Synthesis, structural activity-relationships, and biological evaluation of novel amide-based allosteric binding site antagonists in NR1A/NR2B N-methyl-d-aspartate receptors

Article abstract of DOI:10.1016/j.bmc.2009.05.085

The synthesis and structure-activity relationship analysis of a novel class of amide-based biaryl NR2B-selective NMDA receptor antagonists are presented. Some of the studied compounds are potent, selective, non-competitive, and voltage-independent antagonists of NR2B-containing NMDA receptors. Like the founding member of this class of antagonists (ifenprodil), several interesting compounds of the series bind to the amino terminal domain of the NR2B subunit to inhibit function. Analogue potency is modulated by linker length, flexibility, and hydrogen bonding opportunities. However, unlike previously described classes of NR2B-selective NMDA antagonists that exhibit off-target activity at a variety of monoamine receptors, the compounds described herein show much diminished effects against the hERG channel and α₁-adrenergic receptors. Selections of the compounds discussed have acceptable half-lives in vivo and are predicted to permeate the blood-brain barrier. These data together suggest that masking charged atoms on the linker region of NR2B-selective antagonists can decrease undesirable side effects while still maintaining on-target potency.

Full text of DOI:10.1016/j.bmc.2009.05.085

Bioorganic & Medicinal Chemistry 17 (2009) 6463–6480
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structural activity-relationships, and biological evaluation of novel
amide-based allosteric binding site antagonists in NR1A/NR2B
N-methyl-D-aspartate receptors q
Cara A. Mosley a, Scott J. Myers b, Ernest E. Murray a, Rose Santangelo a, Yesim A. Tahirovic a,
Natalie Kurtkaya b, Praseeda Mullasseril b, Hongjie Yuan b, Polina Lyuboslavsky b, Phuong Le b,
a,
Lawrence J. Wilson a, Manuel Yepes c, Ray Dingledine b, Stephen F. Traynelis b, , Dennis C. Liotta
*
*
a Department of Chemistry, Emory University, Atlanta, GA, USA
b Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA
c Department of Neurology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and structure–activity relationship analysis of a novel class of amide-based biaryl NR2B-
selective NMDA receptor antagonists are presented. Some of the studied compounds are potent, selective,
non-competitive, and voltage-independent antagonists of NR2B-containing NMDA receptors. Like the
founding member of this class of antagonists (ifenprodil), several interesting compounds of the series
bind to the amino terminal domain of the NR2B subunit to inhibit function. Analogue potency is modu-
lated by linker length, flexibility, and hydrogen bonding opportunities. However, unlike previously
described classes of NR2B-selective NMDA antagonists that exhibit off-target activity at a variety of
monoamine receptors, the compounds described herein show much diminished effects against the hERG
Received 13 March 2009
Revised 20 May 2009
Accepted 22 May 2009
Available online 5 July 2009
Keywords:
NMDA
GluN2B
NR2B-selective antagonists
Neuroprotection
channel and
a1-adrenergic receptors. Selections of the compounds discussed have acceptable half-lives
in vivo and are predicted to permeate the blood–brain barrier. These data together suggest that masking
charged atoms on the linker region of NR2B-selective antagonists can decrease undesirable side effects
while still maintaining on-target potency.
Ó 2009 Published by Elsevier Ltd.
1. Introduction
ease,9–12 Huntington’s disease,13–16 Parkinson’s disease,17–19
depression,20 and neuropathic pain.21–24
Ionotropic glutamate receptors mediate excitatory synaptic
transmission in the central nervous system, and can be divided
into three categories based on pharmacology and amino acid se-
Structurally, NMDA receptors are heterooligomeric ligand-
gated cation channels containing two glycine-binding NR1 sub-
units, which exist as eight different RNA splice variants, and two
glutamate-binding NR2 subunits. Simultaneous binding of the co-
agonists glutamate and glycine leads to opening of the receptor
channel, and subsequent influx of cations, including Ca2+. The
resulting membrane depolarization contributes to the propagation
of the excitatory signal, and the influx of Ca2+ can trigger intracel-
lular signaling pathways.
There are numerous sites on the protein that could be pharma-
cologically exploited to modulate NMDA receptor function. For
example, organic cations have been developed as selective blockers
that bind within the ion conduction path in NMDA receptors (e.g.,
MK-801, 1, Fig. 1).25 In addition, competitive antagonists that bind
to the co-agonist glycine site (Licostinel, 2)26 or glutamate site
have been developed (Selfotel, 3).27,28 A number of subtype-selec-
tive non-competitive antagonists that bind to the amino terminal
domain have been developed and potently inhibit receptors con-
taining the NR2B subunit such as the negative allosteric modula-
tors ifenprodil29,30 (4) and CP-101,606 (5).31–36 Initial clinical
quence, which include N-methyl-D-aspartate (NMDA), kainate,
and amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)
receptors.1–3 NMDA receptors are involved in neuronal develop-
ment, learning, motor function, and pain transmission. Excessive
activation of NMDA receptors can lead to neuronal death, and thus
NMDA receptor over-activation has been proposed to occur in con-
ditions in which extracellular glutamate is elevated, such as ische-
mic stroke and traumatic brain injury.4–8 In addition, it has also
been proposed that NMDA receptors play a role in Alzheimer’s dis-
q
Several of the authors (C.A.M., Y.A.T., L.J.W., S.J.M., R.D., S.F.T., D.C.L.) are
inventors of Emory University owned patent-pending technology associated with
these compounds, or have an equity position in companies actively seeking to
license these compounds (D.C.L., S.F.T., R.D.).
* Corresponding authors. Tel.: +1 404 727 1375 (S.F.T.); +1 404 727 6602 (D.C.L.).
E-mail addresses: strayne@emory.edu (S.F. Traynelis), dliotta@emory.edu (D.C.
Liotta).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.05.085

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C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
Figure 1. The structures are shown for six classes of NMDA receptor antagonists.
trials of competitive NMDA receptor blockers and channel blockers
as neuroprotectants failed for several reasons including unfavor-
able side effects, which led to lowering of the dose below
efficacious levels. In addition, the rapidly developing neurodegen-
eration after cerebral ischemia made it problematic to administer
the compounds soon enough after the insult to prevent NMDA
receptor-mediated cell death.37 To date only one NMDA receptor
antagonist, the channel blocker memantine hydrochloride (6,
Namenda Ó), has been approved by the FDA for the treatment of
Alzheimer’s disease.38,39
A number of lines of evidence suggest that NR2B subtype-selec-
tive blockade of NMDA receptors can be beneficial in cerebral
ischemia, epilepsy, Parkinson’s disease, depression, and perhaps
Alzheimer’s disease.20,40–42 In addition, mice over-expressing
NR2B-containing receptors show an increased sensitivity to pain,
and NR2B-selective antagonists are antinociceptive in certain mod-
els of pain.24,43 While NR1 is ubiquitously expressed in the CNS,
NR2B-containing receptors are localized in the forebrain, dorsal
root ganglion, striatum, and the spinal cord.44–46 The differential
distribution of NR2 receptor subtypes17 has supported the hypoth-
esis that targeting specific NMDA receptor subunits, such as NR2B-
containing receptors, will decrease untoward side-effects previ-
ously observed in clinical trials of non-selective NMDA receptor
antagonists.
most potent compounds contain a 3,4-dichlorophenyl A ring and
para-methanesulfonamide B ring.50 The optimal linker in the series
was seven atoms, and a basic amine was required for activity. Com-
pound 8 in that study showed excellent selectivity for NR2B-con-
taining heterodimeric NR1/NR2 NMDA receptors and high
potency in vitro and neuroprotective properties in an in vivo model
of transient focal ischemia. Compound 8 did exhibit off-target
monoamine receptor activity including the a1-adrenergic receptor
and the human-ether-a-go-go (hERG) encoded cardiac potassium
channel. The hERG channel is important in ventricular repolariza-
tion, and channel blockage can lead to an increased QT interval
and potentially deadly ventricular arrhythmias and represents an
off-target limitation addressed through SAR studies for a diverse
set of chemical structures.51,52 Eliminating hERG activity, and
interactions with other polyamine binding receptors including a1
and sigma receptors, has been a significant hurdle in the develop-
ment of NR2B-selective NMDA receptor antagonists.
Recently we explored the structural features of a novel NR2B-
selective antagonist that was identified while screening a focused
library of biaryl compounds (compound 9). This biaryl compound
contains a thiosemicarbazide functionality within a seven-atom
linker that would be non-ionizable under physiological conditions.
We hypothesized that this structural feature would lead to de-
creased activity at off-target receptors compared to the propanol-
amines on which we previously worked. Here we describe the
synthesis and SAR evaluation of compounds with various non-ion-
izable linker regions between the A and B rings of scaffold 7. Our
goal in this study was to achieve selective, potent, and safe com-
pounds through manipulation of the seven-atom linker and incor-
poration of various amides.
Comprehensive SAR studies of NR2B amino terminal domain
(ATD) allosteric binding site antagonists suggest that one family
of compounds (7, Fig. 2) contains a non-polar A ring connected
via a basic amine linker of 9–11 Å to the B ring, which traditionally
contains a hydrogen bond donor substituent.47–49 Previous studies
in our lab involving enantiomeric propanolamines indicated the
Figure 2. Schematic of the NMDA receptor antagonist pharmacophore (7) compared to an NR2B-selective enantiomeric propanolamine (8) and an NR2B-selective screening
hit (9).

C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6465
29–31 were prepared by conversion to the aniline methyl ester
(25, 26), formation of the para-arylmethanesulfonamide ester
(27, 28), and saponification to give the desired carboxylic acids
(Scheme 3).
2. Chemistry
A series of hydrazide derivatives were prepared from the com-
mon starting material fragment propane hydrazide 12. Originating
from para-aminophenylpropionic acid (10, Scheme 1), initial con-
version of the carboxylic acid to methyl ester 11 was achieved
using methanol and thionyl chloride. The ester was treated with
methanesulfonyl chloride to yield the para-arylmethanesulfona-
mide, and finally converted to 13 via hydrazinolysis.53 Thiosemi-
carbazide analogue 14 was formed by reaction of 13 with 3,4-
dichlorophenylisothiocyanate, while the use of the corresponding
isocyanate gave semicarbazide 15. Bishydrazides 16 and 17 were
synthesized using carbodiimide-mediated coupling between 13
and the corresponding carboxylic acid. Phthalamide derivative 18
was prepared via reaction of 13 with 3,4-dichlorophenylmalimide.
Mesylate 21 was obtained from lithium aluminum hydride reduc-
tion of 3,4-dichlorophenylacetic acid (19) and treatment of the
resultant alcohol 20 with methanesulfonyl chloride. The arylethyl
propane hydrazide 22 was obtained by displacement of mesylate
21 by hydrazide 13 (Scheme 2).
Various 3,4-dichlorophenyl alkyl amines were synthesized for
subsequent amide bond formation (Scheme 4). The 3,4-dichloro-
phenyl-1,2-ethanediamine and the corresponding propyldiamine
linkers 33 and 34 were prepared by refluxing excess 3,4-dichloro-
aniline (32) with the corresponding bromoalkyl hydrobromide.54
Mitsunobu conditions with 3,4-dichlorophenol (35) and N-Boc-
ethanolamine followed by TFA deprotection gave amine 37.55 The
sulfur-containing fragment, 3,4-dichlorophenylthioethanamine
(40), was generated by ring opening of 2-oxazolidinone (39) with
3,4-dichlorothiophenol (38) followed by decarboxylation.56 Lith-
ium aluminum hydride reduction of propionamide 42 gave 3,4-
dichlorophenylpropionamine (43). Finally, the arylcinnamylamine
derivative 48 was formed via Heck reaction of allyl phthalamide
(45) and 3,4-dichloroiodobenzene (46) followed by cleavage with
hydrazine.57
Amide 67 was prepared as illustrated in Scheme 5. Mitsunobu
conditions with para-nitrophenol (61) and N-Boc-hydroxyethylcar-
bamate gave 62. The nitro group was hydrogenolized to give ani-
line 63, followed by reaction with methanesulfonyl chloride to
Amides 49–60 (Table 1) were prepared by EDCI-mediated cou-
pling between acids of type 29–31 and amines 33, 34, 37, 40, 43, 48
and commercially available amines. The common carboxylic acids
Scheme 1. Reagents and conditions: (a) SOCl2, MeOH, ꢀ10 °C to rt; (b) MsCl, DCM, pyridine, 0 °C to rt; (c) NHNH2ꢁH2O, MeOH, reflux; (d) 3,4-dichlorophenylisothiocyanate or
3,4-dichlorophenylisocyanate, DMF, rt; (e) EDCI, 3,4-dichlorophenylacetic acid, DMAP, DMF 0 °C to rt; (f) 3,4-dichlorobenzoic acid, DCC, DMF, 0 °C to rt; (g) 5,6-
dichloroisobenzofuran-1,3-dione, DMF, reflux.
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, reflux to rt; (b) pyridine, MsCl, 0 °C to rt; (c) 13, MeOH, reflux.

