Nonpeptide urotensin-II receptor antagonists: A new ligand class based on piperazino-phthalimide and piperazino-isoindolinone subunits

Article abstract of DOI:10.1021/jm900683d

We have discovered two related chemical series of nonpeptide urotensin-II (U-II) receptor antagonists based on piperazino-phthalimide (5 and 6) and piperazino-isoindolinone (7) scaffolds. These structure types are distinctive from those of U-II receptor antagonist series reported in the literature. Antagonist 7a exhibited single-digit nanomolar potency in rat and human cell-based functional assays, as well as strong binding to the human U-II receptor. In advanced pharmacological testing, 7a blocked the effects of U-II in vitro in a rat aortic ring assay and in vivo in a rat ear-flushmodel. Adiscussion of U-II receptor antagonist pharmacophores is presented, and a specifically defined model is suggested from tricycle 13, which has a high degree of conformational constraint.

Full text of DOI:10.1021/jm900683d

7432 J. Med. Chem. 2009, 52, 7432–7445
DOI: 10.1021/jm900683d
Nonpeptide Urotensin-II Receptor Antagonists: A New Ligand Class Based on Piperazino-Phthalimide
and Piperazino-Isoindolinone Subunits
Edward C. Lawson,* Diane K. Luci, Shyamali Ghosh, William A. Kinney, Charles H. Reynolds, Jenson Qi, Charles E. Smith,
Yuanping Wang, Lisa K. Minor, Barbara J. Haertlein, Tom J. Parry, Bruce P. Damiano, and Bruce E. Maryanoff*
Johnson & Johnson Pharmaceutical Research & Development, Welsh & McKean Roads, Spring House, Pennsylvania 19477-0776
Received May 21, 2009
We have discovered two related chemical series of nonpeptide urotensin-II (U-II) receptor antagonists
based on piperazino-phthalimide (5 and 6) and piperazino-isoindolinone (7) scaffolds. These structure
types are distinctive from those of U-II receptor antagonist series reported in the literature. Antagonist
7a exhibited single-digit nanomolar potency in rat and human cell-based functional assays, as well as
strong binding to the human U-II receptor. In advanced pharmacological testing, 7a blocked the effects
of U-II in vitro in a rat aortic ring assay and in vivo in a rat ear-flush model. A discussion of U-II receptor
antagonist pharmacophores is presented, and a specifically defined model is suggested from tricycle 13,
which has a high degree of conformational constraint.
Introduction
failure and atherosclerosis. From the wealth of knowledge
in this area, U-II and its receptor have been implicated in
various cardiorenal and metabolic diseases,1a-e including
hypertension,10 heart failure,11 chronic renal failure,11c,12
diabetes,11c,13 and atherosclerosis.14 Consequently, we3b,15
and other researchers3a,3c,16,17 have been pursuing U-II
receptor antagonists as potential therapeutic agents.
The cyclic peptide urotensin-II (U-IIa) activates the uro-
tensin-II receptor (UT), a G-protein-coupled receptor that
was originally identified as “orphan” receptor GPR14,1 and is
expressed in many tissues, including blood vessels, heart, liver,
kidney, skeletal muscle, and lung.2 Actually, there is a collec-
tion of U-II peptides that range in size from 11 to 14 amino
acids across species, from fish to humans, with 11 amino acids
in humans to 14 amino acids in mice. Residues 5 through 10 of
these U-II peptides comprise a highly conserved CFWKYC
cyclic array, whereas the N-terminal region is variable in
length and constitution. The WKY motif has been identified
as being important for biological activity.3
U-II from the urophysis of the goby fish (Gillichthys
mirabilis) is a 12-mer cyclic peptide that may well be the most
potent vasoconstrictor known.4-6 This compound caused
concentration-dependent contraction of isolated arterial rings
of rats and humans with an EC50 less than 1 nM. However,
the in vivo effects of U-II can be contradictory because of
varied specificity for different tissues and different species. As
a case in point, U-II was reported to cause vasodilation in
conscious rats.7 Nevertheless, relative to the role of U-II in
chronic vascular disease, the goby U-II peptide induced
hypertrophy in cardiomyocytes8 and the proliferation of
smooth muscle cells,9 indicating a potential role in heart
To obtain initial lead structures, we devised and executed a
high-throughput screening (HTS) protocol involving a func-
tional assay based on cells transfected with rat UT, a fluoro-
metric imaging plate reader (FLIPR) to measure intracellular
calcium flux, and the potent, cyclopeptide U-II agonist
Ac-CFWK(2-Nal)C-NH2 (1; rat FLIPR EC50 = 0.54 nM,
rat UT binding Ki = 0.12 nM) (Chart 1; see Experimental
Section).3b Application of this assay to a large compound
library (∼500000 entities) resulted in several confirmed hits,
one of which was moderately potent, nonpeptide antagonist
2 (rat FLIPR IC50 = 1 μM) (Chart 1). Unfortunately, this
compound and its more potent bis-desfluoro analogue, 3 (rat
FLIPR IC50 = 150 nM), turned out to be metabolically
unstable to human liver microsomes (HLM t1/2 < 3 min), pri-
marily because of piperazine debenzylation (Chart 1). As part
of a drug discovery endeavor, we sought to mitigate this
metabolic problem and improve U-II receptor antagonist
potency. This effort led to a new class of potent UT antago-
nists based on piperazino-phthalimide and piperazino-isoin-
dolinone structural subunits (Table 1, compounds 2-4, 5a-n,
6a-j; Table 2, compounds 7a-o). In this paper, we present
our results with these novel series and discuss U-II receptor
antagonist pharmacophores.
*To whom correspondence should be addressed. Phone: 215-628-
5644 (E.C.L.); 215-628-5530 (B.E.M.). Fax: 215-540-4612. E-mail:
elawson@its.jnj.com (E.C.L.); bmaryano@its.jnj.com (B.E.M.).
a Abbreviations: BOP, benzotriazol-1-yloxy-tris(dimethylamino)-
phosphonium; BSA, bovine serum albumin; CHO, Chinese hamster
ovary; dba, (E,E)-dibenzylideneacetone; FLIPR, fluorometric imaging
plate reader; h, human; HBTU, O-benzotriazol-1-yl-N,N,N0,N0-tetra-
methyluronium; HOBt, 1-hydroxybenzotriazole; HLM, human liver
microsomes; HTS, high-throughput screening; PK, pharmacokinetics;
SAR, structure-activity relationship; TFA, trifluoroacetic acid; Tol-
BINAP, 2,20-bis(di-p-tolyl)phosphino-1,10-binaphthyl; U-II, urotensin-
II; UT, urotensin-II receptor.
Results and Discussion
Synthetic Chemistry. Prototype phthalimide 3 was pre-
pared by the route outlined in Scheme 1. Hydroxyphthali-
mide I was coupled with veratrylamine in the presence of
triethylamine, followed by triflate formation to give II in
r
pubs.acs.org/jmc
Published on Web 09/04/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7433
Chart 1
excellent yield. Boc-piperazine was introduced by direct
displacement in the presence of triethylamine at 110 °C in a
sealed tube in good yield. The Boc group was removed and
III was alkylated with benzyl bromide to afford 3 in high
yield.
under palladium catalysis to give XX.22 The Boc group was
removed, and the exposed primary amine was coupled with
2-thienylsulfonyl chloride to furnish target compound 7a
(96% ee; see Experimental Section).
Development of U-II Receptor Antagonists. As an early
approach to metabolic stabilization, we installed an R-meth-
yl group to generate (R)-4 (Chart 1). We realized improved
metabolic stability (HLM t1/2 = 29 min) and improved
potency (rat FLIPR IC50=84 nM);23 also, (R)-4 proved to
be promising in that it exhibited good oral bioavailability in
Compounds of structure type 5 were prepared according
to the general plan in Scheme 2. Ketones V were obtained
from carboxylic acids IV by Weinreb amide formation,
followed by addition of 3,4-dimethoxyphenylmagnesium
bromide.18 Reductive amination with ammonium acetate
and sodium cyanoborohydride gave primary benzylamines
VI, which were converted into phthalimides VII. Activation
as a triflate and piperazine displacement yielded target
piperazino-phthalimides 5 (Table 1). 2-Thienylsulfonami-
doalkyl derivatives 5e, 5g, and 5j were prepared from the
corresponding primary amines and 2-thienylsulfonyl chlor-
ide with K2CO3 as a base. In the case of (R,S)-5a, an
enantioselective synthesis was performed (Scheme 3). Thus,
ketone VIII was reduced asymmetrically with borane and
prolinol IX19 to alcohol X in high yield, with an optical
a pharmacokinetics (PK) experiment in rats (F=33%, t1/2
=
4 h). However, in a whole-cell binding assay involving human
UT (see Experimental Section),24 (R)-4 had just moderate
affinity (hUT Ki=570 nM). Now, the goal became improve-
ment of binding affinity for the human receptor while
retaining favorable rat potency (because rats would serve
the pharmacological test species) and metabolic stability. We
carried out a structure-activity relationship (SAR) study
with respect to the 3,4-dimethoxyphenyl segment and found
that the 3,4-dimethoxyphenyl group is crucial for decent in
vitro potency. Without going into much detail, the following
replacements for 3,4-dimethoxyphenyl in prototype (R)-4
were very unfavorable: phenyl, 3-hydroxy-4-methoxyphe-
nyl, 3-methoxy-4-hydroxyphenyl, 3-ethoxy-4-methoxyphe-
nyl, 3-methoxy-4-ethoxyphenyl, 4-benzoic acid methyl ester,
3-benzoic acid methyl ester, 4-benzoic acid, 3-benzoic acid,
2-pyrimidine, 3-indole, 4-indole, 2-methoxypyridine, and
5,6-dimethoxy-1-indane.
We explored the attachment of substituents onto the
methylene portion of the 3,4-dimethoxybenzyl group to
probe SAR around that benzylic position. Thus, mono-R-
substituted 3,4-dimethoxybenzylamines (VI) were converted
into phthalimide products 5 (Scheme 2). The rat FLIPR
potency of methyl derivative 5a (IC50=70 nM) was in the
range of unsubstituted compound 4 (Table 1). Diastereomer
(R,S)-5a had a rat FLIPR IC50 value of 130 nM, as well as a
3.5-fold potency improvement for the human receptor (hUT
Ki=160 nM) compared to 4.25 Ethyl analogue 5b had a rat
FLIPR IC50 value of 160 nM. On the basis of these promising
results, we introduced more elaborate substituents at that
benzylic position (Table 1). A study of aminoalkyl (5c, 5h,
and 5k) and Boc-aminoalkyl (5d, 5f, 5i, and 5l) groups
indicated that rat UT antagonist potency was optimal with
one to three methylenes in the linker and suggested that
human UT potency was optimal with three methylenes in the
linker. A follow-up study of 2-thienylsulfonamidoalkyl
rotation of [R]25 þ42.0 (c 1.07, CHCl3). Since Ponzo and
D
Kaufman reported an optical rotation for X of [R]25D þ40.5
(c 1.1, CHCl3), corresponding to 97% ee,19 our material was
considered to be essentially g99% ee. The mesylate of X was
reacted with diallylamine to give XI in good yield with
inversion of the stereocenter. The allyl groups were removed
by a palladium catalysis procedure20 and XII was trans-
formed into target (R,S)-5a, [R]25D þ13.8 (c 1.18, CH3OH),
by generation of a phthalimide, formation of a triflate, and
displacement with piperazine XIII (8% yield for four steps).
