity: 97.3%.

(S)-3-Amino-4-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-4-oxobutanamide

1

(GQ129). White solid, yield 65%. H NMR (400 MHz, CD₃OD) δ

7.84 (s, 1H), 7.74−7.69 (m, 2H), 7.54−7.44 (m, 3H), 6.12 (dd, J =

10.8, 2.4 Hz, 1H), 4.87−4.75 (m, 1H), 4.50 (d, J = 15.6 Hz, 1H), 4.38

(ddd, J = 16.8, 12.4, 3.8 Hz, 2H), 4.06−3.93 (m, 1H), 2.99 (dd, J =

16.4, 6.0 Hz, 1H), 2.80 (dd, J = 16.8, 7.2 Hz, 1H), 2.27 (d, J = 12.0

Hz, 1H), 2.13−2.01 (m, 1H), 1.81−1.63 (m, 5H), 1.45−1.35 (m,

1H), 1.34−1.17 (m, 3H), 1.04 (dd, J = 23.6, 11.6 Hz, 2H). ¹³C NMR

(100 MHz, CD₃OD): δ 172.5, 169.2, 145.5, 135.3, 130.7, 130.3 (2C),

127.9, 126.5 (2C), 117.8, 48.1 (2C), 46.2, 41.7, 38.9, 36.2, 34.9, 34.3,

33.1, 27.3, 27.0, 26.7. HRMS: m/z calcd for C₂₃H₃₁N₅O₂[M + H]⁺,

410.2551; found, 410.2535. Purity: 96.2%.

(S)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-(4-hydroxyphenyl)-

propan-1-one (GQ130). White solid, yield 56%. ¹H NMR (400

MHz, CD₃OD) δ 7.75−7.68 (m, 2H), 7.59 (s, 1H), 7.55−7.44 (m,

3H), 7.03 (d, J = 8.4 Hz, 2H), 6.59 (d, J = 8.8 Hz, 2H), 6.06 (dd, J =

10.8, 3.2 Hz, 1H), 4.74 (dd, J = 9.2, 6.4 Hz, 1H), 4.20 (dd, J = 15.2,

4.8 Hz, 1H), 3.93 (dd, J = 12.8, 4.0 Hz, 1H), 3.87−3.74 (m, 1H),

3.13−3.01 (m, 2H), 2.48 (td, J = 12.6, 5.0 Hz, 1H), 2.30 (d, J = 12.4

Hz, 1H), 2.06−1.93 (m, 1H), 1.77−1.61 (m, 5H), 1.45−1.36 (m,

1H), 1.33−1.16 (m, 3H), 1.02 (qd, J = 12.0, 2.8 Hz, 2H). ¹³C NMR

(100 MHz, CD₃OD): δ 170.1, 158.5, 144.8, 135.6, 131.7 (2C), 130.6,

130.2 (2C), 128.2, 126.6 (2C), 125.5, 117.5, 116.6 (2C), 51.9, 48.1,

45.5, 41.3, 38.5, 38.3, 34.9, 34.2, 33.2, 27.3, 27.0, 26.7. HRMS: m/z

calcd for C₂₈H₃₄N₄O₂[M + H]⁺, 459.2755; found, 459.2736. Purity:

95.5%.

1-((S)-4-Amino-5-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-5-oxopentyl)-

1

guanidine (GQ135). White solid, yield 71%. H NMR (400 MHz,

CD₃OD) δ 7.84 (s, 1H), 7.74−7.69 (m, 2H), 7.54−7.44 (m, 3H),

6.13 (dd, J = 10.8, 2.8 Hz, 1H), 4.70 (dd, J = 7.6, 4.0 Hz, 1H), 4.42

(dd, J = 12.8, 3.2 Hz, 2H), 4.29 (ddd, J = 25.6, 12.8, 4.8 Hz, 1H), 4.04

(ddd, J = 26.4, 11.6, 3.6 Hz, 1H), 3.25 (t, J = 6.8 Hz, 2H), 2.26 (d, J =

12.0 Hz, 1H), 2.11−1.96 (m, 2H), 1.93−1.84 (m, 1H), 1.81−1.61

(m, 7H), 1.41−1.19 (m, 4H), 1.08−0.95 (m, 2H). ¹³C NMR (100

MHz, CD₃OD): δ 169.1, 158.6, 145.5, 135.1, 130.7, 130.3 (2C),

127.6, 126.6 (2C), 117.9, 51.6, 48.0, 46.6, 41.9, 41.5, 38.7, 34.9, 34.2,

33.2, 28.9, 27.3, 26.9, 26.6, 24.6. HRMS: m/z calcd for C₂₅H₃₇N₇O

[M + H]⁺, 452.3132; found, 452.3129. Purity: 98.9%.

(S)-1-(8-(Cyclohexylmethyl)-2-phenyl-5,6-dihydroimidazo-

[1,2-a]pyrazin-7(8H)-yl)-2-methylpropan-1-one (GQ136). Yel-

low-white solid, yield 80%. H NMR (400 MHz, CD₃OD) δ 7.84 (s,

1

1H), 7.71 (d, J = 7.2 Hz, 2H), 7.57−7.40 (m, 3H), 6.22 (dd, J = 11.6,

2.8 Hz, 1H), 4.50 (dd, J = 15.2, 3.6 Hz, 1H), 4.34 (dd, J = 12.8, 3.6

Hz, 1H), 4.24 (td, J = 12.4, 4.2 Hz, 1H), 3.97−3.81 (m, 1H), 3.09

(dq, J = 13.2, 6.6 Hz, 1H), 2.17 (d, J = 12.8 Hz, 1H), 2.09−2.00 (m,

1H), 1.80−1.61 (m, 5H), 1.37−1.22 (m, 4H), 1.20 (d, J = 6.8 Hz,

3H), 1.16 (d, J = 6.4 Hz, 3H), 1.09−0.97 (m, 2H). ¹³C NMR (100

MHz, CD₃OD): δ 178.7, 146.1, 134.7, 130.7, 130.3 (2C), 127.6,

126.4 (2C), 117.9, 47.1, 46.6, 41.2, 38.5, 35.1, 35.0, 33.2, 31.4, 27.3,

27.2, 27.0, 20.3, 19.1. HRMS: m/z calcd for C₂₃H₃₁N₃O [M + H]⁺,

366.2540; found, 366.2526. Purity: 98.5%.

(S)-1-((S)-8-(Cyclohexylmethyl)-2-phenyl-5, 6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2-methylbutan-1-one

(GQ137). Yellow-white solid, yield 78%. ¹H NMR (400 MHz,

CD₃OD) δ 7.83 (s, 1H), 7.70 (d, J = 6.8 Hz, 2H), 7.54−7.44 (m,

3H), 6.24 (dd, J = 11.6, 3.2 Hz, 1H), 4.54 (dd, J = 15.2, 4.0 Hz, 1H),

4.35 (dd, J = 13.2, 3.6 Hz, 1H), 4.18 (td, J = 12.6, 4.4 Hz, 1H), 3.97−

3.80 (m, 1H), 2.96−2.85 (m, 1H), 2.21 (d, J = 11.6 Hz, 1H), 2.10−

2.00 (m, 1H), 1.80−1.62 (m, 6H), 1.48 (dt, J = 13.6, 7.0 Hz, 1H),

(S)-7-(^L-Prolyl)-8-(cyclohexylmethyl)-2-phenyl-5,6,7,8-

tetrahydroimidazo[1,2-a]pyrazine (GQ131). White solid, yield

1

59%. H NMR (400 MHz, CD₃OD) δ 7.82 (s, 1H), 7.74−7.65 (m,

2H), 7.55−7.41 (m, 3H), 6.08 (dd, J = 11.6, 3.2 Hz, 1H), 4.86−4.80

(m, 1H), 4.45−4.29 (m, 2H), 4.27−4.16 (m, 1H), 4.07−3.92 (m,

1H), 3.47−3.33 (m, 2H), 2.67−2.54 (m, 1H), 2.28 (d, J = 12.4 Hz,

1H), 2.17−2.01 (m, 4H), 1.82−1.60 (m, 5H), 1.43−1.33 (m, 1H),

1.32−1.18 (m, 3H), 1.12−0.98 (m, 2H). ¹³C NMR (100 MHz,

CD₃OD): δ 169.0, 145.5, 135.8, 130.5, 130.3 (2C), 128.3, 126.4

(2C), 117.7, 59.9, 48.2, 47.0, 46.4, 41.7, 38.5, 35.0, 34.6, 33.1, 29.7,

27.3, 27.2, 26.8, 25.2. HRMS: m/z calcd for C₂₄H₃₂N₄O [M + H]⁺,

393.2649; found, 393.2631. Purity: 99.2%.

(S)-2-Amino-1-((S)-8-(cyclohexylmethyl)-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-(1H-indol-3-yl)-

3145

https://dx.doi.org/10.1021/acs.jmedchem.0c01977

J. Med. Chem. 2021, 64, 3131−3152

Journal of Medicinal Chemistry

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Article

1.32−1.22 (m, 3H), 1.20 (d, J = 7.2 Hz, 3H), 1.14−0.97 (m, 3H),

0.92 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD): δ 178.3,

146.0, 134.9, 130.7, 130.3 (2C), 127.7, 126.5 (2C), 117.9, 47.3, 46.6,

41.3, 38.5 (2C), 35.1, 35.0, 33.3, 27.3 (2C), 27.2, 27.0, 18.3, 12.0.

HRMS: m/z calcd for C₂₄H₃₃N₃O [M + H]⁺, 380.2696; found,

380.2689. Purity: 99.2%.

1.69 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD): δ 167.6,

145.5, 135.6, 130.7, 130.3 (2C), 128.0, 126.5 (2C), 117.7, 53.5, 47.1,

46.8, 39.5, 25.7, 17.5. HRMS: m/z calcd for C₁₆H₂₀N₄OS [M + H]⁺,

317.1431; found, 317.1428. Purity: 96.5%.

