Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of replacement of the 3-hydroxyphenyl group with pyridine or thiophene bioisosteres

Article abstract of DOI:10.1016/j.bmc.2016.06.029

The potent and selective KOR antagonist JDTic was derived from the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class of pure opioid antagonists. In previous studies we reported that compounds that did not have a hydroxyl on the 3-hydroxyphenyl group and did not have methyl groups at the 3- and 4-position of the piperidine ring were still potent and selective KOR antagonists. In this study we report JDTic analogs 2, 3a–b, 4a–b, and 5, where the 3-hydroxyphenyl ring has been replaced by a 2-, 3-, or 4-pyridyl or 3-thienyl group and do not have the 3-methyl or 3,4-dimethyl groups, remain potent and selective KOR antagonists. Of these, (3R)-7-hydroxy-N-(1S)-2-methyl-[4-methyl-4-pyridine-3-yl-carboxamide (3b) had the best overall binding potency and selectivity in a [³⁵S]GTPγS functional assay, with a K_e?=?0.18?nM at the KOR and 273- and 16,700-fold selectivity for the KOR relative to the MOR and DOR, respectively. Calculated physiochemical properties for 3b suggest that it will cross the blood–brain barrier.

Full text of DOI:10.1016/j.bmc.2016.06.029

Accepted Manuscript
Design, synthesis, and pharmacological evaluation of JDTic analogs to examine
the significance of replacement of the 3-hydroxyphenyl group with pyridine or
thiophene bioisosteres
Chad M. Kormos, Moses G. Gichinga, Scott P. Runyon, James B. Thomas, S.
Wayne Mascarella, Ann M. Decker, Hernán A. Navarro, F. Ivy Carroll
PII:
S0968-0896(16)30452-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.029
BMC 13085
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 April 2016
8 June 2016
13 June 2016
Please cite this article as: Kormos, C.M., Gichinga, M.G., Runyon, S.P., Thomas, J.B., Wayne Mascarella, S.,
Decker, A.M., Navarro, H.A., Ivy Carroll, F., Design, synthesis, and pharmacological evaluation of JDTic analogs
to examine the significance of replacement of the 3-hydroxyphenyl group with pyridine or thiophene bioisosteres,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.029
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Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of
replacement of the 3-hydroxyphenyl group with pyridine or thiophene bioisosteres
Chad M. Kormos, Moses G. Gichinga, Scott P. Runyon, James B. Thomas, S. Wayne Mascarella, Ann
M. Decker, Hernán A. Navarro, and F. Ivy Carroll*
Research Triangle Institute, PO Box 12194, Research Triangle Park, NC 27709-2194, United States
*Corresponding author:
Dr. F. Ivy Carroll
Research Triangle Institute
Post Office Box 12194
Research Triangle Park, NC 27709-2194
Telephone: 919 541-6679
Fax: 919 541-8868
E-mail: fic@rti.org

Abstract
The potent and selective KOR antagonist JDTic was derived from the N-substituted trans-3,4-
dimethyl-4-(3-hydroxyphenyl)piperidine class of pure opioid antagonists. In previous studies we
reported that compounds that did not have a hydroxyl on the 3-hydroxyphenyl group and did not have
methyl groups at the 3- and 4-position of the piperidine ring were still potent and selective KOR
antagonists. In this study we report JDTic analogs 2, 3a–b, 4a–b, and 5, where the 3-hydroxyphenyl ring
has been replaced by a 2-, 3-, or 4-pyridyl or 3-thienyl group and do not have the 3-methyl or 3,4-
dimethyl groups, remain potent and selective KOR antagonists. Of these, (3R)-7-hydroxy-N-(1S)-2-
methyl-[4-methyl-4-pyridine-3-yl-carboxamide (3b) had the best overall binding potency and selectivity
in a [35S] GTPγS functional assay, with a Ke = 0.18 nM at the KOR and 273- and 16,700-fold selectivity
for the KOR relative to the MOR and DOR, respectively. Calculated physiochemical properties for 3b
suggest that it will cross the blood-brain barrier.
Keywords: JDTic; Opioids; Kappa antagonist; ADME properties
1.
Introduction
During the last 15-20 years considerable effort has been devoted by academic institutions, research
institutions, and pharmaceutical companies to the development of potent and selective kappa opioid
receptor (KOR) antagonists (see ref 1 for a review).[1] Animal behavioral studies have suggested that
potent and selective KOR antagonists have potential as pharmacotherapies for treating mood disorders
such as depression, anxiety, and substance abuse (nicotine, alcohol, cocaine, and opiates).
Abbreviations: [35S]GTPγS, sulfur-35 guanosine-5′-O-(3-thio)triphosphate; DAMGO, [D-
Ala2,MePhe4,Gly-ol5]enkephalin; DPDPE, [D-Pen2,D-Pen5]enkephalin; U69,593, (5α,7α,8β)-(-)-N-
methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]benzeneacetamide

In our early studies we showed that (3R)-1,2,3,4-tetrahydro-7-hydroxy-Ν-[(1S)-1-[[(3R,4R)-4-(3-
hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl]-2-methylpropyl]-3-isoquinolinecarboxamide
(JDTic) was a potent and selective KOR antagonist.[2, 3] In more recent studies we reported that JDTic
analog 1 (Figure 1), where both the 3- and 4-methyl groups were removed from JDTic, was still a potent
and selective KOR antagonist.[4] In addition we reported that JDTic analogs with the hydroxyl group
removed from the 4-(3-hydroxyphenyl) moiety were still potent and selective KOR antagonists.[5] The
fact that 1 is a potent and selective KOR antagonist suggest that replacement of the 4-phenyl group in 1
with heterocyclic group could also lead to potent and selective KOR antagonist. In this study we report
the synthesis, [35S]GTPγS in vitro binding and calculated physiochemical properties of compounds 2,
3a–b, and 4a–b which have a 2-, 3-, or 4-pyridyl group or 5 which has a thiophene ring replacing the
phenyl ring in previously reported JDTic analogs (Figure 1).
OH
N
HO
HO
HO
N
O
N
O
N
NH
NH
NH
N
H
N
H
N
H
O
N
JDTic
2
1
S
N
R
R
HO
HO
HO
N
O
N
O
N
O
NH
NH
NH
N
H
N
H
N
H
5
3a, R = H
3b, R = CH3
4a, R = H
4b, R = CH3
Figure 1. Structures of JDTic, 1, 2, 3a-b, 4a-b, and 5.