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C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
Table 1
Synthesis of amides 49–60a
Amine
n =
Acid Z
C
Structure
ID
H
N
O
S
O
Cl
Cl
H
N
33
1
49
N
H
O
O
H
N
O
S
O
Cl
H
N
48
1
1
1
1
1
1
1
C
50
51
52
53
54
55
56
Cl
Cl
Cl
H
N
O
S
O
N
1-(3,4-Dichlorophenyl) piperazine
C
N
O
H
O
S
O
Cl
Cl
N
H
33
48
43
37
40
O
O
O
O
O
N
N
O
H
O
H
O
S
O
Cl
Cl
N
H
N
O
O
H
O
S
O
Cl
Cl
N
H
N
O
O
H
O
S
O
Cl
Cl
N
H
N
O
O
O
H
N
O
S
O
Cl
Cl
H
N
S
O
O
Cl
Cl
H
N
O
S
O
N
1-(3,4-Dichlorophenyl) piperazine
3,4-Dichlorophenethylamine
34
1
2
1
1
O
C
O
C
57
58
59
60
N
O
O
O
H
N
O
S
O
Cl
Cl
Cl
Cl
N
H
O
O
O
N
H
N
H
O
S
O
N
H
Cl
Cl
N
H
N
H
O
S
O
34
N
H
a
All molecules were synthesized using EDCI, DMAP, DMF. Acid Z and n refer to atomic detail of compounds 29–31.

C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6467
Scheme 3. Reagents and conditions: (a) SOCl2, MeOH, ꢀ10 °C to rt; (b) H2, 5% Pd/C, MeOH, rt; (c) pyridine, MsCl, 0 °C to rt; (d) 1.0 M NaOH, MeOH, rt.
yield para-arylmethanesulfonamide 64. Deprotection with trifluo-
roacetic acid and formation of the hydrochloride salt afforded com-
pound 65. Acylation of 3,4-dichloroaniline (32) with chloroacetyl
chloride gave 66 which was combined with 65 to yield 67.
Borane reduction of amide 49 resulted in ethanediamine deriv-
ative 68. Subsequent cyclization with N,N0-carbonyldiimidazole
gave imidazolidinone 70. Analogue 71 was prepared in a similar
fashion via amide 52 (Scheme 6).
Scheme 4. Reagents and conditions: (a) bromoethylamine hydrobromide or bromopropylamine HBr, toluene, reflux; (b) N-Boc-aminoethanol, PPh3, DIAD, THF, 0 °C to rt; (c)
TFA, THF, rt; (d) 130 °C; (e) oxalyl choride, THF, DMF, rt; (f) Et2O, NH3 in 1,4-dioxane, rt; (g) LiAlH4, THF, 0 °C to rt; (h) allyl bromide, DMF, 50 °C; (i) 3,4-dichloroiodobenzene
(46), Pd(OAc)2, Et3N, MeCN, 100 °C; (j) NH2NH2ꢁH2O, EtOH, reflux.

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C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
Scheme 5. Reagents and conditions: (a) N-Boc-ethanolamine, PPh3, DIAD, THF, 0 °C to rt; (b) H2, 5% Pd/C, MeOH, rt; (c) MsCl, DIPEA, DCM, 0 °C to rt; (d) TFA, DCM, rt; (e)
chloroacetyl chloride, Et3N, DCM, 0 °C to rt; (f) 65, Et3N THF, reflux.
We subsequently determined the concentration–effect curve
for all compounds against recombinant human NR1/NR2B NMDA
receptors. The concentrations that produced half-maximally effec-
tive inhibition (IC50) for all compounds are presented below in Ta-
ble 3. While screening hit 9 (rat IC50 = 600 nM) contained a
phenolic B ring, our previous studies50 focused on para-arylme-
thanesulfonamide B ring substitution. The hybrid thiosemicarba-
zide structure 14, which replaces the phenol functionality with a
sulfonamide, exhibits increased potency (IC50 of 270 nM), consis-
tent with our previous work with the propanolamine series.
Changing the sulfur to an oxygen, as in semicarbazide 15, signifi-
cantly enhanced potency (IC50 of 28 nM). By contrast, replacement
of the aniline nitrogen with a carbon, as in bishydrazide 16 and
monohydrazide 22, leads to significantly lowered IC50 values
(IC50 5750, 4810 nM, respectively). Rigidifying the A ring into a lar-
ger ring system, such as 18, also decreased potency (IC50
41,800 nM). Compound 17, with a shortened linker (six atoms),
was the only moderately active hydrazide analogue (IC50 521 nM).
Hydrazides are known to lie perpendicular in space at the N-N
bond,58 and further incorporation of the above analogues into the
semicarbazide functionality insures limited bond rotation and flex-
ibility which may be crucial for binding. Therefore, the key aniline
nitrogen required for potency of semicarbazide 15 was incorpo-
rated into various amides. We postulated that reducing chain rigid-
ity and increasing hydrophilicity would lead to potent compounds
3. Results and discussion
3.1. Structure–activity-relationships
The potency and selectivity of all new compounds synthesized
were evaluated using two electrode voltage clamp analysis of the
effect of compounds on recombinant NMDA receptor function.
We first screened the effect of 3 lM of each compound against cur-
rent responses produced by maximally effective concentrations of
the co-agonists glutamate and glycine at rat NR1/NR2A, NR1/NR2B,
NR1/NR2C, NR1/NR2D as well as representative members of the
AMPA-selective class of glutamate receptors (GluR1) and the kai-
nate-selective glutamate receptor class (GluR6, Table 2). Com-
pounds that produced response percentages less than 50% had
marked inhibition of current responses. The data clearly show that
most responses for all compounds in every receptor (NR1/NR2A,
NR1/NR2C, NR1/NR2D, homomeric GluR1, homomeric GluR6) but
the NR1/NR2B sub-type showed little to no effects with percent-
ages greater than 90% in most cases. Furthermore, all but three
compounds (16, 18, and 58) had less than 50% responses against
the NR1/NR2B sub-type. This initial data clearly shows these com-
pounds in general, had minimal effects (<10% change from control)
at receptors comprised of NR1/NR2A, NR1/NR2C, NR1/NR2D,
homomeric GluR1, homomeric GluR6, suggesting high NR2B-selec-
tivity for this class of compounds.
Scheme 6. Reagents and conditions: (a) LiAlH4, THF, 0 °C to rt; (b) CDI, THF, rt.

C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6469
Table 2
In vitro analysis of subunit selectivity
Compound
NR1/NR2A % response
NR1/NR2B % response
NR1/NR2C % response
NR1/NR2D % response
GluR1 % response
GluR6 % response
14
16
15
22
17
18
49
51
50
58
68
52
70
69
57
54
55
56
71
67
53
59
60
104 6.5
104 7.1
101 3.9
106 3.7
102 4.1
101 1.6
99 2.9
103 4.6
99 4.4
99 2.1
89 2.3*
92 1.5*
96 2.2
91 1.4*
89 2.7
99 3.3
97 4.0
96 2.2
105 2.6
108 2.5*
100 2.7
106 2.6
96 5.2
12 1.2*
94 1.0
91 4.4
96 3.1
97 0.9
97 1.4
102 2.0
101 0.9
100 1.8
95 2.3
102 0.7
104 3.1
98 2.4
97 2.0
100 1.2
94 2.3*
96 1.4
96 2.1
95 2.2
98 1.7
94 2.9
95 2.7
97 2.2
90 2.2
92 0.7
94 1.8
91 1.3
95 2.9
96 0.7
93 4.8
84 3.4*
97 1.9
88 2.7*
89 2.4*
91 2.0
91 1.2
91 0.9
100 2.3
106 6.6
79 2.0*
92 2.2
96 3.4
98 1.2
95 1.8
97 2.3
100 2.2
96 2.2
100 0.7
101 0.5
100 0.6
100 0.7
100 0.9
98 1.0
103 1.0
104 1.5
104 1.6
102 0.9
102 1.1
101 0.6
100 0.9
101 0.7
101 0.7
100 0.5
100 0.6
101 0.1
98 3.8
96 0.6
97 1.0
99 1.2
98 1.0
97 1.0
100 0.8
98 2.3
101 2.4
100 0.8
100 0.5
99 1.1
99 3.3
99 0.5
98 0.7
101 0.4
102 1.6
100 1.3
94 2.4
103 0.89
98 1.2
94 4.1
90 2.8
98 7.1
75 2.0*
6
1.4*
44 1.8*
21 2.6*
90 3.2*
14 2.8*
48 5.6*
18 5.0*
62 5.0*
21 0.9*
16 1.2*
17 1.3*
17 1.2*
18 4.0*
15 3.3*
21 2.3*
21 7.7*
20 2.9*
21 3.2*
14 2.3*
12 2.0*
12 1.4*
97 2.6
101 0.4
103 4.0
100 0.4
Data shown are the current response in Xenopus oocytes expressing the indicated recombinant rat glutamate receptors recorded under two electrode voltage clamp (VHOLD
M, 30 M) in the presence of 3 M of the indicated test compound, expressed as a
ꢀ40 mV) in response to maximally effective concentration of glutamate and glycine (50
l
l
l
percent of control response in the absence of test compound. Measurements are the mean SEM from 4 to 10 oocytes for each compound at each receptor subtype. For
*
compounds showing a greater than 10% change from control, indicates p <0.05 (paired t-test).
Table 3
Comparison of in vitro on-target potency with selected off-target data
M)c
hERG Kid (nM)
a1-Adrenergic (% displacement at 3 l
M)e
a
Compound
NR2B IC50 (nM)
NR2B Kib (nM)
hERG (% displacement at 10
13
l
14
16
15
22
17
18
49
51
50
58
68
52
70
69
57
54
55
56
71
67
53
59
60
270
5750
28
4810
521
41,800
65
3600
50
1860
19
54
637
39
166
64
<1
3
5
65
4620
14
1
47*
16
>98
43
81
97
93
<1
42
75
71
67
74
52
<1
<1
49
90
<1
11
5
119
817
380
362
730
1110
72
300
23
21
22
193
750
83
19
a
The IC50 value was determined from two electrode voltage clamp recordings of human NR1/NR2B receptor function, fitted as described in the Methods. For all
experiments, data are the fitted IC50 value for the mean composite averaged from between 16 and 29 oocytes from 3 or more different frogs.
b
Ki values for NR2B receptors determined by displacement of 2 nM [3H] ifenprodil from Wistar rat cerebral cortex membranes.
Binding to the human ether-a-go-go potassium channel (hERG) expressed in HEK293 cells by displacement of 1.5 nM [3H]-astemizole. Each result represents the average
c
of displacement binding experiments done in duplicate at 10 lM of the test compound, except where noted.
d
Ki values for human hERG channels determined by displacement of 1.5 nM [3H] astemizole from HEK-293 cell membranes transfected with human recombinant hERG
channels (MDS Pharma). Data from multipoint displacement curves were fit by a non-linear, least squares regression analysis and the Ki calculated using the Cheng and
Prusoff equation.
e
Percent displacement of 0.25 nM [3H] prazosin from Wistar rat brain membranes. Each result represents the average of displacement binding experiments done in
duplicate at 3
l
M of the test compound.
*
At 3
lM. All binding assays were performed by MDS Pharma, Bothell, WA.
with a more diverse SAR profile. The ethanediamine amide 49
showed excellent potency (IC50 65 nM). Incorporation of a phenoxy
oxygen proximal to the B ring, as in 52, showed similar potency
(IC50 54 nM). Analogues of compounds 49 and 52 with eight-atom
linkers (60 and 59), respectively) were found to be less effective
than their seven-atom counterparts (IC50 values 750, 193 nM,
respectively), suggesting a seven-atom linker is optimal in the
amide series.

