Discovery of Potent and Brain-Penetrant GPR52 Agonist that Suppresses Psychostimulant Behavior

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Discovery of Potent and Brain-Penetrant GPR52 Agonist that

Suppresses Psychostimulant Behavior

Pingyuan Wang,^§Daniel E. Felsing,^§Haiying Chen, Sonja J. Stutz, Ryan E. Murphy,

Kathryn A. Cunningham, John A. Allen,*^,∥and Jia Zhou*^,∥

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ABSTRACT: The G protein-coupled receptor 52 (GPR52) is an orphan receptor that is selectively expressed in the striatum and

regulates various brain functions through activation of cAMP-dependent pathways. GPR52 has been identiﬁed as a promising

therapeutic target for central nervous system disorders including schizophrenia and substance use disorders. Here, a series of novel

GPR52 agonists were designed, synthesized, and evaluated based on compound 4. Several potent and eﬃcacious GPR52 agonists

(12c, 23a, 23d, 23e, 23f, and 23h) were identiﬁed with nanomolar range potency based on a systematic structure−activity

relationship exploration. Further studies of 12c indicate enhanced eﬃcacy, excellent target selectivity, and pharmacokinetic

properties including good brain permeability. In vivo proof-of-concept investigations revealed that 12c displayed antipsychotic-like

activity by signiﬁcantly inhibiting amphetamine-induced hyperlocomotor behavior in mice. Collectively, our ﬁndings have resulted in

an eﬃcacious, brain-penetrant GPR52 agonist as a valuable pharmacological tool for investigating the physiological and therapeutic

potential of GPR52 activation.

GPR52 is an understudied brain orphan receptor selectively

expressed in the striatum and cortex with the highest level of

expression in the nucleus accumbens (NAc).^9,10The selective

expression of GPR52 in the NAc and other striatal regions

suggests an important function for this receptor in primary

striatal neurons and broader corticostriatal circuitry.¹¹More-

over, GPR52 is colocalized exclusively with dopamine D2

receptors in medium spiny neurons (MSNs) in the striatum

and has lesser expression in neurons of the medial prefrontal

cortex.¹⁰Notably in transgenic mice, the overexpression of

GPR52 signiﬁcantly decreased methamphetamine-induced

locomotion, while GPR52 knockout mice showed an

INTRODUCTION

■

G-protein-coupled receptors (GPCRs) are a superfamily of

seven-transmembrane spanning receptor proteins, with over

800 members. GPCRs play critical roles in cellular

communication and regulate a wide range of physiological

and pathological processes.¹To date, GPCRs have accounted

for approximately 30% of all U.S. Food and Drug

Administration (FDA)-approved drugs for the therapies of

numerous human diseases including cancer, central nervous

system (CNS) disorders, metabolic disorders, inﬂammation

diseases, infection, immune diseases, and others.²⁻⁵However,

only a fraction of GPCRs are successfully targeted in drug

development,⁶and roughly one third of the non-sensory

GPCRs remain orphan receptors whose endogenous ligands

and physiological functions are largely unknown. Despite

limited knowledge of these orphan GPCRs, their crucial roles

in physiological signaling pathways and putative druggability

make them attractive novel therapeutic targets.^2,7,8

Received: August 27, 2020

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anxiolytic-like phenotype.¹⁰Studies have indicated that GPR52

couples to G_as/olf- G proteins to activate adenylyl cyclase and

modulate 5′-cyclic adenosine monophosphate (cAMP) signal-

ing and displays high levels of constitutive activity.^12,13Thus,

GPR52 signaling via cAMP could oppose activity of D2

signaling in the striatum while stimulating the D1/N-methyl-^D-

aspartate (NMDA) function in the frontal cortex.¹⁴Therefore,

GPR52 may serve as a promising novel target for psychiatric

disorders¹⁰including schizophrenia¹⁵and substance use

disorders (SUDs).¹⁶

To date, the natural ligand of GPR52 has not been

elucidated, but several surrogate ligands have been re-

ported.^14,16−18Most recently, Lin et al. found that the

orthosteric ligand binding pocket of GPR52 (PDB codes:

6LI1 and 6LI2) was occupied by its extracellular loop 2

(ECL2).¹²They suggested that GPR52 was an unprecedented

self-activating receptor and GPR52 ligands could function as

allosteric agonists to control GPR52 self-activation.¹²Com-

pound 1 (Figure 1) is the ﬁrst reported potent GPR52 agonist

residue Phe300 of TM7, and hydrophobic contacts with

residues Phe117, Thr303, Trp304, and Ile307. The elucidation

of this GPR52 ligand-binding pocket is anticipated to facilitate

further drug discovery of potent and selective GPR52

modulators. Recently, Tokumaru et al. reported another series

of novel GPR52 agonists.¹⁷Among those GPR52 agonists,

compound 3 (Figure 1) was an eﬀective agonist with good

potency and eﬃcacy (EC₅₀of 75 nM and E_maxof 122%).¹⁷

Animal model studies indicated that compound 3 inhibited

MK-801-induced hyperactivity and improved memory recog-

nition.¹⁵Likewise, Arena Pharmaceuticals patented and

disclosed a series of 1-heteroaryl-indoline-4-carboxamines as

GPR52 agonists, among which, compound 4 displayed potent

GPR52 agonist activity.¹⁶Here, we choose compound 4 as a

starting point for our medicinal chemistry eﬀorts due to its

amenability to chemical synthesis, optimal physical−chemical

properties, and lack of previously known structure−activity

relationship (SAR). Taken together, GPR52 is emerging as a

promising neurotherapeutic target of interest for the treatment

of schizophrenia and SUDs, and the reported agonists as well

as the recently disclosed cocrystal structural analysis serve as

new starting points to facilitate further drug discovery of novel

GPR52 ligands as pharmacological tools and potential drug

candidates for preclinical and clinical development.

As part of our ongoing research program in understanding

the GPR52-associated pharmacology and identifying novel

ligands through HTS campaign and structure-based rational

drug design, herein, we report our ﬁndings in the chemical

optimization and pharmacological evaluation of novel GPR52

activators based on compound 4¹⁶as the advanced chemical

lead to understand the key interactions of agonists with the

receptor and to identify improved ligands. Systematic SAR

studies around three pharmacological moieties of compound 4

have been explored. A series of novel 1-(pyrimidin-4-

yl)indoline-4-carboxamide analogues have been identiﬁed as

potent and selective GPR52 agonists with nanomolar range

potency and varied levels of improved eﬃcacy. This work

culminated in discovery of compound 12c (PW0787) with a

new pyrimidine core scaﬀold that is an orally bioavailable,

brain-penetrant, potent, and selective GPR52 agonist. This

novel ligand dose-dependently suppresses amphetamine-

evoked hyperactivity, suggesting a therapeutic potential for

neuropsychiatric diseases.

Figure 1. Structures of representative reported GPR52 agonists.

with a half maximal eﬀective concentration (EC₅₀) of about 30

nM, on the basis of their high-throughput screening (HTS)

hits.¹⁴Particularly, compound 1 had excellent bioavailability (F

= 73%) and brain penetration (brain/plasma AUC ratio =

0.94).¹⁴Additionally, compound 1 signiﬁcantly inhibited

rodent methamphetamine-induced hyperactivity at a dose of

3 mg/kg. However, compound 1 has some drawbacks

including high lipophilicity/poor aqueous solubility, which

limit its application for further use.¹⁸Further modiﬁcations of

compound 1 resulted in optimized compound 2 (Figure 1)

with slightly improved GPR52 potency (EC₅₀= 21 nM and

E_max= 103%) and water solubility (21 μg/mL at pH 6.8) and

similar activity to inhibit stimulant-induced hyperactivity.¹⁸

Interestingly, the cocrystal structure of GPR52 and compound

2 (PDB code: 6LI0) was recently solved.¹²Compound 2 was

found located in a small extracellular pocket of GPR52

surrounded by ECL2, transmembrane helix 1 (TM1), TM2,

and TM7. The agonist formed multiple interactions with

GPR52 including hydrogen bonds with residues Cys40,

Glu191, Ile189, and Asp188 in ECL2, π−π stacking with

RESULTS AND DISCUSSION

■

Design. As described in the Introduction, one of the

drawbacks of previously reported GPR52 agonists is the poor

aqueous solubility, limiting their use as pharmacological tools

or potential drug candidates. The replacement of a CH group

with a N atom in aromatic and heteroaromatic rings of the

pharmacophore could improve its physicochemical properties

for better in vivo pharmacokinetic (PK) properties.¹⁹⁻²¹

Additionally, compared to the CH group, the N atom has an

extra unshared electron pair, which could form hydrogen

bonds to potentially improve binding aﬃnity. On this basis, we

hypothesized that an extra N atom in the core heteroaromatic

ring B of advanced chemical lead 4 could improve its potency

as well as physicochemical properties. Therefore, we ﬁrst

altered moiety B (Figure 2, highlight in magenta) by insertion

of a nitrogen atom in the pyridine ring of compound 4 as the

starting point to systematically pursue SAR studies. Next, we

substituted various moieties onto the aromatic ring of moiety

C (Figure 2, highlight in cyan). Then, the methylene linker

B

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solvent system led to key intermediates 11a−c in excellent

yields.²⁴Coupling of intermediates 11a−c with aminoethanol

produced compounds 12a−c and resulted in good yields (73−

83%).

As outlined in Scheme 2, intermediate 13 was obtained by

reaction of compound 9a with 7a in the presence of NaH.

Reﬂuxing of intermediate 13 in the HCl/AcOH/H₂O solvent

system followed by chlorination with phosphorus oxychloride

produced compound 14. Compound 15 was prepared via

palladium-catalyzed C−N coupling reaction of 14 with 6 in a

yield of 56%. Hydrolysis of intermediate 15 generated key

intermediate acid 16. Further, condensation of compound 16

with aminoethanol aﬀorded compound 12d following the same

procedure as that of preparing compound 12a.

As outlined in Scheme 3, with starting material 9a and

compounds 7d²⁵and 7e,²⁵intermediates 17a and 17b were

obtained following the similar synthetic procedure to that of

preparing compound 13, respectively. Removing the cyano and

the tetrahydro-2H-pyran protection groups of 17a and 17b at

the same time led to compounds 18a and 18b in the HCl/

AcOH/H₂O solvent system. Compounds 18a and 18b were

converted into compounds 19a and 19b by a standard

chlorination reaction. Key intermediates 20a and 20b were

synthesized by C−N coupling reaction of intermediates 19a

and 19b with compound 6, respectively, under the palladium-

catalyzed conditions. Acids 21a and 21b were prepared by

hydrolysis of intermediates 20a and 20b. Compounds 12e and

12f were synthesized following the similar procedure to that of

preparing 12a by coupling 21a and 21b with aminoethanol,

respectively.

Figure 2. Proposed structural modiﬁcations of three substructures (A,

B, and C) based on advanced chemical lead 4 for SAR exploration to

understand the key interactions of agonists with the receptor GPR52

and to identify newer ligands.

between moieties B and C was also investigated. Finally, we

investigated substituents on moiety A (Figure 2, highlight in

green) of compound 4 by replacement of aminoethanol with

various side chains to understand the critical ligand−receptor

interactions.

Chemistry. The synthetic procedures of these newly

synthesized GPR52 agonists are depicted in Schemes 1−7.

As outlined in Scheme 1, intermediate 6 was prepared by

reduction of starting material 5 with triethylsilane and

triﬂuoroacetic acid.²²Intermediates 8a−c were generated by

reaction of compound 6 with commercially available

compounds 7a−c in the presence of Et₃N in yields of 63−

67%.²³Intermediates 10a−c were produced via nucleophilic

substitution of compounds 8a−c with commercially available

2-(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)acetonitrile (9a).²⁴Re-

ﬂuxing of intermediates 10a−c in the HCl/AcOH/H₂O mixed

Compounds 23a−k were synthesized following the same

procedures for preparing 12a from various commercially

available substituted 2-phenylacetonitriles (9b−l) (Scheme

4). As outlined in Scheme 5, intermediate 24a was obtained by

reaction of compound 9a with compound 7a in the presence of

a

Scheme 1. Synthetic Routes of Compounds 12a−c

a

Reagents and conditions: (a) CF₃COOH, Et₃SiH, CH₂Cl₂, overnight, and 89%. (b) Et₃N, EtOH, rt., overnight, and 63−67%. (c) 2-(3-Fluoro-5-

(triﬂuoromethyl)phenyl)acetonitrile (9a), NaH, DMF, 0 °C to rt., 2 h, and 53−66%. (d) Con. HCl/AcOH/H₂O, reﬂux, overnight, and 91−93%.

(e) NH₂CH₂CH₂OH, EDCI, DMAP, DMF, rt., overnight, and 73−83%.

C

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a

Scheme 2. Synthetic Routes of Compound 12d

a

Reagents and conditions: (a) NaH, DMF, 0 °C to rt., 2 h, and 73%. (b) (1) Con. HCl/AcOH/H₂O, reﬂux, and overnight; (2) POCl₃, reﬂux, 3 h,

and 51% for two steps. (c) 6, Pd(OAc)₂, XantPhos, Cs₂CO₃, 1,4-dioxane, 100 °C, overnight, and 56%. (d) (1) 2 N NaOH, MeOH, reﬂux, and 1 h;

(2) 2 N HCl, rt., and 95%. (e) NH₂CH₂CH₂OH, EDCI, DMAP, DMF, rt., overnight, and 83%.

a

Scheme 3. Synthetic Routes of Compounds 12e and 12f

a

Reagents and conditions: (a) NaH, DMF, 0 °C to rt., 2 h, 36% for 17a, and 63% for 17b. (b) HCl, AcOH, H₂O, 120 °C, overnight, 78% for 18a,

and 83% for 18b. (c) POCl₃, 120 °C, 12 h, 83% for 19a, and 80% for 19b. (d) 6, Pd(OAc)₂, XantPhos, Cs₂CO₃, 1,4-dioxnae, 100 °C, overnight,

64% for 20a, and 60% for 20b. (e) (i) 2 N NaOH, MeOH, reﬂux, and 1 h; (ii) 2 N HCl, rt., 92% for 21a, and 90% for 21b. (f) NH₂CH₂CH₂OH,

EDCI, DMAP, DMF, rt., overnight, 79% for 12e, and 59% for 12f.

NaH followed by oxidation of the intermediate and then

coupling with compound 6. Compounds 24b and 24c were

synthesized via reaction of compounds 9a and 9b with

compound 8c, respectively, following similar processes to the

synthesis of compound 24a. Hydrolysis of esters 24a−c

followed by condensation with aminoethanol provided

compounds 25a−c in yields of 63−77% over two steps.

Treatment of 25a−c with NaBH₄as the reducing agent

aﬀorded compounds 25d−f accordingly. Ester intermediates

24a−c were converted into intermediates 26a−c by a

reduction reaction and then a hydrolysis reaction. Fluoridation

of acid intermediates 26a−c by using diethylaminosulfur

triﬂuoride followed by hydrolysis reactions led to acid

compounds 27a−c. Compounds 25g−i were produced

following the same procedure as that of preparing 12a from

compounds 27a−c.

As depicted in Scheme 6, compounds 29a−c were produced

via coupling reaction of 8c with corresponding commercially

available compounds 28a−c in yields of 63−71%. Sub-

sequently, intermediates 29a−c were converted to correspond-

D

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a

Scheme 4. Synthetic Routes of Compounds 23a−k

a

Reagents and conditions: (a) (1) NaH, DMF, 0 °C to rt., and 2 h; (2) Con. HCl/AcOH/H₂O, reﬂux, overnight, and 54−90% for two steps. (b)

NH₂CH₂CH₂OH, EDCI, DMAP, DMF, rt., overnight, and 29−83%.

ing ﬁnal compounds 25j−l by acidolysis and coupling with

aminoethanol, following the similar procedures to that of

preparing 25a. Compounds 30a−g were prepared by coupling

11c with corresponding aminoethanol derivatives following the

similar procedure to that of preparing 12a, as outlined in

Scheme 7.

PK proﬁle. In the further SAR studies, the pyridine ring of

compound 4 was replaced with a pyridazine ring to obtain

compound 12b (EC₅₀= 158 nM and E_max= 158%), which

exhibited comparable potency to that of 4, with improved

eﬃcacy. Next, modiﬁcation of the pyridine ring of compound 4

to a pyrimidine ring with the two N atoms ﬁxed at positions 4

and 6 led to compound 12c (Table 1 and Figure 3A).

Compound 12c showed equal potency as that of lead 4 (135

nM vs 119 nM) but interestingly resulted in a superior eﬃcacy

(E_maxof 136%), which was the best compound among this

series. However, when the two N atoms were moved to

positions 2 and 6 to obtain 12d, the potency decreased slightly

(EC₅₀= 186 nM) compared to 12c. To explore the eﬀect of

adding a substituent on potency, we introduced a methyl or a

chlorine at the 2-position on compound 12b, leading to

compounds 12e and 12f, respectively. However, both

compounds showed substantial loss of potency (>4-fold

decreased EC₅₀), indicating that adding a substituent on core

moiety B was not favorable.

Having identiﬁed an optimal heterocycle in moiety B that

maintains potency while increasing eﬃcacy, we next focused on

probing the role of the substituents on the benzene ring of

moiety C (Figure 2, highlight in cyan), with results

summarized in Table 2. To evaluate the importance of the F

substituent, we removed the F from compound 12c, leading to

compound 23a, resulting in a slight increase in potency (EC₅₀

of 101 nM) and minor decrease in eﬃcacy relative to

compound 12c (127% vs 136%) (Table 2). Compounds 23b

and 23c were then designed and synthesized to explore

whether the CF₃position had an inﬂuence on agonist potency.

CF₃at the ortho-position (23b) resulted in a dramatic loss of

potency (>7-fold loss) compared with CF₃at the meta-position

(23a). Remarkably, when the CF₃moiety was moved to the

para-position (23c), a similar potency and eﬃcacy was

observed to that of 23a (109 nM vs 101 nM for EC₅₀and

136% vs 127% for E_max(Table 2 and Figure 3B)). From these

results, we can conclude that the substituent position on the

benzene ring of moiety C has an important role in compound

potency and their preferred order is the meta > para > ortho

position. Further, we probed the electronic properties of

In Vitro Evaluation of GPR52 Activation. Newly

synthesized compounds were evaluated in a twelve-point

concentration response for GPR52 agonist activity using the

Glosensor cAMP assay in HEK293 cells transiently expressing

human GPR52. Transient expression of GPR52 resulted in a

dramatic increase in basal cAMP levels (>100-fold over an

empty vector, Supporting Information, Figure S1), indicating

that GPR52 displays high constitutive activity for the cAMP

pathway. As summarized in Tables 1−4, the EC₅₀(nM)

indicates the potency of these novel GPR52 agonists, while the

eﬃcacy (E_max) indicates the maximal activity to increase

cAMP. Previously reported GPR52 agonist compound 4 was

used as the reference compound to highlight SAR compar-

isons.¹⁶E_max(%) was evaluated with compound 4 as 100% and

0.5% DMSO (vehicle control) as 0%. Compound 4 exhibited

very good potency and robust eﬃcacy in the cAMP assay

(EC₅₀of 119 nM and E_maxof ∼3-fold over basal values,

Supporting Information, Figure S1). All synthesized ligands

were screened in the assay in a full concentration response (0.1

nM to 30 μM). Some data points, at higher concentrations,

were excluded from pharmacological analysis due to limited

aqueous solubility (as shown in Supporting Information,

Figure S1).