2.
Chemistry
The known piperidines 9a,[6] 9b,[7] 9c,[8] and 9d were prepared according to the route shown in
Scheme 1, affording material consistent with literature characterization. Beginning with vinyl triflate 6,
Negishi or Suzuki cross-coupling afforded the pyridyl and thienyl tetrahydropyridine derivatives 7a–d.
Catalytic hydrogenation yielded the saturated N-Boc-piperidines 8a–d. Treatment of 8a–d with
hydrogen chloride in dioxane deprotected the Boc to afford the amine hydrochloride salts 9a–d, which
were used directly in the next step.
Scheme 1a
Ar
Ar
Ar
OTf
a
c
b
N
Boc
N
Boc
N
H
N
Boc
6
7a, Ar = 2-pyridyl
7b, Ar = 3-pyridyl
7c, Ar = 4-pyridyl
7d, Ar = 3-thienyl
8a, Ar = 2-pyridyl
8b, Ar = 3-pyridyl
8c, Ar = 4-pyridyl
8d, Ar = 3-thienyl
9a, Ar = 2-pyridyl
9b, Ar = 3-pyridyl
9c, Ar = 4-pyridyl
9d, Ar = 3-thienyl
a Reagents: a) Suzuki or Negishi coupling; b) H2, Pd/C; c) 4M HCl in dioxane, CH3CN.
Amines 9e–f were prepared according to the route shown in Scheme 2. Knoevenagel condensation
of the acetylpyridines 10a–b with ethyl cyanoacetate using ammonium acetate and acetic acid followed
by condensation with 2-cyanoacetamide gave the intermediates 11a–b. Hydrolysis and decarboxylation
of 11a–b using sulfuric acid gave glutaric acids which were not isolated. Upon heating the crude diacids
neat with urea as a melt, the glutarimides 12a–b were produced. Reduction of the imides with diborane
in tetrahydrofuran afforded piperidines 9e–f.

Scheme 2a
Ar
Ar
Ar
NC
O
CN
O
Ar
O
a
c
b
N
H
O
N
H
O
N
H
10a, Ar = 3-pyridyl
10b, Ar = 4-pyridyl
11a, Ar = 3-pyridyl
11b, Ar = 4-pyridyl
12a, Ar = 3-pyridyl
12b, Ar = 4-pyridyl
9e, Ar = 3-pyridyl
9f, Ar = 4-pyridyl
a Reagents: a) (1) Ethyl cyanoacetate, NH4OAc, HOAc, (2) NaOEt, 2-cyanoacetamide; b) (1) H2SO4, (2)
urea; c) BH3•S(CH3)2, THF.
Coupling of piperidines 9a–f with N-Boc-L-valine using 3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC•HCl) with catalytic N-hydroxybenzotriazole (HOBt) or 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) in acetonitrile with triethylamine afforded the
desired Boc-protected amides, which were deprotected with 4M hydrogen chloride in dioxane or
hydrochloric acid in aqueous methanol and were directly reduced with diborane or borane
dimethylsulfide in tetrahydrofuran to yield the amines 13a–f (Scheme 3). The amines 13a–f were
coupled with (3R)-2-(tert-butoxycarbonyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic (7-
hydroxy-Boc-D-Tic-OH) using EDC•HCl with a catalytic amount of HOBt in dichloromethane to give
the Boc-protected intermediates. The desired final products 2, 3a–b, 4a–b, and 5 resulted from treatment
of an acetonitrile solution of the Boc-intermediates with 4M hydrogen chloride in dioxane.

Scheme 3a
Ar
N
Ar
N
R
R
Ar
R
HO
a
b
N
H
NH
N
H
NH2
O
9a, Ar = 2-pyridyl, R = H
9b, Ar = 3-pyridyl, R = H
9c, Ar = 4-pyridyl, R = H
9d, Ar = 3-thienyl, R = H
13a, Ar = 2-pyridyl, R = H
13b, Ar = 3-pyridyl, R = H
13c, Ar = 4-pyridyl, R = H
13d, Ar = 3-thienyl, R = H
2, Ar = 2-pyridyl, R = H
3a, Ar = 3-pyridyl, R = H
4a, Ar = 4-pyridyl, R = H
5, Ar = 3-thienyl, R = H
9e, Ar = 3-pyridyl, R = CH3 13e, Ar = 3-pyridyl, R = CH3 3b, Ar = 3-pyridyl, R = CH3
9f, Ar = 4-pyridyl, R = CH3 13f, Ar = 4-pyridyl, R = CH3 4b, Ar = 4-pyridyl, R = CH3
a Reagents: a) (1) N-Boc-L-valine, EDC•HCl, HOBt, Et3N, CH3CN, (2) HCl, (3) BH3, THF; b) (1) 7-
hydroxy-Boc-D-Tic, EDC•HCl, HOBt, Et3N, CH2Cl2, (2) 4M HCl in dioxane.
3.
Results and Discussion
In order to determine the effect of KOR potency and selectivity by replacing the phenyl ring in
previously reported 1 with a 2-, 3-, or 4-pyridyl group or a thiophene ring, compounds 2, 3a–b, 4a–b,
and 5 were synthesized and first evaluated at 10 µM for intrinsic activity in the [35S]GTPγS binding
assay at all three opioid receptors. As none of these compounds displayed measurable intrinsic activity
at this concentration, the compounds were evaluated for antagonist potency and selectivity at the opioid
receptors. Compounds 3b and 4b were synthesized and tested for their KOR [35S]GTPγS binding
potency in order to determine the effect of adding a 4-methyl group to 3a and 4a.
Compounds 2, 3a, and 4a with Ke values at the KOR of 0.43, 0.51, and 0.51 nM, respectively,
were highly potent KOR antagonists but were slightly less potent than 1 which had a Ke = 0.051 nM at
the KOR (Table 1). Similar to 1, all three compounds were highly selective for the KOR relative to the
DOR. Compounds 2, 3a, and 4a were 33-, 89-, and 112-fold selective for the KOR relative to the MOR,
compared to 77-fold selectivity for KOR relative to MOR for 1.

