Design, synthesis and pharmacological evaluation of 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives as GPR88 agonists

Article abstract of DOI:10.1016/j.bmc.2016.11.058

The orphan receptor GPR88 is an attractive therapeutic target because of its implications in a number of basal ganglia-associated disorders. To date, pharmacological characterization of GPR88 has been limited due to the lack of potent and selective agonists and antagonists appropriate for CNS investigations. We have previously reported that GPR88 couples to Gα_iproteins and modulates cAMP levels upon treatment with a small molecule agonist 2-PCCA. Recently, another chemotype of GPR88 agonist, represented by 2-AMPP [(2S)-N-((1R)-2-amino-1-(4-(2-methylpentyloxy)-phenyl)ethyl)-2-phenylpropanamide], has also been discovered. In this report, a new series of 2-AMPP structurally related 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives have been designed and evaluated for agonist activity at GPR88. The structure-activity relationship (SAR) studies suggest that the amine group in 2-AMPP can be replaced by hydroxyl, ester and amide groups, resulting in analogues with good to moderate potency, whereas the phenyl group on the amide cap is essential for activity and has limited size, shape and electronic tolerance.

Full text of DOI:10.1016/j.bmc.2016.11.058

Accepted Manuscript
Design, Synthesis and Pharmacological Evaluation of 4-Hydroxyphenylglycine
and 4-Hydroxyphenylglycinol Derivatives as GPR88 Agonists
Chunyang Jin, Ann M. Decker, Tiffany L. Langston
PII:
S0968-0896(16)31012-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.058
BMC 13425
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 October 2016
29 November 2016
30 November 2016
Please cite this article as: Jin, C., Decker, A.M., Langston, T.L., Design, Synthesis and Pharmacological Evaluation
of 4-Hydroxyphenylglycine and 4-Hydroxyphenylglycinol Derivatives as GPR88 Agonists, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.058
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, Synthesis and Pharmacological Evaluation of 4-Hydroxyphenylglycine and 4-
Hydroxyphenylglycinol Derivatives as GPR88 Agonists
Chunyang Jin,* Ann M. Decker, and Tiffany L. Langston
Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, North Carolina
27709
*Corresponding author:
Dr. Chunyang Jin
Research Triangle Institute
Post Office Box 12194
Research Triangle Park, NC 27709
Telephone: 919 541-6328
Fax: 919 541-8868
E-mail: cjin@rti.org

Abstract
The orphan receptor GPR88 is an attractive therapeutic target because of its implications in a
number of basal ganglia-associated disorders. To date, pharmacological characterization of GPR88
has been limited due to the lack of potent and selective agonists and antagonists appropriate for CNS
investigations. We have previously reported that GPR88 couples to Gαi proteins and modulates
cAMP levels upon treatment with a small molecule agonist 2-PCCA. Recently, another chemotype
of GPR88 agonist, represented by 2-AMPP [(2S)-N-((1R)-2-amino-1-(4-(2-methylpentyloxy)-
phenyl)ethyl)-2-phenylpropanamide], has also been discovered. In this report, a new series of 2-
AMPP structurally related 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives have
been designed and evaluated for agonist activity at GPR88. The structure-activity relationship (SAR)
studies suggest that the amine group in 2-AMPP can be replaced by hydroxyl, ester and amide
groups, resulting in analogues with good to moderate potency, whereas the phenyl group on the
amide cap is essential for activity and has limited size, shape and electronic tolerance.
Keywords: GPR88; 2-PCCA; 2-AMPP; agonist; SAR
Abbreviations: 2-PCCA, (1R,2R)-2-(pyridin-2-yl)cyclopropane carboxylic acid ((2S,3S)-2-amino-
3-methylpentyl)-(4′-propylbiphenyl-4-yl)amide; 2-AMPP, (2S)-N-((1R)-2-amino-1-(4-(2-methyl-
pentyloxy)phenyl)ethyl)-2-phenylpropanamide; SAR, structure activity relationship; PPLS, pre-
prolactin leader sequence; HA, human influenza hemagglutinin; HTRF, homogeneous time resolved
fluorescence; MS, mass spectroscopy; TLC, thin layer chromatography; DIPEA, N,N-
diisopropylethylamine; DCM, dichloromethane; DEAD, diethyl azodicarboxylate; THF,
tetrahydrofuran; DMF, N,N-dimethylformamide; TEA, triethylamine; HBTU, 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; TFA, trifluoroacetic acid; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HOBt, hydroxybenzotriazole.
2

1.
Introduction
The G protein-coupled receptors (GPCRs) represent the largest and most diverse group of
membrane proteins. GPCRs have attracted significant attention for drug discovery due to their
numerous physiological and pathological roles in mediating cellular responses to hormones and
neurotransmitters. Within the large
The key to
discovering the therapeutic potential of orphan GPCRs is to identify highly potent and selective
ligands thereby allowing the elucidation of their biological functions in both physiological and
pathological states.3
GPR88 is an orphan GPCR and has recently attracted considerable interest in studying its
biological functions, mainly through genetic interference. GPR88 exhibits high expression in the
brain, with particularly robust expression in both striatal D1 and D2expressing GABAergic neurons,
suggesting the receptor may play a role in regulating dopaminergic activity.4-8 Genetically-modified
mice that lack GPR88 expression exhibit enhanced response to dopaminergic agonists and altered
performance in models relevant to schizophrenia and anxiety, indicating that GPR88 may have a
potential therapeutic role in treating these CNS disorders.9-11
With the aim of developing potent and selective small molecular ligands to probe GPR88
functions, we previously reported that (1R,2R)-2-PCCA [(1R,2R)-2-(pyridin-2-yl)cyclopropane
carboxylic acid ((2S,3S)-2-amino-3-methylpentyl)-(4′-propylbiphenyl-4-yl)amide (1); Figure 1] was
able to activate GPR88 through a Gαicoupled pathway.12 (1R,2R)-2-PCCA had an EC50 value in the
range of 50-100 nM in Lance cAMP assays using stable PPLS-HA-GPR88 CHO cells.13 Although 2-
PCCA is useful in the characterization of signaling pathways of GPR88, much research remains to
be done before the pharmacology of GPR88 is fully understood. Structurally different chemotypes
3

showing potent and selective GPR88 agonism/antagonism will be particularly helpful. Recently,
another chemotype of GPR88 agonist, 2-AMPP [(2S)-N-((1R)-2-amino-1-(4-(2-methylpentyloxy)-
phenyl)ethyl)-2-phenylpropanamide (2)], was reported to activate GPR88 with an EC50 value of 90
nM in the HTRF cAMP assay.14 Later, 2 was shown to elicit strong [35S]-GTPS binding (EC50 =
940 nM) in mice striatal membranes but was ineffective in the samples from the GPR88 knockout
mice, demonstrating the compound is GPR88-specific in the striatum.11 Early structure-activity
relationship (SAR) studies of 2 suggest that the amine group can be replaced by a hydroxyl group to
give phenylglycinol 3 without altering GPR88 potency, whereas the substitutions on the amide cap
seem to have a profound effect on the activity. In this paper, we designed and synthesized a new
series of 4-hydroxyphenylglycine and 4-hydroxyphenylglycinol derivatives with structural
modifications at sites A and B (Figure 1) in order to determine receptor tolerances at each site and
enhance the potency for GPR88.
Figure 1. Structures of (1R,2R)-2-PCCA (1), 2 and 3, and sites of chemical modification explored.
2.
Chemistry
Synthesis of the designed compounds 2, 3 and 11ac is outlined in Scheme 1. Boc protection
of the amine group in (R)-2-phenylglycine methyl ester (4) afforded 5 in quantitative yield. O-
alkylation of 5 with 2-methylpentanol under Mitsunobu conditions gave 6 in 78% yield. The methyl
ester was reduced to the hydroxyl group, which was then reacted with triphenylphosphine and
4