(S)-2-Amino-3-mercapto-1-((R)-8-methyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

1

(GQ145). White solid, yield 51%. H NMR (400 MHz, CD₃OD) δ

(S)-2-Amino-1-((R)-8-benzyl-2-phenyl-5,6-dihydroimidazo-

7.89 (s, 1H), 7.75−7.70 (m, 2H), 7.55−7.47 (m, 3H), 6.05 (q, J = 6.8

Hz, 1H), 4.76 (dd, J = 6.8, 5.6 Hz, 1H), 4.49 (dd, J = 14.8, 3.6 Hz,

1H), 4.43−4.29 (m, 2H), 3.96 (ddd, J = 15.2, 11.5, 4.0 Hz, 1H), 3.13

(dd, J = 14.8, 5.6 Hz, 1H), 3.00 (dd, J = 14.8, 7.2 Hz, 1H), 1.70 (d, J

= 6.8 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD): δ 167.6, 145.2, 135.4,

130.8, 130.3 (2C), 127.7, 126.5 (2C), 117.9, 53.2, 46.6, 46.3, 39.5,

25.3, 17.9. HRMS: m/z calcd for C₁₆H₂₀N₄OS [M + H]⁺, 317.1431;

found, 317.1428. Purity: 97.9%.

[1,2-a]pyrazin-7(8H)-yl)-3-mercaptopropan-1-one (GQ139).

1

White solid, yield 53%. H NMR (400 MHz, CD₃OD) δ 7.88 (s,

1H), 7.78−7.74 (m, 2H), 7.55−7.51 (m, 2H), 7.49−7.45 (m, 1H),

7.34−7.28 (m, 5H), 6.37 (dd, J = 10.4, 4.4 Hz, 1H), 4.60 (dd, J = 7.6,

4.8 Hz, 1H), 4.38−4.27 (m, 3H), 3.98−3.87 (m, 1H), 3.51−3.46 (m,

1H), 3.39−3.32 (m, 1H), 2.79 (dd, J = 14.8, 4.8 Hz, 1H), 2.42 (dd, J

= 14.8, 7.6 Hz, 1H). ¹³C NMR (100 MHz, CD₃OD): δ 168.1, 144.0,

136.1, 130.6 (2C), 130.6 (2C), 130.3 (2C), 130.3, 129.7 (2C), 128.5,

126.4 (2C), 117.8, 53.3, 50.3, 46.4, 39.4, 38.9, 25.6. HRMS: m/z calcd

for C₂₂H₂₄N₄OS [M + H]⁺, 393.1744; found, 393.1755. Purity:

96.8%.

(S)-1-((R)-8-Isobutyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-3-mercapto-2-(methylamino)propan-1-one

1

(GQ146). White solid, yield 53%. H NMR (400 MHz, CD₃OD) δ

7.87 (s, 1H), 7.72 (d, J = 7.2 Hz, 2H), 7.54−7.47 (m, 3H), 6.21 (dd, J

= 11.6, 2.4 Hz, 1H), 4.84 (t, J = 5.6 Hz, 1H), 4.44−4.33 (m, 3H),

4.05−3.96 (m, 1H), 3.24−3.15 (m, 2H), 2.76 (s, 3H), 2.18 (t, J =

11.6 Hz, 1H), 1.80−1.66 (m, 2H), 1.14 (d, J = 6.4 Hz, 3H), 1.00 (d, J

= 6.4 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 166.5, 143.9, 133.8,

129.6, 129.1 (2C), 126.4, 125.3 (2C), 116.8, 59.4, 47.1, 45.4, 41.5,

37.9, 30.9, 24.4, 23.2, 22.4, 20.3. HRMS: m/z calcd for C₂₀H₂₈N₄OS

[M + H]⁺, 373.2057; found, 373.2053. Purity: 96.7%.

(S)-2-Amino-1-((S)-8-isobutyl-2-phenyl-5,6-dihydroimidazo-

[1,2-a]pyrazin-7(8H)-yl)-3-mercaptopropan-1-one (GQ140).

1

White solid, yield 61%. H NMR (400 MHz, CD₃OD) δ 7.83 (s,

1H), 7.74−7.68 (m, 2H), 7.57−7.41 (m, 3H), 6.09 (d, J = 9.2 Hz,

1H), 4.74 (dd, J = 7.2, 5.2 Hz, 1H), 4.52−4.27 (m, 3H), 4.09−3.96

(m, 1H), 3.10 (dd, J = 14.8, 5.2 Hz, 1H), 2.92 (dd, J = 14.8, 7.2 Hz,

1H), 2.18−2.08 (m, 1H), 1.80−1.66 (m, 2H), 1.16 (d, J = 5.6 Hz,

3H), 0.98 (d, J = 6.0 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD): δ

168.0, 145.4, 135.6, 130.6, 130.3 (2C), 128.1, 126.5 (2C), 117.7, 53.5,

48.2, 46.7, 43.1, 38.7, 25.7, 25.2, 23.6, 21.9. HRMS: m/z calcd for

C₁₉H₂₆N₄OS [M + H]⁺, 359.1900; found, 359.1886. Purity: 98.4%.

(S)-2-Amino-1-((R)-8-isobutyl-2-phenyl-5,6-dihydroimidazo-

[1,2-a]pyrazin-7(8H)-yl)-3-mercaptopropan-1-one (GQ141).

(S)-2-Amino-3-hydroxy-1-((R)-8-isobutyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

1

(GQ147). White solid, yield 73%. H NMR (400 MHz, CD₃OD) δ

7.86 (s, 1H), 7.76−7.68 (m, 2H), 7.54−7.44 (m, 3H), 6.19 (dd, J =

11.8, 2.6 Hz, 1H), 4.71 (t, J = 5.2 Hz, 1H), 4.45 (d, J = 14.0 Hz, 1H),

4.38−4.29 (m, 2H), 4.06−3.92 (m, 2H), 3.88 (dd, J = 11.4, 6.4 Hz,

1H), 2.19−2.07 (m, 1H), 1.78−1.69 (m, 1H), 1.69−1.60 (m, 1H),

1.10 (d, J = 6.4 Hz, 3H), 0.98 (d, J = 6.4 Hz, 3H). ¹³C NMR (100

MHz, CD₃OD) δ 167.2, 144.1, 134.1, 129.5, 129.1 (2C), 126.6, 125.3

(2C), 116.7, 59.9, 53.0, 46.8, 45.4, 41.4, 37.6, 24.2, 22.4, 20.3. HRMS:

m/z calcd for C₁₉H₂₆N₄O₂[M + H]⁺, 343.2129; found, 343.2137.

Purity: 98.5%.

1

White solid, yield 68%. H NMR (400 MHz, CD₃OD) δ 7.87 (s,

1H), 7.74−7.68 (m, 2H), 7.55−7.44 (m, 3H), 6.18 (dd, J = 11.6, 2.4

Hz, 1H), 4.77 (dd, J = 7.2, 5.6 Hz, 1H), 4.51−4.42 (m, 1H), 4.35 (dd,

J = 8.8, 3.0 Hz, 2H), 4.04−3.92 (m, 1H), 3.18 (dd, J = 14.8, 5.6 Hz,

1H), 3.00 (dd, J = 14.8, 7.2 Hz, 1H), 2.18 (dd, J = 18.6, 7.0 Hz, 1H),

1.78−1.61 (m, 2H), 1.12 (d, J = 6.4 Hz, 3H), 0.99 (d, J = 6.4 Hz,

3H). ¹³C NMR (100 MHz, CD₃OD): δ 168.8, 145.1, 135.3, 130.7,

130.3 (2C), 127.8, 126.4 (2C), 117.9, 53.5, 48.2, 46.5, 42.5, 39.0, 25.6

(2C), 23.6, 21.4. HRMS: m/z calcd for C₁₉H₂₆N₄OS [M + H]⁺,

359.1900; found, 359.1893. Purity: 98.0%.

( S ) - 2 - A m i n o - 1 - ( ( S ) - 8 - i s o p r o p y l - 2 - p h e n yl - 5 , 6 -

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-mercaptopropan-1-

one (GQ142). White solid, yield 67%. ¹H NMR (400 MHz,

CD₃OD) δ 7.88 (s, 1H), 7.73 (d, J = 7.2 Hz, 2H), 7.54−7.45 (m,

3H), 5.76 (d, J = 8.0 Hz, 1H), 4.78 (dd, J = 7.2, 5.2 Hz, 1H), 4.56−

4.43 (m, 2H), 4.40−4.30 (m, 1H), 4.10−3.98 (m, 1H), 3.09 (dd, J =

14.8, 5.2 Hz, 1H), 2.91 (dd, J = 14.8, 7.2 Hz, 1H), 2.48 (dq, J = 14.0,

6.8 Hz, 1H), 1.20 (d, J = 6.8 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H). ¹³C

NMR (100 MHz, CD₃OD): δ 168.3, 144.1, 135.6, 130.7, 130.3 (2C),

128.0, 126.7 (2C), 118.2, 55.1, 53.5, 46.4, 39.9, 33.8, 25.7, 19.8, 19.3.

HRMS: m/z calcd for C₁₈H₂₄N₄OS [M + H]⁺, 345.1744; found,

345.1729. Purity: 96.0%.

(2S,3R)-2-Amino-1-((R)-8-isobutyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-methylpentan-1-

one (GQ148). White solid, yield 48%. ¹H NMR (400 MHz,

CD₃OD) δ 7.86 (s, 1H), 7.72 (d, J = 7.6 Hz, 2H), 7.55−7.42 (m,

3H), 6.23 (dd, J = 11.6, 2.8 Hz, 1H), 4.57 (d, J = 4.0 Hz, 1H), 4.43−

4.30 (m, 3H), 4.05−3.91 (m, 1H), 2.25−2.12 (m, 1H), 2.06−1.92

(m, 1H), 1.81−1.69 (m, 1H), 1.63−1.51 (m, 2H), 1.29−1.22 (m,

1H), 1.18 (d, J = 6.8 Hz, 3H), 1.12 (d, J = 6.4 Hz, 3H), 1.01 (t, J = 7.2

Hz, 6H). ¹³C NMR (100 MHz, CD₃OD): δ 168.8, 144.0, 134.2,

129.5, 129.1 (2C), 126.7, 125.3 (2C), 116.7, 55.3, 46.7, 45.4, 41.2,

37.9, 36.5, 24.6, 23.1, 22.3, 20.1, 14.1, 10.5. HRMS (ESI) m/z: calcd

for C₂₂H₃₂N₄O [M + H]⁺, 369.2649; found, 369.2653. Purity: 98.6%.