Table 1. Inhibition of agonist-stimulated [35S]GTPγS binding in cloned human µ, δ, and κ opioid
receptors
Ke (nM)a
Compound
µ, DAMGO
25
δ, DPDPE
74
κ, U69,593
0.02 0.01
0.051 0.01
0.43 0.12
0.51 0.08
0.18 0.01
0.51 0.14
1.75 0.65
0.12 0.02
µ/κ
1,250
77
δ/κ
3,800
5,510
1,372
2,235
>16,700
1,353
1,434
983
JDTic
1
4
2
3.96 1.1
14.5 2.6
45.4 15
49.2 14
57.1 15
154 56
281 44
590 130
1140 70
>3000
33
2
89
3a
3b
4a
4b
5
273
112
88
690 250
2,510 490
118 27
3.93 0.73
33
a Ke values are the mean SEM of at least three independent experiments performed in duplicates.
The addition of a 4-methyl substituent to 3a to give 3b resulted in a significant increase in both
KOR potency and selectivity for the KOR relative to both the MOR and DOR. Compound 3b has a Ke =
0.18 nM at the KOR and is 273- and >16,700-fold selective for the KOR relative to the MOR and DOR,
respectively, whereas compound 3a has a Ke = 0.51 nM at the KOR and has 89- and 2,235-fold
selectively for KOR relative to MOR and DOR, respectively. Compound 4b has a Ke = 1.75 nM at the
KOR and is 88- and 1,434 fold selective for the KOR relative to the MOR and DOR, respectively.
Compound 5, which has a thiophene ring in place of a phenyl ring in 1 has a Ke value of 0.12 nM
at KOR and is 32.5- and 983-fold selective for KOR relative to the MOR and DOR. Thus, 5 is less
potent and less selective than 1.

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 compared
to JDTic and 1 (Table 2). In general, CNS compounds that have TPSA values less than 76 Å2[9], clogP
values in the range of 2-4[10], and derived logBB values greater than -1[11] are predicted to cross the
blood/brain barrier. Compounds 3a–b and 4a–b have TPSA values of 77.49 Å2 compared to 84.83 Å2
for JDTic. Compound 5 has a TPSA value of 64.6, which is identical to that of 1. JDTic, 3a–b, 4a–b and
5 have clogP values in the range of 2–4. All of the compounds have logBB values greater than -1 and
thus, would be predicted to penetrate the brain. In a study of marketed CNS drugs Wager and co-
workers determined that 74% of CNS drugs displayed a CNS MPO (Multi Parameter Optimization)
score greater than or equal to four.[12] All of the compounds in this study had a CNS MPO score greater
than or equal to four. JDTic and 1 had CNS MPO scores of 3.1 and 3.8, respectively.
Table 2. Calculated physiochemical data for compounds 2, 3a–b, 4a–b, and 5 compared to JDTic and
compound 1
Compound
TPSA
84.83
64.60
77.49
77.49
77.49
77.49
77.49
64.60
cLogP
3.60
3.53
2.56
2.42
2.64
2.40
2.63
3.45
ClarkBB
-0.57
-0.28
-0.62
-0.64
-0.61
-0.64
-0.61
-0.29
CNS MPO
3.1
JDTic
1
3.8
4.0
2
4.2
3a
3b
4a
4b
5
4.1
4.2
4.1
4.1
4.
Conclusion

Compounds 2, 3a–b, 4a–b, and 5, which have either a pyridine or thiophene ring in place of the
phenyl groups in JDTic analogs were synthesized and evaluted for their [35S] GTPγS binding potency at
the MOR, DOR, and KOR. All of the compounds had subnanomolar Ke values at the KOR in the [35S]
GTPγS binding assay except 4b which has a Ke = 1.75 nM at the KOR. Compounds 3a–b and 4a–b
were as selective or more selective for the KOR relative to the MOR than previously reported 1. All of
the compounds were highly selective for the KOR relative to the DOR. Compound 3b with a Ke = 0.18
nM at the KOR with 273- and >16,700-fold selectively for the KOR relative to the MOR and DOR
respectively, had the best overall antagonist potency and selectivity in the [35S] GTPγS binding assays.
The calculated logBB values suggest that the compounds will penetrate the blood-brain barrier and the
calculated CNS MPO values are above 4 and thus are in the range for potential useful CNS drugs.
5. Materials and Methods
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 Varian
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 API 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 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 or using EM

Science silica gel 60A (230-400 mesh). Solvent system: CMA80=80:18:2 CHCl3:MeOH:conc. 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.
5.1. (3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-pyridin-2-ylpiperidin-1-yl)methyl]propyl}-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide (2) Trihydrochloride
To a solution of 13a (200 mg, 0.56 mmol) in dichloromethane (25 mL) was added (3R)-2-(tert-
butoxycarbonyl)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (7-hydroxy-Boc-D-Tic)
(176 mg, 0.60 mmol) and triethylamine (0.35 mL, 0.6 mmol) followed by 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) (230 mg, 1.2 mmol) and
hydroxybenzotriazole (HOBt) (8.1 mg, 0.06 mmol). The reaction mixture was stirred overnight at
ambient temperature and then treated with 20 mL of saturated sodium bicarbonate and extracted with 25
mL dichloromethane. The combined organic layers were dried over sodium sulfate, filtered and
evaporated. The resulting residue was subjected to chromatography on silica eluting with CMA 80-
chloroform (1:1) provided 225 mg (77%) of the Boc-intermediate as a white solid. This material was
dissolved in acetonitrile and treated with HCl (4M in dioxane, 1 mL) and stirred for 5 min. The solvent
was evaporated to afford a white precipitate which was subjected to chromatography on silica eluting
with CMA 80-chloroform (1:1) to provide 60 mg (33%) of 2 as the free base: 1H NMR (300 MHz,
METHANOL-d4) δ 8.81 (d, J = 5.27 Hz, 1H), 8.61 (s, 1H), 8.10 (d, J = 8.10 Hz, 1H), 8.00 (d, J = 6.59
Hz, 1H), 7.13 (d, J = 8.48 Hz, 1H), 6.78 (dd, J = 2.45, 8.48 Hz, 1H), 6.68 (d, J = 2.26 Hz, 1H), 4.18–
4.50 (m, 5H), 3.73 (br. s., 1H), 3.04–3.59 (m, 7H), 2.68–2.93 (m, 1H), 2.44–2.66 (m, 1H), 2.21–2.41 (m,
2H), 1.81–1.99 (m, 1H), 1.06 (s, 6H); 13C NMR (75 MHz, METHANOL-d4) δ 171.3, 159.2, 158.1,
148.2, 143.3, 131.2, 129.7, 126.9, 126.5, 121.9, 117.0, 113.7, 61.7, 57.9, 55.7, 52.4, 50.6, 45.5, 39.3,