carbon tetrabromide to produce the bromo intermediate 7b in 76% yield over two steps. Treatment
of 7b with sodium azide yielded 7c quantitatively. Removal of the Boc protecting group in 7a and 7c
with TFA led to amines 8a,b. Amine 8a was coupled with (S)-2-phenylpropionic acid using HBTU
to give the target compound 3 in 53% yield. Following the same conditions, amine 8b was coupled
with (S)-2-phenylpropionic acid to afford azide 9 in 72% yield. Reduction of azide under
hydrogenation provided 2-AMPP (2) in 66% yield. Boc deprotection of 6 led to amine 10, which
was then coupled with (S)-2-phenylpropionic acid to give 11a in 94% yield. Saponification of the
ester group afforded acid 11b in 77% yield. Coupling of the acid with dimethylamine using EDC
hydrochloride and HOBt gave amide 11c in 45% yield.
Scheme 1. Synthesis of target compounds 2, 3, 9 and 11ac. Reagents and conditions: (a) Boc2O, DIPEA,
DCM, rt, overnight, 100%; (b) PPh3, DEAD, 2-methylpentanol, THF, rt, overnight, 78%; (c) NaBH4, LiCl,
THF-EtOH (1:1), rt, 3 h, 91%; (d) PPh3, CBr4, THF, rt, 3 h, 83%; (e) NaN3, DMF, rt, 6 h, 100%; (f) TFA,
DCM, rt, 1 h, 100%; (g) (S)-2-phenylpropionic acid, HBTU, TEA, MeCN, rt, 5 h, 5394%; (h) 10% Pd/C, H2,
EtOH, 40 psi, 1 h, 66%; (i) 1 N NaOH, THF-MeOH (1:1), rt, 1 h, 77%; (j) EDC hydrochloride, HOBt,
DIPEA, Me2N•HCl, DMF, rt, overnight, 45%.
5

Synthesis of target compounds 12ad and 14 was accomplished in 4898% yield by coupling
of amine 8a or 10 with an appropriate acid (Scheme 2). A library of compounds with general
structure 13 were synthesized by acylation of 10 with commercially available carboxylic acids using
HBTU as the coupling agent, which provided clean products as judged by TLC analyses. The
identity of library compounds was confirmed by mass spectroscopy (MS). The library compounds,
used without purification, were screened at 1 µM to evaluate their agonist activity at GPR88. Eight
of the library compounds (13a, 13e–g, 13m, 13p, 13z and 13aw) were resynthesized in pure form to
1
13
determine their potency. All purified target compounds were characterized by H NMR, C NMR
and MS, and the data were in agreement with the assigned structures.
Scheme 2. Synthesis of target compounds 12ad, 14 and library compounds 13. Reagents and conditions: (a)
carboxylic acid, HBTU, TEA, MeCN, rt, 5 h, 4898%.
3.
Results and discussion
Recently, 2-AMPP was discovered as a GPR88 agonist by high throughput screening, followed
by hit-to-lead optimization.14 Given its favorable calculated physiochemical properties (clogP =
4.53, TPSA = 64.35, logBB = -0.12) 15-17 and GPR88-specific activity in the striatum,11 2-AMPP is a
promising lead for further optimization to probe GPR88 in vivo functions. In order to gain the SAR
6

information of 2-AMPP for high potency, we have synthesized a series of analogues with structural
modifications at sites A and B (Figure 1) and evaluated their agonist activity at GPR88. In our Lance
cAMP assay, 2-AMPP had an EC50 of 414 nM (Table 1). Exchange of the amine group (site A) with
a hydroxyl (3) led to a 2-fold increase of potency. The phenylglycinol 3 possessed an EC50 of 194
nM, which was in line with the EC50 value (110 nM) obtained in the HTRF assay.14 Interestingly, an
azide group at site A gave the most potent compound (9, EC50 = 134 nM) in the series. However, a
carboxylic acid group resulted in a completely inactive compound 11b. The methyl ester 11a and
amide 11c were moderately active, with EC50 value of 538 nM and 616 nM, respectively. These
findings suggested that a hydrogen bonding/electrostatic interaction between site A and the receptor
might contribute to the agonist activity, but a carboxylic acid was not favorable.
Table 1. Structural modification at site A.
Compd
2a
R
pEC50 (EC50, nM)b
6.38 ± 0.05 (414)
6.71 ± 0.09 (194)
6.87 ± 0.05 (134)
6.27 ± 0.09 (538)
NAc
CH2NH2
CH2OH
CH2N3
3
9
CO2Me
COOH
CONMe2
11a
11b
11c
6.21 ± 0.08 (616)
b
a
The compound was tested as the HCl salt. pEC50 values are means  standard error of at least three independent
experiments performed in duplicate. c EC50 > 10 µM, mean of two independent experiments.
Substituent effects on the amide cap (site B) of 3 were next investigated and the results are
summarized in Table 2. We first examined the linkage requirement between the phenyl and amide
carbonyl group for receptor activity. The benzyl analogue 12a was approximately 2-fold less potent
than 3 (487 nM vs. 194 nM). Substitution with a phenethyl group led to 12b, which also possessed a
7

moderate potency (EC50 = 345 nM). However, moving the phenyl group farther away from the
amide carbonyl using a propyl link resulted in deteriorated activity. The phenyl substitution on the
amide cap is essential for activity, possibly forming an aromatic stacking interaction with the
binding pocket, as the cyclohexyl analogue 12d was completely inactive compared to the moderately
active 11a (EC50 = 538 nM).
Table 2. Structural modification at site B.
Compd
12a
X
R
pEC50 (EC50, nM)a
6.31 ± 0.04 (487)
6.46 ± 0.11 (345)
5.84 ± 0.02 (1440)
CH2OH
CH2OH
CH2OH
Bn
Ph(CH2)2
Ph(CH2)3
12b
12c
CO2Me
NAb
12d
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
Bn
5.58 ± 0.03 (2630)
5.10 ± 0.06 (7870)
5.41 ± 0.07 (3910)
5.23 ± 0.03 (5860)
5.43 ± 0.07 (3700)
5.02 ± 0.07 (9570)
NAb
13a
13e
13f
2-F-Bn
3-F-Bn
4-F-Bn
13g
13m
13p
13z
4-Me-Bn
4-MeO-Bn
4-CN-Bn
6.23 ± 0.09 (583)
6.84 ± 0.09 (145)
CO2Me
CH2OH
13aw
14
a pEC50 values are means  standard error of at least three independent experiments performed in duplicate. b EC50 > 10
µM, mean of two independent experiments.
To further evaluate the substituent effects of size, polarity, lipophilicity, and electronic
tolerance on the amide cap, a library of 54 analogues with general structure 13 was synthesized and
screened for agonist activity at a final concentration of 1 M using the Lance cAMP assay. Although
8

the methyl ester 11a was approximately 2.5-fold less potent than the hydroxyl analogue 3 (Table 1),
the methyl ester at site A was chosen for library synthesis because of its synthetic feasibility. We
initially attempted to synthesize a library of analogues of 3, but the coupling reaction of amine 8a
with carboxylic acid gave a mixture of crude products, which was not suitable for screening without
purification. The 1 M concentration was used for screening based on the activity of the parent
analogue 11a. The structures of the compounds synthesized are given in Table 3. The agonist
activity is presented in Figure 2 as the percentage of TR-FRET signal of the control compound 11a.
The screening results revealed that 13ay (compound #52), the crude form of 11a, had an activity
similar to 11a. The (R)-isomer 13az with a benzylic methyl group was less potent than the (S)-
isomer (compound #53 vs #52, Figure 2). The benzylic position could also tolerate an ethyl group
(compound #50), but other bulky groups (compounds #4549 and #51) at this position led to
decreased activity compared to 11a. None of the benzyl derivatives bearing a variety of substitutions
on the phenyl ring had a significantly improved activity compared to the unsubstituted analogue
(compounds #344 vs #2). In view of these results, eight of the library compounds (13a, 13eg,
13
13m, 13p, 13z and 13aw) were resynthesized and characterized by 1H NMR, C NMR and MS for
biological testing (Table 2). In general, the SAR trend observed in the library screen was in
agreement with that of the purified compounds. The benzyl analogue 13a, lacking a benzylic methyl
group, was 5-fold less potent than 11a (2630 nM vs 538 nM). All the substitutions on the phenyl ring
led to detrimental activity, possibly due to the limited steric tolerance, as both electron donating
(13p) and electron withdrawing (13z) groups were inactive. The ethyl analogue 13aw was
equipotent with 11a and was 4.5-fold more potent than 13a, validating the screening results. Finally,
we synthesized the phenylglycinol analogue 14, which had an EC50 value of 145 nM.
9

