Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of the 3- and 4-methyl substituents

Article abstract of DOI:10.1016/j.bmc.2015.08.025

The design and discovery of JDTic as a potent and selective kappa opioid receptor antagonist used the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine pharmacophore as the lead structure. In order to determine if the 3-methyl or 4-methyl groups were necessary in JDTic and JDTic analogs for antagonistic activity, compounds 4a-c, and 4d-f which have either the 3-methyl or both the 3- and 4-methyl groups removed, respectively, from JDTic and analogs were synthesized and evaluated for their in vitro opioid receptor antagonist activities using a [³⁵S]GTPγS binding assay. Other ADME properties were also assessed for selected compounds. These studies demonstrated that neither the 3-methyl or 3,4-dimethyl groups present in JDTic and analogs are required to produce potent and selective κ opioid receptor antagonists.

Full text of DOI:10.1016/j.bmc.2015.08.025

Bioorganic & Medicinal Chemistry 23 (2015) 6379–6388
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and pharmacological evaluation of JDTic analogs
to examine the significance of the 3- and 4-methyl substituents
⇑
F. Ivy Carroll , Moses G. Gichinga, Chad M. Kormos, Rangan Maitra, Scott P. Runyon, James B. Thomas,
S. Wayne Mascarella, Ann M. Decker, Hernán A. Navarro
Research Triangle Institute, PO Box 12194, Research Triangle Park, NC 27709-2194, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and discovery of JDTic as a potent and selective kappa opioid receptor antagonist used the
N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine pharmacophore as the lead structure.
In order to determine if the 3-methyl or 4-methyl groups were necessary in JDTic and JDTic analogs
for antagonistic activity, compounds 4a–c, and 4d–f which have either the 3-methyl or both the
3- and 4-methyl groups removed, respectively, from JDTic and analogs were synthesized and evaluated
Received 25 June 2015
Revised 13 August 2015
Accepted 24 August 2015
Available online 25 August 2015
for their in vitro opioid receptor antagonist activities using a [35S]GTP
c
S binding assay. Other ADME prop-
Keywords:
JDTic
Opioids
Kappa antagonist
ADME properties
erties were also assessed for selected compounds. These studies demonstrated that neither the 3-methyl
or 3,4-dimethyl groups present in JDTic and analogs are required to produce potent and selective
receptor antagonists.
j opioid
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
were recently reported, we compare the results from the two
studies5.
The design and discovery of JDTic as a potent and selective
kappa opioid receptor antagonist used the N-substituted trans-
3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (1) pharmacophore
as the lead structure (Fig. 1).1,2 In a recent study we reported that
2. Chemistry
Compound 4a was prepared according to Scheme 1. 3-
Bromiosopropoxybenzene was prepared and distilled from the cor-
responding phenol and isopropyl bromide to be used as substrate
for the lithium–halogen exchange. Addition of the resulting aryl
lithium to N-methylpiperidine 5 gave the alcohol 6 in 42% yield.
Dehydration of 6 with 2 equiv p-toluenesulfonic acid in toluene
gave tetrahydropyridine 7 in 79% yield. Methylation according to
the method reported by Werner et al.6 was followed by NaBH4
reduction of the enamine intermediate to afford 8. Treatment of
8 with 1-chloroethyl chloroformate (ACE-Cl) in refluxing dichlor-
oethane followed by methanolysis yielded piperidine 9. Reductive
N-phenylpropyl-4-methyl-4-(3-hydroxyphenyl)-piperidine
(2a)
where the 3-methyl group was removed from 1 and N-phenyl-
propyl-4-(3-hydroxyphenyl)-piperidine (2b), where both the
3- and 4-methyl groups were removed from 1 were still pure opi-
oid receptor antagonists.3 In another recent study we reported that
the replacement of the hydroxy group in the 4-(3-hydroxyphenyl)
moiety of JDTic with a hydrogen or fluoro group gave compounds
3a and 3b both of which were potent and selective kappa opioid
receptor antagonists (Fig. 1).4 As a continuation of our structural
activity relationship (SAR) studies directed toward the kappa opi-
oid receptor as well as the development of potential pharma-
cotherapies for CNS disorders, compounds 4a–f were synthesized
amination of amine 9 with Boc-
hydride in trifluoroethanol followed by Boc-deprotection afforded
10. HBTU coupling of 10 with Boc-7-hydroxy- -Tic-OH followed
L
-Valinal7 using sodium cyanoboro-
and evaluated in vitro using a [35S]GTP
d, and opioid receptors and their ADME properties were estab-
cS binding assay at the l,
D
j
by deprotection with hydrogen bromide in acetic acid afforded 4a.
Compound 4b was prepared as outlined in Scheme 2. 1-Methyl-
4-(3-fluorophenyl)-1,2,5,6-tetrahydropyridine (11) was prepared
according to the method of Nagai et al.8 The deprotonation and
methylation proceeded analogously to the method described by
Werner.6 The resulting enamine was reduced with sodium borohy-
dride in methanol. Demethylation with ACE-Cl followed by heating
at reflux in methanol to hydrolyze the intermediate carbamate
lished (Fig. 1). A comparison of their in vitro antagonist activity
and ADME properties of selected compounds to those of JDTic
and compounds 3a and 3b are herein presented. In addition, since
the synthesis and [35S]GTP
cS in vitro properties of compounds 4d
⇑
Corresponding author. Tel.: +1 919 541 6679; fax: +1 919 541 8868.
E-mail address: fic@rti.org (F.I. Carroll).
http://dx.doi.org/10.1016/j.bmc.2015.08.025
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
Figure 1. Structures of JDTic, 2a–b, 3a–b, and 4a–f.
Scheme 1. Reagents and conditions: (a) 3-isopropoxyphenyl lithium, THF; (b) TsOH, toluene; (c) (1) n-BuLi, THF, À15 °C; (2) Me2SO4, À50 °C; (3) NaBH4, CH3OH; (d) (1)
1-chloroethyl chloroformate, DCE; (2) CH3OH, reflux; (e) (1) Boc- -Valinal, NaBH3CN; (2) TFA, DCM; (f) (1) Boc-7-hydroxy- -Tic-OH, HBTU, NEt3; (2) HBr, AcOH.
L
D
afforded 4-(3-fluorophenyl)-4-methylpiperidine (12). Piperidine 12
was coupled with Boc- -valine using HBTU in acetonitrile. The tert-
butyloxycarbonyl protecting group was removed with aqueous
hydrogen chloride in methanol so that the amide could be reduced
cleanly with borane dimethylsulfide at reflux in tetrahydrofuran.
Boc-L-isoleucine using EDC and HOBt in acetonitrile followed by
L
removal of the Boc protecting group present with aqueous hydro-
gen chloride in methanol so that the amide could be reduced
cleanly with borane dimethylsulfide at reflux in tetrahydrofuran
to give 14. The resulting amine (14) was coupled with Boc-7-hy-
The resulting amine (13) was coupled with Boc-7-hydroxy-
D
-Tic-OH
droxy-
D
-Tic-OH using EDCÁHCl in dichloromethane followed by
using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride
(EDCÁHCl) in dichloromethane followed by treatment with hydrogen
chloride in dioxane and acetonitrile to afford 4b.
treatment with hydrogen chloride in dioxane and acetonitrile to
afford 4c.
The synthesis of 4f is outlined in Scheme 4. Coupling of N-Boc-L-
Compound 4c was prepared from 12 via a procedure analogous
valine with 4-(3-fluorophenyl)piperidine 15 using HBTU in acetoni-
trile followed by cleavage of the tert-butyloxycarbonyl protecting
group in intermediate tert-butyl [(1S)-1-{[4-(3-fluorophenyl)-
to that used for 4b by substituting Boc-L-isoleucine for Boc-L-valine
as outlined in Scheme 3. Piperidine 12 was first coupled to

F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
6381
Scheme 2. Reagents and conditions: (a) (1) n-BuLi, THF, À78 °C; (2) Me2SO4; (b) NaBH4, CH3OH; (c) (1) ACE-Cl, DCE; (2) CH3OH, reflux; (d) HBTU, NEt3, Boc-
L-valine, CH3CN;
(e) (1) HCl; (2) BH3ÁSMe2; (f) Boc-7-hydroxy-
D
-Tic-OH, EDCÁHCl, HOBt, DIPEA, THF; (g) HCl, CH3CN, dioxane.