32.3, 29.9, 29.0, 28.9, 19.9, 18.3; MS (ESI) m/z 423.5 (M+H)+. A solution of the free base in CH2Cl2
was treated with HCl (2 M in ether) to afford the trihydrochloride salt (2•3HCl): mp 225–229 °C (dec);
[α]25D = +69.2 (c 1.08, MeOH). Anal. Calcd for C25H37Cl3N4O2•2H2O: C, 52.87; H, 7.28; N, 9.86.
Found: C, 52.95; H, 7.21; N, 10.12.
5.2. (3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-pyridin-3-ylpiperidin-1-yl)methyl]propyl}-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide (3a) Trihydrochloride
Compound 3a was prepared according to procedure analogous to that of 2 from (2S)-3-methyl-1-
(4-pyridin-3-ylpiperidin-1-yl)butan-2-amine (13b) (200 mg, 0.56 mmol), which in turn was prepared
from amine 9b according to procedure analogous to that of 13a, to afford 109 mg (60%) of the 3a free
1
base. The free base was converted to the trihydrochoride salt (3a•3HCl): H NMR (300 MHz,
METHANOL-d4) δ 8.90 (s, 1H), 8.81 (d, J = 5.65 Hz, 1H), 8.61–8.74 (m, 1H), 8.11 (dd, J = 5.93, 8.01
Hz, 1H), 7.11 (d, J = 8.29 Hz, 1H), 6.75 (dd, J = 2.26, 8.29 Hz, 1H), 6.67 (d, J = 2.07 Hz, 1H), 4.25–
4.48 (m, 4H), 4.17 (d, J = 11.68 Hz, 1H), 3.70 (d, J = 11.49 Hz, 1H), 2.99–3.55 (m, 8H), 2.57–2.77 (m,
1H), 2.33–2.56 (m, 1H), 2.17 (d, J = 9.42 Hz, 2H), 1.77–2.01 (m, 1H), 1.03 (t, J = 5.75 Hz, 6H); 13C
NMR (126 MHz, METHANOL-d4) δ 171.2, 158.0, 146.8, 145.8, 141.7, 141.4, 131.2, 129.7, 128.7,
121.9, 116.9, 113.7, 61.5, 57.8, 56.1, 52.8, 50.5, 45.4, 37.9, 32.3, 30.0, 29.9, 19.9, 18.4; MS (ESI) m/z
423.5 (M+H)+ mp 223–227 °C; [α]25D = +70.0 (c 0.95, MeOH). Anal. Calcd for C25H37Cl3N2O2•1.5H2O:
;
C, 53.72; H, 7.21; N, 10.02. Found: C, 53.57; H, 7.37; N, 9.83.
5.3. (3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-methyl-4-pyridin-3-ylpiperidin-1-yl)methyl]propyl}-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (3b) Trihydrochloride
Compound 3b was prepared according to procedure analogous to that of 2 from amine 13e (117
1
mg, 0.45 mmol) to afford the 3b freebase: H NMR (300 MHz, CHLOROFORM-d) δ 8.50–8.65 (m,
1H), 8.43 (dd, J = 1.32, 4.71 Hz, 1H), 7.54–7.71 (m, 1H), 7.19–7.33 (m, 1H), 7.11 (d, J = 9.42 Hz, 1H),

6.82–6.97 (m, 1H), 6.60 (dd, J = 2.35, 8.19 Hz, 1H), 6.38–6.51 (m, 1H), 4.03–4.18 (m, 1H), 3.71–3.81
(m, 2H), 3.38 (dd, J = 5.27, 10.17 Hz, 1H), 2.94 (dd, J = 5.27, 16.39 Hz, 1H), 2.20–2.81 (m, 7H), 1.99–
2.19 (m, 2H), 1.67–1.92 (m, 3H), 1.20–1.25 (m, 3H), 0.79–0.99 (m, 6H); 13C NMR (75 MHz,
CHLOROFORM-d) δ 173.2, 155.2, 147.6, 146.7, 144.3, 136.9, 133.9, 130.4, 125.0, 123.5, 114.1, 112.4,
60.0, 56.6, 50.3, 49.9, 47.5, 36.4, 36.2, 35.1, 31.2, 29.6, 19.1, 17.9. The free base was converted to the
trihydrochloride salt affording 62.5 mg (25% over two steps) 3b•3HCl as a white powder: MS (ESI) m/z
437.7 (M+H)+, mp 215–219 °C (fusion), [α]25D +69. (c 0.10, CH3OH). Anal. Calcd for
C26H39Cl3N4O2•H2O: C, 53.66; H, 7.45; N, 9.63. Found: C, 53.49; H, 7.41; N, 9.50.
5.4. (3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-pyridin-4-ylpiperidin-1-yl)methyl]propyl}-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide (4a) Trihydrochloride
Compound 4a was prepared according to procedure analogous to that of 2 from amine 13c (280
mg, 1.13 mmol) to afford 113 mg (24%) of 4a as the free base: 1H NMR (300 MHz, METHANOL-d4) δ
8.85 (d, J = 6.59 Hz, 2H), 8.12 (d, J = 6.59 Hz, 2H), 7.13 (d, J = 8.48 Hz, 1H), 6.78 (dd, J = 2.45, 8.29
Hz, 1H), 6.68 (d, J = 2.26 Hz, 1H), 4.30–4.45 (m, 4H), 4.23 (br. s., 1H), 3.71 (br. s., 1H), 3.04–3.54 (m,
7H), 2.63–2.87 (m, 1H), 2.37–2.61 (m, 1H), 2.08–2.32 (m, 2H), 1.91 (d, J = 6.59 Hz, 1H), 1.06 (t, J =
6.40 Hz, 6H); 13C NMR (75 MHz, METHANOL-d4) δ 171.3, 166.5, 158.1, 143.0, 131.2, 129.6, 127.3,
121.9, 117.0, 113.7, 61.7, 57.9, 56.1, 52.7, 50.5, 45.5, 40.9, 32.3, 29.9, 29.6, 29.5, 19.9, 18.3; MS (ESI)
m/z 423.4 (M+H)+. The free base was converted to the trihydrochloride salt (4a•3HCl): mp 238–241 °C
(dec); [α]25D = +60.0 (c 0.98, MeOH). Anal. Calcd for C25H37Cl3N4O2•H2O: C, 54.60; H, 7.15; N, 10.19.
Found: C, 54.31; H, 7.16; N, 9.91.
5.5. (3R)-7-Hydroxy-N-{(1S)-2-methyl-1-[(4-methyl-4-pyridin-4-ylpiperidin-1-yl)methyl]propyl}-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (4b) Trihydrochloride