Table 3. Structures of library compounds 13.
#
Compd
11a
13a
13b
13c
13d
13e
13f
R1
Control
H
R2
X
#
Compd
13ab
13ac
13ad
13ae
13af
R1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Me
R2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
X
3,5-diF
2,5-diF
2,4-diF
2,3-diF
2,6-diF
2,4-diCl
2,6-diCl
3,4-diCl
2,3-diMeO
2,4-diMeO
2,5-diMeO
3,4-diMeO
3,4-MD
3,4,5-triMeO
2,5-diMe
2,4,6-triMe
H
1
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3
H
2-Cl
4
H
3-Cl
5
H
4-Cl
6
H
2-F
13ag
13ah
13ai
7
H
3-F
8
H
4-F
13g
13h
13i
9
H
2-Br
13aj
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
H
3-Br
13ak
13al
H
4-Br
13j
H
2-Me
3-Me
4-Me
2-MeO
3-MeO
4-MeO
2-NO2
3-NO2
4-NO2
2-CF3
3-CF3
4-CF3
4-EtO
4-BuO
4-Me2N
13k
13l
13am
13an
13ao
13ap
13aq
13ar
13as
13at
H
H
13m
13n
13o
13p
13q
13r
13s
H
H
H
H
H
H
Cyclopropyl
H
H
Cyclobutyl
Cyclohexyl
H
13au
13av
13aw
13ax
13ay
13az
13ba
H
H
13t
H
(S)-Et
H
H
H
H
H
13u
13v
13w
13x
13y
H
(S)-MeO
(S)-Me
(R)-Me
H
H
H
H
H
H
27
28
H
H
H
H
4-CN
55
13z
13bb
3,4-diF
13aa
10

Figure 2. Screening of library compounds. The library compounds were screened at 1 µM final. 11a was used
as control (compound #1) and its TR-FRET signal set as 100%. Representative data from two separate
experiments are demonstrated. Compounds with red columns were resynthesized to determine the potency.
4. Conclusions
In summary, we have designed and synthesized a series of 4-hydroxyphenylglycine and 4-
hydroxyphenylglycinol analogues to determine SAR requirements at GPR88. The target compounds
were evaluated in a Lance cAMP assay using stable PPLS-HA-GPR88 CHO cells. SAR studies
suggest that the amine group in 2-AMPP can be replaced by azide, hydroxyl, ester and amide
groups, resulting in analogues with good to moderate potency. The phenyl group on the amide cap is
essential for activity, possibly due to the formation of an aromatic stacking interaction with the
receptor. However, the substitutions on the phenyl ring have limited size, shape and electronic
tolerance.
Recently, we reported a homology model of GPR88 and docking study of (1R,2R)-PCCA,
which suggested the presence of a small hydrophobic binding pocket housing the aromatic group on
the amide cap.13 Superposition of (1R,2R)-PCCA with 2-AMPP, as well as the similar SAR trends18
11

observed in the region of the amide cap for both scaffolds, suggests that the amide cap in 2-AMPP
may position in the same binding pocket as that of (1R,2R)-2-PCCA. Docking of 2-AMPP analogues
will help refine the model and explore structural features for optimization. Studies including
chemical modifications of compounds 3, 9 and 14 to further improve the potency and evaluation of
receptor specificity are currently under investigation.
5.
Experimental procedures
5.1. Chemistry
5.1.1. General. Reagents and starting materials were obtained from commercial suppliers and were
used without purification. All moisture- and air-sensitive reactions and reagent transfers were carried
out under dry nitrogen. Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were obtained
on a Bruker Avance DPX-300 MHz NMR spectrometer. Chemical shifts are reported in parts per
million (ppm) with reference to internal solvent. Mass spectra (MS) were run on a Waters Alliance
HT/Micromass ZQ system (ESI). Analytical thin-layer chromatography (TLC) was carried out using
EMD silica gel 60 F254 TLC plates. TLC visualization was achieved with a UV lamp or in an iodine
chamber. Flash column chromatography was done on a CombiFlash Companion system using Isco
prepacked silica gel columns.
5.1.2. (2R)-Methyl 2-(tert-butoxycarbonylamino)-2-(4-hydroxyphenyl)acetate (5). To a solution
of (R)-2-phenylglycine methyl ester hydrochloride (5 g, 23 mmol) in DCM (175 mL) at 0 °C under
nitrogen was added DIPEA (12 mL, 69 mmol), followed by di-tert-butyldicarbonate (5.2 g, 23
mmol). The reaction mixture was stirred at room temperature overnight, and concentrated under
reduced pressure. The residue was dissolved in EtOAc (200 mL), washed with 10% citric acid (3 x
50 mL), brine (3 x 50 mL) and dried (Na2SO4). Removal of the solvent under reduced pressure
afforded the crude 5 (6.45 g, 100%) as a colorless oil. 1H NMR (300 MHz; CDCl3)  7.16 (d, J = 9.0
12

Hz, 2H), 7.13 (d, J = 9.0 Hz, 2H), 6.44 (s, 1H), 5.60 (br d, J = 6.0 Hz, 1H), 5.22 (d, J = 6.0 Hz, 1H),
13
3.71 (s, 3H), 1.44 (s, 9H); C NMR (75 MHz; CDCl3)  172.0, 156.3, 155.0, 128.5, 128.4, 115.8,
80.5, 57.1, 52.7, 28.3; MS (ESI) m/z 282.3 [M + H]+.
5.1.3. (2R)-Methyl 2-(tert-butoxycarbonylamino)-2-[4-(2-methylpentyloxy)phenyl]acetate (6).
To a solution of 5 (2.81 g, 10 mmol), 2-methylpentanol (2.48 mL, 20 mmol), and PPh3 (4.45 g, 17
mmol) in THF (70 mL) at room temperature under nitrogen was slowly added DEAD (7.8 mL, 17
mmol) dropwise, while keeping the reaction temperature below 35 °C. After addition, the reaction
mixture was stirred at room temperature overnight and quenched with H2O (50 mL). The layers were
separated and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic
layers were washed with brine (3 x 50 mL), dried (Na2SO4) and concentrated under reduced
pressure. Flash column chromatography of the crude product on silica gel using 020% EtOAc in
1
hexanes afforded 6 (2.85 g, 78%) as a colorless oil. H NMR (300 MHz; CDCl3)  7.26 (d, J = 9.0
Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 5.46 (br d, J = 6.0 Hz, 1H), 5.23 (d, J = 6.0 Hz, 1H), 3.853.65
(m, 2H), 3.71 (s, 3H), 2.001.88 (m, 1H), 1.501.15 (m, 4H), 1.43 (s, 9H), 1.01 (d, J = 6.0 Hz, 3H),
13
0.92 (t, J = 7.5 Hz, 3H); C NMR (75 MHz; CDCl3)  171.9, 159.5, 154.8, 128.7, 128.3, 114.9,
80.0, 73.3, 57.1, 52.5, 35.7, 32.9, 28.3, 20.0, 17.0, 14.3; MS (ESI) m/z 388.6 [M + Na]+.
5.1.4. tert-Butyl (1R)-2-hydroxy-1-[4-(2-methylpentyloxy)phenyl]ethylcarbamate (7a). To a
suspension of NaBH4 (0.62 g, 16.4 mmol) in EtOH (23 mL) at 0 °C under nitrogen was added LiCl
(0.7 g, 16.4 mmol). After stirring at 0 °C for 10 min, a solution of 6 (2.3 g, 6.3 mmol) in THF (23
mL) was added. The reaction mixture was stirred at room temperature for 3 h and quenched with
saturated NH4Cl solution (10 mL), followed by addition of H2O (50 mL). The mixture was extracted
with EtOAc (3 x 100 mL). The combined organic layers were washed with brine (50 mL), dried
(Na2SO4) and concentrated under reduced pressure. Flash column chromatography of the crude
13