As summarized in Table 1, to begin a systematic SAR study

of the scaﬀold of compound 4,¹⁶we ﬁrst investigated the core

ring B (Figure 2, highlight in magenta) by insertion of an

additional N atom in the pyridine ring of compound 4.

Replacement of the pyridine ring by a pyrimidine ring with two

N atoms at positions 2 and 4, leading to compound 12a,

resulted in ∼3-fold loss of potency with an EC₅₀value of 373

nM relative to compound 4. However, compared to compound

4, the eﬃcacy of compound 12a increased with an E_maxof

144%. In addition, 12a has a more favorable ClogP relative to

that of 4 (3.64 vs 4.11), suggesting that 12a may have a better

E

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a

Scheme 5. Synthetic Routes of Compounds 25a−i

a

Reagents and conditions: (a) For 24a, (i) 7a, NaH, DMF, 0 °C to rt., and 2 h; (ii) mCPBA, 0 °C to rt., 10 min, and 47% for two steps; (iii) 6,

XantPhos, Cs₂CO₃, Pd(OAc)₂, 1,4-dioxane, 100 °C, overnight, and 82%; for 24b and 24c, (i) 8c, NaH, DMF, 0 °C to rt., and 2 h; (ii) mCPBA, 0

°C to rt., 10 min, 49% over two steps for 24b, and 79% over two steps for 24c. (b) (i) Con. HCl/AcOH/H₂O, reﬂux, and overnight; (ii)

NH₂CH₂CH₂OH, EDCI, DMAP, DMF, rt., overnight, and 63−77% for two steps. (c) NaBH₄, DMF/MeOH, 0 °C to rt., 30 min, and 68−90%. (d)

(i) NaBH₄, DMF/MeOH, 0 °C to rt., and 30 min; (ii) con. HCl/AcOH/H₂O, reﬂux, overnight, and 78−90%. (e) (i) DAST, CH₂Cl₂, 0 °C to rt.,

and 10 min; (ii) THF/H₂O, reﬂux, overnight, and 60−76% for two steps. (f) NH₂CH₂CH₂OH, EDCI, DMAP, DMF, rt., overnight, and 68−86%.

a

Scheme 6. Synthetic Routes of Compounds 25j−l

a

Reagents and conditions: (a) For 29a, Pd(OAc)₂, XantPhos, Cs₂CO₃, 1,4-dioxnae, 100 °C, overnight, and 69%; for 29b and 29c, Cs₂CO₃, DMF,

120 °C, overnight, 63% for 29b, and 71% for 29c. (b) (i) Con. HCl/AcOH/H₂O, reﬂux, and overnight; (ii) NH₂CH₂CH₂OH, EDCI, DMAP,

DMF, rt., overnight, and 43−73% for two steps.

substituents on the benzene ring of moiety C. Replacement of

meta-CF₃of 23a with a methyl group led to compound 23d,

which displayed the best potency among this series with an

EC₅₀of 90 nM while maintaining excellent eﬃcacy with an

E_maxof 144% (Table 2 and Figure 3B). Similarly, compound

23e was obtained by substitution of meta-CF₃of 23a with a F

atom, which displayed no change in potency, an EC₅₀of 106

nM, and maintained excellent eﬃcacy, an E_maxof 148%, (Table

2). Next, we probed whether a second additional electron-

withdrawing moiety in the 5-position would further improve

potency and eﬃcacy. Disubstituted variants of 23d and 23e,

leading to compounds 23f and 23g, respectively, displayed

F

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a

that the addition of an extra electron-withdrawing substituent

at the 5-position of the benzene ring C has limited inﬂuence on

the potency and eﬃcacy. However, substitution of meta-CF₃of

23a with an electron-donating group such as OCF₃(23h),

OMe (23i), and 3,5-di-OMe (23j) dramatically decreased the

potency as well as the eﬃcacy (Table 2 and Figure 3B).

Moreover, compound 23k was synthesized without any

substituent on the benzene ring C, resulting in ∼3-fold loss

of potency relative to 23a, suggesting that an electron-

withdrawing substituent (e.g., 23a, 23c, and 23e) on the

benzene ring of moiety C is favorable for the potency. Taken

together, these ﬁndings suggest that electron-withdrawing or

small alkyl substituents at the 3-position are favorable for

improved potency, with an additional small substituent at the

5-position also well-tolerated, while compounds without any

substituent on the benzene ring or with electron-donating

groups are not tolerated.

Scheme 7. Synthetic Routes of Compounds 30a−g

a

To probe the impact of the ﬂexibility and steric availability

around the linker between moieties B and C, compounds 25a−

l were designed, synthesized, and evaluated, with the results

summarized in Table 3. We ﬁrst replaced the methylene linker

with a carbonyl moiety to yield compounds 25a−c, which were

Reagents and conditions: (a) EDCI, DMAP, DMF, rt., overnight,

and 37−78%.

identical pharmacologic proﬁles as their monosubstituted

counter parts (Table 2 and Figure 3B). These data suggest

Table 1. EC₅₀and E_maxof Compounds 12a−f

a

b

cLogP: http://biosig.unimelb.edu.au/pkcsm/prediction. The values are the mean SEM of at least three independent experiments. E_max(%) is

the eﬃcacy maximum of the compounds in the cAMP assay relative to compound 4 as 100% and 0.5% DMSO as 0%.

G

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either an NH, O, or S led to corresponding compounds 25j−l;

however, none of these three compounds showed favorable

potency and eﬃcacy (Table 3 and Figure 3C). Taken together,

these results suggest that GPR52 ligands of this scaﬀold need

to have a ﬂexible and sterically limited linker to achieve good

potency. These SAR results are consistent with the recently

disclosed cocrystal structure of compound 2 and GPR52,

suggesting that the GPR52 binding region for these molecules

is a narrow sterically limited pocket.¹²

Table 2. EC₅₀and E_maxof Compounds 23a−k

Finally, to detect the impact of an amino alcohol side chain

in moiety A, compounds with various lengths and ﬂexibilities

were designed through diﬀerent strategies, including introduc-

ing extra hydroxyl groups (30a and 30g), modiﬁcation of the

terminal hydroxyl group (30b and 30f), and changing the

length of the alkyl linker between amino and hydroxyl groups

(30c−e) (Figure 3D). Unfortunately, these seven compounds

substantially lost GPR52 potency compared to compound 12c

(Table 4), indicating that moiety A is critical and unlikely

amenable for further optimization.

a

b

compound

R²

3-CF₃

2-CF₃

4-CF₃

ClogP

EC₅₀(nM)

E_max(%)

100

127 10

4

4.11

3.50

2.79

2.62

3.10

2.76

3.38

2.49

2.50

2.48

119 18

101 31

5

23a

23b

23c

23d

23e

23f

23g

23h

23i

23j

23k

711 160

109 19

90 19

115

136

144

6

7

9

3-Me

3-F

106 35

97 20

115 19

131 24

148 14

140 11

142 10

113 11

3,5-di-Me

3,5-di-F

3-OCF₃

3-OMe

3,5-di-OMe

H

Molecular Docking Study of Compound 12c with

GPR52. To understand the potential binding mode and the

receptor−ligand interactions that are important for agonist

351

6

122

113

134

8

2

4

850 35

292 17

̈

activity at GPR52, molecular docking using Schrodinger Drug

a

b

cLogP: http://biosig.unimelb.edu.au/pkcsm/prediction. The values

Discovery Suite was employed. Interestingly, the cocrystal

structure of GPR52 bound to compound 2 was recently solved

(PDB code: 6LI0), aﬀording an unprecedented opportunity to

pursue computational docking studies with an orphan

GPCR.¹²Compound 12c was selected as the example of our

optimized compounds to further explore the binding

interactions with GPR52. As shown in Figure 4, the docking

results indicated that compound 12c has energetically

favorable interactions with the narrow pocket surrounded by

ECL2, TM1, TM2, and TM7 and forms a similar binding pose

to compound 2 with GPR52 in the solved cocrystal structure.¹²

Notably, there are three critical hydrogen bonding interactions

are the mean SEM of at least three independent experiments. E_max

(%) is the eﬃcacy maximum of the compounds in the cAMP assay

relative to compound 4 as 100% and 0.5% DMSO as 0%.

found to dramatically decrease the potency and eﬃcacy, likely

due to limited ﬂexibility. Reduction of the carbonyl moiety of

compounds 25a−c produced compounds 25d−f, and this

restored a certain degree of the potency and eﬃcacy but still at

least 3-fold less potency than compound 23a. Fluorination of

compounds 25d−f yielded compounds 25g−i, which failed to

improve the potency or eﬃcacy (Table 3 and Figure 3C).

Replacement of the methylene linker of compound 23a with

Table 3. EC₅₀and E_maxof Compounds 25a−l

a

b

compound

R²

Y

W

Z

ClogP

EC₅₀(nM)

119 18

E_max(%)

4

4.11

3.28

3.14

3.13

2.99

4.10

3.97

3.66

3.70

4.06

100

96

22

130

5

7

3

4

9

25a

25b

25c

25d

25e

25f

25g

25h

25i

25j

25k

25l

F

H

F

H

F

H

CO

CHOH

CHF

NH

N

CH

N

CH

N

CH

N

1589 166

1693 437

1008 237

399 68

CH

N

CH

N

CH

338 26

560 19

134 28

89

144

85

83

81

4

9

5

2

4

6

329 113

330 38

557 273

1105 24

2404 292

371 47

O

S

99

123

N

a

b

cLogP: http://biosig.unimelb.edu.au/pkcsm/prediction. The values are the mean SEM of at least three independent experiments. E_max(%) is

the eﬃcacy maximum of the compounds in the cAMP assay relative to compound 4 as 100% and 0.5% DMSO as 0%.

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Table 4. EC₅₀and E_maxof Compounds 30a−g

a

b

cLogP: http://biosig.unimelb.edu.au/pkcsm/prediction. The values are the mean SEM of at least three independent experiments. E_max(%) is

the eﬃcacy maximum of the compounds in the cAMP assay relative to compound 4 as 100% and 0.5% DMSO as 0%.

Figure 3. Concentration responses of select GPR52 agonists on cAMP signaling in HEK293 cells. HEK293 cells expressing human GPR52 and the

cAMP Glosensor reporter were subjected to twelve-point concentration response (0.3 nM−30 μM) testing. (A) Modiﬁcations around ring B

(magenta): comparison with compounds 4, 12a, 12b, 12c, and 12e. (B) Modiﬁcations around ring C (cyan): comparison with compounds 4, 23a,

23d, 23f, 23i, 23j. (C) Modiﬁcations around carbon linker (cyan): comparison with compounds 4, 25c, 25e, 25g, 25i, and 25l. (D) Modiﬁcations

around head group A (green): comparison with compounds 4, 30a, 30c, 30d, and 30g. All results are presented as the mean SEM from triplicate

testing in a representative experiment, with similar results observed in 3−17 experiments.

between compound 12c and the residues of the ECL2 and

TM1 of the receptor: the 12c terminal hydroxyl group forms

hydrogen bonds with residues Glu191 and Ile189 of ECL2,

and the 12c carbonyl group of the amide forms a hydrogen

bond with residue Cys40 from TM1. This binding model may

explain the signiﬁcant activity loss when replacing the amino

alcohol side chain into other side chains. The pyrimidine ring

(core ring B) forms a π−π stacking interaction with the residue

Phe300 of TM7. Moreover, the benzene ring (moiety C) of

compound 12c pointed into a narrow hydrophobic pocket and

formed hydrophobic interactions with residues Phe117, Cys94,

Tyr185, Ile47, Ile121, Ile307, and Trp304. This may explain

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Figure 4. Putative binding mode and molecular docking of compound 12c with GPR52 (PDB code: 6LI0). (A) Docking of compound 12c

(magenta) into the binding pocket of GPR52. Important residues are drawn as sticks. Hydrogen bonds are shown as dashed red lines. (B) Docking

of compound 12c into the binding pocket of GPR52 in 2D view. Hydrogen bonds are shown as magenta lines, and π−π interaction is shown as a

green line.

why modiﬁcations with only small substitutions on moiety C

are tolerated, and why the hydrophobic groups (e.g., F and

CF₃) are more favorable for the binding to drive potency. This

docking also indicates that the binding pocket near ECL2 is a

hot spot of interaction for both previously identiﬁed GPR52

agonists as well as 12c and likely other indoline-carboxamide-

based ligands.

Table 5. Broad-Panel Counter Screening of 12c against

Other GPCRs and Transporters

a

%

GPCRs,

%

inhibition

K_i

(μM)

transporters, ion inhibition

K_i

b

GPCRs

(10 μM)

channels

(10 μM) (μM)

5-HT1A

5-HT1B

5-HT1D

5-HT1E

5-HT2A

5-HT2B

5-HT2C

5-HT3

8.68

6.3

29.64

7.71

10.62

28.69

52.62

26.82

13.95

7.98

ND

>10

ND

D1

D2

D3

D4

12.29

2.51

ND

In Vitro Assessment of Oﬀ-Target Eﬀects of Com-

pound 12c. Considering the potential drug-like properties of

chemical scaﬀolds and overall in vitro activities of GPR52

activation, compound 12c was selected as a representative

chemical probe among the identiﬁed active analogs to pursue

further pharmacological evaluations in a proof-of-concept

study. To evaluate if the cAMP signaling activity of optimized

compound 12c is indeed due to GPR52 agonist activation, 12c

was further tested in control studies using HEK293 cells

lacking GPR52. Results determined that 12c did not increase

cAMP in the cells lacking GPR52 (Supporting Information,

Figure S2). Therefore, compound 12c regulates cAMP

increases through a direct interaction with GPR52 in this

cellular system. To determine the selectivity of 12c against

other GPCRs, a broad-panel counter screening was tested by

the National Institute of Mental Health Psychoactive Drug

Screening Program (NIMH-PDSP).²⁶This testing determined

that compound 12c at a concentration of 10 μM displayed no

signiﬁcant binding aﬃnity (K_i) at over 30 brain receptors or

channels, including important GPCRs that are current targets

of antipsychotic medications (e.g., 5-HT_2Aand D₂receptors),

as shown in Table 5. In addition, compound 12c at 10 μM did

not show any human ether-a-go-go-related gene (hERG)

potassium channel binding activity (Table 5), indicating that

compound 12c should not have undesirable hERG inhibition

known to cause cardiotoxicity.²⁷

10.64

29.91

−7.17

24.83

5.98

6.43

22.36

1.48

D5

DAT

GABAA

H1

H2

KOR

M1

M2

M3

M4

M5

5-HT5A

5-HT6

5-HT7A

Alpha1A

Alpha1B

Alpha2A

Beta1

5.16

2.86

13.56

−5.42

4.45

17.2

−14.07

−1.87

45.38

29.26

0.82

16.24

11.04

Beta2

MOR

hERG

a

The broad-panel counter screening of 12c against a panel of GPCRs

and transporters was generously provided by the NIMH Psychoactive

b

Drug Screening Program. The values are the mean percent inhibition

of binding from at least three independent experiments, SEM < 20%.

“ND” means that a K_ibinding aﬃnity was not detected.

rats after a single dose of 20 mg/kg by oral (PO) or 10 mg/kg

by intravenous (IV) administration, and the results are

summarized in Table 6. Compound 12c has excellent plasma

exposure after PO (AUC_0‑inf= 13,749 ng·h/mL) and IV dosing

(AUC_0‑inf= 9030 ng·h/mL), as well as high maximum serum

concentration following PO (C_max= 3407 ng/mL) and IV

administration (C_max= 6726 ng/mL). Additionally, compound

12c displayed good volume of plasma distribution (V_ss= 1.5

L/kg) and acceptable plasma clearance (CL = 1.1 L/h/kg)

In Vivo PK Proﬁle of Compound 12c. Based on its

potency and eﬃcacy as well as potential drug-like properties,

compound 12c was selected as the representative compound

for further PK evaluations. Compound 12c was evaluated in

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a

Table 6. In Vivo Pharmacokinetic Parameters of Compound 12c in Rats

route

AUC_0‑∞(ng·h/mL)

t_1/2(h)

C_max(ng/mL)

CL (L/h/kg)

V_ss(L/kg)

F (%)

76 15

PO

IV

13,749 2710

9030 1018

2.5 0.2

1.0 0.1

3407 179

6726 727

1.5 0.3

1.1 0.1

5.5 1.16

1.5 0.2

a

AUC_0‑∞, area under the curve (t = 0 to 24 h); t_1/2, terminal half-life; C_max, maximum concentration of the drug in plasma; CL, plasma clearance;

V_ss, volume of distribution at steady state; F, absolute oral bioavailability. Experiments were studied in biological triplicates, and data values are

shown as the mean SEM.

after 10 mg/kg IV. Excellent oral bioavailability (F) with the

value of 76% was observed. Compound 12c was evaluated in

rats for brain permeability and concentration after a single dose

of 10 mg/kg by IV administration, and the concentrations of

12c in the brain and plasma were measured at 0.25 and 1 h

after administration. As shown in Table 7, compound 12c

conducted a comprehensive structural optimization campaign

based on previously reported advanced chemical lead 4

through a systematic SAR study by introduction of various

amino alcohol side chains and modiﬁcations of numerous

substituents on the core B ring and C ring. Among them,

several compounds 12c (PW0787), 23a (PW0860), 23d

(PW0878), 23e (PW0885), 23f (PW0888), and 23h

(PW0890) were identiﬁed as novel GPR52 agonists that

elevated cAMP signaling and were more potent than reference

compound 4. Compound 12c has a favorable ClogP and a

good in vivo PK proﬁle. Therefore, compound 12c was selected

for further in vivo eﬃcacy determination. Molecular docking

studies of compound 12c and GPR52 suggest a binding mode

with three critical hydrogen bond pairs, π−π stacking with ring

B, and hydrophobic interactions with ring C. This binding

model oﬀers theoretical studies for further SAR studies of

compound 12c. Compound 12c was also counter screened and

displayed no oﬀ-target aﬃnities at other important brain

GPCRs and ion channels. In addition, compound 12c has

excellent oral plasma exposure, maximum serum concentration,

bioavailability, and brain concentration. In vivo studies showed

that compound 12c signiﬁcantly inhibited amphetamine-

induced hyperactivity in mice. Therefore, newly discovered

GPR52 agonist 12c provides a useful pharmacological tool for

studying GPR52 functions and for evaluating GPR52 agonist

therapeutic potential for the treatment of brain diseases such as

neuropsychiatric disorders.

a

Table 7. Brain Permeability of Compound 12c in Rats

time

(h)

brain conc.

(ng/g)

plasma conc.

(ng/g)

brain/plasma

ratio

route

IV

0.25

1

1807 198

1006 43

6324 271

2589 241

0.28 0.02

0.39 0.02

a

Concentrations of compound 12c in the brain and plasma were

determined at 0.25 and 1 h after a single dose of 10 mg/kg by IV.

Experiments were studied in biological triplicates, and data values are

shown as the mean SEM.

exhibits a good concentration in the brain at 0.25 h with the

value of 1807 ng/g and also exhibits an acceptable brain

permeability with a brain/plasma ratio of 0.28. The high brain

concentration and brain permeability of 12c persisted for at

least 1 h, with a brain concentration over 1000 ng/g and a

brain/plasma ratio of 0.39 after 1 h injection. Based upon these

collective ﬁndings, compound 12c was selected for further in

vivo evaluation.