On the basis of the chemistry used, (R,S)-5a should have
been >99% ee.
We developed an enantioselective synthesis of key diamine
enantiomers (R)-XVI and (S)-XVI to provide individual
diastereomers for testing (Scheme 4; exemplied for (R)-XVI).
Weinreb amide XIV16 was subjected to asymmetric reductive
amination by using ammonium formate and a chiral Binap-
ruthenium complex to give XV (96% ee).21 The primary
amine in (R)-XV was protected as a trifluoroacetamide, the
Cbz group was removed, a Boc group was installed, and
the trifluoroacetyl group was removed to afford (R)-XVI.
The incorporation of this intermediate into a final product is
illustrated for 7a in Scheme 5. Benzoic acid XVII was
esterified and R-brominated to give XVIII, which was
coupled with (R)-XVI and cyclized thermally to indolinone
XIX. The aryl halide was displaced by N-ethylpiperazine

7434 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lawson et al.
Table 1. Urotensin-II Receptor Functional and Binding Data for Phthalimidesa
compd
R1
R2
rat FLIPRb IC50, nM
hUT bdgcKi, nM
human FLIPRd IC50, nM
2
H
2,6-F2C6H3CH2
PhCH2
1000
150
84
310
160
570
3
(R)-4
H
H
(R)-Ph(Me)CH
(S)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
H
1900
(S)-4
5a
H
Me
490
70
310
230
(R,R)-5a
(R,S)-5a
(R)-Me
(S)-Me
Et
150
130
180
300
120
75
160
310
5b
5c
CH2NH2
CH2NH-Boc
700
430
5d
5e
CH2NH-SO2-Th
(CH2)2NH-Boc
290
540
5f
5g
110
120
200
200
80
(CH2)2NH-SO2-Th
(CH2)3NH2
(CH2)3NH-Boc
270
5h
5i
3200
200
5j
(CH2)3NH-SO2-Th
(CH2)3NH-SO2-Th
(CH2)3NH-SO2-Th
(CH2)4NH-Boc
76
120
250
260
240
(R,S)-5j
(R,R)-5je
5l
69
29
49
>10000
1400
65000
110
1700
40
5m
5n
(CH2)3NH-SO2Me
(CH2)3NH-SO2-ClTh
(CH2)3NH-SO2-Th
(CH2)3NH-SO2-Th
(CH2)3NH-SO2-Th
(CH2)3NH-SO2-Th
(CH2)3NH-SO2-Isox
(CH2)3NH-C(O)-Isox
(CH2)3NH-SO2-Pyzl
(CH2)3NH-C(O)-Pyzl
(CH2)3NH-SO2-Imid
(CH2)3NH-SO2-Imid
6a
6b
150
59
500
220
150
150
130
760
47
Me
Et
16
6c
6d
4.8
3.5
35
45
i-Pr
6e
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
(R)-Ph(Me)CH
Et
20
11
32
6f
6g
130
13
11
11
6h
6i
87
16
37
14
220
810
8.0
6j
140
34
97
8 f
8.0
41
9g
>75000
320
a Target compounds were purified either by silica gel flash-column chromatography (230-400 mesh) or HPLC on a Gilson instrument equipped
with a reverse-phase Kromasil C18 column (10 μ, 250 mm ꢀ 50 mm). All compounds were characterized by ESI-MS and 300 MHz 1H NMR.
Abbreviations: Th, 2-thienyl; ClTh, 5-chlorothien-2-yl; Isox, 5-methylisoxazol-4-yl; Imid, 1,2-dimethylimidazol-4-yl; Pyzl, 3,5-dimethylpyrazol-4-yl.
b Inhibition of calcium mobilization in CHO-K1 cells transfected with the rat UT, as measured by FLIPR (N = 2-6). c Inhibition of [125I]U-II binding
to UT in cultured human rhabdomyosarcoma cells (RMS13) (N = 2). d Inhibition of calcium mobilization in human rhabdomyosarcoma cells,
as measured by FLIPR (N = 2-6). e Corresponds to structure type 6. f Reference standard SB-706375 (ref 26; also Dhanak, D.; Gallagher, T. F.;
Knight, S. D. PCT Int. Appl. WO 2002089792, 2002). g Reference standard palosuran (ref 17i; also Aissaoui, H., et al. PCT Int. Appl. WO 2003048154,
2003).
groups with one to three methylenes (5e, 5g, and 5j) con-
firmed that a three-carbon linker leads to better human UT
antagonist potency (hUT Ki = 290, 270, and 76 nM, re-
spectively). Methylsulfonamide 5m and, especially, (5-chloro-
thienyl)sulfonamide 5n showed attenuated potency. Given
that such derivatization could yield good antagonist potency
against both rat and human receptors, numerous sulfon-
amides with a three-carbon linker were examined in our rat
and human FLIPR-based functional assays and for human
UT binding (see Experimental Section). Two notable sub-
units were (5-Me-oxazol-4-yl)SO2NH(CH2)3- (rat FLIPR
IC50/hUT Ki/human FLIPR IC50 = 14/31/46 nM) and
MeSO2CH2SO2NH(CH2)3- (19/25/250 nM), which im-
proved upon 5j (80/76/250 nM).
At this juncture, we pursued the evaluation of single
diastereomers only. Although the enantiomers (R,S)-5j and
(R,R)-5j did not have distinctly different potencies, there was
a potency bias for the (R,R)-isomer over the (R,S)-isomer
(Table 1). Thus, diverse derivatives with the (R,R) structure
were synthesized and tested, and a sampling of our large
number of analogues is presented in Table 1. It was found
that ethyl (6c) and isopropyl (6d) are optimal groups for the
piperazine nitrogen, thereby attaining single-digit nanomo-
lar rat UT antagonist potency. A carboxamide functionality
could replace the sulfonamide, albeit with an attenuation
in potency (cf. 6e and 6f; 6g and 6h). The piperazine
N-substituent and the sulfonamide group did not necessarily
combine in an additive manner (cf. 6i and 6j).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7435
Table 2. Urotensin-II Receptor Functional and Binding Data for Isoindolinonesa
compd
R1
R2
rat FLIPRbIC50, nM
hUT bdgcKi, nM
human FLIPRdIC50, nM
7a
7b
7c
7d
7e
7f
(CH2)3NH-SO2-Th
(CH2)3NH-SO2-Th
(CH2)3NH-C(O)-Th
(CH2)3NH-CH2-Th
(CH2)3NMe-SO2-Th
(CH2)3NMe-SO2-Th
(CH2)3NMe-SO2-Th
(CH2)3NMe-SO2-Th
(CH2)3NMe-SO2-Th
(CH2)3NMe-SO2-Th
(CH2)3NH-C(O)-Pyzl
(CH2)3NH-C(O)-Isox
(CH2)3NH-SO2-Imid
(CH2)3NH-ClPyrm
(CH2)3NH-MeOPyrm
Et
1.0
3.0
4.0
4.0
18
8.0
53
i-Pr
Et
7.0
6.0
140
59
Et
Et
18
7.0
<5
49
88
H
39
16
410
340
500
1200
510
130
320
51
7g
7h
7i
Me
i-Pr
<6
11
2.0
cyclopropyl
cyclobutyl
13
59
44
7j
4.0
7k
7l
Et
(R)-Ph(Me)CH
16
11
15
6.0
7m
7n
7o
8e
Et
Et
Et
23
25
62
1.0
4.0
500
62
8.0
8.0
41
34
>75000
8.0
9 f
320
a Target compounds were purified either by silica gel flash-column chromatography (230-400 mesh) or HPLC on a Gilson instrument equipped with
a reverse-phase Kromasil C18 column (10 μ, 250 mm ꢀ 50 mm). All compounds were characterized by ESI-MS and 300 MHz 1H NMR. Abbreviations:
Th, 2-thienyl; ClTh, 5-chlorothien-2-yl; Isox, 5-methylisoxazol-4-yl; Imid, 1,2-dimethylimidazol-4-yl; Pyzl, 3,5-dimethylpyrazol-4-yl. Additional
abbreviations: ClPyrm, 6-chloropyrimidin-2-yl; MeOPyrm, 6-methoxypyrimidin-2-yl. b Inhibition of calcium mobilization in CHO-K1 cells transfected
with the rat UT, as measured by FLIPR (N = 2-6). c Inhibition of [125I]U-II binding to UT in cultured human rhabdomyosarcoma cells (RMS13)
(N = 2). d Inhibition of calcium mobilization in human rhabdomyosarcoma cells, as measured by FLIPR (N = 2-6). e Reference standard SB-706375
(ref 26; also Dhanak, D.; Gallagher, T. F.; Knight, S. D. PCT Int. Appl. WO 2002089792, 2002). f Reference standard palosuran (ref 17i; also Aissaoui, H.,
et al. PCT Int. Appl. WO 2003048154, 2003).
Scheme 1. Synthesis of 3
We extended our investigation to the corresponding iso-
indolinone analogues, 7 (Table 2), which were assembled
from diamine (R)-XVI according to the route in Scheme 5.
The SAR information gained from the phthalimide series
was used as a starting point for the isoindolinone series.
Thus, we readily obtained some very potent antagonists,
several with single-digit nanomolar potencies in the rat
FLIPR assay (viz. 7a-e, 7h, 7j, 7l, 7n, and 7o) and some
with single-digit nanomolar affinities in the hUT binding
assay (viz. 7a, 7b, 7e, 7g, and 7o). Remarkably, 7a also
exhibited very potent functional antagonism in the human
calcium flux assay (IC50 = 8.0 nM) (see Experimental
Section). The next tier of compounds in this human cellular
functional assay were 7b, 7d, and 7m with IC50 values of
50-60 nM. Across our three assays, ethyl was the optimal
N-alkyl group (R2) for antagonist potency. It is noteworthy

7436 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Scheme 2. Synthesis of 5
Lawson et al.