product on silica gel using 050% EtOAc in hexanes afforded 7a (1.93 g, 91%) as a white semi-
solid. 1H NMR (300 MHz; CDCl3)  7.20 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 5.17 (br d, J
= 6.0 Hz, 1H), 4.71 (br s, 1H), 3.863.72 (m, 3H), 3.723.65 (m, 1H), 2.48 (br s, 1H), 2.001.88 (m,
13
1H), 1.541.13 (m, 4H), 1.43 (s, 9H), 1.01 (d, J = 6.0 Hz, 3H), 0.92 (t, J = 7.5 Hz, 3H); C NMR
(75 MHz; CDCl3)  159.2, 156.4, 131.5, 127.9, 115.0, 80.2, 73.5, 67.2, 56.8, 36.0, 33.1, 28.6, 20.3,
17.2, 14.5; MS (ESI) m/z 338.6 [M + H]+.
5.1.5. tert-Butyl (1R)-2-bromo-1-[4-(2-methylpentyloxy)phenyl]ethylcarbamate (7b).
A
solution of 7a (1.05 g, 3.12 mmol), Ph3P (1.25 g, 4.67 mmol) and CBr4 (1.55 g, 4.67 mmol) in THF
(20 mL) was stirred at room temperature under nitrogen for 3 h. The mixture was filtered and the
filtrate was concentrated under reduced pressure. Flash column chromatography of the crude product
1
on silica gel using 030% EtOAc in hexanes afforded 7b (1.03 g, 83%) as a white solid. H NMR
(300 MHz; CDCl3)  7.20 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 5.08 (br s, J = 6.0 Hz, 1H),
4.92 (br s, 1H), 3.853.76 (m, 1H), 3.753.60 (m, 3H), 2.021.90 (m, 1H), 1.601.12 (m, 4H), 1.44
13
(s, 9H), 1.02 (d, J = 6.0 Hz, 3H), 0.92 (t, J = 7.5 Hz, 3H); C NMR (75 MHz; CDCl3)  159.1,
155.0, 131.3, 127.5, 114.7, 80.0, 73.3, 54.5, 37.2, 35.8, 32.9, 28.3, 20.0, 17.0, 14.3; MS (ESI) m/z
400.01 [M + H]+ (79Br), 402.3 [M + H]+ (81Br).
5.1.6. tert-Butyl (1R)-2-azido-1-[4-(2-methylpentyloxy)phenyl]ethylcarbamate (7c). To a
solution of 7b (0.9 g, 2.25 mmol) in DMF (10 mL) at room temperature under nitrogen was added
NaN3 (0.59 g, 9 mmol). After stirring for 6 h, the reaction was quenched by H2O (30 mL) and
extracted with Et2O (3 x 30 mL). The combined organic layers were washed with brine (50 mL) and
dried (Na2SO4). Removal of the solvent under reduced pressure afforded the crude 7c (0.81 g, 100%)
1
as a white semi-solid. H NMR (300 MHz; CDCl3)  7.21 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz,
2H), 4.98 (br s, 1H), 4.81 (br s, 1H), 3.863.78 (m, 1H), 3.783.55 (m, 3H), 2.001.90 (m, 1H),
14

1.551.13 (m, 4H), 1.43 (s, 9H), 1.01 (d, J = 6.0 Hz, 3H), 0.92 (t, J = 7.5 Hz, 3H); MS (ESI) m/z
363.2 [M + H]+.
5.1.7. (2R)-2-Amino-2-[4-(2-methylpentyloxy)phenyl]ethanol hydrochloride (8a). To a solution
of 7a (400 mg, 1.2 mmol) in DCM (2 mL) at room temperature under nitrogen was added TFA (2
mL). After stirring for 1 h, the reaction was quenched with saturated NaHCO3 (10 mL). The mixture
was extracted with DCM (3 x 20 mL). The combined organic layers were washed with brine (20
mL) and dried (Na2SO4). Removal of the solvent under reduced pressure afforded the desired amine,
which was then converted into the hydrochloride salt 8a (325 mg, 100%) as a yellow foamy solid.
1H NMR (300 MHz; CDCl3)  8.32 (s, 3H), 7.39 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 4.44
(br s, 1H), 3.983.82 (m, 1H), 3.803.58 (m, 3H), 2.21 (br s, 1H), 2.001.80 (m, 1H), 1.521.12 (m,
4H), 0.99 (d, J = 6.0 Hz, 3H), 0.92 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CDCl3)  159.0, 129.0,
128.0, 115.2, 73.6, 68.2, 56.9, 36.0, 32.5, 20.3, 17.2, 14.5; MS (ESI) m/z 238.4 [M + H]+.
5.1.8. (1R)-2-Azido-1-[4-(2-methylpentyloxy)phenyl]ethanamine (8b). The procedure for 8a was
followed using 810 mg (2.2 mmol) of 7c to give 575 mg (100%) of 8b as a yellow oil. 1H NMR (300
MHz; CDCl3)  7.26 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 4.144.05 (m, 1H), 3.903.68 (m,
2H), 3.563.32 (m, 2H), 2.26 (br s, 2H), 2.021.86 (m, 1H), 1.541.13 (m, 4H), 1.01 (d, J = 6.0 Hz,
3H), 0.92 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CDCl3)  159.0, 133.5, 127.7, 114.9, 73.5, 59.2,
55.1, 35.9, 33.1, 20.2, 17.2, 14.5; MS (ESI) m/z 263.1 [M + H]+.
5.1.9. (2S)-N-{(1R)-2-Hydroxy-1-[4-(2-methylpentyloxy)phenyl]ethyl}-2-phenylpropanamide
(3). To a solution of 8a (90 mg, 0.33 mmol) in MeCN (10 mL) at room temperature under nitrogen
was added TEA (0.14 mL, 0.99 mmol), followed by (S)-2-phenylpropionic acid (50 mg, 0.33 mmol)
and HBTU (152 mg, 0.4 mmol). After stirring for 5 h, the reaction was quenched by H2O (5 mL),
followed by addition of EtOAc (50 mL). The layers were separated. The organic layer was washed
15