Eﬃcacy Evaluation of Compound 12c In Vivo.

Compound 12c exhibited potent GPR52 activity in vitro and

a good PK proﬁle to support further in vivo assessment.

Amphetamine-induced hyperlocomotion in rodents is a well-

characterized task with high translational validity for predicting

preclinical antipsychotic-like activity.^28,29Given that GPR52

agonists inhibit hyperactivity induced by psychostimulants,^10,30

we employed this assay to investigate the eﬃcacy of compound

12c in vivo. Mice were injected intraperitoneally (IP) with a

vehicle or compound 12c (0.3, 1, 3, or 10 mg/kg in vehicle;

Figure 5A); automated activity monitoring began immediately

and continued for 30 min. Mice were then injected with

amphetamine (AMPH; 3 mg/kg IP), and activity was

monitored for the next 90 min (Figure 5A). A one-way

ANOVA indicated that compound 12c suppressed horizontal

activity (F4,54 = 3.287, P < 0.05); Dunnett’s a priori

comparisons indicated that 3 mg/kg (P < 0.05) and 10 mg/

kg (P < 0.05) suppressed horizontal activity (Figure 5B).

Additionally, compound 12c suppressed AMPH-induced

horizontal activity (F4,54 = 4.736, P < 0.05; Figure 5C) at

both 3 mg/kg (P < 0.05) and 10 mg/kg doses (P < 0.05).

Taken together, compound 12c is an orally bioavailable and

brain-penetrant GPR52 agonist, which dose-dependently

inhibits amphetamine-evoked hyperactivity, suggesting ther-

apeutic potential for neuropsychiatric diseases.

EXPERIMENTAL SECTION

■

General. All commercially available starting materials and solvents

were reagent grade and used without further puriﬁcation. Reactions

were performed under a nitrogen atmosphere in dry glassware with

magnetic stirring. Preparative column chromatography was performed

using silica gel 60, particle size 0.063−0.200 mm (70−230 mesh,

ﬂash). Analytical TLC was carried out by employing silica gel 60 F254

plates (Merck, Darmstadt). Visualization of the developed chromato-

grams was performed with detection by UV (254 nm). NMR spectra

were recorded on a Bruker-600 (¹H, 300 MHz; ¹³C, 75 MHz)

1

spectrometer. H and ¹³C NMR spectra were recorded with TMS as

an internal reference. Chemical shifts downﬁeld from TMS were

expressed in ppm, and J values were given in Hz. High-resolution

mass spectra (HRMS) were obtained from a Thermo Fisher LTQ

Orbitrap Elite mass spectrometer. Parameters include the following:

nano ESI spray voltage was 1.8 kV, capillary temperature was 275 °C,

and the resolution was 60,000; ionization was achieved by positive

mode. Purity of ﬁnal compounds was determined by analytical HPLC,

which was carried out on a Shimadzu HPLC system (model: CBM-

20A LC-20AD SPD-20A UV/vis). HPLC analysis conditions were a

Waters μBondapak C18 (300 mm × 3.9 mm), a ﬂow rate 0.5 mL/

min, UV detection at 270 and 254 nm, and linear gradient from 10%

acetonitrile in water (0.1% TFA) to 100% acetonitrile (0.1% TFA) in

20 min followed by 30 min of the last-named solvent. Compounds 4

and 6 were resynthesized in-house following reported synthetic

procedures.^16,22All biologically evaluated compounds are >95% pure.

Methyl 1-(2-Chloropyrimidin-4-yl)indoline-4-carboxylate

(8a). To a solution of 6 (354 mg, 2 mmol) and 2,4-

dichloropyrimidine (7a) (300 mg, 2 mmol) in EtOH (5 mL) was

CONCLUSIONS

To understand the key ligand−receptor interactions and

identify potent and eﬃcacious GPR52 agonists, we have

■

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Figure 5. Compound 12c shows antipsychotic-like activity in mice. (A) Time course of horizontal activity (counts/5 min; mean SEM) following

injection of the vehicle (black circles) or compound 12c (0.3 (blue squares), 1 (burgundy triangles), 3 (green inverted triangles), or 10 mg/kg IP

(red diamonds) is shown during automated activity monitoring for 30 min (blue dotted box)). Mice were then injected with amphetamine

(AMPH; 3 mg/kg IP), and activity was monitored for the next 90 min. Horizontal activity (counts/5 min; mean SEM) during the ﬁrst 30 min

following AMPH injection is illustrated (purple dotted box). (B) Bar graph showing horizontal activity/30 min (mean SEM) following injection

of the vehicle (Veh) or compound 12c. (C) Bar graph showing horizontal activity (mean SEM) in the ﬁrst 30 min period after AMPH injection.

Filled circles indicate horizontal activity for individual animals. n = 12 mice/dose except for the 1 mg/kg AMPH (n = 11 mice/dose). *P < 0.05 vs

Veh.

added DIPEA (387 mg, 3 mmol) at rt., and the mixture solution was

stirred at rt. overnight. After the reaction was completed (detected by

TLC), the white solid was ﬁltered and washed with water and cold

EtOH. The cake was collected and dried to aﬀord the product as

solution at 0 °C, and the mixture solution was stirred at 0 °C for 30

min. Then, 8a (480 mg, 1.6 mmol) was added to the solution at 0 °C,

and the mixture solution was stirred at rt. for 2 h. After the reaction

was completed (detected by TLC), the reaction was quenched with

NH₄Cl_{(sat. aq.)}. The solution was worked up by the addition of water

and then extracted with EtOAc (20 mL × 3). The combined EtOAc

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and

condensed by rotary evaporation to yield a yellow oil. The residue was

puriﬁed by silica gel chromatography (gradient: 1 to 5% MeOH in

CH₂Cl₂) and provided product 10a (300 mg, 66%) as a yellow solid.

¹H NMR (300 MHz, chloroform-d) δ 8.67 (d, J = 8.3 Hz, 1H), 8.40

1

white solid 8a (387 mg, 67%). H NMR (300 MHz, DMSO-d₆) δ

8.53 (d, J = 8.1 Hz, 1H), 8.34 (d, J = 6.0 Hz, 1H), 7.57 (d, J = 7.8 Hz,

1H), 7.40 (t, J = 8.0 Hz, 1H), 6.87 (d, J = 6.1 Hz, 1H), 4.08 (t, J = 8.6

Hz, 2H), 3.85 (s, 3H), 3.50 (s, 2H).

Methyl 1-(5-Chloropyridazin-3-yl)indoline-4-carboxylate

(8b). Compound 8b (364 mg, 63%) was synthesized by a procedure

similar to that used to prepare compound 8a as a light yellow solid.

¹H NMR (300 MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.68 (d, J = 8.2 Hz,

(d, J = 6.1 Hz, 1H), 7.71 (dd, J = 8.0, 1.0 Hz, 2H), 7.54 (dt, J = 8.8,

2.0 Hz, 1H), 7.36 (t, J = 8.0 Hz, 2H), 6.53 (d, J = 6.1 Hz, 1H), 5.40

(s, 1H), 4.06 (dd, J = 9.3, 7.8 Hz, 2H), 3.94 (s, 3H), 3.66 (t, J = 8.5

Hz, 2H).

1H), 7.55−7.47 (m, 2H), 7.37 (s, 1H), 4.11 (t, J = 8.7 Hz, 2H), 3.86

(s, 3H), 3.53 (t, J = 8.7 Hz, 2H).

Methyl 1-(6-Chloropyrimidin-4-yl)indoline-4-carboxylate

(8c). Compound 8c (370 mg, 64%) was synthesized by a procedure

Methyl 1-(5-(Cyano(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)-

methyl)pyridazin-3-yl)indoline-4-carboxylate (10b). Com-

pound 10b (120 mg, 53%) was synthesized by a procedure similar

1

similar to that used to prepare compound 8a as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 8.69 (dd, J = 8.2, 1.1 Hz, 1H), 8.61

(d, J = 0.8 Hz, 1H), 7.56 (dd, J = 7.9, 1.0 Hz, 1H), 7.37 (t, J = 8.0 Hz,

1H), 6.97 (d, J = 0.9 Hz, 1H), 4.07 (t, J = 8.5 Hz, 2H), 3.85 (s, 3H),

3.50 (t, J = 8.5 Hz, 2H).

Methyl 1-(2-(Cyano(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)-

methyl)pyrimidin-4-yl)indoline-4-carboxylate (10a). 2-(3-Fluo-

ro-5-(triﬂuoromethyl)phenyl)acetonitrile (9a) (336 mg, 1.6 mmol)

was dissolved in 5 mL of DMF, and the mixture solution was cooled

to 0 °C with an ice bath. NaH (141 mg, 3.5 mmol) was added to the

1

to that used to prepare compound 10a as a yellow solid. H NMR

(300 MHz, chloroform-d) δ 8.74−8.62 (m, 2H), 7.66 (dd, J = 7.9, 1.0

Hz, 1H), 7.48 (s, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.33 (ddd, J = 8.1,

4.9, 3.0 Hz, 2H), 7.01 (dd, J = 1.9, 0.8 Hz, 1H), 5.25 (s, 1H), 4.14 (t,

J = 8.6 Hz, 2H), 3.94 (s, 3H), 3.69 (t, J = 8.5 Hz, 2H).

Methyl 1-(6-(Cyano(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)-

methyl)pyrimidin-4-yl)indoline-4-carboxylate (10c). Com-

pound 10c (130 mg, 57%) was synthesized by a procedure similar

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to that used to prepare compound 10a as a yellow solid. H NMR

(300 MHz, chloroform-d) δ 8.83−8.70 (m, 2H), 7.71 (dd, J = 7.9, 1.0

Hz, 1H), 7.60 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.36 (t, J = 8.1 Hz,

2H), 6.80 (s, 1H), 5.22 (s, 1H), 4.19−4.07 (m, 2H), 3.95 (s, 3H),

3.69 (t, J = 8.5 Hz, 2H).

chloroform-d) δ 8.70 (s, 1H), 8.57 (d, J = 8.1 Hz, 1H), 7.37 (s, 1H),

7.30−7.16 (m, 3H), 7.10 (d, J = 7.7 Hz, 1H), 6.71 (t, J = 5.6 Hz, 1H),

6.33 (s, 1H), 4.04 (s, 2H), 3.91 (t, J = 8.6 Hz, 2H), 3.83 (t, J = 4.9 Hz,

2H), 3.60 (d, J = 5.1 Hz, 2H), 3.49 (d, J = 8.4 Hz, 3H). ¹³C NMR (75

MHz, DMSO) δ 167.5, 166.7, 163.9, 160.7, 159.5, 157.9, 144.3,

143.8, 143.7, 132.6, 132.5, 131.2 (qd, J = 32.1, 8.3 Hz), 127.6, 122.5

(p, J = 3.6 Hz), 123.8 (qd, J = 270.8, 3.0 Hz), 121.1, 120.9, 120.6,

117.9, 111.4 (q, J = 4.0 Hz), 111.1 (q, J = 3.7 Hz), 105.0, 60.2, 48.9,

42.7, 42.4, 27.7. HRMS (ESI): (M + H)⁺calcd for C₂₃H₂₁F₄N₄O₂,

461.1595; found, 461.1591.

1-(2-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-

indoline-4-carboxylic acid (11a). To a solution of 10a (300 mg,

0.7 mmol) in con. HCl (4 mL), H₂O (1 mL) and AcOH (1 mL) were

added, and the mixture solution was stirred at reﬂux overnight. The

reaction was cooled to room temperature, and a yellow solid

precipitated from the solution. The yellow solid was ﬁltered and

washed with water, and the cake was collected and dried to aﬀord the

2-(2-Chloropyrimidin-4-yl)-2-(3-ﬂuoro-5-(triﬂuoromethyl)-

phenyl)acetonitrile (13). 2-(3-Fluoro-5-(triﬂuoromethyl)phenyl)-

acetonitrile (9a) (406 mg, 2 mmol) was dissolved in 5 mL of DMF,

and the mixture solution was cooled to 0 °C with an ice bath. NaH

(190 mg, 4.4 mmol) was added to the solution at 0 °C, and the

mixture solution was stirred at 0 °C for 30 min. Then, 2,4-

dichloropyrimidine (7a) (450 mg, 3 mmol) was added to the solution

at 0 °C, and the mixture solution was stirred at rt. for 2 h. After the

reaction was completed (detected by TLC), the reaction mixture was

worked up by the addition of water and then extracted with EtOAc

(20 mL × 3). The combined EtOAc extracts were washed with brine,

dried over Na₂SO₄, ﬁltered, and condensed by rotary evaporation to

yield a yellow oil. The residue was puriﬁed by silica gel

chromatography (gradient: 10 to 20% EtOAc in hexane) to provide

1

product as light yellow solid 11a (256 mg, 93%). H NMR (300

MHz, DMSO-d₆) δ 13.12 (s, 1H), 8.62−8.51 (m, 1H), 7.89 (d, J =

8.3 Hz, 1H), 7.83−7.55 (m, 4H), 7.10−6.93 (m, 2H), 4.51 (s, 2H),

4.16 (t, J = 8.4 Hz, 2H), 3.52 (t, J = 8.3 Hz, 4H).

1-(5-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyridazin-3-yl)-

indoline-4-carboxylic acid (11b). Compound 11b (101 mg, 92%)

was synthesized by a procedure similar to that used to prepare

1

compound 11a as a white solid. H NMR (300 MHz, DMSO-d₆) δ

8.99 (d, J = 1.4 Hz, 1H), 8.48 (d, J = 8.1 Hz, 1H), 7.74−7.69 (m,

2H), 7.69−7.63 (m, 1H), 7.57 (dd, J = 10.7, 7.6 Hz, 2H), 7.35 (d, J =

8.0 Hz, 1H), 4.23 (s, 2H), 4.17 (t, J = 8.5 Hz, 2H), 3.54 (s, 2H).

1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-

indoline-4-carboxylic Acid (11c). Compound 11c (100 mg, 91%)

was synthesized by a procedure similar to that used to prepare

1

product 13 (460 mg, 73%) as a yellow oil. H NMR (300 MHz,

chloroform-d) δ 8.76 (d, J = 5.0 Hz, 1H), 7.57 (s, 1H), 7.52 (d, J =

5.0 Hz, 1H), 7.43 (ddd, J = 14.3, 7.2, 1.8 Hz, 2H), 5.30 (s, 1H).

2-Chloro-4-(3-ﬂuoro-5-(triﬂuoromethyl)benzyl)pyrimidine

(14). To a solution of 13 (460 mg, 1.5 mmol) in con. HCl (4 mL),

H₂O (1 mL) and AcOH (1 mL) were added, and the mixture solution

was stirred at reﬂux overnight. The reaction was cooled to room

temperature, and a yellow solid precipitated from the solution. The

yellow solid was ﬁltered and washed with water, and the cake was

collected and dried to aﬀord the product as a light yellow solid. The

yellow solid was dissolved in 2 mL of POCl₃, and the mixture solution

was stirred at reﬂux overnight. The reaction was cooled to room

temperature. After the reaction was completed (detected by TLC),

the reaction was poured into ice water, and the pH of the mixture

solution was adjusted to pH = 8 with Na₂CO_{3(sat. aq.)}solution. The

solution was extracted with EtOAc (20 mL × 3). The organic phase

was washed with brine, dried over Na₂SO₄, and then concentrated

under reduced pressure. The residue was puriﬁed by silica gel

chromatography (gradient: 10 to 20% EtOAc in hexane) to provide

product 14 as a brown colorless oil (222 mg, 51% for two steps). ¹H

NMR (300 MHz, chloroform-d) δ 8.57 (d, J = 5.0 Hz, 1H), 7.36 (s,

1H), 7.30−7.26 (m, 1H), 7.22 (dd, J = 8.8, 1.9 Hz, 1H), 7.09 (d, J =

5.0 Hz, 1H), 4.17 (s, 2H).

Methyl 1-(4-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-

2-yl)indoline-4-carboxylate (15). A mixture of 14 (145 mg, 0.5

mmol), Pd(OAc)₂(6 mg, 0.025 mmol), XantPhos (29 mg, 0.05

mmol), Cs₂CO₃(325 mg, 1 mmol), and 6 (89 mg, 0.5 mmol) in 1,4-

dioxane (3 mL) was subjected to three rounds of vacuum evacuation

followed by introduction of nitrogen. The reaction mixture was then

stirred at 100 °C overnight. The reaction was cooled to room

temperature and poured in water and then extracted with EtOAc (10

mL × 3). The organic phase was washed with brine, dried over

Na₂SO₄, and then concentrated under reduced pressure. The residue

was puriﬁed by silica gel chromatography (gradient: 10 to 20% EtOAc

in hexane) to provide product 15 (120 mg, 56%) as a yellow solid. ¹H

NMR (300 MHz, chloroform-d) δ 8.52−8.42 (m, 2H), 7.60 (dd, J =

7.8, 1.1 Hz, 1H), 7.45 (s, 1H), 7.26 (dd, J = 8.4, 3.5 Hz, 3H), 6.62 (d,

J = 5.0 Hz, 1H), 4.28 (dd, J = 9.3, 8.2 Hz, 2H), 4.10 (s, 2H), 3.93 (s,

3H), 3.60−3.53 (m, 2H).

1

compound 11a as a white solid. H NMR (300 MHz, DMSO-d₆) δ

8.92 (s, 1H), 8.71 (d, J = 8.2 Hz, 1H), 7.73 (s, 1H), 7.71−7.65 (m,

2H), 7.62 (dd, J = 8.8, 1.9 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.21 (s,

1H), 4.28 (s, 2H), 4.21 (t, J = 8.3 Hz, 2H), 3.59 (t, J = 8.3 Hz, 2H).

1-(2-(3-Fuoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-N-

(2-hydroxyethyl)indoline-4-carboxamide (12a). Compound 11a

(42 mg, 0.1 mmol) and 2-aminoethan-1-ol (13 mg, 0.2 mmol) were

dissolved in 2 mL of DMF, and the mixture solution was cooled to 0

°C with an ice bath. HOBt (14 mg, 0.1 mmol), EDCI (39 mg, 0.2

mmol), and DMAP (24 mg, 0.2 mmol) were added to the solution at

0 °C. Then, the ice bath was removed, and the mixture solution was

stirred at room temperature overnight. After the reaction was

completed (detected by TLC), the reaction was worked up by the

addition of water and then extracted with EtOAc (20 mL × 3). The

combined EtOAc extracts were washed with brine, dried over

Na₂SO₄, ﬁltered, and condensed by rotary evaporation to yield a

yellow oil. This material was further puriﬁed by preparative TLC

plates using CH₂Cl₂/MeOH = 50:1 as the eluent to yield 12a as a

1

white solid (33 mg, 73%). H NMR (300 MHz, chloroform-d and

MeOD) δ 8.26 (dd, J = 6.3, 2.6 Hz, 1H), 8.14 (dd, J = 7.9, 2.4 Hz,

1H), 7.45 (s, 1H), 7.29−7.04 (m, 5H), 6.39 (dd, J = 6.5, 2.5 Hz, 1H),

4.20 (s, 2H), 3.93 (t, J = 8.6 Hz, 2H), 3.71 (t, J = 5.0 Hz, 2H), 3.48

(qd, J = 8.7, 7.3, 2.6 Hz, 4H). ¹³C NMR (75 MHz, chloroform-d and

MeOD) δ 168.9, 167.3, 159.0, 155.9, 144.1, 142.0, 132.4, 130.9,

127.5, 122.4−122.1 (m), 120.3, 120.0, 110.8 (d, J = 24.5 Hz), 103.1,

61.2, 49.2, 45.0, 42.3, 27.5. HRMS (ESI): (M + H)⁺calcd for

C₂₃H₂₁F₄N₄O₂, 461.1595; found, 461.1591.