Scheme 3. Synthesis of (R,S)-5a
Scheme 4. Synthesis of Intermediate (R)-XVI
that 7a displayed better potency than two reference U-II
receptor antagonists that we tested, 8 (SB-706375)26 and
palosuran17i (9) (Chart 2). In a rat aortic ring assay,27 7a
blocked the effect of rat U-II with 74% inhibition at 100 nM,
whereas 8 exhibited inhibition of 97% at 100 nM.
Compound 7a was examined in the U-II-induced ear-flush
model in rats to determine if it could block a U-II-mediated
effect in vivo (Figure 1).28 Subcutaneous administration
of U-II causes an increase in ear pinna temperature, with
a maximum effect at 30 min, which can be blocked by

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7437
Scheme 5. Synthesis of 7a
Chart 2. U-II Receptor Antagonist Molecules
antagonist molecules. Compound 7a, administered iv 15 min
prior to administration of U-II, attenuated the increase in
ear pinna temperature (whereas vehicle had no effect).
Complete reversal with 7a in this assay was attained at
10 mg/kg (Figure 1), and a similar response was observed
with intravenously administered 8. An area for improvement
for 7a and its congeners would be oral bioavailability, as
F generally ranged between 10 and 15% in oral PK studies
in rats.
To obtain preliminary insight into selectivity for UT over
other molecular targets, 7a was profiled for binding affinity
in 50 receptors and ion channels at a high concentration of
10 μM by CEREP.29 Less than 50% inhibition of the binding
of a reference ligand was observed in 90% of the individual
assays.30 The following assays showed inhibition values of
>50%: R1 adrenergic receptor (68%), human M1 muscari-
nic receptor (65%), human M3 muscarinic receptor (55%),
human 5-HT1A serotonin receptor (93%), 5-HT1B seroto-
nin receptor (66%), and Naþ channel site 2 (59%).
Our earlier series revolved around phenylpiperidine-benzoxa-
zinones, such as 10, which also bears a piperazine group with
one basic nitrogen center (Chart 2).15 The series from Actelion
was anchored by palosuran (9), in which a 4-quinoline is linked
to a 4-substituted piperidine,17i and GlaxoSmithKline com-
pound 8 contains a 3,4-dimethoxybenzenesulfonamide linked
to a pyrrolidine.26 Some other classes of potent U-II antago-
nists are represented by structures 1117g and 1217f (Chart 2).
There appears to be some analogy between the subunits of
chemotypes 10 and 11, but their key basic amine centers do not
correlate, as discussed further below.
A cursory inspection of structures 7a and 8-12 suggests
that there is no clear-cut structural pattern to describe a
common pharmacophore for potent UT antagonist ligands.
However, to gain a better appreciation of the pharmaco-
phores for these six series, we generated energy-minimized
structures for each representative compound (see Supporting
Information) with Maestro31 by employing the OPLS-AA
force-field32 and a GB/SA water model.33 Each structure
was also subjected to a limited amount of conformational
analysis by using the mixed-torsion low-mode algorithm34 in
Different Classes of Nonpeptide U-II Receptor Antagonists.
Various U-II receptor antagonist series have been reported.15-17

7438 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lawson et al.
Figure 2. Spatial arrangement and distances for loci in the ground-
state conformation of tricycle 13. The basic piperazine nitrogen (N),
carbonyl oxygen (O), and center of the isoindolinone benzene ring
(A) are coplanar, while the center of the dimethoxybenzene (B)
projects forward from that defined plane.
Figure 1. Effects of 7a on ear pinna temperature over time. A
solution of the test compound in 20% aqueous hydroxypropyl-β-
cyclodextrin (HPBDC) was administered intravenously at 3 mg/kg
(filled squares) or 10 mg/kg (filled, inverted triangles). The admin-
istration of vehicle is depicted by open squares. Data are mean
temperature changes from baseline ( SEM (N = 5 per group).
˚
˚
∼17 A from the benzamide ring; the amide carbonyl is 4.6 A
˚
fromthe pyrrolidinenitrogenand∼14.5 A fromthepiperidine
nitrogen. This comparative analysis, albeit less than rigorous,
suggests that several pharmacophore models can satisfy the
requirements for U-II receptor binding and antagonism. A
caveat to this analysis would be that we have only considered
unbound, ground-state conformations (global energy mini-
ma), whereas receptor-bound conformations may be different
and more relevant. Given this assessment, it would appear
that effective UT antagonists are likely to emanate from
diverse chemical entities with specific pharmacophore com-
ponents related to those that are highlighted.
Maestro. To assess the relative distances between key inter-
action sites, a common pharmacophore was generated by
using Phase.35 It was only possible to obtain very simple
three-point pharmacophores because of the great diversity
between these six structures. The distances in the following
discussion were derived from low energy conformations
aligned according to this rudimentary pharmacophore. All
of the chemotypes possess at least one basic amine nitrogen,
which was used as a reference frame, and at least two
benzenoid subunits, which were used as secondary reference
elements. Compound 7a contains a basic nitrogen center in
A key issue in dealing with ligands 7a and 8-12 to
establish understanding about UT pharmacophore models
is the conformational freedom available to these molecules.
Their wide structural variation also poses a problem. It
would be useful to consider a UT ligand with much less
conformational dimensionality. Fortunately, we identified
tricyclic compound 13 as a potent UT antagonist (rat FLIPR
IC50=14 nM; human UT binding Ki=64 nM), which is quite
conformationally constrained (Chart 2).36 In its ground-
˚
the piperazine ring that is 5.5 A from the isoindolinone
˚
benzene ring (center of ring), 8.3 A from the amide carbonyl,
˚
9.9 A from the key dimethoxybenzene ring (center of ring),
˚
and 6.7 A from the sulfonamide NH, with the dimethoxy-
˚
benzene ring (center of ring) ∼6.5 A from the other benzene
˚
˚
˚
˚
state conformation, 13 has its basic amine nitrogen 5.5 A
ring, 3.6 A from the amide carbonyl, and 6.4 A from the
sulfonamide NH. Considering 8-12, they all contain a basic
amine nitrogen, as a pyrrolidine, piperidine, piperidine/
piperazine, pyrrolidine, and piperidine/pyrrolidine, respec-
tively, and they all contain at least one aromatic ring that is
proximal to the amine center and nearby amide groups. Our
series needed the 3,4-dimethoxybenzene subunit for strong
antagonist potency, and this subunit is also present in 8;
however, it is absent from the series represented by 9-12.
from the isoindolinone benzene ring (center of ring), ∼9 A
˚
from the amide carbonyl, and ∼10 A from the dimethoxy-
benzene ring (center of ring), with the dimethoxybenzene
˚
ring spaced 4.5 A from the amide carbonyl and the two
˚
benzene rings (centers) separated by 6.5 A (Figure 2). Many
of these dimensions correspond to the dimensions in 7a
(disregarding the sulfonamide group).
On the basis of antagonist structures known through 2005,
Carotenuto et al. described two general pharmacophores in
terms of a basic amine linked to an aryl-1 and to an aryl-2
group, or an aryl-1 linked to an aryl-2 that is then linked to a
basic amine.16b Lescot et al. also developed more than
one antagonist pharmacophore, which suggests a lack of
convergence between the antagonist ligands that were con-
sidered.37 They advanced two different models comprised of
two aromatic rings, one hydrophobic group, a basic amine
center, and a hydrogen-bond acceptor, each with a different
spatial arrangement.37 Consequently, there would seem to be
general agreement that UT antagonist structures can be
achieved through more than one pharmacophore model.
Given that 13 has a fairly well-defined spatial geometry,
because of its conformational constraints,36 it serves to
define one type of UT antagonist pharmacophore.
˚
The basic center in 8 is 5.6 A from the dimethoxybenzene ring
˚
(center of ring), 8.4 A from the sulfonamide NH, and 6.0 A
˚
˚
from another aromatic ring. The basic center in 9 is 6.4 A
˚
from an aromatic ring (center of ring), 8.9 A from the bicyclic
˚
aromatic group in the opposite direction, and 4.5 A from the
urea carbonyl. There are two basic amine centers in 10,
˚
separated by ∼10 A. The benzoxazinone benzene ring is
˚
˚
5.4 A from the piperidine nitrogen and ∼15 A from the basic
˚
piperazine nitrogen; the benzamide benzene ring is 5.2 A
˚
from the piperidine nitrogen and 7.3 A from the piperazine
˚
amine nitrogen; the piperidine nitrogen is 4.5 A from the
˚
benzoxazine carbonyl and 5.7 A from the amide carbonyl;
˚
the piperazine amine is 5.0 A from the Boc carbonyl and
˚
4.5 A from the amide carbonyl. The basic center in 11 is 7.8 A
˚
˚
from the benzoxazolidinone benzene ring and ∼6 A from the
benzenesulfonyl ring; each amide carbonyl is 5.7, 5.8, and
˚
6.7 A from the amine center. The pyrrolidine nitrogen in 12 is
Conclusion
˚
7.3 A from the benzamide ring and 3.8 A from the benzyl
˚
We have discovered two related series of nonpeptide
U-II receptor antagonists based on piperazino-phthalimide
˚
ring; the piperidine nitrogen is 6.6 A from the benzyl ring and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7439
(5 and 6) and piperazino-isoindolinone (7) scaffolds. Com-
pound 7a was found to be an exceedingly potent U-II receptor
antagonist in vitro, and it also blocked the effects of U-II in
vivo in the rat ear-flushmodel. Structure types 6 and 7 are very
distinctive relative to the structure types for other nonpeptide
U-II receptor antagonist series, such as those represented by
gradient 10-90% (MeCN þ 0.16% TFA) in (water þ 0.2% TFA)
over 4 min, tR=3.38 min (98%). A 500 mL round-bottom flask
containing 4-hydroxy-2-(3,4-dimethoxybenzyl)-2,3-dihydro-1H-
isoindole-1,3-dione (5.90 g, 0.019 mol), CH2Cl2 (80 mL), and
Et3N (3.6 mL, 0.026 mol) was cooled using an ice-water bath. A
solution of trifluoromethanesulfonyl chloride (2.2 mL, 0.021 mol)
in CH2Cl2 (20 mL) was added dropwise via an addition funnel.
The mixture was then stirred for 1 h in an ice-water bath. The
mixture was then diluted with CH2Cl2 (200 mL) and washed with
1 N HCl (100 mL). The organic layer was dried using MgSO4,
filtered through celite, and concentrated in vacuo to give 8.20 g
(97%) of II as a yellow solid. 1H NMR δ 7.78-7.87 (m, 2 H), 7.53
(d, J = 8.3 Hz, 1 H), 6.99-7.02 (m, 2 H), 6.79 (d, J =
8.1 Hz, 1 H), 4.79 (s, 2 H), 3.87 (s, 3 H), 3.84 (s, 3 H). MS (ESþ)
446 (M þ 1). HPLC: Kromasil C18 column (50 mm ꢀ 2 mm;
3.5 μ), gradient 10-90% (MeCN þ 0.16% TFA) in (water þ
0.2% TFA) over 4 min, tR=4.14 min (96%).