with saturated NaHCO3 (10 mL), brine (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 3 (65 mg, 53%) as an off-white solid. 1H NMR (300 MHz; CDCl3)  7.407.22 (m,
5H), 6.96 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 5.97 (d, J = 6.0 Hz, 1H), 5.024.93 (m, 1H),
3.853.72 (m, 3H), 3.703.60 (m, 2H), 2.60 (br s, 1H), 1.981.86 (m, 1H), 1.52 (d, J = 6.0 Hz, 3H),
1.501.12 (m, 4H), 0.99 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CDCl3) 
174.6, 158.9, 141.3, 130.6, 128.9, 127.6, 127.5, 127.3, 114.8, 73.3, 66.7, 55.4, 47.1, 35.8, 32.9, 20.0,
18.5, 17.0, 14.3; MS (ESI) m/z 370.5 [M + H]+.
5.1.10. (2S)-N-{(1R)-2-Azido-1-[4-(2-methylpentyloxy)phenyl]ethyl}-2-phenylpropanamide (9).
The procedure for 3 was followed using 291 mg (1.1 mmol) of 8b to give 326 mg (72%) of 9 as an
1
off-white semi-solid. H NMR (300 MHz; CDCl3)  7.427.26 (m, 5H), 6.97 (d, J = 9.0 Hz, 2H),
6.79 (d, J = 9.0 Hz, 2H), 5.76 (d, J = 6.0 Hz, 1H), 5.155.06 (m, 1H), 3.823.75 (m, 1H), 3.753.50
(m, 4H), 2.001.88 (m, 1H), 1.53 (d, J = 6.0 Hz, 3H), 1.501.15 (m, 4H), 0.99 (d, J = 6.0 Hz, 3H),
13
0.91 (t, J = 7.5 Hz, 3H); C NMR (75 MHz; CDCl3)  172.2, 157.6, 139.6, 128.9, 127.6, 126.2,
126.1, 126.0, 113.3, 71.8, 53.7, 50.6, 45.7, 34.3, 31.5, 18.6, 16.9, 15.6, 12.9; MS (ESI) m/z 395.5 [M
+ H]+.
5.1.11. (2S)-N-{(1R)-2-Amino-1-[4-(2-methylpentyloxy)phenyl]ethyl}-2-phenylpropanamide
hydrochloride (2). A mixture of 9 (316 mg, 0.8 mmol) and 10% Pd/C (80 mg) in EtOH (5 mL) was
hydrogenated under 40 psi for 1 h. The mixture was filtered through a short pad of Celite and the
filtrate was concentrated. Flash column chromatography of the crude product on silica gel using
010% MeOH in DCM afforded 195 mg of a semi-solid, which was then treated with 2 M HCl in
Et2O to give the hydrochloride salt 2 (210 mg, 65%) as an off-white solid. Free base: 1H NMR (300
MHz; CDCl3)  7.407.15 (m, 5H), 6.92 (d, J = 9.0 Hz, 2H), 6.76 (d, J = 9.0 Hz, 2H), 6.30 (d, J =
16

6.0 Hz, 1H), 4.964.82 (m, 1H), 3.813.58 (m, 3H), 3.122.82 (m, 2H), 2.021.86 (m, 1H), 1.51 (d,
J = 6.0 Hz, 3H), 1.501.12 (m, 6H), 0.99 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR (75
MHz; CDCl3)  173.6, 158.5, 141.6, 132.0, 128.8, 127.6, 127.3, 127.2, 114.6, 73.2, 54.3, 47.1, 46.8,
35.8, 32.9, 20.0, 18.5, 17.0, 14.3; MS (ESI) m/z 369.3 [M + H]+.
5.1.12. Methyl (2R)-2-amino-2-[4-(2-methylpentyloxy)phenyl]acetate hydrochloride (10). The
procedure for 8a was followed using 3.86 g (10.5 mmol) of 6 to give 3.15 g (100%) of 10 as an off-
white foamy solid. 1H NMR (300 MHz; CDCl3)  9.02 (s, 3H), 7.48 (d, J = 9.0 Hz, 2H), 6.86 (d, J =
9.0 Hz, 2H), 5.225.12 (m, 1H), 3.803.60 (m, 2H), 3.66 (s, 3H), 2.001.85 (m, 1H), 1.521.12 (m,
4H), 1.00 (d, J = 6.0 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CDCl3)  169.0, 160.3,
130.2, 123.2, 115.0, 73.2, 56.6, 53.3, 35.7, 32.8, 20.0, 17.0, 14.3; MS (ESI) m/z 266.3 [M + H]+.
5.1.13. Methyl
(2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[(S)-2-phenylpropanamido]acetate
(11a). The procedure for 3 was followed using 512 mg (2 mmol) of 10 to give 750 mg (94%) of 11a
as a white solid. 1H NMR (300 MHz; CDCl3)  7.327.20 (m, 5H), 7.09 (d, J = 9.0 Hz, 2H), 6.78 (d,
J = 9.0 Hz, 2H), 6.46 (d, J = 6.0 Hz, 1H), 5.44 (d, J = 6.0 Hz, 1H), 3.783.58 (m, 3H), 3.65 (s, 3H),
1.981.86 (m, 1H), 1.501.16 (m, 4H), 1.48 (d, J = 6.0 Hz, 3H), 0.97 (d, J = 6.0 Hz, 3H), 0.91 (t, J =
13
7.5 Hz, 3H); C NMR (75 MHz; CDCl3)  173.4, 171.6, 159.4, 141.1, 128.8, 128.3, 128.1, 127.6,
127.2, 114.8, 73.2, 56.0, 52.6, 46.7, 35.7, 32.9, 20.0, 18.5, 17.0, 14.3; MS (ESI) m/z 398.4 [M + H]+.
5.1.14. (2R)-2-[4-(2-Methylpentyloxy)phenyl]-2-[(S)-2-phenylpropanamido]acetic acid (11b). A
solution of 11a (550 mg, 1.39 mmol) and 1 N NaOH (2 mL) in 1:1 THF-MeOH (5 mL) was stirred
at room temperature for 1 h. The mixture was acidified with 1 N HCl and extracted with EtOAc (3 x
30 mL). The combined organic layers were washed with brine (10 mL), dried (Na2SO4) and
concentrated under reduced pressure. Crystallization of the crude product from EtOAc/hexanes
1
afforded 11b (410 mg, 77%) as a white solid. H NMR (300 MHz; CDCl3)  8.91 (br s, 1H),
17

7.407.20 (m, 5H), 7.10 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 6.32 (d, J = 6.0 Hz, 1H), 5.43
(d, J = 6.0 Hz, 1H), 3.803.60 (m, 3H), 2.001.86 (m, 1H), 1.501.15 (m, 4H), 1.47 (d, J = 6.0 Hz,
3H), 0.98 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CDCl3)  174.4, 174.1,
159.6, 140.7, 128.9, 128.3, 127.6, 127.4, 127.3, 114.9, 73.3, 56.1, 46.7, 35.7, 32.9, 20.0, 18.3, 17.0,
14.3; MS (ESI) m/z 384.4 [M + H]+.
5.1.15. N-Dimethyl
(2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[(S)-2-phenylpropanamido]-
acetamide (11c). To a solution of 11b (115 mg, 0.3 mmol) in DMF (5 mL) under nitrogen were
added EDC hydrochloride (63 mg, 0.33 mmol), HOBt (45 mg, 0.33 mmol) and DIPEA (104 L, 0.6
mmol). After cooling to 0 °C, dimethylamine hydrochloride (27 mg, 0.33 mmol) was added and the
reaction mixture was stirred at room temperature overnight. The reaction was quenched with 10%
citric acid solution (5 mL) and extracted with EtOAc (3 x 20 mL). The combined organic layers
were washed with saturated NaHCO3 (10 mL) and brine (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 11c (55 mg, 45%) as a colorless oil. 1H NMR (300 MHz; CDCl3)
 7.327.10 (m, 7H), 6.98 (d, J = 9.0 Hz, 1H), 6.78 (d, J = 9.0 Hz, 2H), 5.75 (d, J = 9.0 Hz, 1H),
3.803.55 (m, 3H), 2.94 (s, 3H), 2.88 (s, 3H), 1.981.86 (m, 1H), 1.521.10 (m, 4H), 1.48 (d, J =
6.0 Hz, 3H), 0.99 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CDCl3)  173.0,
170.0, 159.1, 141.2, 129.1, 128.9, 128.7, 127.5, 127.0, 114.7, 73.2, 53.4, 46.8, 36.8, 35.9, 35.7, 32.9,
20.0, 18.5, 17.0, 14.3; MS (ESI) m/z 411.6 [M + H]+.
5.1.16. N-{(1R)-2-Hydroxy-1-[4-(2-methylpentyloxy)phenyl]ethyl}-2-phenylacetamide
(12a).
The procedure for 3 was followed using 68 mg (0.25 mmol) of 8a to give 45 mg (51%) of 12a as a
white solid. 1H NMR (300 MHz; CDCl3)  7.387.20 (m, 5H), 7.05 (d, J = 9.0 Hz, 2H), 6.81 (d, J =
9.0 Hz, 2H), 6.23 (d, J = 9.0 Hz, 1H), 5.014.93 (m, 1H), 3.803.62 (m, 4H), 3.55 (s, 2H), 2.98 (br
18

