Evaluation of Amide Bioisosteres Leading to 1,2,3-Triazole Containing Compounds as GPR88 Agonists: Design, Synthesis, and Structure-Activity Relationship Studies

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Evaluation of Amide Bioisosteres Leading to 1,2,3-Triazole

Containing Compounds as GPR88 Agonists: Design, Synthesis, and

Structure−Activity Relationship Studies

Md Tou ﬁ qur Rahman, Ann M. Decker, Lucas Laudermilk, Rangan Maitra, Weiya Ma, Sami Ben Hamida,

Emmanuel Darcq, Brigitte L. Kie ﬀ er, and Chunyang Jin *

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ABSTRACT: The orphan receptor GPR88 has been implicated in

a number of striatal-associated disorders, yet its endogenous ligand

has not been discovered. We have previously reported that the

amine functionality in the 2-AMPP-derived GPR88 agonists can be

replaced with an amide (e.g., 4) without losing activity. Later, we

have found that the amide can be replaced with a bioisosteric 1,3,4-

oxadiazole with improved potency. Here, we report a further study

of amide bioisosteric replacement with a variety of azoles

containing three heteroatoms, followed by a focused structure−

activity relationship study, leading to the discovery of a series of

novel 1,4-disubstituted 1H-1,2,3-triazoles as GPR88 agonists.

Collectively, our medicinal chemistry eﬀorts have resulted in a

potent, eﬃcacious, and brain-penetrant GPR88 agonist 53 (cAMP EC₅₀= 14 nM), which is a suitable probe to study GPR88

functions in the brain.

Despite the continuous eﬀorts and progress on multiple

fronts, GPR88 has not been deorphanized to date.²²In this

vein, our medicinal chemistry campaign to understand the

signaling mechanism and biological functions of GPR88

showed that 2-PCCA [(1R,2R)-2-(pyridin-2-yl)cyclopropane

carboxylic acid ((2S,3S)-2-amino-3-methylpentyl)-(4′-propyl-

biphenyl-4-yl)amide, 1, Figure 1] can activate GPR88 via a

Gα_i-coupled signaling pathway in the functional cAMP assay in

GPR88 overexpressing CHO cells.^23,24Afterward, our system-

atic modiﬁcations of the 2-PCCA scaﬀold have resulted in the

discovery of a brain-penetrant agonist, RTI-13951-33 (2), that

enabled in vivo studies of GPR88 in operant alcohol self-

administration in rats, demonstrating that GPR88 agonists are

potential drug candidates for the treatment of alcohol

addiction.²⁵Other than the 2-PCCA scaﬀold, the 2-AMPP

[(2S)-N-((1R)-2-amino-1-(4-(2-methylpentyloxy)phenyl)-

ethyl)-2-phenylpropanamide (3, Figure 1)] scaﬀold has also

shown promise for GPR88 agonist development.^15,26,27It has

been illustrated that the 2-AMPP scaﬀold can tolerate

INTRODUCTION

■

GPR88 is a class A G-protein-coupled receptor (GPCR) for

which the endogenous ligand is yet to be discovered.¹Since its

discovery two decades ago, the GPR88 receptor has evoked

signiﬁcant interest due to its predominant expression in both

striatonigral and striatopallidal pathways of the striatum.²

GPR88 transcriptional proﬁling³⁻⁷and genetic knockout

(KO)⁸⁻¹⁹studies have provided evidence of GPR88’s

involvement in a variety of striatal-associated disorders,

including Parkinson’s disease, schizophrenia, and drug

addiction. For example, GPR88’s expression in rodents was

altered upon their exposure to antipsychotic,^3,4antidepres-

sant,⁵or drugs of abuse.⁶Additionally, GPR88 KO studies

found its key role as a regulator of the dopaminergic system

that linked this receptor to the motor-control and reward

system.^8,9In this regard, there is a direct link between GPR88

and alcohol addiction as GPR88 KO mice were prone to

higher voluntary alcohol intake compared to the wild-type

(WT) mice, while their consumptions of water and palatability

were intact.¹¹Furthermore, human genetic studies revealed

that the GPR88 gene is linked to schizophrenia and several

childhood cognitive genetic disorders including speech delay,

learning disabilities, and chorea.^20,21Collectively, these

research studies demonstrate that GPR88 is a novel drug

target that can potentially meet current unmet demands for

treating various neuropsychiatric and neurodegenerative

diseases.

Received: June 15, 2021

Published: August 13, 2021

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Figure 1. Selected GPR88 agonists 2-PCCA (1), RTI-13951-33 (2), 2-AMPP (3), and 4−7 and their cAMP EC₅₀values.

a

Scheme 1. Synthesis of Target Compounds 9, 10, and 12

a

Reagents and conditions: (a) NaH, N′-hydroxyethanimidamide, tetrahydrofuran (THF), 0 °C to rt, 4 h; (b) hydrazine, EtOH, reﬂux, 3 h; (c)

CH₃CN, K₂CO₃, n-BuOH, 150 °C, 5 h.

modiﬁcations at three distinct sites and the amino group can

be replaced by amide (4), among other functional groups, with

improved potency.^26,28Recently, we have reported our rational

modiﬁcations of 4 by investigating the structure−activity

relationship (SAR) at the alkoxy side chain and the N-methyl

amide functionality.²⁸Although replacement of the N-methyl

amide group with a bioisosteric 1,3,4-oxadiazole moiety

provided 5 with a moderate potency, notable improvement

in potency was achieved upon the addition of a methylene

linker between the oxadiazole moiety and the benzylic carbon

(e.g., 6 and 7, Figure 1). 5-Amino-1,3,4-oxadiazole 7 had an

EC₅₀of 59 nM in the cAMP assay using CHO-GPR88 cells,

which is 5-fold more potent than 4. Additionally, 7 had

GPR88-speciﬁc agonist signaling activity when tested in the

[³⁵S]GTPγS binding assay using striatal membranes prepared

from WT mice and GPR88 KO mice.²⁸However, in vitro

ADME and pharmacokinetic (PK) assessments revealed that

1,3,4-oxadiazole analogues have poor aqueous solubility and

limited blood−brain barrier (BBB) permeability, thus requiring

further optimization to improve druglike properties. It is

worthy to note that 7 has high lipophilicity (clog P = 4.2) and

polar surface area (PSA = 103.3), both of which are above the

recommended threshold (CNS drugs usually have clog P in the

range 2−4²⁹and PSA less than 90 Å²).³⁰Given that both

potency and brain bioavailability continue to be the major

challenges for developing an in vivo agonist probe for the

GPR88 receptor, we planned to further modify the 2-AMPP

scaﬀold by ﬁne-tuning potency and physicochemical proper-

ties, such as clog P and PSA, to overcome the aforementioned

challenges.

Bioisosteric replacement continues to prevail as an

indispensable tool in medicinal chemists’ toolbox.³¹Due to

the prevalence of amide functionality in a wide range of

chemical probes and drugs, amide chemistry engenders

tremendous interest to medicinal chemists. Further, due to

the enzymatic lability of the amide functional group in vivo, the

improvement of metabolic stability of this moiety using a

number of bioisosteric replacements has been docu-

mented.^31,32In addition to our initially explored 1,3,4-

oxadiazoles, several other heterocycles such as 1,2,4-oxadiazole,

1,2,4-triazole, 1,2,3-triazole, and tetrazole have also been

shown to perform as amide-mimetics.³¹

Both 1,2,4- and 1,2,3-triazoles are well documented as

nonclassical ﬁve-membered heterocyclic amide bioisosteres,

although application of 1,2,3-triazoles is much more preva-

lent.^31,33Depending on the substitution pattern, 1,2,3-triazoles

can mimic both trans- and cis-amide bonds.³⁴Because 1,4-

disubstituted 1,2,3-triazoles imitate the trans-amide bond, their

applications as amide replacements are much more widespread

than the 1,5-disubstituted 1,2,3-triazoles that mimic the cis-

amide.³¹The 1,2,3-triazoles can be easily synthesized via “click

reaction” of the readily accessible alkynes and azides using mild

reaction conditions.³⁵⁻³⁸In addition, the 1,2,3-triazole motif is

capable of forming hydrogen-bond, dipole−dipole or π-

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a

Scheme 2. Synthesis of Target Compounds 16 and 17

a

Reagents and conditions: (a) NaBH₄, LiCl, THF, 0 °C to rt, 3 h; (b) DMP, DCM, rt, 3 h; (c) dimethylacetyldiazophosphonoacetate, K₂CO₃,

MeOH, rt, 3 h; (d) TMS−N₃, CuSO₄·5H₂O, sodium ascorbate, N,N-dimethylformamide (DMF)/H₂O (9:1), 90 °C, 5 h; (e) CH₃I, K₂CO₃, DMF,

rt, overnight.

a

Scheme 3. Synthesis of Target Compounds 21, 25, 26, and 28

a

Reagents and conditions: (a) PPh₃, diethyl azodicarboxylate (DEAD), alcohol, THF, rt, overnight; (b) NaBH₄, LiCl, EtOH/THF (1:1), rt, 5 h;

(c) PPh₃, CBr₄, THF, rt, overnight; (d) 1,2,4-triazole, K₂CO₃, DMF, rt, overnight; (e) 4 M HCl in dioxane, DCM, rt, overnight; (f) (S)-2-

phenylpropionic acid, HBTU, TEA, MeCN, rt, 5 h; (g) NaN₃, DMF, rt, overnight; (h) acetylene, CuSO₄·5H₂O, sodium ascorbate, t-BuOH/H₂O,

rt, overnight; (i) Bu₄N⁺CN⁻, THF, 50 °C, 2 h; (j) NaN₃, ZnBr₂, 1,4-dioxane, reﬂux, 48 h.

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a

Scheme 4. Synthesis of Target Compounds 29−53

a

(a) Alkyne, CuSO₄·5H₂O, sodium ascorbate, t-BuOH/H₂O, rt, overnight or alkyne, toluene, heat; (b) Dess−Martin periodinane (DMP),

dichloromethane (DCM), rt, 2 h; (c) (i) NaOH, THF/H₂O, rt, 2 h, then HCl; (ii) MeNH₂·HCl or Me₂NH·HCl, 2-(1H-benzotriazol-1-yl)-1,1,3,3-

tetramethyluronium hexaﬂuorophosphate (HBTU), triethylamine (TEA), MeCN, rt, overnight.

stacking interactions with the receptor and is not prone to

metabolic degradation.^33,34As a result, the 1,2,3-triazole motif

oﬀers a rapid and eﬃcient way to expand SAR for hit-to-lead

optimization. Herein, we report our further optimization of the

2-AMPP scaﬀold by replacing the 1,3,4-oxadiazole moiety in 6

(Figure 1) with other azole heterocycles, especially 1,4-

disubstituted 1H-1,2,3-triazoles, leading to highly potent and

eﬃcacious brain-penetrant GPR88 agonists.

triazole 17 as the major product. The structure of compound

17 was conﬁrmed by NMR studies, including the hetero-

nuclear multiple bond correlation (HMBC) experiment (see

the Supporting Information).

Synthesis of 1-substituted 1H-1,2,4-triazole 21, 1-substituted

1H-1,2,3-triazoles 25 and 26, and 5-substituted 2H-1,2,3,4-

tetrazole 28 is depicted in Scheme 3. O-alkylation of 18²⁸with

2-methylpentanol, cyclobutylmethanol, or (S)-2-methylbutanol

under Mitsunobu conditions aﬀorded ethers 19a, 19b, and

19c, respectively. The ester in 19a−c was reduced with freshly

prepared LiBH₄to give the corresponding alcohol, which was

converted into bromides 20a−c by treatment with CBr₄. The

bromo group in 20b was substituted with 1,2,4-triazole

followed by Boc deprotection with 4 M HCl in dioxane and

subsequent amide coupling with (S)-2-phenylpropionic acid to

furnish 1,2,4-triazole 21. An S_N2 displacement of the bromo

group in 20a−c with NaN₃in DMF provided azides 22a−c.

Removal of the Boc group with HCl led to the ammonium

salts 23a−c, which were subsequently subjected to HBTU-

mediated coupling with (S)-2-phenylpropionic acid to give

amides 24a−c. Click reaction of 24b and 24a with acetylene

provided 1,2,3-triazoles 25 and 26, respectively. Bromide 20a

was subjected to an S_N2 reaction with tetrabutylammonium

cyanide followed by Boc deprotection and HBTU-mediated

coupling of the resulting amine with (S)-2-phenylpropionic

acid to give nitrile 27. Compound 27 was reacted with NaN₃in

the presence of ZnBr₂to provide tetrazole 28.

RESULTS AND DISCUSSION

■

Chemistry. The newly designed GPR88 agonists were

synthesized following procedures depicted in Schemes 1−4. All

ﬁnal products were characterized by H NMR, ¹³C NMR, and

1

high-resolution mass spectrometry (HRMS) and determined

to be >95% pure by high-performance liquid chromatography

(HPLC) analyses. The reaction yield is presented in the

Experimental Section. As shown in Scheme 1, reaction of esters

8a,b with N′-hydroxyethanimidamide in the presence of NaH

aﬀorded 1,2,4-oxadiazoles 9 and 10. On the other hand,

treatment of 8b with hydrazine aﬀorded the corresponding

hydrazide 11, which was then reacted with acetonitrile in the

presence of K₂CO₃in n-butanol at 150 °C to furnish 3-

substituted 5-methyl-4H-1,2,4-triazole 12.

1,2,3-Triazoles 16 and 17 were synthesized in accordance

with the reactions in Scheme 2. Reduction of ester 8b with

LiBH₄, prepared freshly from NaBH₄and LiCl, aﬀorded

alcohol 13, which was oxidized subsequently by Dess−Martin

periodinane (DMP) in dichloromethane (DCM) to give

aldehyde 14. The aldehyde was converted into the terminal

alkyne by treating 14 with Ohira’s reagent to provide 15. The

alkyne was subjected to standard copper-catalyzed azide-alkyne

cycloaddition (CuAAC) conditions with TMS−N₃to provide

the desired 4-substituted 1H-1,2,3-triazole 16. Methylation of

the triazole N−H with iodomethane gave 2-methyl-2H-1,2,3-

Synthesis of 1,4-disubstituted 1H-1,2,3-triazoles 29−53 is

depicted in Scheme 4. Cyclization of azide 24b with an

appropriate alkyne under CuAAC or thermal conditions

provided 1,2,3-triazoles 29−45 and 47−49. Dess−Martin

oxidation of the 4-hydroxymethyl group in 39 gave aldehyde

46. The ester in 48 was converted into the corresponding

carboxylic acid, which was subjected to HBTU-mediated

coupling with methylamine or dimethylamine to furnish

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c

Table 1. Biological Data of Amide Bioisosteres

a

b

clog P was calculated using Instant JChem 5.4.0 (ChemAxon Ltd.). pEC₅₀values are mean standard error of at least three independent

c

experiments performed in duplicate. E_maxvalue is % of RTI-13951-33 maximal signal (mean standard error).

amides 50 and 51, respectively. Likewise, cyclization of azide

24c with propargyl alcohol under CuAAC conditions gave 52.

Finally, cyclization of azide 24c with ethyl propiolate, followed

by functional group transformations of the ester yielded the N-

methyl amide 53 by employing conditions analogous to the

synthesis of 50.