Scheme 3. Reagents and conditions: (a) Boc-
THF; (e) HCl, CH3CN, dioxane.
L
-isoleucine, HOBt, EDC, DIPEA, CH3CN; (b) HCl, CH3OH; (c) BH3ÁS(CH3)2; (d) Boc-7-hydroxy- -Tic-OH, EDCÁHCl, DIPEA, HOBt,
D
piperidin-1-yl]carbonyl}-2-methylpropyl]carbamate 16 using
concentrated hydrochloric acid in methanol gave 17. Reduction
of 17 with diborane in tetrahydrofuran gave 18. Coupling of 18
Replacement of the 3-OH in 4a with a fluoro group gave 4b
which was also a pure opioid antagonist in the [35S]GTP
S test
(Table 1). Compound 4b, which has a Ke = 0.18 nM at the receptor
is only 9- and 18-fold less potent than JDTic and 3b as a opioid
antagonist (Table 1). With Ke values of 38 and 1480 nM at the
and d receptors, respectively, the compound 4b remains highly
selective for the relative to the and d opioid receptors. Com-
pound 4c, which has the isopropyl group in 4b replaced with an
isobutyl group, has Ke values of 11.3, 903, and 0.033 nM at the
d, and receptors, respectively. Thus, 4c is 6-fold more potent than
4b as a opioid receptor antagonist.
c
j
with Boc-7-hydroxy-
D
-Tic-OH using EDC and catalytic amount
j
of N-hydroxybenzotriazole (HOBt) in dichloromethane provided
tert-butyl (3R)-3-{[(1S)-1-{[4-(3-fluorophenyl)piperidin-1-yl]methyl}-
2-methylpropyl]carbamoyl}-7-hydroxy-3,4-dihydroisoquinoline-2
(1H)-carboxylate 19. Compound 4f was obtained by treating 19
with concentrated hydrochloric acid in methanol.
l
j
l
l,
Compounds 4d and 4e were synthesized by procedures analo-
j
gous to the reported synthesis.5
j
Compounds 4d, 4e, and 4f have both the 3- and 4-methyl
groups removed from the piperidine ring, relative to JDTic, 3a
and 3b, respectively (Table 1). Compound 4d with a Ke = 0.1 nM
3. Results and discussion
In order to determine if the 3-methyl group on the piperidine
ring of JDTic was required to obtain a pure antagonist, we synthe-
sized and tested 4a for its opioid antagonism properties. We found
at the
in this assay (Table 1). Having Ke values of 12.3 and 240 nM at the
and d receptors, respectively, 4d is 123-fold and 2400-fold selec-
tive for the receptor relative to the and d receptors, respec-
tively. Compound 4e also has the 3-OH removed. With
Ke = 0.051 nM at the receptor, this compound is only 2.6- and
2-times less potent as a antagonist than JDTic and 3a, respec-
tively. Compound 4e has Ke values of 3.96 and 281 nM at the
and d receptors, respectively, and is 77- and 5500-fold selective
j receptor is 5-times less potent than JDTic as a j antagonist
l
that 4a had no agonist activity at 10
assay and was indeed a pure opioid receptor antagonist (Table 1).
With Ke values of 67.5, 927, and 0.69 nM at the , d, and opioid
l cS binding
M in the [35S]GTP
j
l
a
l
j
j
receptors, respectively, it was a potent and selective kappa opioid
receptor antagonist. However, it was 35-times less potent than
JDTic as a kappa opioid receptor antagonist (Table 1).
j
l

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F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
Scheme 4. Reagents: (a) N-Boc-L-valine, HBTU, CH3CN, TEA; (b) HCl, MeOH; (c) B2H6, THF; (d) EDC, HOBt, TEA, Boc-7-hydroxy-D-Tic-OH, DCM.
for the
j
receptor relative to the
l
and d receptors, respectively.
ratio-metric number that is calculated from the rightward shift
in the agonist EC50 in the presence of antagonist.9 However, differ-
ences in our Ke assay methods could affect receptor conformation
altering the manner in which this ligand interacts with these
receptors.
Compound 4f like 3b has a 3-fluoro substituent in place of the
3-OH in JDTic and 4d. Similar to JDTic, 4f with a Ke = 0.023 nM at
the
Ke = 3.55 and 128 nM at the
4f is 7- and 4-times more potent as a
and 3b, respectively, and is 154- and 5600-fold selective for the
receptor relative to the and d receptors, respectively.
Compound 4d has a structure identical to that of a compound
named AT-076 reported by Zaveri and coworkers.5 These authors
report Ke values of 0.48, 24.95, and 4.3 nM at the , d, and opioid
receptors for AT-076 compared to Ke values of 12.3, 240, and
0.1 nM determined in this study for 4d using a [35S]GTP
S binding
j
receptor is a potent
j
l
antagonist. Compound 4f has a
and d receptors, respectively. Thus,
antagonist than JDTic
l
In order to determine if the compounds would be predicted to
cross the blood/brain barrier, their topological polar surface area
(TPSA), clogP, and derived logBB values were calculated and com-
pared to JDTic, 3a and 3b. In general CNS compounds that have
TPSA values less than 76 Å2,10 clogP values in the range of 2–4,11
and derived logBB values greater than À112 are predicted to cross
the blood/brain barrier. Compounds 3b, 4f and 4c have TPSA values
less than 76 Å2. JDTic and 4a with TPSA values of 84.83 Å2 are a
little greater than 76 Å2. JDTic, 4a, and 4b have clogP values in
the range of 2–4. Compounds 3b and 4c have clogP values a little
above 4. All of the compounds have logBB values greater than À1
and thus, would be predicted to penetrate the brain.
j
l
l
j
c
assay. Although the Zaveri et al. Ke values for the mu and delta
receptors are lower than those for 4d, both compounds show
approximately the same relative selectivity for these receptors,
but their Ke value is 43-fold higher for the
determined for 4d. Differences in opioid receptor–effector coupling
j receptor than that
Compound 4d has a calculated TPSA value of 84.83 Å2, which is
identical to that of JDTic. Compounds 4e and 4f have TPSA values of
efficiency cannot explain this difference because the Ke is a

F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
6383
Table 1
Inhibition of agonist-stimulated [35S]GTP
cS binding by compounds in cloned human l, d, and j opioid receptors
a
Compd
Ke (nM)
d, DPDPE
X
R1
R2
l, DAMGO
j, U69,593
l/
j
d/
j
nor-BNI
JDTic
3a
3b
4a
4b
4c
4d
4e
—
—
—
—
OH
F
F
OH
H
—
—
—
—
CH3
CH3
CH3
H
H
H
—
—
—
—
H
H
CH3
H
26
25
8.9
14.8
67.5 10.4
38.0 6.4
11.3 4.3
12.3 1.0
3.96 1.1
3.55 1.1
29
74
442
249
927 350
1480 350
903 200
240 73
281 44
128 36
0.05
0.02
0.024
0.01
0.69 0.21
0.18 0.04
0.033 0.01
0.10 0.03
0.051 0.01
0.023 0.01
520
1255
370
1480
98
211
342
123
77
580
3800
18,400
24,900
1344
8211
27,363
2400
5500
5600
4
2
H
H
4f
F
154
a
Ke values are the mean SEM of at least three independent experiments performed in duplicate.