(S)-2-Amino-3-(4-hydroxyphenyl)-1-((R)-8-isobutyl-2-phe-

nyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

1

(GQ149). White solid, yield 67%. H NMR (400 MHz, CD₃OD) δ

7.80 (s, 1H), 7.70−7.67 (m, 2H), 7.52−7.44 (m, 3H), 7.23−7.15 (m,

2H), 6.86−6.75 (m, 2H), 6.11 (dd, J = 11.2, 3.2 Hz, 1H), 4.81 (dd, J

= 8.6, 6.8 Hz, 1H), 4.29−4.18 (m, 2H), 4.01 (dd, J = 15.2, 3.6 Hz,

1H), 3.46 (ddd, J = 15.2, 9.6, 4.2 Hz, 1H), 3.12 (qd, J = 13.8, 7.6 Hz,

2H), 1.98−1.90 (m, 1H), 1.67−1.59 (m, 1H), 1.51−1.40 (m, 1H),

1.13 (d, J = 6.4 Hz, 3H), 0.97 (d, J = 6.4 Hz, 3H). ¹³C NMR (100

MHz, CD₃OD): δ 169.0, 157.2, 143.9, 133.9, 130.5 (2C), 129.6,

129.1 (2C), 126.4, 125.3 (2C), 124.0, 116.7, 115.5 (2C), 51.3, 47.0,

45.3, 41.3, 37.3, 36.7, 24.1, 22.4, 20.1. HRMS (ESI) m/z: calcd for

C₂₅H₃₀N₄O₂[M + H]⁺, 419.2442; found, 419.2440. Purity: 96.9%.

(S)-2,6-Diamino-1-((R)-8-isobutyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)hexan-1-one (GQ150).

White solid, yield 62%. ¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H),

7.76−7.68 (m, 2H), 7.54−7.43 (m, 3H), 6.18 (dd, J = 11.6, 2.8 Hz,

1H), 4.65 (dd, J = 8.0, 4.8 Hz, 1H), 4.35 (dd, J = 9.2, 3.0 Hz, 3H),

3.99 (dt, J = 16.4, 7.6 Hz, 1H), 2.97 (t, J = 8.0 Hz, 2H), 2.22−2.09

(m, 1H), 2.06−1.84 (m, 2H), 1.82−1.68 (m, 3H), 1.65−1.51 (m,

3H), 1.12 (d, J = 6.4 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H). ¹³C NMR

( S ) - 2 - A m i n o - 1 - ( ( R )- 8 - i s o p r o p y l - 2 - p h e n y l - 5 , 6 -

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-mercaptopropan-1-

one (GQ143). White solid, yield 61%. ¹H NMR (400 MHz,

CD₃OD) δ 7.89 (s, 1H), 7.75−7.70 (m, 2H), 7.55−7.45 (m, 3H),

5.80 (d, J = 8.0 Hz, 1H), 4.77 (dd, J = 7.2, 5.2 Hz, 1H), 4.49 (dd, J =

15.6, 4.8 Hz, 1H), 4.43−4.32 (m, 2H), 4.08−3.97 (m, 1H), 3.22 (dd,

J = 14.4, 5.2 Hz, 1H), 3.02 (dd, J = 14.8, 7.2 Hz, 1H), 2.49 (dq, J =

13.6, 6.8 Hz, 1H), 1.20 (d, J = 6.8 Hz, 3H), 1.05 (d, J = 6.8 Hz, 3H).

¹³C NMR (100 MHz, CD₃OD): δ 169.0, 143.9, 135.6, 130.7, 130.3

(2C), 127.9, 126.7 (2C), 118.3, 54.8, 53.6, 46.3, 40.2, 33.6, 25.7, 19.8,

19.2. HRMS: m/z calcd for C₁₈H₂₄N₄OS [M + H]⁺, 345.1744; found,

345.1731. Purity: 98.3%.

(S)-2-Amino-3-mercapto-1-((S)-8-methyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

1

(GQ144). White solid, yield 64%. H NMR (400 MHz, CD₃OD) δ

7.87 (s, 1H), 7.74−7.70 (m, 2H), 7.54−7.44 (m, 3H), 5.91 (q, J = 6.8

Hz, 1H), 4.74−4.67 (m, 1H), 4.52−4.29 (m, 3H), 4.01−3.87 (m,

1H), 3.09 (dd, J = 14.8, 5.6 Hz, 1H), 2.94 (dd, J = 14.8, 7.2 Hz, 1H),

3146

https://dx.doi.org/10.1021/acs.jmedchem.0c01977

J. Med. Chem. 2021, 64, 3131−3152

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(100 MHz, CD₃OD): δ 170.3, 145.2, 135.6, 130.6, 130.3 (2C), 128.1,

126.4 (2C), 117.8, 51.9, 48.0, 46.5, 42.5, 40.0, 38.8, 31.4, 27.9, 25.8,

23.6, 22.7, 21.5. HRMS: m/z calcd for C₂₂H₃₃N₅O [M + H]⁺,

384.2758; found, 384.2752. Purity: 99.0%.

2.37 (qd, J = 13.6, 6.8 Hz, 1H), 1.19 (d, J = 6.8 Hz, 3H), 1.07 (d, J =

6.8 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 169.1, 157.3, 142.2,

133.9, 130.5 (2C), 129.7, 129.1 (2C), 126.4, 125.8 (2C), 124.2,

117.1, 115.4 (2C), 53.8, 50.6, 44.1, 37.8, 37.1, 31.5, 18.5, 18.0.

HRMS: m/z calcd for C₂₄H₂₈N₄O₂[M + H]⁺, 405.2285; found,

405.2287. Purity: 96.9%.

(R)-1-(8-Isobutyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-2-methylpropan-1-one (GQ151). Yellow-white

1

solid, yield 48%. H NMR (400 MHz, CD₃OD) δ 7.84 (s, 1H), 7.71

(S)-2,6-Diamino-1-((S)-8-isopropyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)hexan-1-one (GQ157).

White solid, yield 59%. ¹H NMR (400 MHz, CD₃OD) δ 7.88 (s, 1H),

7.74 (d, J = 7.1 Hz, 2H), 7.55−7.45 (m, 3H), 5.78 (d, J = 7.8 Hz,

1H), 4.68 (dd, J = 7.8, 4.0 Hz, 1H), 4.45 (ddd, J = 20.2, 14.2, 4.0 Hz,

2H), 4.35−4.26 (m, 1H), 4.11−3.98 (m, 1H), 2.94 (t, J = 7.6 Hz,

2H), 2.49 (dq, J = 13.6, 6.8 Hz, 1H), 2.00−1.83 (m, 2H), 1.77−1.66

(m, 2H), 1.61−1.48 (m, 2H), 1.19 (d, J = 6.8 Hz, 3H), 1.06 (d, J =

6.8 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 168.4, 143.0, 134.2,

129.5, 129.1 (2C), 126.6, 125.6 (2C), 117.1, 53.8, 50.7, 45.2, 38.8,

38.7, 32.8, 30.0, 26.7, 21.1, 18.6, 18.1. HRMS: m/z calcd for

C₂₁H₃₁N₅O [M + H]⁺, 370.2601; found, 370.2600. Purity: 96.6%.

(S)-1-(8-Isopropyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-2-methylpropan-1-one (GQ158). White solid,

yield 60%. ¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H), 7.72 (d, J =

7.2 Hz, 2H), 7.54−7.44 (m, 3H), 5.82 (d, J = 8.2 Hz, 1H), 4.53 (dd, J

= 15.0, 4.0 Hz, 1H), 4.39 (dd, J = 13.0, 3.8 Hz, 1H), 4.26 (td, J = 12.4,

4.8 Hz, 1H), 4.02−3.85 (m, 1H), 3.16−3.04 (m, 1H), 2.44 (dq, J =

13.8, 6.8 Hz, 1H), 1.22 (d, J = 6.8 Hz, 3H), 1.18 (d, J = 6.8 Hz, 3H),

1.15 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H). ¹³C NMR (100

MHz, CD₃OD) δ 177.6, 143.6, 134.1, 129.5, 129.1 (2C), 126.8, 125.5

(2C), 117.0, 52.5, 45.5, 38.5, 32.3, 30.2, 19.0, 18.6, 17.9 (2C). HRMS:

m/z calcd for C₁₉H₂₅N₃O [M + H]⁺, 312.2070; found, 312.2063.

Purity: 96.6%.

(S)-1-((S)-8-Isopropyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-2-methylbutan-1-one (GQ159). White solid,

yield 66%. ¹H NMR (400 MHz, CD₃OD) δ 7.87 (s, 1H), 7.73 (d, J =

7.2 Hz, 2H), 7.55−7.44 (m, 3H), 5.85 (d, J = 8.2 Hz, 1H), 4.57 (dd, J

= 15.0, 4.2 Hz, 1H), 4.42 (dd, J = 13.2, 3.8 Hz, 1H), 4.22 (td, J = 12.6,

4.8 Hz, 1H), 4.02−3.86 (m, 1H), 2.98−2.80 (m, 1H), 2.45 (dq, J =

13.8, 6.8 Hz, 1H), 1.80−1.67 (m, 1H), 1.54−1.37 (m, 1H), 1.20 (t, J

= 8.0 Hz, 6H), 1.01 (d, J = 6.8 Hz, 3H), 0.91 (t, J = 7.4 Hz, 3H). ¹³C

NMR (100 MHz, CD₃OD) δ 177.2, 143.5, 134.0, 129.5, 129.1 (2C),

126.7, 125.6 (2C), 117.1, 52.5, 45.7, 38.5, 37.3, 32.4, 26.2, 18.6, 18.0,

17.0, 10.8. HRMS: m/z calcd for C₂₀H₂₇N₃O [M + H]⁺, 326.2227;

found, 326.2216. Purity: 98.2%.

(d, J = 6.8 Hz, 2H), 7.53−7.44 (m, 3H), 6.20 (dd, J = 11.6, 2.4 Hz,

1H), 4.50 (dd, J = 15.2, 3.6 Hz, 1H), 4.35 (dd, J = 13.2, 3.6 Hz, 1H),

4.24 (td, J = 12.6, 4.4 Hz, 1H), 3.97−3.84 (m, 1H), 3.15−3.02 (m,

1H), 2.17−2.06 (m, 1H), 1.75−1.66 (m, 1H), 1.65−1.50 (m, 1H),

1.19 (d, J = 6.8 Hz, 3H), 1.16 (d, J = 6.4 Hz, 3H), 1.12 (d, J = 6.4 Hz,

3H), 0.97 (d, J = 6.4 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD): δ

178.7, 145.9, 134.7, 130.7, 130.3 (2C), 127.5, 126.4 (2C), 117.9, 47.2,

47.0, 42.7, 38.5, 31.4, 25.6, 23.6, 21.8, 20.2, 19.1. HRMS: m/z calcd

for C₂₀H₂₇N₃O [M + H]⁺, 326.2227; found, 326.2216. Purity: 97.0%.