General Synthesis of Compounds 2 and 3. 4-[4-(Benzyl)-
piperazin-1-yl]-2-(3,4-dimethoxybenzyl)-2,3-dihydro-1H-isoindole-
1,3-dione (3). A 10 mL sealed-tube reactor was charged with II
(0.50 g, 1.12 mmol), 1-Boc-piperazine (0.22 g, 1.18 mmol), toluene
(1.3 mL), and Et3N (0.2 mL, 1.43 mmol). The tube was sealed
under argon and heated to 110 °C for 18 h. The mixture was
cooled to 23 °C and purified via flash silica gel chromatography
(230-400 mesh silica gel 60, 80:20 hexanes:ethyl acetate) to give
0.33 g(61%) ofa yellowsolid. 1H NMR (300 MHz, MeOH) δ7.67
(overlapping dd, J=7.2 Hz, 1 H), 7.38 (d, J=7.2 Hz, 1 H), 7.28 (d,
J=8.2 Hz, 1 H), 6.99 (s, 1 H), 6.86-6.93 (m, 2 H), 4.71 (s, 2 H),
3.81 (s, 3 H), 3.79 (s, 3 H), 3.61-3.65 (m, 4 H), 3.27-3.31 (m, 4 H),
1.48 (s, 9 H). MS (ESþ) 482 (M þ 1). A 50 mL round-bottom
flask containing 4-[2-(3,4-dimethoxybenzyl)-1,3-dioxo-2,3-dihy-
dro-1H-isoindol-4-yl]-piperazine-1-carboxylic acid tert-butyl es-
ter (0.33 g, 0.69 mmol) and CH2Cl2 (3.0 mL) was treated with
TFA (0.7 mL) and the mixture was stirred at 23 °C for 1 h. The
mixture was concentrated in vacuo, and the crude oil was dis-
solved in THF (3.4 mL). Benzyl bromide (0.12 mL, 0.97 mmol)
and diethylisopropylamine (0.26 mL, 1.49 mmol) were added. The
mixture was heated at reflux for 24 h and evaporated. The crude
oil was purified on a Gilson HPLC with a reversed phaseKromasil
C18 column (gradient 80:20-0:100 H2O:MeCN) to give 369 mg
(85%) of 3 as a yellow solid. 1H NMR δ 7.60 (overlapping dd, J=
7.4 Hz, 1 H), 7.39-7.50 (m, 6 H), 7.12 (d, J = 8.2 Hz, 1 H), 6.90-
6.97 (m, 2 H), 6.37 (d, J=8.7 Hz, 1 H), 4.72 (s, 2 H), 4.27 (s, 2 H),
3.87 (s, 3 H), 3.83 (s, 3 H), 3.66-3.77 (m, 4 H), 3.41-3.48 (m, 2 H),
3.14-3.18 (m, 2 H). MS (ESþ) 472 (M þ 1). HPLC: Kromasil
C18 column (50 mm ꢀ 2 mm; 3.5 μ), gradient 10-90% (MeCN þ
0.16% TFA) in (water þ 0.2% TFA) over 4 min, tR=3.21 min
(99%).
General Synthesis of Compounds 4 and 5b-n. N-(2-Thio-
phenesulfonyl)-[4-(3,4-dimethoxyphenyl)]-4-{1,3-dioxo-4-[4-(1-
phenylethyl)piperazin-1-yl]-2,3-dihydro-1H-isoindol-2-yl}butyl)-
amide) (5j). A 500 mL round-bottom flask containing DMF
(207 mL) and 4-tert-butoxycarbonylaminobutyric acid (4.2 g,
20.6 mmol) was cooled using an ice-water bath. Triethylamine
(8.8 mL, 63.1 mmol) was added, followed by the benzotriazol-
1-yloxy-tris(dimethylamino)phosphonium (BOP) hexafluoro-
phosphate (10.0 g, 22.6 mmol) and N,O-dimethylhydroxylamine
hydrochloride (3.1 g, 31.8 mmol). The mixture was stirred at
room temperature for 24 h, diluted with CH2Cl2 (500 mL), and
transferred to a separatory funnel. The organic layer was washed
with 1 N HCl (300 mL) and water (2 ꢀ 300 mL). The organic
layer was dried using MgSO4, filtered through celite, concen-
trated in vacuo, and purified via flash chromatography (230-400
mesh silica gel 60, gradient 100:0-90:10 CH2Cl2:MeOH) to give
5.08 g of [3-(methoxymethylcarbamoyl)propyl]carbamic acid tert-
butyl ester as a white solid (91%). 1H NMR δ 3.65 (s, 3 H), 3.22-
3.21 (m, 2 H), 3.16 (s, 3 H), 2.45-2.54 (m, 2 H), 1.80-1.89 (m, 2 H),
1.42 (s, 9 H). HPLC: Kromasil C18 column (50 mm ꢀ 2 mm; 3.5 μ),
gradient 10-90% (MeCN þ 0.16% TFA) in (water þ 0.2% TFA)
over 4 min, tR = 2.74 min (97%). A 1 L round-bottom flask
8
26 and palosuran (9).17i Our series required the 3,4-dimethoxy-
benzene subunit for robust antagonist potency, and this
structural element is also present in 8. However, the series
represented by 9-12 do not possess this subunit. In any case,
potent UT antagonists contain at least one basic amine nitro-
gen and at least two benzenoid subunits, with one aromatic
ring being proximal to the amine center, as well as amide
groups near the amine center. Interestingly, such structural
elements are contained within the U-II agonist pharmaco-
phore, although with much different distances.3 At this junc-
ture, we can conclude that several pharmacophore models will
satisfy the requirements for U-II receptor binding and antago-
nist action.
Experimental Section
1
General Chemical Procedures. H NMR spectra were gener-
ally acquired at 300.14 MHz on a Bruker Avance-300 spectro-
meter in CDCl3 with Me4Si as an internal standard, unless
indicated otherwise. The 400-MHz 1H NMR spectra were
recorded with a Bruker AC-400 spectrometer with Me4Si as
an internal standard. Electrospray (ES) mass spectra were
obtained on a Micromass Platform LC single quadrupole mass
spectrometer in the positive mode. The structures of all new
1
compounds were consistent with their H NMR and ES-MS
mass spectra. Analytical HPLC analyses were performed on an
Agilent series 1100 HPLC instrument eluting with a gradient of
water/MeCN/TFA (10:90:0.2 to 90:10:0.2) over 4 min with a
flow rate of 0.75 mL/min on a Kromasil C18 column (50 mm ꢀ
2.0 mm; 3.5 μm particle size) or on a Supelcosil ABZþPlus
column (50 mm ꢀ 2.0 mm; 3.5 μm particle size) at 32 °C, with
signals being recorded simultaneously at 220 and 254 nm with a
diode-array detector. These HPLC analyses served as the prin-
cipal determinant of compound purity, which was >95% for
target compounds. The measured purity for target compounds
is given in parentheses throughout the Experimental Section.
Normal-phase preparative chromatography was performed on
an Isco Combiflash Separation System Sg 100c equipped with a
Biotage FLASH Si 40 M silica gel cartridge (KP-Sil Silica, 32-
˚
63 μ, 60 A; 4 cm ꢀ 15 cm) eluting at 35 mL/min with detection at
254 nm. Reversed-phase preparative chromatography was per-
formed on a Gilson HPLC with a reversed-phase Kromasil
˚
column (10 μ, 100 A C18, column length 250 mm ꢀ 50 mm).
Optical rotations were measured on a Perkin-Elmer 241 polari-
meter. Elemental analysis and Karl Fischer water analysis were
determined for certain specific compounds by Quantitative
Technologies Inc., Whitehouse, NJ.
Synthesis of 4-Hydroxy-2-(3,4-dimethoxybenzyl)-1,3-dioxo-2,3-
dihydro-1H-isoindol-4-yl Trifluoromethanesulfonic Acid Ester (II).
A 200 mL round-bottom flask was charged with 4-hydroxy-2,3-
dihydro-1H-isobenzofuran-1,3-dione (3.07 g, 0.019 mol) and dry
toluene (94 mL). Veratrylamine (2.8 mL, 0.019 mol) and triethyl-
amine (3.6 mL, 0.026 mol) were added to the mixture. A Dean-
Stark trap filled with 4A molecular sieves was attached to the flask
and the mixture was refluxed for 24 h. The mixture was cooled to
room temperature and diluted with CH2Cl2 (200 mL) and washed
with 1 N HCl (100 mL). The organic layers were combined and
dried using MgSO4, filtered through celite, and concentrated in
vacuo to give 5.6 g (96%) of a yellow solid. 1H NMR δ 7.70-7.86
(m, 1 H), 7.57 (overlapping dd, J=7.5 Hz, 1 H), 7.37 (d, J=7.3Hz,
1 H), 7.15 (d, J=8.4 Hz, 1 H), 6.91-7.01 (m, 2 H), 6.81 (d, J=
7.8 Hz, 1 H), 4.74 (s, 2 H), 3.88 (s, 3 H), 3.85 (s, 3 H). MS (ESþ)
314 (M þ 1). HPLC: Kromasil C18 column (50 mm ꢀ 2 mm; 3.5 μ),

7440 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lawson et al.
containing this material (5.09 g, 20.7 mmol) and tetrahydrofuran
(415 mL) was cooled by using an ice-water bath. A solution of 3,4-
dimethoxyphenylmagnesium bromide in THF (207 mL, 104 mmol)
was added dropwise via an addition funnel over 30 min. The
mixture was allowed to warm to room temperature and stirred
for 20 h. Water (150 mL) was added, and the mixture was
concentrated in vacuo, extracted with CH2Cl2 (600 mL), and
washed with water (2 ꢀ 300 mL). The organic layer was dried with
MgSO4, filtered through celite, concentrated in vacuo, and purified
via flash chromatography (230-400 mesh silica gel 60, gradient
90:10-50:50 hexanes:ethyl acetate) to give 4.0 g of [4-(3,4-
dimethoxyphenyl)-4-oxo-butyl]carbamic acid tert-butyl ester [V,
R1=(CH2)3NH-Boc], as a white solid (60%). 1H NMR δ 7.57 (d,
J=8.2 Hz, 1 H), 7.52 (s, 1 H), 6.88 (d, J=8.6 Hz, 1 H), 3.95 (s, 3 H),
3.92 (s, 3 H), 3.27-3.34 (m, 2 H), 2.97-3.02 (m, 2 H), 1.92-1.99
(m, 2 H), 1.42 (s, 9 H). HPLC: Kromasil C18 column (50 mm ꢀ
2mm;3.5μ), gradient 10-90% (MeCN þ 0.16% TFA) in (water þ
0.2% TFA) over 4 min, tR=3.48 min (98%). A 300 mL round-
bottom flask was charged with this material (4.0 g, 12.4 mmol),
ammonium acetate (9.5 g, 123.2 mmol), and methanol (42.0 mL).