Table 2
In vitro ADME data for 3a, 3b, and 4a–f
Compd
hERG
(Ki, M)
MDCK-mdr1
(% transported, A–B)
Solubility (lM)
Plasma stability
(% of parent)
S9 stability
(% of parent)
PAMPA (%
transported)
TPSA
cLogP
LogBB
l
pH 7.4
pH 3
pH 7.4
pH 5.5
JDTic
3a
3b
4a
4b
4c
4d
4e
8.820
7.048
6.251
>10
4.237
1.666
1.732
0.777
0.436
11
27
6
<1
1.3
11
11
10
44
34
42
47
97
76
26.8
91.4
19.6
1
57.9
67.3
3
84.83
64.60
64.60
84.83
64.60
64.60
84.83
64.6
3.60
3.89
4.15
3.43
3.97
4.38
3.23
3.53
3.75
À0.57
À0.23
À0.19
À0.59
À0.21
À0.15
À0.63
À0.28
À0.25
67.0
49.6
62.5
82.0
77.5
62.5
101
2.6
4f
64.60
64.60 Å2, which are identical to those of 3a and 3b. Compounds 4d,
4e and 4f all have clogP values less than 4 and greater than 2. Com-
pounds 4d, 4e and 4f with logBB values of greater than À1 would
be predicted to have good brain penetration.
PAMPA permeability properties of 4a were determined and com-
pared to the properties of JDTic (Table 2). The permeability of 4a
is negligible across MDCK-mdr1 monolayers compared to 11% for
JDTic. However, 4a with solubility of 44 and 101 lM at pH 7.4
ADME properties of these compounds were assessed (Table 2).
Compounds that interact with the human ether-a-go-go gene
(hERG) product, which is a potassium channel, can produce QT pro-
longation and cardiotoxic effects. The hERG Ki values of 4a–f were
compared to the Ki values for JDTic, 3a and 3b (Table 2). Compound
and 3, respectively, is more soluble than JDTic. With plasma and
S9 stabilities of 62.5% following incubation in both assays, it is
not as metabolically stable as JDTic but is above the 50% threshold
considered to be desirable. The percent transported values of 1%
and 2.6% at pH 7.4 and 5.5 in the PAMPA assay are well below
the 26.8 and 57.9 percent values for JDTic. Due to the low Ki values
for 4b–f in the hERG assay the ADME properties of the compounds
were not determined.
4a had a Ki = >10
lM which is larger than the Ki value for JDTic
(Ki = 8.820 M). All the other compounds (4b–f) had Ki values
l
lower than JDTic.
Compounds that have >5% permeability in the Madin Derby
Canine Kidney cells stably expressing the human multidrug resis-
tant gene (mdr1) product P-glycoprotein (MDCK-mdr1) monolayer
permeability assay, >50% stability in the plasma and hepatic S9
stability assay, >25% transported in the Parallel Artificial
4. Conclusions
In conclusion, JDTic analogs 4a–c and 4d–f which have the
3-methyl or both the 3- and 4-methyl groups removed, respectively,
from JDTic and analogs were synthesized and evaluated for their
Membrane Permeability Assay (PAMPA), and >20 lM solubility
are considered desirable for further development by our group.
ability to antagonize [35S]GTP
c
S binding at the
l, d, and j opioid
The MDCK-mdr1 permeability, solubility, plasma, S9 stability and
receptors. The data showed that neither the 3- or 3,4-dimethyl

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F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
groups present in JDTic and analogs are required to obtain potent
and selective kappa opioid receptor antagonists. Compound 4c
5.2. (3R)-N-[(1S)-1-{[4-(3-Fluorophenyl)-4-methylpiperidin-1-
yl]methyl}-2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroiso-
quinoline-3-carboxamide (4b) dihydrochloride
with a Ke = 0.033 nM at the
selectivity for the relative to the
was the most potent and selective compound having only the
3-methyl group removed. Compound 4f with a Ke = 0.023 nM at
the receptor and 154- and 5600-fold selectivity for the relative
to the and d receptors was the most potent and selective com-
pound having both the 3- and 4-methyl groups removed. However,
all of the compounds 4a–f have subnanomolar potency for the
receptor and are selective for the receptor relative to the and
d receptors and may be useful pharmacological tools for studying
the opioid receptor.
j
receptor and 342- and 27,360-fold
j
l
and d receptors, respectively,
j
(2S)-1-[4-(3-Fluorophenyl)-4-methylpiperidin-1-yl]-3-methyl-
butan-2-amine (13) (274 mg, 0.98 mmol), Boc-7-hydroxy-D-Tic
j
j
(300 mg, 1.02 mmol), hydroxybenzotriazole hydrate (20 mg,
0.12 mmol), and EDCÁHCl (390 mg, 2.0 mmol) were combined in
THF (10 mL). Diisopropylethylamine (0.75 mL, 4.3 mmol) was
slowly added. The resulting solution was stirred at room tempera-
ture for 12 h then concentrated. The residue was dissolved in CH2-
Cl2 (15 mL) then washed with saturated aqueous NaHCO3 (5 mL).
The aqueous layer was extracted once with EtOAc (15 mL). The
combined organic layers were washed with brine (5 mL), dried
(Na2SO4), and concentrated. The residue was purified by
chromatography on silica gel using a gradient up to 40% CMA80
in CH2Cl2. The product containing fractions were combined and
concentrated then dissolved in acetonitrile (5 mL) to which HCl
in dioxane (4 N, 5 mL) was added. The resulting solution was evap-
orated under a stream of N2 to afford a white powder. The powder
dissolved in a minimum of MeOH was purified by chromatography
on silica gel using a gradient up to 50% CMA80 in CH2Cl2 to afford
4b as the free base: 1H NMR (300 MHz, CDCl3) d 7.22–7.32 (m, 1H),
7.03–7.14 (m, 2H), 6.99 (td, J = 2.03, 11.21 Hz, 1H), 6.82–6.92 (m,
2H), 6.55 (dd, J = 2.35, 8.19 Hz, 1H), 6.42 (d, J = 2.26 Hz, 1H), 4.12–
4.25 (m, 1H), 3.57–3.77 (m, 2H), 3.24 (dd, J = 5.18, 11.40 Hz, 1H),
2.78–2.98 (m, 2H), 2.46–2.69 (m, 3H), 2.06–2.43 (m, 5H), 1.73–1.91
(m, 3H), 1.19 (s, 3H), 0.84–0.96 (m, 6H); 19F NMR (282 MHz, CDCl3)
d À112.84 (s, 1F); 13C NMR (75 MHz, CDCl3) d 173.4, 163.1 (d,
J = 245 Hz), 155.0, 151.2 (broad), 137.3, 130.7, 129.9 (d, J = 8.3 Hz),
125.1, 121.3 (d, J = 1.8 Hz), 113.8, 112.9 (d, J = 25.6 Hz), 112.6 (d,
J = 24.8 Hz), 112.2, 59.9, 56.7, 50.5, 50.0, 49.8, 48.2, 36.2, 36.1, 31.6,
29.5, 19.0, 17.9. The free base was converted to the dihydrochloride
salt, affording 142 mg (26% over two steps) of a white powder: MS
l
j
j
j
l
j
5. Experimental
Melting points were determined using a MEL-TEMP II capillary
melting point apparatus and are uncorrected. Nuclear magnetic
resonance (1H NMR and 13C NMR) spectra were obtained on a Var-
ian Avance DPX-300 MHz NMR spectrometer or a Bruker Unity
Inova 500 MHz NMR spectrometer. Chemical shifts are reported
in parts per million (ppm) with reference to internal solvent. Mass
spectra (MS) were run on a Perkin–Elmer Sciex AP1 150 EX mass
spectrometer equipped with APCI (atmospheric pressure chemical
ionization) or ESI (turbospray) sources or on a Hewlett Packard
5989A instrument by electron impact. Elemental analyses were
performed by Atlantic Microlab Inc., Atlanta, GA. Optical rotations
were measured on an AutoPol III polarimeter, purchased from
Rudolf Research. Analytical thin-layer chromatography (TLC) was
carried out using EMD silica gel 60 F254 TLC plates. TLC visualiza-
tion 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 or using EM
Science silica gel 60A (230–400 mesh). Solvent system:
CMA80 = 80:18:2 CHCl3–MeOH–concd NH4OH. Unless otherwise
stated, reagent-grade chemicals were obtained from commercial
sources and were used without further purification. All moisture-
and air-sensitive reactions and reagent transfers were carried out
under dry nitrogen.