1-(5-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyridazin-3-yl)-N-

(2-hydroxyethyl)indoline-4-carboxamide (12b). Compound

12b (35 mg, 76%) was synthesized by a procedure similar to that

used to prepare compound 12a as a white solid. ¹H NMR (300 MHz,

methanol-d₄) δ 8.58 (s, 1H), 8.45 (dd, J = 7.5, 1.7 Hz, 1H), 7.53 (s,

1H), 7.39 (tt, J = 6.3, 2.0 Hz, 2H), 7.29−7.17 (m, 2H), 7.11 (d, J =

1.6 Hz, 1H), 4.14 (s, 2H), 4.03 (t, J = 8.6 Hz, 2H), 3.74 (t, J = 5.8 Hz,

2H), 3.51 (t, J = 5.8 Hz, 2H), 3.45 (t, J = 8.6 Hz, 2H). ¹³C NMR (75

MHz, MeOD) δ 169.6, 164.4, 161.1, 144.6, 142.7 (d, J = 7.7 Hz),

131.7, 131.6, 127.1, 121.6−121.4 (m), 119.7, 119.4, 116.7, 114.1,

111.1 (d, J = 4.1 Hz), 111.0, 110.7 (d, J = 4.0 Hz), 60.23, 48.81,

41.92, 37.30, 27.06. HRMS (ESI): (M + H)⁺calcd for C₂₃H₂₁F₄N₄O₂,

461.1595; found, 461.1591.

1-(4-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-2-yl)-

indoline-4-carboxylic Acid (16). To a solution of 14 (120 mg, 0.28

mmol) in MeOH (4 mL) was added a 2 N solution of NaOH (1 mL),

and the mixture solution was stirred at reﬂux for 1 h. The pH of the

mixture solution was adjusted to pH = 1 with 2 N HCl solution, and

then, a yellow solid precipitated from the solution. The yellow solid

was ﬁltered and washed with water, and the cake was collected and

1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-N-

(2-hydroxyethyl)indoline-4-carboxamide (12c). Compound 12c

(38 mg, 83%) was synthesized by a procedure similar to that used to

prepare compound 12a as a white solid. ¹H NMR (300 MHz,

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dried to aﬀord the product as light yellow solid 16 (110 mg, 95%). ¹H

NMR (300 MHz, DMSO-d₆) δ 8.51 (d, J = 5.0 Hz, 1H), 8.23 (d, J =

8.1 Hz, 1H), 7.67 (s, 1H), 7.59 (dt, J = 9.5, 2.0 Hz, 2H), 7.44 (dd, J =

7.8, 1.1 Hz, 1H), 7.13 (t, J = 8.0 Hz, 1H), 6.91 (d, J = 5.0 Hz, 1H),

4.23 (s, 2H), 4.14 (t, J = 8.7 Hz, 2H), 3.43 (t, J = 8.6 Hz, 2H).

1-(4-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-2-yl)-N-

(2-hydroxyethyl)indoline-4-carboxamide (12d). Compound

12d (92 mg, 83%) was synthesized by a procedure similar to that

used to prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d) δ 8.37 (d, J = 5.0 Hz, 1H), 8.30 (dd, J = 7.4, 1.5 Hz,

1H), 7.43 (s, 1H), 7.24 (dd, J = 8.9, 1.4 Hz, 2H), 7.16−7.05 (m, 2H),

6.77 (t, J = 5.6 Hz, 1H), 6.57 (d, J = 5.0 Hz, 1H), 4.17 (t, J = 8.7 Hz,

2H), 4.05 (s, 2H), 3.81 (t, J = 4.9 Hz, 2H), 3.58 (d, J = 5.1 Hz, 2H),

3.40 (q, J = 9.6, 8.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 169.2,

167.6, 164.1, 160.8, 159.0, 157.9, 144.6, 141.7 (d, J = 7.7 Hz), 132.6,

130.8, 127.5, 122.1 (dd, J = 7.1, 3.5 Hz), 120.1, 119.8, 119.2, 117.6,

111.3 (q, J = 3.9 Hz), 111.0 (q, J = 3.8 Hz), 110.6, 62.1, 49.0, 43.5,

42.6, 27.3. HRMS (ESI): (M + H)⁺calcd for C₂₃H₂₁F₄N₄O₂,

461.1595; found, 461.1591.

2-(3-Fluoro-5-(triﬂuoromethyl)phenyl)-2-(5-methyl-6-oxo-

1-(tetrahydro-2H-pyran-2-yl)-1,6-dihydropyridazin-4-yl)-

acetonitrile (17a). Compound 17a (150 mg, 36%) was synthesized

by a procedure similar to that used to prepare compound 10a as a

yellow oil. ¹H NMR (300 MHz, chloroform-d) δ 7.75 (d, J = 8.8 Hz,

1H), 7.46−7.37 (m, 2H), 7.27 (dt, J = 8.5, 2.3 Hz, 1H), 6.06 (dt, J =

10.6, 1.9 Hz, 1H), 5.30 (d, J = 6.2 Hz, 1H), 4.17−4.09 (m, 1H), 3.75

(ddd, J = 11.7, 9.0, 2.7 Hz, 1H), 2.18 (dd, J = 10.7, 4.2 Hz, 1H),

1.85−1.58 (m, 4H).

2-(5-Chloro-6-oxo-1-(tetrahydro-2H-pyran-2-yl)-1,6-dihy-

dropyridazin-4-yl)-2-(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)-

acetonitrile (17b). Compound 17b (520 mg, 63%) was synthesized

by a procedure similar to that used to prepare compound 10a as a

yellow oil. ¹H NMR (300 MHz, chloroform-d) δ 7.90 (d, J = 6.1 Hz,

1H), 7.51 (s, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.39−7.33 (m, 1H), 6.07

(dt, J = 10.6, 2.7 Hz, 1H), 5.59 (d, J = 6.9 Hz, 1H), 4.13 (td, J = 6.9,

3.2 Hz, 1H), 3.77 (ddd, J = 11.5, 8.7, 3.0 Hz, 1H), 2.24−2.06 (m,

2H), 1.87−1.62 (m, 4H).

5-(3-Fluoro-5-(triﬂuoromethyl)benzyl)-4-methylpyridazin-

3(2H)-one (18a). To a solution of 17a (830 mg, 2 mmol) in con.

HCl (8 mL), H₂O (2 mL) and AcOH (2 mL) were added, and the

mixture solution was stirred at reﬂux overnight. After the reaction was

completed (detected by TLC), the reaction was poured into ice water.

The pH of the mixture solution was adjusted to pH = 8 with

Na₂CO_{3(sat. aq.)}solution. Then, the solution was extracted with EtOAc

(20 mL × 3). The organic phase was washed with brine, dried over

Na₂SO₄, and then concentrated under reduced pressure. The residue

puriﬁcation by silica gel chromatography (gradient: 30 to 50% EtOAc

in hexane) provided product 18a (480 mg, 78%) as a white solid. ¹H

NMR (300 MHz, chloroform-d) δ 11.67 (s, 1H), 7.58 (s, 1H), 7.25

(d, J = 7.0 Hz, 2H), 7.07−6.99 (m, 1H), 3.97 (s, 2H), 2.23 (s, 3H).

4-Chloro-5-(3-ﬂuoro-5-(triﬂuoromethyl)benzyl)pyridazin-

3(2H)-one (18b). Compound 18b (410 mg, 83%) was synthesized

by a procedure similar to that used to prepare compound 18a as a

yellow oil. ¹H NMR (300 MHz, chloroform-d) δ 7.65 (s, 1H), 7.38−

7.23 (m, 3H), 7.17−7.10 (m, 1H), 4.12 (s, 2H).

brown solid. H NMR (300 MHz, chloroform-d) δ 8.87 (s, 1H),

7.35−7.29 (m, 2H), 7.16−7.06 (m, 1H), 4.23 (s, 2H).

Methyl 1-(5-(3-Fluoro-5-(triﬂuoromethyl)benzyl)-4-methyl-

pyridazin-3-yl)indoline-4-carboxylate (20a). Compound 20a

(142 mg, 64%) was synthesized by a procedure similar to that used

1

to prepare compound 15 as a brown solid. H NMR (300 MHz,

chloroform-d) δ 8.76 (s, 1H), 7.50 (dd, J = 7.9, 0.9 Hz, 1H), 7.27 (d, J

= 7.9 Hz, 4H), 7.10 (q, J = 9.6, 8.7 Hz, 2H), 6.46 (d, J = 7.8 Hz, 1H),

4.27 (t, J = 8.2 Hz, 2H), 4.13 (s, 2H), 3.94 (s, 3H), 3.57 (t, J = 8.2 Hz,

2H), 2.18 (s, 3H).

Methyl 1-(4-Chloro-5-(3-ﬂuoro-5-(triﬂuoromethyl)benzyl)-

pyridazin-3-yl)indoline-4-carboxylate (20b). Compound 20b

(280 mg, 60%) was synthesized by a procedure similar to that used

to prepare compound 15 as a white solid. ¹H NMR (300 MHz,

chloroform-d) δ 8.71 (s, 1H), 7.59 (dd, J = 7.9, 1.0 Hz, 1H), 7.37−

7.29 (m, 2H), 7.25−7.12 (m, 2H), 7.00 (dd, J = 8.0, 0.9 Hz, 1H), 4.34

(t, J = 8.3 Hz, 2H), 4.23 (s, 2H), 3.94 (s, 3H), 3.60 (t, J = 8.3 Hz,

2H).

1-(5-(3-Fluoro-5-(triﬂuoromethyl)benzyl)-4-methylpyrida-

zin-3-yl)indoline-4-carboxylic Acid (21a). Compound 21a (126

mg, 92%) was synthesized by a procedure similar to that used to

prepare compound 16 as a yellow solid. ¹H NMR (300 MHz, DMSO-

d₆) δ 8.91 (d, J = 1.8 Hz, 1H), 7.66−7.47 (m, 3H), 7.38 (d, J = 7.9

Hz, 1H), 7.14 (t, J = 7.9 Hz, 1H), 6.64 (d, J = 7.9 Hz, 1H), 4.29 (s,

2H), 4.12 (t, J = 8.3 Hz, 2H), 3.45 (t, J = 8.0 Hz, 2H), 2.20 (d, J = 1.9

Hz, 3H).

1-(4-Chloro-5-(3-ﬂuoro-5-(triﬂuoromethyl)benzyl)-

pyridazin-3-yl)indoline-4-carboxylic Acid (21b). Compound 21a

(243 mg, 90%) was synthesized by a procedure similar to that used to

prepare compound 16 as a yellow solid. ¹H NMR (300 MHz, DMSO-

d₆) δ 9.00 (s, 1H), 7.65−7.51 (m, 3H), 7.44 (dd, J = 7.8, 1.0 Hz, 1H),

7.18 (t, J = 7.9 Hz, 1H), 6.95 (dd, J = 8.0, 1.0 Hz, 1H), 4.34 (s, 2H),

4.21 (t, J = 8.3 Hz, 4H), 3.46 (t, J = 8.2 Hz, 2H).

1-(5-(3-Fluoro-5-(triﬂuoromethyl)benzyl)-4-methylpyrida-

zin-3-yl)-N-(2-hydroxyethyl)indoline-4-carboxamide (12e).

Compound 12e (107 mg, 79%) was synthesized by a procedure

1

similar to that used to prepare compound 12a as a white solid. H

NMR (300 MHz, chloroform-d) δ 8.71 (s, 1H), 7.29 (d, J = 9.7 Hz,

1H), 7.24 (s, 1H), 7.07 (d, J = 9.0 Hz, 1H), 7.02−6.84 (m, 3H), 6.29

(dd, J = 5.4, 3.4 Hz, 1H), 4.25−4.08 (m, 4H), 3.86 (t, J = 4.8 Hz,

2H), 3.63 (q, J = 5.1 Hz, 2H), 3.43 (t, J = 8.1 Hz, 3H), 2.15 (s, 3H).

¹³C NMR (75 MHz, CDCl₃) δ 168.9, 158.9, 149.1, 147.8, 140.9 (d, J

= 7.3 Hz), 138.7, 132.0, 131.8, 131.2, 126.8, 121.0 (t, J = 3.3 Hz),

119.3, 119. 0, 118.4, 111.9, 62.1, 53.6, 42.8, 36.0, 28.8, 14.5. HRMS

(ESI): (M + H)⁺calcd for C₂₄H₂₃F₄N₄O₂, 475.1752; found,

475.1748.

1-(4-Chloro-5-(3-ﬂuoro-5-(triﬂuoromethyl)benzyl)-

pyridazin-3-yl)-N-(2-hydroxyethyl)indoline-4-carboxamide

(12f). Compound 12f (29 mg, 59%) was synthesized by a procedure

1

similar to that used to prepare compound 12a as a white solid. H

NMR (300 MHz, chloroform-d) δ 8.66 (s, 1H), 7.36−7.24 (m, 2H),

7.14 (d, J = 9.0 Hz, 1H), 7.11−7.02 (m, 2H), 6.89 (dd, J = 6.4, 3.6

Hz, 2H), 4.34−4.15 (m, 4H), 3.81 (t, J = 4.9 Hz, 2H), 3.60 (t, J = 5.1

Hz, 2H), 3.47 (t, J = 8.2 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ

168.8, 164.4, 161.0, 156.8, 148.2, 146.2, 139.9 (d, J = 7.4 Hz), 138.7,

132.1, 131.6, 130.2, 126.8, 121.5−121.4 (m), 119.6, 119.4, 119.3,

115.3, 112.2 (d, J = 3.6 Hz), 111.9 (d, J = 3.9 Hz), 62.1, 53.6, 42.7,

36.1, 29.1. HRMS (ESI): (M + H)⁺calcd for C₂₃H₂₀ClF₄N₄O₂,

495.1205; found, 495.1201.

1-(6-(3-(Triﬂuoromethyl)benzyl)pyrimidin-4-yl)indoline-4-

carboxylic Acid (22a). 2-(3-(Triﬂuoromethyl)phenyl)acetonitrile

(9b) (370 mg, 2 mmol) was dissolved in 5 mL of DMF, and the

mixture solution was cooled to 0 °C with an ice bath. NaH (176 mg,

4.4 mmol) was added to the solution at 0 °C, and the mixture solution

was stirred at 0 °C for 30 min. Then, 8c (580 mg, 2 mmol) was added

to the solution at 0 °C, and the mixture solution was stirred at rt. for 2

h. After the reaction was completed (detected by TLC), the reaction

mixture was worked up by the addition of water and then extracted

with EtOAc (20 mL × 3). The combined EtOAc extracts were

washed with brine, dried over Na₂SO₄, ﬁltered, and condensed by

rotary evaporation to yield a yellow solid. ¹H NMR (300 MHz,

3-Chloro-5-(3-ﬂuoro-5-(triﬂuoromethyl)benzyl)-4-methyl-

pyridazine (19a). A solution of 18a (400 mg, 1.3 mmol) in POCl₃

(3 mL) was heated to reﬂux for 3 h. After the reaction was completed

(detected by TLC), the reaction was cooled to room temperature and

poured in ice water. The pH of the mixture solution was adjusted to

pH = 8 with 2 N NaOH solution and then extracted with EtOAc (30

mL × 3). The organic phase was washed with brine, dried over

Na₂SO₄, and then concentrated under reduced pressure. The residue

puriﬁcation by silica gel chromatography (gradient: 10 to 20% EtOAc

in hexane) provided product 19a (350 mg, 83%) as a brown solid. ¹H

NMR (300 MHz, chloroform-d) δ 8.83 (s, 1H), 7.29 (d, J = 9.7 Hz,

2H), 7.22 (s, 1H), 6.99 (d, J = 8.8 Hz, 1H), 4.12 (s, 2H), 2.39 (s,

3H).

3,4-Dichloro-5-(3-ﬂuoro-5-(triﬂuoromethyl)benzyl)-

pyridazine (19b). Compound 19b (350 mg, 80%) was synthesized

by a procedure similar to that used to prepare compound 19a as a

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DMSO-d₆) δ 8.75 (s, 1H), 8.67 (d, J = 8.0 Hz, 1H), 7.93−7.85 (m,

2H), 7.77 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.54 (dd, J =

7.9, 1.0 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.07 (s, 1H), 6.00 (s, 1H),

4.08 (dd, J = 8.8, 2.0 Hz, 2H), 3.85 (s, 3H), 3.51 (t, J = 8.6 Hz, 2H).

To a solution of the yellow solid (100 mg, 0.23 mmol) in con. HCl

(2 mL), H₂O (0.5 mL) and AcOH (0.5 mL) were added, and the

mixture solution was stirred at reﬂux overnight. The reaction was

cooled to room temperature, and a yellow solid precipitated from the

solution. The yellow solid was ﬁltered and washed with water, and the

cake was collected and dried to aﬀord the product as light yellow solid

1-(6-(3,5-Dimethoxybenzyl)pyrimidin-4-yl)indoline-4-car-

boxylic Acid (22j). Compound 22j (94 mg, 80% for two steps) was

synthesized by a procedure similar to that used to prepare compound

22a as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 13.12 (s, 1H),

8.94 (s, 1H), 8.71 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.43

(t, J = 8.1 Hz, 1H), 7.15 (d, J = 9.7 Hz, 1H), 6.44 (d, J = 19.6 Hz,

2H), 6.24 (d, J = 11.6 Hz, 1H), 4.20 (t, J = 8.4 Hz, 2H), 4.02 (s, 2H),

3.71 (d, J = 14.4 Hz, 6H), 3.58 (t, J = 8.2 Hz, 2H).

1-(6-Benzylpyrimidin-4-yl)indoline-4-carboxylic Acid (22k).

Compound 22k (76 mg, 77% for two steps) was synthesized by a

procedure similar to that used to prepare compound 22a as a white

solid. ¹H NMR (300 MHz, DMSO-d₆) δ 13.19 (s, 1H), 8.96 (s, 1H),

8.71 (d, J = 8.2 Hz, 1H), 7.70 (dd, J = 7.9, 1.0 Hz, 1H), 7.48−7.42

(m, 2H), 7.41−7.27 (m, 3H), 7.17 (s, 1H), 4.25−4.16 (m, 4H), 3.57

(t, J = 8.2 Hz, 2H).

N-(2-Hydroxyethyl)-1-(6-(3-(triﬂuoromethyl)benzyl)-

pyrimidin-4-yl)indoline-4-carboxamide (23a). Compound 23a

(52 mg, 59%) was synthesized by a procedure similar to that used to

prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d and MeOD) δ 8.62 (s, 1H), 8.48 (d, J = 7.6 Hz, 1H),

7.52−7.37 (m, 4H), 7.21−7.09 (m, 2H), 6.32 (s, 1H), 4.01 (s, 2H),

3.87 (t, J = 8.6 Hz, 2H), 3.70 (t, J = 5.2 Hz, 2H), 3.53−3.37 (m, 4H).