(S)-1-((R)-8-Isobutyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-2-methylbutan-1-one (GQ152). Yellow-white

1

solid, yield 66%. H NMR (400 MHz, CD₃OD) δ 7.73 (s, 1H), 7.60

(d, J = 7.2 Hz, 2H), 7.44−7.32 (m, 3H), 6.14 (dd, J = 11.6, 2.8 Hz,

1H), 4.44 (dd, J = 15.2, 4.0 Hz, 1H), 4.25 (dd, J = 13.2, 4.0 Hz, 1H),

4.12 (td, J = 12.6, 4.2 Hz, 1H), 3.79 (ddd, J = 15.6, 11.8, 4.0 Hz, 1H),

2.91−2.79 (m, 1H), 2.07−1.95 (m, 1H), 1.68−1.56 (m, 2H), 1.54−

1.45 (m, 1H), 1.38 (dt, J = 20.8, 7.0 Hz, 1H), 1.08−0.99 (m, 6H),

0.92−0.80 (m, 6H). ¹³C NMR (100 MHz, CD₃OD): δ 178.1, 145.9,

134.7, 130.7, 130.3 (2C), 127.5, 126.4 (2C), 117.9, 47.2, 47.1, 42.6,

38.6, 38.1, 28.4, 25.6, 23.6, 21.6, 17.2, 11.6. HRMS: m/z calcd for

C₂₁H₂₉N₃O [M + H]⁺, 340.2383; found, 340.2380. Purity: 95.8%.

(S)-1-((S)-8-Isopropyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-3-mercapto-2-(methylamino)propan-1-one

1

(GQ153). White solid, yield 52%. H NMR (400 MHz, CD₃OD) δ

7.91 (s, 1H), 7.75 (d, J = 7.4 Hz, 2H), 7.54−7.47 (m, 3H), 5.86 (d, J

= 8.0 Hz, 1H), 4.86 (t, J = 5.6 Hz, 1H), 4.58−4.47 (m, 2H), 4.45−

4.37 (m, 1H), 4.13−4.01 (m, 1H), 3.10 (qd, J = 15.0, 5.6 Hz, 2H),

2.77 (s, 3H), 2.52 (td, J = 13.8, 7.0 Hz, 1H), 1.22 (d, J = 6.8 Hz, 3H),

1.07 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 166.2,

142.8, 134.0, 129.7, 129.1 (2C), 126.3, 125.6 (2C), 117.1, 59.6, 53.9,

45.3, 38.8, 32.6, 31.1, 23.3, 18.6, 18.1. HRMS: m/z calcd for

C₁₉H₂₆N₄OS [M + H]⁺, 359.1900; found, 359.1895. Purity: 95.9%.

(S)-2-Amino-3-hydroxy-1-((S)-8-isopropyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

1

(GQ154). White solid, yield 62%. H NMR (400 MHz, CD₃OD) δ

7.90 (s, 1H), 7.73 (d, J = 7.0 Hz, 2H), 7.57−7.46 (m, 3H), 5.79 (d, J

= 8.0 Hz, 1H), 4.73 (t, J = 5.0 Hz, 1H), 4.58−4.42 (m, 2H), 4.39−

4.28 (m, 1H), 4.13−3.96 (m, 1H), 3.91 (d, J = 5.4 Hz, 2H), 2.48 (dq,

J = 13.6, 6.8 Hz, 1H), 1.21 (d, J = 6.8 Hz, 3H), 1.06 (d, J = 6.8 Hz,

3H). ¹³C NMR (100 MHz, CD₃OD) δ 167.0, 142.9, 133.9, 129.7,

129.1 (2C), 126.3, 125.6 (2C), 117.2, 60.0, 53.6, 52.7, 45.4, 38.4,

32.4, 18.6, 18.0. HRMS: m/z calcd for C₁₈H₂₄N₄O₂[M + H]⁺,

329.1972; found, 329.1966. Purity: 96.1%.

(2S,3R)-2-Amino-1-((S)-8-isopropyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-methylpentan-1-

one (GQ155). White solid, yield 60%. ¹H NMR (400 MHz,

CD₃OD) δ 7.89 (s, 1H), 7.74 (d, J = 7.2 Hz, 2H), 7.56−7.45 (m,

3H), 5.79 (d, J = 7.8 Hz, 1H), 4.58 (d, J = 4.0 Hz, 1H), 4.46 (ddd, J =

19.8, 14.0, 4.2 Hz, 2H), 4.29−4.20 (m, 1H), 4.14−3.98 (m, 1H), 2.48

(dq, J = 13.8, 6.8 Hz, 1H), 2.04−1.86 (m, 1H), 1.52 (ddd, J = 13.4,

7.6, 3.4 Hz, 1H), 1.25−1.21 (m, 1H), 1.19 (d, J = 6.8 Hz, 3H), 1.16

(d, J = 6.8 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H).

¹³C NMR (100 MHz, CD₃OD) δ 168.1, 143.0, 134.2, 129.6, 129.1

(S)-1-((R)-8-Isopropyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-3-mercapto-2-(methylamino)propan-1-one

1

(GQ160). White solid, yield 51%. H NMR (400 MHz, CD₃OD) δ

7.90 (s, 1H), 7.73 (d, J = 7.0 Hz, 2H), 7.55−7.47 (m, 3H), 5.82 (d, J

= 8.0 Hz, 1H), 4.83 (t, J = 5.6 Hz, 1H), 4.55−4.45 (m, 2H), 4.42−

4.34 (m, 1H), 4.11−4.02 (m, 1H), 3.08 (qd, J = 15.0, 5.6 Hz, 2H),

2.76 (s, 3H), 2.51 (td, J = 13.8, 6.8 Hz, 1H), 1.22 (d, J = 6.8 Hz, 3H),

1.07 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 166.2,

142.8, 134.1, 129.7, 129.1 (2C), 126.4, 125.6 (2C), 117.1, 59.6, 53.9,

45.2, 38.8, 32.5, 31.1, 23.3, 18.6, 18.1. HRMS: m/z calcd for

C₁₉H₂₆N₄OS [M + H]⁺, 359.1900; found, 359.1896. Purity: 96.6%.

(S)-2-Amino-3-hydroxy-1-((R)-8-isopropyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

1

(GQ161). White solid, yield 67%. H NMR (400 MHz, CD₃OD) δ

7.91 (s, 1H), 7.73 (d, J = 7.0 Hz, 2H), 7.55−7.45 (m, 3H), 5.84 (d, J

= 7.8 Hz, 1H), 4.72 (t, J = 6.0 Hz, 1H), 4.50 (dd, J = 15.2, 3.8 Hz,

1H), 4.38 (qd, J = 13.2, 4.8 Hz, 2H), 4.01 (ddd, J = 17.6, 9.4, 5.0 Hz,

2H), 3.93 (dd, J = 11.6, 6.4 Hz, 1H), 2.49 (dq, J = 13.8, 6.8 Hz, 1H),

1.19 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H). ¹³C NMR (100

MHz, CD₃OD) δ 167.5, 142.8, 134.1, 129.7, 129.1 (2C), 126.4, 125.6

(2C), 117.2, 59.9, 53.2, 52.9, 45.1, 38.9, 32.5, 18.5, 17.8. HRMS: m/z

calcd for C₁₈H₂₄N₄O₂[M + H]⁺, 329.1972; found, 329.1962. Purity:

96.3%.

(2S,3R)-2-Amino-1-((R)-8-isopropyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-3-methylpentan-1-

one (GQ162). White solid, yield 69%. ¹H NMR (400 MHz,

CD₃OD) δ 7.90 (s, 1H), 7.76−7.69 (m, 2H), 7.55−7.45 (m, 3H),

5.86 (d, J = 8.0 Hz, 1H), 4.57 (d, J = 4.2 Hz, 1H), 4.44−4.33 (m,

3H), 4.08−3.96 (m, 1H), 2.51 (dq, J = 13.6, 6.8 Hz, 1H), 2.04 (ddd, J

(2C), 126.6, 125.6 (2C), 117.1, 55.6, 53.8, 45.2, 38.7, 35.8, 32.8, 22.4,

18.7, 18.1, 14.5, 10.5. HRMS: m/z calcd for C₂₁H₃₀N₄O [M + H]⁺,

355.2492; found, 355.2495. Purity: 97.9%.

(S)-2-Amino-3-(4-hydroxyphenyl)-1-((S)-8-isopropyl-2-phe-

nyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

1

(GQ156). White solid, yield 65%. H NMR (400 MHz,CD₃OD) δ

7.80−7.75 (m, 2H), 7.64 (s, 1H), 7.59−7.49 (m, 3H), 7.02 (d, J = 8.6

Hz, 2H), 6.57 (d, J = 8.6 Hz, 2H), 5.66 (d, J = 9.2 Hz, 1H), 4.79 (dd,

J = 10.0, 5.8 Hz, 1H), 4.23 (dd, J = 15.4, 5.8 Hz, 1H), 4.04 (dd, J =

13.2, 4.8 Hz, 1H), 3.93−3.79 (m, 1H), 3.13 (dd, J = 13.0, 5.8 Hz,

1H), 3.04 (dd, J = 12.8, 10.0 Hz, 1H), 2.64 (td, J = 12.6, 5.8 Hz, 1H),

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= 14.0, 7.0, 3.6 Hz, 1H), 1.64 (ddd, J = 14.8, 7.4, 2.8 Hz, 1H), 1.27

(ddd, J = 10.8, 6.8, 3.6 Hz, 1H), 1.20 (dd, J = 6.8, 3.6 Hz, 6H), 1.03

(d, J = 7.2 Hz, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 169.0, 142.7,

134.2, 129.6, 129.1 (2C), 126.5, 125.6 (2C), 117.2, 55.3, 53.3, 45.2,

39.0, 36.4, 32.2, 23.1, 18.6, 18.0, 14.2, 10.5. HRMS: m/z calcd for

C₂₁H₃₀N₄O [M + H]⁺, 355.2492; found, 355.2493. Purity: 96.9%.