(ESI) m/z 454.4 (M+H)+, mp 205–209 °C (fusion), [a 2D5
+122 (c 0.1,
]
CH3OH). Anal. Calcd for C27H38Cl2FN3O2Á1.5H2O: C, 58.59; H, 7.47;
N, 7.59. Found: C, 58.73; H, 7.43, N, 7.30.
5.3. (3R)-N-[(1S,2S)-1-{[4-(3-Fluorophenyl)-4-methylpiperidin-
1-yl]methyl}-2-methylbutyl]-7-hydroxy-1,2,3,4-tetrahydroiso-
quinoline-3-carboxamide (4c) dihydrochloride
5.1. (3R)-7-Hydroxy-N-[(1S)-1-{[4-(3-hydroxyphenyl)-4-methyl-
piperidin-1-yl]methyl}-2-methylpropyl]-1,2,3,4-tetrahydroiso-
quinoline-3-carboxamide (4a) dihydrochloride
(2S,3S)-1-[4-(3-Fluorophenyl)-4-methylpiperidin-1-yl]-3-methyl-
pentan-2-amine (14) (266 mg, 0.91 mmol), Boc-7-hydroxy-D-Tic
(293 mg, 1.00 mmol), hydroxybenzotriazole hydrate (20 mg,
0.12 mmol), and EDCÁHCl (390 mg, 2.0 mmol) were combined in
10 mL THF. Diisopropylethylamine (0.75 mL, 4.3 mmol) was slowly
added. The resulting solution was stirred at room temperature for
12 h then concentrated. The residue was dissolved in CH2Cl2
(15 mL) then washed with saturated aqueous NaHCO3 (5 mL). The
aqueous layer was extracted once with EtOAc (15 mL). The combined
organic layers were washed with brine (5 mL), dried (Na2SO4), and
concentrated. The residue was purified by chromatography on silica
gel using a gradient up to 40% CMA80 in CH2Cl2. The product con-
taining fractions were combined and concentrated then dissolved
in acetonitrile (10 mL) to which aq HCl (6 N, 2 mL) was added. The
resulting solution was evaporated under a stream of N2 to afford a
powder. The powder dissolved in a minimum of MeOH was purified
by chromatography on silica gel using a gradient up to 50% CMA80 in
CH2Cl2 to afford 4c as the free base: 1H NMR (300 MHz, CDCl3) d
7.22–7.33 (m, 1H), 7.14 (d, J = 9.04 Hz, 1H), 7.06 (d, J = 8.10 Hz, 1H),
6.98 (td, J = 2.03, 11.21 Hz, 1H), 6.83–6.92 (m, 2H), 6.50 (dd,
J = 2.45, 8.29 Hz, 1H), 6.40 (d, J = 2.26 Hz, 1H), 4.26 (t, J = 9.98 Hz,
1H), 3.52–3.75 (m, 2H), 3.20 (dd, J = 5.18, 11.40 Hz, 1H), 2.89 (dd,
J = 5.18, 16.48 Hz, 2H), 2.45–2.74 (m, 3H), 2.05–2.43 (m, 5H),
1.74–1.95 (m, 2H), 1.55–1.71 (m, 1H), 1.42 (ddd, J = 4.90, 7.54,
The amine 10 and Boc-7-hydroxy-D-Tic-OH (1.1 equiv) were
combined with HBTU (1.1 equiv) and NEt3 (4 equiv) in DCM
(30 mL). The reaction mixture was stirred overnight then concen-
trated. The residue was subjected to chromatography using EtOAc
as the eluent to afford the protected product. The residue resulting
from concentration of the desired fractions was refluxed in 1:1 48%
HBr/glacial AcOH overnight. The resulting solution was concen-
trated and the resulting residue was subjected to a gradient of
CMA80:CH2Cl2 through silica gel to afford 128 mg of 4a free base
(21% over two steps): 1H NMR (CDCl3) d 7.16 (t, 1H, J = 7.9 Hz),
6.95 (d, 1H, J = 8.1 Hz), 6.85–6.78 (m, 2H), 6.69–6.62 (m, 2H),
6.54–6.49 (m, 1H), 4.07–3.97 (m, 1H), 3.96–3.90 (m, 1H), 3.10–
2.95 (m, 1H), 2.83–2.29 (m, 6H), 2.17–2.00 (m, 2H), 1.86–1.67
(m, 2H), 1.22–1.16 (m, 3H), 0.96–0.80 (m, 6H); 13C NMR (CDCl3)
d 174.3, 173.7, 156.9, 155.1, 155.0, 136.3, 136.2, 130.2, 129.9,
129.5, 124.8, 124.7, 117.2, 114.0, 113.0, 112.7, 112.3, 112.1, 60.1,
59.8, 57.3, 56.9, 47.3, 47.1, 36.5, 36.4, 36.3, 36.0, 31.3, 31.2, 30.4,
19.8, 17.7, 17.5; MS (ESI) m/z 452.6 (M+H)+; The free base was con-
25
verted into the dihydrochloride salt: mp 180–184 °C (fusion); [a]
D
+45.6° (c 0.50, CH3OH). Anal. Calcd for C27H39Cl2N3O3Á1.5H2O: C,
58.80; H, 7.68; N, 7.62. Found: C, 59.08; H, 7.34; N, 7.34.

F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
6385
13.00 Hz, 1H), 1.17–1.23 (m, 3H), 1.04–1.17 (m, 1H), 0.83–0.96 (m,
6H); 19F NMR (282 MHz, CDCl3) d À112.82 (s, 1F); 13C NMR
(75 MHz, CDCl3) d 173.2, 163.1 (d, J = 245 Hz), 154.9, 151.1 (broad),
137.5, 130.8, 129.9 (d, J = 8.4 Hz), 125.3, 121.3 (d, J = 2.0 Hz), 113.6,
112.9 (d, J = 21.9 Hz), 112.6 (d, J = 21.2 Hz), 112.1, 112.1, 58.8, 56.6,
50.5, 50.1, 48.8, 48.3, 38.3, 36.2, 36.1, 29.2, 25.7, 14.8, 11.9. The free
base was converted to the dihydrochloride salt, affording 216 mg
(38% over two steps) of a white powder: MS (ESI) m/z 468.3 (M
(m, 4H), 6.52 (dd, J = 2.45, 8.10 Hz, 1H), 6.37 (d, J = 2.07 Hz, 1H),
4.08–4.23 (m, 1H), 3.53–3.74 (m, 2H), 3.30 (d, J = 11.11 Hz, 1H),
3.20 (dd, J = 5.18, 11.21 Hz, 1H), 2.97 (d, J = 11.30 Hz, 1H), 2.86
(dd, J = 5.09, 16.58 Hz, 1H), 2.59 (t, J = 12.06 Hz, 1H), 2.38–2.52 (m,
1H), 2.21–2.36 (m, 2H), 2.06–2.20 (m, 1H), 1.90–2.03 (m, 1H),
1.60–1.88 (m, 5H), 0.87 (d, J = 6.78 Hz, 6H); 13C NMR (75 MHz,
CHLOROFORM-d) d 173.4, 164.6, 161.3, 154.9, 148.4, 148.3, 137.3,
130.6, 129.9, 129.8, 125.4, 122.4, 122.3, 113.9, 113.8, 113.5, 113.2,
112.9, 112.2, 60.0, 56.7, 55.9, 52.7, 50.0, 48.1, 42.0, 32.7, 32.4, 31.5,
29.6, 19.1, 17.9; ESI MS (M+H)+ 440.5. The free base was converted
to the dihydrochloride salt by adding 2 M HCl in ether to solution of
+H)+, mp 191–195 °C (fusion), [a D25 +65.2 (c 0.50, CH3OH). Anal. Calcd
]
for C28H40Cl2FN3O2Á1.25H2O: C, 59.73; H, 7.61; N, 7.46. Found: C,
59.76; H, 7.69; N, 7.21.