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C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
A collection of amides were synthesized to probe the effect of
to the hERG channel (estimated Ki ꢂ3000 nM) without actions at
the 1-adrenergic receptor.
Rigidifying the linker region also has a profound influence on
off-target effects. While amide 52 exhibited minimal hERG and
the aniline nitrogen proximal to the A ring on activity. In contrast
to the semicarbazide series where replacement of the aniline nitro-
gen with a carbon led to a 200-fold decrease in activity, such a
replacement in the amide series, particularly propaneamide 54,
had little effect (IC50 64 nM). Substitution of the aniline nitrogen
with oxygen (55) or sulfur (56) however, did result in significant
decreases in potency (IC50 380, 362 nM, respectively). Analogues
with carbons containing a sp2 orientation, cinnamyl amides 50
and 53, both showed good activity (IC50 50, 72 nM, respectively),
suggesting that conformation might be more important than
hydrogen bonding interactions for binding to the allosteric site.
The location of the amide in the linker region is also important
for potency. For example, aniline acetamide 67 reduced NR2B po-
tency to ꢂ1000 nM IC50 value compared with 52, whose amide is
centrally located in the linker.
a
a1 binding, the corresponding acyl piperazine 57 showed more po-
tent affinity for hERG (93% displacement) and the highest 1 activ-
a
ity of any compound tested (90% ligand displacement).
Imidazolidinones 70 and 71 have hERG receptor activity at levels
between linear amides and acyl piperazines but negligible a1 activ-
ity. This suggests that in addition to the presence of charge within
the linker region, conformation and hydrogen bonding opportuni-
ties are important to off-target activity.
We also determined plasma half-life for a select group of com-
pounds (50, 52, and 55) based on the overall in vitro data. Com-
pound 52 showed a half-life of 0.92 0.13 h (n = 3) when dosed
at 3 mg/kg iv in rats. When the aniline nitrogen was replaced with
an oxygen (55) or a carbon (50) the plasma half-life was shortened
by half to 0.4 0.16 (n = 3) h and 0.45 0.07 (n = 3) h, respectively.
The effect of rigidifying the ethanediamine linker by incorpora-
tion into various heterocyclic structures was also probed. Both
acyl piperazines 51 (Z = C) and 57 (Z = O) showed decreased po-
tency from the parent ethanediamine amides. However 57
showed only a modest decrease in potency (IC50 166 nM) while
3.3. In vitro analysis of NMDA receptor antagonism
51 had an IC50 value over 3
l
M, indicative of the varying effect
From these experiments we determined that compound 52
shows high potency and strong NR2B subunit selectivity. In addi-
tion, compound 52 was easily synthesized on large scale and
exhibited the best half-life in rats (0.92 0.13 h). We thus initiated
a series of experiments to further probe the site and mechanism of
action for this compound.
Compound 52 inhibits NR1/NR2B current responses with a half-
maximally effective concentration of 36 nM (mouse subunits,
n = 11 oocytes from 2 frogs), 57 nM (rat, n = 5–18 oocytes from 3
frogs), and 54 nM (human, n = 6–24 oocytes from 4 frogs). For all
inhibition curves, the Hill slope ranged between 0.81 and 0.94.
The amino acid identity between murine and human NR1 and
NR2B subunits is ꢂ98% with most changes occurring in a region
of the receptor not expected to be part of the binding pocket for
NR2B-selective inhibitors. Compound 52 did not inhibit receptor
function fully, but rather showed a maximal inhibition of 88% in
rat NR1/NR2B, 78% in mouse NR1/NR2B, and 90% inhibition in hu-
man (Fig. 3A and B). This result is consistent with other NR2B-
selective antagonists, which act by a non-competitive mechanism
to bring about incomplete inhibition.33,59–62 Compound 52
of the phenoxy oxygen on activity depending on the particular
scaffold. Imidazolidinone activity was also dependent on the pres-
ence of a phenoxy oxygen in the linker region of the molecule.
Unlike the piperazine amides, cyclic ureas 70 (Z = C) and 71
(Z = O) had similar potency (IC50 637, 730 nM, respectively),
although both were less active than their parent ethanediamine
amides.
3.2. Off-target effects
Because previous classes of NR2B antagonists have shown off-
target interactions with hERG potassium channels and
gic receptors, we also evaluated the actions of selected new com-
pounds with IC50 values less than at NR1/NR2B
recombinant NMDA receptors on these targets (Table 3). Thiosem-
icarbazide analogues exhibited minimal hERG and 1 activity. This
confirmed our original hypothesis that charged nitrogen atoms are
a strong structural determinant for off-target activity; elimination
of the charge in the linker region attenuates affinity of the NR2B
antagonists for the hERG channel.52
While amine 68 has a Ki value of 14 nM against hERG, the cor-
responding amide 49 shows a greater than 300-fold decrease in the
measured Ki value to 4620 nM. A similar case is seen with amine 69
(estimated Ki of 300 nM based on 97% displacement of [3H]astem-
a1-adrener-
1 lM
a
(3
receptors that contain other rat NR2 subunits (Fig. 3A). Determina-
tion of the IC50 value at rat NR1/NR2A (82 M, n = 7), NR1/NR2C
(58 M, n = 6), NR1/NR2D (107 M, n = 5) suggested that com-
lM) had minimal effects on recombinant heterodimeric NMDA
l
l
l
pound 52 was more than 1000-fold selective for rat NR1/NR2B over
all other NR1/NR2 NMDA receptors. There were no detectable ef-
fects on recombinant kainate receptors (GluR6), recombinant
AMPA (GluR1) receptors, or voltage-gated Na+ or K+ currents re-
corded from cultured cortical neurons (Fig. 3A). Furthermore, con-
sistent with other NR2B-selective ligands, inhibition of NR1/NR2B
receptor responses by compound 52 was not surmountable by
increasing the concentrations of glycine or glutamate 10-fold
(Fig. 3C), suggesting inhibition is non-competitive. The inhibition
produced by 52 was voltage-independent (n = 5; Fig. 3D).
izole at 10 lM) and corresponding amide 52 (estimated Ki
ꢂ12,000 nM). Also important is the 40-fold decrease in hERG bind-
ing observed with the incorporation of the phenoxy oxygen prox-
imal to the B ring. Similar to the trend seen with hERG activity,
neither amide 49 or 52 showed any
mated as displacement of ligand at 3
responding amines. Within the linear amide series, hERG activity
was also influenced by the functionality proximal to the A ring.
Sulfur-containing analogue 56 showed significant binding to the
a1-adrenergic binding (esti-
lM) compared with the cor-
hERG channel (75% displacement at 10
ment consistent with low affinity for the
The corresponding oxygen-containing analogue 55 reduced hERG
channel binding (42% displacement at 10 M) but exhibited little
change in displacement of a1 ligands. This reinforces the value of
introducing a phenoxy oxygen proximal to the B ring discussed
above. It is clear that higher number of oxygen atoms in the linker
l
M) and ligand displace-
These data are consistent with compound 52 exerting a nega-
tive allosteric effect on NR2B receptor function through direct
interaction with at least a portion of the ifenprodil binding site,
which has been proposed to be contained within the amino termi-
nal domain of the NR2B receptor.29,63,64 To test whether compound
52 and its analogues bind to the amino terminal domain, we eval-
uated a rat NR2B subunit in which residues within the amino ter-
minal domain between S28 and H405 were deleted. We
hypothesized that deletion of the amino terminal domain from
a1-adrenergic receptor.
l
leads to decreased off-target effects at hERG channels and a1
adrenergic receptors. While the sp3-carbon-containing amide ana-
logue 54 exhibited negligible hERG and 1-adrenergic receptor
binding, the sp2-carbon analogue 50 showed low potency binding
-
a
the NR2B subunit, NR2B(DATD), would render the receptors insen-
sitive to inhibition by compound 52 and its analogues. Functional

C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6471
Figure 3. Compound 52 is an NR2B-selective, non-competitive, voltage-independent NMDA receptor inhibitor. (A) Two electrode voltage clamp recordings from Xenopus
oocytes were used to measure the mean SEM response to glutamate and glycine coapplied with 3 M compound 52 on various glutamate receptors expressed as a percent
of the maximal response evoked by saturating concentrations of agonists (50 M glutamate plus 30 M glycine) for the indicated NMDA receptors. Similar experiments were
performed on recombinant GluR1 AMPA receptors (100 M glutamate), and recombinant GluR6 kainate receptors pre-treated with 10 M concanavalin-A (100 lM
l
l
l
l
l
glutamate). The effect of 3 lM compound 52 was also evaluated on voltage-dependent sodium and potassium whole cell currents recorded from cultured cortical neurons
and elicited by incremental 10 mV step depolarizations from ꢀ90 mV to +50 mV. The amplitudes of Na+ currents and K+ currents in the absence and presence of 3
lM
compound 52 were measured at ꢀ10 mV and +50 mV, respectively. The numbers in parenthesis are the number of oocytes or neurons tested under each condition. *p <0.05,
paired-t-test. (B) Concentration–effect curves for compound 52 were generated in oocytes expressing wild type and mutant NR1/NR2B receptors. The IC50 values of
compound 52 were 0.057
lM on rat wild type receptors (n = 18), 52
l
M on mouse amino terminal domain D101A point mutants (n = 4) and 563
lM on rat amino terminal
domain deletion mutants (n = 3). (C) Increasing the concentration of glutamate does not surmount the inhibition by 0.054
l
M of compound 52 (n = 4), suggesting compound
52 inhibits the receptor by a non-competitive mechanism. The mean SEM response as a percent control is summarized for experiments raising either glutamate or glycine
concentration in the right panel. (D) Inhibition by compound 52 is independent of membrane potential. A representative recording from an oocyte expressing rat NR1/NR2B
shows the current–voltage relationship for responses evoked by of 50
lM glutamate plus 30 lM glycine as closed circles and 0.054 lM compound 52 coapplied with 50 lM
glutamate plus 30 M glycine as open circles (representative of eight experiments).
l
properties of this deletion construct are similar to that of wild type
receptors (e.g., glutamate and glycine EC50s are not altered, data
not shown). As expected, compound 52 had virtually no effect on
3.4. Brain penetration
To evaluate how this class of molecules might penetrate the
brain, we estimated brain penetration for a number of compounds
using the MDR1-MDCK in vitro assay (Table 4). It was determined
that 52 was not expected to appreciably pass the blood–brain bar-
rier (Papp(A–B) = 1.80) and is deemed a substrate for P-glycoprotein
(Pgp) efflux (efflux ratio = 35). Acyl piperazine 57 shows improved
brain penetration potential (Papp(A–B) = 4.8) but is also deemed a
the current response of NR1/NR2B(DATD) when activated by max-
imal concentrations of glutamate and glycine (Fig. 3B). In addition,
Asp101 (D101) within NR2B has been suggested to play an impor-
tant role in ifenprodil binding. We find that mutation of this resi-
due to alanine NR2B(D101A) increased the IC50 to ꢂ30
lM (n = 4
oocytes). This ꢂ500-fold reduction in potency by NR2B(D101A) is
consistent with compound 52 binding to the ifenprodil site on
the NR2B subunit. Similar results were found for compounds 15,
49, and 68, which were 51-, 102-, 177-fold less potent when eval-
uated at NR2B(D101A) (n = 7, 13, 20 oocytes, respectively). A re-
cently published homology model suggests the protonated
piperidine of ifenprodil makes an electrostatic interaction with
D101.65 Presumably, the amide compounds described here could
make no such salt bridge. However, since D101A mutation reduces
the potency (increases the IC50 value) for 15 and 52, we hypothe-
size that the amide compounds make H-bond(s) to D101 and are
situated similarly to ifenprodil within the ligand binding site of
the NR2B ATD.66
Table 4
MDR1-MDCK permeability
Compound
Papp (A–B)a
Papp (B–A)
Efflux ratiob
52
57
71
1.80
4.76
12.9
63.2
42.4
40.2
35
8.9
3.1
a
Papp is the apparent permeability for the apical to basal (A–B) and the basal to
apical (B–A) direction across MDR1-MDCK cell monolayers in TranswellÒ wells. Papp
units are ꢃ10ꢀ6 cm/s.
b
Efflux ratio = Papp (A–B)/Papp (B–A).