Compound 4b was prepared according to procedure analogous to that of 2 from amine 13f (78 mg,
0.30 mmol) to afford the 4b freebase: 1H NMR (300 MHz, METHANOL-d4) δ 8.44 (dd, J = 1.60, 4.62
Hz, 2H), 7.43 (dd, J = 1.60, 4.62 Hz, 2H), 6.91 (d, J = 8.10 Hz, 1H), 6.52–6.68 (m, 1H), 6.48 (br. s.,
1H), 3.79–4.16 (m, 3H), 3.44–3.65 (m, 1H), 2.72–3.00 (m, 2H), 2.63 (br. s., 1H), 2.19–2.56 (m, 5H),
1.94–2.17 (m, 2H), 1.65–1.91 (m, 3H), 1.05–1.35 (m, 4H), 0.75–1.04 (m, 6H); 13C NMR (75 MHz,
METHANOL-d4) δ 175.4, 161.2, 156.8, 150.1, 137.3, 130.8, 125.6, 123.2, 114.9, 113.2, 61.4, 58.0,
52.5, 51.3, 51.2, 47.7, 37.5, 37.2, 37.1, 32.4, 32.1, 19.9, 17.9. The free base was converted to the
trihydrochloride salt (4b•3HCl) affording 19.2 mg (11% over two steps) of a white powder: MS (ESI)
m/z 437.5 (M+H)+; mp 220–224 °C (fusion); [α]25D +75. (c 0.21, CH3OH). Anal. Calcd for
C26H39Cl3N4O2•3.25H2O: C, 51.66; H, 7.59; N, 8.85. Found: C, 51.88; H, 7.42; N, 9.27.
5.6 (3R)-7-Hydroxy-N-[(1S)-2-methyl-1-{[4-(3-thienyl)piperidin-1-yl]methyl}propyl]-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide (5)
Compound 5 was prepared according to procedure analogous to that of 2 from amine 13d (680 mg,
2.7 mmol) to afford the 5 free base as a viscous, colorless oil (220 mg, 19%): 1H NMR (300 MHz,
CHLOROFORM-d) δ 7.35 (br. s., 1H), 7.14 (dd, J = 2.83, 4.71 Hz, 1H), 6.68–6.90 (m, 3H), 6.53 (d, J =
7.91 Hz, 1H), 6.39 (br. s., 1H), 4.12 (br. s., 1H), 3.62 (br. s., 2H), 3.21–3.45 (m, 2H), 3.06 (br. s., 1H),
2.84 (t, J = 15.16 Hz, 2H), 2.29–2.67 (m, 4H), 2.19 (br. s., 1H), 1.59–2.01 (m, 5H), 0.86 (d, J = 6.03 Hz,
6H); 13C NMR (75 MHz, CHLOROFORM-d) δ 172.5, 154.1, 144.8, 135.5, 129.3, 125.6, 124.6, 123.7,
118.2, 113.1, 111.3, 58.4, 55.8, 54.3, 51.4, 48.7, 46.7, 35.5, 30.4, 28.9, 18.2, 17.0. The free base was
converted to the trihydrochloride salt (5•3HCl): MS (ESI) m/z 428.2 (M+H)+; mp 196–200 °C (fusion);
[α]25D = +75.2 (c 1.0, MeOH). Anal. Calcd for C24H36Cl2N3O2S•H2O: C, 55.11; H, 7.23; N, 8.03.
Found: C, 55.09; H, 7.00; N, 7.80.
5.7. tert-Butyl 4-thiophen-3-ylpiperidine-1-carboxylate (8d)

A mixture of 6 (3.2 g, 9.6 mmol), thiophene-3-boronic acid (1.72 g, 13.5 mmol),
tetrakis(triphenylphosphine)palladium (1.1 g, 0.96 mmol), cesium carbonate (9.4 g, 28.6 mmol),
dimethoxyethane (34 mL) and water (16 mL) was stirred at 80 °C under nitrogen overnight. The cooled
mixture was partitioned between water and EtOAc. The aqueous was further extracted with EtOAc (100
mL). The combined organic layers were dried (Na2SO4), filtered and evaporated to obtain intermediate
tert-butyl 4-thiophen-3-yl-3,6-dihydropyridine-1(2H)-carboxylate 7d: 1H NMR (300 MHz,
CHLOROFORM-d) δ 7.10–7.26 (m, 2H), 7.05 (br. s., 1H), 5.98 (br. s., 1H), 3.99 (d, J = 2.64 Hz, 2H),
3.55 (t, J = 5.75 Hz, 2H), 2.44 (br. s., 2H), 1.36–1.46 (m, 9H). Intermediate 7d was hydrogenated with
palladium hydroxide on carbon (1.0 g, 20 wt%) in ethanol under H2 (50 psi) at room temperature for 2 h.
The solids were filtered and solvent evaporated to provide 1.25 g (35% over 2 steps) of 8d: 1H NMR
(300 MHz, CHLOROFORM-d) δ 7.17–7.23 (m, 1H), 6.80–6.97 (m, 2H), 3.92–4.27 (m, 2H), 2.71 (d, J
= 12.06 Hz, 3H), 1.82 (br. s., 2H), 1.52 (br. s., 2H), 1.36–1.43 (m, 9H).
5.8. 4-Thiophen-3-ylpiperidine (9d)
A solution of 8d (1.25 g, 4.67 mmol) in acetonitrile (20 mL) was treated with 4M HCl in dioxane
(4 mL) and the mixture stirred for 1 h at room temperature. The solvent was evaporated to obtain 0.86 g
(90%) of 9d as the hydrochloride salt: 1H NMR (300 MHz, DMSO-d6) δ 8.66–9.67 (m, 2H), 7.40–7.59
(m, 1H), 7.14–7.28 (m, 1H), 6.97–7.11 (m, 1H), 3.14–3.39 (m, 2H), 2.81–3.06 (m, 3H), 1.93–2.12 (m,
2H), 1.67–1.91 (m, 2H).
5.9. 3-(4-Methylpiperidin-4-yl)pyridine (9e)
A solution of 12a (3.95 g, 19.3 mmol) in THF (50 mL) was treated with BH3•SMe2 (10 mL, 100
mmol) at 0°C, then heated to reflux for 12 h. The reaction mixture was quenched by addition of EtOAc,
then methanol. The concentrated residue was dissolved in aq. HCl (2.0 M, 10 mL). The pH was adjusted
with NaOH (2 M, 12 mL) and the resulting aqueous layer was extracted with EtOAc (3 x 50 mL). The