s, 1H), 1.981.85 (m, 1H), 1.521.12 (m, 4H), 0.99 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C
NMR (75 MHz; CDCl3)  171.6, 158.9, 134.8, 130.6, 129.3, 129.0, 127.6, 127.4, 114.8, 73.3, 66.5,
55.5, 43.7, 35.8, 32.9, 20.0, 17.0, 14.3; MS (ESI) m/z 356.3 [M + H]+.
5.1.17. N-{(1R)-2-Hydroxy-1-[4-(2-methylpentyloxy)phenyl]ethyl}-3-phenylpropanamide (12b).
The procedure for 3 was followed using 68 mg (0.25 mmol) of 8a to give 47 mg (51%) of 12b as a
white solid. 1H NMR (300 MHz; CDCl3)  7.327.12 (m, 5H), 7.04 (d, J = 9.0 Hz, 2H), 6.82 (d, J =
9.0 Hz, 2H), 6.10 (d, J = 6.0 Hz, 1H), 4.984.88 (m, 1H), 3.803.65 (m, 4H), 2.95 (t, J = 7.5 Hz,
3H), 2.51 (t, J = 7.5 Hz, 2H), 2.001.88 (m, 1H), 1.521.12 (m, 4H), 1.00 (d, J = 6.0 Hz, 3H), 0.92
13
(t, J = 7.5 Hz, 3H); C NMR (75 MHz; CDCl3)  172.7, 159.0, 140.6, 130.6, 128.6, 128.4, 127.8,
126.3, 114.8, 73.3, 66.5, 55.4, 18.5, 35.8, 32.9, 31.7, 20.0, 17.0, 14.3; MS (ESI) m/z 370.3 [M + H]+.
5.1.18. N-{(1R)-2-Hydroxy-1-[4-(2-methylpentyloxy)phenyl]ethyl}-4-phenylbutanamide (12c).
The procedure for 3 was followed using 68 mg (0.25 mmol) of 8a to give 55 mg (57%) of 12c as a
white solid. 1H NMR (300 MHz; CDCl3)  7.307.10 (m, 7H), 6.85 (d, J = 9.0 Hz, 2H), 6.22 (d, J =
6.0 Hz, 1H), 5.014.92 (m, 1H), 3.823.65 (m, 3H), 3.24 (br s, 1H), 2.62 (t, J = 7.5 Hz, 3H), 2.19 (t,
J = 7.5 Hz, 2H), 2.001.87 (m, 3H), 1.521.12 (m, 4H), 0.99 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz,
13
3H); C NMR (75 MHz; CDCl3)  173.4, 159.0, 141.4, 130.8, 128.5, 128.4, 127.9, 126.0, 114.9,
73.3, 66.6, 55.5, 35.8, 35.7, 35.1, 32.9, 27.0, 20.0, 17.0, 14.3; MS (ESI) m/z 384.5 [M + H]+.
5.1.19. Methyl
(2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[(R,S)-2-cyclohexylpropanamido]-
acetate (12d). The procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 61 mg (76%)
of 12d as a white semi-solid. 1H NMR (300 MHz; CDCl3)  7.26 (d, J = 9.0 Hz, 2H), 6.87 (d, J = 9.0
Hz, 2H), 6.30 (d, J = 9.0 Hz, 1H), 5.49 (d, J = 6.0 Hz, 1H), 3.823.66 (m, 2H), 3.72 (s, 3H),
2.051.88 (m, 2H), 1.821.58 (m, 5H), 1.561.00 (m, 10H), 1.08 (d, J = 6.0 Hz, 3H), 0.98 (d, J =
6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CDCl3)  175.6, 171.8, 159.5, 128.4,
19

114.9, 73.3, 55.7, 52.6, 47.3, 40.9, 35.7, 32.9, 31.3, 29.9, 26.4, 26.3, 20.0, 17.0, 14.7, 14.3; MS (ESI)
m/z 404.3 [M + H]+.
5.1.20. Synthesis of library compounds 13. Amine 10 (0.02 mmol x the number of derivatives
being prepared) was dissolved in anhydrous THF (0.5 mL x the number of derivatives being
prepared). HBTU (0.024 mmol x the number of derivatives being prepared) was dissolved in
anhydrous MeCN (1 mL x the number of derivatives being prepared). To the prelabeled 20 mL
scintillation vial containing a stir bar was added one of the chosen carboxylic acid (0.022 mmol)
solution in MeCN (0.5 mL). To this was added the appropriate fraction of amine solution (0.5 mL)
followed by TEA (0.006 mL, 0.04 mmol) and the appropriate fraction of HBTU solution (1 mL).
The vial was then capped with a Teflon-lined lid and stirred at room temperature. After TLC
analysis indicated no amine left, Et2O (4 mL) and H2O (2 mL) were added to the vial. After the vial
was shaken and the layers were allowed to settle, the aqueous layer was withdrawn with a pipet.
Next, saturated NaHCO3 (2 x 2 mL) was added and the procedure repeated. This was followed by
washing with brine (2 x 2 mL). Na2SO4 was added to the vial, and after drying, the mixture was
pipetted into a preweighed, prelabeled 20 mL scintillation vials using a 6 in. Pasteur pipet containing
a small cotton plug. Following this, Et2O (2 mL) was added to the drying agent and the vial was
shaken, after which the Et2O rinse was filtered as above. The vials were placed under a nitrogen
outlet and allowed to evaporate. Once the solvent was removed, the vials were placed in a high
vacuum desiccator and allowed to remain overnight. The vials were reweighed, and the crude yield
was determined by difference. The products were characterized by MS to confirm the identity and
were used without further purification.
5.1.21. Methyl (2R)-2-[4-(2-methylpentyloxy)phenyl]-2-(2-phenylacetamido)acetate (13a). The
procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 72 mg (94%) of 13a as a white
20