¹³C NMR (75 MHz, CDCl₃and MeOD) δ 168.8, 166.7, 159.4, 157.5,

144.2, 138.6, 132.5, 132.4, 131.1 (d, J = 5.2 Hz), 131.0, 129.2, 127.7,

125.9−125.6 (m), 123.7 (d, J = 3.9 Hz), 120.3, 118.6, 103.9, 61.1,

48.7, 43.5, 42.2, 27.4. HRMS (ESI): (M + H)⁺calcd for

C₂₃H₂₂F₃N₄O₂, 443.1689; found, 443.1685.

N-(2-Hydroxyethyl)-1-(6-(2-(triﬂuoromethyl)benzyl)-

pyrimidin-4-yl)indoline-4-carboxamide (23b). Compound 23b

(36 mg, 61%) was synthesized by a procedure similar to that used to

prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d and MeOD) δ 8.65 (s, 1H), 8.48 (d, J = 7.8 Hz, 1H),

7.66 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 8.1 Hz,

2H), 7.21 (ddd, J = 28.3, 13.0, 6.9 Hz, 3H), 6.19 (s, 1H), 4.18 (s,

2H), 3.83 (t, J = 8.5 Hz, 2H), 3.70 (t, J = 5.3 Hz, 2H), 3.53−3.39 (m,

4H). ¹³C NMR (75 MHz, CDCl₃and MeOD) δ 168.9, 166.9, 159.4,

157.3, 144.3, 135.7, 132.4, 132.1, 131.0, 127.7, 127.1, 126.2 (d, J = 5.6

Hz), 120.2, 118.5, 104.0, 61.1, 48.6, 42.3, 40.1, 27.4. HRMS (ESI):

(M + H)⁺calcd for C₂₃H₂₂F₃N₄O₂, 443.1689; found, 443.1685.

N-(2-Hydroxyethyl)-1-(6-(4-(triﬂuoromethyl)benzyl)-

pyrimidin-4-yl)indoline-4-carboxamide (23c). Compound 23c

(31 mg, 53%) was synthesized by a procedure similar to that used to

prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d and MeOD) δ 8.65 (s, 1H), 8.52 (d, J = 7.8 Hz, 1H),

7.55 (d, J = 7.9 Hz, 2H), 7.38 (d, J = 7.9 Hz, 2H), 7.27−7.12 (m,

3H), 6.35 (s, 1H), 4.03 (s, 2H), 3.91 (t, J = 8.6 Hz, 2H), 3.72 (t, J =

5.2 Hz, 2H), 3.56−3.41 (m, 4H). ¹³C NMR (75 MHz, CDCl₃and

MeOD) δ 168.9, 166.8, 159.4, 157.5, 144.3, 141.8, 132.4, 131.0,

129.4, 127.7, 125.6 (q, J = 3.7 Hz), 120.3, 118.6, 104.0, 61.2, 48.7,

43.6, 42.3, 27.5. HRMS (ESI): (M + H)⁺calcd for C₂₃H₂₂F₃N₄O₂,

443.1689; found, 443.1685.

N-(2-Hydroxyethyl)-1-(6-(3-methylbenzyl)pyrimidin-4-yl)-

indoline-4-carboxamide (23d). Compound 23d (43 mg, 83%) was

synthesized by a procedure similar to that used to prepare compound

12a as a white solid. ¹H NMR (300 MHz, chloroform-d and MeOD)

δ 8.63 (s, 1H), 8.49 (d, J = 7.9 Hz, 1H), 7.34−7.09 (m, 4H), 7.03 (d,

J = 7.7 Hz, 3H), 6.33 (s, 1H), 3.99−3.82 (m, 4H), 3.71 (t, J = 5.1 Hz,

2H), 3.56−3.34 (m, 5H), 2.30 (s, 3H). ¹³C NMR (75 MHz, CDCl₃

and MeOD) δ 168.9, 168.1, 159.4, 157.3, 144.4, 138.3, 137.5, 132.4,

131.0, 129.8, 128.6, 127.7, 127.5, 126.1, 120.2, 118.5, 103.9, 61.2,

49.1, 43.8, 42.2, 27.4, 21.2. HRMS (ESI): (M + H)⁺calcd for

C₂₃H₂₅N₄O₂, 389.1972; found, 389.1968.

1

22a (82 mg, 90% for two steps). H NMR (300 MHz, DMSO-d₆) δ

8.86 (s, 1H), 8.70 (d, J = 8.1 Hz, 1H), 7.81 (s, 1H), 7.73 (d, J = 7.5

Hz, 1H), 7.68−7.53 (m, 3H), 7.40 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H),

4.26−4.11 (m, 4H), 3.57 (t, J = 8.4 Hz, 2H).

1-(6-(2-(Triﬂuoromethyl)benzyl)pyrimidin-4-yl)indoline-4-

carboxylic Acid (22b). Compound 22b (65 mg, 54% for two steps)

was synthesized by a procedure similar to that used to prepare

1

compound 22a as a white solid. H NMR (300 MHz, DMSO-d₆) δ

8.86 (s, 1H), 8.70 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.64

(dd, J = 10.4, 7.6 Hz, 2H), 7.55 (d, J = 7.4 Hz, 1H), 7.46 (d, J = 7.8

Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 6.77 (s, 1H), 4.32 (s, 2H), 4.07 (t,

J = 8.4 Hz, 2H), 3.53 (t, J = 8.4 Hz, 2H).

1-(6-(4-(Triﬂuoromethyl)benzyl)pyrimidin-4-yl)indoline-4-

carboxylic Acid (22c). Compound 22c (68 mg, 57% for two steps)

was synthesized by a procedure similar to that used to prepare

1

compound 22a as a white solid. H NMR (300 MHz, DMSO-d₆) δ

8.82 (s, 1H), 8.69−8.66 (m, 1H), 7.71 (d, J = 8.1 Hz, 2H), 7.64−7.59

(m, 3H), 7.40 (d, J = 8.2 Hz, 1H), 7.08 (s, 1H), 4.19−4.12 (m, 4H),

3.54 (d, J = 8.7 Hz, 2H).

1-(6-(3-Methylbenzyl)pyrimidin-4-yl)indoline-4-carboxylic

Acid (22d). Compound 22d (86 mg, 83% for two steps) was

synthesized by a procedure similar to that used to prepare compound

22a as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.86 (s, 1H),

8.70 (d, J = 8.1 Hz, 1H), 7.81 (s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.68−

7.53 (m, 3H), 7.40 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 4.26−4.11 (m,

4H), 3.57 (t, J = 8.4 Hz, 2H), 2.49 (s, 3H).

1-(6-(3-Fluorobenzyl)pyrimidin-4-yl)indoline-4-carboxylic

Acid (22e). Compound 22e (71 mg, 68% for two steps) was

synthesized by a procedure similar to that used to prepare compound

22a as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.91 (s, 1H),

8.71 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.47−7.38 (m,

2H), 7.31−7.23 (m, 2H), 7.16−7.06 (m, 2H), 4.24−4.13 (m, 4H),

3.58 (t, J = 8.4 Hz, 2H).

1-(6-(3,5-Dimethylbenzyl)pyrimidin-4-yl)indoline-4-carbox-

ylic Acid (22f). Compound 22f (88 mg, 81% for two steps) was

synthesized by a procedure similar to that used to prepare compound

22a as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.95 (s, 1H),

8.74 (d, J = 5.7 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.58 (s, 1H), 7.43

(d, J = 8.3 Hz, 1H), 7.17 (s, 1H), 7.02 (s, 2H), 4.24 (d, J = 8.1 Hz,

2H), 4.08 (s, 2H), 3.60 (d, J = 8.4 Hz, 2H), 2.25 (s, 6H).

1-(6-(3,5-Diﬂuorobenzyl)pyrimidin-4-yl)indoline-4-carbox-

ylic Acid (22g). Compound 22g (79 mg, 72% for two steps) was

synthesized by a procedure similar to that used to prepare compound

22a as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.93 (s, 1H),

8.72 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 7.6 Hz, 1H), 7.43 (t, J = 8.0 Hz,

1H), 7.29−7.11 (m, 4H), 4.20 (d, J = 8.3 Hz, 4H), 3.58 (t, J = 8.3 Hz,

2H).

1-(6-(3-(Triﬂuoromethoxy)benzyl)pyrimidin-4-yl)indoline-

4-carboxylic Acid (22h). Compound 22h (87 mg, 70% for two

steps) was synthesized by a procedure similar to that used to prepare

1

compound 22a as a white solid. H NMR (300 MHz, DMSO-d₆) δ

8.95 (s, 1H), 8.71 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.50

(d, J = 4.7 Hz, 3H), 7.43 (t, J = 8.0 Hz, 1H), 7.33−7.27 (m, 1H), 7.20

(s, 1H), 4.25−4.16 (m, 4H), 3.58 (t, J = 8.1 Hz, 2H).

1-(6-(3-Methoxybenzyl)pyrimidin-4-yl)indoline-4-arboxylic

Acid (22i). Compound 22i (90 mg, 83% for two steps) was

synthesized by a procedure similar to that used to prepare compound

22a as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.89 (s, 1H),

8.70 (d, J = 8.0 Hz, 1H), 7.66 (dt, J = 7.8, 1.2 Hz, 2H), 7.45−7.38 (m,

2H), 7.13 (t, J = 7.8 Hz, 1H), 7.08 (d, J = 7.1 Hz, 1H), 6.79 (d, J = 2.0

Hz, 1H), 4.13−4.04 (m, 4H), 3.75 (s, 3H), 3.58 (d, J = 8.2 Hz, 2H).

1-(6-(3-Fluorobenzyl)pyrimidin-4-yl)-N-(2-hydroxyethyl)-

indoline-4-carboxamide (23e). Compound 23e (38 mg, 72%) was

synthesized by a procedure similar to that used to prepare compound

12a as a white solid. ¹H NMR (300 MHz, chloroform-d and MeOD)

δ 8.68−8.57 (m, 1H), 8.51 (d, J = 7.8 Hz, 1H), 7.32−7.11 (m, 4H),

7.02 (d, J = 7.7 Hz, 1H), 6.98−6.86 (m, 2H), 6.34 (s, 1H), 4.02−3.86

(m, 4H), 3.71 (t, J = 5.2 Hz, 2H), 3.55−3.40 (m, 4H). ¹³C NMR (75

MHz, CDCl₃and MeOD) δ 168.9, 168.9, 167.1, 164.5, 161.2, 159.5,

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157.4, 144.3, 140.0 (d, J = 7.4 Hz), 132.4, 131.0 (2C), 130.2 (d, J =

8.3 Hz), 127.7, 124.8 (d, J = 2.9 Hz), 120.3, 118.6, 115.9 (d, J = 21.4

Hz), 113.73 (d, J = 21.0 Hz), 103.9, 61.1, 48.7, 43.5, 43.4, 42.2, 27.5.

HRMS (ESI): (M + H)⁺calcd for C₂₂H₂₂FN₄O₂, 393.1721; found,

393.1716.

1-(6-(3,5-Dimethylbenzyl)pyrimidin-4-yl)-N-(2-

hydroxyethyl)indoline-4-carboxamide (23f). Compound 23f (42

mg, 78%) was synthesized by a procedure similar to that used to

prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d and MeOD) δ 8.59 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H),

7.40−7.05 (m, 3H), 6.82 (s, 3H), 6.32 (s, 1H), 3.87 (d, J = 5.0 Hz,

4H), 3.73−3.56 (m, 4H), 3.46 (d, J = 6.6 Hz, 2H), 2.23 (s, 6H). ¹³C

NMR (75 MHz, CDCl₃and MeOD) δ 169.0, 168.1, 159.4, 157.2,

144.3, 138.2, 137.4, 132.3, 131.0, 128.4, 127.6, 126.8, 120.2, 118.4,

103.9, 61.0, 48.7, 43.7, 42.3, 27.4, 21.1 (2C). HRMS (ESI): (M + H)⁺

calcd for C₂₄H₂₇N₄O₂, 403.2129; found, 403.2125.

1-(6-(3, 5-Diﬂuorobenzyl)pyrimidin-4-yl)-N-(2-

hydroxyethyl)indoline-4-carboxamide (23g). Compound 23g

(37 mg, 67%) was synthesized by a procedure similar to that used to

prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d) δ 8.81−8.40 (m, 2H), 7.63−7.05 (m, 3H), 6.96−6.55

(m, 3H), 6.43 (s, 1H), 3.97 (d, J = 9.2 Hz, 4H), 3.89−3.66 (m, 4H),

3.54 (s, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 173.0, 172.9, 170.1,

168.6 (d, J = 13.3 Hz), 165.4, 163.4, 161.4, 148.1, 145.4, 136.3, 135.1,

131.7, 124.4, 122.6, 116.0, 115.7, 108.0, 106.6, 106.2, 105.9, 65.0,

52.7, 47.2, 46.2, 46.1, 31.4. HRMS (ESI): calcd for C₂₂H₂₁F₂N₄O₂,

411.1627; found, 411.1623.

N-(2-Hydroxyethyl)-1-(6-(3-(triﬂuoromethoxy)benzyl)-

pyrimidin-4-yl)indoline-4-carboxamide (23h). Compound 23h

(41 mg, 67%) was synthesized by a procedure similar to that used to

prepare compound 12a as a white solid. ¹H NMR (300 MHz,

methanol-d₄) δ 8.57 (d, J = 3.4 Hz, 2H), 7.43 (t, J = 7.9 Hz, 1H), 7.33

(d, J = 7.8 Hz, 1H), 7.29−7.22 (m, 3H), 7.20−7.13 (m, 1H), 6.65 (s,

1H), 4.05 (s, 2H), 3.94 (t, J = 8.6 Hz, 2H), 3.74 (t, J = 5.8 Hz, 2H),

3.51 (t, J = 5.8 Hz, 2H), 3.41 (t, J = 8.5 Hz, 2H). ¹³C NMR (75 MHz,

MeOD) δ 169.4, 166.6, 159.6, 156.9, 149.3, 144.0, 140.7, 132.1,

131.6, 129.9, 127.6, 127.1, 121.4, 120.6, 118.8, 118.4, 104.0, 60.2,

48.4, 42.5, 41.9, 26.9. HRMS (ESI): (M + H)⁺calcd for

C₂₃H₂₂F₃N₄O₃, 459.1639; found, 459.1636.

5.3 Hz, 2H), 3.44−3.30 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ

168.9, 167.9, 166.6, 159.3, 157.2, 144.3, 137.6, 132.3, 131.0, 129.1,

128.7, 127.6, 126.8, 120.2, 118.5, 103.9, 61.2, 48.6, 43.8, 42.3, 27.4.

HRMS (ESI): (M + H)⁺calcd for C₂₂H₂₃N₄O₂, 375.1816; found,

375.1812.

Methyl 1-(4-(3-Fluoro-5-(triﬂuoromethyl)benzoyl)-

pyrimidin-2-yl)indoline-4-carboxylate (24a). Compound 9a

(406 mg, 2 mmol) was dissolved in 5 mL of DMF, and the mixture

solution was cooled to 0 °C with an ice bath. NaH (190 mg, 4.4

mmol) was added to the solution at 0 °C, and the mixture solution

was stirred at 0 °C for 30 min. Then, 2,4-dichloropyrimidine (450 mg,

3 mmol) was added to the solution at 0 °C. After the mixture solution

was stirred at rt. for 2 h, the mixture was cooled to 0 °C with an ice

bath. Followed by adding mCPBA (516 mg, 3 mmol), the mixture

solution was stirred at 0 °C for 30 min. After the reaction was

completed (detected by TLC), the reaction was worked up by the

addition of water and EtOAc extraction. The combined EtOAc

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and

condensed by rotary evaporation to yield a yellow oil. The residue

puriﬁcation by silica gel chromatography (gradient: 10 to 20% EtOAc

in hexane) provided product (2-chloropyrimidin-4-yl)(3-ﬂuoro-5-

(triﬂuoromethyl)phenyl)methanone (280 mg, 47%) as a yellow solid.

¹H NMR (300 MHz, chloroform-d) δ 8.98 (d, J = 4.9 Hz, 1H), 8.30

(s, 1H), 8.14 (dt, J = 8.9, 1.9 Hz, 1H), 7.96 (d, J = 4.9 Hz, 1H), 7.63

(dt, J = 8.1, 2.0 Hz, 1H).

A

mixture of (2-chloropyrimidin-4-yl)(3-ﬂuoro-5-

(triﬂuoromethyl)phenyl)methanone (91 mg, 0.3 mmol), Pd(OAc)₂

(3 mg, 0.015 mmol), XantPhos (17 mg, 0.03 mmol), Cs₂CO₃(196

mg, 0.6 mmol), and 6 (53 mg, 0.3 mmol) in 1,4-dioxane (3 mL) was

subjected to three rounds of vacuum evacuation followed by

introduction of nitrogen. The reaction mixture was then stirred at

100 °C overnight. The reaction mixture was cooled to room

temperature and poured into water and then extracted with EtOAc

(10 mL × 3). The organic phase was washed with brine, dried over

Na₂SO₄, and then concentrated under reduced pressure. The residue

puriﬁcation by silica gel chromatography (gradient: 10 to 20% EtOAc

in hexane) provided product 24a (110 mg, 82%) as a yellow solid. ¹H

NMR (300 MHz, chloroform-d) δ 8.84 (d, J = 4.8 Hz, 1H), 8.43 (s,

2H), 8.15 (d, J = 8.8 Hz, 1H), 7.64 (dd, J = 7.9, 1.2 Hz, 2H), 7.39 (d,

J = 4.8 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 4.32 (t, J = 8.7 Hz, 2H),

3.94 (s, 3H), 3.62 (t, J = 8.7 Hz, 2H).

N-(2-Hydroxyethyl)-1-(6-(3-methoxybenzyl)pyrimidin-4-yl)-

indoline-4-carboxamide (23i). Compound 23i (26 mg, 48%) was

synthesized by a procedure similar to that used to prepare compound

Methyl 1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzoyl)-

pyrimidin-4-yl)indoline-4-carboxylate (24b). 2-(3-Fluoro-5-

(triﬂuoromethyl)phenyl)acetonitrile (9a) (406 mg, 2 mmol) was

dissolved in 5 mL of DMF, and the mixture solution was cooled to 0

°C with an ice bath. NaH (190 mg, 4.4 mmol) was added to the

solution at 0 °C, and the mixture solution was stirred at 0 °C for 30

min. Then, compound 8c (580 mg, 2 mmol) was added to the

solution at 0 °C. After the mixture solution was stirred at rt. for 2 h,

the mixture was cooled to 0 °C with an ice bath. Followed by adding

mCPBA (516 mg, 3 mmol), the mixture solution was stirred at 0 °C

for 10 min. After the reaction was completed (detected by TLC), the

reaction was worked up by the addition of water and then extracted

with EtOAc (20 mL × 3). The combined EtOAc extracts were

washed with brine, dried over Na₂SO₄, ﬁltered, and condensed by

rotary evaporation to yield a yellow oil. The residue puriﬁcation by

silica gel chromatography (gradient: 20 to 50% EtOAc in hexane)

1

12a as a white solid. H NMR (300 MHz, chloroform-d) δ 8.72 (s,

1H), 8.59 (d, J = 8.2 Hz, 1H), 7.28−7.19 (m, 2H), 7.05 (d, J = 7.7

Hz, 1H), 6.95−6.79 (m, 3H), 6.51 (t, J = 5.4 Hz, 1H), 6.36 (s, 1H),

4.01 (s, 2H), 3.94 (t, J = 8.6 Hz, 2H), 3.87 (t, J = 4.9 Hz, 2H), 3.82 (s,

3H), 3.63 (q, J = 5.3 Hz, 2H), 3.54 (t, J = 8.5 Hz, 2H), 2.91 (s, 1H).