Pharmacological Evaluation and SAR Study. Our

Lance time-resolved ﬂuorescence resonance energy transfer

(TR-FRET) cAMP assay with stable CHO-GPR88 cells was

optimal balance between potency and lipophilicity. The data

are summarized in Table 1. The previously reported 1,3,4-

oxadiazole 6²⁸is included to highlight SAR comparison. In this

SAR, both 2-methylpentyl and cyclobutylmethyl groups were

used in the alkoxy side chain as the cyclobutylmethyl analogue

has a lower lipophilicity (in general an order of magnitude of

clog P) than that of the 2-methylpentyl counterpart. As it can

be seen from Table 1, replacement of the 5-methyl-1,3,4-

oxadiazole moiety in 6 with a 3-methyl-1,2,4-oxadizaole led to

an equally potent compound 9 (EC₅₀= 132 nM); however, it

has a signiﬁcantly higher clog P than that of 6 (5.7 vs 4.5).

Attempts to lower clog P by substituting the 2-methylpentyl

side chain with a cyclobutylmethyl group led to a 2-fold less

potent compound 10. Holding the cyclobutylmethyl side chain

in analogue 10 constant, we next introduced 5-methyl-4H-

1,2,4-triazole to give 12, resulting in further loss of activity

(EC₅₀= 501 nM). On the contrary, 1H-1,2,3-triazole 16 and 2-

methyl-2H-1,2,3-triazole 17 were moderately active with EC₅₀

values of 355 and 372 nM, respectively, but oﬀered no

advantage in terms of lipophilicity. An improvement in potency

was realized when a 1H-1,2,4-triazole moiety was used as the

amide bioisostere to give 21, which had a comparable potency

used to assess the agonist potency (EC₅₀) and eﬃcacy (E_max

)

at the GPR88 receptor as described previously.^24,28In these

assays, RTI-13951-33 (2, Figure 1) was used as the reference

compound and had a collectively average EC₅₀of 45 nM (n >

100). E_maxvalues (%) of test compounds were evaluated with

RTI-13951-33 as 100%. In the previous SAR study of 2-AMPP,

we revealed that GPR88 agonist activity correlates with

lipophilicity of the compounds;²⁸thus, the clog P values of

all newly designed compounds were also calculated. Given the

improvement in potency with 1,3,4-oxadiazole as the

bioisostere of amide (6 vs 4, Figure 1),²⁸we ﬁrst explored

whether the 1,3,4-oxadiazole moiety can be replaced with other

ﬁve-membered heterocycles with the goal of identifying the

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c

Table 2. Biological Data of 1,2,3-Triazoles

a

b

clog P was calculated using Instant JChem 5.4.0 (ChemAxon Ltd.). pEC₅₀values are mean standard error of at least three independent

c

experiments performed in duplicate. E_maxvalue is % of RTI-13951-33 maximal signal (mean standard error).

(EC₅₀= 178 nM) to the 5-methyl-1,3,4-oxadiazole 6.

Exchanging of 1H-1,2,4-triazole in 21 with a 1H-1,2,3-triazole

led to further improvement of potency. The 1H-1,2,3-triazole

analogues 25 and 26 with EC₅₀values of 95 and 60 nM,

respectively, emerged as the most potent and eﬃcacious (both

with E_max= 101% relative to RTI-13951-33) agonists from the

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heterocyclic bioisosteres investigated. Finally, tetrazole 28 was

completely inactive which was expected as 1,2,3,4-tetrazoles

are better known as carboxylic acid bioisosteres. Overall, this

SAR suggests that a variety of azoles containing three

heteroatoms can serve as amide bioisosteres in the 2-AMPP

scaﬀold with moderate to high GPR88 agonist activity.

Considering both potency and synthetic feasibility for a rapid

SAR expansion, the 1H-1,2,3-triazole 25 was chosen as the

starting point for further optimization.

The SAR of the modiﬁed 1H-1,2,3-triazole is shown in Table

2, again holding the cyclobutylmethyl side chain constant. The

1,2,3-triazole moiety in 25 tolerated a substitution at the 4-

position as the 4-methyl analogue 29 (EC₅₀= 89 nM) was

equipotent to 25 but did not tolerate a substitution at the 5-

position as the 4,5-dimethyl analogue 30 (EC₅₀= 457 nM) was

5-fold less potent. The C-5 hydrogen atom of 1,2,3-triazoles is

known to be a hydrogen-bond donor.³¹In this case, the lack of

tolerance of a substitution at C-5 is presumably due to the

necessity of a hydrogen-bond donor at this position.

Consequently, a range of alkyl and aryl functional substitutions

at C-4 were synthesized keeping the C-5 unsubstituted. In

general, the GPR88 activity decreased with the increase in size

of the substitutions with a preferred rank order of methyl (29)

> ethyl (31) ≈ n-propyl (32) > i-propyl (33) > t-butyl (34) >

n-hexyl (35). Interestingly, the C-4 position was well tolerated

with a cycloalkyl group (e.g., cyclopropyl 36 and cyclohexyl

37) or a phenyl group (38), all of which had EC₅₀values less

than 350 nM. In the further SAR studies, substitutions with

diﬀerent electronic properties (e.g., hydroxyl, amino, electron-

donating, and electron-withdrawing groups) were probed.

Addition of a hydroxymethyl group at C-4 (39) had similar

activity to 25 (EC₅₀= 91 vs 95 nM) but reduced the clog P by

almost a unit. On the other hand, when the hydroxyl group in

39 was capped with a methyl ether (40) or an acetyl ester

(41), it led to approximately 2- to 3-fold loss of activity. In

Figure 2. Concentration−response curves of RTI-13951-33 and 53 in

the GPR88 Lance TR-FRET cAMP assay. The TR-FRET signal (665

nm) was converted to fmol cAMP by interpolating from the standard

cAMP curve. Percent inhibition of forskolin-induced (300 nM) cAMP

levels was plotted against the log of compound concentration, and

data were ﬁt to a three-parameter logistic curve to generate EC₅₀

values. Each data point is the mean standard deviation (S.D.) of at

least three independent experiments performed in duplicate.

Because GPR88 is highly expressed in the striatum, we

further characterized 53 in the [³⁵S]GTPγS binding assays

using striatal membrane preparations from WT mice and

GPR88 KO mice to conﬁrm its agonist activity and GPR88-

speciﬁcity. RTI-13951-33 was used as the reference compound

and had an EC₅₀of 2.9 μM, which is 5-fold less potent than the

previously reported EC₅₀of 535 nM.²⁵The variation in

potency is likely due to the diﬀerent batch of striatal

membranes with diﬀerent GPR88 expression levels. Never-

theless, compound 53 also signiﬁcantly enhanced [³⁵S]GTPγS

binding activity to a similar extent as RTI-13951-33, with an

EC₅₀of 8.9 μM (E_max= 270%, n = 3) in WT mouse striatal

membranes (Figure 3). This activity was conﬁrmed using

addition, all of the aminomethyl analogues 42−44 (EC₅₀

=

700−2000 nM) suﬀered from substantial loss of activity

compared with 25 (EC₅₀= 95 nM). An ethoxy group directly

linked to the C-4 (45, EC₅₀= 407 nM) was also not favored,

indicating that incorporation of an electron-donating group

(EDG) has a detrimental eﬀect on the GPR88 activity. On the

contrary, substitution of C-4 with an electron-withdrawing

group (EWG), such as aldehyde (46), ketone (47), ester (48),

and amides (primary amide 49 and N-methyl amide 50 but not

N,N-dimethyl amide 51), had a favorable eﬀect on activity

leading to compounds with EC₅₀values either comparable or

better than that of 25. Among these, the N-methyl amide 50,

with an EC₅₀of 40 nM, was the most potent compound. In

addition, compound 50 has a favorable clog P of 3.7 which is

within the 2−4 range for potential BBB penetration.²⁹Given

that both hydroxymethyl 39 (EC₅₀= 91 nM, clog P = 3.6) and

N-methyl amide 50 (EC₅₀= 40, clog P = 3.7) have a good

balance of potency and lipophilicity, we designed and

synthesized compounds 52 and 53, in which an (S)-2-

methylbutyl group was used as the alkoxy side chain. The (S)-

2-methylbutyl analogues have been previously shown to have

superior agonist activity compared to the cyclobutylmethyl

analogues but have lower lipophilicity than the 2-methylpentyl

analogues.²⁸In agreement with the previously reported SAR,

compounds 52 and 53 were more potent than 39 and 50,

respectively. Compound 53 had an EC₅₀of 14 nM, being the

most potent compound in this study and is 3-fold more potent

than RTI-13951-33 in the functional cAMP assay (Figure 2).

Figure 3. [³⁵S]GTPγS binding of compound 53 in wild-type (WT)

mouse striatal membranes vs GPR88 KO mouse striatal membranes.

The data are the means of triplicate measurements with standard

deviation shown as error bars and are representative from three

independent experiments.

striatal membranes from commercial C57BL/6J mice with an

EC₅₀of 6.5 μM (E_max= 320%, n = 4). Importantly, the

compound at a concentration of 100 μM was inactive in striatal

membranes prepared from GPR88 KO mice, demonstrating a

GPR88-speciﬁc G-protein signaling activity in the striatum.

BBB Permeability and PK Proﬁle of Compound 53.

Considering both GPR88 agonist activity and druglike

properties (e.g., clog P and PSA, Table 3), compounds 39,

50, 52, and 53 were selected for further characterization. The

BBB penetration remains a signiﬁcant challenge for the

development of GPR88 modulators since suﬃcient brain

exposure is necessary to modulate the target receptor which is

predominantly expressed in the brain. Thus, selected

compounds were ﬁrst screened through a bidirectional

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Table 3. Physicochemical Properties and BBB Permeability

of Compounds 39, 50, 52, and 53

EXPERIMENTAL SECTION

■

Chemistry. General Methods. All solvents and chemicals were

reagent grade. Unless otherwise mentioned, all reagents and solvents

were purchased from commercial vendors and used as received. Flash

column chromatography was carried out on a Teledyne ISCO

CombiFlash Rf system using prepacked columns. Solvents used

include EtOAc, hexanes, MeOH, and DCM. ¹H and ¹³C NMR spectra

were recorded on a Bruker Avance DPX-300 (300 MHz) or a Bruker

AVANCE III HD 700 MHz spectrometer and were determined in

CDCl₃or CD₃OD with tetramethylsilane (TMS) (0.00 ppm) or

solvent peaks as the internal reference. Chemical shifts are reported in

ppm relative to the reference signal and coupling constant (J) values

are reported in hertz (Hz). Nominal mass spectra were obtained using

an Agilent InﬁnityLab MSD single quadrupole mass spectrometer

system (ESI). High-resolution mass spectra (HRMS) were obtained

using Agilent 1290 Inﬁnity UHPLC-6230 TOF mass spectrometer

(ESI). Thin-layer chromatography (TLC) was performed on EMD

precoated silica gel 60 F254 plates, and spots were visualized with UV

light or iodine staining. CMA80 for column chromatography is a

mixture of 80:18:2 chloroform/MeOH/NH₄OH. All ﬁnal compounds

were greater than 95% pure as determined by HPLC on a Waters

2695 Separation Module equipped with a Waters 2996 Photodiode

Array Detector and a Phenomenex Synergi 4 mm Hydro-RP 80A C18

250 mm × 4.6 mm column using a ﬂow rate of 1 mL/min starting

with 1 min at 5% solvent B, followed by a 15 min gradient of 5−95%

solvent B, followed by 9 min at 95% solvent B (solvent A, H₂O with

0.1% TFA; solvent B, acetonitrile with 0.1% TFA and 5% H₂O;

absorbance monitored at 280 nm). All of the synthesized target

desired value

39

91

50

40

52

77

53

14

cAMP EC₅₀(nM)

a

clog P

PSA

2−4

<90

>3

3.6

89.3

6.7

35.1

5.2

3.7

4.1

4.2

a

98.1

10.9

23.6

2.2

89.3

10.6

31.9

3.0

98.1

13.3

23.5

1.8

P_app(10⁻⁶cm/s) A-to-B

P_app(10⁻⁶cm/s) B-to-A

eﬄux ratio

<2.5

a

clog P and PSA were calculated using Instant JChem 5.4.0

(ChemAxon Ltd.).

transport assay using Madin−Darby canine kidney (MDCK)

cells expressing the human multidrug resistance protein 1

(MDR1) gene before selecting and testing a lead compound in

the PK assessment. As shown in Table 3, all compounds

demonstrated good apical to basolateral apparent permeability

P_app(A → B) values of 6−13 × 10⁻⁶cm/s, which is higher

than the recommended threshold of 3 × 10⁻⁶cm/s.³⁹Both

hydroxymethyl analogues 39 and 52 are likely P-glycoprotein

(Pgp) substrates as indicated by their high eﬄux ratios (P_B→A

_A→B). On the other hand, both N-methyl amides 50 and 53

have eﬄux ratios within the desired value of <2.5.⁴⁰

/

P

Compound 53, with the best potency and BBB permeability,

was further evaluated in a mouse PK study using a single dose

of 20 mg/kg by intraperitoneal (ip) injection. The results are

summarized in Table 4. Compound 53 had a good plasma

1

compounds were characterized by H NMR, ¹³C NMR, and HRMS,

and determined to be >95% pure by HPLC analyses.

(2S)-N-[(1S)-2-(3-Methyl-1,2,4-oxadiazol-5-yl)-1-{4-[(2-

methylpentyl)oxy]phenyl}ethyl]-2-phenylpropanamide (9). To a

solution of 8a²⁸(80 mg, 0.19 mmol) in dry THF (10 mL) at 0 °C

under nitrogen were added N′-hydroxyethanimidamide (57.6 mg,

0.78 mmol), NaH (8.5 mg, 0.21 mmol), and molecular sieves (3 Å,

300 mg). After stirring at room temperature for 3 h, the reaction was

quenched with saturated aqueous NH₄Cl solution and diluted with

EtOAc. The organic layer was separated, and the aqueous layer was

extracted with EtOAc (2 × 15 mL). The combined organic layers

were washed with brine (2 × 30 mL), dried (Na₂SO₄), and

concentrated under reduced pressure. Flash column chromatography

of the crude product on silica gel using 0−30% EtOAc in hexanes

Table 4. PK Proﬁle of Compound 53 in Mice

mouse PK (20 mg/kg, ip)

plasma

brain

C_max

(ng/mL)

t_1/2

(h)

CL

C_max

(ng/mL)

B/P

compound

(mL/min/kg)

ratio

53

1670

5.6

21

576

0.34

exposure after ip and reached plasma C_maxof 1670 ng/mL at 2

h. The compound had acceptable metabolic stability with a

half-life of 5.6 h and clearance (CL) of 21 mL/mg/kg. The

C_maxin the brain was 576 ng/mL (1.2 μM), which is ∼100

times its cAMP EC₅₀of 14 nM, indicating that 53 has suﬃcient

brain penetration to modulate GPR88 functions when

administered intraperitoneally. The brain-to-plasma ratio was

0.34.