1
solid. H NMR (300 MHz; CDCl3)  7.487.20 (m, 5H), 7.16 (d, J = 9.0 Hz, 2H), 6.82 (d, J = 9.0
Hz, 2H), 6.46 (d, J = 6.0 Hz, 1H), 5.45 (d, J = 9.0 Hz, 1H), 3.823.62 (m, 2H), 3.67 (s, 3H), 3.58 (s,
2H), 1.981.85 (m, 1H), 1.521.12 (m, 4H), 0.99 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C
NMR (75 MHz; CDCl3)  171.5, 170.3, 159.5, 134.5, 129.4, 129.0, 128.4, 128.1, 127.4, 114.9, 73.3,
56.0, 52.7, 43.4, 35.7, 32.9, 20.0, 17.0, 14.3; MS (ESI) m/z 384.4 [M + H]+.
5.1.22. Methyl (2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[2-(2-fluorophenyl)acetamido]acetate
(13e). The procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 75 mg (93%) of 13e
1
as a white solid. H NMR (300 MHz; CDCl3)  7.267.16 (m, 2H), 7.12 (d, J = 9.0 Hz, 2H),
7.066.95 (m, 2H), 6.76 (d, J = 9.0 Hz, 2H), 6.50 (d, J = 6.0 Hz, 1H), 5.40 (d, J = 6.0 Hz, 1H),
3.723.56 (m, 2H), 3.60 (s, 3H), 3.53 (s, 2H), 1.901.78 (m, 1H), 1.451.05 (m, 4H), 0.91 (d, J =
13
6.0 Hz, 3H), 0.83 (t, J = 7.5 Hz, 3H); C NMR (75 MHz; CDCl3)  170.4, 168.1, 159.9 (d, JC-F
=
245 Hz), 158.5, 130.6 (d, JC-F = 3.8 Hz), 128.3 (d, JC-F = 8.3 Hz), 127.3, 127.1, 123.5 (d, JC-F = 3.3
Hz), 120.8 (d, JC-F = 15.8 Hz), 114.6 (d, JC-F = 21.8 Hz), 113.9, 72.2, 55.0, 51.6, 35.4 (d, JC-F = 2.3
Hz), 34.7, 31.8, 19.0, 16.0, 13.3; MS (ESI) m/z 402.0 [M + H]+.
5.1.23. Methyl (2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[2-(3-fluorophenyl)acetamido]acetate
(13f). The procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 73 mg (92%) of 13f
1
as a white solid. H NMR (300 MHz; CDCl3)  7.347.22 (m, 1H), 7.18 (d, J = 9.0 Hz, 2H),
7.066.90 (m, 3H), 6.83 (d, J = 9.0 Hz, 2H), 6.61 (d, J = 6.0 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H),
3.803.62 (m, 2H), 3.68 (s, 3H), 3.55 (s, 2H), 1.981.88 (m, 1H), 1.521.10 (m, 4H), 0.99 (d, J =
13
6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); C NMR (75 MHz; CDCl3)  171.5, 169.6, 162.9 (d, JC-F
=
245 Hz), 159.6, 136.9 (d, JC-F = 7.5 Hz), 130.3 (d, JC-F = 8.3 Hz), 128.4, 128.0, 125.0 (d, JC-F = 3.0
Hz), 116.3 (d, JC-F = 21.8 Hz), 114.9, 114.2 (d, JC-F = 21.0 Hz), 73.3, 56.1, 52.7, 42.9 (d, JC-F = 1.5
Hz), 35.7, 32.9, 20.0, 17.0, 14.3; MS (ESI) m/z 402.2 [M + H]+.
21

5.1.24. Methyl (2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[2-(4-fluorophenyl)acetamido]acetate
(13g). The procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 75 mg (93%) of 13g
1
as a white solid. H NMR (300 MHz; CDCl3)  7.287.12 (m, 4H), 7.056.96 (m, 2H), 6.83 (d, J =
9.0 Hz, 2H), 6.54 (d, J = 6.0 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H), 3.803.62 (m, 2H), 3.68 (s, 3H), 3.53
(s, 2H), 1.981.87 (m, 1H), 1.521.14 (m, 4H), 0.99 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C
NMR (75 MHz; CDCl3)  171.5, 170.4, 162.1 (d, JC-F = 245 Hz), 159.5, 130.9 (d, JC-F = 8.3 Hz),
130.3 (d, JC-F = 3.0 Hz), 128.4, 128.0, 115.8 (d, JC-F = 21.0 Hz), 114.9, 73.3, 56.0, 52.7, 42.4, 35.7,
32.9, 20.0, 17.0, 14.3; MS (ESI) m/z 402.2 [M + H]+.
5.1.25. Methyl (2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[2-(4-methylphenyl)acetamido]acetate
(13m). The procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 72 mg (91%) of
1
13m as a white solid. H NMR (300 MHz; CDCl3)  7.217.10 (m, 6H), 6.82 (d, J = 9.0 Hz, 2H),
6.50 (d, J = 9.0 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H), 3.803.62 (m, 2H), 3.66 (s, 3H), 3.53 (s, 2H), 2.33
(s, 3H), 1.961.85 (m, 1H), 1.521.12 (m, 4H), 0.99 (d, J = 6.0 Hz, 3H), 0.93 (t, J = 7.5 Hz, 3H); 13C
NMR (75 MHz; CDCl3)  171.6, 170.5, 159.5, 136.9, 131.4, 129.6, 129.3, 128.4, 128.2, 114.9, 73.2,
55.9, 52.6, 43.0, 35.7, 32.9, 21.1, 20.3, 17.0, 14.3; MS (ESI) m/z 398.3 [M + H]+.
5.1.26. Methyl (2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[2-(4-methoxyphenyl)acetamido]acetate
(13p). The procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 78 mg (94%) of 13p
1
as a white solid. H NMR (300 MHz; CDCl3)  7.227.12 (m, 4H), 6.926.78 (m, 4H), 6.46 (d, J =
6.0 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H), 3.813.62 (m, 2H), 3.79 (s, 3H), 3.67 (s, 3H), 3.52 (s, 2H),
1.981.88 (m, 1H), 1.521.12 (m, 4H), 0.99 (d, J = 6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR
(75 MHz; CDCl3)  171.6, 170.7, 159.5, 159.9, 130.5, 128.4, 128.2, 126.5, 114.9, 114.4, 73.2, 55.9,
55.3, 52.6, 42.5, 35.7, 32.9, 20.0, 17.0, 14.3; MS (ESI) m/z 414.4 [M + H]+.
22

5.1.27. Methyl (2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[2-(4-cyanophenyl)acetamido]acetate
(13z). The procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 80 mg (98%) of 13z
1
as a white solid. H NMR (300 MHz; CDCl3)  7.62 (d, J = 9.0 Hz, 2H), 7.39 (d, J = 9.0 Hz, 2H),
7.19 (d, J = 9.0 Hz, 2H), 6.85 (d, J = 9.0 Hz, 2H), 6.48 (d, J = 6.0 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H),
3.863.55 (m, 2H), 3.71 (s, 3H), 3.63 (s, 2H), 2.001.88 (m, 1H), 1.521.12 (m, 4H), 1.00 (d, J =
6.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz; CDCl3)  171.5, 168.7, 159.6, 140.0,
132.4, 130.1, 128.4, 127.8, 118.7, 115.0, 111.2, 73.3, 56.1, 52.8, 43.0, 35.7, 32.9, 20.0, 17.0, 14.3;
MS (ESI) m/z 409.6 [M + H]+.
5.1.28. Methyl
(2R)-2-[4-(2-methylpentyloxy)phenyl]-2-[(S)-2-phenylbutanamido]acetate
(13aw). The procedure for 3 was followed using 60 mg (0.2 mmol) of 10 to give 78 mg (95%) of
1
13aw as a white solid. H NMR (300 MHz; CDCl3)  7.357.20 (m, 5H), 7.09 (d, J = 9.0 Hz, 2H),
6.78 (d, J = 9.0 Hz, 2H), 6.34 (d, J = 6.0 Hz, 1H), 5.42 (d, J = 6.0 Hz, 1H), 3.803.62 (m, 2H), 3.68
(s, 3H), 3.31 (t, J = 7.5 Hz, 1H), 2.222.08 (m, 1H), 1.981.70 (m, 2H), 1.521.15 (m, 4H), 0.98 (d,
J = 6.0 Hz, 3H), 0.950.85 (m, 6H); 13C NMR (75 MHz; CDCl3)  172.8, 171.6, 159.4, 139.7, 128.7,
128.2, 128.0, 127.2, 114.8, 73.2, 56.0, 54.8, 52.6, 35.7, 32.9, 26.4, 20.0, 17.0, 14.3, 12.2; MS (ESI)
m/z 412.3 [M + H]+.
5.1.29. (2S)-N-{(1R)-2-Hydroxy-1-[4-(2-methylpentyloxy)phenyl]ethyl}-2-phenylbutanamide
(14). The procedure for 3 was followed using 68 mg (0.25 mmol) of 8a to give 46 mg (48%) of 14 as
a white solid. 1H NMR (300 MHz; CDCl3)  7.357.20 (m, 5H), 6.94 (d, J = 9.0 Hz, 2H), 6.75 (d, J
= 9.0 Hz, 2H), 6.22 (d, J = 6.0 Hz, 1H), 5.004.90 (m, 1H), 3.803.70 (m, 3H), 3.683.60 (m, 1H),
3.30 (t, J = 7.5 Hz, 1H), 2.97 (br s, 1H), 2.222.10 (m, 1H), 1.981.72 (m, 2H), 1.521.14 (m, 4H),
13
0.98 (d, J = 6.0 Hz, 3H), 0.970.85 (m, 6H); C NMR (75 MHz; CDCl3)  174.1, 158.8, 139.8,
23