¹³C NMR (75 MHz, CDCl₃) δ 168.6, 168.0, 159.9, 159.4, 157.5,

144.7, 139.4, 132.6, 130.7, 129.7, 127.8, 121.6, 119.6, 118.6, 115.1,

112.1, 103.8, 62.4, 55.2, 48.7, 44.3, 42.7, 27.7. HRMS (ESI): (M +

H)⁺calcd for C₂₃H₂₅N₄O₃, 405.1921; found, 405.1917.

1-(6-(3,5-Dimethoxybenzyl)pyrimidin-4-yl)-N-(2-

hydroxyethyl)indoline-4-carboxamide (23j). Compound 23j (17

mg, 29%) was synthesized by a procedure similar to that used to

prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d) δ 8.67 (d, J = 1.0 Hz, 1H), 8.55 (d, J = 8.1 Hz, 1H),

7.18 (t, J = 7.9 Hz, 1H), 6.98 (dd, J = 7.8, 1.0 Hz, 1H), 6.56 (t, J = 5.6

Hz, 1H), 6.48 (d, J = 2.3 Hz, 2H), 6.39 (t, J = 2.3 Hz, 1H), 6.32 (d, J

= 1.1 Hz, 1H), 3.94 (s, 2H), 3.91 (d, J = 8.5 Hz, 2H), 3.87−3.83 (m,

2H), 3.80 (s, 6H), 3.62 (t, J = 5.1 Hz, 2H), 3.50 (t, J = 8.6 Hz, 2H).

¹³C NMR (75 MHz, CDCl₃) δ 168.5, 167.6, 161.0, 159.3, 157.4,

144.6, 140.0, 132.5, 130.7, 127.7, 119.7, 118.5, 107.4, 103.8, 98.6,

62.2, 55.4 (2C), 48.7, 44.5, 42.7, 27.6. HRMS (ESI): (M + H)⁺calcd

for C₂₄H₂₇N₄O₄, 435.2027; found, 435.2024.

1-(6-Benzylpyrimidin-4-yl)-N-(2-hydroxyethyl)indoline-4-

carboxamide (23k). Compound 23k (39 mg, 78%) was synthesized

by a procedure similar to that used to prepare compound 12a as a

white solid. ¹H NMR (300 MHz, chloroform-d and MeOD) δ 8.60 (s,

1H), 8.47 (d, J = 7.9 Hz, 1H), 7.33−7.07 (m, 8H), 6.27 (s, 1H), 3.96

(s, 2H), 3.82 (t, J = 8.5 Hz, 2H), 3.71 (t, J = 5.1 Hz, 2H), 3.49 (q, J =

1

provided product 24b (440 mg, 49%) as a yellow solid. H NMR

(300 MHz, chloroform-d) δ 8.97 (d, J = 1.1 Hz, 1H), 8.82 (d, J = 8.1

Hz, 1H), 8.31 (s, 1H), 8.17 (d, J = 9.0 Hz, 1H), 7.73 (dd, J = 7.9, 1.0

Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.31 (s,

1H), 4.21 (t, J = 8.6 Hz, 2H), 3.96 (s, 3H), 3.72 (t, J = 8.6 Hz, 2H).

Methyl 1-(6-(3-(Triﬂuoromethyl)benzoyl)pyrimidin-4-yl)-

indoline-4-carboxylate (24c). Compound 24c (162 mg, 79%)

was synthesized by a procedure similar to that used to prepare

1

compound 24b as a white solid. H NMR (300 MHz, DMSO-d₆) δ

8.75 (d, J = 0.9 Hz, 1H), 8.67 (d, J = 8.1 Hz, 1H), 7.94−7.84 (m,

2H), 7.77 (d, J = 7.9 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.54 (dd, J =

7.9, 1.0 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.08 (s, 1H), 6.01 (s, 1H),

4.10 (t, J = 9.8 Hz, 2H), 3.85 (s, 3H), 3.51 (t, J = 8.5 Hz, 2H).

P

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1-(4-(3-Fluoro-5-(triﬂuoromethyl)benzoyl)pyrimidin-2-yl)-

N-(2-hydroxyethyl)indoline-4-carboxamide (25a). To a solution

of 24a (45 mg, 0.1 mmol) in con. HCl (4 mL), H₂O (1 mL) and

AcOH (1 mL) were added, and the mixture solution was stirred at

reﬂux overnight. The reaction mixture was cooled to room

temperature, and a yellow solid precipitated from the solution. The

yellow solid was ﬁltered and washed with water, and the cake was

collected and dried to aﬀord the product as a light yellow solid. The

yellow solid and 2-aminoethan-1-ol (13 mg, 0.2 mmol) were dissolved

in 2 mL of DMF, and the mixture solution was cooled to 0 °C with an

ice bath. HOBt (14 mg, 0.1 mmol), EDCI (39 mg, 0.2 mmol), and

DMAP (24 mg, 0.2 mmol) were added to the solution at 0 °C. Then,

the ice bath was removed, and the mixture solution was stirred at

room temperature overnight. After the reaction was completed

(detected by TLC), the reaction was worked up by the addition of

water and then extracted with EtOAc (20 mL × 3). The combined

EtOAc extracts were washed with brine, dried over Na₂SO₄, ﬁltered,

and condensed by rotary evaporation to yield a yellow oil. This

material was further puriﬁed by preparative TLC plates using

CH₂Cl₂/MeOH = 50:1 as the eluent to yield 25a as a yellow solid

8.1 Hz), 132.5 (d, J = 8.0 Hz), 130.9, 127.7, 125.0 (d, J = 3.0 Hz),

121.3 (d, J = 3.0 Hz), 119.5−119.24 (m), 117.7, 117.3 (d, J = 22.2

Hz), 112.5 (ddd, J = 24.2, 7.1, 3.4 Hz), 108.0, 73.8, 62.3, 49.2, 42.6,

27.2. HRMS (ESI): (M + H)⁺calcd for C₂₃H₂₁F₄N₄O₃, 477.1544;

found, 477.1540.

1-(6-((3-Fluoro-5-(triﬂuoromethyl)phenyl)(hydroxy)-

methyl)pyrimidin-4-yl)-N-(2-hydroxyethyl)indoline-4-carbox-

amide (25e). Compound 25e (32 mg, 78%) was synthesized by a

procedure similar to that used to prepare compound 25d as a white

1

solid. H NMR (300 MHz, chloroform-d and MeOD) δ 8.66−8.27

(m, 2H), 7.51 (s, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.26−7.01 (m, 3H),

6.68 (s, 1H), 5.60 (s, 1H), 3.90 (t, J = 8.6 Hz, 2H), 3.70 (s, 2H),

3.46−3.27 (m, 4H). ¹³C NMR (75 MHz, CDCl₃and MeOD) δ 168.7

(d, J = 36.5 Hz), 164.0, 159.4, 156.8, 146.2, 144.1, 132.4, 131.0, 127.6,

120.5, 119.2, 118.7, 117.2 (d, J = 22.5 Hz), 112.4−111.7 (m), 101.0,

73.9, 61.1, 42.3, 42.2, 27.3. HRMS (ESI): (M + H)⁺calcd for

C₂₃H₂₁F₄N₄O₃, 477.1544; found, 477.1540.

1-(6-(Hydroxy(3-(triﬂuoromethyl)phenyl)methyl)pyrimidin-

4-yl)-N-(2-hydroxyethyl)indoline-4-carboxamide (25f). Com-

pound 25f (41 mg, 90%) was synthesized by a procedure similar to

1

that used to prepare compound 25d as a white solid. H NMR (300

(36 mg, 77% for two steps). H NMR (300 MHz, DMSO-d₆) δ 8.94

MHz, DMSO-d₆) δ 8.64 (s, 1H), 8.54 (d, J = 7.0 Hz, 1H), 8.25 (t, J =

5.8 Hz, 1H), 7.89−7.72 (m, 2H), 7.69−7.52 (m, 2H), 7.28 (d, J = 6.6

Hz, 2H), 7.09 (s, 1H), 6.45 (d, J = 4.5 Hz, 1H), 5.72 (d, J = 4.5 Hz,

1H), 4.71 (t, J = 5.6 Hz, 1H), 4.11 (q, J = 7.9 Hz, 2H), 3.60−3.33 (m,

6H). ¹³C NMR (75 MHz, DMSO) δ 170.9, 167.5, 159.7, 157.4,

145.2, 144.4, 132.6, 131.5, 129.6, 129.2 (d, J = 31.4 Hz), 127.6, 126.5,

124.5 (t, J = 3.9 Hz), 123.7 (t, J = 3.9 Hz), 121.1, 117.9, 101.3, 74.5,

60.3, 49.0, 42.4, 27.7. HRMS (ESI): (M + H)⁺calcd for

C₂₃H₂₂F₃N₄O₃, 459.1639; found, 459.1635.

1-(4-((3-Fluoro-5-(triﬂuoromethyl)phenyl)(hydroxy)-

methyl)pyrimidin-2-yl)indoline-4-carboxylic Acid (26a). To a

solution of 24a (427 mg, 1 mmol) in DMF (5 mL) and MeOH (5

mL) was added NaBH₄(38 mg, 1 mmol) at 0 °C, and the mixture

solution was stirred at rt. for 30 min. After the reaction was completed

(detected by TLC), the reaction mixture was worked up by the

addition of water and then extracted with EtOAc (20 mL × 3). The

combined EtOAc extracts were washed with brine, dried over

Na₂SO₄, ﬁltered, and condensed by rotary evaporation to yield a

colorless oil. The colorless oil was dissolved in con. HCl (4 mL), H₂O

(1 mL), and AcOH (1 mL), and the mixture solution was stirred at

reﬂux overnight. The reaction was cooled to room temperature, and a

yellow solid precipitated from the solution. The yellow solid was

ﬁltered and washed with water, and the cake was collected and dried

to aﬀord the product as a light yellow solid (210 mg, 78%). ¹H NMR

(300 MHz, chloroform-d) δ 8.63 (s, 1H), 8.51 (d, J = 4.8 Hz, 1H),

7.74 (dd, J = 7.9, 1.0 Hz, 1H), 7.57 (s, 1H), 7.36 (t, J = 10.4 Hz, 3H),

6.67 (d, J = 5.1 Hz, 1H), 5.73 (s, 1H), 4.39 (t, J = 8.3 Hz, 2H), 3.66

(t, J = 8.6 Hz, 2H).

(d, J = 4.9 Hz, 1H), 8.33 (s, 1H), 8.17 (dd, J = 26.9, 8.6 Hz, 3H), 7.44

(d, J = 4.9 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.10 (s, 1H), 4.71 (t, J =

5.6 Hz, 1H), 4.19 (t, J = 8.6 Hz, 2H), 3.51 (q, J = 6.1 Hz, 2H), 3.39

(t, J = 8.7 Hz, 2H), 3.30 (s, 2H). ¹³C NMR (75 MHz, DMSO) δ

159.9, 143.8, 132.7, 127.4, 122.0, 120.9, 116.7, 110.5, 60.2, 49.4, 42.4,

42.3, 40.8, 27.3. HRMS (ESI): (M + H)⁺calcd for C₂₃H₁₉F₄N₄O₃,

475.1388; found, 475.1385.

1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzoyl)pyrimidin-4-yl)-

N-(2-hydroxyethyl)indoline-4-carboxamide (25b). Compound

25b (35 mg, 75%) was synthesized by a procedure similar to that used

1

to prepare compound 25a as a yellow solid. H NMR (300 MHz,

chloroform-d and MeOD) δ 8.86 (d, J = 1.1 Hz, 1H), 8.63 (d, J = 7.7

Hz, 1H), 8.21 (s, 1H), 8.07 (dt, J = 8.8, 2.0 Hz, 1H), 7.55 (dt, J = 8.0,

2.0 Hz, 1H), 7.28−7.23 (m, 2H), 7.22−7.20 (m, 1H), 4.10 (t, J = 8.5

Hz, 2H), 3.74 (t, J = 5.1 Hz, 2H), 3.54 (dd, J = 9.3, 7.1 Hz, 4H). ¹³C

NMR (75 MHz, CDCl₃and MeOD) δ 190.3, 168.8, 166.6, 163.7,

160.4, 159.5 (d, J = 59.6 Hz), 157.3, 143.9, 138.0, 132.7, 131.2, 127.8,

123.9−123.3 (m), 121.3 (d, J = 23.2 Hz), 120.9, 119.0, 117.34 (dd, J

= 24.4, 3.2 Hz), 105.1, 61.2, 49.0, 42.3, 27.5. HRMS (ESI): (M + H)⁺

calcd for C₂₃H₁₉F₄N₄O₃, 475.1388; found, 475.1386.

N-(2-Hydroxyethyl)-1-(6-(3-(triﬂuoromethyl)benzoyl)-

pyrimidin-4-yl)indoline-4-carboxamide (25c). Compound 25c

(360 mg, 63%) was synthesized by a procedure similar to that used to

prepare compound 25a as a yellow solid. ¹H NMR (300 MHz,

DMSO-d₆) δ 8.92 (d, J = 1.1 Hz, 1H), 8.63 (dd, J = 6.6, 2.6 Hz, 1H),

8.38−8.25 (m, 3H), 8.13−8.05 (m, 1H), 7.88−7.77 (m, 1H), 7.39−

7.26 (m, 3H), 4.73 (t, J = 5.6 Hz, 1H), 4.17 (t, J = 8.5 Hz, 2H), 3.59−

3.41 (m, 4H), 3.34 (m, 2H). ¹³C NMR (75 MHz, DMSO) δ 192.0,

167.4, 160.0, 159.9 (d, J = 8.6 Hz), 157.5, 143.9, 136.4, 135.0, 132.9

(d, J = 14.2 Hz), 132.7, 130.2, 129.4, 127.7, 127.3 (d, J = 3.6 Hz),

127.2, 121.8, 118.3, 105.5, 60.2, 49.0, 42.4, 27.7. HRMS (ESI): (M +

H)⁺calcd for C₂₃H₂₀F₃N₄O₃, 457.1482; found, 457.1480.

1-(6-((3-Fluoro-5-(triﬂuoromethyl)phenyl)(hydroxy)-

methyl)pyrimidin-4-yl)indoline-4-carboxylic Acid (26b). Com-

pound 26b (390 mg, 90%) was synthesized by a procedure similar to

that used to prepare compound 26a as a yellow solid. ¹H NMR (300

MHz, DMSO-d₆) δ 8.86 (s, 1H), 8.71 (d, J = 7.8 Hz, 1H), 7.84 (s,

1H), 7.77 (d, J = 9.5 Hz, 1H), 7.64 (d, J = 7.8 Hz, 2H), 7.45−7.40

(m, 1H), 7.32 (d, J = 2.9 Hz, 1H), 6.00 (s, 1H), 4.24 (dt, J = 9.0, 3.9

Hz, 2H), 3.61−3.50 (m, 3H).

1-(4-((3-Fluoro-5-(triﬂuoromethyl)phenyl)(hydroxy)-

methyl)pyrimidin-2-yl)-N-(2-hydroxyethyl)indoline-4-carbox-

amide (25d). To a solution of 25a (10 mg, 0.02 mmol) in MeOH (1

mL) was added NaBH₄(1 mg, 0.02 mmol) at 0 °C, and the mixture

solution was stirred at rt. for 1 h. After the reaction was completed

(detected by TLC), the reaction mixture was worked up by the

addition of water and EtOAc extraction. The combined EtOAc

extracts were washed with brine, dried over Na₂SO₄, ﬁltered, and

condensed by rotary evaporation to yield a colorless oil. This material

was further puriﬁed by preparative TLC plates using CH₂Cl₂/MeOH

1-(6-(Hydroxy(3-(triﬂuoromethyl)phenyl)methyl)pyrimidin-

4-yl)indoline-4-carboxylic Acid (26c). Compound 26c (117 mg,

68%) was synthesized by a procedure similar to that used to prepare

1

compound 26a as a yellow solid. H NMR (300 MHz, DMSO-d₆) δ

8.86 (d, J = 3.4 Hz, 1H), 8.71 (d, J = 7.8 Hz, 1H), 7.96 (s, 1H), 7.87

(d, J = 7.6 Hz, 1H), 7.73−7.58 (m, 3H), 7.42 (t, J = 8.0 Hz, 1H), 7.31

(d, J = 2.3 Hz, 1H), 5.99 (s, 1H), 4.32−4.14 (m, 2H), 3.58 (t, J = 8.2

Hz, 2H).

1-(4-(Fluoro(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)methyl)-

pyrimidin-2-yl)indoline-4-carboxylic Acid (27a). To a solution

of 26a (210 mg, 0.6 mmol) in DCM (5 mL) was added DAST (322

mg, 1.8 mmol) at 0 °C, and the mixture solution was stirred at 0 °C

for 10 min. After the reaction was completed (detected by TLC), the

1

= 50:1 as the eluent to yield 25d as a white solid (9 mg, 93%). H

NMR (300 MHz, chloroform-d) δ 8.50−8.35 (m, 2H), 7.57 (s, 1H),

7.37 (d, J = 9.1 Hz, 1H), 7.24 (dd, J = 15.9, 7.8 Hz, 2H), 7.12 (d, J =

7.7 Hz, 1H), 6.66 (t, J = 4.5 Hz, 2H), 5.67 (s, 1H), 4.94 (s, 1H), 4.24

(t, J = 8.8 Hz, 2H), 3.85 (t, J = 4.9 Hz, 2H), 3.63 (q, J = 5.2 Hz, 2H),

3.44 (t, J = 8.6 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 169.1, 168.7,

164.2, 160.9, 158.4, 158.1, 145.8 (d, J = 6.9 Hz), 144.4, 132.9 (d, J =

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c01498

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

reaction mixture was worked up by the addition of water and then

extracted with EtOAc (20 mL × 3). The combined EtOAc extracts

were washed with brine, dried over Na₂SO₄, ﬁltered, and condensed

by rotary evaporation to yield a yellow oil. The residue was dissolved

in THF (2 mL) and H₂O (2 mL), and the mixture solution was

stirred at reﬂux overnight. The reaction was cooled to room

temperature, and a yellow solid precipitated from the solution. The

yellow solid was ﬁltered and washed with water, and the cake was

collected and dried to aﬀord the product as light yellow solid 27a

144.1, 138.4 (d, J = 20.5 Hz), 132.5, 131.2, 131.1, 130.8, 130.3 (d, J =

6.5 Hz), 129.2, 127.7, 125.9, 123.5 (dt, J = 6.1, 2.8 Hz) 123.5, 120.5,

118.9, 100.2 (d, J = 8.1 Hz), 92.7 (d, J = 177.7 Hz), 61.3, 48.9, 42.4,

27.5. ¹⁹F NMR (282 MHz, CDCl₃) δ −62.8, −181.1. HRMS (ESI):

(M + H)⁺calcd for C₂₃H₂₁F₄N₄O₂, 461.1595; found, 461.1590.