product in dichloromethane: mp 215–219 °C, [a D +61.6 (c 1.04,
]
5.4. N-[(1S)-1-{[4-(3-Hydroxyphenyl)piperidin-1-yl]methyl}-2-
methylpropyl]-3-methyl-4-(3-methylphenoxy)benzamide (4d)
dihydrochloride
MeOH). Anal. Calcd for C26H36Cl2FN3O2ÁH2O: C, 58.87; H, 7.22; N,
7.92. Found C, 59.20; H, 7.39; N, 7.70.
5.7. 1-Methyl-4-[3-(1-methylethoxy)phenyl]piperidin-4-ol (6)
Compound 4d was synthesized by a procedure analogous to the
reported synthesis.7 1H NMR (300 MHz, CHLOROFORM-d) d 6.94–
7.11 (m, 2H), 6.75 (d, J = 8.29 Hz, 1H), 6.45–6.62 (m, 4H), 6.38 (s,
1H), 4.08 (d, J = 9.61 Hz, 1H), 3.75 (d, J = 16.58 Hz, 1H), 3.61 (d,
J = 16.39 Hz, 1H), 3.33 (t, J = 7.06 Hz, 1H), 3.09 (d, J = 9.98 Hz, 1H),
2.86 (d, J = 10.36 Hz, 1H), 2.74 (br s, 1H), 2.58 (t, J = 11.59 Hz,
1H), 2.24 (d, J = 10.74 Hz, 2H), 2.08 (br s, 1H), 1.72 (dd, J = 6.31,
11.96 Hz, 2H), 1.60 (br s, 2H), 1.38 (br s, 2H), 0.86 (t, J = 6.69 Hz,
6H); 13C NMR (75 MHz, CHLOROFORM-d) d 173.4, 171.6, 156.7,
154.7, 147.7, 136.2, 132.7, 130.4, 129.5, 124.7, 119.0, 114.5,
113.5, 112.7, 60.2, 56.4, 52.4, 50.2, 49.3, 46.8, 42.0, 32.7, 32.1,
31.7, 29.5, 19.3, 17.9; ESI MS (M+H)+ 438.6. The product was con-
verted to the dihydrochloride salt by adding 2 M HCl in ether to
solution of the free base in dichloromethane: mp >220 °C (dec),
A solution of n-butyl lithium (2.5 M in hexanes, 8.7 mL, 22 mmol)
was slowly added to a solution of 1-bromo-3-isopropoxybenzene
(5.2 g, 24 mmol) in THF (14 mL) at À78 °C. After 30 min, N-methyl-
4-piperidinone (5) (2.49 g, 22.0 mmol) was added dropwise. The
solution warmed to rt overnight. Hydrochloric acid (6 M, 8 mL)
was added and the resulting biphasic mixture was extracted with
hexanes. The organic layer was discarded and the aqueous layer
was adjusted to pH 10 with NH4OH (2 M). Extraction with hexanes,
drying with Na2SO4 and concentration to afford 2.29 g (42%) of crude
6. 1H NMR (CDCl3) d 7.25 (t, J = 7.9 Hz, 1H), 7.01–7.10 (m, 2H), 6.74–
6.83 (m, 1H), 4.56 (spt, J = 6.0 Hz, 1H), 2.75 (d, J = 11.3 Hz, 1H), 2.38–
2.54 (m, 2H), 2.35 (s, 3H), 2.17 (dt, J = 4.5, 13.0 Hz, 1H), 1.69–1.81 (m,
1H), 1.33 (d, J = 6.0 Hz, 6H).
[
a
]
+58.5 (c 0.62, MeOH). Anal. Calcd for C26H37Cl2N3O3Á1.25H2O:
D
C, 58.59; H, 7.47; N, 7.88. Found C, 58.45; H, 7.26; N, 7.78.
5.8. 1-Methyl-4-[3-(1-methylethoxy)phenyl]-1,2,3,6-tetrahydro-
pyridine (7)
5.5. (3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-phenylpiperidin-1-
yl)methyl]propyl}-1,2,3,4-tetrahydroisoquinoline-3-carboxamide
(4e) dihydrochloride
Crude 6 was refluxed in toluene (15 mL) with TsOHÁH2O
(2 equiv) for 3 h. The product was extracted into water. The aque-
ous layer was adjusted to pH 10 with NaOH (2 M) and then
extracted with hexanes. The combined organic layer was washed
with NaOH (2 M) and dried (Na2SO4). Concentration afforded
1.67 g (79%) of crude 7. 1H NMR (300 MHz, CDCl3) d 7.14–7.25
(m, 2H), 6.96 (d, J = 7.9 Hz, 1H), 6.87–6.93 (m, 1H), 6.77 (dd,
J = 2.5, 8.1 Hz, 1H), 6.00–6.08 (m, 1H), 4.55 (spt, J = 6.1 Hz, 1H),
3.10 (q, J = 2.8 Hz, 2H), 2.62–2.70 (m, 2H), 2.51–2.62 (m, 2H),
2.40 (s, 3H), 2.36 (s, 1H), 1.67 (s, 1H), 1.33 (d, J = 6.0 Hz, 7H).
Compound 4e was synthesized by the reported procedure.7 1
H
NMR (300 MHz, CHLOROFORM-d) d 7.29–7.24 (m, 2H), 7.20–7.10
(m, 4H), 6.91 (d, J = 8.3 Hz, 1H), 6.61 (dd, J = 2.5, 8.2 Hz, 1H), 6.45
(d, J = 2.5 Hz, 1H), 4.26 (br s, 1H), 3.71 (q, J = 15.5 Hz, 2H), 3.45
(d, J = 10.9 Hz, 1H), 3.26 (dd, J = 5.2, 11.2 Hz, 1H), 3.07 (d,
J = 11.7 Hz, 1H), 2.94 (dd, J = 5.2, 16.5 Hz, 1H), 2.70 (t, J = 12.1 Hz,
1H), 2.61–2.48 (m, 1H), 2.40–2.17 (m, 3H), 2.06 (m, 1H), 1.99–
1.76 (m, 5H), 0.95 (d, J = 7.0 Hz, 6H); 13C NMR (75 MHz, CHLORO-
FORM-d) d 173.3, 154.8, 145.6, 137.6, 130.8, 128.5, 126.7, 126.3,
125.5, 113.7, 112.1, 60.0, 56.6, 56.2, 52.6, 49.8, 48.3, 42.2, 32.9,
32.4, 31.7, 29.4, 19.0, 18.0; ESI MS (M+H)+ 422.5. The free base
was converted to the dihydrochloride salt by adding 2 M HCl in
5.9. 1,4-Dimethyl-4-[3-(1-methylethoxy)phenyl]piperidine (8)
Crude 7 in THF (18 mL) was treated at À15 °C with butyl lithium
(2.5 M, 4.5 mL) to afford a blood-red solution. After cooling to
À50 °C, dimethylsulfate (0.8 mL) was cautiously added. After
30 min, NH4OH (2 M, 10 mL) was added and the resulting biphasic
mixture was extracted with hexanes. The combined organic layer
was washed with water, dried (Na2SO4), and concentrated to a resi-
due which was dissolved in MeOH (20 mL) and treated at 0 °C with
NaBH4 (0.42 g). After warming to room temperature the solution was
concentrated and subjected to silica gel chromatography using a gra-
dient of CMA80 in CH2Cl2 as the eluent to afford 1.44 g (81%) of 8. 1H
NMR (300 MHz, CHLOROFORM-d) d 7.20–7.31 (m, 1H), 7.01–7.10 (m,
2H), 6.73–6.83 (m, 1H), 4.56 (spt, J = 5.97 Hz, 1H), 2.75 (d,
J = 11.30 Hz, 2H), 2.38–2.51 (m, 2H), 2.35 (s, 3H), 2.17 (dt, J = 4.52,
13.00 Hz, 2H), 1.68–1.81 (m, 2H), 1.33 (d, J = 6.03 Hz, 6H).
ether to solution of product in dichloromethane: [
a
]
D
+62.7 (c
1.01, MeOH). Anal. Calcd for C26H37Cl2N3O2Á1.5H2O: C, 59.88; H,
7.73; N, 8.06. Found C, 59.67; H, 7.40; N, 8.01.