Sodium cyanoborohydride (0.55 g, 8.8 mmol) was added, and the
mixture was heated at 40 °C for 22 h. The mixture was cooled to
room temperature, and 1 N NaOH (200 mL) was added. The
mixture was transferred to a separatory funnel and extracted with
CH2Cl2 (3 ꢀ 200 mL). The organic layer was dried with MgSO4,
filtered through celite, concentrated in vacuo, and purified via flash
chromatography (230-400 mesh silica gel 60, gradient 100:0-90:10
CH2Cl2:MeOH) to give 2.0 g of [4-amino-4-(3,4-dimethoxyphe-
nyl)butyl]carbamic acid tert-butyl ester [VI, R1=(CH2)3NH-Boc],
as a white solid (50%). 1H NMR δ 6.83-6.87 (m, 3 H), 4.53-4.60
(m, 1 H), 3.90 (s, 3 H), 3.87 (s, 3 H), 3.10-3.15 (m, 2 H), 1.63-1.71
(m, 5 H), 1.43 (s, 9 H). HPLC: Kromasil C18 column (50 mm ꢀ
2mm;3.5μ), gradient 10-90% (MeCN þ 0.16% TFA) in (water þ
0.2% TFA) over 4 min, tR=2.84 min (97%). To a 50 mL round-
bottom flask containing 4-hydroxy-2,3-dihydro-1H-isobenzofur-
an-1,3-dione (1.1 g, 6.70 mmol) and dry toluene (30 mL) was added
[4-amino-4-(3,4-dimethoxyphenyl)butyl]carbamic acid tert-butyl
ester (2.2 g, 6.79 mmol) and triethylamine (1.2 mL, 8.61 mmol).
A Dean-Stark trap filled with 4A molecular sieves was attached to
the flask and the mixture was refluxed for 24 h. The mixture was
cooled to room temperature, diluted with CH2Cl2 (200 mL), and
washed with 1 N HCl (100 mL). The organic layer was dried
(MgSO4), filtered through celite, and concentrated in vacuo to give
3.1 g (98%) of [4-(3,4-dimethoxyphenyl)-4-(4-hydroxy-1,3-dioxo-
2,3-dihydro-1H-isoindol-2-yl)butyl]carbamic acid tert-butyl ester
[VII, R1=(CH2)3NH-Boc], as a yellow solid. 1H NMRδ7.66-7.70
(m, 1 H), 7.52-7.57 (m, 1 H), 7.06-7.16 (m, 1 H), 6.79-6.88 (m,
3 H), 5.16-5.21 (m, 1 H), 3.88 (s, 3 H), 3.85 (s, 3 H), 3.06-3.19 (m,
1 H), 2.49-2.56 (m, 1 H), 2.26-2.33 (m, 1 H), 1.46-1.56 (m, 2 H),
1.42 (s, 9 H). HPLC: Kromasil C18 column (50 mm ꢀ 2 mm; 3.5 μ),
gradient 10-90% (MeCN þ 0.16% TFA) in (water þ 0.2% TFA)
over 4 min, tR=3.94 min (97%). A 500 mL round-bottom flask
containing this material (6.60 g, 0.014 mol), CH2Cl2 (60 mL), and
Et3N (2.3 mL, 0.017 mol) was cooled using an ice-water bath. A
solution of trifluoromethanesulfonyl chloride (1.6 mL, 0.015 mol)
in CH2Cl2 (10 mL) was added dropwise via an addition funnel. The
mixture was stirred for 1 h in an ice-water bath. The mixture was
diluted with CH2Cl2 (200 mL), washedwith1N HCl(100mL), and
saturated aqueous sodium bicarbonate (1 ꢀ 100 mL). The organic
layer was dried using MgSO4, filtered through celite, and concen-
trated in vacuo to give 7.06 g (84%) of triflate as a yellow solid. 1H
NMR δ7.76-7.83 (m, 2 H), 7.51 (d, J=7.8 Hz, 1 H), 7.07-7.12 (m,
2H), 6.80(d, J=8.0 Hz, 1 H), 5.22-5.27 (m, 1 H), 3.88 (s, 3 H), 3.85
(s, 3 H), 3.16-3.26 (m, 2 H), 2.42-2.59 (m, 1 H), 2.29-2.40 (m,
1 H), 1.55-1.66 (m, 2 H), 1.42 (s, 9 H). MS (ESþ) 603 (M þ 1).
HPLC: Kromasil C18 column (50 mm ꢀ 2 mm; 3.5 μ), gradient
10-90% (MeCN þ 0.16% TFA) in (water þ 0.2% TFA)
over 4 min, tR=4.72 min (96%). A 50 mL sealed-tube vessel was
charged with this material (7.53 g, 0.013 mol), 1-[(R)-1-phenyl-
ethyl]piperazine (2.50 g, 0.013 mol), toluene (13 mL), and Et3N (2.4
mL, 0.017 mol). The tube was sealed under argon and heated to 110
°C for 21 h. The mixture was cooled to room temperature and
purified via flash silica gel chromatography (230-400 mesh silica
gel 60, 80:20 hexanes:ethyl acetate) to give 4.61 g (57%) of (4-(3,
4-dimethoxyphenyl)-4-{1,3-dioxo-4-[4-(1-phenylethyl)piperazin-1-
yl]-2,3-dihydro-1H-isoindol-2-yl}butyl)carbamic acid tert-butyl es-
ter (5i) as a yellow solid. 1H NMR δ 7.48-7.54 (m, 1 H), 7.23-7.36
(m, 7 H), 7.07-7.11 (m, 2 H), 6.78 (d, J=8.2 Hz, 1 H), 5.18-5.23
(m, 1 H), 4.08-4.16 (m, 1 H), 3.87 (s, 3 H), 3.83 (s, 3 H), 3.26-3.45
(m, 4 H), 3.14-3.22 (m, 2 H), 2.72-2.76 (m, 2 H), 2.55-2.64 (m, 2
H), 2.44-2.52 (m, 1 H), 2.23-2.30 (m, 1 H), 1.38-1.54 (m, 14 H).
MS (ESþ) 643 (M þ 1). HPLC: Kromasil C18 column (50 mm ꢀ
2 mm; 3.5 μ), gradient 10-90% (MeCN þ 0.16% TFA) in (water þ
0.2% TFA) over 4 min, tR=3.31 min (97%). A 50 mL round-
bottom flask was charged with 5i (1.90 g, 2.95 mmol) and 1,4-
dioxane (15 mL). A solution of 4 N HCl in 1,4-dioxane (10 mL) was
added, and the mixture was stirred at room temperature for 4 h. The
mixture was concentrated in vacuo to give 5h. 1H NMR (300 MHz,
CD3OD) δ 7.63-7.68 (m, 1 H), 7.51-7.61 (m, 5 H), 7.41 (d, J=
7.2 Hz, 1 H), 7.28 (d, J=7.9 Hz, 1 H), 7.13 (s, 1 H), 7.04 (d, J=
6.5 Hz, 1 H), 6.89 (d, J=8.4 Hz, 1 H), 5.22-5.27 (m, 1 H), 4.46-
4.57 (m, 1 H), 3.82 (s, 3 H), 3.80 (s, 3 H), 3.47-3.66 (m, 4 H), 3.13-
3.26 (m, 2 H), 2.96-3.07 (m, 2 H), 2.57-2.67 (m, 1 H), 2.32-2.42
(m, 1 H), 1.80 (d, J=6.8 Hz, 1 H), 1.64-1.74 (m, 2 H), 1.29-1.35
(m, 2 H). MS (ESþ) 543 (M þ 1). HPLC: Kromasil C18 column
(50 mm ꢀ 2 mm; 3.5 μ), gradient 10-90% (MeCN þ 0.16% TFA)
in (water þ 0.2% TFA) over 4 min, tR=2.44 min (98%). The solid
(1.0 g, 1.63 mmol) was dissolved in CH2Cl2 (8.2 mL). Triethy-
lamine (0.7 mL, 5.02 mmol) was added followed by 2-thiophe-
nesulfonyl chloride (0.37 g, 2.03 mmol). The mixture was
stirred at room temperature for 24 h and concentrated in
vacuo. The crude oil was purified on a Gilson HPLC with a
reverse-phase Kromasil C18 column (gradient 80:20-0:100
H2O:MeCN). The purified material was dissolved in CH2Cl2
(50 mL), treated with 1 N HCl in diethyl ether (10 mL), and
concentrated in vacuo. This procedure was repeated two more
1
times to give 1.18 g (96%) of 5j as a yellow solid. H NMR δ
7.71-7.74 (m, 1 H), 7.51-7.61 (m, 3 H), 7.29-7.38 (m, 6 H),
7.02-7.18 (m, 3 H), 6.73-6.80 (m, 1 H), 5.10-5.17 (m, 1 H),
4.50-4.57 (m, 1 H), 3.86 (s, 3 H), 3.83 (s, 3 H), 3.31-3.39 (m,
4 H), 3.06-3.11 (m, 4 H), 2.65-2.74 (m, 2 H), 2.45-2.55 (m,
1 H), 2.20-2.31 (m, 1 H), 1.58-1.63 (m, 2 H), 1.49 (d, J=6.8 Hz,
3 H). MS (ESþ) 689 (M þ 1). HPLC: Kromasil C18 column
(50 mm ꢀ 2 mm; 3.5 μ), gradient 10-90% (MeCN þ 0.16%
TFA) in (water þ 0.2% TFA) over 4 min, tR=2.54 min (100%).