6472
C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
Pgp efflux substrate (efflux ratio = 8.9). The compound with the
highest brain penetration potential tested in this in vitro model as-
say was imidazolidinone 71, which showed both the highest pas-
sive diffusion potential (Papp(A–B) = 12.9), and lowest efflux ratio
of 3.1. Thus, tying up the chain nitrogen atoms into a cyclic func-
tionality, such as a piperazine or imidazolidinone, appears to in-
crease the likelihood of crossing the blood brain barrier probably
due to increased lipophilicity as well as decreasing the number
of free H-bond donors. Furthermore, the ability of these com-
pounds to act as a substrate for Pgp efflux is significantly decreased
based on the Papp(B–A) results.
line that splits in a Y-configuration to perfuse two oocytes. Dual
recordings were made at room temperature (23 °C) using two
Warner OC725B or OC725C two electrode voltage clamp amplifi-
ers, arranged as recommended by the manufacturer. Glass micro-
electrodes (1–10 Megaohms) were filled with 300 mM KCl
(voltage electrode) or 3 M KCl (current electrode). The bath
clamps communicated across silver chloride wires placed into
each side of the recording chamber, both of which were assumed
to be at a reference potential of 0 mV. Oocytes were perfused with
a solution comprised of (in mM) 90 NaCl, 1 KCl, 10 HEPES, and 0.5
BaCl2; pH was adjusted to 7.4 or 7.6 by addition of 1 M NaOH. Oo-
cytes expressing NR1/NR2A were pre-incubated before recording
in recording solution supplemented with 50 lM BAPTA-AM at
room temperature. Oocytes were recorded under voltage clamp
4. Conclusions
at ꢀ40 mV. Final concentrations for control application of gluta-
A novel series of amide-based NR2B-selective NMDA receptor
antagonists was developed with high on-target potency and
attractive off-target profiles. Compounds 52 and 71 in particular
show an increased drug-like characteristics over the previous
enantiomeric propanolamine series, including concomitant high
potency with minimal hERG and a1 binding. In addition, 52 has
an acceptable half-life in vivo while 71 is expected to cross the
blood–brain barrier. These amide-based analogues provide new
insight into ways by which off-target effects can be mitigated
while retaining high potency and selectivity at NR2B containing
receptors, and represent a starting point for further development
of the potential therapeutic uses of NR2B-selective NMDA recep-
tor antagonists.
mate (50–100 lM) plus glycine (30 lM) to oocytes expressing
NMDA receptors were achieved by dilution from 100 and 30–
100 mM stock solutions, respectively. In addition, 10 M final
l
EDTA was obtained by adding a 1:1000 dilution of 10 mM EDTA,
in order to chelate contaminant divalent ions such as extracellular
Zn2+
100
.
l
Homomeric GluR1 AMPA receptors were activated by
M glutamate. Homomeric GluR6 kainate receptors were
incubated in concanavalin-A (10
lM) for 5 min, and activated by
100 M glutamate. Concentration–response curves for experi-
l
mental compounds acting on NMDA receptors were obtained by
applying in successive fashion a maximally effective concentra-
tion of glutamate/glycine, followed by glutamate/glycine plus var-
iable concentrations of experimental compounds. Concentration–
response curves consisting of 5–8 concentrations were obtained
in this manner. The baseline leak current at ꢀ40 mV was mea-
sured before and after recording, and the full recording linearly
corrected for any change in leak current. Oocytes with gluta-
mate-evoked responses smaller than 50 nA were not included in
the analysis. The level of inhibition produced by experimental
compounds was expressed as a percent of the initial glutamate
response, and averaged together across oocytes from a single frog.
Each experiment consisted of recordings from 3 to 10 oocytes ob-
tained from a single frog. Results from P3 experiments using oo-
cytes from three different frogs were pooled, and the percent
responses at antagonist concentrations for each oocyte were fitted
by the equation,
5. Experimental procedures-biology
5.1. Expression of glutamate receptors in Xenopus laevis
oocytes
All protocols involving the use of animals were approved by the
Emory University IACUC. cRNA was synthesized from linearized
template cDNA for rat glutamate receptor subunits according to
manufacturer specifications (Ambion). Quality of synthesized
cRNA was assessed by gel electrophoresis, and quantity was esti-
mated by spectroscopy and gel electrophoresis. Stage V and VI oo-
cytes were surgically removed from the ovaries of large, well-fed
and healthy Xenopus laevis anesthetized with 3-amino-benzoic
acid ethyl ester (1–3 gm/l) as previously described.67 Clusters of
isolated oocytes were incubated with 292 U/mL Worthington
(Freehold, NJ) type IV collagenase or 1.3 mg/mL collagenase (Life
Technologies, Gaithersburg, MD; 17018–029) for 2 h in Ca2+-free
solution comprised of (in mM) 89 NaCl, 1 KCl, 2.4 NaHCO3, 0.82
MgSO4, 10 HEPES, with slow agitation to remove the follicular cell
layer. Oocytes were then washed extensively in the same solution
and maintained in Barth’s solution comprised of (in mM) 88 NaCl, 1
KCl, 2.4 NaHCO3, 10 HEPES, 0.82 MgSO4, 0.33 Ca(NO3)2, and 0.91
nH
Percent response ¼ ð100 ꢀ minimumÞ=ð1 þ ð½concdꢄ=IC50
þ minimum
Þ Þ
where minimum is the residual percent response in saturating con-
centration of the experimental compounds, IC50 is the concentra-
tion of antagonist that causes half of the achievable inhibition,
and nH is a slope factor describing steepness of the inhibition curve.
Minimum was constrained to be greater than or equal to 0.
CaCl2 and supplemented with 100
lg/mL gentamycin, 10 lg/mL
5.3. Whole cell patch clamp recording of voltage-activated
currents in neurons
streptomycin, and 10 g/mL penicillin. Oocytes were manually
l
defolliculated and injected within 24 h of isolation with 3–5 ng
of NR1–1a (hereafter NR1) subunit and 7–10 ng of NR2 subunit
in a 50 nl volume, or 5–10 ng in 50 nl of AMPA or kainate receptor
cRNAs, and incubated in Barth’s solution at 15 °C for 1–7 d. Glass
Neuronal cultures were derived from E17 Sprague-Dawley rat
pups. Briefly, cortical tissue was dissected, transferred into saline
containing penicillin/streptomycin and 10 mM HEPES, and incu-
bated in trypsin containing 0.02% DNase at 37 °C for 15 min. Tissue
was then triturated and the supernatant resuspended in B27-sup-
injection pipettes had tip sizes ranging from 10 to 20
lm, and were
backfilled with mineral oil.
plemented Neurobasal medium (Gibco) containing 2 mM
L-gluta-
mine and 5% fetal bovine serum. Cells were plated onto poly-
D
-
5.2. Two electrode voltage clamp recording from Xenopus
laevis oocytes
lysine-coated coverslips and after three days the media was re-
placed with serum-free medium. Cultures were maintained at
37 °C in a humidified 5% CO2-containing atmosphere. Whole cell
patch clamp recordings (voltage clamp, holding potential
ꢀ60 mV) from 5- to 10-day cultured cortical neurons were made
Two electrode voltage clamp recordings were made 2–7 days
post-injection as previously described.67 Oocytes were placed in
a dual-track plexiglass recording chamber with a single perfusion

C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6473
with an Axopatch 200B amplifier (Axon Instruments, Union City,
6.2. Chemistry: experimental synthetic procedures
6.2.1. Methyl 3-(4-aminophenyl)propanoate (11)
CA) at room temperature (23 °C). The recording chamber was con-
tinually perfused with recording solution composed of (in mM)
150 NaCl, 3 KCl, 2 CaCl2, 1.5 MgCl2, 5.5 glucose and 10 HEPES
(pH 7.4 by NaOH; osmolality adjusted to 315 mOsm with sucrose).
Thionyl chloride (1.65 mL, 22.6 mmol, 3.3 equiv) was added
dropwise to a solution of dry methanol (6.65 mL, 164 mmol,
24 equiv) at ꢀ10 °C. After stirring for 10 min, the propionic acid
10 (1.13 g, 6.84 mmol) was added to give a yellow suspension.
The solution stirred for 1 h and warmed to room temperature.
The resulting solution was concentrated in vacuo to give a
yellow solid. The solid was suspended in EtOAc, and solid
NaHCO3 was added until the salt dissolved fully. The layers were
separated and the organics were washed with brine, dried over
Thin wall glass pipettes were filled with (in mM) 110 D-gluconate
(50% w/w), 110 CsOH (50% w/w), 30 CsCl, 5 HEPES, 4 NaCl, 0.5
CaCl2, 2 MgCl2, 5 BAPTA, 2 NaATP, and 0.3 NaGTP (pH adjusted
to 7.3 with CsOH and osmolality adjusted to 300 mOsm with su-
crose). Recordings were made in the presence of 10
lM bicuculline,
10 M CNQX, and 100 M DL-APV to block both excitatory and
l
l
inhibitory synaptic transmission. Drugs were applied by gravity
and controlled by manual valves. Voltage-gated macroscopic
whole cell currents were activated by 100 ms voltage steps from
a holding potential of ꢀ60 mV to between ꢀ90 and +50 mV. The
MgSO4, and concentrated in vacuo to give
a yellow solid
(1.06 g, 98%). 1H NMR (300 MHz, CDCl3) d 7.00 (d, J = 8.3 Hz,
2H,) 6.60 (d, J = 8.3 Hz, 2H), 3.67 (s, 3H), 3.59 (br s, 2H),
2.85 (t, J = 7.6 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H). 13C NMR
(150 MHz, CDCl3) d 173.8, 144.9, 130.7, 129.3, 115.5, 51.8, 36.4,
30.4. m/z (EI+) calcd for C10H13NO2, 179.09; found, 180.30
[M+H]+.
sensitivity of Na+ currents to 0.5
lM tetrodotoxin was confirmed
at the end of each experiment; K+ channels were recorded in the
presence of 0.5 l
M tetrodotoxin to block Na+ channels. We evalu-
ated the mean Na+ current from a number of whole cell recordings
at ꢀ10 mV, and the mean K+ current at +50 mV using a paired t-
test.
6.2.2. Methyl 3-(4-(methylsulfonamido)phenyl)propanoate (12)
The ester 11 (7.38 g, 41.2 mmol) was dissolved in pyridine
(17.0 mL, excess). After cooling to 0 °C, methanesulfonyl chloride
(4.55 mL, 57.7 mmol, 1.4 equiv) was added dropwise. The reaction
was warmed to room temperature and stirred for 20 h. The reac-
tion was quenched with water and diluted with DCM. The layers
were separated and the organics were washed with brine and con-
centrated in vacuo to give a red solid. The crude material was puri-
fied using silica gel chromatography (1 EtOAc/1 hexanes) to give a
white solid (9.26 g, 87%). 1H NMR (400 MHz, CDCl3) d 7.20 (d,
J = 8.6 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 6.45 (br s, NH,1H), 3.68 (s,
3H), 3.00 (s, 3H), 2.94 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H).13C
NMR (100 MHz, CDCl3) d 173.4, 137.6, 135.2, 129.4, 121.4, 51.7,
38.5, 35.5, 30.1. m/z (APCI) calcd for C11H15NO4S, 257.07; found,
257.56 [M]+.
5.4. Estimation of brain penetration
TranswellÒ wells containing MDR1-MDCK cell monolayers
were used for measuring the percent recovery of compound after
dosing both sides of a cell monolayer with the test article (per-
formed by Absorption Systems, Exton PA, USA). Briefly, monolay-
ers were grown for 7–11 days at which time 5 lM of the test
article was made by dilution from DMSO stocks into a Hank’s
balanced salt solution (pH 7.4), final DMSO not greater than
1%, and added to: (a) the apical side for A–B permeability assess-
ment, or separately (b) the basal side for the B–A permeability
assessment, all at pH 7.4. After a 2 h incubation (37C) both the
apical and the basal compartments were sampled and the
amount of test article present determined by generic LC–MS/
MS methods against a >4 point calibration curve. Apparent per-
6.2.3. 3-(4-(Methylsulfonamido)phenyl)propanehydrazide (13)
Compound 12 (1.34 g, 5.21 mmol) was dissolved in MeOH
(30 mL) and hydrazine monohydrate (0.49 mL, 15.6 mmol,
3.0 equiv) was added. The solution was refluxed for 12 h. The
resulting solution was concentrated in vacuo to give a white solid.
The crude material was purified using silica gel chromatography
(10–20% MeOH/DCM gradient) to give a white solid. 1H NMR
(DMSO-d6) d 9.59 (s, 1H), 8.95 (s, 1H), 7.15 (d, J = 8.6 Hz, 2H),
7.10 (d, J = 8.6 Hz, 2H), 4.16 (br s, 2H), 2.93 (s, 3H), 2.76 (t,
J = 7.3 Hz, 2H), 2.28 (t, J = 7.3 Hz, 2H).
meability (Papp
were done in duplicate.
)
units are reported ꢃ 10–6 cm/s. Experiments
6. Experimental procedures: chemistry
6.1. General experimental procedures
All reagents were obtained from commercial suppliers and
used without further purification. Reaction progress was moni-
tored by thin layer chromatography (TLC) on precoated glass
plates (Silica Gel 60 F254, 0.25 mm). Proton and carbon NMR
spectra were recorded on an INOVA-400 (400 MHz), VNMRS
400 (400 MHz), INOVA-600 (600 MHz), or Mercury 300 Vx
(300 MHz). The spectra obtained were referenced to the residual
solvent peak. Mass spectra were performed by the Emory
University Mass Spectroscopy Center on either a VG 70-S Nier
Johnson or JEOL instrument. Elemental analyses were performed
by Atlantic Microlab Inc. C, H, N agreed with proposed structures
within 0.4% of theoretical values. Flash chromatography was
performed on
(Varian) was used to determine the purity of some compounds.
HPLC was performed on C18 analytical column with UV
detection using methanol and acetonitrile at 1 mL/min according
to the following ratios: (a) 50% acetonitrile/50% methanol
over 10 min, (b) 30% acetonitrile/70% methanol-70% acetoni-
trile/30% methanol gradient over 10 min and (c) 20% acetoni-
trile/80% methanol-80% acetonitrile/20% methanol gradient over
10 min.
6.2.4. 4-(3,4-Dichlorophenyl)-1-(3-(4-(methylsulfonamido)-
phenyl)propanoyl)thiosemicarbazide (14)
Hydrazide 13 (0.499 g, 1.94 mmol) was dissolved in DMF
(15.0 mL). To this solution, 3,4-dichlorophenylisothiocyanate
(0.278 mL, 1.94 mmol, 1.0 equiv) was added. The orange solution
was stirred for 12 h at room temperature. The solution was con-
centrated in vacuo to give an orange residue which was purified
using silica gel chromatography (3 EtOAc/1 hexanes) to give a
white foam (0.670 g, 75%). 1H NMR (600 MHz, CD3OD) d 7.96
(s, 1H), 7.76 (s, 1H), 7.44 (d, J = 9.1 Hz, 1H), 7.35 (dd,
J1 = 9.1 Hz, J2 = 1.9 Hz, 1H), 7.22 (d, J = 8.6 Hz, 2H), 7.16 (d,
J = 8.6 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H), 2.91 (s, 3H), 2.61 (t,
a Teledyne Isco Combiflash Companion. HPLC
a
J = 8.1 Hz, 2H).13C NMR (150 MHz, CD3OD)
d 183.7, 174.9,
164.9, 140.1, 138.7, 137.6, 132.8, 131.3, 131.1, 130.6, 130.5,
129.9, 122.3, 122.2, 39.2, 36.6, 31.4. HRMS calcd for
C17H18Cl2N4O3S2, 460.0197; found, 459.01074 [MꢀH]+. Anal.
(C17H18Cl2N4O3S2) C, H, N.