combined organics were washed with brine, dried (Na2SO4) and concentrated. The resulting residue was
subjected to chromatography on silica gel eluting with a gradient up to 100% CMA80 in CH2Cl2 to
afford 567 mg (17%) of piperidine 9e: 1H NMR (300 MHz, CHLOROFORM-d) δ 8.54–8.67 (m, 1H),
8.44 (d, J = 3.77 Hz, 1H), 7.56–7.71 (m, 1H), 7.18–7.33 (m, 1H), 3.57 (br. s., 1H), 2.89–3.04 (m, 1H),
2.74–2.89 (m, 1H), 1.52–2.70 (m, 7H), 1.28 (d, J = 7.72 Hz, 2H).
5.10. 4-(4-Methylpiperidin-4-yl)pyridine (9f)
Compound 9f was prepared according to procedure analogous to that of 9e from glutarimide 12b
(652 mg, 3.2 mmol) to afford 100 mg (18%) of piperidine 9f: 1H NMR (300 MHz, CDCl3) δ 8.39–8.63
(m, 2H), 7.25 (dd, J = 1.9, 4.1 Hz, 2H), 2.86–3.02 (m, 2H), 2.71–2.86 (m, 2H), 1.93–2.12 (m, 2H), 1.60–
1.79 (m, 2H), 1.17–1.31 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 158.4, 149.7, 121.1, 42.6, 37.2, 36.6,
28.7; MS (ESI) m/z 177.3 (M+H)+.
5.11. 4-Methyl-2,6-dioxo-4-(pyridin-3-yl)piperidine-3,5-dicarbonitrile (11a)
A solution of 3-acetylpyridine (10a) (10.0 g, 82.5 mmol, ethyl cyanoacetate (9.33 g, 82.5 mmol)
and ammonium acetate (1.3 g, 16.9 mmol) was refluxed in glacial acetic acid (4.0 mL) and benzene
(16.5 mL) with a Dean-Stark apparatus for 12 h. Ethyl acetate was added to the cooled solution. The
solution was washed with aq. NaHCO3 then brine, dried (Na2SO4) and concentrated. The residue was
eluted through a plug of silica gel using ethyl acetate to afford 16.32 g (92%) of the Knoevenagel
product. The oil was combined with 2-cyanoacetamide (7.36 g, 87.5 mmol) in a solution of NaOH (3.5
g, 87.5 mmol) in ethanol (60 mL). Within 5 minutes, a clear solution had formed which was left to stir
for 12 h, after which aq. NaHSO4 (1 M, 90 mL) was added. The resulting solids were collected by
filtration and the mother liquor was concentrated to approximately half the original volume of kept in a
refrigerator overnight. A second filtration yielded more solids, which were combined with the first crop
and dried under vacuum to afford 11.6 g (55% over two steps) of 11a: 1H NMR (300 MHz, DMSO-d6) δ

12.40 (br. s., 1H), 8.48–9.02 (m, 2H), 8.08 (d, J = 6.59 Hz, 1H), 7.57 (dd, J = 4.71, 7.91 Hz, 1H), 5.42
(s, 2H), 1.72 (br. s., 3H).
5.12. 4-Methyl-2,6-dioxo-4-(pyridin-4-yl)piperidine-3,5-dicarbonitrile (11b)
A solution of 4-acetylpyridine (10b) (10.0 g, 82.5 mmol), ethyl cyanoacetate (9.33 g, 82.5 mmol)
and ammonium acetate (1.3 g, 16.9 mmol) was refluxed in glacial acetic acid (4.0 mL) and benzene
(16.5 mL) with a Dean-Stark apparatus for 12 h. Ethyl acetate was added to the cooled solution. The
solution was washed with aq. NaHCO3 then brine, dried (Na2SO4) and concentrated. The residue was
eluted through a plug of silica gel using ethyl acetate to afford 13.76 g of an amber oil. The oil was
combined with cyanoacetamide (5.35 g, 63.6 mmol) in methanol (200 mL) at 0 °C. Ammonia gas was
bubbled in to the solution until it was saturated, then the dark red solution was left to warm to room
temperature and stir 1 week. The solution was brought to reflux for 1 h, at which point the solution
became cloudy. The solution was concentrated to a thick oil which was dissolved in EtOAc. Diethyl
ether was slowly added, causing a precipitate to form. Filtration afforded 8.81 g (54%) of 11b as a pale
yellow powder. 1H NMR (300 MHz, DMSO-d6) δ 9.39 (d, J = 4.1 Hz, 1H), 8.50 (ddd, J = 1.6, 3.2, 4.6
Hz, 2H), 7.20–7.60 (m, 2H), 4.31–4.74 (m, 1H), 1.28–1.79 (m, 3H); MS (ESI) m/z 253.3 (M–H)-.
5.13. 4-Methyl-4-(pyridin-3-yl)piperidine-2,6-dione (12a)
Sulfuric acid (6 mL) was slowly added to a slurry of 11a (11.6 g, 11.8 mmol) in water (6 mL). The
resulting solution was refluxed for 12 h, cooled, quenched with 50% NaOH (6 mL), diluted with
methanol (40 mL) then CH2Cl2 (160 mL). The white solids were filtered and washed with 10%
methanol in CH2Cl2. The orange filtrate was concentrated to afford 11.03 g of crude material, which was
combined with urea (7.1 g, 0.12 mol) and heated as a melt for 3 h until no further gas evolution was
observed. The mixture was partitioned between aq. NaHCO3 and EtOAc, and the resulting emulsion was
filtered through Celite. The aqueous layer was further extracted with EtOAc. The combined organic