(S)-2-Amino-3-(4-hydroxyphenyl)-1-((R)-8-isopropyl-2-phe-

nyl-5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

(GQ163). White solid, yield 73%. ¹H NMR (400 MHz, DMSO-d₆) δ

8.29 (s, 3H), 7.97 (s, 1H), 7.75 (d, J = 7.2 Hz, 2H), 7.48−7.33 (m,

3H), 7.16 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 8.6 Hz, 2H), 5.50 (d, J =

7.8 Hz, 1H), 4.84−4.71 (m, 1H), 4.22−4.08 (m, 3H), 3.50−3.38 (m,

1H), 3.07−2.95 (m, 2H), 2.23 (ddd, J = 26.0, 12.0, 5.2 Hz, 1H), 1.06

(d, J = 6.8 Hz, 3H), 0.76 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz,

DMSO-d₆) δ 169.6, 159.10, 157.3, 143.1, 131.2 (2C), 130.7, 129.4

(2C), 129.3, 125.4 (2C), 124.5, 115.9 (2C), 115.3, 54.6, 51.0, 45.2,

39.3, 36.8, 32.1, 20.0, 19.3. HRMS: m/z calcd for C₂₄H₂₈N₄O₂[M +

H]⁺, 405.2285; found, 405.2283. Purity: 96.3%.

(S)-2,6-Diamino-1-((R)-8-isopropyl-2-phenyl-5,6-

dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)hexan-1-one (GQ164).

White solid, yield 61%. ¹H NMR (400 MHz, CD₃OD) δ 7.91 (s, 1H),

7.76−7.70 (m, 2H), 7.55−7.47 (m, 3H), 5.82 (d, J = 8.0 Hz, 1H),

4.66 (dd, J = 8.0, 4.4 Hz, 1H), 4.45−4.33 (m, 3H), 4.03 (ddd, J =

16.8, 10.8, 5.8 Hz, 1H), 2.98 (t, J = 7.8 Hz, 2H), 2.50 (dq, J = 13.6,

6.8 Hz, 1H), 2.11−2.00 (m, 1H), 1.99−1.88 (m, 1H), 1.83−1.72 (m,

2H), 1.68−1.55 (m, 2H), 1.19 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8

Hz, 3H). ¹³C NMR (100 MHz, CD₃OD) δ 169.3, 142.7, 134.1, 129.7,

129.1 (2C), 126.4, 125.6 (2C), 117.2, 53.2, 50.7, 45.1, 38.8, 38.7,

32.3, 30.2, 26.7, 21.5, 18.5, 17.9. HRMS: m/z calcd for C₂₁H₃₁N₅O

[M + H]⁺, 370.2601; found, 370.2597. Purity: 97.2%.

to achieve a cell density of 4 million cells mL⁻¹. Compounds and cells

(2 × 10⁴cells per well) were incubated at 37 °C for 1 h. After the

incubation, carbamoylcholine chloride (carbachol (Sigma)) was

dissolved in assay buﬀer supplemented with 200 mM LiCl (ﬁnal

concentration of LiCl in the assay was 20 mM). Carbachol was added

to each well at a ﬁnal concentration of 9 μM (∼EC₈₀(drug

concentration that induces 80% of the maximal response)), and the

plate was incubated at 37 °C for 1 h followed by 1 h of incubation at

room temperature. The detection solution was prepared as follows:

IP₁conjugate and lysis buﬀer (Cisbio) + 2.5% anti-IP₁cryptate Tb

conjugate (Cisbio) + 2.5% D-myo-IP₁-d2 conjugate (Cisbio).

Cell Proliferation Inhibition Assay. The human uveal

melanoma cell lines 92.1 and MP41 were maintained in RPMI-

1640 medium containing 10% (v/v) FBS at 37 °C in a 5% (v/v) CO₂

humidiﬁed incubator. Cell proliferation assay was determined by the

CCK-8 assay. Cells were seeded in 96-well plates (3 × 10³cells/well),

then exposed to diﬀerent concentrations of compounds for 72 h at 37

°C, 5% CO₂. After incubation, an amount of 10 μL of CCK-8 was

added to each well of the plates and incubated at 37 °C for 1−4 h.

The absorption at 450 nm was measured by a microplate

spectrophotometer. IC₅₀values were calculated by concentration−

response curve-ﬁtting using GraphPad Prism 7.0 software. Each IC₅₀

value was expressed as the mean SD.

Cells were seeded in six-well plates (1 × 10⁵cells/well), then

exposed to diﬀerent concentrations of compounds for 24, 48, 72, or

96 h at 37 °C, 5% CO₂. After incubation, the cell numbers were

determined with blood corpuscle counting meter at 24, 48, 72, or 96

h, respectively.

BRET Measurements. HEK293 cells were transiently transfected

to express AT₁R, Gαq-RLuc8, Gγ2-Venus, and unlabeled Gβ1.

Twenty-four hours after transfection, cells were washed and

resuspended in PBS. Then cells were seeded in 96-well plates and

incubated overnight in incubator. Cells were washed with HBS,

starved for 10 min, and treated with diﬀerent concentrations of 3, 4,

and GQ127 for 1 h at 37 °C, 5% CO₂. After agonist AngII addition,

the luciferase substrate (coelenterazine) was added. BRET signals

were read using a Flex Station 3 (Moleculardevices). The BRET ratio

was calculated based on the light emitted by the Venus emission

(520−570 nm) and the light emitted by RLuc8 (370−480 nm).

Rescue Assay. For conﬁrming that the GQ127 is an inhibitor for

Gαq/11 proteins, 92.1 cells were seeded into six-well plates, and after

18−24 h for cell adherence, cells grew to 60−70%. According to the

manufacturer’s protocol of DharmaFECT (T-2001-03, Dharmacon,

USA), cells were transfected with negative control or siRNA targeting

GNAQ. Two days after transfecting, cells with si#control (UUCUCC-

GAACGUGUCACGUTT) or si#GNAQ (GCACAAUUAGUUC-

GAGAAGUU) were seeded into six-well plates (2 × 10⁵cells/

well), and after 18−24 h for cell adherence, the GQ127 was added

into cells with diﬀerent concentrations and continuously cultured for

72 h. Then, the amount of cells treated with diﬀerent concentrations

of GQ127 were counted to test the cells’ proliferation.

Intracellular Calcium Release Measurements. HEK293 cells

were transfected with the AT₁R to overexpress AT₁R. Cells were

washed with 0.1% BSA Hanks’ balanced salt solution (HBSS) and

loaded with the calcium-sensitive dye Fluo-4 AM (1 mM; Molecular

Probes). After 1 h of incubation at 37 °C, the cells were washed and

50 μL of 0.1% BSA HBSS, 3, 4, or GQ127 was added into each well

for 30 min. An application plate was prepared with diﬀerent

concentrations of AngII, 3, 4, or GQ127, and twice concentrated

drug solution (50 mL) was added into each well 20 s after the start of

the recording. Both excitation at 485 nm and emission at 525 nm

were measured with a Flex Station 3 (Moleculardevices) every 1.5 s

for 1 min.

(R)-1-(8-Isopropyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-2-methylpropan-1-one (GQ165). Yellow-white

1

solid, yield 69%. H NMR (400 MHz, CD₃OD) δ 7.88 (s, 1H), 7.73

(d, J = 7.2 Hz, 2H), 7.55−7.45 (m, 3H), 5.84 (d, J = 8.0 Hz, 1H),

4.54 (dd, J = 15.0, 4.2 Hz, 1H), 4.41 (dd, J = 13.0, 3.8 Hz, 1H), 4.27

(td, J = 12.6, 4.8 Hz, 1H), 4.04−3.87 (m, 1H), 3.17−3.05 (m, 1H),

2.45 (dq, J = 13.8, 6.8 Hz, 1H), 1.22 (d, J = 6.8 Hz, 3H), 1.18 (d, J =

6.8 Hz, 3H), 1.15 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H). ¹³C

NMR (100 MHz, CD₃OD) δ 177.6, 143.5, 133.7, 129.6, 129.1 (2C),

126.4, 125.6 (2C), 117.2, 52.4, 45.6, 38.5, 32.4, 30.2, 19.0, 18.6, 17.9

(2C). HRMS: m/z calcd for C₁₉H₂₅N₃O [M + H]⁺, 312.2070; found,

312.2075. Purity: 98.2%.

(S)-1-((R)-8-Isopropyl-2-phenyl-5,6-dihydroimidazo[1,2-a]-

pyrazin-7(8H)-yl)-2-methylbutan-1-one (GQ166). Yellow-white

1

solid, yield 70%. H NMR (400 MHz, CD₃OD) δ 7.88 (s, 1H), 7.73

(d, J = 7.2 Hz, 2H), 7.55−7.45 (m, 3H), 5.88 (d, J = 8.0 Hz, 1H),

4.58 (dd, J = 15.2, 4.4 Hz, 1H), 4.42 (dd, J = 13.2, 4.0 Hz, 1H), 4.26

(td, J = 12.6, 4.8 Hz, 1H), 4.02−3.87 (m, 1H), 2.97 (dd, J = 13.4, 6.6

Hz, 1H), 2.46 (td, J = 13.8, 6.8 Hz, 1H), 1.82−1.70 (m, 1H), 1.57−

1.43 (m, 1H), 1.19 (d, J = 6.8 Hz, 3H), 1.13 (d, J = 6.8 Hz, 3H),

1.05−0.93 (m, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 177.1, 143.5,

133.7, 129.6, 129.1 (2C), 126.3, 125.6 (2C), 117.2, 52.5, 45.7, 38.6,

37.0, 32.3, 27.1, 18.7, 18.1, 16.0, 10.5. HRMS: m/z calcd for

C₂₀H₂₇N₃O [M + H]⁺, 326.2227; found, 326.2232. Purity: 97.9%.

Cell Culture. The CHO cell line that stably expresses the

muscarinic M₁receptor was a generous gift from Xinmiao Liang

(Dalian Institute of Chemical Physics, Chinese Academy of Sciences).