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C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6.2.5. 4-(3,4-Dichlorophenyl)-1-(3-(4-(methylsulfonamido)-
phenyl)propanoyl)semicarbazide (15)
(0.220 g, 50%).1H NMR (400 MHz, CDCl3) d 8.11 (s, 1H), 8.07 (s,
1H), 7.89 (s, 1H), 7.23–7.24 (mult, 2H), 7.19–7.17 (mult, 2H), 3.35
(br s, 2H), 3.00–2.95 (mult, 2H), 2.91 (s, 3H), 2.70 (t, J = 7.6 Hz,
1H), 2.60 (t, J = 7.6 Hz, 1H).13C NMR (150 MHz, CDCl3) d 189.0,
174.2, 138.7, 137.9, 137.7, 137.4, 135.6, 133.2, 130.6, 122.3, 39.1,
36.7, 31.9. HRMS calcd for C18H15Cl2N3O5S, 455.0109; found,
456.01001 [M+H]+. Anal. (C18H15Cl2N3O5Sꢁ0.25 CH3OH) C, H, N.
Hydrazide 13 (0.100 g, 0.39 mmol) was dissolved in DMF
(3.0 mL). To this solution, 3,4-dichlorophenyisocyanate (0.073 g,
0.39 mmol, 1.0 equiv) was added and the mixture was stirred at
room temperature for 12 h. The resulting solution was concen-
trated in vacuo to give a white solid. The crude material was puri-
fied using silica gel chromatography (10–50% MeOH/DCM
gradient) to give a white solid (0.114 g, 66%). 1H NMR (400 MHz,
DMSO-d6) d 9.69 (br s, 1H), 9.60 (br s, 1H), 9.12 (br s, 1H), 8.27
(br s, 1H), 7.85 (d, J = 2.4 Hz, 1H), 7.51 (d, J = 8.8 Hz), 7.41 (m,
1H), 7.20 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 2.94 (s, 3H),
2.80 (t, J = 8.0 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) d 172.2, 155.8, 140.7, 137.4, 136.9, 131.5, 131.1, 129.7,
123.8, 120.9, 120.6, 119.3, 35.5, 30.6. HRMS calcd for
C17H18Cl2N4O4S, 444.0426; found 444.04663 [M]+. Ana-
l.(C17H18Cl2N4O4S) C, H, N.
6.2.9. 2-(3,4-Dichlorophenyl)ethanol (20)
Lithium aluminum hydride (0.90 g, 24 mmol, 1.0 equiv) was
dissolved in THF (50.0 mL) at 0 °C, and then warmed to room tem-
perature for 30 min. To this suspension, a solution of 3,4-dichloro-
phenyl acetic acid (19, 5.0 g, 24 mmol) and triethyl amine (3.0 mL,
24 mmol, 1.0 equiv) in THF (37.5 mL) was added dropwise. The
reaction mixture was refluxed for 1 h. The reaction was cooled to
room temperature and poured into water (125 mL) to give a bright
yellow solution. The mixture was extracted with EtOAc (2ꢃ). The
organics were dried over Na2SO4 and concentrated in vacuo to give
a yellow oil (3.76 g, 81%). The crude material was carried on with-
out further purification. 1H NMR (300 MHz, CDCl3) d 7.38 (d,
J = 8.3 Hz, 1H), 7.35 (d, J = 1.9 Hz, 1H), 7.08 (dd, J1 = 8.3 Hz,
J2 = 1.9 Hz), 3.87 (quart, J = 6.6 Hz, 2H), 2.83 (t, J = 6.6 Hz, 2H),
1.45 (br s, 1H).
6.2.6. N0-(2-(3,4-Dichlorophenyl)acetyl)-3-(4-
(methylsulfonamido)phenyl)propanehydrazide (16)
3,4-Dichlorophenylacetic acid (0.800 g, 3.9 mmol), EDCI
(0.750 g, 3.9 mmol, 1.0 equiv) and DMAP (0.470 g, 3.9 mmol,
1.0 equiv) were dissolved in DMF (10.0 mL) at 0 °C and stirred for
45 min. Hydrazide 13 (1.0 g, 3.9 mmol, 1.0 equiv) was added and
the mixture was warmed to room temperature and stirred for
12 h. The reaction was quenched with 1.0 N HCl and extracted with
EtOAc. The organics were dried over MgSO4 and concentrated in
vacuo to give an off-white powder. The crude material was purified
using silica gel chromatography (5% MeOH/DCM) to give a white
solid which was further purified by recrystallization with EtOAc
(0.103 g, 6.0%). 1H NMR (400 MHz, DMSO-d6) d 10.89 (s, 1H), 9.88
(s, 1H), 9.60 (s, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 1.9 Hz,
1H), 7.28 (dd, J1 = 8.6 Hz, J2 = 1.9 Hz, 1H), 7.18 (d, J = 8.3 Hz, 2H),
7.10 (d, J = 8.6 Hz, 2H), 2.94 (s, 3H), 2.78 (t, J = 7.3 Hz, 2H), 2.40
(t, J = 7.3 Hz). 13C NMR (150 MHz, DMSO-d6) d 170.2, 168.1,
136.8, 136.3, 131.1, 130.8, 130.4, 129.6, 129.4, 129.3, 129.0,
120.2, 34.7, 30.0, 24.4. HRMS calcd for C18H19Cl2N3O4S, 444.0473;
found, 444.04802 [M]+. Anal. (C18H19Cl2N3O4S) C, H, N.
6.2.10. 3,4-Dichlorophenethyl methanesulfonate (21)
Crude alcohol 20 (3.76 g, 2.79 mmol) was dissolved in pyridine
(30.0 mL). Methanesulfonyl chloride (1.7 mL, 30 mmol, 1.1 equiv)
was added dropwise at room temperature and the reaction was
stirred for 1 h. The mixture was poured into ice water and warmed
to room temperature. The organics were extracted with EtOAc
(3ꢃ), dried over Na2SO4, and concentrated in vacuo to give a yellow
liquid. The crude material was purified using silica gel chromatog-
raphy (1 EtOAc/1 hexanes) to give a yellow oil (4.25 g, 80%). 1H
NMR (300 MHz, CDCl3)
d 7.41 (d, J = 8.1 Hz, 1H), 7.35 (d,
J = 1.9 Hz, 1H), 7.10 (dd, J1 = 8.1 Hz, J2 = 1.9 Hz, 1H), 4.41 (t,
J = 6.6 Hz, 2H), 3.03 (t, J = 6.6 Hz, 2H), 2.95 (s, 3H).
6.2.11. N0-(3,4-Dichlorophenethyl)-3-(4-
(methylsulfonamido)phenyl)propanehydrazide (22)
6.2.7. 3,4-Dichloro-N0-(3-(4-
Hydrazide 13 (0.300 g, 1.11 mmol) was dissolved in methanol
(11.0 mL). To this solution, mesylate 21 was added dropwise
(0.287 g, 1.11 mmol, 1.0 equiv). The solution was refluxed for
48 h, cooled to room temperature, and concentrated in vacuo to
give a white solid. The crude material was purified using silica
gel chromatography (10% MeOH/DCM) to give a white foam
(0.065 g, 14%). 1H NMR (400 MHz, CDCl3) d 7.37 (d, J = 8.3 Hz,
1H), 7.29 (d, J = 1.90 Hz, 1H), 7.20 (d, J = 8.6 Hz, 2H), 7.14 (d,
J = 8.8 Hz, 2H), 7.04 (dd, J1 = 8.1 Hz, J2 = 2.2 Hz, 2H), 6.73 (br s,
1H), 6.25 (br s, 1H), 4.60 (br s, 1H), 3.04 (mult, 2H), 2.99 (s, 3H),
2.96 (t, J = 7.3 Hz, 2H), 2.69 (t, J = 7.3 Hz, 2H), 2.41 (t, J = 7.6 Hz,
2H). 13C NMR (150 MHz, CDCl3) d 172.0, 139.8, 137.7, 135.4,
132.4, 130.8, 130.6, 130.4, 129.7, 128.3, 121.4, 52.6, 39.4, 36.2,
33.6, 30.9. HRMS calcd for C18H21Cl2N3O3S, 429.0681; found,
430.07001 [M+H]+ Anal. (C18H21Cl2N3O3S) C, H, N.
(methylsulfonamido)phenyl)propanoyl)benzohydrazide (17)
The hydrazide 13 (0.500 g, 1.9 mmol) was dissolved in DMF
(10.0 mL). To this solution, 3,4-dichlorobenzoic acid (0.370 g,
1.9 mmol, 1.0 equiv) was added. Finally, DCC (1.0 M in DCM,
1.9 mL, 1.9 mmol, 1.0 equiv) was added. The mixture was stirred
at room temperature for 12 h. The white precipitate was filtered
off, and the resulting solution was concentrated in vacuo to give
a brown residue. The crude material was purified using silica gel
chromatography (10% MeOH/DCM) to give a white solid (0.648 g,
77%). 1H NMR: (600 MHz, CD3OD) d 8.03 (d, J = 1.9 Hz,1H), 7.86
(dd, J1 = 8.1 Hz, J2 = 1.0 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.25 (d,
J = 8.6 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H), 2.97 (t, J = 7.6 Hz, 2H), 2.92
(s, 3H), 2.61 (t, J = 8.1 Hz, 2H). 13C NMR: (150 MHz, CD3OD) d
174.4, 166.8, 138.8, 137.8, 137.5, 134.1, 134.1, 132.1, 131.0,
130.6, 128.6, 122.4, 39.1, 36.7, 31.8. HRMS calcd for
C17H17Cl2N3O4S, 429.0317; found, 430.03129 [M+H]+. Anal.
(C17H17Cl2N3O4S) C, H, N.
General method for preparation of esters 25 and 26, exemplified
by 25.
6.2.12. Methyl 2-(4-nitrophenoxy)acetate (25a)
6.2.8. N-(5,6-Dichloro-1,3-dioxoisoindolin-2-yl)-3-(4-
Thionyl chloride (17 mL, 234 mmol, 3.3 equiv) was added drop-
wise to a solution of dry methanol (70 mL, 1704 mmol, 24 equiv) at
ꢀ10 °C. After stirring for 10 min, 4-nitrophenoxyacetic acid (14.0 g,
71 mmol) was added to give a white precipitate in an orange sus-
pension. The solution stirred for 1 h and slowly warmed to room
temperature. The resulting solution was concentrated in vacuo to
give an off-white solid. The solid was dissolved in EtOAc, and
(methylsulfonamido)phenyl)propanamide (18)
The hydrazide 13 (0.250 g, 0.972 mmol) was dissolved in DMF
(5.0 mL). To this solution, 5,6-dichloroisobenzofuran-1,3-dione
(0.211 g, 0.972 mmol, 1.0 equiv) was added. The mixture was re-
fluxed for 12 h. The resulting solution was concentrated in vacuo
to give a yellow residue. The crude material was purified using silica
gel chromatography (10% MeOH/DCM) to give a yellow solid