1

aﬀorded 9 (65 mg, 77% yield) as a waxy solid: H NMR (300 MHz,

CDCl₃) δ 7.49−7.19 (m, 5H), 6.91 (d, J = 8.7 Hz, 2H), 6.72 (d, J =

8.7 Hz, 2H), 6.45 (d, J = 8.3 Hz, 1H), 5.43 (dd, J = 14.2, 6.0 Hz, 1H),

3.72 (dt, J = 15.0, 7.5 Hz, 1H), 3.68−3.55 (m, 2H), 3.29 (qd, J = 15.4,

6.0 Hz, 2H), 2.29 (s, 3H), 2.00−1.71 (m, 1H), 1.50 (t, J = 7.1 Hz,

3H), 1.47−1.09 (m, 4H), 0.98 (d, J = 6.7 Hz, 3H), 0.91 (t, J = 7.0 Hz,

3H); ¹³C NMR (75 MHz, CDCl₃) δ 176.2, 173.4, 166.8, 158.8, 141.1,

131.4, 128.9, 127.7, 127.3, 127.0, 114.7, 73.2, 49.9, 47.1, 35.7, 32.8,

32.6, 20.0, 18.2, 17.0, 14.3, 11.5; HRMS (ESI) m/z calcd for

C₂₆H₃₃N₃O₃[M + H]⁺436.2595, m/z found 436.2596; HPLC, t_R

18.56 min.

CONCLUSIONS

■

In summary, our optimization eﬀorts to develop novel and

potent small molecule GPR88 agonists based on the 2-AMPP

scaﬀold in this study have unraveled interesting SAR trends.

We have demonstrated that 1,2,3-triazoles can bioisosterically

replace the amide functionality as the key pharmacophore in

the 2-AMPP scaﬀold with a good balance of potency and

lipophilicity. Compound 53 emerged as the most potent

compound in this study and had an EC₅₀of 14 nM in the cell-

based functional cAMP assay and GPR88-speciﬁc agonist

activity in the mouse striatal tissues. In addition, 53 exhibited a

good BBB permeability and had a good in vivo PK proﬁle.

Taken together, our data suggest that 53 is a suitable agonist

probe for elucidating GPR88 functions and for evaluating

GPR88’s therapeutic potential for the treatment of striatal-

associated disorders such as Parkinson’s disease, schizophrenia,

and drug addiction.

(2S)-N-[(1S)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(3-methyl-1,2,4-

oxadiazol-5-yl)ethyl]-2-phenylpropanamide (10). The procedure

for the synthesis of 9 was followed starting with 8b²⁸to aﬀord 10

1

(75% yield) as a white waxy solid: H NMR (300 MHz, CDCl₃) δ

7.50−7.19 (m, 5H), 6.91 (d, J = 8.7 Hz, 2H), 6.79−6.64 (m, 2H),

6.46 (d, J = 8.3 Hz, 1H), 5.53−5.32 (m, 1H), 3.84 (d, J = 6.7 Hz,

2H), 3.61 (q, J = 7.2 Hz, 1H), 3.29 (qd, J = 15.4, 6.0 Hz, 2H), 2.72

(dt, J = 14.7, 7.5 Hz, 1H), 2.29 (s, 3H), 2.20−2.01 (m, 2H), 2.01−

1.67 (m, 4H), 1.51 (d, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃)

δ 176.2, 173.4, 166.8, 158.8, 141.1, 131.4, 128.9, 127.7, 127.3, 127.0,

114.7, 72.1, 49.9, 47.1, 34.6, 32.6, 24.8, 18.5, 18.2, 11.5; HRMS (ESI)

m/z calcd for C₂₅H₂₉N₃O₃[M + H]⁺420.2282, m/z found 420.2280;

HPLC, t_R17.22 min.

(2S)-N-[(1S)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(5-methyl-4H-

1,2,4-triazol-3-yl)ethyl]-2-phenylpropanamide (12). To a solution

of 11²⁸(50 mg, 0.13 mmol) in n-BuOH (5 mL) at room temperature

were added K₂CO₃(52.4 mg, 0.38 mmol) and CH₃CN (0.2 mL). The

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reaction was heated at 150 °C in a sealed tube for 5 h. After cooling to

room temperature, H₂O (10 mL) and the organic layer was separated.

The aqueous layer was extracted with EtOAc (2 × 10 mL). The

combined organic layers were washed with brine (2 × 10 mL), dried

(Na₂SO₄), and concentrated under reduced pressure. Flash column

chromatography of the crude product on silica gel using 0−100%

EtOAc in hexanes aﬀorded 12 (26 mg, 49% yield) as a waxy white

solid. The product was a tautomeric mixture as judged by NMR

Hz, 1H), 5.10 (dt, J = 8.2, 5.6 Hz, 1H), 3.88 (d, J = 6.7 Hz, 2H), 3.62

(q, J = 7.2 Hz, 1H), 2.85−2.48 (m, 3H), 2.21−2.02 (m, 2H), 2.02−

1.73 (m, 5H), 1.53 (d, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃)

δ 172.5, 157.9, 140.4, 131.5, 128.1, 126.9, 126.5, 113.7, 79.1, 76.4,

71.3, 70.5, 49.3, 46.4, 33.8, 24.8, 24.0, 17.7, 17.4; MS (ESI) m/z 362.4

[M + H]⁺.

(2S)-N-[(1S)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(1H-1,2,3-tria-

zol-4-yl)ethyl]-2-phenylpropanamide (16). To a stirred solution of

15 (29 mg, 0.08 mmol) in DMF/H₂O (3 mL, 9:1, v/v) at room

temperature were added CuSO₄·5H₂O (10 mg, 0.03 mmol), sodium

ascorbate (20 mg, 0.08 mmol), and TMS−N₃(21 μL, 0.16 mmol).

The reaction was heated at 90 °C for 5 h. After cooling to room

temperature, the mixture was concentrated under reduced pressure.

Flash column chromatography of the crude product on silica gel using

0−100% EtOAc in hexanes aﬀorded 16 (24 mg, 75% yield) as a white

1

studies. H NMR (300 MHz, CDCl₃) δ 7.38−7.17 (m, 5H), 7.17−

7.09 (m, 1H), 7.04 (d, J = 8.6 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.75

(d, J = 8.7 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 5.46−5.13 (m, 1H),

3.82 (dd, J = 11.9, 6.7 Hz, 2H), 3.54 (dq, J = 14.4, 7.1 Hz, 1H), 3.10

(dd, J = 15.0, 6.1 Hz, 2H), 2.70 (dq, J = 14.6, 7.5 Hz, 1H), 2.32 (s,

1.6H), 2.25 (s, 1.4H), 2.18−2.01 (m, 2H), 2.01−1.69 (m, 4H), 1.43

(dd, J = 7.1, 3.7 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 174.1,

173.9, 158.6, 158.4, 141.3, 141.1, 133.0, 132.9, 128.7, 128.6, 127.6,

127.5, 127.3, 127.1, 127.0, 114.6, 114.5, 72.1, 72.1, 51.4, 51.2, 47.1,

46.9, 34.6, 33.7, 33.6, 24.8, 18.5, 18.4, 18.2, 12.7, 12.7; HRMS (ESI)

m/z calcd for C₂₅H₃₀N₄O₂[M + H]⁺419.2442, m/z found 419.2434;

HPLC, t_R14.07 min.

1

solid: H NMR (300 MHz, CD₃OD) δ 7.44 (s, 1H), 7.30−7.11 (m,

5H), 7.07 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 8.7 Hz, 2H), 5.17 (t, J = 7.6

Hz, 1H), 3.86 (d, J = 6.6 Hz, 2H), 3.64 (q, J = 7.1 Hz, 1H), 3.17 (d, J

= 7.6 Hz, 2H), 2.82−2.59 (m, 1H), 2.18−2.02 (m, 2H), 2.02−1.70

(m, 4H), 1.35 (d, J = 7.1 Hz, 3H); ¹³C NMR (75 MHz, CD₃OD) δ

176.2, 160.0, 142.8, 134.7, 129.5, 128.6, 128.4, 127.9, 115.5, 73.2,

53.8, 47.4, 36.1, 32.5, 25.7, 19.4, 18.7; HRMS (ESI) m/z calcd for

C₂₄H₂₈N₄O₂[M + H]⁺405.2285, m/z found 405.2277; HPLC, t_R

15.37 min.

(2S)-N-[(1S)-1-[4-(Cyclobutylmethoxy)phenyl]-3-hydroxypropyl]-

2-phenylpropanamide (13). To a stirred suspension of NaBH₄(120

mg, 3.16 mmol) in EtOH (5 mL) at 0 °C was added LiCl (134 mg,

3.16 mmol). The mixture was stirred at 0 °C for 30 min. After that, a

solution of 8b (500 mg, 1.26 mmol) in THF (5 mL) was added to the

above reaction at 0 °C. After stirring at room temperature for 6 h, the

reaction was quenched with saturated NH₄Cl solution (10 mL)

followed by addition of H₂O. The mixture was extracted with EtOAc

(3 × 10 mL). The combined organic layers were washed with brine (2

× 10 mL), dried (Na₂SO₄), and concentrated under reduced pressure.

Flash column chromatography of the crude product on silica gel using

0−50% EtOAc in hexanes aﬀorded 13 (465 mg, quantitative yield) as

(2S)-N-[(1S)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(2-methyl-2H-

1,2,3-triazol-4-yl)ethyl]-2-phenylpropanamide (17). To a solution

of 16 (16 mg, 0.04 mmol) in DMF (2 mL) at room temperature were

added K₂CO₃(11 mg, 0.08 mmol) and CH₃I (2.5 μL, 0.04 mmol).

After stirring at room temperature overnight, the reaction was

quenched with H₂O (1 mL) and diluted with EtOAc (5 mL). The

organic layer was separated, and the aqueous layer was extracted with

EtOAc (2 mL). The combined organic layers were washed with H₂O

(3 × 5 mL), brine (3 × 10 mL), dried (Na₂SO₄), and concentrated

under reduced pressure. Flash column chromatography of the crude

product on silica gel using 0−100% EtOAc in hexanes aﬀorded 17 (7

mg, 42% yield) as a waxy white solid: ¹H NMR (300 MHz, CDCl₃) δ

7.42−7.14 (m, 5H), 7.02 (s, 1H), 6.85 (d, J = 8.6 Hz, 2H), 6.77−6.64

(m, 2H), 6.10 (d, J = 7.9 Hz, 1H), 5.21 (dd, J = 13.9, 6.3 Hz, 1H),

4.05 (s, 3H), 3.86 (d, J = 6.7 Hz, 2H), 3.58 (q, J = 7.2 Hz, 1H), 3.03

(qd, J = 14.8, 6.1 Hz, 2H), 2.83−2.65 (m, 1H), 2.25−2.04 (m, 2H),

2.02−1.73 (m, 4H), 1.48 (d, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz,

CDCl₃) δ 173.2, 158.4, 144.2, 141.4, 133.5, 133.1, 128.8, 127.8, 127.2,

127.1, 114.5, 72.1, 51.8, 47.1, 41.4, 34.6, 32.0, 24.8, 18.5, 18.2; HRMS

(ESI) m/z calcd for C₂₅H₃₀N₄O₂[M + H]⁺419.2442, m/z found

419.2434; HPLC, t_R16.96 min.

1

a colorless oil: H NMR (300 MHz, CDCl₃) δ 7.41−7.13 (m, 5H),

6.99 (d, J = 8.7 Hz, 2H), 6.86−6.61 (m, 2H), 5.87 (d, J = 7.5 Hz,

1H), 5.10 (td, J = 10.3, 3.9 Hz, 1H), 3.88 (d, J = 6.7 Hz, 2H), 3.71−

3.45 (m, 3H), 3.35 (m, 1H), 2.92−2.49 (m, 1H), 2.24−1.62 (m, 8H),

1.54 (d, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 174.7,

158.6,140.9, 133.2, 128.9, 127.6, 127.4, 127.3, 114.7, 72.1, 58.9, 49.9,

47.1, 38.4, 34.6, 24.8, 18.6, 18.4; MS (ESI) m/z 368.0 [M + H]⁺.

(2S)-N-[(1S)-1-[4-(Cyclobutylmethoxy)phenyl]-3-oxopropyl]-2-

phenylpropanamide (14). To a solution of 13 (300 mg, 0.85 mmol)

in DCM (10 mL, saturated with H₂O) at 0 °C was added Dess−

Martin periodinane (756 mg, 1.78 mmol) in two portions over 15

min. After stirring at 0 °C for 14 h, the reaction was diluted with H₂O

and poured into a mixture of Na₂S₂O₃(1.5 g) in NaHCO₃(20 mL,

80% saturated in H₂O). The layers were separated, and the aqueous

layer was extracted with DCM (3 × 10 mL). The combined organic

layers were washed with ice-cold NaHCO₃(10 mL), brine (2 × 15

mL), dried (Na₂SO₄), and concentrated under reduced pressure.

Flash column chromatography of the crude product on silica gel using

0−50% EtOAc in hexanes aﬀorded 14 (95 mg, 31% yield) as a waxy

Methyl (2R)-2-{[(tert-Butoxy)carbonyl]amino}-2-{4-[(2-

methylpentyl)oxy]phenyl}acetate (19a). To a solution of 18²⁸(5.0

g, 17.78 mmol) in THF (100 mL) at room temperature were added

PPh₃(7.93 g, 30.21 mmol) and 2-methylpentanol (4.41 mL, 35.55

mmol). This was followed by slow addition of DEAD (6.13 mL, 30.21

mmol) maintaining the reaction temperature below 35 °C. After

stirring at room temperature overnight, the reaction was quenched

with H₂O (50 mL) and diluted with EtOAc (100 mL). The layers

were separated, and the aqueous layer was extracted with EtOAc (2 ×

30 mL). The combined organic layers were washed with brine (3 ×

50 mL), dried (Na₂SO₄), and concentrated under reduced pressure.

Flash column chromatography of the crude product on silica gel using

0−30% EtOAc in hexanes aﬀorded 19a (6.50 g, quantitative yield) as

1

white solid: H NMR (300 MHz, CDCl₃) δ 9.82−9.49 (m, 1H),

7.48−7.14 (m, 5H), 6.98 (d, J = 8.6 Hz, 2H), 6.83−6.69 (m, 2H),

5.85 (d, J = 7.9 Hz, 1H), 5.40 (dd, J = 14.3, 6.8 Hz, 1H), 3.87 (d, J =

6.7 Hz, 2H), 3.55 (q, J = 7.1 Hz, 1H), 3.00−2.79 (m, 2H), 2.73 (dt, J

= 14.9, 7.5 Hz, 1H), 2.19−2.04 (m, 2H), 2.04−1.75 (m, 4H), 1.50 (d,

J = 7.2 Hz, 3H); MS (ESI) m/z 366.4 [M + H]⁺.

(2S)-N-[(1S)-1-[4-(Cyclobutylmethoxy)phenyl]but-3-yn-1-yl]-2-

phenylpropanamide (15). A solution of 14 (73.5 mg, 0.20 mmol)

and dimethylacetyldiazophosphonoacetate (82 μL, 0.54 mmol) in

anhydrous MeOH (5 mL) at room temperature was treated with

K₂CO₃(97.3 mg, 0.70 mmol) and stirred for 2 h. The reaction was

partitioned between EtOAc and aqueous NaHCO₃(10 mL). The

layers were separated, and the aqueous layer was extracted with

EtOAc (2 × 10 mL). The combined organic layers were washed with

brine (2 × 10 mL), dried (Na₂SO₄), and concentrated under reduced

pressure. Flash column chromatography of the crude product on silica

gel using 0−30% EtOAc in hexanes aﬀorded 15 (65 mg, 89% yield) as

1

a colorless oil: H NMR (300 MHz, CDCl₃) δ 7.25 (d, J = 8.6 Hz,

2H), 6.85 (d, J = 8.7 Hz, 2H), 5.63 (d, J = 7.0 Hz, 1H), 5.25 (d, J =

7.2 Hz, 1H), 3.78 (dd, J = 8.9, 5.8 Hz, 1H), 3.73−3.60 (m, 4H),

1.97−1.79 (m, 1H), 1.55−1.28 (m, 12H), 1.28−1.07 (m, 1H), 1.00

(d, J = 6.7 Hz, 3H), 0.91 (t, J = 7.0 Hz, 3H); ¹³C NMR (75 MHz,

CDCl₃) δ 171.8, 159.4, 154.8, 128.7, 128.3, 114.8, 79.8, 73.1, 57.0,

52.4, 35.7, 32.8, 28.2, 20.0, 16.9, 14.2; MS (ESI) m/z 388.4 [M +

Na]⁺.