CHO-M₁cells were maintained in Ham’s F12K media (zqxzbio)

supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), 1%

(v/v) penicillin−streptomycin (10 000 units mL⁻¹(Gibco)), and 0.25

mg mL⁻¹G418 (Beyotime). MP41 cell line was a generous gift from

Jingxuan Pan (Zhongshan Ophthalmic Center, Sun Yat-sen

University). MP41 cells were cultured in RPMI-1640 (Roswell Park

Memorial Institute) supplemented with 10% (v/v) FBS. 92.1 cells

purchased from Shanghai FuHeng BioLogy Co., Ltd. 92.1 cells were

cultured in RPMI-1640 supplemented with 10% (v/v) FBS. All the

cells were kept at 37 °C in a humidiﬁed atmosphere (95% air and 5%

CO₂).

Flow Cytometer Analysis of Cell Apoptosis. 92.1 and MP41

cells were seeded in six-well plates (3 × 10⁵cells/well) and incubated

overnight at 37 °C, 5% CO₂. Cells were exposed to 3, 4, and GQ127

with diﬀerent concentrations for 48 h. After treatment, cells were

washed with PBS and collected by using standard protocols. Cells

were resuspended in 1× binding buﬀer and stained with annexin V-

FITC (2.5 μL) for 15 min. Then, cells were stained with propidium

IP₁Assay of the M₁Receptor. CHO-M₁cells were resuspended

in assay buﬀer supplemented with 0.2% bovine serum albumin (BSA)

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iodide (5 μL) for 5 min. After being stained, each sample was

analyzed by ﬂow cytometer (Beckman Coulter, Brea, CA, USA).

Clonogenic Assay. 92.1 and MP41 cells were seeded in 12-well

plates (4 × 10³cells/well) and incubated overnight at 37 °C, 5% CO₂.

Cells were exposed to GQ127 with diﬀerent concentrations for 9 days

until colonies were visible. Cell media and the inhibitor were changed

every 3 days. Cells were ﬁxed in 4% paraformaldehyde for 15 min.

Colonies were stained with crystal violet (Solarbio, Beijing, China) for

30 min and counted. The experiments were repeated twice with

consistent results. Colonies were scored using a colony counter.

Transwell Migration and Invasion Assay. 92.1 and MP41 cells

were maintained in RPMI-1640 medium containing 10% (v/v) FBS at

37 °C, 5% CO₂. Migration of 92.1 and MP41 cells was quantiﬁed

using transwell assays. Invasion of 92.1 and MP41 cells was tested in

Transwell chambers. Cells were seeded into the upper chamber with a

serum-free medium in 24-well plates (1 × 10⁴cells/well), and the

medium supplemented with 20% FBS was added in the lower

chamber. Subsequently, cells were incubated overnight at 37 °C, 5%

CO₂. Cells were treated with 3, 4, and GQ127 at diﬀerent

concentrations for 48 h. After treatment, cells in the upper chamber

were removed by a cotton swab. Cells in the lower chamber were

stained with crystal violet (Solarbio, Beijing, China) for 30 min.

Transmigrated cells were viewed by microscope (Nikon). Cells were

counted under the light microscope (magniﬁcation of ×40 and

×100).

Western Blot. 92.1 and MP41 cells were placed in six-well plates

and incubated overnight. Then cells were exposed to diﬀerent

concentrations of GQ127 for 48 h. Cells were washed with PBS and

collected in ice-cold lysis buﬀer. Lysates were centrifuged at 15 000

rpm for 15 min at 4 °C, and supernatants were removed. Protein

concentration was measured by the Pierce BCA protein assay kit

(Thermo Scientiﬁc). Equal amounts of protein were loaded on 8−

12% SDS−PAGE gels and separated by electrophoresis. The target

proteins were transferred to polyvinylidene diﬂuoride membranes

(Millipore, USA), followed by being blocked with 5% BSA for 1 h.

Membranes were sequentially incubated with primary antibodies

phospho-YAP (p-YAP-Ser127), total YAP, phospho-ERK1/2 (p-

ERK1/2-Thr202/Tyr204), total ERK1/2, and GAPDH (CST) in

primary antibody dilution buﬀer overnight at 4 °C. Membranes were

then washed three times with TBST and incubated with secondary

antibodies at room temperature for 1 h. Protein detection was

performed by using enhanced chemiluminescence (ECL).

MP41 Xenograft Models and Study. Male and female nude

(nu/nu) BALB/c mice weighing 20−25 g at 6−8 weeks old were

purchased from the Gempharmatech Co, Ltd. (Nanjing, China)

(SCXK, 2018-0008). The animal procedures were approved by the

Research Ethics Committee of Sun Yat-sen University (Approval Nos.

SYSU-IACUC-2020-000477 and 2020-001005). All animals were

housed in a controlled environment at 23 2 °C under a 12 h dark/

light cycle with free access to food and water (Certiﬁcation No.

SYXK(Guangdong)2016-0112). The animal maintenance and experi-

ments were performed in accordance with the guidelines of the

Animal Care and Use Committee. MP41 cells (3 × 10⁶cells/mouse)

were subcutaneously inoculated in the ﬂank of mice to allow

formation of the primary tumors. When ∼1000 mm³was reached,

xenografted tumors were removed and cut into ∼30 mm³slices and

consecutively implanted into the secondary recipient mice for rounds

of mouse-to-mouse passage. The tertiary recipient mice bearing MP41

PDX were randomly grouped (n = 8/group). The mice were treated

by intraperitoneal injection (ip) with 10 or 30 (mg/kg)/d of GQ127,

10 (mg/kg)/d of 3 or vehicle for 21 days. During the treatment,

tumor size and body weight were monitored every 3 days. Tumor

volume (V) was calculated via the equation V = A × B²× π/6 where

A and B stand for the longest and shortest diameters measured by

vernier caliper, respectively. After 21 days, the tumor blocks were

obtained and weighted. Tumor growth inhibition (TGI) was

treatment, and V_tumor‑c0is tumor volume in the control group on day

0. Data were analyzed using GraphPad Prism 7.0 software.

Haematoxylin and Eosin (H&E) Staining. Tissue specimens

were obtained from sacriﬁcing mice, ﬁxed in 4% paraformaldehyde for

24 h, progressively dehydrated, and embedded in paraﬃn. H&E

staining was implemented according to standard protocol with

hematoxylin and eosin to indicate nucleus and cytoplasm,

respectively.

Pharmacokinetics Studies. The pharmacokinetics of GQ127

were determined in ICR mice following iv dosing at 10 mg/kg or po

dosing at 30 mg/kg. The tested compound was dissolved in a vehicle

containing 5% (v/v) DMSO, 95% (v/v) saline. Brieﬂy, blood samples

(30 μL) were collected from mice with a catheter at 0, 5, 15, and 30

min and 1, 2, 4, 6, 8, and 24 h after administration of GQ127. The

blood samples were centrifuged at 6800g for 6 min at 2−8 °C within 1

h after collection and stored frozen at −80 °C until analysis. Plasma

concentrations of the compound were determined by the liquid

chromatography−tandem mass spectrometry (LC−MS/MS). All

pharmacokinetic parameters were calculated by noncompartmental

methods using WinNonlin, version 7.0 (Pharsight, USA). Pharmaco-

kinetic parameters are shown as the mean SD.

Statistical Analysis. All experiments were performed in triplicate

and repeated at least twice. We expressed data as the mean SD.

Signiﬁcance between groups was determined by Student’s test using

GraphPad Prism 7.0. For animal experiments, we used one-way

ANOVA.

ASSOCIATED CONTENT

* Supporting Information

■

sı

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01977.

¹H NMR and ¹³C NMR spectra of 10a−10i; H NMR

1

and ¹³C NMR spectra of GQ111-GQ166; HPLC

spectra of GQ127; schematic of G proteins signaling

cycle (Figure S1); retrosynthetic analysis of 1 and 2

(Figure S2); the stability of GQ127 in medium (Figure

S3); dose-dependent curves of 3, GQ127, and GQ130

in IP₁accumulation (Figure S4); eﬀects of 3 and

GQ127 on morphological changes of CHO-M₁cells

(Figure S5); dose- and time-dependent curves of

GQ127 in cell proliferative inhibition (Figure S6);

eﬀects of 3 and 4 on UM cell apoptosis (Figure S7);

eﬀects of GQ127 on inhibiting UM cell colony

formation (Figure S8); eﬀects of 3 and 4 on UM cell

migration (Figure S9); eﬀects of 3 and 4 on UM cell

invasion (Figure S10); eﬀects of 3 and GQ127 on tumor

tissues, heart, kidney, and spleen of nude mice (Figure

S11); metabolite mean plasma concentration−time

curve of GQ127 in ICR mice (Figure S12); and eﬀects

of GQ127 on suppressing the downstream signaling

eﬀectors of Gαq/11 proteins in UM cells and MP41

xenografts (Figure S13) (PDF)

Molecular formula strings and some data (CSV)

Additional data for tables and ﬁgures in the main text of

this manuscript (XLSX)

AUTHOR INFORMATION

Corresponding Authors

■

Xiaolei Zhang − Guangdong Key Laboratory of Chiral

Molecule and Drug Discovery, School of Pharmaceutical

Sciences, Sun Yat-sen University, 510006 Guangzhou,

Guangdong, P. R. China; Email: zhangxlei5@

mail.sysu.edu.cn

calculated via the formula TGI = [1 − (V_tumor‑d/V_tumor‑d0)/(V_tumor‑c

/

V

_tumor‑c0)] × 100%, where V_tumor‑dis tumor volume in the treatment

Xiao-Feng Xiong − Guangdong Key Laboratory of Chiral

Molecule and Drug Discovery, School of Pharmaceutical

group on day 21, V_tumor‑d0is tumor volume in the treatment group on

day 0, V_tumor‑cis tumor volume in control group on day 21 of

3149

https://dx.doi.org/10.1021/acs.jmedchem.0c01977

J. Med. Chem. 2021, 64, 3131−3152

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Sciences, Sun Yat-sen University, 510006 Guangzhou,

Guangdong, P. R. China; orcid.org/0000-0001-7868-

8448; Email: xiongxf7@mail.sysu.edu.cn

coupled receptor; GDP, guanosine diphosphate; GTP,

guanosine trisphosphate; GTPase, guanosine triphosphatase;