C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6475
NaHCO3 (satd) was added. The layers were separated and the
organics were washed with brine, dried over MgSO4, and concen-
trated in vacuo to give a white solid (14.3 g, 95%). 1H NMR
(300 MHz, CDCl3) d 8.23 (d, J = 9.2 Hz, 2H), 6.98 (d, J = 9.2 Hz),
4.75 (s, 2H), 3.84 (s, 3H). 13C NMR (100 MHz, CDCl3) d 168.4,
162.7, 142.5, 126.2, 114.9, 65.5, 52.8.
with a solution of aqueous HCl. The volume of methanol was
reduced by rotary evaporation (40 mbar), upon which the product
crashed out of solution. The yellow crystals were collected by fil-
tration and dried in vacuo (0.900 g, 82%). 1H NMR (400 MHz,
CD3OD) d 7.21 (d, J = 8.6 Hz, 2H), 7.17 (d, J = 8.6 Hz, 2H), 2.91 (s,
3H), 2.89 (t, J = 7.6 Hz, 2H), 2.59 (t, J = 7.6 Hz, 2H). 13C NMR
(100 MHz, CD3OD) d 176.7, 139.0, 137.7, 130.5, 122.3, 39.1, 36.8,
31.4. m/z (APCI) calcd for C10H13NO4S, 243.06; found, 242.05 [M-
H]+.
6.2.13. Methyl 2-(4-aminophenoxy)acetate (25)
Ester 25a (14.3 g, 68 mmol) was dissolved in dry methanol
(100 mL) and 5% palladium on carbon catalyst (1.43 g, 10 wt %)
was added. The suspension was hydrogenolyzed using a balloon
for 12 h. The mixture was filtered over a pad of Celite, and the
resulting solution was concentrated in vacuo to give a pink oil
which turned to a solid upon trituration with chloroform. The
crude material was purified using silica gel chromatography (1
EtOAc/2 hexanes) to give a pink solid (11.7 g, 95%). 1H NMR
(400 MHz, CDCl3) d 6.74 (d, J = 8.6 Hz, 2H), 6.60 (d, J = 8.6 Hz),
4.54 (s, 2H), 3.77 (s, 3H), 3.50 (br s, 2H). 13C NMR (100 MHz, CDCl3)
d 169.9, 150.9, 141.2, 116.3, 116.0, 66.4, 51.2. m/z calcd for
C9H11NO3, 182.18; found, 182.081 [M]+.
6.2.19. 2-(4-(Methylsulfonamido)phenoxy)acetic acid (29)
Compound 29 was obtained from compound 27 (pink crystals,
5.6 g, 97%). 1H NMR (300 MHz, CD3OD) d 7.20 (d, J = 9.0 Hz, 2H),
6.92 (d, J = 9.0 Hz, 2H), 4.65 (s, 2H), 2.88 (s, 3H). 13C NMR
(75 MHz, CD3OD) d 172.7, 157.3, 132.8, 125.0, 116.5, 66.2, 38.8.
m/z (APCI) calcd for C9H11NO5S, 245.0358; found, 489.064 [Mꢃ2]+.
6.2.20. 4-(4-(Methylsulfonamido)phenyl)butanoic acid (31)
Compound 31 was obtained from compound 28 (off-white so-
lid, 3.24 g, 53%). 1H NMR (300 MHz, DMSO-d6) d 12.04 (br s, 1H),
9.58 (br s, 1H), 7.12 (mult, 4H), 2.91 (s, 3H), 2.51 (t, J = 7.3 Hz,
2H), 2.18 (t, J = 7.3 Hz, 2H), 1.74 (quint, J = 7.3 Hz, 2H). 13C NMR
(75 MHz, DMSO-d6) d 175.0, 138.0, 136.8, 129.8, 121.0, 34.4, 33.7,
28.3, 27.0. HRMS calcd for C11H15NO4S, 257.0722; found,
256.06500 [M+H]+.
6.2.14. Methyl 4-(4-nitrophenyl)butanoate (26a)
Compound 26a was obtained from 4-(4-nitrophenyl)butanoic
acid (off-white solid, 5.01 g, 94%). 1H NMR (600 MHz, CDCl3) d
7.98 (d, J = 9.1 Hz, 2H), 7.22 (d, J = 9.1 Hz, 2H), 3.53 (s, 3H), 2.63
(t, J = 7.6 Hz, 2H), 2.22 (t, J = 7.1 Hz, 2H), 1.85 (quint, J = 7.6 Hz,
2H). 13C NMR (150 MHz, CDCl3) d 173.3, 149.4, 146.3, 129.2,
123.5, 51.4, 34.7, 32.9, 25.4.
General method for preparation of amines 33 and 34, exempli-
fied by 33.
6.2.21. N1-(3,4-Dichlorophenyl)ethane-1,2-diamine (33)
Bromoethylamine hydrobromide salt (5.0 g, 25 mmol) was sus-
pended in toluene (100 mL) and 3,4-dichloroaniline (32, 12 g,
74 mmol, 3.0 equiv) was added. The brown solution was refluxed
for 1 h. The resulting mixture was cooled to room temperature to
give a brown precipitate which was filtered off and washed with
toluene. The solid was then treated with 20% w/v NaOH (100 mL)
and extracted with DCM (2ꢃ). The organics were extracted with
an acetic acid/sodium acetate buffer (300 mL of 0.1 M pH 5.5).
The resulting aqueous layer was then basified with 20% w/v NaOH
(100 mL) and extracted with DCM (2ꢃ). The combined organics
were collected and washed with water, dried over MgSO4, and con-
centrated in vacuo to give a brown oil. The crude material was
purified using silica gel chromatography (100% DCM to 10%
MeOH/DCM + 1% Et3N gradient) to give a brown residue (3.60 g,
72%). 1H NMR (400 MHz, CDCl3) d 7.17 (d, J = 8.6 Hz, 1H), 6.69 (s,
1H), 6.46 (d, J = 8.6 Hz, 1H), 4.25 (br s, 1H), 3.13 (t, J = 5.1 Hz,
2H), 2.96 (t, J = 5.1 Hz, 2H), 1.41 (br s, 2H). 13C NMR (150 MHz,
CDCl3) d 148.1, 133.0, 131.8, 114.0, 113.0, 46.2, 40.9. HRMS calcd
for C8H10Cl2N2, 204.0221; found, 205.02914 [M+H]+.
6.2.15. Methyl 4-(4-aminophenyl)butanoate (26)
Compound 26 was obtained from compound 26a (pink solid,
4.34 g, 100%). 1H NMR (400 MHz, CDCl3) d 6.93 (d, J = 7.0 Hz, 2H),
6.57 (d, J = 6.7 Hz, 2H), 3.68 (br s, 2H), 3.63 (s, 3H), 2.51 (t,
J = 7.3 Hz, 2H), 2.30 (t, J = 7.3 Hz, 2H), 1.88 (quint, J = 7.3 Hz, 2H).
13C NMR (100 MHz, CDCl3) d 173.9, 114.5, 130.8, 129.0, 115.0,
51.2, 34.0, 33.1, 26.6. HRMS calcd for C11H15NO2, 193.1103; found,
194.11743 [M+H]+.
Sulfonamides 27 and 28 were obtained in the same manner as
compound 12.
6.2.16. Methyl 2-(4-(methylsulfonamido)phenoxy)acetate (27)
Compound 27 was obtained from compound 25 (orange solid,
15.0 g, 90%). 1H NMR (300 MHz, CDCl3) d 7.22 (d, J = 9.0 Hz, 2H),
6.88 (d, J = 9.0 Hz, 2H), 4.65 (s, 2H), 3.83 (s, 3H), 2.96 (s, 3H). 13C
NMR (75 MHz, CDCl3) d 169.5, 155.8, 130.6, 124.2, 115.6, 65.5,
52.5, 38.7. HRMS calcd for C10H13NO5S, 259.0514; found,
264.00073 [M+Li]+.
6.2.22. N1-(3,4-Dichlorophenyl)propane-1,3-diamine (34)
Compound 34 was obtained from compound 32 and bromopro-
pylamine hydrobromide (brown residue, 45%). 1H NMR (400 MHz,
CDCl3) d 7.06 (d, J = 8.0 Hz, 1H), 6.54 (d, J = 2.9 Hz, 1H), 6.32 (dd,
J1 = 8.0 Hz, J2 = 2.9 Hz, 1H), 4.76–4.27 (br s, 1H), 3.01 (t, J = 6.7 Hz,
2H), 2.73 (t, J = 6.7 Hz, 2H), 2.25–1.84 (br s, 2H), 1.64 (quint,
J = 6.7 Hz, 2H). 13C NMR (100 MHz, CDCl3) d 148.0, 132.3, 130.3,
118.6, 113.2, 112.3, 41.9, 39.9, 31.7. HRMS calcd for C9H12Cl2N2,
218.0378; found, 219.04479 [M+H]+.
6.2.17. Methyl 4-(4-(methylsulfonamido)phenyl)butanoate (28)
Compound 28 was obtained from compound 26 (pink solid,
6.5 g, 107%). The crude material was used without further purifica-
tion. 1H NMR (300 MHz, CDCl3) d 7.43 (br s, 1H), 7.16 (d, J = 8.5 Hz,
2H), 7.10 (d, J = 8.5 Hz, 2H), 3.62 (s, 3H), 2.94 (s, 3H), 2.57 (t,
J = 7.3 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.88 (quint, J = 7.6 Hz, 2H).
13C NMR (75 MHz, CDCl3) d 174.1, 138.7, 134.9, 129.6, 121.5,
51.6, 39.3, 38.5, 33.3, 26.4.
General method for preparation of carboxylic acids 29–31,
exemplified by 30.
6.2.23. tert-Butyl 2-(3,4-dichlorophenoxy)ethylcarbamate (36)
To a solution of dry THF (40.0 mL), 3,4-dichlorophenol (35,
1.11 g, 6.82 mmol, 1.1 equiv), N-Boc-aminoethanol (0.962 mL,
6.20 mmol) and triphenyl phosphine (2.28 g, 8.68 mmol,
1.40 equiv) were added. Diisopropylazodicarboxylate (1.71 mL,
8.68 mmol, 1.4 equiv) was added dropwise over 5 min at 0 °C.
6.2.18. 3-(4-(Methylsulfonamido)phenyl)propanoic acid (30)
The sulfonamide ester 12 (1.16 g, 4.5 mmol) was dissolved in
methanol (50 mL). To this solution, 1.0 N NaOH (17.0 mL,
17.0 mmol, 3.8 equiv) was added. The mixture was stirred at room
temperature for 12 h. The pH of the solution was adjusted to ꢂ3