Synthesis of (R,S)-5a and (R,R)-5a. A 50 mL round-bottom
flask was charged with borane-dimethyl sulfide solution in
toluene (0.4 mL, 0.8 mmol) and toluene (6.0 mL). Octanol
(0.32 mL, 2.01 mmol) was added dropwise to the mixture, and
stirring was continued at 34 °C for 1 h. (S)-R,R-Diphenyl-2-
pyrrolidinemethanol (0.14 g, 0.55 mmol) was dissolved in
toluene (6.0 mL) and added to the mixture, followed by stirring
for 1 h at 34 °C. Borane-dimethyl sulfide solution in toluene (2.8
mL, 1.40 mmol) was added, followed by dropwise addition of a
solution of 3,4-dimethoxyacetophenone (1.0 g, 5.55 mmol) in
toluene (6.0 mL) over 1 h via addition funnel. The mixture
was cooled to room temperature and quenched with 1 N HCl
(20 mL). The mixture was transferred to a separatory funnel and
extracted with ethyl acetate (200 mL). The organic layer was
washed with sodium bicarbonate (50 mL) and sodium chloride
(50 mL). The organic layer was dried using MgSO4, filtered
through celite, and concentrated in vacuo to give 820 mg of (R)-
1-(3,4-dimethoxyphenyl)ethanol (X) as a white solid. 1H NMR
δ 6.82-6.95 (m, 3 H), 4.86 (q, J=3.3 Hz, 2 H), 3.90 (s, 3 H), 3.88
(s, 3 H), 1.83 (s, 1 H), 1.49 (d, J=6.4 Hz, 3 H); [R]25 þ42.0
D
(c 1.07, CHCl3). A 100 mL round-bottom flask containing
X (0.40 g, 2.20 mmol) and CH2Cl2 (11.0 mL) was cooled using
an ice-water bath. Triethylamine (0.37 mL, 2.65 mmol) was
added to the mixture, followed by the dropwise addition metha-
nesulfonyl chloride (0.19 mL, 2.45 mmol). The mixture was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7441
stirred for 4 h at 0 °C and then washed with 1 N HCl (5 mL). The
organic layer was dried with MgSO4 and filtered through celite.
Diallylamine (0.27 mL, 2.19 mmol) and 2,2,6,6-tetramethylpi-
peridine (0.82 mL, 4.83 mmol) were added to the crude CH2Cl2
solution. The mixture was refluxed for 24 h, cooled to room
temperature, and concentrated in vacuo. The crude oil was
purified via flash chromatography (230-400 mesh silica gel
60, gradient 100:0-98:2 CH2Cl2:MeOH) to give 0.4 g of di-
allyl-[1-(R)-(3,4-dimethoxyphenyl)ethyl]amine (XI) as a white
solid. 1H NMR δ 6.78-6.87 (m, 3 H), 5.78-5.91 (m, 2 H),
5.09-5.20 (m, 4 H), 4.18 (q, J=6.4 Hz, 2 H), 3.90 (m, 3 H), 3.86
(m, 3 H), 3.00-3.17 (m, 4 H), 1.45 (d, J=6.4 Hz, 3 H). A 50 mL
round-bottom flask was charged with XI (0.40 g, 1.53 mmol)
heated at 85 °C for 22 h. The mixture was cooled to room
temperature, and the sealed tube was opened carefully due to the
release of pressure from excess ammonia. The mixture was
concentrated in vacuo, diluted with 1 N HCl (300 mL) and
ethanol (150 mL), heated at reflux for 2 h, cooled to room
temperature, and washed with diethyl ether (1 ꢀ 500 mL). The
aqueous layer was basified with 3 N NaOH to pH > 10. It was
extracted by using CH2Cl2 (3 ꢀ 400 mL), and the organic
solution was dried (MgSO4), filtered through celite, and con-
centrated in vacuo to give 15.66 g of [4-(R)-amino-4-(3,4-
dimethoxyphenyl)butyl]carbamic acid benzyl ester XV as a
white solid. 1H NMR δ 7.30-7.34 (m, 5 H), 6.81-6.87 (m,
3 H), 5.08 (s, 2 H), 3.83-3.90 (m, 7 H), 3.16-3.22 (m, 2 H),
1.38-1.71 (m, 6 H). MS (ESþ) 359 (M þ 1). Daicel Chiralpak
AD-H, 4.6 mmꢀ15 cm, hexanes:2-propanol:Et2NH (86:14:0.1),
1.0 mL/min; (S)-enantiomer 13.57 min, (R)-enantiomer 15.67
min; 96% ee. A 500 mL round-bottom flask containing
XV (15.66 g, 0.044 mol) and CH2Cl2 (220 mL) was cooled using
an ice-water bath. Triethylamine (7.4 mL, 0.053 mol) was
added, followed by the dropwise addition of trifluoroacetic
anhydride (6.8 mL, 0.049 mol). After 3 h, the mixture was
extracted with CH2Cl2 (200 mL) and washed with 1 N HCl
(1 ꢀ 100 mL), 1 N NaOH (1 ꢀ 100 mL), and water (1 ꢀ 100 mL).
The organic layer was dried with MgSO4, filtered through celite,
and concentrated in vacuo to give 19.49 g (98%) of [4-(3,4-
dimethoxyphenyl)-4-(R)-(2,2,2-trifluoroacetylamino)butyl]car-
bamic acid benzyl ester as a white solid. 1H NMR δ 7.31-7.35
(m, 5 H), 6.80-6.89 (m, 3 H), 5.10 (s, 2 H), 4.81-4.93 (m, 1 H),
3.87 (overlapping s, 6 H), 3.13-3.28 (m, 2 H), 1.80-2.00 (m, 2
H), 1.42-1.59 (m, 2 H). MS (ESþ) 455 (M þ 1). HPLC:
Kromasil C18 column (50 mm ꢀ 2 mm; 3.5 μ), gradient 10-
90% (MeCN þ 0.16% TFA) in (water þ 0.2% TFA) over 4 min,
tR=3.82 min (96%). A 500 mL hydrogenation vessel was charge
with this material (19.49 g, 0.043 mol), ethyl acetate (80 mL),
ethanol (70 mL), 1 N HCl (20 mL), and 10% palladium on
carbon (2.0 g). The mixture was hydrogenated at 50 psig of
hydrogen for 24 h, filtered through celite, and concentrated
in vacuo to give 13.77 g (90%) of N-[4-amino-1-(R)-(3,4-
dimethoxyphenyl)butyl]-2,2,2-trifluoroacetamide as a white so-
lid. 1H NMR δ 7.29-7.33 (m, 5 H), 6.81-6.86 (m, 3 H), 5.07 (s,
2 H), 4.80-4.89 (m, 1 H), 3.86 (overlapping s, 6 H), 3.15-3.25
(m, 2 H), 1.35-1.64 (m, 6 H). MS (ESþ) 321 (M þ 1). A 500 mL
round-bottom flask containing this material (16.85 g,
0.047 mol), CH2Cl2 (220 mL), and triethylamine (14.0 mL,
0.10 mol) was cooled using an ice-water bath. Di-tert-butyl
dicarbonate (9.77 g, 0.045 mol) was added in one portion. The
mixture was stirred at room temperature for 18 h, diluted with
CH2Cl2 (300 mL), and washed with 1 N HCl (1 ꢀ 100 mL), 1 N
NaOH (1 ꢀ 100 mL), and water (1 ꢀ 100 mL). The organic layer
was dried (MgSO4), filtered through celite, and concentrated in
vacuo to give 12.78 g (64%) of [4-(3,4-dimethoxyphenyl)-
4-(R)-(2,2,2-trifluoroacetylamino)butyl]carbamic acid tert-bu-
tyl ester as a white solid. 1H NMR δ 6.81-6.85 (m, 2 H), 6.78 (s,
1 H), 4.86-4.94 (m, 1 H), 3.89 (s, 3 H), 3.87 (s, 1 H), 3.09-3.24
(m, 2 H), 1.82-2.00 (m, 2 H), 1.47-1.57 (m, 2 H), 1.44 (s, 9 H).
HPLC: Kromasil C18 column (50 mm ꢀ 2 mm; 3.5 μ), gradient
10-90% (MeCN þ 0.16% TFA) in (water þ 0.2% TFA) over
4 min, tR=3.95 min (99%). A 500 mL round-bottom flask was
charged with [4-(3,4-dimethoxyphenyl)-4-(R)-(2,2,2-trifluoro-
acetylamino)butyl]carbamic acid tert-butyl ester (12.78 g,
0.030 mol), tetrahydrofuran (150 mL), methanol (40 mL), and
3 N NaOH (30 mL). After 3 h, the mixture was diluted with
CH2Cl2 (500 mL) and washed with water (1 ꢀ 100 mL). The
organic layer was dried with MgSO4, filtered through celite, and
concentrated in vacuo. The crude material was purified via flash
silica gel chromatography (230-400 mesh silica gel 60, gradient
90:10-40:60 hexanes:ethyl acetate) to give 9.73 g (99%) of XVI
as a white solid. 1H NMR δ 6.87 (s, 1 H), 6.82-6.84 (m, 2 H),
3.84-3.90 (m, 7 H), 3.08-3.14 (m, 2 H), 1.62-1.71 (m, 4 H),
1.43 (s, 9 H). MS (ESþ) 325 (M þ 1). HPLC: Kromasil C18
and CH2Cl2 (8.0 mL). Pd2(dba)3 CHCl3 (0.16 g, 0.15 mmol),
3
triphenylphosphine (0.16 g, 6.1 mmol), and 1,3-dimethylbarbi-
turic acid (0.79 g, 5.06 mmol) were added, and the mixture was
heated at reflux for 3 h. The mixture was cooled to room
temperature, transferred to a separatory funnel, and extracted
with 1 N HCl (50 mL). The aqueous layer was basified with 3 N
NaOH and extracted with ethyl acetate (200 mL). The organic
layer was dried with MgSO4 and filtered through celite to give
1-(R)-(3,4-dimethoxyphenyl)ethylamine (XII) as a white solid.
1H NMR δ 6.74-6.85 (m, 3 H), 3.96-4.08 (m, 1 H), 3.83 (m,
3 H), 3.80 (m, 3 H), 1.87-1.97 (broad s, 2 H), 1.31 (d, J=6.6 Hz,
3 H). Compound (R,S)-5a was prepared by the general methods
for compounds 4 and 5b-n (vide supra) but employing enan-
tiomerically pure benzylamine XII (0.14 g). Compound (R,S)-5a
was isolated as a yellow solid. 1H NMR δ 7.35-7.54 (m, 7 H),
6.93-7.07 (m, 3 H), 6.73 (d, J=8.6 Hz, 1 H), 5.36 (q, J=7.2 Hz, 1
H), 4.21 (q, J=5.2 Hz, 1 H), 3.79 (s, 3 H), 3.77 (s, 3 H), 3.36-3.64
(m, 6 H), 3.79-3.16 (m, 2 H), 1.83 (d, J=7.0 Hz, 3 H), 1.79 (d, J=
7.4 Hz, 3 H). MS (ESþ) 500 (M þ 1); [R]25 þ13.8 (c 1.18,
D
CH3OH). HPLC: Kromasil C18 column (50 mm ꢀ 2 mm; 3.5 μ),
gradient 10-90% (MeCN þ 0.16% TFA) in (water þ 0.2%
TFA) over 4 min, tR=3.43 min (97%).