MAPK, mitogen-activated protein kinase; ERK, extracellular

signal-regulated kinase; PLCβ, phospholipase Cβ; DAG,

diacylglycerol; PKC, protein kinase C; PI3K, phosphatidylino-

sitol-4,5-bisphosphate 3-kinase; AKT, protein kinase B;

mTOR, mammalian target of rapamycin; TRIO, trio rho

guanine nucleotide exchange factor; Rho, ras homolog family

member; RAC, rac family small GTPase 1; YAP, yes-associated

protein; MOA, mode of action; SAR, structure−activity

relationship; Cbz, carbobenzoxy; DMF, N,N-dimethylforma-

mide; rt, room temperature; THF, tetrahydrofuran; HATU, 2-

(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-

ﬂuorophosphate; DIPEA, N,N-diisopropylethylamine; TFA,

triﬂuoroacetic acid; IP₁, inositol monophosphate; HTRF,

homogeneous time-resolved ﬂuorescence; CCK-8, cell count-

ing kit-8; BRET, bioluminescence resonance energy transfer;

HEK, human embryonic kidney; AT₁R, angiotensin type 1

receptor; PI, propidium iodide; TGI, tumor growth inhibition;

H&E, hematoxylin and eosin; ip, intraperitoneal injection; PK,

pharmacokinetic; ICR, Institute of Cancer Research; iv,

intravenous injection; po, oral administration; F, oral

bioavailability; AUC_0−t, area under the curve; C_max, maximum

plasma concentration; T_max, time at maximum plasma

concentration; t_1/2, half-life; MRT, mean residence time; V_ss,

apparent volume of distribution at steady state; CL, clearance;

NMR, nuclear magnetic resonance; HRMS, high-resolution

mass spectrometry; TMS, tetramethylsilane; TLC, thin-layer

chromatography; HPLC, high-performance liquid chromatog-

raphy; FBS, fetal bovine serum; BSA, bovine serum albumin;

HBSS, Hanks’ balanced salt solution; LC−MS/MS, liquid

chromatography−tandem mass spectrometry

Authors

Yang Ge − Guangdong Key Laboratory of Chiral Molecule

and Drug Discovery, School of Pharmaceutical Sciences, Sun

Yat-sen University, 510006 Guangzhou, Guangdong, P. R.

China; orcid.org/0000-0002-0415-0933

Shuo Shi − Guangdong Key Laboratory of Chiral Molecule

and Drug Discovery, School of Pharmaceutical Sciences, Sun

Yat-sen University, 510006 Guangzhou, Guangdong, P. R.

China

Jun-Jie Deng − Guangdong Key Laboratory of Chiral

Molecule and Drug Discovery, School of Pharmaceutical

Sciences, Sun Yat-sen University, 510006 Guangzhou,

Guangdong, P. R. China

Xue-Ping Chen − Guangdong Key Laboratory of Chiral

Molecule and Drug Discovery, School of Pharmaceutical

Sciences, Sun Yat-sen University, 510006 Guangzhou,

Guangdong, P. R. China

Zhendong Song − Guangdong Key Laboratory of Chiral

Molecule and Drug Discovery, School of Pharmaceutical

Sciences, Sun Yat-sen University, 510006 Guangzhou,

Guangdong, P. R. China

Lu Liu − Guangdong Key Laboratory of Chiral Molecule and

Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-

sen University, 510006 Guangzhou, Guangdong, P. R. China

Linlin Lou − Guangdong Key Laboratory of Chiral Molecule

and Drug Discovery, School of Pharmaceutical Sciences, Sun

Yat-sen University, 510006 Guangzhou, Guangdong, P. R.

China

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01977

REFERENCES

■

(1) Smit, K. N.; Jager, M. J.; de Klein, A.; Kilic,

towards a molecular understanding. Prog. Retinal Eye Res. 2020, 75,

100800.

̧

E. Uveal melanoma:

Author Contributions

^†Y.G. and S.S. contributed equally. The manuscript was written

through contributions of all authors. All authors have given

approval to the ﬁnal version of the manuscript. .

(2) Singh, A. D.; Bergman, L.; Seregard, S. Uveal melanoma:

epidemiologic aspects. Ophthalmol. Clin. N. Am. 2005, 18, 75−84.

(3) Jager, M. J.; Shields, C. L.; Cebulla, C. M.; Abdel-Rahman, M.

H.; Grossniklaus, H. E.; Stern, M. H.; Carvajal, R. D.; Belfort, R. N.;

Jia, R.; Shields, J. A.; Damato, B. E. Uveal melanoma. Nat. Rev. Dis.

Primers 2020, 6, 24.

(4) Yonekawa, Y.; Kim, I. K. Epidemiology and management of

uveal melanoma. Hematol. Oncol. Clin. N. Am. 2012, 26, 1169−1184.

(5) Balch, C. M.; Gershenwald, J. E.; Soong, S.-J.; Thompson, J. F.;

Atkins, M. B.; Byrd, D. R.; Buzaid, A. C.; Cochran, A. J.; Coit, D. G.;

Ding, S.; Eggermont, A. M.; Flaherty, K. T.; Gimotty, P. A.;

Kirkwood, J. M.; McMasters, K. M.; Mihm, M. C., Jr; Morton, D. L.;

Ross, M. I.; Sober, A. J.; Sondak, V. K. Final version of 2009 AJCC

melanoma staging and classification. J. Clin. Oncol. 2009, 27, 6199−

6206.

(6) Wierenga, A. P. A.; Cao, J.; Luyten, G. P. M.; Jager, M. J.

Immune checkpoint inhibitors in uveal and conjunctival melanoma.

Int. Ophthalmol. Clin. 2019, 59, 53−63.

(7) Zoroquiain, P.; Esposito, E.; Logan, P.; Aldrees, S.; Dias, A. B.;

Mansure, J. J.; Santapau, D.; Garcia, C.; Saornil, M. A.; Belfort Neto,

R.; Burnier, M. N. Programmed cell death ligand-1 expression in

tumor and immune cells is associated with better patient outcome and

decreased tumor-infiltrating lymphocytes in uveal melanoma. Mod.

Pathol. 2018, 31, 1201−1210.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors acknowledge ﬁnancial support by National

Natural Science Foundation of China (Grants 22077144,

81602972, and 81973359), Guangdong Natural Science Funds

for Distinguished Young Scholar (Grant 2018B030306017),

Guangdong Province Universities and Colleges Pearl River

Scholar Funded Scheme (2018), The Fundamental Research

Funds for the Central Universities (Grants 19ykpy125 and

20ykzd15), Guangdong Provincial Key Laboratory of Chiral

Molecule and Drug Discovery (Grant 2019B030301005), Key

Reasearch and Development Program of Guangdong Province

(Grant 2020B1111110003), Guangdong Basic and Applied

Basic Research Foundation (Grant 2019A1515011215), and

Key Lab of Advanced Drug Preparation Technologies,

Ministry of Education of China (Grant ZKF202002).

ABBREVIATIONS USED

■

(8) Van Raamsdonk, C. D.; Bezrookove, V.; Green, G.; Bauer, J.;

Gaugler, L.; O’Brien, J. M.; Simpson, E. M.; Barsh, G. S.; Bastian, B.

C. Frequent somatic mutations of GNAQ in uveal melanoma and blue

naevi. Nature 2009, 457, 599−602.

UM, uveal melanoma; CM, cutaneous melanoma; BRAF, v-raf

murine sarcoma viral oncogene homolog B1; NRAS, neuro-

blastoma rat sarcoma viral oncogene; GPCR, G-protein-

3150

https://dx.doi.org/10.1021/acs.jmedchem.0c01977

J. Med. Chem. 2021, 64, 3131−3152

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(9) Van Raamsdonk, C. D.; Griewank, K. G.; Crosby, M. B.;

Garrido, M. C.; Vemula, S.; Wiesner, T.; Obenauf, A. C.;

Wackernagel, W.; Green, G.; Bouvier, N.; Sozen, M. M.;

Baimukanova, G.; Roy, R.; Heguy, A.; Dolgalev, I.; Khanin, R.;

Busam, K.; Speicher, M. R.; O’Brien, J.; Bastian, B. C. Mutations in

GNA11 in uveal melanoma. N. Engl. J. Med. 2010, 363, 2191−2199.

(10) Rimoldi, D.; Salvi, S.; Liénard, D.; Lejeune, F. J.; Speiser, D.;

Zografos, L.; Cerottini, J.-C. Lack of BRAF mutations in uveal

melanoma. Cancer Res. 2003, 63, 5712−5715.

(27) Nishimura, A.; Kitano, K.; Takasaki, J.; Taniguchi, M.; Mizuno,

N.; Tago, K.; Hakoshima, T.; Itoh, H. Structural basis for the specific

inhibition of heterotrimeric Gq protein by a small molecule. Proc.

Natl. Acad. Sci. U. S. A. 2010, 107, 13666−13671.

(28) Schrage, R.; Schmitz, A. L.; Gaffal, E.; Annala, S.; Kehraus, S.;

Wenzel, D.; Bullesbach, K. M.; Bald, T.; Inoue, A.; Shinjo, Y.;

̈

Galandrin, S.; Shridhar, N.; Hesse, M.; Grundmann, M.; Merten, N.;

Charpentier, T. H.; Martz, M.; Butcher, A. J.; Slodczyk, T.; Armando,

S.; Effern, M.; Namkung, Y.; Jenkins, L.; Horn, V.; Stößel, A.; Dargatz,

(11) Campbell, A. P.; Smrcka, A. V. Targeting G protein-coupled

receptor signalling by blocking G proteins. Nat. Rev. Drug Discovery

2018, 17, 789−803.

H.; Tietze, D.; Imhof, D.; Galés, C.; Drewke, C.; Muller, C. E.; Hölzel,

̈

M.; Milligan, G.; Tobin, A. B.; Gomeza, J.; Dohlman, H. G.; Sondek,

J.; Harden, T. K.; Bouvier, M.; Laporte, S. A.; Aoki, J.; Fleischmann,

̈

B. K.; Mohr, K.; König, G. M.; Tuting, T.; Kostenis, E. The

experimental power of FR900359 to study Gq-regulated biological

processes. Nat. Commun. 2015, 6, 10156.

(12) Li, J.; Ge, Y.; Huang, J.-X.; Strømgaard, K.; Zhang, X.; Xiong,

X.-F. Heterotrimeric G proteins as therapeutic targets in drug

discovery. J. Med. Chem. 2020, 63, 5013−5030.

(13) Maziarz, M.; Leyme, A.; Marivin, A.; Luebbers, A.; Patel, P. P.;

Chen, Z.; Sprang, S. R.; Garcia-Marcos, M. Atypical activation of the

G protein Galphaq by the oncogenic mutation Q209P. J. Biol. Chem.