Methyl (2R)-2-{[(tert-Butoxy)carbonyl]amino}-2-[4-

(cyclobutylmethoxy)phenyl]acetate (19b). The procedure for the

synthesis of 19a was followed starting with 18 and cyclo-

butylmethanol to aﬀord 19b (98% yield) as a colorless oil: ¹H

1

a waxy white solid: H NMR (300 MHz, CDCl₃) δ 7.51−7.20 (m,

5H), 7.02 (d, J = 8.6 Hz, 2H), 6.83−6.70 (m, 2H), 5.84 (d, J = 8.3

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NMR (300 MHz, CDCl₃) δ 7.25 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.6

Hz, 2H), 5.90 (d, J = 7.3 Hz, 1H), 5.25 (d, J = 7.2 Hz, 1H), 3.86 (d, J

= 6.6 Hz, 2H), 3.63 (s, 3H), 2.86−2.61 (m, 1H), 2.25−2.01 (m, 2H),

1.96−1.69 (m, 4H), 1.40 (s, 9H); MS (ESI) m/z 372.4 [M + Na]⁺.

Methyl (2R)-2-{[(tert-Butoxy)carbonyl]amino}-2-{4-[(2S)-2-

methylbutoxy]phenyl}acetate (19c). The procedure for the synthesis

of 19a was followed starting with 18 and (S)-2-methylbutanol to

aﬀord 19c (quantitative yield) as a colorless oil: ¹H NMR (300 MHz,

CDCl₃) δ 7.26 (d, J = 8.7 Hz, 2H), 6.92−6.78 (m, 2H), 5.49 (t, J =

23.9 Hz, 1H), 5.25 (d, J = 7.3 Hz, 1H), 3.79 (dd, J = 9.0, 6.0 Hz, 1H),

3.75−3.64 (m, 4H), 1.94−1.73 (m, 1H), 1.67−1.49 (m, 1H), 1.43 (s,

9H), 1.33−1.12 (m, 2H), 1.00 (d, J = 6.7 Hz, 3H), 0.93 (t, J = 7.5 Hz,

3H); ¹³C NMR (75 MHz, CDCl₃) δ 171.9, 159.4, 154.8, 128.7, 128.3,

114.8, 80.0, 72.9, 57.0, 52.5, 34.6, 28.3, 26.1, 16.5, 11.2; MS (ESI) m/

z 374.4 [M + Na]⁺.

a suspension of the salt in MeCN (2 mL) at room temperature were

added HBTU (61 mg, 0.14 mmol), N,N-diisopropylethylamine

(DIPEA) (56 μL, 0.27 mmol), and (S)-2-phenylpropionic acid (18

mg, 0.14 mmol). After stirring at room temperature overnight, the

reaction was quenched with H₂O (5 mL) and diluted with EtOAc (10

mL). The layers were separated, and the aqueous layer was extracted

with EtOAc (5 mL). The combined EtOAc layers were washed with

brine (3 × 10 mL), dried (Na₂SO₄), and concentrated under reduced

pressure. Flash column chromatography of the crude product on silica

gel using 0−100% EtOAc in hexanes aﬀorded 21 (25 mg, 46% yield

1

over three steps) as a white solid: H NMR (300 MHz, CDCl₃) δ

7.81 (s, 1H), 7.54 (s, 1H), 7.45−7.16 (m, 5H), 6.95−6.52 (m, 5H),

5.46−5.14 (m, 1H), 4.41 (ddd, J = 19.2, 14.0, 4.6 Hz, 2H), 3.83 (d, J

= 6.6 Hz, 2H), 3.63 (q, J = 7.2 Hz, 1H), 2.89−2.53 (m, 1H), 2.25−

2.03 (m, 2H), 2.03−1.76 (m, 4H), 1.50 (d, J = 7.2 Hz, 3H); ¹³C

NMR (75 MHz, CDCl₃) δ 173.6, 158.9, 152.1, 144.0, 141.0, 129.7,

128.9, 127.7, 127.4, 126.9, 114.8, 72.1, 53.9, 52.9, 47.1, 34.6, 24.8,

18.5, 17.9; HRMS (ESI) m/z calcd for C₂₄H₂₈N₄O₂[M + H]⁺

405.2285, m/z found 405.2269; HPLC, t_R15.72 min.

tert-Butyl N-[(1R)-2-Bromo-1-{4-[(2-methylpentyl)oxy]phenyl}-

ethyl]carbamate (20a). This compound was synthesized according

to our previously reported procedure.²⁶

tert-Butyl N-[(1R)-2-Bromo-1-[4-(cyclobutylmethoxy)phenyl]-

ethyl]carbamate (20b). To a suspension of NaBH₄(541 mg, 14.30

mmol) in EtOH (20 mL) at 0 °C under nitrogen was added LiCl

(607 mg, 14.30 mmol). After stirring at 0 °C for 10 min, a solution of

19b (2.0 g, 5.72 mmol) in THF (20 mL) was added. The reaction

mixture was stirred at room temperature for 3 h and quenched with

saturated NH₄Cl solution (30 mL), followed by addition of H₂O (30

mL). The mixture was extracted with EtOAc (3 × 100 mL). The

combined organic layers were washed with brine (2 × 50 mL), dried

(Na₂SO₄), and concentrated under reduced pressure. Flash column

chromatography of the crude product on silica gel using 0−100%

EtOAc in hexanes aﬀorded the intermediate alcohol (1.7 g, 92% yield)

as a waxy white solid: MS (ESI) m/z 344.2 [M + Na]⁺. To a solution

of the intermediate alcohol (1.6 g, 4.98 mmol) in THF (40 mL) at

room temperature were added PPh₃(1.96 g, 7.47 mmol) and CBr₄

(2.48 g, 7.47 mmol). After stirring at room temperature overnight, the

mixture was ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. Flash column chromatography of the crude product on silica

gel using 0−30% EtOAc in hexanes aﬀorded 20b (1.1 g, 58% yield) as

tert-Butyl N-[(1R)-2-Azido-1-{4-[(2-methylpentyl)oxy]phenyl}-

ethyl]carbamate (22a). This compound was synthesized according

to our previously reported procedure.²⁶

tert-Butyl N-[(1R)-2-Azido-1-[4-(cyclobutylmethoxy)phenyl]-

ethyl]carbamate (22b). To a solution of 20b (1.0 g, 2.60 mmol)

in DMF (15 mL) at room temperature was added NaN₃(677 mg,

10.41 mmol). After stirring at room temperature overnight, the

reaction was quenched by H₂O (30 mL) and extracted with EtOAc (3

× 50 mL). The combined organic layers were washed with brine (2 ×

50 mL), dried (Na₂SO₄), and concentrated under reduced pressure.

Flash column chromatography of the crude product on silica gel using

0−100% EtOAc in hexanes aﬀorded 22b (870 mg, 97% yield) as a

waxy white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.20 (d, J = 8.6 Hz,

2H), 6.88 (d, J = 8.7 Hz, 2H), 5.04 (br s, 1H), 4.93 (br s, 1H), 3.91

(d, J = 6.7 Hz, 2H), 3.74−3.45 (m, 2H), 2.93−2.53 (m, 1H), 2.26−

2.04 (m, 2H), 2.04−1.77 (m, 4H), 1.44 (s, 9H); MS (ESI) m/z 369.4

[M + Na]⁺.

tert-Butyl N-[(1R)-2-Azido-1-{4-[(2S)-2-methylbutoxy]phenyl}-

ethyl]carbamate (22c). The procedure for the synthesis of 22b

was followed starting with 20c to aﬀord 22c (92% yield) as a waxy

white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.20 (d, J = 8.7 Hz, 2H),

6.96−6.79 (m, 2H), 4.97 (br s, 1H), 4.80 (br s, 1H), 3.76 (ddd, J =

26.1, 9.0, 6.3 Hz, 2H), 3.60 (d, J = 5.4 Hz, 2H), 1.97−1.74 (m, 1H),

1.69−1.49 (m, 1H), 1.44 (s, 9H), 1.36−1.14 (m, 1H), 1.00 (d, J = 6.7

Hz, 3H), 0.94 (t, J = 7.5 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ

159.1, 155.0, 131.1, 127.6, 114.8, 80.0, 72.9, 55.7, 53.7, 34.7, 28.3,

26.1, 16.5, 11.3; MS (ESI) m/z 371.2 [M + Na]⁺.

(1R)-2-Azido-1-{4-[(2-methylpentyl)oxy]phenyl}ethan-1-amine

Hydrochloride (23a). This compound was synthesized according to

our previously reported procedure.²⁶

(1R)-2-Azido-1-[4-(cyclobutylmethoxy)phenyl]ethan-1-amine

Hydrochloride (23b). To a solution of 22b (870 mg, 2.51 mmol) in

DCM (20 mL) at room temperature was added 4 M HCl in dioxane

(6.3 mL). After stirring at room temperature overnight, the mixture

was concentrated under reduced pressure to aﬀord 23b (740 mg,

quantitative yield) as a residue, which was used for the next step

without puriﬁcation.

(1R)-2-Azido-1-{4-[(2S)-2-methylbutoxy]phenyl}ethan-1-amine

Hydrochloride (23c). The procedure for the synthesis of 23b was

followed starting with 22c to aﬀord 23c (quantitative yield) as a

residue, which was used for the next step without puriﬁcation.

(2S)-N-[(1R)-2-Azido-1-{4-[(2-methylpentyl)oxy]phenyl}ethyl]-2-

phenylpropanamide (24a). This compound was synthesized

according to our previously reported procedure.²⁶

(2S)-N-[(1R)-2-Azido-1-[4-(cyclobutylmethoxy)phenyl]ethyl]-2-

phenylpropanamide (24b). To a solution of 23b (0.8 g, 2.83 mmol)

in MeCN (30 mL) at 0 °C were added HBTU (1.61 g, 4.24 mmol),

Et₃N (2.37 mL, 16.98 mmol), and (S)-2-phenylpropionic acid (425

mg, 2.83 mmol). After stirring at room temperature 5 h, the reaction

was quenched with saturated NaHCO₃(20 mL) and diluted with

EtOAc (30 mL). The organic layer was separated. The aqueous layer

was extracted with EtOAc (2 × 10 mL). The combined organic layers

1

a white solid: H NMR (300 MHz, CDCl₃) δ 7.20 (d, J = 8.6 Hz,

2H), 6.96−6.81 (m, 2H), 5.07 (s, 1H), 4.93 (s, 1H), 3.91 (d, J = 6.7

Hz, 2H), 3.76−3.43 (m, 2H), 2.95−2.61 (m, 1H), 2.27−2.03 (m,

2H), 2.03−1.77 (m, 4H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃)

δ 159.0, 155.0, 131.3, 127.5, 114.7, 80.0, 72.1, 54.5, 37.1, 34.6, 28.3,

24.8, 18.6; MS (ESI) m/z 406.2 [M + Na]⁺(⁷⁹Br), 408.2 [M + Na]⁺

(⁸¹Br).

tert-Butyl N-[(1R)-2-Bromo-1-{4-[(2S)-2-methylbutoxy]phenyl}-

ethyl]carbamate (20c). The procedure for the synthesis of 20b

was followed starting with 19c to aﬀord 20c (60% yield over 2 steps)

as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.24−7.10 (m, 2H),

6.95−6.79 (m, 2H), 5.08 (br s, 1H), 4.93 (br s, 1H), 3.87−3.51 (m,

4H), 1.98−1.75 (m, 1H), 1.66−1.48 (m, 1H), 1.44 (s, 9H), 1.35−

1.17 (m, 1H), 1.00 (d, J = 6.7 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H); ¹³C

NMR (75 MHz, CDCl₃) δ 159.1, 155.0, 131.3, 127.5, 114.7, 80.0,

72.9, 54.6, 37.1, 34.7, 28.3, 26.1, 16.5, 11.3; MS (ESI) m/z 408.2 [M

+ Na]⁺(⁷⁹Br), 410.2 [M + Na]⁺(⁸¹Br).

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(1H-1,2,4-tria-

zol-1-yl)ethyl]-2-phenylpropanamide (21). To a solution of 20b (35

mg, 0.09 mmol) in DMF (2 mL) at room temperature were added

1,2,4-triazole (12.4 mg, 0.14 mmol) and K₂CO₃(38 mg, 0.27 mmol).

After stirring at room temperature overnight, the reaction was

quenched with H₂O (2 mL) and diluted with EtOAc (10 mL). The

layers were separated, and the aqueous layer was extracted with

EtOAc (5 mL). The combined organic layers were washed with brine

(3 × 10 mL), dried (Na₂SO₄), and concentrated under reduced

pressure. Flash column chromatography of the crude product on silica

gel using 0−100% EtOAc in hexanes aﬀorded the intermediate N-Boc

triazole (33 mg, 97% yield) as a white solid: MS (ESI) m/z 373.4 [M

+ H]⁺. Removal of the Boc protecting group of this material (33 mg,

0.09 mmol) was achieved by treatment with 4 M HCl in dioxane

(0.27 mL) at room temperature overnight. The solvent was removed

under reduced pressure to give the corresponding amine HCl salt. To

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were washed with brine (2 × 30 mL), dried (Na₂SO₄), and

concentrated under reduced pressure. Flash column chromatography

of the crude product on silica gel using 0−50% EtOAc in hexanes

and (S)-2-phenylpropionic acid (17 mg, 0.11 mmol). After stirring at

room temperature for 5 h, the reaction was quenched with H₂O and

diluted with EtOAc (10 mL). The layers were separated, and the

organic layer was washed with brine (2 × 10 mL), dried (Na₂SO₄),

and concentrated under reduced pressure. Flash column chromatog-

raphy of the crude product on silica gel using 0−50% EtOAc in

hexanes aﬀorded 27 (30 mg, 77% yield) as a brown residue: ¹H NMR

(300 MHz, CDCl₃) δ 7.45−7.21 (m, 5H), 7.05 (d, J = 8.7 Hz, 2H),

6.82 (d, J = 8.7 Hz, 2H), 5.81 (d, J = 7.0 Hz, 1H), 5.24−4.99 (m,

1H), 3.88−3.51 (m, 3H), 3.12−2.91 (m, 1H), 2.81 (dd, J = 16.7, 4.6

Hz, 1H), 1.92 (dd, J = 12.6, 6.2 Hz, 1H), 1.53 (d, J = 7.2 Hz, 3H),

1.50−1.10 (m, 4H), 0.99 (d, J = 6.7 Hz, 3H), 0.91 (t, J = 7.0 Hz, 3H);

¹³C NMR (75 MHz, CDCl₃) δ 174.0, 159.5, 140.7, 129.7, 129.0,

1

aﬀorded 24b (0.97 g, 91% yield) as a colorless oil: H NMR (300

MHz, CDCl₃) δ 7.45−7.17 (m, 5H), 6.96 (d, J = 8.6 Hz, 2H), 6.84−

6.64 (m, 2H), 5.91 (d, J = 7.9 Hz, 1H), 5.23−4.95 (m, 1H), 3.87 (d, J

= 6.6 Hz, 2H), 3.69−3.42 (m, 3H), 2.90−2.57 (m, 1H), 2.21−2.03

(m, 2H), 2.03−1.71 (m, 4H), 1.51 (d, J = 7.2 Hz, 3H); ¹³C NMR (75

MHz, CDCl₃) δ 173.6, 159.0, 141.1, 130.5, 129.0, 127.6, 127.5, 127.4,

114.8, 72.1, 55.1, 52.1 47.1, 34.6, 24.8, 18.6, 18.3; MS (ESI) m/z

379.2 [M + H]⁺.