Synthesis of [(R)-4-Amino-4-(3,4-dimethoxyphenyl)butyl]car-
bamic Acid tert-Butyl Ester (XVI). A 1 L round-bottom flask
containing [3-(methoxymethylcarbamoyl)propyl]carbamic acid
benzyl ester (5.08 g, 20.7 mmol) and tetrahydrofuran (415 mL)
was cooled using an ice-water bath. A solution of 3,4-dime-
thoxyphenylmagnesium bromide in THF (207 mL, 104 mmol)
was added dropwise via addition funnel over 30 min. The
mixture was allowed to warm to room temperature and stirred
for 20 h. Water (150 mL) was added to the mixture and
concentrated in vacuo. The mixture was extracted with CH2Cl2
(600 mL) and washed with water (2 ꢀ 300 mL). The organic
layer was dried with MgSO4, filtered through celite, concen-
trated in vacuo, and purified via flash chromatography (230-
400 mesh silica gel 60, gradient 90:10-50:50 hexanes:ethyl
acetate) to give 4.0 g of [4-(3,4-dimethoxyphenyl)-4-oxo-bu-
tyl]carbamic acid benzyl ester as a white solid. 1H NMR
δ 7.57 (d, J=8.4 Hz, 1 H), 7.51 (s, 1 H), 7.29-7.44 (m, 5 H),
6.88 (d, J=8.4 Hz, 1 H), 5.08 (s, 2 H), 3.95 (s, 3 H), 3.93 (s, 3 H),
3.27-3.34 (m, 2 H), 2.97-3.02 (m, 2 H), 1.92-2.01 (m, 2 H). MS
(ESþ) 358 (M þ 1). HPLC: Kromasil C18 column (50 mm ꢀ
2 mm; 3.5 μ), gradient 10-90% (MeCN þ 0.16% TFA) in
(water þ 0.2% TFA) over 4 min, tR=3.62 min (98%). A 200 mL
Schlenk tube was charged with [RuCl2(benzene)]2 (2.0 g,
4.0 mmol) and (R)-Tol-BINAP (5.7 g, 8.4 mmol). The tube
was put under vacuum for 15 min and then backflushed with
argon. DMF (133 mL, degassed with argon) was added to the
tube, and the mixture was flushed with argon. The tube was
sealed off and heated to 100 °C for 10 min (with stirring). The
DMF was then removed under high vacuum at 70 °C to give
[((R)-Tol-BINAP)RuCl2(DMF)x] as a reddish/brown solid.38
A
200 mL sealed tube was charged with [4-(3,4-dimethoxyphenyl)-
4-oxo-butyl]carbamic acid benzyl ester (8.66 g, 24.2 mmol),
[((R)-Tol-BINAP)RuCl2(DMF)x] (2.1 g, 2.5 mmol), ammonium
formate (15.3 g, 243 mmol), and a 2.0 M solution of ammonia in
methanol (97 mL). The tube was flushed with argon, sealed, and

7442 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Lawson et al.
column (50 mm ꢀ 2 mm; 3.5 μ), gradient 10-90% (MeCN þ
0.16% TFA) in (water þ 0.2% TFA) over 4 min, tR=3.40 min
(98%).
g, 2.95 mmol) and 1,4-dioxane (15.0 mL). A solution of 4 N HCl
in 1,4-dioxane (10 mL) was added, and the mixture was stirred at
23 °C for 4 h. The mixture was concentrated in vacuo to give
solid (770 mg, 1.7 mmol), which was dissolved in CH2Cl2 (9 mL),
cooled to 5 °C, and treated with triethylamine (0.8 mL, 6 mmol)
and 2-thiophenesulfonyl chloride (365 mg, 2.0 mmol) for 1 h.
The reaction mixture was diluted with CH2Cl2 (200 mL), washed
with 1 N HCl (50 mL), washed with saturated sodium bicarbo-
nate (50 mL), dried (MgSO4), and concentrated in vacuo to give
an oil that was dissolved in CH2Cl2 (20 mL), treated with 1 N
anhydrous HCl in diethyl ether, and concentrated in vacuo
Synthesis of N-(2-Thiophenesulfonyl)-{4-(3,4-dimethoxyphenyl)-
4-[4-(4-ethylpiperazin-1-yl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]-
butyl}amide (7a). 3-Bromo-2-methylbenzoic acid (15.3 g,
71 mmol) was dissolved in methanol (75 mL) and CH2Cl2
(425 mL), cooled to -5 °C, and treated with 2 M trimethylsi-
lyldiazomethane in hexanes (100 mL, 200 mmol) dropwise from
an addition funnel. One hour after the addition of reagent, the
reaction was complete by LC/MS. Evaporation of volatiles
afforded methyl 3-bromo-2-methylbenzoate (16.6 g) as an oil.
1H NMR δ 7.71 (t, J=8 Hz, 2 H), 7.10 (d, J=8 Hz, 1 H), 3.90 (s,
3 H), 2.63 (s, 3 H). HPLC: Kromasil C18 column (50 mm ꢀ
2 mm; 3.5 μ), gradient 10-90% (MeCN þ 0.16% TFA) in (water
þ 0.2% TFA) over 4 min, tR=4.25 (98%). This ester (16.6 g,
71 mmol), N-bromosuccinimide (13.09 g, 74 mmol), and benzoyl
peroxide (0.56 g, 2.3 mmol) were dissolved in carbon tetrachlor-
ide (180 mL) and heated at 82 °C (bath) overnight. The reaction
was cooled to 23 °C, diluted with ethyl acetate (600 mL), washed
with water (300 mL), dried (MgSO4), filtered, and concentrated
in vacuo to afford XVIII as a solid (20.7 g, 95%), which was
stored under argon in a freezer. 1H NMR δ 7.89 (dd, J=7.8 and
1.3 Hz, 1 H), 7.76 (dd, J=8.0 and 1.3 Hz, 1 H), 7.23 (t, J=8.0 Hz,
1 H), 5.13 (s, 2 H), 3.96 (s, 3 H). HPLC: Kromasil C18 column
(50 mm ꢀ 2 mm; 3.5 μ), gradient 10-90% (MeCN þ 0.16%
TFA) in (water þ 0.2% TFA) over 4 min, tR=4.16 min (99%).
(R)-XVI (3.04 g, 8.5 mmol) and triethylamine (1.2 mL,
8.6 mmol) were combined in toluene (50 mL) and treated with
XVIII (2.4 g, 7.8 mmol) in toluene (100 mL) via an addition
funnel. The reaction mixture was stirred at 23 °C for 1 h, refluxed
for 5 h, and evaporated. Purification on silica gel with an
Analogix system (SF40-150 g, gradient elution with 0-2%
methanol in CH2Cl2) afforded XIX (3.9 g, 82%) as a solid. 1H
NMR δ 7.79 (d, J=8.0 Hz, 1 H), 7.39-7.49 (m, 2 H), 6.84-6.98
(m, 3 H), 5.53 (t, J=7.9 Hz, 1 H), 4.26 and 3.96 (pair of d, J=
17 Hz, 1 H each), 3.87 (s, 3 H), 3.85 (s, 3 H), 3.18-3.25 (m, 2 H),
2.08-2.16 (m, 2 H), 1.50-1.60 (m, 2 H), 1.42 (s, 9 H). LC/MS
(ESþ) m/z 520 (M þ 1). HPLC: Kromasil C18 column (50 mm ꢀ
2 mm; 3.5 μ), gradient 10-90% (MeCN þ 0.16% TFA) in
(water þ 0.2% TFA) over 4 min, tR=4.07 min (97%). Com-
pound XIX (3.30 g, 6.0 mmol) was suspended in dry toluene
(20 mL) under argon and treated with bis-(tri-tert-butylphos-
phine)palladium(0) (307 mg, 0.60 mmol), cetyl trimethylammo-
nium bromide (120 mg, 0.33 mmol), and N-ethylpiperazine
(2.75 g, 24 mmol). After adding 45% aqueous KOH solution
(1.12 g, 9 mmol), the reaction mixture was purged well with
argon, sealed with a Teflon screw cap, and heated to 105 °C
(bath) for 2 h. Analysis by LC/MS indicated 50% XX and 33%
des-bromo compound. The reaction mixture was cooled to
23 °C, decanted from solids, washed with additional toluene
(25 mL), and evaporated in vacuo to afford a brown oil, which
was dissolved in CH2Cl2 (200 mL), washed with saturated
sodium bicarbonate (50 mL), water (50 mL), and brine (50
mL), dried (Na2SO4), and concentrated in vacuo to obtain a
solid. The solid was dissolved in ethyl acetate, treated with silica
gel (10 g), evaporated, and loaded onto a prepared column of
silica gel (2 in. diameter, 200 g, 40:4:0.5 ethyl acetate/methanol/
ammonium hydroxide) and eluted with the same. The product-
containing fractions were evaporated, dissolved in ethyl acetate,
dried (Na2SO4), and evaporated to give XX as a foamy solid
(1.46 g, 44%). 1H NMR δ 7.51 (d, J=7.1 Hz, 1 H), 7.37-7.43 (m,
1 H), 7.08 (d, J=7.4 Hz, 1 H), 6.82-6.96 (m, 3 H), 5.53 (t, J=7.8
Hz, 1 H), 4.22 and 3.92 (pair of d, J=17 Hz, 1 H each), 3.87 (s, 3
H), 3.84 (s, 3 H), 3.18-3.24 (m, 2H), 3.05-3.12 (m, 4 H), 2.52-
2.61 (m, 4 H), 2.05-2.15 (m, 2 H), 1.51-1.63 (m, 2 H), 1.42 (s, 9
H). LC/MS (ESþ) m/z 553 (M þ 1). HPLC: Kromasil C18
column (50 mm ꢀ 2 mm; 3.5 μ), gradient 10-90% (MeCN þ
0.16% TFA) in (water þ 0.2% TFA) over 4 min, tR=3.15 min
(96%). A 50 mL round-bottom flask was charged with XX (1.63
1
overnight to afford 7a as a solid HCl salt (0.90 g, 88%). H
NMR δ 7.40-7.57 (m, 3 H), 7.37 (t, J=7 Hz, 1 H), 7.06 (d, J=
8 Hz, 1 H), 7.00 (t, J=4 Hz, 1 H), 6.80-6.92 (m, 3 H), 5.68 (broad
s, 1 H, NH), 5.49 (t, J=8 Hz, 1 H), 4.22 and 3.90 (pair of d, J=
17 Hz, 1 H each), 3.86 (s, 3 H), 3.82 (s, 3 H), 3.0 (m, 6 H), 2.61 (m,
4 H), 2.50 (m, 2 H), 2.16 (q, J=7 Hz, 2 H), 1.59 (m, 2 H), 1.12 (t,
J=7 Hz, 3 H). LC/MS (ESþ) m/z 599 (M þ 1). Anal. calcd for
C30H38N4O5S2 1.8HCl 0.5H2O: C, 53.51; H, 6.11; N, 8.32; Cl,
3
3
9.48; H2O, 1.34%. Found: C, 53.27; H, 6.04; N, 7.95; Cl, 9.44;
H2O, 1.46%. Compound 7a was determined to be 96% ee
[Daicel Chiralpak AD-H 25 cm ꢀ 0.46 cm, heptane:2-propa-
nol:Et2NH (70:30:0.1), 0.7 mL/min, tR=20.6 min; its enantio-
mer had tR=12.2 min].