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C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
The mixture was warmed to room temperature and stirred for
12 h. The resulting solution was washed with 1.0 N NaOH
(20 mL), water (20 mL), and brine (20 mL). The organics were dried
over MgSO4 and concentrated in vacuo to give a yellow oil. The
crude material was purified using silica gel chromatography (1
EtOAc/1 hexanes) to give a colorless oil (1.30 g, 68%). 1H NMR
(400 MHz, CDCl3,) d 7.33 (d, J = 8.9 Hz, 1H), 7.00 (d, J = 2.9 Hz,
1H), 6.76 (dd, J1 = 8.9 Hz, J2 = 2.9 Hz, 1H), 4.96 (br s, 1H), 3.99 (t,
J = 5.1 Hz, 2H), 3.55–3.50 (mult, 2H), 1.46 (s, 9H).
THF, 3.1 mL, 6.1 mmol, 3.0 equiv) was added dropwise. The mix-
ture was stirred at room temperature for 12 h. The mixture was di-
luted with EtOAc and water, filtered over a pad of Celite, and the
resulting layers were separated. The organics were washed with
brine, dried over MgSO4, and concentrated in vacuo to give a yel-
low residue (0.400 g, 96%). 1H NMR (400 MHz, CD3O) d 7.40–7.36
(mult, 2H), 7.13 (dd, J1 = 8.9 Hz, J2 = 2.0 Hz, 1H), 2.68–2.61 (mult,
4H), 1.80–1.73 (mult, 2H). 13C NMR (100 MHz, CD3OD) d 143.0,
131.9, 130.3, 130.2, 129.4, 128.2, 40.5, 33.5, 32.0. HRMS calcd for
C9H11Cl2N, 203.0269; found, 204.03458 [M+H]+.
6.2.24. 2-(3,4-Dichlorophenoxy)ethanamine (37)
Carbamate 36 (1.30 g, 4.25 mmol) was dissolved in THF (30.0 mL)
and cooled to 0 °C. To this solution trifluoroacetic acid (10.0 mL,
130 mol, 31 equiv) was added dropwise to give a brown solution,
which was warmed to room temperature and stirred for 2 days.
The mixture was basified with solid sodium bicarbonate and parti-
tioned between water and ethyl acetate. The layers were separated
and the organics were washed with brine, dried over MgSO4, and con-
centrated in vacuo to give a yellow residue. The crude material was
purified using silica gel chromatography (10% MeOH/DCM) to give
a yellow oil (0.826 g, 94%). 1H NMR (300 MHz, CDCl3) d 7.28 (d,
J = 9.0 Hz, 1H), 6.92 (d, J = 2.8 Hz, 1H), 6.67 (dd, J1 = 9.0 Hz,
J2 = 2.8 Hz), 4.05 (t, J = 4.7 Hz, 2H), 3.52 (t, J = 4.3 Hz, 2H). 13C NMR
(75 MHz, CDCl3) d 156.6, 133.2, 125.5, 116.7, 114.4, 64.3, 39.5.
6.2.28. 2-Allylisoindoline-1,3-dione (45)
Potassium phthalimide (44, 2.59 g, 14 mmol, 1.4 equiv) was
added to anhydrous DMF (100 mL). To this solution allyl bromide
(0.86 mL, 10.0 mmol) was added dropwise. The reaction was
heated to 50 °C and stirred for 12 h. The DMF was removed in
vacuo and the remaining yellow liquid was dissolved in DCM.
The solution was washed with water (2ꢃ) and brine (2ꢃ). The
organics were dried over MgSO4 and concentrated in vacuo to
give an off-white solid. The crude material was purified using sil-
ica gel chromatography (50% DCM/50% hexanes) to give a white
solid (1.80 g, 96%). 1H NMR (400 MHz, CDCl3)
d 7.87 (dd,
J1 = 5.7 Hz, J2 = 2.9 Hz, 2H), 7.73 (dd, J1 = 5.7 Hz, J2 = 2.9 Hz, 2H),
5.90 (mult, 1H), 5.28–5.29 (mult, 2H), 4.31 (d, J = 5.4 Hz, 2H).
13C NMR (100 MHz, CDCl3) d 168.2, 134.2, 132.3, 131.7, 123.5,
118.0, 40.2.
6.2.25. 2-(3,4-Dichlorophenylthio)ethanamine (40)
Thiophenol (38, 1.00 g, 5.58 mmol) and oxazolidinone (38,
0.486 g, 5.58 mmol, 1.0 equiv) were heated to 130 °C, and stirred
for 2 h. After cooling to room temperature, the resulting green solid
was taken up in 10% HCl and heated for several minutes. The mix-
ture was filtered and the filtrate was made basic (pH 10) with solid
NaOH and extracted with DCM. The combined extracts were
washed with water and brine, dried over MgSO4, and concentrated
in vacuo to give a colorless residue. The crude material was puri-
fied using silica gel chromatography (10% MeOH/DCM) to give a
colorless residue (0.338 g, 27%). 1H NMR (600 MHz, CDCl3) d 7.43
(d, J = 1.9 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.18 (dd, J1 = 8.6 Hz,
J2 = 1.9 Hz, 1H), 3.02 (t, J = 6.7 Hz, 2H), 2.94 (t, J = 6.2 Hz, 2H),
1.59 (br s, 2H). HRMS calcd for C8H9Cl2NS, 220.9833; found,
221.99072 [M+H]+.
6.2.29. 2-(3,4-Dichlorocinnamyl)isoindoline-1,3-dione (47)
N-Allylphthalimide 45 (0.660 g, 3.53 mmol, 1.25 equiv) and
3,4-dichloroiodobenzene (46, 0.770 g, 2.82 mmol) were dissolved
in triethylamine (10.0 mL) and acetonitrile (10 mL). Palladium
acetate (6 mg, 0.028 mmol, 0.01 equiv) was added. The yellow
solution was heated at 100 °C in a thick-walled glass container
for 20 h. The reaction was cooled to room temperature, upon
which precipitation occurred to give a brown solid. The solid
was filtered and washed with water. The solid was then dissolved
in hot DMF to give an orange liquid which was filtered through
Celite to remove Pd(0). The resulting hot DMF filtrate was diluted
with an equal volume of water, upon which precipitation oc-
curred. The yellow solid was filtered off and dried in vacuo
(6.9 g, 85%) 1H NMR (400 MHz, DMSO-d6) d 7.92–7.84 (mult,
4H), 7.73 (d, J = 1.9 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.44 (dd,
J1 = 1.9 Hz, J1 = 8.3 Hz, 1H), 6.55 (d, J = 15.9 Hz, 1H), 6.50–6.43
(mult, 1H), 4.35 (d, J = 4.1 Hz, 2H). 13C NMR (100 MHz, DMSO-
d6) d 167.6, 137.1, 134.4, 131.7, 131.4, 130.6, 129.7, 128.7,
128.5, 128.2, 126.9, 126.3. HRMS calcd for C17H11Cl2NO2,
331.0167; found, 332.01368 [M+H]+.
6.2.26. 3-(3,4-Dichlorophenyl)propanamide (42)
3-(3,4-Dichlorophenyl)propionic acid (41, 3.00 g, 14 mmol)
was dissolved in benzene (30.0 mL) and treated with oxalyl cho-
ride (5.8 mL, 68 mmol, 5.0 equiv) and DMF (catalytic). The mix-
ture was stirred for 12 h at room temperature. The resulting
solution was concentrated in vacuo to give an opaque liquid
and dried in vacuo overnight. 1H, 13C NMR confirms the presence
of the acid chloride. The acid chloride was dissolved in ether
(24.0 mL), and ammonia (0.5 M solution in 1,4-dioxane, 82 mL,
41 mmol, 3.0 equiv) was added dropwise to give a white precipi-
tate. The mixture was stirred at room temperature for 2 h. The
reaction was quenched with water and diluted with ether, and
the layers were separated. The organics were washed with 2.0 N
HCl, 10% NaHCO3 solution, and brine. They were then dried over
MgSO4 and concentrated in vacuo to give a colorless oil which
solidified upon trituration with DCM and hexanes (1.40 g, 47%).
1H NMR (400 MHz, CDCl3) d 7.33 (d, J = 8.3 Hz, 1H), 7.29 (d,
J = 1.9 Hz, 1H), 7.04 (dd, J1 = 8.3 Hz, J2 = 1.9 Hz, 1H), 6.04 (br s,
1H), 5.61 (br s, 1H), 2.90 (t, J = 7.6 Hz, 2H), 2.49 (t, J = 7.6 Hz,
2H). 13C NMR (100 MHz, CDCl3) d 174.3. 141.1, 132.5, 130.1,
128.1, 37.0, 30.4.
6.2.30. (E)-3-(3,4-Dichlorophenyl)prop-2-en-1-amine (48)
Compound 47 (0.660 g, 1.99 mmol) was suspended in ethanol
(7.5 mL) and hydrazine hydrate (0.1 mL, 2.0 mmol, 1.1 equiv) was
added. The solution was refluxed (heating dissolved to give an or-
ange solution) overnight to give a yellow precipitate. The suspen-
sion was cooled to room temperature, and the precipitate was
filtered off and washed with ethanol. The yellow solid was then
suspended in water, and 1.0 N NaOH was added until the solid
mostly dissolved. DCM was added and the layers were separated.
The organics were washed with water, dried over MgSO4, and con-
centrated in vacuo to give a yellow residue (0.221 g, 55%). 1H NMR
(400 MHz, CDCl3) d 7.42 (d, J = 2.2 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H),
7.17 (dd, J1 = 8.5 Hz, J2 = 2.1 Hz, 1H), 6.41 (d, J = 15.9 Hz, 1H), 6.31
(mult, J1 = 16.0 Hz, J2 = 5.1 Hz, 1H), 3.47 (d, J = 5.4 Hz, 2H), 1.23
(br s, 2H). 13C NMR (150 MHz, CDCl3) d 137.6, 133.7, 130.6,
128.2, 127.2, 125.6, 44.3. m/z (APCI) calcd for C9H9Cl2N, 202.0;
found, 202.1[ M]+.
6.2.27. 3-(3,4-Dichlorophenyl)propan-1-amine (43)
Amide 42 (0.446 g, 2.0 mmol) was dissolved in THF (15.0 mL).
After cooling to 0 °C, lithium aluminum hydride (2.0 M sol’n in