(2S)-N-[(1R)-2-Azido-1-{4-[(2S)-2-methylbutoxy]phenyl}ethyl]-2-

phenylpropanamide (24c). The procedure for the synthesis of 24b

was followed starting with 23c to aﬀord 24c (1.96 g, quantitative

127.6, 127.5, 127.2, 117.0, 115.1, 73.3, 49.3, 47.0, 35.7, 32.8, 24.4,

1

20.0, 18.3, 16.9, 14.3; MS (ESI) m/z 379.2 [M + H]⁺.

yield) as a waxy white solid: H NMR (300 MHz, CDCl₃) δ 7.37−

7.23 (m, 5H), 6.96 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.7 Hz, 2H), 5.80

(d, J = 7.8 Hz, 1H), 5.22−4.94 (m, 1H), 3.87−3.41 (m, 5H), 1.90−

1.75 (m, 1H), 1.63−1.41 (m, 4H), 1.33−1.15 (m, 1H), 1.04−0.85

(m, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 173.6, 159.0, 141.0, 130.4,

129.0, 127.6, 127.5, 127.4, 114.8, 72.9, 55.1, 52.1, 47.1, 34.7, 26.1,

18.3, 16.5, 11.3; MS (ESI) m/z 381.2 [M + H]⁺.

(2S)-N-[(1S)-1-{4-[(2-Methylpentyl)oxy]phenyl}-2-(2H-1,2,3,4-tet-

razol-5-yl)ethyl]-2-phenylpropanamide (28). To a solution of 27

(20 mg, 0.05 mmol) in 1,4-dioxane (3 mL) at room temperature were

added NaN₃(4.1 mg, 0.06 mmol) and ZnBr₂(18 mg, 0.08 mmol).

The reaction was reﬂuxed for 48 h. After cooling to room

temperature, the mixture was acidiﬁed to pH ∼1 with 1 N HCl.

The solution was extracted with EtOAc (4 × 10 mL). The combined

organic layers were dried (Na₂SO₄) and concentrated under reduced

pressure. Flash column chromatography of the crude product on silica

gel using 7% MeOH in DCM aﬀorded 28 (15 mg, 68% yield) as an

oﬀ-white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.31−7.13 (m, 5H),

7.05 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 8.6 Hz, 2H), 5.31 (t, J = 7.1 Hz,

1H), 3.86−3.53 (m, 3H), 3.47−3.35 (m, 2H), 1.96−1.73 (m, 1H),

1.58−1.09 (m, 7H), 0.98 (d, J = 6.7 Hz, 3H), 0.92 (t, J = 7.0 Hz, 3H);

¹³C NMR (75 MHz, CD₃OD) δ 176.2, 160.2, 156.1, 142.8,

133.6,129.5, 128.6, 128.4, 127.9, 115.5, 74.2, 52.8, 47.3, 36.9, 34.1,

31.4, 21.1, 18.7, 17.3, 14.6; HRMS (ESI) m/z calcd for C₂₄H₃₁N₅O₂

[M + H]⁺422.2551, m/z found 422.2540; HPLC, t_R16.35 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-methyl-1H-

1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (29). To a solution

of 24b (80 mg, 0.21 mmol) in t-BuOH:H₂O (5 mL, 1:1, v/v) at room

temperature were added CuSO₄·5H₂O (21.1 mg, 0.08 mmol), sodium

ascorbate (41.8 mg, 0.21 mmol), and propyne bubbled into the

reaction via a needle. After stirring at room temperature overnight, the

reaction was treated with NH₄OH/NH₄Cl (5 mL, 1:9, v/v) and

extracted with EtOAc (3 × 10 mL). The combined organic layers

were washed with brine (2 × 10 mL), dried (Na₂SO₄), and

concentrated under reduced pressure. Flash column chromatography

of the crude product on silica gel using 0−30% CMA80 in DCM

aﬀorded 29 (71 mg, 80% yield) as a waxy white solid: ¹H NMR (300

MHz, CDCl₃) δ 7.42−7.07 (m, 5H), 6.96 (s, 1H), 6.83 (d, J = 8.7 Hz,

2H), 6.73 (d, J = 8.7 Hz, 2H), 6.41 (d, J = 7.9 Hz, 1H), 5.43−5.12

(m, 1H), 4.56 (qd, J = 14.0, 5.8 Hz, 2H), 3.85 (d, J = 6.6 Hz, 2H),

3.56 (q, J = 7.1 Hz, 1H), 2.87−2.54 (m, 1H), 2.25 (s, 3H), 2.19−2.02

(m, 2H), 2.02−1.74 (m, 4H), 1.45 (d, J = 7.2 Hz, 3H); ¹³C NMR (75

MHz, CDCl₃) δ 173.8, 159.0, 143.1, 140.9, 129.7, 128.9, 127.5, 127.3,

122.2, 114.8, 72.1, 54.1, 52.8, 47.0, 34.6, 24.8, 18.5, 18.1, 10.7; HRMS

(ESI) m/z calcd for C₂₅H₃₀N₄O₂[M + H]⁺419.2442, m/z found

419.2435; HPLC, t_R16.07 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(1H-1,2,3-tria-

zol-1-yl)ethyl]-2-phenylpropanamide (25). To a stirred solution of

24b (100 mg, 0.26 mmol) in MeOH/H₂O (5 mL, 1:1, v/v) at room

temperature were added K₂CO₃(55 mg, 0.40 mmol), CuSO₄·5H₂O

(66 mg, 0.26 mmol), sodium ascorbate (53 mg, 0.26 mmol), and

trimethylsilyl acetylene (56 μL, 0.40 mmol). After stirring at room

temperature for 14 h, the reaction was treated with NH₄OH/NH₄Cl

(5 mL, 1:9, v/v) and extracted with EtOAc (3 × 10 mL). The

combined organic layers were washed with brine (2 × 10 mL), dried

(Na₂SO₄), and concentrated under reduced pressure. Flash column

chromatography of the crude product on silica gel using 0−30%

CMA80 in DCM aﬀorded 25 (59 mg, 55% yield) as a waxy white

1

solid: H NMR (300 MHz, CDCl₃) δ 7.56 (s, 1H), 7.39−7.16 (m,

6H), 6.81 (d, J = 8.6 Hz, 2H), 6.72 (d, J = 8.6 Hz, 2H), 6.47 (d, J =

7.9 Hz, 1H), 5.36 (dd, J = 12.2, 7.0 Hz, 1H), 4.65 (qd, J = 14.0, 5.7

Hz, 2H), 3.84 (d, J = 6.6 Hz, 2H), 3.57 (q, J = 7.1 Hz, 1H), 2.88−

2.57 (m, 1H), 2.21−2.04 (m, 2H), 2.00−1.70 (m, 4H), 1.45 (d, J =

7.1 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.8, 159.0, 140.9,

133.6, 129.5, 128.9, 127.5, 127.3, 127.2, 124.5, 114.9, 72.1, 54.2, 52.7,

47.0, 34.6, 24.8, 18.5, 18.1; HRMS (ESI) m/z calcd for C₂₄H₂₈N₄O₂

[M + H]⁺405.2285, m/z found 405.2278; HPLC, t_R16.08 min.

(2S)-N-[(1R)-1-{4-[(2-Methylpentyl)oxy]phenyl}-2-(1H-1,2,3-tria-

zol-1-yl)ethyl]-2-phenylpropanamide (26). The procedure for the

synthesis of 25 was followed starting with 24a to aﬀord 26 (20%

yield) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.57 (d, J = 0.7

Hz, 1H), 7.40−7.08 (m, 6H), 6.88−6.60 (m, 4H), 6.30 (d, J = 7.9 Hz,

1H), 5.46−5.26 (m, 1H), 4.66 (qd, J = 14.0, 5.6 Hz, 2H), 3.82−3.49

(m, 3H), 2.00−1.77 (m, 1H), 1.50−1.14 (m, 7H), 0.98 (d, J = 6.7 Hz,

3H), 0.91 (t, J = 7.0 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.7,

159.1, 140.8, 133.6, 129.3, 128.9, 127.6, 127.3, 127.2, 124.5, 114.8,

73.3, 54.2, 52.8, 47.0, 35.7, 32.8, 20.0, 18.1, 16.9, 14.2; HRMS (ESI)

m/z calcd for C₂₅H₃₂N₄O₂[M + H]⁺421.2598, m/z found 421.2594;

HPLC, t_R17.37 min.

(2S)-N-[(1S)-2-Cyano-1-{4-[(2-methylpentyl)oxy]phenyl}ethyl]-2-

phenylpropanamide (27). To a solution of 20a (200 mg, 0.50 mmol)

in THF (5 mL) at room temperature was added tetrabutylammonium

cyanide (336 mg, 1.25 mmol). The reaction was heated at 50 °C for 2

h. After cooling to room temperature, the mixture was concentrated

under reduced pressure. Flash column chromatography of crude

product on silica gel using 0−50% EtOAc in hexanes to aﬀord N-Boc

protected cyano intermediate (150 mg, 87% yield). Removal of the

Boc protecting group of this material (35 mg, 0.10 mmol) was

achieved by treatment with TFA:DCM (1:2, 1 mL). After stirring at 0

°C for 1 h, the reaction was quenched with cold saturated aqueous

NaHCO₃(10 mL) and extracted with EtOAc (2 × 20 mL). The

organic layer was washed with brine (2 × 20 mL), dried (Na₂SO₄),

and concentrated to provide the intermediate amine (25.5 mg). To a

solution of the amine (25.5 mg, 0.10 mmol) in MeCN (2 mL) at 0 °C

were added HBTU (59 mg, 0.15 mmol), Et₃N (43 μL, 0.31 mmol),

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4,5-dimethyl-

1H-1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (30). A solution

of 24b (50 mg, 0.13 mmol) and 2-butyne (0.1 mL, 1.3 mmol) in

toluene (2 mL) was heated at 125 °C for 18 h in a sealed tube. After

cooling to room temperature, the mixture was concentrated under

reduced pressure. Flash column chromatography of the crude product

on silica gel using 0−100% EtOAc in hexanes aﬀorded 30 (38 mg,

1

63% yield) as a white waxy solid: H NMR (300 MHz, CDCl₃) δ

7.35−7.15 (m, 5H), 6.83 (d, J = 7.8 Hz, 1H), 6.77−6.61 (m, 4H),

5.40−5.18 (m, 1H), 4.41 (ddd, J = 20.8, 14.2, 5.3 Hz, 2H), 3.84 (d, J

= 6.7 Hz, 2H), 3.58 (q, J = 7.1 Hz, 1H), 2.89−2.50 (m, 1H), 2.17 (s,

3H), 2.15−1.80 (m, 6H), 1.79 (d, J = 4.8 Hz, 3H), 1.45 (d, J = 7.1

Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.6, 159.0, 141.1, 140.4,

130.1, 128.8, 127.5, 127.2, 127.1, 114.8, 72.2, 52.3, 52.1, 46.9, 34.6,

24.8, 18.5, 18.1, 10.2, 7.2; HRMS (ESI) m/z calcd for C₂₆H₃₂N₄O₂

[M + H]⁺433.2598, m/z found 433.2592; HPLC, t_R16.06 min.

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(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-ethyl-1H-

1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (31). To a solution

of 24b (51 mg, 0.13 mmol) in MeOH (5 mL) were added sodium

ascorbate (30 mg, 0.16 mmol), CuSO₄·5H₂O (30 mg, 0.13 mmol), 2-

pentynoic acid (15 μL, 0.15 mmol), and Cs₂CO₃(33 mg, 0.1 mmol).

The reaction was heated at 60 °C for 2 h. After cooling to room

temperature, the mixture was ﬁltered through a short of pad of Celite,

the ﬁltrate was concentrated under reduced pressure. The residue was

puriﬁed by preparative TLC on silica gel eluted with 30% EtOAC/

3H), 1.28 (s, 6H), 0.88 (t, J = 6.5 Hz, 3H); ¹³C NMR (75 MHz,

CDCl₃) δ 173.7, 159.0, 148.2, 141.0, 129.7, 128.9, 127.5, 127.2, 127.2,

121.8, 114.8, 72.1, 54.3, 52.7, 47.0, 34.6, 31.5, 29.4, 28.8, 25.5, 24.8,

22.5, 18.5, 18.1, 14.0; HRMS (ESI) m/z calcd for C₃₀H₄₀N₄O₂[M +

H]⁺489.3224, m/z found 489.3218; HPLC, t_R19.07 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-cyclopropyl-

1H-1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (36). The proce-

dure for the synthesis of 29 was followed starting with 24b and

1

cyclopropyl acetylene to aﬀord 36 (80% yield) as a white solid: H

1

NMR (300 MHz, CDCl₃) δ 7.40−7.18 (m, 5H), 6.92 (s, 1H), 6.82

(d, J = 8.6 Hz, 2H), 6.73 (d, J = 8.7 Hz, 2H), 6.36 (d, J = 7.7 Hz, 1H),

5.30 (dd, J = 12.0, 7.1 Hz, 1H), 4.54 (qd, J = 14.0, 5.8 Hz, 2H), 3.86

(d, J = 6.6 Hz, 2H), 3.55 (q, J = 7.0 Hz, 1H), 2.86−2.56 (m, 1H),

2.24−2.03 (m, 2H), 2.03−1.69 (m, 5H), 1.45 (d, J = 7.1 Hz, 3H),

1.03−0.82 (m, 2H), 0.82−0.52 (m, 2H); ¹³C NMR (75 MHz,

CDCl₃) δ 173.7, 159.0, 150.1, 140.9, 129.7, 128.9, 127.5, 127.3, 120.7,

114.8, 72.1, 54.2, 52.7, 47.0, 34.6, 24.8, 18.5, 18.2, 7.7, 7.6, 6.5; HRMS

(ESI) m/z calcd for C₂₇H₃₂N₄O₂[M + H]⁺445.2598, m/z found

445.2592; HPLC, t_R16.73 min.