layers were dried (Na2SO4) and concentrated. The resulting residue was eluted through a plug of silica
gel using 5–7.5% MeOH in CH2Cl2 as the eluent. The concentrated residue, upon trituration with
EtOAc, afforded 3.95 g (42% over two steps) of 12a as a solid: 1H NMR (300 MHz, DMSO-d6) δ 10.80
(br. s., 1H), 8.63 (d, J = 2.64 Hz, 1H), 8.36–8.52 (m, 1H), 7.74–7.90 (m, 1H), 7.37 (dd, J = 4.71, 8.10
Hz, 1H), 2.98–3.17 (m, 1H), 2.75–2.94 (m, 1H), 1.33 (br. s., 1H).
5.14. 4-Methyl-4-(pyridin-4-yl)piperidine-2,6-dione (12b)
Sulfuric acid (1.5 mL) was slowly added to a slurry of 11b (3.0 g, 11.8 mmol) in water (1.5 mL).
The resulting solution was refluxed for 12 h, cooled, quenched with 50% NaOH (1.5 mL), diluted with
methanol (10 mL) then CH2Cl2 (40 mL). The white solids were filtered and washed with 10% methanol
in CH2Cl2. The orange filtrate was concentrated to afford 2.23 g (85%) of 3-methyl-3-(pyridin-4-
yl)pentanedioic acid. 1H NMR (300 MHz, CD3OD) δ 8.23 – 8.62 (m, 2H), 7.30 – 7.62 (m, 2H), 2.70 –
3.01 (m, 4H), 1.58 (br. s., 3H). The 3-methyl-3-(pyridin-4-yl)pentanedioic acid (2.23 g, 10.0 mmol) was
combined with urea (2.0 g, 33 mmol) and heated as a melt for 1 h. The resulting material was subjected
to chromatography on silica gel. Although the product had Rf =0.3 in EtOAc, the solubility in pure
EtOAc was limited, so a gradient up to 10% methanol in EtOAc was used to afford 652 mg (32%) of
12b as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.59 (dd, J = 1.60, 4.4 Hz, 2H), 7.25 (dd, J = 1.6,
4.4 Hz, 2H), 2.97–3.19 (m, 2H), 2.80 (d, J = 17.0 Hz, 2H), 1.45 (s, 3H); MS (ESI) m/z 205.3 (M+H)+.
5.15. (2S)-3-methyl-1-(4-pyridin-2-ylpiperidin-1-yl)butan-2-amine (13a)
A solution of 9a (4.66 g, 19.8 mmol) in acetonitrile (150 mL) was treated with N-Boc-L-valine
(4.55 g, 21.0 mmol), HBTU (7.98 g, 21.0 mmol) and triethylamine (8.4 g, 84 mmol). The reaction
mixture was stirred overnight at ambient temperature then evaporated. The residue was partitioned
between sat. aq. NaHCO3 (100 mL) and EtOAc (100 mL). The aqueous was extracted further with
EtOAc (2 x 100 mL). The combined organic layer was dried (Na2SO4) then evaporated to obtain the

crude Boc intermediate which was subjected to silica gel chromatography eluting with a gradient up to
10% MeOH in CH2Cl2: MS (ESI) m/z 362.3 (M+H)+. The purified Boc intermediate was dissolved in
acetonitrile (100 mL), treated with 4M HCl in dioxane (20 mL) and stirred for 2 h. The reaction was
evaporated to obtain a solid which was subjected to chromatography on silica gel using a gradient of 10-
50% CMA80 in CHCl3 to afford 5.5 g (83% over two steps) of the intermediate amide (2S)-3-methyl-1-
oxo-1-(4-pyridin-2-ylpiperidin-1-yl)butan-2-amine as an off white solid: MS (ESI) m/z 262.2 (M+H)+
.
A solution of the intermediate amide (5.5 g, 21 mmol) in THF (40 mL) at 0 °C was treated with borane
dimethyl sulfide complex (3.3 mL, 33 mmol). The reaction mixture was warmed to ambient temperature
then heated at reflux overnight. The reaction was cooled in an ice bath, treated with 20 mL methanol
then warmed to ambient temperature over 1 h. The resulting solution was again cooled in an ice bath,
treated with 2M HCl in ether (100 mL) and then heated at reflux for 1 h. The solvent was evaporated
and the residue was subjected to chromatography on silica gel eluting with a gradient of CMA80 in
CHCl3 to afford 2.2 g (42%) of 13a as a white solid.
5.16. (2S)-3-methyl-1-(4-pyridin-4-ylpiperidin-1-yl)butan-2-amine (13c)
Compound 13c was prepared according to the general procedure of 13a from the amine 9c (1.42 g,
5.9 mmol) to afford 0.77 g (53% yield) of the desired 13c free base: 1H NMR (300 MHz, METHANOL-
d4) δ 8.42–8.52 (m, 2H), 7.35–7.46 (m, 2H), 3.21 (q, J = 7.35 Hz, 2H), 3.00 (d, J = 11.11 Hz, 1H), 2.42–
2.77 (m, 3H), 2.19 (dt, J = 4.33, 10.83 Hz, 1H), 1.84–1.97 (m, 4H), 1.32 (t, J = 7.35 Hz, 2H), 0.98–1.11
(m, 6H).
5.17. (2S)-3-Methyl-1-[4-(thiophen-3-yl)piperidin-1-yl]butan-2-amine (13d)
A solution of 4-(thiophen-3-yl)piperidine (9d) (850 mg, 4.17 mmol) in CH3CN (30 mL) was
treated with N-Boc-L-valine(1.08 g, 5.0 mmol), HBTU (1.9 g, 5.0 mmol) and NEt3 (1.5 g, 15 mmol).
The reaction mixture was stirred overnight at ambient temperature. The concentrated residue was