5.6. (3R)-N-[(1S)-1-{[4-(3-Fluorophenyl)piperidin-1-yl]methyl}-
2-methylpropyl]-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-
carboxamide (4f) dihydrochloride
To a solution of tert-butyl (3R)-3-{[(1S)-1-{[4-(3-fluorophenyl)-
piperidin-1-yl]methyl}-2-methylpropyl]carbamoyl}-7-hydroxy-3,4-
dihydroisoquinoline-2(1H)-carboxylate (19) (150 mg, 0.28 mmol) in
4 mL methanol was added 4 mL concd HCl and the reaction mixture
stirred for overnight. Evaporation of solvents led to a white solid
that was purified by chromatography on silica gel with CMA80/
chloroform (1:1) as the eluent to provide 98 mg (79%) of the title
compound as a white solid. 1H NMR (300 MHz, CHLOROFORM-d) d
7.19 (s, 1H), 7.08–7.16 (m, 1H), 7.03 (d, J = 9.61 Hz, 1H), 6.72–6.90
5.10. 4-Methyl-4-[3-(1-methylethoxy)phenyl]piperidine (9)
The N-methyl amine 8 was concentrated from toluene then dis-
solved in 1,2-dichloroethane (9 mL) and treated with freshly

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F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
distilled 1-chloroethyl chloroformate (1.8 mL, 3.0 equiv). The
resulting dark solution was refluxed 12 h then concentrated. The
residue was dissolved in methanol (10 mL), refluxed 24 h and then
concentrated. The residue was subjected to chromatography on sil-
ica gel using a gradient of CMA80 in CH2Cl2 as the eluent to afford
0.677 g (50%) of 9. 1H NMR (300 MHz, CHLOROFORM-d) d 7.14–
7.31 (m, 1H), 6.96 (d, J = 7.91 Hz, 1H), 6.87–6.93 (m, 1H), 6.77
(dd, J = 2.45, 8.10 Hz, 1H), 5.99–6.10 (m, 1H), 4.55 (spt,
J = 6.06 Hz, 1H), 3.10 (q, J = 2.83 Hz, 2H), 2.61–2.70 (m, 2H), 2.52–
2.61 (m, 2H), 2.40 (s, 3H), 1.30–1.36 (m, 6H).
J = 2.17, 8.05 Hz, 1H), 2.74–3.00 (m, 4H), 1.93–2.10 (m, 2H), 1.70
(ddd, J = 3.39, 7.25, 13.09 Hz, 3H), 1.20–1.30 (m, 3H).
5.13.
(2S)-1-[4-(3-Fluorophenyl)-4-methylpiperidin-1-yl]-3-
methylbutan-2-amine (13)
A solution of amine 12 (740 mg, 3.8 mmol) and Boc-L-valine
(0.86 g, 4.0 mmol) in acetonitrile (20 mL) was cooled in an ice-
bath, then treated with HBTU (1.51 g, 4.0 mmol) and diisopropy-
lethylamine (2.1 mL, 12 mmol). The flask was removed from the
ice bath, and the reaction mixture was stirred overnight. The solu-
tion was concentrated then partitioned between concentrated
aqueous NaHCO3 and EtOAc. The mixture was extracted three
times with EtOAc (25 mL). The combined organic extracts were
washed with brine, dried over MgSO4, filtered, and evaporated to
leave a residue which was dissolved in acetonitrile (20 mL) and
treated with HCl in dioxane (20 mL). The solvent was concentrated
under a stream of nitrogen. The remaining residue was subjected
to chromatography on silica gel using a gradient of CMA80 in
DCM as the eluent to afford the intermediate amide. This amide
was dissolved in THF (20 mL) and treated with borane dimethyl-
sulfide (3 mL, 30 mmol). The solution was stirred at reflux over-
night and then quenched with methanol. The residue was treated
with HCl (6 M, 10 mL) and stirred for 1 h. Solid NaHCO3 was added
to adjust the solution to a pH of 8, and the mixture was extracted
with CH2Cl2 (3 Â 25 mL), washed with brine, and dried over
MgSO4. The concentrated residue was subjected to chromatogra-
phy on silica gel eluting with a gradient of CMA80 in DCM to afford
0.64 g (61% from 12) of 13: 1H NMR (300 MHz, CHLOROFORM-d) d
7.22–7.33 (m, 1H), 7.11 (d, J = 8.10 Hz, 1H), 7.03 (td, J = 2.14,
11.35 Hz, 1H), 6.82–6.92 (m, 1H), 2.45–2.74 (m, 3H), 1.98–2.44
(m, 5H), 1.65–1.87 (m, 5H), 1.51 (qd, J = 6.66, 12.97 Hz, 1H),
1.18–1.23 (m, 3H), 0.83–0.95 (m, 6H); 19F NMR (282 MHz,
CHLOROFORM-d) d À133.33.
5.11. (2S)-3-Methyl-1-{4-methyl-4-[3-(1-methylethoxy)phenyl]-
piperidin-1-yl}butan-2-amine (10)
Boc-L-Valinal was prepared by LAH reduction of the Weinreb
amide.7 The aldehyde (319 mg, 1.6 mmol) was combined with
amine 9 (572 mg, 2.5 mmol) in trifluoroethanol (12 mL) and stirred
for 15 min prior to addition of NaCNBH3 (154 mg, 2.5 mmol). The
resulting mixture was stirred overnight, concentrated, and the
residue partitioned between 2 M NH4OH and EtOAc. The aqueous
layer was extracted further with EtOAc. The dried (Na2SO4) com-
bined organics were concentrated and the residue subjected to
chromatography on silica gel using a gradient of CMA80 in CH2Cl2
as the eluent. 1H NMR (CDCl3) d 7.23 (t, J = 8.0 Hz, 1H), 6.82–6.93
(m, 2H), 6.72 (dd, J = 1.9, 8.1 Hz, 1H), 4.74 (br s, 1H), 4.54 (spt,
J = 6.0 Hz, 1H), 3.55–3.74 (m, 1H), 2.75 (br s, 1H), 2.58 (br s, 2H),
2.26–2.51 (m, 3H), 2.05–2.24 (m, 2H), 1.70–1.92 (m, 3H), 1.45 (s,
9H), 1.34 (d, J = 6.0 Hz, 6H), 1.21 (s, 3H), 0.81–0.99 (m, 6H). The iso-
lated product was dissolved in CH2Cl2 (5 mL) and treated with TFA
(10 mL) at rt for 12 h. The concentrated residue was partitioned
between CHCl3 and 2 M NH4OH. The organic layer was dried (Na2-
SO4) and concentrated to afford 439 mg of 10 (1.38 mmol, 56% over
two steps), which was used in the next step without further
purifications.
5.12. 4-Methyl-4-(3-fluorophenyl)piperidine (12)
5.14.