N-(2-Thiophenecarbonyl)-{4-(3,4-dimethoxyphenyl)-4-[4-(4-
ethylpiperazin-1-yl)-1-oxo-2,3-dihydro-1H-isoindol-2-yl]butyl}-
amide (7c). The crude BOC-deprotected solid (30 mg, 0.044
mmol) mentioned above (from XX in preparation of 7a) was
dissolved in DMF (2 mL) under nitrogen and treated with
N-methylmorpholine (0.022 mL, 0.20 mmol), 1-methylimida-
zole-2-carboxylic acid (10 mg, 0.079 mmol), 1-hydroxybenzo-
triazole (HOBt; 4 mg, 0.03 mmol), O-benzotriazol-1-yl-N,
N,N0,N0-tetramethyluronium (HBTU) hexafluorophosphate
(30 mg, 0.079 mmol) at 23 °C overnight. The reaction mixture
was diluted with water (4 mL) and acetonitrile (3 mL), filtered,
and purified by reversed-phase HPLC [Phenomenex, Kromasil
˚
C18, 5 μ, 100 A, 100 mm ꢀ 21 mm, gradient elution with 10-
50% acetonitrile (0.16% TFA) in water (0.2% TFA)] to yield 7c
1
(39 mg, quantitative). H NMR δ 7.36-7.59 (m, 4 H), 6.81-
7.09 (m, 5 H), 5.46-5.51 (m, 1 H), 4.22 and 3.88 (pair of d, J=
17.9 and 17 Hz, 1 H each), 3.87 (s, 3 H), 3.84 (s, 3 H), 3.49-3.60
(m, 4 H), 3.21-3.37 (m, 5 H), 2.97-3.12 (m, 2 H), 2.17-2.28
(m, 2 H), 1.72-1.85 (m, 2 H), 1.52-1.58 (m, 3 H). MS (ESþ) 563
(M þ 1). Anal. calcd for C31H38N4O6S 2.1HCl 1.0H2O: C,
3
3
56.65; H, 6.46; N, 8.52; Cl, 11.33; H2O, 2.74. Found: C, 56.30;
H, 6.45; N, 8.34; Cl, 11.22; H2O, 2.57.
Calcium Flux Assay. A calcium mobilization assay based on
a Fluorescence Imaging Plate Reader (FLIPR; Molecular
Devices, Sunnyvale, CA) was used to determine the agonist
activity of the peptides in CHO cells transfected with rat GPR14
(U-II receptor).39 To derive these cells, the complete coding
sequence of rat U-II (Genbank Accession no. U32673) was
amplified by nested PCR from rat heart marathon-ready
cDNA. PCR was carried out by using the DNA polymerase
PFU (Stratagene) following conditions suggested by the manu-
facturer. The PCR products were cloned into pcDNA3
(Invitrogen) digested with EcoR I and Xba I. Clones containing
rat UT were verified by complete sequencing of the U-II
receptor insert to ensure a lack of PCR-introduced errors. The
constructed vector was transfected into CHO cells by using
lipofectamine (GIBCO BRL). CHO cells with high expression of
rat UT (determined by response to U-II in FLIPR-based
calcium mobilization) were selected and established as stable
cell lines by using G418. CHO cells were seeded at 40000 cells per
well into 96-well, black-wall, clear-bottom microtiter plates 24 h
before running the assay. Cells in culture media (DMEM/F12
containing 15 mM HEPES, L-glutamine, pyridoxine hydro-
chloride, 10% fetal bovine serum, 1 mg/mL G418 sulfate,
antibiotic/antimycotic, pH 7.4) were loaded with proprietary
dye, from the FLIPR calcium assay kit (Molecular Devices),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7443
prepared in assay buffer (Hanks Balanced Salts Solution,
20 mM HEPES, 0.1% bovine serum albumin (BSA), 2.5 mM
probenecid, pH 7.4), and incubated for 1 h at 37 °C. Calcium
mobilization determinations were performed at 23 °C. EC50
values were calculated relative to the maximum calcium iono-
phore (A23187) response at 10 μM. Antagonist activity
of inactive compounds was assessed, after a 10 min of incuba-
tion, in response to an agonist, such as goby U-II (0.1 nM) or
peptide 1 (0.3 or 1 nM). The use of rat GPR14 and the goby
peptide was considered acceptable because human and goby
U-II have similar affinity for human or rat GPR14 in the
transfected cells.6
(n=2/test compound) until a stable response was obtained or for
a maximum of 30 min. The degree of contraction was expressed
as a percentage of the KCl-induced maximal contraction.
^
ꢀ
Acknowledgment. We thank Benoit Liegault for initial
development of the palladium-catalyzed aryl amination reac-
tion and Andrew Mahan for chiral HPLC analyses.
Supporting Information Available: Characterization data
for compounds 2, (R)-4, (S)-4, 5a-g, 5k-m, (R,R)-5a, (R)-5j,
(S)-5j, 6a-j, 7b, 7d-o. Energy-minimized structures for 7a and
8-12. This material is available free of charge via the Internet
at http://pubs.acs.org.
Radioligand Binding Assay.24 CHO cells stably expressing rat
GPR1439 (UT) were harvested and washed with ice-cold phos-
phate buffered saline (PBS, pH 7.4) and resuspended in 20 mM
Tris-HCl (pH 7.4), 250 mM sucrose, and 1 mM ethyleneglycol
tetraacetic acid. Cells were homogenized in a glass-Teflon
homogenizer and, after centrifugation at 1000g for 15 min, the
resulting supernatant was obtained and recentrifuged at 30000g
for 60 min at 4 °C. The resulting pellet was passed through a
25 gauge needle five times, and membranes were stored at -80 °C.
Membranes were precoupled to wheat germ agglutinin-coated
scintillation-proximity assay (SPA) beads (Amersham Pharma-
cia Biotech, England, UK) at the concentration of 15 μg
membrane/mg beads. The coupling was carried out for 30 min
at room temperature in buffer A (50 mM Tris-HCl, 5 mM
MgCl2, and 0.1% BSA adjusted to pH 7.4) with continual shak-
ing. After a brief spin, the supernatant was discarded. The assay
was conducted in 96-well microtiter plates, with a final concen-
tration of 1.0 mg of precoupled beads per well, 0.17 nM
monoiododinated 125I-[Tyr]-rat U-II (Phoenix Pharmaceuti-
cals, Belmont, CA), and various concentrations of compounds
in a total volume of 100 μL buffer A. The plate was shaken for
3 h at 22 °C, and after a brief spin, the radioactivity was counted
by using a Topcount instrument (Packard Bioscience Company,
Meriden, CT). Nonspecific binding, determined using 1 μM rat
U-II, was always less than 15% of the total binding. The
GraphPad Prism software package (GraphPad Software, Inc.,
San Diego, CA) was used to analyze the binding data, and Ki
values were calculated (n=2) by the Cheng-Prussoff equation.
Functional Assay in Rat Aortic Rings.27 Selected compounds
were tested for contractile effects on rat thoracic aorta smooth
muscle at a concentration of 30 nM. The response (n=2) was
measured relative to that produced by 80 mM KCl. These
studies were conducted by CEREP (Celle L’Evescault, France).
The aortas were obtained from male Wistar rats (250 g; CERJ,
Le Genest St. Isle, France). The animals were sacrificed by
cervical dislocation followed by exsanguination, and the aortas
were quickly removed, cleaned, and dissected into rings. The
rings were suspended between two stainless steel hooks in organ
baths containing 20 mL of an oxygenated and prewarmed
physiological salt solution. They were connected to force-dis-
placement transducers (UF1, Phymep) for isometric tension
recordings. Data were continuously recorded on a multichannel
data acquisition system. The physiological salt solution con-
tained the following (mM): NaCl, 118.0; KCl, 4.7; MgSO4, 1.2;
CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.0. The
high-Kþ (80 mM) solutions were prepared by equimolar repla-
cement of NaCl with KCl. These solutions were continuously
aerated with a 95% O2/5% CO2 gas mixture and maintained
at pH 7.4 and 37 °C. Goby U-II (Palomar Research, Inc., cat.
no. U4832, lot no. A34001-298M) and test compounds were
freshly prepared in stock solutions by using physiological
buffer. The aortic rings were stretched to an optimal resting
tension and then allowed to equilibrate for at least 30 min,
during which time they were washed repeatedly. Each ring was
contracted using a high KCl solution (80 mM) to verify respon-
siveness and to obtain a maximal contractile response. The
rings were again washed until resting tension returned to base-
line and then they were exposed to 30 nM of the test compound
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(29) Cerep (Seattle, WA; www.cerep.com), an independent contract
laboratory, performed standardized binding assays on receptors
and ion channels. Each results was expressed as percent inhibition
of a control specific binding (mean values, n=2; h, human).
(30) Receptors and channels, <50% inhibition at 10 μM: adenosine A1
(h), A2A (h), A3 (h); adrenergic R-2, β (h); angiotensin AT1 (h);
central benzodiazepine; bradykinin B2 (h); cholecytokinin CCK-A
(h); dopamine D1 (h), D2S (h); endothelin ET-A (h); γ-aminobu-
tyric acid (GABA); galanin GAL2 (h); CXCR2 (h); CCR1 (h);
histamine H1 (h), H2 (h); melanocortin MC4 (h); melatonin MT1
(h); muscarinic M2; neuokinin NK2 (h), NK3 (h); neuropeptide Y
NPY1 (h), NPY2 (h); neurotensin NT1 (h); opioid δ2 (h), κ, μ (h);
ORL1 (NOP) (h); serotonin 5HT2A (h), 5-HT3 (h), 5-HT-5A (h),
5-HT6 (h), 5-HT7 (h); somatostatin; vasoactive intestinal peptide
VIP (h); vasopressin V1a (h); L, SK Ca2þ channels; Kþ channel;
Cl- channel; norepinephrine transporter; dopamine transporter.

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