partitioned between sat. aq. NaHCO3 (15 mL) and EtOAc (15 mL). The aqueous layer was further
extracted with EtOAc (2 x 15 mL). The combined organic layer was dried (Na2SO4), filtered, and
evaporated. The residue was subjected to chromatography on silica gel eluting with 50% EtOAc in
hexanes to afford the intermediate tert-butyl {(1S)-2-methyl-1-[(4-thiophen-3-ylpiperidin-1-
yl)carbonyl]propyl}carbamate as a light yellow oil (1.34 g, 88%): 1H NMR (300 MHz,
CHLOROFORM-d) δ 7.20–7.38 (m, 1H), 6.89–7.07 (m, 2H), 5.40 (d, J = 9.04 Hz, 1H), 4.63–4.84 (m,
1H), 4.54 (br. s., 1H), 3.92–4.20 (m, 1H), 3.10–3.29 (m, 1H), 2.90 (t, J = 11.68 Hz, 1H), 2.65–2.80 (m,
1H), 1.85–2.18 (m, 3H), 1.52–1.77 (m, 1H), 1.45 (s, 9H), 0.82–1.07 (m, 7H). The Boc-intermediate was
dissolved in CH3CN (10 mL) then treated with HCl (4 N in dioxane, 10 mL). After 1 h at ambient
temperature, the solution was evaporated to afford a quantitative yield (1.09 g) of the (2S)-3-methyl-1-
1
oxo-1-(4-thiophen-3-ylpiperidin-1-yl)butan-2-amine hydrochloride salt: H NMR (300 MHz, DMSO-
d6) δ 8.21 (d, J = 11.30 Hz, 3H), 7.38–7.58 (m, 1H), 7.21 (d, J = 2.83 Hz, 1H), 6.96–7.14 (m, 1H), 4.50
(d, J = 12.62 Hz, 1H), 4.27 (br. s., 1H), 4.04 (d, J = 10.36 Hz, 1H), 3.17 (t, J = 12.43 Hz, 1H), 2.91 (t, J
= 10.46 Hz, 1H), 2.75 (t, J = 12.53 Hz, 1H), 1.81–2.14 (m, 3H), 1.30–1.78 (m, 2H), 0.83–1.09 (m, 6H);
MS (ESI) m/z 267.2 (M+H)+. The intermediate amide was dissolved in THF (25 mL) then treated with
borane dimethyl sulfide complex (1.8 mL, 18 mmol) at 0 °C. The reaction mixture was warmed to
ambient temperature and stirred overnight. The reaction mixture was refluxed for 2 h, cooled in ice,
treated with methanol (20 mL) and stirred for 1 h at ambient temperature. The reaction mixture was
cooled in ice and treated with 2M hydrogen chloride in ether (40 mL) then refluxed overnight. The
solvent was evaporated and the resulting residue was subjected to chromatography on silica gel eluting
with a gradient up to 30% CMA80 in CHCl3 to afford 680 mg (75%) of 13d as a white solid: 1H NMR
(300 MHz, CHLOROFORM-d) d 7.13–7.25 (m, 1H), 6.82–6.98 (m, 2H), 4.91 (br. s., 2H), 2.78–3.03
(m, 3H), 2.48–2.62 (m, 1H), 2.36–2.45 (m, 2H), 2.29 (dt, J = 2.54, 11.54 Hz, 1H), 1.53–2.07 (m, 6H),

1.03 (d, J = 6.78 Hz, 3H), 0.93 (d, J = 6.78 Hz, 3H); 13C NMR (75 MHz, CHLOROFORM-d) d 146.9,
126.8, 125.3, 118.9, 59.7, 55.6, 54.4, 52.9, 37.4, 33.3, 32.9, 30.3, 19.3, 18.8; MS (ESI) m/z 253.2
(M+H)+.
5.18. (2S)-3-Methyl-1-(4-methyl-4-pyridin-3-ylpiperidin-1-yl)butan-2-amine (13e)
A solution of 9e (152 mg, 0.86 mmol) and Boc-L-valine (230 mg, 1.06 mmol) in acetonitrile (30
mL) was treated with EDC•HCl (340 mg, 1.8 mmol) and triethylamine (0.7 mL, 5 mmol) and stirred 12
h. The concentrated residue was partitioned between EtOAc and water. The organic layer was washed
(aq. NaHCO3 then brine) then dried (Na2SO4). The concentrated residue was eluted from silica gel using
ethyl acetate to afford an oil which was dissolved in methanol (10 mL) and treated with HCl (12 N, 2
mL), stirred 1 h and then concentrated. The residue was subjected to chromatography on silica gel
eluting with a gradient up to 50% CMA80 in CH2Cl2 to afford 201 mg (84% over two steps) of the
intermediate amide: 1H NMR (300 MHz, CHLOROFORM-d) δ 8.64 (br. s., 1H), 8.40–8.57 (m, 1H),
7.55–7.74 (m, 1H), 7.18–7.39 (m, 1H), 1.60–4.27 (m, 12H), 1.33 (d, J = 11.87 Hz, 3H), 0.83–1.11 (m,
6H); MS (ESI) m/z 276.5 (M+H)+. A solution of the intermediate amide (201 mg, 0.73 mmol) in THF (3
mL) was treated with BH3•THF (1 M, 10 mL) then heated to reflux overnight. The reaction mixture was
quenched with methanol and concentrated. The residue was subjected to chromatography on silica gel
eluting with 25% CMA80 in CH2Cl2 to afford 117 mg (62%) of the desired 13e: 1H NMR (300 MHz,
CHLOROFORM-d) δ 8.55 (s, 1H), 8.39–8.50 (m, 1H), 7.95 (d, J = 7.91 Hz, 1H), 7.50 (dd, J = 5.93,
7.82 Hz, 1H), 4.56 (br. s., 2H), 3.58–3.72 (m, 1H), 2.73–2.96 (m, 1H), 2.49–2.73 (m, 2H), 2.21–2.50 (m,
3H), 1.95–2.21 (m, 2H), 1.54–1.94 (m, 3H), 1.15–1.42 (m, 3H), 0.72–1.11 (m, 6H).
5.19. (2S)-3-Methyl-1-[4-methyl-4-(pyridin-4-yl)piperidin-1-yl]butan-2-amine (13f)
Compound 13f was prepared according to procedure analogous to that of 13e from amine 9f (100
mg, 0.57 mmol) to afford 78 mg (52% over three steps) of 13f: 1H NMR (300 MHz, CDCl3) δ 8.53 (dd,

J = 2.3, 4.0 Hz, 2H), 7.25 (d, J = 3.2 Hz, 2H), 2.33–2.76 (m, 4H), 1.96–2.33 (m, 7H), 1.67–1.88 (m,
2H), 1.41–1.61 (m, 1H), 1.15–1.27 (m, 3H), 0.81–0.99 (m, 6H).
5.20. [35S]GTPγS assay
The [35S]GTPγS assays were conducted using the methods previously reported.[13]
5.21. 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 Pickett model (Eq.3).[11, 14]
Acknowledgements
This research was supported by the National Institute on Drug Abuse Grant No. DA09045. We
thank Tiffany Langston and Keith Warner for conducting the in vitro testing.
Supplementary data
Supplementary data ssociated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.
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Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of
replacement of the 3-hydroxyphenyl group with pyridine or thiophene bioisosteres
Chad M. Kormos, Moses G. Gichinga, Scott P. Runyon, James B. Thomas, S. Wayne Mascarella, Ann
M. Decker, Hernán A. Navarro, and F. Ivy Carroll