(2S)-1-[4-(3-Fluorophenyl)-4-methylpiperidin-1-yl]-3-
methylpentan-2-amine (14)
A solution of n-BuLi (7.2 mL, 2.5 M in hexanes, 18 mmol) was
added dropwise to a solution of 11 (2.46 g, 12.9 mmol) in THF
(22 mL) maintained between À10 and À20 °C. After 15 min, the
solution was cooled to À50 °C and dimethyl sulfate (1.75 mL,
18.5 mmol) was slowly and cautiously added. The reaction mixture
was stirred an additional 30 min, then 2 M NH4OH (10 mL) was
added. The resulting mixture was extracted with hexanes. The
combined organic layer was washed with water, dried (Na2SO4),
and concentrated to a residue. The residue was dissolved in CH3OH
(20 mL), cooled in an ice bath, and treated with an excess of NaBH4
(0.5 g, 13 mmol). The reaction mixture was stirred 3 h at room
temperature and then was quenched with the addition of acetone
and saturated NaHCO3. The concentrated residue was dissolved in
water and EtOAc. The aqueous layer was extracted again with
EtOAc before the combined organic layer was washed with water
then concentrated to afford 1.72 g (8.3 mmol, 64%) of the 1,4-
dimethylpiperidine intermediate. This residue was concentrated
thrice from toluene then dissolved in 1,2-dichloroethane (12 mL).
A freshly distilled aliquot of 1-chloroethyl chloroformate (1.3 mL,
12 mmol) was added under inert atmosphere and the resulting
solution was heated at reflux overnight. The concentrated residue
was then dissolved in CH3OH and refluxed 1 h. The concentrated
residue was dissolved in 2 M NaOH and extracted with CH2Cl2.
The combined organic layers were dried (Na2SO4), concentrated,
and subjected to chromatography on silica gel using a gradient of
CMA80 in DCM as the eluent to afford 0.74 g (30% from 11) of
12. 1H NMR (300 MHz, CHLOROFORM-d) d 7.22–7.36 (m, 1H),
7.12 (d, J = 7.91 Hz, 1H), 7.04 (td, J = 2.14, 11.35 Hz, 1H), 6.88 (dt,
A solution of amine 12 (233 mg, 1.2 mmol) and Boc-L-isoleucine
(275 g, 1.2 mmol) in acetonitrile (7 mL) was cooled in an ice-bath,
then treated with HBTU (0.50 g, 1.3 mmol) and diisopropylethy-
lamine (0.65 mL, 3.7 mmol). The flask was removed from the ice
bath, and the reaction was stirred overnight. The solution was con-
centrated then partitioned between concentrated aqueous NaHCO3
and EtOAc. The mixture was extracted three times with EtOAc
(25 mL). The combined organic extracts were washed with brine,
dried over MgSO4, filtered, and evaporated to leave a residue which
was dissolved in methanol (10 mL) and treated with HCl (6 M,
10 mL). The solution was concentrated to a solid from toluene.
The remaining residue was subjected to chromatography on silica
gel using a gradient of CMA80 in DCM to afford the intermediate
amide. This amide was dissolved in THF (15 mL) and treated with
borane dimethylsulfide (2.5 mL, 25 mmol). The solution was stir-
red at reflux overnight, then quenched with and concentrated from
methanol. The residue was treated with HCl (6 M, 10 mL) and
methanol (10 mL), stirred for 1 h, and then concentrated. The resi-
due was combined with 2 M NaOH (10 mL) and extracted with
EtOAc (3 Â 25 mL). The combined extracts were washed with
brine, and dried (MgSO4). The concentrated residue was subjected
to chromatography on silica gel eluting with a gradient of CMA80
in DCM to afford 0.27 g (77% from 12) of the intermediate amine
14: 1H NMR (300 MHz, CHLOROFORM-d) d 7.22–7.33 (m, 1H),
7.08–7.14 (m, 1H), 7.03 (td, J = 2.14, 11.35 Hz, 1H), 6.87 (ddt,
J = 0.75, 2.45, 8.29 Hz, 1H), 2.71–2.84 (m, 1H), 2.44–2.67 (m, 2H),
1.96–2.43 (m, 5H), 1.57–1.83 (m, 6H), 1.48 (ddd, J = 3.96, 7.58,

F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
6387
12.95 Hz, 1H), 1.24–1.37 (m, 1H), 1.18–1.24 (m, 3H), 0.78–0.95 (m,
6H).
148.9, 148.8, 129.8, 129.7, 122.6, 122.5, 113.9, 113.6, 113.0,
112.7, 57.8, 55.5, 54.9, 53.1, 42.1, 33.3, 32.8, 29.3, 19.4, 18.5; ESI
MS (M+H)+ 265.5.
5.15. tert-Butyl [(1S)-1-{[4-(3-fluorophenyl)piperidin-1-yl]carbonyl}-
2-methylpropyl]carbamate (16)
5.18. tert-Butyl (3R)-3-{[(1S)-1-{[4-(3-fluorophenyl)piperidin-1-
yl]methyl}-2-methylpropyl]carbamoyl}-7-hydroxy-3,4-dihydro-
isoquinoline-2(1H)-carboxylate (19)
To a solution of 4-(3-fluorophenyl)piperidine (15) (5.0 mg,
27.9 mmol) in dry acetonitrile (150 mL) was added Boc-L-valine
(6.5 g, 30 mmol) followed by 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) (11.4 g, 30 mmol)
and triethylamine (8.3 g, 82.5 mmol). The reaction mixture was
stirred overnight then concentrated. The resulting solid was trea-
ted with 50 mL saturated NaHCO3. The aqueous layer was
extracted with EtOAc (2 Â 50 mL). Combined organic layers were
dried (Na2SO4), filtered then concentrated. The resulting residue
was purified by column chromatography on silica gel with hex-
anes/ethyl acetate (1:1) to provide 7.93 g (75%) of the title com-
pound. 1H NMR (300 MHz, CHLOROFORM-d) d 7.14–7.24 (m, 1H),
6.78–6.92 (m, 2H), 5.32 (d, J = 9.04 Hz, 1H), 4.70 (d, J = 12.81 Hz,
1H), 4.44 (d, J = 6.97 Hz, 1H), 3.90–4.21 (m, 1H), 3.03–3.17 (m,
1H), 2.55–2.75 (m, 2H), 1.77–2.04 (m, 3H), 1.43–1.68 (m, 2H),
1.31–1.40 (m, 9H), 0.69–0.96 (m, 6H); 13C NMR (75 MHz, CHLORO-
FORM-d) d 170.6, 164.7, 161.4, 155.9, 147.5, 130.1, 130.0, 122.4,
113.8, 113.7, 113.5, 113.3, 79.4, 54.8, 46.5, 46.1, 42.8, 42.6, 42.2,
33.7, 33.6, 32.8, 31.8, 31.5, 28.4, 19.8, 19.6, 17.2, 17.1; ESI MS (M
+H)+ 379.6.
To a solution of (2S)-1-[4-(3-fluorophenyl)piperidin-1-yl]-3-
methylbutan-2-amine (70 mg, 0.27 mmol) (18) in dichloro-
methane (15 mL) was added Boc-7-hydroxy-D-Tic-OH (117 mg,
0.40 mmol) and triethylamine (80 mg, 0.8 mmol) followed by
EDCÁHCl (153 mg, 0.8 mmol) and HOBt (5.4 mg, 0.04 mmol). The
reaction mixture was stirred overnight at ambient temperature.
The reaction mixture was treated with saturated NaHCO3 and
extracted with dichloromethane. Combined organic layers were
dried (Na2SO4), filtered then evaporated to obtain crude product.
Purification of the crude product by chromatography on silica gel
with CMA80/chloroform (1:1) as the eluent provided 150 mg
(74%) of the title compound as a white solid. 1H NMR (300 MHz,
CHLOROFORM-d) d 7.09–7.31 (m, 2H), 6.68–6.97 (m, 4H), 6.37–
6.65 (m, 2H), 5.66–5.95 (m, 1H), 4.55–4.94 (m, 1H), 4.25–4.53
(m, 2H), 3.76 (br s, 1H), 3.15 (dd, J = 2.92, 15.35 Hz, 1H), 2.88 (dd,
J = 6.03, 15.26 Hz, 1H), 2.66 (br s, 2H), 2.22–2.43 (m, 1H), 1.90–
2.21 (m, 2H), 1.69–1.90 (m, 2H), 1.49–1.70 (m, 3H), 1.26–1.49
(m, 9H), 0.76 (dd, J = 6.88, 17.61 Hz, 6H); 13C NMR (75 MHz,
CHLOROFORM-d) d 171.4, 164.6, 161.3, 155.6, 149.0, 148.9, 129.8,
129.7, 129.3, 124.4, 122.5, 122.5, 114.9, 113.8, 113.5, 113.0,
112.7, 59.7, 54.5, 51.3, 44.8, 42.0, 33.3, 33.0, 30.3, 28.4, 19.1, 17.3.