2018, 293, 19586−19599.

(14) Onken, M. D.; Worley, L. A.; Long, M. D.; Duan, S.; Council,

M. L.; Bowcock, A. M.; Harbour, J. W. Oncogenic mutations in

GNAQ occur early in uveal melanoma. Invest. Ophthalmol. Visual Sci.

2008, 49, 5230−5234.

(15) Annala, S.; Feng, X.; Shridhar, N.; Eryilmaz, F.; Patt, J.; Yang, J.;

Pfeil, E. M.; Cervantes-Villagrana, R. D.; Inoue, A.; Häberlein, F.;

Slodczyk, T.; Reher, R.; Kehraus, S.; Monteleone, S.; Schrage, R.;

Heycke, N.; Rick, U.; Engel, S.; Pfeifer, A.; Kolb, P.; König, G.;

(29) Xiong, X.-F.; Zhang, H.; Underwood, C. R.; Harpsøe, K.;

Gardella, T. J.; Wöldike, M. F.; Mannstadt, M.; Gloriam, D. E.;

Bräuner-Osborne, H.; Strømgaard, K. Total synthesis and structure-

activity relationship studies of a series of selective G protein

inhibitors. Nat. Chem. 2016, 8, 1035−1041.

(30) Crusemann, M.; Reher, R.; Schamari, I.; Brachmann, A. O.;

̈

Ohbayashi, T.; Kuschak, M.; Malfacini, D.; Seidinger, A.; Pinto-

Carbó, M.; Richarz, R.; Reuter, T.; Kehraus, S.; Hallab, A.; Attwood,

M.; Schiöth, H. B.; Mergaert, P.; Kikuchi, Y.; Schäberle, T. F.;

Kostenis, E.; Wenzel, D.; Muller, C. E.; Piel, J.; Carlier, A.; Eberl, L.;

̈

König, G. M. Heterologous expression, biosynthetic studies, and

ecological function of the selective Gq-signaling inhibitor FR900359.

Angew. Chem., Int. Ed. 2018, 57, 836−840.

Bunemann, M.; Tuting, T.; Vázquez-Prado, J.; Gutkind, J. S.; Gaffal,

̈ ̈

E.; Kostenis, E. Direct targeting of G α q and G α 11 oncoproteins in

cancer cells. Sci. Signaling 2019, 12, eaau5948.

(31) Zhang, H.; Xiong, X.-F.; Boesgaard, M. W.; Underwood, C. R.;

Bräuner-Osborne, H.; Strømgaard, K. Structure-activity relationship

studies of the cyclic depsipeptide natural product YM-254890,

targeting the Gq protein. ChemMedChem 2017, 12, 830−834.

(32) Xiong, X.-F.; Zhang, H.; Boesgaard, M. W.; Underwood, C. R.;

Bräuner-Osborne, H.; Strømgaard, K. Structure-activity relationship

studies of the natural product Gq/11 protein inhibitor YM-254890.

ChemMedChem 2019, 14, 865−870.

̀

(16) Larribere, L.; Utikal, J. Update on GNA alterations in cancer:

implications for uveal melanoma treatment. Cancers 2020, 12, 1524.

(17) Gresset, A.; Sondek, J.; Harden, T. K. The phospholipase C

isozymes and their regulation. Subcell. Biochem. 2012, 58, 61−94.

(18) Hubbard, K. B.; Hepler, J. R. Cell signalling diversity of the

Gq α family of heterotrimeric G proteins. Cell. Signalling 2006, 18,

135−150.

(33) Schmitz, A.-L.; Schrage, R.; Gaffal, E.; Charpentier, T. H.;

Wiest, J.; Hiltensperger, G.; Morschel, J.; Hennen, S.; Häußler, D.;

(19) Kadamur, G.; Ross, E. M. Mammalian phospholipase C. Annu.

Rev. Physiol. 2013, 75, 127−154.

Horn, V.; Wenzel, D.; Grundmann, M.; Bu

R.; Brewitz, H. H.; Schmidt, J.; Gomeza, J.; Galés, C.; Fleischmann, B.

K.; Tuting, T.; Imhof, D.; Tietze, D.; Gutschow, M.; Holzgrabe, U.;

̈

llesbach, K. M.; Schröder,

(20) Arnold, J. J.; Blinder, K. J.; Bressler, N. M.; Bressler, S. B.;

Burdan, A.; Haynes, L.; Lim, J. I.; Miller, J. W.; Potter, M. J.; Reaves,

A.; Rosenfeld, P. J.; Sickenberg, M.; Slakter, J. S.; Soubrane, G.;

Strong, H. A.; Stur, M. Acute severe visual acuity decrease after

photodynamic therapy with Verteporfin: case reports from random-

ized clinical trials-TAP and VIP report no. 3. Am. J. Ophthalmol. 2004,

137, 683−696.

̈

Sondek, J.; Harden, T. K.; Mohr, K.; Kostenis, E. A cell-permeable

inhibitor to trap G α q proteins in the empty pocket conformation.

Chem. Biol. 2014, 21, 890−902.

(34) Prévost, G. P.; Lonchampt, M. O.; Holbeck, S.; Attoub, S.;

Zaharevitz, D.; Alley, M.; Wright, J.; Brezak, M. C.; Coulomb, H.;

Savola, A.; Huchet, M.; Chaumeron, S.; Nguyen, Q.-D.; Forgez, P.;

Bruyneel, E.; Bracke, M.; Ferrandis, E.; Roubert, P.; Demarquay, D.;

Gespach, C.; Kasprzyk, P. G. Anticancer activity of BIM-46174, a new

inhibitor of the heterotrimeric Galpha/Gbetagamma protein complex.

Cancer Res. 2006, 66, 9227−9234.

(21) Singh, A. D.; Turell, M. E.; Topham, A. K. Uveal melanoma:

trends in incidence, treatment, and survival. Ophthalmology 2011, 118,

1881−1885.

(22) Yu, F.-X.; Luo, J.; Mo, J. S.; Liu, G.; Kim, Y. C.; Meng, Z.;

Zhao, L.; Peyman, G.; Ouyang, H.; Jiang, W.; Zhao, J.; Chen, X.;

Zhang, L.; Wang, C.-Y.; Bastian, B. C.; Zhang, K.; Guan, K.-L. Mutant

Gq/11 promote uveal melanoma tumorigenesis by activating YAP.

Cancer Cell 2014, 25, 822−830.

(35) Ku

E.; Gutschow, M. BIM-46174 fragments as potential ligands of G

proteins. MedChemComm 2019, 10, 1838−1843.

(36) Kuppers, J.; Benkel, T.; Annala, S.; Kimura, K.; Reinelt, L.;

Fleischmann, B. K.; Kostenis, E.; Gutschow, M. Tetrahydroimidazo-

̈

ppers, J.; Benkel, T.; Annala, S.; Schnakenburg, G.; Kostenis,

̈

(23) Chua, V.; Lapadula, D.; Randolph, C.; Benovic, J. L.;

Wedegaertner, P.; Aplin, A. E. Dysregulated GPCR signaling and

therapeutic options in uveal melanoma. Mol. Cancer Res. 2017, 15,

501−506.

̈

[1,2-a]pyrazine derivatives: synthesis and evaluation as Galphaq-

protein ligands. Chem. - Eur. J. 2020, 26, 12615−12623.

(37) Mukai, H.; Munekata, E.; Higashijima, T. G protein

antagonists. A novel hydrophobic peptide competes with receptor

for G protein binding. J. Biol. Chem. 1992, 267, 16237−16243.

(38) Charpentier, T. H.; Waldo, G. L.; Lowery-Gionta, E. G.;

Krajewski, K.; Strahl, B. D.; Kash, T. L.; Harden, T. K.; Sondek, J.

Potent and selective peptide-based inhibition of the G protein

Galphaq. J. Biol. Chem. 2016, 291, 25608−25616.

(24) Takasaki, J.; Saito, T.; Taniguchi, M.; Kawasaki, T.; Moritani,

Y.; Hayashi, K.; Kobori, M. A novel Galphaq/11-selective inhibitor. J.

Biol. Chem. 2004, 279, 47438−47445.

(25) Fujioka, M.; Koda, S.; Morimoto, Y.; Biemann, K. S. Structure

of FR900359, a cyclic depsipeptide from Ardisia Crenata Sims. J. Org.

Chem. 1988, 53, 2820−2825.

(26) Ayoub, M. A.; Damian, M.; Gespach, C.; Ferrandis, E.;

̀

Lavergne, O.; De Wever, O.; Baneres, J.-L.; Pin, J.-P.; Prévost, G. P.

(39) Zhang, H.; Nielsen, A. L.; Strømgaard, K. Recent achievements

in developing selective Gq inhibitors. Med. Res. Rev. 2020, 40, 135−

157.

Inhibition of heterotrimeric G protein signaling by a small molecule

acting on Galpha subunit. J. Biol. Chem. 2009, 284, 29136−29145.

3151

https://dx.doi.org/10.1021/acs.jmedchem.0c01977

J. Med. Chem. 2021, 64, 3131−3152

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(40) Meister, S.; Plouffe, D. M.; Kuhen, K. L.; Bonamy, G. M. C.;

Wu, T.; Barnes, S. W.; Bopp, S. E.; Borboa, R.; Bright, A. T.; Che, J.;

Cohen, S.; Dharia, N. V.; Gagaring, K.; Gettayacamin, M.; Gordon,

P.; Groessl, T.; Kato, N.; Lee, M. C. S.; McNamara, C. W.; Fidock, D.

A.; Nagle, A.; Nam, T.; Richmond, W.; Roland, J.; Rottmann, M.;

Zhou, B.; Froissard, P.; Glynne, R. J.; Mazier, D.; Sattabongkot, J.;

Schultz, P. G.; Tuntland, T.; Walker, J. R.; Zhou, Y.; Chatterjee, A.;

Diagana, T. T.; Winzeler, E. A. Imaging of plasmodium liver stages to

drive next-generation antimalarial drug discovery. Science 2011, 334,

1372−1377.

3152

https://dx.doi.org/10.1021/acs.jmedchem.0c01977

J. Med. Chem. 2021, 64, 3131−3152

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Design, Synthesis, and Evaluation of Small Molecule Gαq/11 Protein Inhibitors for the Treatment of Uveal Melanoma

DOI: 10.1021/acs.jmedchem.0c01977

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