(2S)-N-[(1R)1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-cyclohexyl-

1H-1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (37). The proce-

dure for the synthesis of 29 was followed starting with 24b and

cyclohexyl acetylene to furnish 37 (80% yield) as a waxy white solid:

¹H NMR (300 MHz, CDCl₃) δ 7.41−7.14 (m, 5H), 6.91 (s, 1H),

6.75 (q, J = 8.8 Hz, 4H), 6.41 (d, J = 7.9 Hz, 1H), 5.43−5.18 (m,

1H), 4.56 (ddd, J = 21.0, 14.0, 5.7 Hz, 2H), 3.85 (d, J = 6.7 Hz, 2H),

3.55 (q, J = 7.1 Hz, 1H), 2.90−2.56 (m, 2H), 2.21−2.04 (m, 2H),

2.03−1.58 (m, 10H), 1.45 (d, J = 7.1 Hz, 3H), 1.40−1.19 (m, 4H);

¹³C NMR (75 MHz, CDCl₃) δ 173.7, 159.0, 153.5, 141.0, 129.8,

128.8, 127.5, 127.2, 120.6, 114.8, 72.2, 54.3, 52.8, 47.0, 35.1, 34.6,

32.9, 32.9, 26.0, 24.8, 18.5, 18.2; HRMS (ESI) m/z calcd for

C₃₀H₃₈N₄O₂[M + H]⁺487.3068, m/z found 487.3062; HPLC, t_R

18.26 min.

hexanes to aﬀord 31 (36 mg, 60% yield) as a white waxy solid: H

NMR (300 MHz, CDCl₃) δ 7.37−7.14 (m, 5H), 6.96 (s, 1H), 6.81

(d, J = 8.7 Hz, 2H), 6.73 (d, J = 8.8 Hz, 2H), 6.40 (d, J = 7.8 Hz, 1H),

5.32 (td, J = 7.3, 4.7 Hz, 1H), 4.57 (qd, J = 14.0, 5.8 Hz, 2H), 3.85 (d,

J = 6.6 Hz, 2H), 3.56 (q, J = 7.2 Hz, 1H), 2.85−2.49 (m, 3H), 2.21−

2.01 (m, 2H), 2.01−1.65 (m, 4H), 1.45 (d, J = 7.1 Hz, 3H), 1.19 (t, J

= 7.6 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.7, 159.0, 149.7,

140.9, 129.7, 128.9, 127.5, 127.2, 121.3, 114.8, 72.1, 54.22, 52.8, 47.0,

34.6, 24.8, 18.9, 18.5, 18.1, 13.7; HRMS (ESI) m/z calcd for

C₂₆H₃₂N₄O₂[M + H]⁺433.2598, m/z found 433.2591; HPLC, t_R

16.40 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-propyl-1H-

1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (32). The procedure

for the synthesis of 29 was followed starting with 24b and 1-pentyne

to aﬀord 32 (80% yield) as a white solid: ¹H NMR (300 MHz,

CDCl₃) δ 7.34−7.14 (m, 5H), 6.94 (s, 1H), 6.80 (d, J = 8.7 Hz, 2H),

6.71 (d, J = 8.8 Hz, 2H), 6.52 (d, J = 7.9 Hz, 1H), 5.48−5.14 (m,

1H), 4.56 (qd, J = 14.0, 5.7 Hz, 2H), 3.84 (d, J = 6.6 Hz, 2H), 3.57

(q, J = 7.1 Hz, 1H), 2.82−2.64 (m, 1H), 2.59 (t, J = 7.5 Hz, 2H),

2.23−2.03 (m, 2H), 2.04−1.77 (m, 4H), 1.70−1.50 (m, 2H), 1.44 (d,

J = 7.1 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H); ¹³C NMR (75 MHz,

CDCl₃) δ 173.7, 159.0, 148.0, 141.0, 129.7, 128.8, 127.5, 127.2, 121.8,

114.8, 72.1, 54.2, 52.7, 46.9, 34.6, 27.5, 24.8, 22.6, 18.5, 18.2, 13.6;

HRMS (ESI) m/z calcd for C₂₇H₃₄N₄O₂[M + H]⁺447.2755, m/z

found 447.2750; HPLC, t_R17.17 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-[4-(propan-2-

yl)-1H-1,2,3-triazol-1-yl]ethyl]-2-phenylpropanamide (33). The

procedure for the synthesis of 29 was followed starting with 24b

and 3-methyl-1-butyne to aﬀord 33 (56% yield) as a waxy white solid:

¹H NMR (300 MHz, CDCl₃) δ 7.46−7.12 (m, 5H), 6.92 (s, 1H),

6.83−6.65 (m, 4H), 6.34 (d, J = 7.8 Hz, 1H), 5.39−5.23 (m, 1H),

4.62 (dd, J = 14.0, 4.4 Hz, 1H), 4.50 (dd, J = 14.0, 6.9 Hz, 1H), 3.85

(d, J = 6.7 Hz, 2H), 3.55 (q, J = 7.1 Hz, 1H), 3.09−2.91 (m, 1H),

2.82−2.60 (m, 1H), 2.19−2.02 (m, 2H), 2.02−1.74 (m, 4H), 1.45 (d,

J = 7.2 Hz, 3H), 1.23 (d, J = 6.9 Hz, 6H); ¹³C NMR (75 MHz,

CDCl₃) δ 173.7, 159.0, 154.5, 141.0, 129.7, 128.9, 127.5, 127.2, 127.2,

120.4, 114.8, 72.1, 54.3 52.8, 47.0, 34.6, 25.7, 24.8, 22.5, 22.5, 18.5,

18.1; HRMS (ESI) m/z calcd for C₂₇H₃₄N₄O₂[M + H]⁺447.2755,

m/z found 447.2748; HPLC, t_R17.08 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-phenyl-1H-

1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (38). The procedure

for the synthesis of 29 was followed starting with 24b and

1

phenylacetylene to aﬀord 38 (74% yield) as a white solid: H NMR

(300 MHz, CDCl₃) δ 7.73 (d, J = 7.1 Hz, 2H), 7.47−7.37 (m, 3H),

7.37−7.15 (m, 6H), 6.86 (d, J = 8.7 Hz, 2H), 6.75 (d, J = 8.7 Hz,

2H), 6.26 (d, J = 7.7 Hz, 1H), 5.38 (dd, J = 12.1, 6.8 Hz, 1H), 4.68

(qd, J = 14.1, 5.8 Hz, 2H), 3.85 (d, J = 6.7 Hz, 2H), 3.58 (q, J = 7.1

Hz, 1H), 2.72 (dt, J = 14.5, 7.3 Hz, 1H), 2.21−2.01 (m, 2H), 2.01−

1.66 (m, 4H), 1.47 (d, J = 7.2 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃)

δ 173.8, 159.1, 147.6, 140.8, 130.4, 129.4, 128.9, 128.8, 128.2, 127.5,

127.3, 127.3, 125.8, 120.6, 115.0, 72.1, 54.4, 52.9, 47.1, 34.6, 24.8,

18.5, 18.1; HRMS (ESI) m/z calcd for C₃₀H₃₂N₄O₂[M + H]⁺

481.2598, m/z found 481.2591; HPLC, t_R17.70 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-[4-(hydroxy-

methyl)-1H-1,2,3-triazol-1-yl]ethyl]-2-phenylpropanamide (39).

The procedure for the synthesis of 29 was followed starting with

24b and propargyl alcohol to aﬀord 39 (75% yield) as a white solid:

¹H NMR (300 MHz, CDCl₃) δ 7.52−7.14 (m, 6H), 6.85 (d, J = 8.7

Hz, 2H), 6.75 (d, J = 8.7 Hz, 2H), 6.28 (d, J = 7.9 Hz, 1H), 5.33 (dd,

J = 12.6, 7.2 Hz, 1H), 4.77−4.49 (m, 4H), 3.85 (d, J = 6.6 Hz, 2H),

3.56 (q, J = 7.1 Hz, 1H), 2.73 (dt, J = 14.8, 7.5 Hz, 1H), 2.51 (br s,

1H), 2.22−2.02 (m, 2H), 2.02−1.77 (m, 4H), 1.45 (d, J = 7.2 Hz,

3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.9, 159.1, 147.5, 140.8, 129.3,

128.9, 127.5, 127.3, 127.3, 122.6, 114.9, 72.1, 56.5, 54.2, 52.8, 47.0,

34.6, 24.8, 18.5, 18.1; HRMS (ESI) m/z calcd for C₂₅H₃₀N₄O₃[M +

H]⁺435.2391, m/z found 435.2385; HPLC, t_R14.49 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-[4-(methoxy-

methyl)-1H-1,2,3-triazol-1-yl]ethyl]-2-phenylpropanamide (40).

The procedure for the synthesis of 29 was followed starting with

24b and methoxyethyne to aﬀord 40 (75% yield) as a white solid: ¹H

NMR (700 MHz, CDCl₃) δ 7.31 (t, J = 7.5 Hz, 2H), 7.28−7.24 (m,

1H), 7.22 (d, J = 5.9 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 6.73 (d, J =

8.6 Hz, 2H), 6.36 (d, J = 7.8 Hz, 1H), 5.41−5.29 (m, 1H), 4.65 (dd, J

= 14.1, 4.6 Hz, 1H), 4.58 (dd, J = 14.1, 7.0 Hz, 1H), 4.52−4.44 (m,

2H), 3.85 (d, J = 6.6 Hz, 2H), 3.57 (q, J = 7.1 Hz, 1H), 3.32 (s, 3H),

2.82−2.64 (m, 1H), 2.16 − 2.05 (m, 2H), 2.00−1.87 (m, 2H), 1.87−

(2S)-N-[(1R)-2-(4-tert-Butyl-1H-1,2,3-triazol-1-yl)-1-[4-

(cyclobutylmethoxy)phenyl]ethyl]-2-phenylpropanamide (34). The

procedure for the synthesis of 29 was followed starting with 24b and

3,3-dimethyl-1-butyne to aﬀord 34 (80% yield) as a waxy white solid:

¹H NMR (300 MHz, CDCl₃) δ 7.35−7.20 (m, 5H), 6.91 (s, 1H),

6.82−6.66 (m, 4H), 6.43−6.27 (m, 1H), 5.41−5.23 (m, 1H), 4.55

(ddd, J = 21.0, 14.0, 5.7 Hz, 2H), 3.85 (d, J = 6.7 Hz, 2H), 3.55 (q, J

= 7.1 Hz, 1H), 2.83−2.60 (m, 1H), 2.21−2.02 (m, 2H), 2.02−1.76

(m, 4H), 1.45 (d, J = 7.1 Hz, 3H), 1.27 (d, J = 6.3 Hz, 9H); ¹³C NMR

(75 MHz, CDCl₃) δ 173.7, 159.0, 157.5, 141.0, 129.8, 128.8, 127.5,

127.2, 119.8, 114.8, 72.2, 54.3, 52.8, 47.0, 34.6, 30.6, 30.3, 24.8, 18.5,

18.2; HRMS (ESI) m/z calcd for C₂₈H₃₆N₄O₂[M + H]⁺461.2911,

m/z found 461.2905; HPLC, t_R17.56 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-hexyl-1H-

1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (35). The procedure

for the synthesis of 29 was followed starting with 24b and 1-octyne to

1

aﬀord 35 (90% yield) as a waxy white solid: H NMR (300 MHz,

CDCl₃) δ 7.39−7.15 (m, 5H), 6.93 (s, 1H), 6.75 (dd, J = 21.9, 8.8

Hz, 4H), 6.44 (d, J = 7.9 Hz, 1H), 5.44−5.21 (m, 1H), 4.56 (ddd, J =

20.8, 14.0, 5.6 Hz, 2H), 3.84 (d, J = 6.6 Hz, 2H), 3.57 (q, J = 7.1 Hz,

1H), 2.84−2.66 (m, 1H), 2.61 (t, J = 7.6 Hz, 2H), 2.23−2.02 (m,

2H), 2.02−1.69 (m, 4H), 1.63−1.48 (m, 2H), 1.45 (d, J = 7.1 Hz,

12408

https://doi.org/10.1021/acs.jmedchem.1c01075

J. Med. Chem. 2021, 64, 12397−12413

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1.75 (m, 2H), 1.46 (d, J = 7.2 Hz, 3H); ¹³C NMR (175 MHz, CDCl₃)

δ 173.8, 159.0, 144.9, 140.9, 129.4, 128.9, 127.5, 127.3, 127.2, 123.5,

114.8, 72.1, 65.7, 58.1, 54.3, 52.7, 47.0, 34.5, 24.8, 18.5, 18.2; HRMS

(ESI) m/z calcd for C₂₆H₃₂N₄O₃[M + H]⁺449.2547, m/z found

449.2543; HPLC, t_R15.60 min.

52.6, 47.0, 34.6, 24.8, 18.5, 18.2, 14.7; HRMS (ESI) m/z calcd for

C₂₆H₃₂N₄O₃[M + H]⁺449.2547, m/z found 449.2541; HPLC, t_R

16.69 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-formyl-1H-

1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (46). To a solution

of alcohol 39 (30 mg, 0.07 mmol) in DCM (2 mL, saturated with

H₂O) was added Dess−Martin periodinane (44 mg, 0.10 mmol).

After stirring at room temperature for 3 h, the reaction was treated

with saturated NaHCO₃(3 mL) and extracted with DCM (3 × 5

mL). The combined organic layers were washed with brine, dried

(Na₂SO₄), and concentrated under reduced pressure. Flash column

chromatography of the crude product on silica gel using 0−10%

{1-[(2R)-2-[4-(Cyclobutylmethoxy)phenyl]-2-[(2S)-2-

phenylpropanamido]ethyl]-1H-1,2,3-triazol-4-yl}methyl Acetate

(41). The procedure for the synthesis of 29 was followed starting

with 24b and propargyl acetate to aﬀord 41 (70% yield) as a waxy

white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.38−7.10 (m, 5H), 6.82

(d, J = 8.7 Hz, 2H), 6.77−6.64 (m, 2H), 6.28 (d, J = 7.9 Hz, 1H),

5.41−5.25 (m, 1H), 5.11 (s, 2H), 4.62 (qd, J = 14.0, 5.8 Hz, 2H),

3.85 (d, J = 6.6 Hz, 2H), 3.56 (q, J = 7.1 Hz, 1H), 2.90−2.51 (m,

1H), 2.22−2.06 (m, 2H), 2.04 (s, 3H), 1.99−1.75 (m, 4H), 1.46 (d, J

= 7.2 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.8, 170.7, 159.1,

142.7, 140.8, 128.9, 127.5, 127.3, 127.2, 124.8, 114.9, 72.1, 57.4, 54.3,

52.8, 47.0, 34.6, 24.8, 20.8, 18.5, 18.1; HRMS (ESI) m/z calcd for

C₂₇H₃₂N₄O₄[M + H]⁺477.2496, m/z found 477.2489; HPLC, t_R

16.20 min.

1

MeOH in DCM aﬀorded 46 (21 mg, 70% yield) as a thick oil: H

NMR (300 MHz, CDCl₃) δ 10.06 (s, 1H), 7.74 (s, 1H), 7.45−7.12

(m, 5H), 6.78 (dd, J = 20.6, 8.8 Hz, 4H), 6.06 (d, J = 7.5 Hz, 1H),

5.34 (dd, J = 12.9, 6.2 Hz, 1H), 4.88−4.52 (m, 2H), 3.85 (d, J = 6.6

Hz, 2H), 3.59 (q, J = 7.2 Hz, 1H), 2.81−2.55 (m, 1H), 2.20−2.01 (m,

2H), 2.01−1.73 (m, 4H), 1.49 (d, J = 7.2 Hz, 3H); ¹³C NMR (75

MHz, CDCl₃) δ 184.6, 174.0, 159.4, 147.3, 140.6, 129.0, 128.5, 127.5,

127.2, 126.2, 115.1, 72.1, 54.6, 53.0, 47.1, 34.5, 24.8, 18.5, 18.0;

HRMS (ESI) m/z calcd for C₂₅H₂₈N₄O₃[M + H]⁺433.2230, m/z

found 433.2230; HPLC, t_R15.85 min.