5.16. (2S)-2-Amino-1-[4-(3-fluorophenyl)piperidin-1-yl]-3-methyl-
butan-1-one (17)
To a solution of tert-butyl [(1S)-1-{[4-(3-fluorophenyl)piperi-
din-1-yl]carbonyl}-2-methylpropyl]carbamate (16) (7.93 g, 21.0 mmol)
in methanol (100 mL) was added concd HCl (8 mL) and the reac-
tion mixture stirred for 4 h at ambient temperature. Evaporation
of solvents led to a white solid that was purified by chromatogra-
phy on silica gel with CMA80/chloroform (1:1) as the eluent to pro-
vide 6.05 g (92%) of the title compound as a white solid. 1H NMR
(300 MHz, CHLOROFORM-d) d 8.43 (br s, 2H), 7.11–7.23 (m, 2H),
6.76–6.97 (m, 2H), 4.70 (d, J = 13.19 Hz, 1H), 4.51 (br s, 1H),
3.76–4.12 (m, 1H), 3.09 (br s, 1H), 2.67 (d, J = 9.04 Hz, 2H), 2.09–
2.29 (m, 1H), 1.98 (s, 2H), 1.66–1.81 (m, 1H), 1.47 (br s, 1H),
0.89–1.19 (m, 5H); 13C NMR (75 MHz, CHLOROFORM-d) d 167.4,
166.9, 164.6, 161.3, 147.7, 147.6, 147.3, 130.1, 130.0, 129.9,
122.8, 122.4, 114.1, 113.8, 113.7, 113.4, 113.1, 55.5, 55.2, 47.2,
46.4, 43.7, 43.0, 42.9, 41.8, 33.2, 32.5, 30.2, 30.1, 19.3, 18.9, 17.8,
17.5.
5.19. [35S]GTP
c
S assay
S assays were conducted using the methods pre-
The [35S]GTP
c
viously reported.3,4
5.20. Calculated pharmacokinetic properties
The topological polar surface area (TPSA) and calculated
lipophilicity (clogP) values were calculated using the ChemAxon
Instant JChem package. Predictions of the logarithm of the
in vivo blood–brain ratio (logBB) were based on the Clark and Pick-
ett model (Eq. 3).13
5.21. MDCK-mdr1 permeability assays
MDCK-mdr1 cells obtained from the Netherlands Cancer Insti-
tute were grown on Transwell type filters (Corning) for 4 days to
confluence in DMEM/F12 media containing 10% fetal bovine serum
and antibiotics as has been described previously.14 Compounds
5.17. (2S)-1-[4-(3-Fluorophenyl)piperidin-1-yl]-3-methylbutan-
2-amine (18)
were added to the apical side at a concentration of 10
transport buffer comprising of 1Â Hank’s balanced salt solution,
25 mM -glucose and buffered with HEPES to pH 7.4. Samples were
lM in a
To a cooled (0 °C) solution of (2S)-2-amino-1-[4-(3-fluoro-
phenyl)-1-piperidin-1-yl]-3-methyl-butan-1-one (17) hydrochlo-
ride (6.05 g, 19.2 mmol) in THF (40 mL) was added borane
dimethyl sulfide complex (3.8 mL, 38 mmol) (10 M as BH3). The
reaction mixture was warmed to ambient temperature and stirred
overnight. The reaction mixture was refluxed for 3 h, cooled in ice,
quenched with methanol and stirred for 1 h at ambient tempera-
ture. The reaction mixture was cooled in ice bath and treated with
2 M HCl in ether and refluxed for 2 h. The solvent was evaporated
and the resulting material purified by chromatography on silica gel
using chloroform/CMA80 gradient to provide 2.5 g (49%) of the
desired product as a white solid. 1H NMR (300 MHz, CDCl3) d
7.68 (br s, 2H), 7.17–7.25 (m, 1H), 6.93–7.02 (m, 2H), 6.82–6.89
(m, 1H), 3.01–3.12 (m, 3H), 2.44–2.66 (m, 3H), 2.31–2.37 (m,
1H), 2.03–2.23 (m, 2H), 1.75–1.93 (m, 4H), 1.17 (d, J = 7 Hz, 3H),
1.09 (d, J = 7 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 164.5, 161.3,
D
incubated for 1 h at 37 °C and carefully collected from both the
apical and basal side of the filters. Compounds selected for
MDCK-mdr1 cell assays were infused on an Applied Biosystems
API-4000 mass spectrometer to optimize for analysis using multi-
ple reaction monitoring (MRM). Flow injection analysis was also
conducted to optimize for mass spectrometer parameters. Samples
from the apical and basolateral side of the MDCK cell assay were
dried under nitrogen on a Turbovap LV. The chromatography was
conducted with an Agilent 1100 binary pump with a flow rate of
0.5 mL/min. Mobile phase solvents were A, 0.1% formic acid in
water, and B, 0.1% formic acid in methanol. The initial solvent con-
ditions were 10% B for 1 min, then a gradient was used by increas-
ing to 95% B over 5 min, then returning to initial conditions. Data
reported are average values from 2 to 3 measurements.

6388
F. I. Carroll et al. / Bioorg. Med. Chem. 23 (2015) 6379–6388
5.22. In vitro stability testing
5.25. Solubility determination
Stability of compounds to plasma and S9 fraction was per-
formed as previously described.15 In vitro testing for metabolic sta-
bility was conducted in mixed gender pooled hepatic S9 fraction
supplied by Xenotech, LLC, Lenexa, KS. Identity of the donors was
unknown.
For the hepatic S9 metabolism studies, all samples were tested
at 10 lM final concentration in a 1 mL volume containing 1 mg/mL
S9. Samples were incubated in a buffer containing 50 mM potas-
sium phosphate, pH 7.4 with 3 mM MgCl2 and a NADPH regenera-
tion system comprising of NADP (1 mM), glucose-6-phosphate
(5 mM) and glucose-6-phosphate dehydrogenase (1 unit/mL).
Triplicate samples were incubated for 60 min. Reactions were
terminated by addition of 3 volumes of acetonitrile and processed
as described for the MDCK-mdr1 assays, but standard curves
were prepared in blank matrix for each compound for quantita-
For these experiments, 10 mM DMSO stocks of compounds
were directly diluted into 10 mM phosphate buffer at pH 7.4 or 3
and shaken for 90 min at room temperature. The final concentra-
tion of DMSO was 1%. After the incubation, samples were filtered
through a 0.4 micron filterplate (Millipore). Filtrates were carefully
collected. Analysis of compounds was performed by LC/MS using
previously available methods and concentrations determined. Data
are reported as mean values from three determinations.
Acknowledgements
This research was supported by the National Institute on Drug
Abuse Grant DA09045. We thank Tiffany Langston, Keith Warner,
and Rodney Snyder for conducting the in vitro testing and
in vitro preclinical studies. R.M. was supported by AA022235 and
DK100414 from NIH.
tive assessment. Data reported are average values from
measurements.
3
Supplementary data
5.23. PAMPA
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.08.025.
PAMPA was conducted using a 96-well plate based kit from BD
Biosciences (RTP, NC) and manufacturer’s instructions were fol-
lowed closely. Briefly, all samples were tested at 10 lM final con-
References and notes
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methods for analytical evaluation were similar to those described
for MDCK-mdr1 transport assays. Data reported are average values
from 3 measurements.
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