Methyl 1-(6-((3-(Triﬂuoromethyl)phenyl)amino)pyrimidin-

4-yl)indoline-4-carboxylate (29a). A mixture of 28a (48 mg, 0.3

mmol), Pd(OAc)₂(3 mg, 0.015 mmol), XantPhos (17 mg, 0.03

mmol), Cs₂CO₃(196 mg, 0.6 mmol), and 8c (87 mg, 0.3 mmol) in

1,4-dioxane (3 mL) was subjected to three rounds of vacuum

evacuation followed by introduction of nitrogen. The reaction mixture

was then stirred at 100 °C overnight. The reaction mixture was cooled

to room temperature and poured into water and then extracted with

EtOAc (10 mL × 3). The organic phase was washed with brine, dried

over Na₂SO₄, and then concentrated under reduced pressure. The

residue was puriﬁed by silica gel chromatography (gradient: 10 to

20% EtOAc in hexane) to provide product 29a (86 mg, 69%) as a

1

(140 mg, 54% for two steps). H NMR (300 MHz, chloroform-d) δ

8.63 (d, J = 5.0 Hz, 1H), 8.49 (d, J = 8.2 Hz, 1H), 7.73−7.64 (m,

2H), 7.46 (d, J = 8.9 Hz, 1H), 7.40−7.29 (m, 2H), 7.04 (dd, J = 5.1,

1.7 Hz, 1H), 6.41 (d, J = 46.4 Hz, 1H), 4.28 (dd, J = 8.3, 3.1 Hz, 2H),

3.61 (t, J = 8.8 Hz, 2H).

1-(6-(Fluoro(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)methyl)-

pyrimidin-4-yl)indoline-4-carboxylic Acid (27b). Compound

27b (260 mg, 60%) was synthesized by a procedure similar to that

1

used to prepare compound 27a as a yellow solid. H NMR (300

yellow solid. H NMR (300 MHz, DMSO-d₆) δ 9.72 (s, 1H), 8.65

MHz, DMSO-d₆) δ 13.01 (s, 1H), 8.71 (d, J = 7.6 Hz, 2H), 7.80−

7.71 (m, 3H), 7.56 (ddd, J = 7.8, 3.3, 1.2 Hz, 1H), 7.35 (dd, J = 9.1,

6.9 Hz, 1H), 7.11 (s, 1H), 6.75 (d, J = 45.7 Hz, 1H), 4.26−4.13 (m,

2H), 3.55 (t, J = 8.4 Hz, 2H).

(dd, J = 8.2, 1.1 Hz, 1H), 8.49 (s, 1H), 8.24 (s, 1H), 7.86 (d, J = 8.0

Hz, 1H), 7.55−7.46 (m, 2H), 7.36−7.27 (m, 2H), 6.11 (s, 1H), 4.03

(t, J = 8.7 Hz, 2H), 3.86 (s, 3H), 3.52 (t, J = 8.7 Hz, 2H).

Methyl 1-(6-(3-(Triﬂuoromethyl)phenoxy)pyrimidin-4-yl)-

indoline-4-carboxylate (29b). To a solution of 28b (49 mg, 0.3

mmol) in DMF (2 mL) were added cesium carbonate (195 mg, 0.6

mmol) and 8c (87 mg, 0.3 mmol), and the reaction was heated to 120

°C overnight. At this point, the reaction mixture was cooled to rt.,

poured into water (15 mL), and extracted with ethyl acetate (3 × 10

mL). The combined organic extracts were washed with water (10

mL) and brine (10 mL), dried over anhydrous sodium sulfate, ﬁltered,

and concentrated in vacuo. The residue was puriﬁed by chromatog-

raphy on silica gel (eluent: 1% MeOH in CH₂Cl₂) and yielded

1-(6-(Fluoro(3-(triﬂuoromethyl)phenyl)methyl)pyrimidin-4-

yl)indoline-4-carboxylic Acid (27c). Compound 27c (110 mg,

76%) was synthesized by a procedure similar to that used to prepare

1

compound 27a as a yellow solid. H NMR (300 MHz, DMSO-d₆) δ

8.71 (s, 1H), 7.90 (s, 1H), 7.83−7.74 (m, 1H), 7.69 (d, J = 7.7 Hz,

1H), 7.56 (dd, J = 7.3, 3.1 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.10 (s,

1H), 6.72 (d, J = 45.7 Hz, 1H), 4.20 (dd, J = 9.4, 4.7 Hz, 2H), 3.55 (t,

J = 8.6 Hz, 2H).

1-(4-(Fluoro(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)methyl)-

pyrimidin-2-yl)-N-(2-hydroxyethyl)indoline-4-carboxamide

(25g). Compound 25g (140 mg, 86%) was synthesized by a

procedure similar to that used to prepare compound 12a as a light

1

product 29b (79 mg, 63%) as a white solid. H NMR (300 MHz,

DMSO-d₆) δ 8.69 (t, J = 8.2 Hz, 1H), 8.47 (s, 1H), 7.73−7.50 (m,

5H), 7.36 (t, J = 8.2 Hz, 1H), 6.47 (d, J = 5.4 Hz, 1H), 4.10 (t, J = 8.5

Hz, 2H), 3.86 (s, 3H), 3.54 (t, J = 8.6 Hz, 2H).

1

yellow solid. H NMR (300 MHz, chloroform-d and MeOD) δ 8.54

(d, J = 5.0 Hz, 1H), 8.27 (d, J = 7.7 Hz, 1H), 7.63 (s, 1H), 7.41 (d, J =

8.9 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.21−7.09 (m, 2H), 7.07−6.90

(m, 2H), 6.35 (d, J = 46.3 Hz, 1H), 4.17 (tt, J = 9.1, 3.5 Hz, 2H), 3.75

(t, J = 5.1 Hz, 2H), 3.54 (q, J = 5.3 Hz, 2H), 3.41 (t, J = 8.7 Hz, 2H).

¹³C NMR (75 MHz, CDCl₃and MeOD) δ 169.2, 169.1, 166.6 (d, J =

Methyl 1-(6-((3-(Triﬂuoromethyl)phenyl)thio)pyrimidin-4-

yl)indoline-4-carboxylate (29c). Compound 29c (92 mg, 71%)

was synthesized by a procedure similar to that used to prepare

1

compound 29b as a white solid. H NMR (300 MHz, DMSO-d₆) δ

8.63−8.50 (m, 2H), 8.01−7.85 (m, 3H), 7.75 (t, J = 7.8 Hz, 1H),

7.52 (dd, J = 7.9, 1.1 Hz, 1H), 7.32 (t, J = 8.1 Hz, 1H), 6.52 (d, J =

1.1 Hz, 1H), 3.91 (t, J = 8.6 Hz, 2H), 3.84 (s, 3H), 3.45 (t, J = 8.6 Hz,

2H).

28.5 Hz), 164.0, 160.7, 159.0, 158.4 (d, J = 3.6 Hz), 144.3, 141.5 (dd,

J = 21.8, 7.5 Hz), 132.7, 132.5, 131.0, 131.0, 127.5, 119.13−118.56

(m), 116.8 (dd, J = 22.9, 7.7 Hz), 113.2 (d, J = 23.1 Hz), 106.5 (d, J =

6.7 Hz), 91.95 (d, J = 179.2 Hz), 61.5, 49.1, 42.4, 27.2. HRMS (ESI):

(M + H)⁺calcd for C₂₃H₂₀F₅N₄O₂, 479.1501; found, 479.1498.

1-(6-(Fluoro(3-ﬂuoro-5-(triﬂuoromethyl)phenyl)methyl)-

pyrimidin-4-yl)-N-(2-hydroxyethyl)indoline-4-carboxamide

(25h). Compound 25h (162 mg, 68%) was synthesized by a

procedure similar to that used to prepare compound 12a as a white

solid. ¹H NMR (300 MHz, chloroform-d) δ 8.74 (s, 1H), 8.66 (d, J =

8.1 Hz, 1H), 7.60 (s, 1H), 7.44 (d, J = 8.9 Hz, 1H), 7.37−7.31 (m,

1H), 7.28 (t, J = 4.0 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 6.86 (s, 1H),

6.61 (t, J = 5.8 Hz, 1H), 6.39 (d, J = 46.4 Hz, 1H), 4.11 (t, J = 8.6 Hz,

2H), 3.87 (q, J = 4.8 Hz, 2H), 3.72−3.53 (m, 4H), 2.73 (t, J = 5.0 Hz,

1H). ¹³C NMR (75 MHz, CDCl₃) δ 168.6, 164.9 (d, J = 26.3 Hz),

164.1, 160.7, 159.6, 157.7 (d, J = 3.4 Hz), 144.4, 141.4, 132.8, 130.9,

127.9, 124.8, 121.2, 120.2, 119.1, 117.3 (d, J = 15.5 Hz), 113.4 (d, J =

23.9 Hz), 100.1 (d, J = 8.2 Hz), 92.07 (d, J = 179.4 Hz), 77.4, 62.3,

49.0, 42.6, 27.7. ¹⁹F NMR (282 MHz, chloroform-d) δ −62.8, −109.5

(t, J = 8.7 Hz), −183.9 (d, J = 46.4 Hz). HRMS (ESI): (M + H)⁺

calcd for C₂₃H₂₀F₅N₄O₂, 479.1501; found, 479.1498.

1-(6-(Fluoro(3-(triﬂuoromethyl)phenyl)methyl)pyrimidin-4-

yl)-N-(2-hydroxyethyl)indoline-4-carboxamide (25i). Com-

pound 25i (240 mg, 72%) was synthesized by a procedure similar

to that used to prepare compound 12a as a white solid. ¹H NMR (300

MHz, chloroform-d and MeOD) δ 8.59 (dd, J = 15.6, 5.0 Hz, 2H),

7.76−7.42 (m, 4H), 7.18 (dt, J = 12.7, 7.0 Hz, 3H), 6.80 (d, J = 4.2

Hz, 1H), 6.35 (dd, J = 46.3, 4.4 Hz, 1H), 4.00 (q, J = 7.3 Hz, 2H),

3.74 (d, J = 5.6 Hz, 2H), 3.49 (dt, J = 20.7, 6.7 Hz, 4H). ¹³C NMR

(75 MHz, CDCl₃) δ 168.8, 165.3 (d, J = 26.5 Hz), 159.5, 157.4,

N-(2-Hydroxyethyl)-1-(6-((3-(triﬂuoromethyl)phenyl)-

amino)pyrimidin-4-yl)indoline-4-carboxamide (25j). To a sol-

ution of 29a (41 mg, 0.1 mmol) in con. HCl (4 mL), H₂O (1 mL)

and AcOH (1 mL) were added, and the mixture solution was stirred

at reﬂux overnight. The reaction was cooled to room temperature, and

a yellow solid precipitated from the solution. The yellow solid was

ﬁltered and washed with water, and the cake was collected and dried

to aﬀord the product as a light yellow solid. The yellow solid and 2-

aminoethan-1-ol (13 mg, 0.2 mmol) were dissolved in 2 mL of DMF,

and the mixture solution was cooled to 0 °C with an ice bath. HOBt

(14 mg, 0.1 mmol), EDCI (39 mg, 0.2 mmol), and DMAP (24 mg,

0.2 mmol) were added to the solution at 0 °C. Then, the ice bath was

removed, and the mixture solution was stirred at rt. overnight. After

the reaction was completed (detected by TLC), the reaction mixture

was worked up by the addition of water and then extracted with

EtOAc (20 mL × 3). The combined EtOAc extracts were washed

with brine, dried over Na₂SO₄, ﬁltered, and condensed by rotary

evaporation to yield a yellow oil. This material was further puriﬁed by

preparative TLC plates using CH₂Cl₂/MeOH = 50:1 as the eluent to

yield 25j as a white solid (43 mg, 73% for two steps). ¹H NMR (300

MHz, chloroform-d and MeOD) δ 8.43−8.25 (m, 2H), 7.66 (s, 1H),

7.55 (d, J = 8.2 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.32−7.23 (m, 2H),

7.19−7.03 (m, 2H), 5.89 (s, 1H), 3.80 (t, J = 8.5 Hz, 2H), 3.70 (t, J =

5.2 Hz, 2H), 3.49 (t, J = 5.3 Hz, 2H), 3.34 (t, J = 8.5 Hz, 2H). ¹³C

NMR (75 MHz, CDCl₃and MeOD) δ 169.2, 166.6, 160.5, 159.7,

157.4, 153.0, 144.8, 139.9, 132.0, 131.6, 131.2, 131.0, 129.7, 127.6,

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124.1, 119.6, 117.8, 117.5, 85.9, 61.1, 42.3, 27.4. HRMS (ESI): (M +

H)⁺calcd for C₂₂H₂₁F₃N₅O₂, 444.1642; found, 444.1639.

= 8.5 Hz, 2H), 3.61 (t, J = 5.8 Hz, 2H), 3.47 (t, J = 8.5 Hz, 2H), 3.38

(t, J = 6.2 Hz, 2H), 1.63 (dq, J = 12.9, 6.7 Hz, 4H). ¹³C NMR (75

MHz, CDCl₃and MeOD) δ 168.5, 165.9, 164.1, 160.8, 159.5, 157.7,

144.2, 141.6 (d, J = 7.7 Hz), 132.3, 131.5, 127.7, 121.6, 120.3, 119.7,

119.4, 118.5, 111.4 (d, J = 3.8 Hz), 111.1 (d, J = 3.7 Hz), 104.0, 61.7,

48.8, 43.3, 39.6, 29.6, 27.4, 25.9. HRMS (ESI): (M + H)⁺calcd for

C₂₅H₂₅F₄N₄O₂, 489.1908; found, 489.1904.

N-(2-Hydroxyethyl)-1-(6-(3-(triﬂuoromethyl)phenoxy)-

pyrimidin-4-yl)indoline-4-carboxamide (25k). Compound 25k

(48 mg, 43%) was synthesized by a procedure similar to that used to

prepare compound 25j as a white solid. ¹H NMR (300 MHz,

chloroform-d and MeOD) δ 8.50 (d, J = 7.8 Hz, 1H), 8.39 (d, J = 3.5

Hz, 1H), 7.59−7.08 (m, 7H), 6.05 (d, J = 3.5 Hz, 1H), 3.93 (t, J = 8.8

Hz, 2H), 3.69 (q, J = 4.5 Hz, 2H), 3.48 (s, 4H). ¹³C NMR (75 MHz,

CDCl₃and MeOD) δ 169.3, 169.0, 161.2, 157.4, 152.9, 144.4, 132.3,

131.1, 130.3, 127.7, 124.9, 122.1, 120.3, 118.4, 89.3, 61.1, 42.3, 27.5.

HRMS (ESI): (M + H)⁺calcd for C₂₂H₂₀F₃N₄O₃, 445.1482; found,

445.1480.

(1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-

indolin-4-yl)(3-hydroxyazetidin-1-yl)methanone (30e). Com-

pound 30e (17 mg, 37%) was synthesized by a procedure similar to

1

that used to prepare compound 12a as a white solid. H NMR (300

MHz, chloroform-d) δ 8.76 (d, J = 1.0 Hz, 1H), 8.56 (d, J = 8.2 Hz,

1H), 7.38 (s, 1H), 7.24 (d, J = 8.4 Hz, 3H), 7.00 (dd, J = 7.7, 1.0 Hz,

1H), 6.45−6.39 (m, 1H), 4.79−4.66 (m, 1H), 4.41 (s, 2H), 4.11−

3.94 (m, 6H), 3.52−3.39 (m, 2H), 3.06 (s, 1H). ¹³C NMR (75 MHz,

CDCl₃) δ 169.9, 166.2, 164.2, 160.9, 159.5, 158.0, 144.5, 141.7 (d, J =

7.6 Hz), 132.0, 129.5, 127.5, 121.8−121.6 (m), 120.9, 119.6 (d, J =

21.7 Hz), 118.3, 111.3 (d, J = 28.0 Hz), 103.9, 62.2, 61.7, 58.2, 48.7,

43.7, 27.2. HRMS (ESI): (M + H)⁺calcd for C₂₄H₂₁F₄N₄O₂,

473.1595; found, 473.1593.

N-(2-Hydroxyethyl)-1-(6-((3-(triﬂuoromethyl)phenyl)thio)-

pyrimidin-4-yl)indoline-4-carboxamide (25l). Compound 25l

(64 mg, 56%) was synthesized by a procedure similar to that used

1

to prepare compound 25j as a white solid. H NMR (300 MHz,

chloroform-d and MeOD) δ 8.47 (d, J = 3.9 Hz, 1H), 8.42−8.28 (m,

1H), 7.92−7.52 (m, 4H), 7.31−7.07 (m, 3H), 6.03 (d, J = 3.8 Hz,

1H), 3.71 (d, J = 5.7 Hz, 4H), 3.49 (t, J = 5.0 Hz, 2H), 3.40 (d, J = 9.4

Hz, 2H). ¹³C NMR (75 MHz, CDCl₃and MeOD) δ 169.0, 168.8,

158.5, 157.1, 144.1, 138.6, 132.4, 132.3, 131.9, 131.1, 130.2, 130.2,

127.6, 126.5, 120.4, 118.4, 100.4, 61.2, 48.6, 42.3, 42.2, 27.4. HRMS

(ESI): (M + H)⁺calcd for C₂₂H₂₀F₃N₄O₂S, 461.1254; found,

461.1250.

N-(1,3-Dihydroxypropan-2-yl)-1-(6-(3-ﬂuoro-5-

(triﬂuoromethyl)benzyl)pyrimidin-4-yl)indoline-4-carboxa-

mide (30a). Compound 30a (28 mg, 57%) was synthesized by a

procedure similar to that used to prepare compound 12a as a white

solid. ¹H NMR (300 MHz, chloroform-d and MeOD) δ 8.68 (s, 1H),

8.55 (d, J = 7.5 Hz, 1H), 7.33 (s, 1H), 7.28−7.11 (m, 4H), 6.40 (s,

1H), 4.10−3.90 (m, 5H), 3.82 (dd, J = 11.3, 4.3 Hz, 2H), 3.70 (dd, J

= 11.3, 5.1 Hz, 2H), 3.49 (t, J = 8.5 Hz, 2H). ¹³C NMR (75 MHz,

CDCl₃and MeOD) δ 168.5, 166.6, 166.0, 159.5, 157.7, 144.3, 141.6,

132.3, 130.9, 127.8, 121.8−121.5 (m), 120.5, 119.7, 119.4, 118.8,

111.5 (d, J = 3.9 Hz), 111.13 (d, J = 3.8 Hz), 104.0, 61.6, 52.6, 48.8,

43.4, 27.5. HRMS (ESI): (M + H)⁺calcd for C₂₄H₂₃F₄N₄O₃,

491.1701; found, 491.1697.

1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-N-

(2-methoxyethyl)indoline-4-carboxamide (30b). Compound

30b (30 mg, 65%) was synthesized by a procedure similar to that

used to prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d) δ 8.78 (s, 1H), 8.63 (d, J = 8.1 Hz, 1H), 7.38 (s, 1H),

7.31−7.21 (m, 3H), 7.17 (d, J = 7.7 Hz, 1H), 6.47 (d, J = 6.2 Hz,

1H), 6.43 (s, 1H), 4.08 (s, 2H), 4.02 (t, J = 8.6 Hz, 2H), 3.61 (ddd, J

= 16.4, 10.7, 6.9 Hz, 6H), 3.42 (s, 3H). ¹³C NMR (75 MHz, CDCl₃)

δ 167.6, 166.2, 164.2, 160.9, 159.5, 158.0, 144.5, 141.8 (d, J = 7.6 Hz),

132.9, 132.5, 132.5, 131.2, 127.8, 125.0, 121.7 (t, J = 3.6 Hz), 120.0,

119.6 (d, J = 21.4 Hz), 118.6, 111.3 (dd, J = 24.6, 3.9 Hz), 103.9,

77.4, 77.0, 76.6, 71.2, 48.8, 43.8, 39.5, 27.7. HRMS (ESI): (M + H)⁺

calcd for C₂₄H₂₃F₄N₄O₂, 475.1752; found, 475.1749.