(2S)-N-[(1R)-2-[4-(Aminomethyl)-1H-1,2,3-triazol-1-yl]-1-[4-

(cyclobutylmethoxy)phenyl]ethyl]-2-phenylpropanamide Hydro-

chloride (42). The procedure for the synthesis of 29 was followed

starting with 24b and propargylamine to aﬀord 42 (75% yield) as a

(2S)-N-[(1R)-2-(4-Acetyl-1H-1,2,3-triazol-1-yl)-1-[4-

(cyclobutylmethoxy)phenyl]ethyl]-2-phenylpropanamide (47). The

procedure for the synthesis of 29 was followed starting with 24b and

1

white solid: H NMR [free base] (300 MHz, CDCl₃) δ 7.43−7.16

(m, 5H), 7.12 (s, 1H), 6.83 (d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.7 Hz,

2H), 6.30 (d, J = 7.8 Hz, 1H), 5.33 (dd, J = 11.9, 6.9 Hz, 1H), 4.60

(qd, J = 14.0, 5.8 Hz, 2H), 3.98−3.73 (m, 4H), 3.57 (q, J = 7.2 Hz,

1H), 2.91−2.55 (m, 1H), 2.23−2.02 (m, 2H), 2.02−1.70 (m, 4H),

1.46 (d, J = 7.1 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.7, 159.1,

149.5, 140.9, 129.5, 128.9, 127.5, 127.3, 127.2, 121.7, 114.9, 72.1,

54.3, 52.8, 47.0, 37.5, 34.6, 24.8, 18.5, 18.2; HRMS (ESI) m/z calcd

for C₂₅H₃₁N₅O₂[M + H]⁺434.2551, m/z found 434.2542; HPLC, t_R

13.06 min. The sample was converted into the HCl salt.

1

but-3-yn-2-one to aﬀord 47 (50% yield) as a waxy white solid: H

NMR (300 MHz, CDCl₃) δ 7.73 (s, 1H), 7.37−7.15 (m, 5H), 6.83

(d, J = 8.7 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H), 6.10 (d, J = 7.8 Hz, 1H),

5.34 (dd, J = 12.6, 6.8 Hz, 1H), 4.84−4.44 (m, 2H), 3.85 (d, J = 6.6

Hz, 2H), 3.58 (q, J = 7.2 Hz, 1H), 2.83−2.68 (m, 1H), 2.65 (s, 3H),

2.21−2.02 (m, 2H), 2.02−1.77 (m, 4H), 1.48 (d, J = 7.2 Hz, 3H); ¹³C

NMR (75 MHz, CDCl₃) δ 192.4, 173.9, 159.3, 147.8, 140.7, 129.0,

127.5, 127.4, 127.2, 126.3, 115.1, 72.1, 54.5, 52.9, 47.1, 34.5, 27.1,

24.8, 18.5, 18.1; HRMS (ESI) m/z calcd for C₂₆H₃₀N₄O₃[M + H]⁺

447.2391, m/z found 447.2381; HPLC, t_R16.33 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-{4-

[(methylamino)methyl]-1H-1,2,3-triazol-1-yl}ethyl]-2-phenylpropa-

namide (43). The procedure for the synthesis of 29 was followed

starting with 24b and N-methylpropargylamine to aﬀord 43 (80%

yield) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.44−7.20 (m,

5H), 7.16 (s, 1H), 6.81 (d, J = 8.7 Hz, 2H), 6.72 (d, J = 8.8 Hz, 2H),

6.37 (d, J = 7.9 Hz, 1H), 5.50−5.25 (m, 1H), 4.60 (qd, J = 14.0, 5.7

Hz, 2H), 3.84 (d, J = 6.6 Hz, 2H), 3.78 (s, 2H), 3.58 (q, J = 7.1 Hz,

1H), 2.80−2.63 (m, 1H), 2.38 (s, 3H), 2.23−2.04 (m, 2H), 2.04−

1.76 (m, 4H), 1.51−1.39 (m, 3H); ¹³C NMR (75 MHz, CDCl₃) δ

173.7, 159.0, 146.2, 140.9, 129.5, 128.9, 127.5, 127.3, 127.2, 122.8,

114.9, 72.1, 54.3, 52.7, 47.0, 46.3, 35.6, 34.6, 24.8, 18.5, 18.2; HRMS

(ESI) m/z calcd for C₂₆H₃₃N₅O₂[M + H]⁺448.2707, m/z found

448.2700; HPLC, t_R13.41 min.

Ethyl 1-[(2R)-2-[4-(Cyclobutylmethoxy)phenyl]-2-[(2S)-2-

phenylpropanamido]ethyl]-1H-1,2,3- triazole-4-carboxylate (48).

The procedure for the synthesis of 29 was followed starting with 24b

1

and ethyl propiolate to aﬀord 48 (77% yield) as a white solid: H

NMR (300 MHz, CDCl₃) δ 7.80 (s, 1H), 7.38−7.12 (m, 5H), 6.84

(d, J = 8.7 Hz, 2H), 6.75 (d, J = 8.7 Hz, 2H), 6.21 (d, J = 7.8 Hz, 1H),

5.34 (dd, J = 12.6, 6.9 Hz, 1H), 4.85−4.59 (m, 2H), 4.39 (q, J = 7.1

Hz, 2H), 3.85 (d, J = 6.6 Hz, 2H), 3.57 (q, J = 7.1 Hz, 1H), 2.86−

2.59 (m, 1H), 2.22−2.03 (m, 2H), 2.03−1.72 (m, 4H), 1.47 (d, J =

7.2 Hz, 3H), 1.39 (t, J = 7.1 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ

174.0, 160.5, 159.2, 140.7, 140.0, 129.0, 127.5, 127.4, 127.3, 115.1,

72.1, 61.3, 54.5, 52.9, 47.0, 34.5, 24.8, 18.5, 18.1, 14.3; HRMS (ESI)

m/z calcd for C₂₇H₃₂N₄O₄[M + H]⁺477.2496, m/z found 477.2489;

HPLC, t_R16.72 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-{4-

[(dimethylamino)methyl]-1H-1,2,3-triazol-1-yl}ethyl]-2-phenylpro-

panamide (44). The procedure for the synthesis of 29 was followed

starting with 24b and N,N-dimethylpropargylamine to aﬀord 44 (75%

yield) as a white solid: ¹H NMR (300 MHz, CDCl₃) δ 7.34−7.11 (m,

5H), 7.07 (s, 1H), 6.67 (dd, J = 20.0, 8.7 Hz, 4H), 6.31 (d, J = 7.9 Hz,

1H), 5.42−5.14 (m, 1H), 4.53 (ddd, J = 20.6, 14.0, 5.5 Hz, 2H), 3.77

(d, J = 6.6 Hz, 2H), 3.60−3.31 (m, 3H), 2.78−2.49 (m, 1H), 2.13 (s,

6H), 2.10−1.98 (m, 2H), 1.93−1.64 (m, 4H), 1.40 (d, J = 7.1 Hz,

3H); ¹³C NMR (75 MHz, CDCl₃) δ 171.9, 157.3, 143.0, 139.2, 127.7,

127.2, 125.8, 125.5, 125.4, 122.0, 113.1, 70.4, 52.8, 52.4, 50.9, 45.3,

43.1, 32.8, 23.1, 16.8, 16.4; HRMS (ESI) m/z calcd for C₂₇H₃₅N₅O₂

[M + H]⁺462.2864, m/z found 462.2855; HPLC, t_R14.07 min.

(2S)-N-[(1R)-1-[4-(Cyclobutylmethoxy)phenyl]-2-(4-ethoxy-1H-

1,2,3-triazol-1-yl)ethyl]-2-phenylpropanamide (45). The procedure

for the synthesis of 29 was followed starting with 24b and

1-[(2R)-2-[4-(Cyclobutylmethoxy)phenyl]-2-[(2S)-2-

phenylpropanamido]ethyl]-1H-1,2,3-triazole-4-carboxamide (49).

The procedure for the synthesis of 29 was followed starting with

1

24b and propiolamide to aﬀord 49 (76% yield) as a white solid: H

NMR (300 MHz, CD₃OD) δ 8.25 (s, 1H), 7.32−7.08 (m, 7H), 6.82

(d, J = 8.7 Hz, 2H), 5.42 (dd, J = 9.1, 5.7 Hz, 1H), 4.83−4.63 (m,

2H), 3.88 (d, J = 6.6 Hz, 2H), 3.62 (q, J = 7.2 Hz, 1H), 2.89−2.51

(m, 1H), 2.22−2.03 (m, 2H), 2.03−1.73 (m, 4H), 1.32 (d, J = 7.0 Hz,

3H); ¹³C NMR (75 MHz, CD₃OD) δ 176.5, 164.7, 160.6, 143.6,

142.6, 131.2, 129.5, 128.9, 128.3, 128.2, 128.0, 115.8, 73.2, 55.3, 54.1,

47.4, 36.1, 25.7, 19.3, 18.7; HRMS (ESI) m/z calcd for C₂₅H₂₉N₅O₃

[M + H]⁺448.2343, m/z found 448.2341; HPLC, t_R15.68 min.

1-[(2R)-2-[4-(Cyclobutylmethoxy)phenyl]-2-[(2S)-2-

phenylpropanamido]ethyl]-N-methyl-1H-1,2,3-triazole-4-carboxa-

mide (50). A solution of ester 48 (50 mg, 0.10 mmol) and NaOH

(4.6 mg, 0.11 mmol) in MeOH/H₂O (1 mL, 1:1) was stirred at room

temperature for 2 h. The reaction was acidiﬁed with 1 N HCl and

extracted with EtOAc (3 × 5 mL). The combined organic layers were

dried (Na₂SO₄) and concentrated under reduced pressure to aﬀord

the corresponding acid (45 mg, 95% yield) as a white solid: MS (ESI)

m/z 449 [M + H]⁺. The carboxylic acid (50 mg, 0.11 mmol) was then

1

ethoxyethyne to aﬀord 45 (80% yield) as a white solid: H NMR

(300 MHz, CDCl₃) δ 7.40−7.09 (m, 5H), 6.84 (d, J = 8.7 Hz, 2H),

6.79−6.70 (m, 2H), 6.68 (s, 1H), 6.29 (d, J = 7.9 Hz, 1H), 5.42−5.19

(m, 1H), 4.51 (qd, J = 14.0, 5.9 Hz, 2H), 4.28−4.06 (m, 2H), 3.85 (d,

J = 6.6 Hz, 2H), 3.56 (q, J = 7.1 Hz, 1H), 2.85−2.50 (m, 1H), 2.24−

2.03 (m, 2H), 2.03−1.73 (m, 4H), 1.46 (d, J = 7.2 Hz, 3H), 1.37 (t, J

= 7.0 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 173.7, 160.6, 159.1,

140.9, 129.5, 128.9, 127.5, 127.3, 127.2, 114.9, 107.2, 72.1, 66.3, 55.0,

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dissolved in DMF (4 mL), followed by addition of EDCI (32.1 mg,

0.17 mmol), HOBt (22.6 mg, 0.17 mmol), DIPEA (99.6 μL, 0.55

mmol) and methylamine hydrochloride (11.3 mg, 0.17 mmol). After

stirring at room temperature for 24 h, the mixture was diluted with

EtOAc (5 mL) and treated with saturated NaHCO₃(2 mL). The

organic layer was separated, and the aqueous layer was extracted with

EtOAc (2 × 5 mL). The combined organic layers were washed with

brine (4 × 5 mL), dried (Na₂SO₄), and concentrated under reduced

pressure. Flash column chromatography of the crude product on silica

gel using 0−30% CMA80 in DCM aﬀorded 50 (42 mg, 82% yield) as

sulfonic acid) (HEPES), 0.1% bovine serum albumin (BSA) stabilizer,

and 0.5 mM ﬁnal IBMX was prepared and titrated to pH 7.4 at room

temperature. Serial dilutions of the test compounds (5 μL) and 300

nM forskolin (5 μL), both prepared at 4× the desired ﬁnal

concentration in 2% DMSO/stimulation buﬀer, were added to a

96-well white 1/2 area microplate (PerkinElmer). A cAMP standard

curve was prepared at 4× the desired ﬁnal concentration in

stimulation buﬀer and 5 μL was added to the assay plate. Stable

PPLS-HA-GPR88 CHO cells were lifted with versene and spun at

270g for 10 min. The cell pellet was resuspended in stimulation buﬀer

and 4000 cells (10 μL) were added to each well except wells

containing the cAMP standard curve. After incubating for 30 min at

room temperature, Eu-cAMP tracer and uLIGHT-anti-cAMP working

solutions were added per the manufacturer’s instructions. After

incubation at room temperature for 1 h, the TR-FRET signal (ex 337

nm) was read on a CLARIOstar multimode plate reader (BMG

Biotech, Cary, NC).

Data Analysis. The TR-FRET signal (665 nm) was converted to

fmol cAMP by interpolating from the standard cAMP curve. Fmol

cAMP was plotted against the log of compound concentration and

data were ﬁt to a three-parameter logistic curve to generate EC₅₀

values (Prism, version 6.0, GraphPad Software, Inc., San Diego, CA).

The E_maxvalue for each test compound relative to the control

compound RTI-13951-33 was calculated with the equation % control

E_max= (maximal test compound signal/maximal control signal) ×

100.

1

a white solid: H NMR (700 MHz, CD₃OD) δ 8.21 (s, 1H), 7.27−

7.16 (m, 5H), 7.15−7.10 (m, 2H), 6.82 (d, J = 8.7 Hz, 2H), 5.42 (dd,

J = 9.4, 5.5 Hz, 1H), 4.74 (ddd, J = 23.4, 13.9, 7.5 Hz, 2H), 4.58 (s,

1H), 3.89 (d, J = 6.5 Hz, 2H), 3.63 (q, J = 7.1 Hz, 1H), 2.93 (d, J =

4.9 Hz, 3H), 2.76−2.65 (m, 1H), 2.23−2.07 (m, 2H), 2.06−1.77 (m,

4H), 1.32 (d, J = 7.1 Hz, 3H); ¹³C NMR (175 MHz, CD₃OD) δ

173.6, 160.1, 157.6, 140.8, 139.7, 128.2, 126.6, 126.0, 125.4, 125.0,

124.7, 112.8, 70.2, 52.3, 51.1, 44.4, 33.1, 23.1, 22.8, 16.4, 15.8; HRMS

(ESI) m/z calcd for C₂₆H₃₁N₅O₃[M + H]⁺462.2500, m/z found

462.2649; HPLC, t_R15.14 min.