130.6, 128.8, 128.0, 127.5, 127.2, 114.7, 73.3, 66.6, 55.4, 55.1, 35.8, 32.9, 26.3, 20.0, 17.0, 14.3,
12.3; MS (ESI) m/z 384.4 [M + H]+.
5.2. Pharmacology
5.2.1. Materials. Cell culture materials were purchased from Fisher SSI. Forskolin was purchased
from Sigma-Aldrich. The Lance Ultra kit (TRF0262) was purchased from PerkinElmer.
5.2.2. LanceTM cAMP assay using stable PPLS-HA-GPR88 CHO cells. Stimulation buffer
containing 1X Hank’s Balanced Salt Solution (HBSS), 5 mM HEPES, 0.1% 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 4x the desired final concentration
in 2% DMSO/stimulation buffer, were added to a 96-well white ½ area microplate (PerkinElmer). A
cAMP standard curve was prepared at 4x 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 minutes. The cell pellet was resuspended in stimulation buffer and 4,000 cells
(10 µL) were added to each well except wells containing the cAMP standard curve. After incubating
for 30 min at RT, Eu-cAMP tracer and uLIGHT-anti-cAMP working solutions were added per the
manufacturer’s instructions. After incubation at RT for 1 hour, the TR-FRET signal (ex 337 nm) was
read on a CLARIOstar multimode plate reader (BMG Biotech, Cary, NC). For the screen, the same
procedure was followed except that instead of adding serial dilutions of the test compounds, a single
concentration (1 µM, prepared at 4x final) of each test compound was added to the assay plates.
5.2.3. 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,
24

GraphPad Software, Inc., San Diego, CA). For the screen, the raw TR-FRET signal was expressed as
percent of control (11a) signal.
Acknowledgments
We are grateful to National Institute of Mental Health, National Institutes of Health, U.S.
(Grant MH103708 to C.J.) for the financial support of this research. We thank Drs. Bruce Blough,
Danni Harris and Rangan Maitra for valuable discussions during the course of this study.
References
1.
2.
3.
Rask-Andersen, M.; Almen, M. S.; Schioth, H. B. Nat. Rev. Drug Discov. 2011, 10, 579.
Armbruster, B. N.; Roth, B. L. J. Biol. Chem. 2005, 280, 5129.
Civelli, O.; Reinscheid, R. K.; Zhang, Y.; Wang, Z.; Fredriksson, R.; Schioth, H. B. Annu. Rev.
Pharmacol. Toxicol. 2013, 53, 127.
4.
5.
Mizushima, K.; Miyamoto, Y.; Tsukahara, F.; Hirai, M.; Sakaki, Y.; Ito, T. Genomics 2000,
69, 314.
Ghate, A.; Befort, K.; Becker, J. A.; Filliol, D.; Bole-Feysot, C.; Demebele, D.; Jost, B.; Koch,
M.; Kieffer, B. L. Neuroscience 2007, 146, 1182.
6.
7.
8.
Massart, R.; Guilloux, J. P.; Mignon, V.; Sokoloff, P.; Diaz, J. Eur. J. Neurosci. 2009, 30, 397.
Van Waes, V.; Tseng, K. Y.; Steiner, H. Basal Ganglia 2011, 1, 83.
Becker, J. A.; Befort, K.; Blad, C.; Filliol, D.; Ghate, A.; Dembele, D.; Thibault, C.; Koch, M.;
Muller, J.; Lardenois, A.; Poch, O.; Kieffer, B. L. Neuroscience 2008, 156, 950.
Logue, S. F.; Grauer, S. M.; Paulsen, J.; Graf, R.; Taylor, N.; Sung, M. A.; Zhang, L.; Hughes,
Z.; Pulito, V. L.; Liu, F.; Rosenzweig-Lipson, S.; Brandon, N. J.; Marquis, K. L.; Bates, B.;
Pausch, M. Mol. Cell. Neurosci. 2009, 42, 438.
9.
25

10. Quintana, A.; Sanz, E.; Wang, W.; Storey, G. P.; Guler, A. D.; Wanat, M. J.; Roller, B. A.; La
Torre, A.; Amieux, P. S.; McKnight, G. S.; Bamford, N. S.; Palmiter, R. D. Nat. Neurosci.
2012, 15, 1547.
11. Meirsman, A. C.; Le Merrer, J.; Pellissier, L. P.; Diaz, J.; Clesse, D.; Kieffer, B. L.; Becker, J.
A. Biol. Psychiatry 2016, 79, 917.
12. Jin, C.; Decker, A. M.; Huang, X. P.; Gilmour, B. P.; Blough, B. E.; Roth, B. L.; Hu, Y.; Gill,
J. B.; Zhang, X. P. ACS Chem. Neurosci. 2014, 5, 576.
13. Jin, C.; Decker, A. M.; Harris, D. L.; Blough, B. E. ACS Chem. Neurosci. 2016, 7, 1418.
14. Dzierba, C. D.; Bi, Y.; Dasgupta, B.; Hartz, R. A.; Ahuja, V.; Cianchetta, G.; Kumi, G.; Dong,
L.; Aleem, S.; Fink, C.; Garcia, Y.; Green, M.; Han, J.; Kwon, S.; Qiao, Y.; Wang, J.; Zhang,
Y.; Liu, Y.; Zipp, G.; Liang, Z.; Burford, N.; Ferrante, M.; Bertekap, R.; Lewis, M.; Cacace,
A.; Grace, J.; Wilson, A.; Nouraldeen, A.; Westphal, R.; Kimball, D.; Carson, K.; Bronson, J.
J.; Macor, J. E. Bioorg. Med. Chem. Lett. 2015, 25, 1448.
15. Summerfield, S. G.; Read, K.; Begley, D. J.; Obradovic, T.; Hidalgo, I. J.; Coggon, S.; Lewis,
A. V.; Porter, R. A.; Jeffrey, P. J. Pharmacol. Exp. Ther. 2007, 322, 205.
16. Ghose, A. K.; Herbertz, T.; Hudkins, R. L.; Dorsey, B. D.; Mallamo, J. P. ACS Chem.
Neurosci. 2012, 3, 50.
17. Clark, D. E.; Pickett, S. D. Drug Discov. Today 2000, 5, 49.
18. Bi, Y.; Dzierba, C. D.; Fink, C.; Garcia, Y.; Green, M.; Han, J.; Kwon, S.; Kumi, G.; Liang, Z.;
Liu, Y.; Qiao, Y.; Zhang, Y.; Zipp, G.; Burford, N.; Ferrante, M.; Bertekap, R.; Lewis, M.;
Cacace, A.; Westphal, R. S.; Kimball, D.; Bronson, J. J.; Macor, J. E. Bioorg. Med. Chem. Lett.
2015, 25, 1443.
26

Table of Contents Graphic
27