1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-N-

(3-hydroxypropyl)indoline-4-carboxamide (30c). Compound

30c (26 mg, 56%) was synthesized by a procedure similar to that

used to prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d) δ 8.77 (s, 1H), 8.63 (d, J = 8.1 Hz, 1H), 7.38 (s, 1H),

7.29−7.20 (m, 3H), 7.14 (d, J = 7.7 Hz, 1H), 6.64 (t, J = 6.1 Hz, 1H),

6.43 (s, 1H), 4.07 (s, 2H), 4.00 (t, J = 8.6 Hz, 2H), 3.76 (t, J = 5.5 Hz,

2H), 3.69−3.50 (m, 4H), 3.23 (s, 1H), 1.82 (p, J = 5.7 Hz, 2H). ¹³C

NMR (75 MHz, CDCl₃) δ 168.7, 166.2, 164.2, 160.9, 159.5, 158.0,

144.6, 141.8 (d, J = 7.5 Hz), 132.6, 131.0, 127.9, 121.8−121.6 (m),

119.8, 119.5, 118.8, 111.7−111.0 (m), 103.9, 59.9, 48.8, 43.8, 37.0,

32.1, 27.7. HRMS (ESI): (M + H)⁺calcd for C₂₄H₂₃F₄N₄O₂,

475.1752; found, 475.1749.

1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-N-

(2-ﬂuoroethyl)indoline-4-carboxamide (30f). Compound 30f

(36 mg, 78%) was synthesized by a procedure similar to that used

1

to prepare compound 12a as a white solid. H NMR (300 MHz,

chloroform-d) δ 8.83−8.73 (m, 1H), 8.66 (d, J = 8.1 Hz, 1H), 7.39 (s,

1H), 7.33−7.15 (m, 4H), 6.45 (d, J = 9.4 Hz, 2H), 4.71 (t, J = 4.7 Hz,

1H), 4.55 (t, J = 4.7 Hz, 1H), 4.14−3.95 (m, 4H), 3.83 (q, J = 5.1 Hz,

1H), 3.74 (q, J = 5.1 Hz, 1H), 3.58 (t, J = 8.5 Hz, 2H). ¹³C NMR (75

MHz, CDCl₃) δ 167.8, 166.3, 164.2, 160.9, 159.5, 158.0, 144.7, 141.8

(d, J = 7.6 Hz), 132.6, 130.8, 128.0, 121.7 (t, J = 3.5 Hz), 119.9,

119.8, 119.5, 118.9, 111.5 (d, J = 3.7 Hz), 111.2 (d, J = 3.8 Hz),

103.9, 82.8 (d, J = 166.7 Hz), 48.8, 43.8, 40.2 (d, J = 19.5 Hz), 27.7.

¹⁹F NMR (282 MHz, chloroform-d) δ −62.7, −110.6 (t, J = 8.8 Hz),

−224.2 (tt, J = 47.4, 28.3 Hz). HRMS (ESI): (M + H)⁺calcd for

C₂₃H₂₀F₅N₄O, 473.1595; found, 473.1593.

1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-

N,N-bis(2-hydroxyethyl)indoline-4-carboxamide (30g). Com-

pound 30g (27 mg, 53%) was synthesized by a procedure similar to

1

that used to prepare compound 12a as a white solid. H NMR (300

MHz, chloroform-d) δ 8.71 (s, 1H), 8.45 (d, J = 8.2 Hz, 1H), 7.37 (s,

1H), 7.29−7.17 (m, 3H), 6.97 (d, J = 7.6 Hz, 1H), 6.40 (s, 1H),

4.10−3.57 (m, 12H), 3.44 (t, J = 5.0 Hz, 2H), 3.26 (t, J = 8.5 Hz,

2H). ¹³C NMR (75 MHz, CDCl₃) δ 172.3, 166.2, 164.2, 160.9, 159.5,

158.0, 144.0, 141.7 (d, J = 7.5 Hz), 133.0, 129.7, 127.8, 122.0−121.5

(m), 120.2, 119.8, 119.5, 117.0, 111.3 (d, J = 24.4 Hz), 103.8, 61.1,

60.1, 52.9, 49.1, 48.5, 43.6, 26.1. HRMS (ESI): (M + H)⁺calcd for

C₂₅H₂₅F₄N₄O₃, 505.1857; found, 505.1853.

Molecular Docking Protocol. The molecular docking study was

̈

performed using Schrodinger Small-Molecule Drug Discovery Suite

̈

(Schrodinger, LLC, New York, NY, 2020). The cocrystal structures of

GPR52 and compound 2 (PDB code: 6LI0) were downloaded from

the RCS PDB bank. The cocrystal structure was preprocessed and

̈

minimized with Schrodinger Protein Preparation Wizard using default

settings. The grid center was chosen on the centroid of an existing

ligand, and the size of the grid box was set to 30 Å on each side. The

̈

3D structure of ligand 12c was created using Schrodinger Maestro,

and a low energy conformation was calculated using LigPrep. Docking

was employed with Glide using the SP precision. Docked poses were

̈

incorporated into Schrodinger Maestro for visualization and analysis

of binding site interactions.

In Vitro Pharmacology. Cell Culture and Plasmids. Wildtype

(WT) human embryonic kidney 293 (HEK293) cells were a generous

gift from Dr. Asuka Inoue, Tohoku University.³¹The wildtype

HEK293 cells were cultured in DMEM (Gibco, Carlsbad, CA) with

10% FBS (Omega Scientiﬁc, Tarzana, CA) and 10,000 U/mL

penicillin−streptomycin (Gibco, Carlsbad, CA) in a humidiﬁed

incubator at 37 °C in 5% CO₂. A GPR52 TANGO construct was

purchased from Addgene (Watertown, MA). A stop codon was placed

in the frame immediately after the receptor coding sequence to

generate a human GPR52 WT coding construct. Point mutation of

1-(6-(3-Fluoro-5-(triﬂuoromethyl)benzyl)pyrimidin-4-yl)-N-

(4-hydroxybutyl)indoline-4-carboxamide (30d). Compound

30d (30 mg, 61%) was synthesized by a procedure similar to that

used to prepare compound 12a as a white solid. ¹H NMR (300 MHz,

chloroform-d and MeOD) δ 8.66 (s, 1H), 8.52 (d, J = 8.1 Hz, 1H),

7.33 (s, 1H), 7.27−7.06 (m, 5H), 6.39 (s, 1H), 4.02 (s, 2H), 3.94 (t, J

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GPR52 WT from GPR52 TANGO was made by polymerase chain

reaction (PCR) using the QuikChange II Site-Directed Mutagenesis

Kit (Agilent Technologies, Santa Clara, CA) according to the

manufacturer’s protocol. Mutagenesis and sequencing primers were

obtained from ThermoFisher (Pittsburgh, PA). The primers used to

make the GPR52 WT point mutant were 5′−gctaacagctgttccatctaa-

gataccggtggacgcacc−3′ (sense) and 5′−cgattgtcgacaaggtagattctatggc-

cacctgcgtgg−3′ (antisense). Parental DNA in the reaction mixture

was digested using restriction endonuclease DpnI (from diplococcus

pneumoniae) at 37 °C for one hour. A digestion mixture (2 μL) was

transformed into XL1-Blue supercompetent cells by heat shock at 42

°C for 45 s. The reaction was incubated in Super Optimal broth with

a catabolite repression medium (Sigma-Aldrich) and then plated onto

Luria−Bertani (LB) agar plates containing 100 μg/mL ampicillin. A

single colony was selected for sequencing, and the point mutation was

veriﬁed prior to use (Molecular Genomics Core, University of Texas

Medical Branch, Galveston, TX).

The study and the related standard operating procedures were

reviewed and approved by the Sundia Institutional Animal Care and

Use Committee. The Sundia animal facility is approved with yearly

inspection by the Shanghai Laboratory Animal Management

Committee. All treatment assignments were blinded to investigators

who performed PK assays and end point statistical analyses.

The PK parameters of compound 12c were calculated according to

a non-compartmental model using WinNonlin (Pharsight Corpo-

ration, ver 5.3, Mountain View, CA, USA). The peak concentration

(C_max) was directly obtained by visual inspection of the plasma

concentration−time proﬁle. The elimination rate constant (λ) was

obtained by the least-squares ﬁtted terminal log-linear portion of the

slope of the plasma concentration−time proﬁle. The elimination half-

life (t_1/2) was evaluated according to 0.693/λ. The area under the

plasma concentration−time curve from 0 to time t (AUC_0−t) was

evaluated using the linear trapezoidal rule and further extrapolated to

Transfections. For the cAMP Glosensor assays, 6.5 × 10⁵wildtype

HEK293 cells were plated in 6-well plates (Corning, Oneonta, NY)

24 h before transfection to achieve 70−80% conﬂuency the following

day. Each well of wildtype HEK293 cells was transiently transfected

with 0.10 μg of wildtype human GPR52 plasmid cDNA and 1 μg of

the 22F Glosensor plasmid (Promega, Madison, WI) using 10 μL of

Lipofectamine 2000 (Invitrogen, Waltham, MA) in 1 mL of

OptiMEM (Gibco, Carlsbad, CA) and 1 mL of growth media.

cAMP Glosensor Assay. Eighteen hours after transfection, wildtype

HEK293 cells with GPR52 and Glosensor constructs (see Trans-

fections above), cells were seeded at 60,000 cells/well in growth

media into poly-^L-lysine (Trevigen, Gaithersburg, MD) coated 96-well

white clear-bottom cell culture plates (Greiner Bio-One, Monroe,

NC). Four hours later, complete media was aspirated and replaced

with 90 μL of a 1% Glosensor reagent (Promega, Madison, WI) in 1×

Hanks’ balanced salt solution (HBSS) and 20 mM HEPES. Cells were

serum-starved for 2 h at room temperature in the dark. Test

compounds were weighed and diluted to 2 mM in DMSO. Serial

dilutions of each test compound were prepared at 10× ﬁnal

concentration and transferred to a 96-well source plate. Cells were

incubated with 10 μL of 10× drug solutions for 15 min. LCPS were

recorded on a Microbeta2 Microplate counter (PerkinElmer,

Waltham, MA). Data from at least three independent experiments

(n = 3 to 21) conducted in technical triplicate are presented as

percentage of compound 4 response. All in vitro pharmacological data

were analyzed using GraphPad Prism 7.05 software (La Jolla, CA).

Data from cAMP ligand dose response assays are presented as the half

maximum (EC₅₀, nM) and maximum eﬀect (E_max) (means SEM) as

computed by GraphPad using a four-parameter nonlinear regression

curve-ﬁtting algorithm. All E_maxligand responses were scaled relative

to 3 μM compound 4 in cells treated for each assay (3 μM compound

4 response set to 100%).

In Vivo PK and Brain Permeability Studies. Male Sprague Dawley

rats (n = 3 per treatment group; Beijing Vital River Laboratory,

Animal Technology Co., Ltd., Beijing, China) weighing 200−250 g at

the beginning of the experiment were housed three per cage in a

pathogen-free, temperature-controlled (20−26 °C), and humidity-

controlled (40−70%) environment with a 12 h light−dark cycle and

ad libitum access to food and ﬁltered water. Rats were randomly

assigned to treatment groups. A vehicle [10% dimethyl sulfoxide

(DMSO) and 90% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD);

Cyclodextrin Technologies Development, Inc., High Springs, FL,

USA] or compound 12c dissolved in the vehicle was administered to

rats IV at 10 mg/kg or PO at 20 mg/kg. The rat was restrained

manually at the designated time points (0.08, 0.25, 0.5, 1.0, 2.0, 4.0,

8.0, and 24 h post-dosing for IV; 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h

post-dosing for PO). Blood (yielding ∼50 μL of plasma) samples

(100 μL) are taken from the animal’s jugular vein and placed into

micro K₂EDTA tubes. Blood samples were placed on ice and

centrifuged at 8000 rpm for 5 min at 4 °C to generate plasma samples

within 0.5 h of collection. Brain samples were collected at 0.25 and 1

h post-dosing. All samples were stored at −20 °C. The concentration

of 12c in each sample was analyzed by Sundia MediTech Co., Ltd.

inﬁnity (AUC_0−∞) according to the following equation: AUC_0−∞=

AUC_0−t+ C_last/λ. The pharmacokinetic parameters were presented as

mean SEM.

̈

In Vivo Pharmacology of Compound 12c. Animals. Naive

male C57/BL6 mice (n = 60; Jackson Laboratory, Bar Harbor, ME)

weighing between 24 and 31 g (∼60 days of age) at the beginning of

the experiment were housed four/cage in a temperature- (21−23 °C)

and humidity-controlled (40−50%) environment on a 12 h light/dark

cycle (lights on at 0600). Mice were provided ad libitum access to

chow and water for 14−20 days prior to experiments, which were

carried out in accordance with the National Institutes of Health Guide

for the Care and Use of Laboratory Animals (2011) and with the

approval of the Institutional Animal Care and Use Committee at the

University of Texas Medical Branch.

Drugs. Compound 12c was dissolved in 0.9% saline containing

20% HP-β-CD with a ﬁnal pH of the solution adjusted to 7.4.

Dextroamphetamine (Sigma-Aldrich, St Louis, MO, USA) was

prepared in 0.9% saline. Compounds were then administered IP at

2 mL/1 kg body weight.

Locomotor Activity Assessment. A modiﬁed open ﬁeld activity

system (San Diego Instruments, San Diego, California, U.S.A.) was

employed to quantify activity under low-light conditions.³²⁻³⁴Clear

Plexiglas chambers (40 × 40 × 40 cm) were surrounded by a 4 × 4

photobeam matrix positioned ∼1 cm above the chamber ﬂoor.

Horizontal activity was quantiﬁed as the sum of photobeam breaks

that occurred within the inner central 16 × 16 cm area and in the

surrounding outer 12 × 12 cm perimeter of the activity monitor.

A between-subjects design was employed to study the eﬃcacy of

compound 12c to impact horizontal activity. Mice were handled three

times/week for two weeks prior to the study and were habituated to

the motor activity monitors 1 h/day for three days. Mice were

transported in their home cages and allowed to acclimate to the

testing room for 1 h prior to evaluation. On the day of the test, mice

were weighed and randomized into ﬁve treatment groups for

compound 12c administration (n = 12/dose). One animal was

excluded as an outlier (i.e., horizontal activity two standard deviations

from the mean) with n = 11 for 1 mg/kg compound 12c). Following

IP administration of the vehicle or compound 12c (0.3, 1, 3, and 10

mg/kg), automated activity monitoring began immediately and

continued for 30 min. Mice were then injected with AMPH (3 mg/

kg IP), and activity was monitored for the next 90 min. All testings

occurred between 0800 and 1700 h.

Statistical Analyses. Locomotor activities are presented as mean

horizontal activity (mean SEM). A one-way ANOVA for a between-

subjects design was employed to analyze horizontal activity. A priori

comparisons were deﬁned prior to the start of experimentation and

conducted by Dunnett’s procedure,³⁵with an experiment-wise error

rate set at α = 0.05. Investigators who performed compound

administration were blinded to treatment assignments and endpoint

statistical analyses.

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partially supported by the John D. Stobo, M. D., Distinguished

Chair Endowment Fund. The authors would like to acknowl-

edge Robert Fox for in vivo assay assistance and the

preparation of amphetamine solutions for in vivo injections.

The authors would also like to acknowledge Dr. Asuka Inoue,

Tohoku University, for the generous gift of the HEK cell line,

Dr. Lawrence C. Sowers at the UTMB Department of

Pharmacology and Toxicology as well as Dr. Tianzhi Wang

at the NMR core facility of UTMB for the NMR spectroscopy

assistance, and Dr. Xuemei Luo at the UTMB Mass

Spectrometry Core with the funding support from the

University of Texas System Proteomics Network for the

HRMS analysis.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01498.

■

sı

Figures S1 and S2, HPLC analysis spectra of

representative compounds, and ¹H and ¹³C NMR

spectra of all new compounds (PDF)

Molecular formula strings and data (CSV)

Docking compound 12c with GPR52 (PDB)

AUTHOR INFORMATION

Corresponding Authors

■

John A. Allen − Chemical Biology Program, Department of

Pharmacology and Toxicology and Center for Addiction

Research, University of Texas Medical Branch, Galveston,

Texas 77555, United States; Phone: (409) 772-9621;

Email: joaallen@utmb.edu; Fax: 409-747-7050

Jia Zhou − Chemical Biology Program, Department of

Pharmacology and Toxicology and Center for Addiction

Research, University of Texas Medical Branch, Galveston,

Texas 77555, United States; orcid.org/0000-0002-2811-

1090; Phone: (409) 772-9748; Email: jizhou@utmb.edu;

Fax: (409) 772-9648

ABBREVIATIONS USED

■

GPCRs, G-protein-coupled receptors; FDA, U.S. Food and

Drug Administration; CNS, central nervous system; NAc,

nucleus accumbens; MSNs, medium spiny neurons; cAMP,

cyclic adenosine monophosphate; NMDA, N-methyl-^D-aspar-

tate; SUD, substance use disorder; ECL2, extracellular loop 2;

EC₅₀, half maximal eﬀective concentration; HTS, high-

throughput screening; TM, transmembrane helix; SAR,

structure−activity relationship; PK, pharmacokinetic; DMF,

N,N-dimethylformamide; EDCI, N-(3-dimethylaminopropyl)-

N′-ethylcarbodiimide hydrochloride; DMAP, 4-dimethylami-

nopyridine; XantPhos, 4,5-bis(diphenylphosphino)-9,9-dime-

thylxanthene; mCPBA, meta-chloroperoxybenzoic acid; DAST,

diethylaminosulfur triﬂuoride; DMSO, dimethyl sulfoxide;

NIMH, National Institute of Mental Health; PDSP, psycho-

active drug screening program; hERG, human ether-a-go-go-

related gene; PO, oral; IV, intravenous; AMPH, amphetamine;

WT, wildtype; HEK293, human embryonic kidney 293; PCR,

polymerase chain reaction; HBSS, Hanks’ balanced salt

solution; IP, intraperitoneal

Authors

Pingyuan Wang − Chemical Biology Program, Department of

Pharmacology and Toxicology, University of Texas Medical

Branch, Galveston, Texas 77555, United States

Daniel E. Felsing − Center for Addiction Research, University

of Texas Medical Branch, Galveston, Texas 77555, United

States

Haiying Chen − Chemical Biology Program, Department of

Pharmacology and Toxicology, University of Texas Medical

Branch, Galveston, Texas 77555, United States

Sonja J. Stutz − Center for Addiction Research, University of

Texas Medical Branch, Galveston, Texas 77555, United

States

Ryan E. Murphy − Center for Addiction Research, University

of Texas Medical Branch, Galveston, Texas 77555, United

States

Kathryn A. Cunningham − Chemical Biology Program,

Department of Pharmacology and Toxicology and Center for

Addiction Research, University of Texas Medical Branch,

Galveston, Texas 77555, United States; orcid.org/0000-

0002-4257-1739

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The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by grants T32 DA 007287 and U18

DA052543 from the National Institutes of Health, the PhRMA

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Discovery of Potent and Brain-Penetrant GPR52 Agonist that Suppresses Psychostimulant Behavior

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