1-[(2R)-2-[4-(Cyclobutylmethoxy)phenyl]-2-[(2S)-2-

phenylpropanamido]ethyl]-N,N-dimethyl-1H-1,2,3-triazole-4-car-

boxamide (51). The procedure for the synthesis of 50 was followed

starting with 48 and dimethylamine hydrochloride to aﬀord 51 (75%

1

yield) as a sticky solid: H NMR (700 MHz, CDCl₃) δ 7.79 (s, 1H),

7.34−7.28 (m, 2H), 7.27−7.19 (m, 4H), 6.83 (d, J = 8.6 Hz, 2H),

6.74 (d, J = 8.7 Hz, 2H), 6.23 (d, J = 7.8 Hz, 1H), 5.38−5.27 (m,

1H), 4.66 (ddd, J = 20.9, 14.0, 5.8 Hz, 2H), 3.85 (d, J = 6.6 Hz, 2H),

3.58 (q, J = 7.2 Hz, 1H), 3.48 (s, 3H), 3.09 (d, J = 6.1 Hz, 3H), 2.80−

2.64 (m, 1H), 2.17−2.05 (m, 2H), 2.01−1.88 (m, 2H), 1.88−1.73

(m, 2H), 1.47 (d, J = 7.2 Hz, 3H); ¹³C NMR (175 MHz, CDCl₃) δ

173.8, 161.2, 159.2, 144.3, 140.9, 128.9, 128.8, 127.6, 127.4, 127.2,

114.9, 72.0, 54.3, 52.8, 47.0, 38.7, 36.3, 34.5, 24.8, 18.6, 18.5, 18.2;

HRMS (ESI) m/z calcd for C₂₇H₃₃N₅O₃[M + H]⁺476.2656, m/z

found 476.2649; HPLC, t_R15.47 min.

(2S)-N-[(1R)-2-[4-(Hydroxymethyl)-1H-1,2,3-triazol-1-yl]-1-{4-

[(2S)-2-methylbutoxy]phenyl} ethyl]-2-phenylpropanamide (52).

The procedure for the synthesis of 29 was followed starting with

24c and propargyl alcohol to aﬀord 52 (63% yield) as a waxy white

solid: ¹H NMR (300 MHz, CDCl₃) δ 7.44−7.11 (m, 6H), 6.90 (d, J =

8.7 Hz, 2H), 6.73 (d, J = 8.7 Hz, 2H), 6.61 (d, J = 8.1 Hz, 1H), 5.35

(dd, J = 14.2, 6.4 Hz, 1H), 4.75−4.50 (m, 4H), 3.83−3.59 (m, 3H),

3.54 (q, J = 7.1 Hz, 1H), 2.33 (br s, 1H), 1.97−1.68 (m, 1H), 1.67−

1.46 (m, 1H), 1.40 (d, J = 7.1 Hz, 3H), 1.33−1.14 (m, 1H), 0.98 (d, J

= 6.7 Hz, 3H), 0.92 (t, J = 7.4 Hz, 3H); ¹³C NMR (175 MHz,

CDCl₃) δ 174.1, 159.1, 147.8, 140.9, 129.4, 128.8, 127.5, 127.4, 127.3,

122.8, 114.8, 72.90, 56.2, 54.2, 52.8, 46.8, 34.7, 26.1, 18.2, 16.5, 11.3;

HRMS (ESI) m/z calcd for C₂₅H₃₂N₄O₃[M + H]⁺437.2547, m/z

found 437.2529; HPLC, t_R15.47 min.

[³⁵S]GTPγS Binding Assay. [³⁵S]GTPγS binding assays were

performed on membrane preparations from wild-type (WT) mice

or GPR88 KO mice, following our previously published methods.^15,16

To assess [³⁵S]GTPγS binding in the whole striatal region, brains

were quickly removed after cervical dislocation and the whole striatal

region was dissected out, frozen, and stored at −80 °C until use.

Membranes were prepared by homogenizing brain samples in ice-cold

0.25 M sucrose solution 10 vol (mL/g wet weight of tissue). The

obtained suspensions were then centrifuged at 2500g for 10 min.

Supernatants were collected and diluted 10 times in buﬀer containing

50 mM TrisHCl (pH 7.4), 3 mM MgCl₂, 100 mM NaCl, and 0.2 mM

EGTA, and then centrifuged at 23 000g for 30 min. The pellets were

homogenized in 800 μL of ice-cold sucrose solution (0.32 M),

aliquoted, and kept at −80 °C. For [³⁵S]GTPγS binding assays, 2 μg

of protein was used per well. Samples were incubated with and

without the test compound for 1 h at 25 °C in an assay buﬀer

containing 30 mM GDP and 0.1 nM [³⁵S]GTPγS. Bound radioactivity

was quantiﬁed using a liquid scintillation counter. Nonspeciﬁc binding

was deﬁned as binding in the presence of 10 μM GTPγS; basal

binding refers to binding in the absence of the agonist. Data were

expressed as a mean percentage of activation above the basal binding.

GTPγS binding by agonist was plotted with X axis representing

concentration and Y axis representing the percentage of activation

against background. EC₅₀values were calculated using GraphPad

Prism software. The E_maxis expressed as percentage of activation

above the basal binding, which is set as 100%, and the basal binding

refers to binding in the absence of the agonist.

Bidirectional MDCK-MDR1 Permeability Assay. MDCK-MDR1

cells at passage 17 were seeded onto permeable polycarbonate

supports in 12-well Costar Transwell plates and allowed to grow and

diﬀerentiate for 3 days. On day 3, culture medium (Dulbecco’s

modiﬁed Eagle’s medium (DMEM) supplemented with 10% fetal

bovine serum (FBS)) was removed from both sides of the transwell

inserts, and cells were rinsed with warm HBSS. After the rinse step,

the chambers were ﬁlled with warm transport buﬀer (HBSS

containing 10 mM HEPES, 0.25% BSA, pH 7.4), and the plates

were incubated at 37 °C for 30 min prior to Trans Epithelial Electric

Resistance (TEER) measurements.

N-Methyl-1-[(2R)-2-{4-[(2S)-2-methylbutoxy]phenyl}-2-[(2S)-2-

phenylpropanamido]ethyl]-1H-1,2,3-triazole-4-carboxamide (53).

The procedure for the synthesis of 50 was followed starting with

24c and ethyl propiolate to aﬀord 53 (27% yield over 3 steps) as a

1

white solid: H NMR (300 MHz, CD₃OD) δ 8.21 (d, J = 1.4 Hz,

1H), 7.18 (ddd, J = 15.8, 9.6, 6.8 Hz, 7H), 6.91−6.66 (m, 2H), 5.41

(dd, J = 9.1, 5.7 Hz, 1H), 4.83−4.64 (m, 3H), 3.87−3.67 (m, 2H),

3.62 (q, J = 7.0 Hz, 1H), 2.92 (s, 3H), 1.91−1.68 (m, 1H), 1.65−1.42

(m, 1H), 1.29 (dd, J = 13.4, 7.4 Hz, 4H), 0.99 (d, J = 6.7 Hz, 3H),

0.94 (t, J = 7.5 Hz, 3H); ¹³C NMR (75 MHz, CD₃OD) δ 176.5,

160.6, 143.8, 142.6, 131.1, 129.5, 128.9, 128.3, 128.0, 127.6, 115.8,

73.9, 55.2, 54.1, 47.4, 36.0, 27.1, 26.1, 18.7, 16.8, 11.6; HRMS (ESI)

m/z calcd for C₂₆H₃₃N₅O₃[M + H]⁺464.2656, m/z found 464.2650;

HPLC, t_R15.97 min.

Pharmacology. Materials. Cell culture materials were purchased

from Fisher SSI. Forskolin was purchased from Sigma-Aldrich. The

Lance Ultra kit (TRF0262) was purchased from PerkinElmer.

Lance Ultra cAMP Assay Using Stable PPLS-HA-GPR88 CHO

Cells. All cAMP assays were performed using our previously published

methods.²⁴Stimulation buﬀer containing 1X Hank’s balanced salt

solution (HBSS), 5 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethane-

The buﬀer in the donor chamber (apical side for A-to-B assay,

basolateral side for B-to-A assay) was removed and replaced with the

working solution (10 μM test article in transport buﬀer). The plates

were then placed at 37 °C under light agitation. At designated time

points (30, 60, and 90 min), an aliquot of transport buﬀer from the

receiver chamber was removed and replenished with fresh transport

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buﬀer. Samples were quenched with ice-cold acetonitrile containing

internal standard and then centrifuged to pellet protein. Resulting

supernatants are further diluted with 50/50 acetonitrile/water (water

only for atenolol) and submitted for LC-MS/MS analysis. Reported

apparent permeability (P_app) values were calculated from single

determination. Atenolol and propranolol were tested as low and

moderate permeability references. Bidirectional transport of digoxin

was assessed to demonstrate Pgp activity/expression.

Ann M. Decker − Center for Drug Discovery, Research

Triangle Institute, Research Triangle Park, North Carolina

27709, United States

Lucas Laudermilk − Center for Drug Discovery, Research

Triangle Institute, Research Triangle Park, North Carolina

27709, United States

Rangan Maitra − Center for Drug Discovery, Research

Triangle Institute, Research Triangle Park, North Carolina

27709, United States; orcid.org/0000-0001-6663-6800

Weiya Ma − Douglas Research Center, Department of

Psychiatry, McGill University, Montréal, Quebec H4H 1R3,

Canada

Sami Ben Hamida − Douglas Research Center, Department of

Psychiatry, McGill University, Montréal, Quebec H4H 1R3,

Canada; INSERM U1114, University of Strasbourg,

Strasbourg 67085, France

Emmanuel Darcq − Douglas Research Center, Department of

Psychiatry, McGill University, Montréal, Quebec H4H 1R3,

Canada; INSERM U1114, University of Strasbourg,

Strasbourg 67085, France

Brigitte L. Kieﬀer − Douglas Research Center, Department of

Psychiatry, McGill University, Montréal, Quebec H4H 1R3,

Canada; INSERM U1114, University of Strasbourg,

Strasbourg 67085, France

The apparent permeability (P_app, measured in cm/s) of a

compound is determined according to the following formula: P_app

=

(dQ/dt)/(AC_i× 60), where dQ/dt is the net rate of appearance in the

receiver compartment, A is the area of the Transwell measured in cm²

(1.12 cm²), C_iis the initial concentration of compound added to the

donor chamber, and 60 is the conversion factor for minute to seconds.

Pharmacokinetic Analysis. Male C57BL/6NCrl mice were

procured from Charles River Laboratories at 8−9 weeks of age.

The animals were allowed to acclimate to the vivarium for a period of

1 week. Each mouse was administered a single 20 mg/kg dose of 53

through IP injection in a 10 mL/kg dosing volume. Doses were

formulated in 2% NMP in canola oil. Three animals were sacriﬁced at

each of the selected time points (0.25, 0.5, 1, 2, 4, 6, 8, and 24 s) post-

dose. Blood was collected through the abdominal vena cava for

plasma analysis, and whole-body perfusion was performed with

phosphate-buﬀered saline (pH 7.4). Brains were collected following

perfusion.

Sample Preparations. Plasma (40 μL), MeCN (10 μL), and

Reserpine (150 μL, 100 ng/mL) in MeCN with 0.1% formic acid

were vortexed and centrifuged at 4000 RPM for 10 min. The

supernatant (50 μL) was diluted with 50 μL of H₂O prior to LC/MS/

MS analysis. The brain was homogenized with 50:50 EtOH:H₂O

(1:5, v/v) using a Geno Grinder. 40 μL of the homogenate, 10 μL of

MeCN, and 150 μL of 100 ng/mL Reserpine in MeCN with 0.1%

formic acid were vortexed and centrifuged at 4000 RPM for 10 min.

The supernatant (50 μL) was diluted with 50 μL of H₂O prior to LC/

MS/MS analysis.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c01075

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

LC/MS/MS Analysis. LC/MS/MS was conducted using an Applied

Biosystems API 4000 coupled with an Agilent 1100 HPLC system.

Chromatography was performed with a Phenomenex Luna C18 (50

mm × 2 mm, 5 μm) column with C18 guard cartridge. Mobile phases

were (A) 0.1% formic acid and 10 mM ammonium formate in H₂O

and (B) 0.1% formic acid and 10 mM ammonium formate in MeOH.

Initial conditions were 10% B and held for 0.5 min, followed by a

linear gradient to 95% B over 3.5 min; 95% B was held for 2.9 min

before returning to initial conditions.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by the National Institute on Alcohol

Abuse and Alcoholism (NIAAA, grants R01AA026820 to C.J.

and B.K., and R03AA029013 to C.J.), National Institutes of

Health. The authors thank Tiﬀany Langston and Rodney

Snyder for conducting the in vitro testing and PK analysis.

ASSOCIATED CONTENT

■

ABBREVIATIONS USED

sı

* Supporting Information

■

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01075.

ADME, absorption, distribution, metabolism, and excretion; 2-

AMPP, (2S)-N-((1R)-2-amino-1-(4-(2-methyl-pentyloxy)-

phenyl)ethyl)-2-phenylpropanamide; cAMP, cyclic adenosine

monophosphate; BBB, blood−brain barrier; CHO, Chinese

hamster ovary; CL, clearance; CNS, central nervous system;

CuAAC, copper-catalyzed azide-alkyne cycloaddition; DEAD,

diethyl azodicarboxylate; DIPEA, N,N-diisopropylethylamine;

DMF, N,N-dimethylformamide; DMP, Dess−Martin period-

inane; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

EDG, electron-donating group; EWG, electron-withdrawing

group; ESI, electrospray ionization; GPCR, G-protein-coupled

receptor; GPR88, G-protein-coupled receptor 88; HA, human

inﬂuenza hemagglutinin; HBSS, Hanks balanced salt solution;

HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

hexaﬂuorophosphate; HEPES, N-(2-hydroxyethyl)piperazine-

N′-(2-ethanesulfonic acid); HMBC, heteronuclear multiple

bond correlation; HOBt, hydroxybenzotriazole; HPLC, high-

performance liquid chromatography; HRMS, high-resolution

mass spectrometry; IP, intraperitoneal; KO, knockout; MDCK,

Madin−Darby canine kidney; MDR1, multidrug resistance

Molecular formula strings with biological data (CSV)

¹H NMR, ¹³C NMR, and HPLC analysis results of target

compounds; structural assignment of compound 17

(PDF)

AUTHOR INFORMATION

■

Corresponding Author

Chunyang Jin − Center for Drug Discovery, Research Triangle

Institute, Research Triangle Park, North Carolina 27709,

United States; orcid.org/0000-0001-6733-3094;

Phone: 919 541-6328; Email: cjin@rti.org

Authors

Md Touﬁqur Rahman − Center for Drug Discovery, Research

Triangle Institute, Research Triangle Park, North Carolina

27709, United States; orcid.org/0000-0003-2752-6822

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knockout mice showed by automatized home cage phenotyping.

Genes, Brain Behav. 2018, 17, No. e12473.

protein 1; NMR, nuclear magnetic resonance; MS, mass

spectroscopy; 2-PCCA, (1R,2R)-2-(pyridin-2-yl)cyclopropane

carboxylic acid ((2S,3S)-2-amino-3-methylpentyl)-(4′-propyl-

biphenyl-4-yl)amide; Pgp, P-glycoprotein; PK, pharmacoki-

netic; PPLS, pre-prolactin leader sequence; PSA, polar surface

area; SAR, structure−activity relationship; TEA, triethylamine;

TFA, triﬂuoroacetic acid; THF, tetrahydrofuran; TLC, thin-

layer chromatography; TMS, trimethylsilyl; TR-FRET, time-

resolved ﬂuorescence resonance energy transfer; WT, wild-type

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Article Doi

Evaluation of Amide Bioisosteres Leading to 1,2,3-Triazole Containing Compounds as GPR88 Agonists: Design, Synthesis, and Structure-Activity Relationship Studies

DOI: 10.1021/acs.jmedchem.1c01075

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