C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6477
General method for preparation of amides 49–60, exemplified
by 49.
131.5, 129.0, 124.4, 118.5, 116.4, 113.9, 113.5, 68.4, 43.8, 38.9,
38.7. HRMS calcd for
C17H19Cl2N3O4S, 431.0473; found:
432.05383 [M+H]+. Anal. (C17H19Cl2N3O4S) C, H, N.
6.2.31. N-(2-(3,4-Dichlorophenylamino)ethyl)-3-(4-
(methylsulfonamido)phenyl)propanamide (49)
6.2.35. N-(3,4-Dichlorocinnamyl)-2-(4-(methylsulfonamido)-
phenoxy)acetamide (53)
Carboxylic acid 30 (0.700 g, 2.88 mmol) was dissolved in DMF
(30.0 mL) and cooled to 0°C. To this solution, DMAP (0.352 g,
2.28 mmol, 1.1 equiv), and EDCI (0.552 g, 2.88 mmol, 1.0 equiv)
were added to give a clear suspension. After stirring for 45 min,
amine 33 (0.590 g, 2.88 mmol, 1.0 equiv) in THF (5.0 mL) was
added dropwise. The mixture was warmed to room temperature
and stirred for 12 h. The mixture was concentrated in vacuo, and
the resulting residue was partitioned between 1.0 N HCl, and
EtOAc. After extracting with EtOAc (2ꢃ), the combined organic lay-
ers were dried over MgSO4 and concentrated in vacuo. The crude
material was purified by taking the residue up in DCM and stirring.
Immediately a white powder precipitated, which was collected by
filtration (0.920 g, 74%). 1H NMR (400 MHz, CD3OD) d 7.14–7.10
(mult, 5H), 6.72 (d, J = 2.9 Hz, 1H), 6.51 (dd, J1 = 8.9 Hz,
J2 = 2.6 Hz, 1H), 3.27 (t, 2H), 3.10 (t, J = 6.4 Hz, 2H), 2.88 (s, 3H),
2.87 (t, J = 6.5 Hz, 2H), 2.46, (t, J = 6.4 Hz, 2H). 13C NMR (75 MHz,
CDCl3) d 175.8, 150.0, 138.7, 138.3, 131.7, 130.5, 122.3, 114.4,
113.6, 44.0, 39.8, 39.2, 39.0, 32.3. HRMS calcd for C18H21Cl2N3O3S,
429.0681; found, 431.08143 [M+H]+. Anal. (C18H21Cl2N3O3S) C, H,
N.
Compound 53 was obtained using carboxylic acid 29 and amine
48. The crude material was purified using silica gel chromatogra-
phy (2 EtOAc/1 hexanes) to give a white solid (0.247, 71%). 1H
NMR (300 MHz, DMSO-d6) d 9.43 (s, 1H), 8.40 (br t, J = 5.7 Hz,
1H), 7.68 (d, J = 1.9 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.39 (dd,
J1 = 8.2 Hz, J2 = 1.9 Hz, 1H), 7.17 (d, J = 9.0 Hz, 2H), 8.96 (d,
J = 9.0 Hz, 2H), 6.41–6.39 (mult, 2H), 4.52 (s, 2H), 3.94–3.92 (mult,
2H), 2.89 (s, 3H). 13C NMR (75 MHz, DMSO-d6) d 167.6, 154.9,
137.5, 131.5, 131.4, 130.4, 129.7, 129.5, 127.9, 127.4, 126.2,
123.0, 115.4, 67.2, 39.0. HRMS calcd for C18H18Cl2N2O4S,
428.0364; found, 427.0296 [M-H]+. Anal. (C18H18Cl2N2O4S) C,H,N.
6.2.36. N-(3-(3,4-Dichlorophenyl)propyl)-2-(4-(methylsulfo-
namido)phenoxy)acetamide (54)
Compound 54 was obtained using carboxylic acid 29 and amine
43. The crude material was purified using silica gel chromatogra-
phy (3 EtOAc/1 hexanes) to give a yellow oil (0.380 g, 43%). 1H
NMR (400 MHz, CDCl3) d 7.37 (br s, 1H), 7.31 (d, J = 8.3 Hz, 1H),
7.26–7.23 (mult, 3H), 7.00 (dd, J1 = 8.1 Hz, J2 = 2.2 Hz, 1H), 6.89
(d, J = 8.8 Hz, 2H), 6.67 (br t, J = 5.71 Hz, 1H), 4.46 (s, 2H), 3.38 (q,
J = 6.7 Hz, 2H), 2.95 (s, 3H), 2.60 (t, J = 7.3 Hz, 2H), 1.87 (quint,
J = 7.3 Hz, 2H). 13C NMR (100 MHz, CDCl3) d 168.4, 155.3, 141.6,
132.4, 131.2, 130.5, 130.4, 130.1, 128.0, 124.3, 115.8, 67.7, 39.1,
38.7, 34.4, 30.9. HRMS calcd for C18H20Cl2N2O4S, 430.0521; found,
431.05898 [M+H]+. HPLC (a) 99.7% (b) 98.6%
6.2.32. N-(3,4-Dichlorocinnamyl)-3-(4-(methylsulfonamido)-
phenyl)propanamide (50)
Compound 50 was obtained using carboxylic acid 30 and amine
48. The crude material was purified using silica gel chromatogra-
phy (100% EtOAc) to give yellow crystals (1.05 g, 80%). 1H NMR
(400 MHz, DMSO-d6) d 9.61 (s, 1H), 8.11 (br t, J = 5.7 Hz, 1H),
7.69 (d, J = 1.9 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.38 (dd,
J1 = 8.6 Hz, J2 = 1.9 Hz, 1H), 7.18 (d, J = 8.3 Hz, 2H), 7.11 (d,
J = 8.6 Hz, 2H), 6.37–6.34 (mult, 2H), 3.83 (t, J = 5.1 Hz, 2H), 2.80
(t, J = 7.6 Hz, 2H), 2.92 (s, 3H), 2.41 (t, J = 8.2 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6) d 171.2, 137.5, 137.0, 136.2, 131.4, 130.7,
130.1, 129.4, 129.1, 127.9, 127.3, 126.2, 120.2, 39.0, 36.9, 30.4.
HRMS calcd for C19H20Cl2N2O3S, 426.0672; found, 427.06418
[M+H]+. Anal. (C19H20Cl2N2O3S) C, H, N.
6.2.37. N-(2-(3,4-Dichlorophenoxy)ethyl)-2-(4-(methylsulfo-
namido)phenoxy)acetamide (55)
Compound 55 was obtained using carboxylic acid 29 and amine
37.The crude material was purified using silica gel chromatography
(2 EtOAc/1 hexanes) to give a white foam (0.366 g, 41%). 1H NMR
(300 MHz, CDCl3) d 7.32 (s, 1H), 7.23 (d, J = 9.0 Hz, 2H), 7.05 (br t,
J = 5.7 Hz, 1H), 6.96 (d, J = 2.8 Hz, 1H), 6.89 (d, J = 9.0 Hz, 2H),
6.73 (dd, J1 = 9.0 Hz, J2 = 2.8 Hz, 1H), 4.50 (s, 2H), 4.04 (t,
J = 5.2 Hz, 2H), 3.76 (q, J = 5.5 Hz, 2H), 2.94 (s, 3H). 13C NMR
(75 MHz, CDCl3) d 168.6, 157.5, 155.2, 133.0, 131.1, 130.9, 124.6,
124.2, 116.6, 115.8, 114.5, 67.7, 67.2, 39.1, 38.6. HRMS calcd for
C17H18Cl2N2O5S, 432.0313; found, 433.03824 [M+H]+. Anal.
(C18H18Cl2N2O4S) C, H, N.
6.2.33. N-(4-(3-(4-(3,4-Dichlorophenyl)piperazin-1-yl)-3-oxo-
propyl)phenyl)methanesulfonamide (51)
Compound 51 was obtained using carboxylic acid 30 and 1-
(3,4-dichlorophenyl)piperazine. The crude material was purified
using silica gel chromatography (100% EtOAc) to give an off-white
foam (0.320 g, 28%). 1H NMR (600 MHz, CDCl3) d 7.37 (s, 1H), 7.34
(d, J = 8.6 Hz, 1H), 7.15 (s, 4H), 6.88 (d, J = 2.4 Hz, 1H), 6.67 (dd,
J1 = 8.8 Hz, J2 = 2.8 Hz, 1H), 3.71 (t, J = 4.8 Hz, 2H), 3.51 (t,
J = 4.8 Hz, 2H), 3.06 (t, J = 5.2 Hz, 2H), 3.01 (t, J = 5.2 Hz, 2H), 2.92
(t, J = 7.6 Hz, 2H), 2.91 (s, 3H), 2.62 (t, J = 7.6 Hz, 2H). 13C NMR
(150 MHz, CDCl3) d 170.9, 150.4, 138.5, 135.4, 133.1, 130.8,
129.9, 123.2, 121.6, 118.0, 116.0, 49.1, 49.0, 45.3, 41.5, 39.3, 34.9,
30.9. HRMS calcd for C20H23Cl2N3O3S, 455.0837; found,
456.09066 [M+H]+. HPLC (a) 99.5% (c) 99.6%.
6.2.38. N-(2-(3,4-Dichlorophenylthio)ethyl)-2-(4-(methylsulfo-
namido)phenoxy)acetamide (56)
Compound 56 was obtained using carboxylic acid 29 and amine
40. The crude material was purified using silica gel chromatogra-
phy (3 EtOAc/1 hexanes) to give a white foam (0.389 g, 69%). 1H
NMR (600 MHz, CDCl3)
d 7.44 (d, J = 2.4 Hz, 1H), 7.32 (d,
J = 8.6 Hz, 1H), 7.29 (br s, 1H), 7.24 (d, J = 8.6 Hz, 2H), 7.19 (dd,
J1 = 8.3 Hz, J2 = 2.4 Hz, 1H), 6.98 (br t, J = 5.7 Hz, 1H), 6.88 (d,
J = 9.0 Hz), 4.45 (s, 2H), 3.58 (quart, J = 6.2 Hz, 2H), 3.10 (t,
J = 6.7 Hz, 2H), 2.96 (s, 3H). 13C NMR (150 MHz, CDCl3) d 168.6,
155.3, 135.6, 133.2, 131.2, 131.0, 130.9, 130.8, 128.8, 124.4,
115.9, 67.7, 39.2, 39.1, 38.5, 33.3. HRMS calcd for C17H18Cl2N2O4S2,
448.0085; found, 449.01536 [M+H]+. Anal. (C17H18Cl2N2O4S2) C, H,
N.
6.2.34. N-(2-(3,4-Dichlorophenylamino)ethyl)-2-(4-(meth-
ylsulfonamido)phenoxy)acetamide (52)
Compound 52 was obtained using carboxylic acid 29 and amine
33. The crude material was purified using silica gel chromatogra-
phy (100% EtOAc) to give a white powder (0.970 g, 55%). 1H NMR
(400 MHz, acetone-d6)
d
7.28 (d, J = 8.8 Hz, 2H), 7.20 (d,
6.2.39. N-(4-(2-(4-(3,4-Dichlorophenyl)piperazin-1-yl)-2-oxo-
ethoxy)phenyl)methanesulfonamide (57)
Compound 57 was obtained using carboxylic acid 29 and 1-
(3,4-dichlorophenyl)piperazine. Upon addition of 1.0 N HCl a white
powder precipitated out which was obtained by filtration and
J = 8.8 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.79 (d, J = 2.9 Hz, 1H),
6.61 (dd, J1 = 8.8 Hz, J2 = 2.9 Hz, 1H), 4.47 (s, 2H), 3.78 (s, 1H),
3.49 (quint, J = 6.3 Hz, 2H), 3.28 (quint, J = 6.3 Hz, 2H), 2.89 (s,
3H). 13C NMR (100 MHz, acetone-d6) d 169.3, 156.3, 149.7, 132.9,

6478
C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
washing with hexanes (0.775 g, 85%). 1H NMR (600 MHz, DMSO-
d6) d 9.39 (s, 1H), 7.42 (d, J = 9.1 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H),
7.13 (d, J = 9.1 Hz, 2H), 6.96 (dd, J1 = 9.1 Hz, J2 = 2.3 Hz, 1H), 6.92
(d, J = 9.1 Hz, 2H), 4.84 (s, 2H), 3.27–3.18 (mult, 4H), 2.88 (s, 3H).
13C NMR (150 MHz, DMSO-d6) d 165.9, 155.3, 150.4, 131.5, 131.2,
130.5, 123.2, 120.0, 116.6, 115.6, 115.3, 59.8, 47.7, 47.4, 43.7,
40.7, 38.7. HRMS calcd for C19H21Cl2N3O4S, 457.0630; found,
458.07034 [M+H]+. Anal. (C19H21Cl2N3O4Sꢁ0.8H2O) C, H, N.
extracted once more with EtOAc. The organic layers were com-
bined, dried over MgSO4 and volatiles removed in vacuo. The
remaining yellow residue was purified using silica gel chromatog-
raphy in 2:1 EtOAc/hexanes to yield 62 (3.0 g, 82%) as a yellow so-
lid. 1H NMR (300 MHz, CDCl3) d 8.21 (d, J = 9.3 Hz, 2H), 6.96 (d,
J = 9.3 Hz, 2H), 4.95 (br s, 1H), 4.11 (t, J = 5.4 Hz, 2H), 3.56 (t,
J = 5.4 Hz, 2H), 1.45 (s, 9H).
6.2.44. tert-Butyl N-2-(4-aminophenoxy)ethylcarbamate (63)
Nitro compound, 62, (3.0 g, 11.0 mmol, 1.0 equiv) was dis-
solved in 20 mL of MeOH. 5% palladium on carbon catalyst
(0.6 g, 10 wt %) was added to the reaction mixture. The suspension
was hydrogenolyzed using a balloon for 16 h. The reaction mix-
ture was filtered over a pad of Celite and washed with DCM,
EtOAc, and MeOH. The volatiles were removed in vacuo to yield
63 (2.7 g, 100%) as a brown-red oil. 1H NMR (300 MHz, CD3OD)
d 6.88 (mult, 4H), 3.90 (t, J = 5.7 Hz, 2H), 3.36 (t, J = 5.7 Hz, 2H),
1.41 (s, 9H).
6.2.40. N-(3,4-Dichlorophenethyl)-4-(4-(methylsulfonamido)-
phenyl)butanamide (58)
Compound 58 was obtained using carboxylic acid 31 and 3,4-
dichlorophenylethylamine. The crude material was purified using
silica gel chromatography (50% EtOAc/50% hexanes to 100% EtOAc
gradient) to give a white foam which solidified in vacuo (0.571 g,
68%). 1H NMR (600 MHz, CDCl3) d 7.57 (s, 1H), 7.29 (d, J = 8.1 Hz,
1H), 7.23 (d, J = 1.9 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.03 (d,
J = 8.1 Hz, 2H), 6.99 (dd, J1 = 8.1 Hz, J2 = 1.9 Hz, 1H), 5.82 (br t,
J = 6.2 Hz, 1H), 3.44 (quart, J = 6.2 Hz, 2H), 2.92 (s, 3H), 2.73 (t,
J = 7.1 Hz, 2H), 2.52 (t, J = 7.1 Hz, 2H), 2.11 (t, J = 7.1 Hz, 2H), 1.84
(quint, J = 8.1 Hz, 2H). 13C NMR (150 MHz, CDCl3) d 173.3, 139.4,
138.9, 135.2, 132.5, 130.9, 130.7, 130.6, 129.7, 128.5, 121.6, 40.4,
39.3, 35.9, 35.0, 34.6, 27.3. HRMS calcd for C19H22Cl2N2O3S,
428.0728; found, 429.08027 [M+H]+. Anal. (C19H22Cl2N2O3S) C, H,
N.
6.2.45. tert-Butyl N-2-(4-
(methylsulfonamido)phenoxy)ethylcarbamate (64)
Aniline 63, (2.5 g, 10 mmol, 1.0 equiv) was suspended in DCM
(30 mL). Diisopropylethylamine (2 mL, 12 mmol, 1.2 equiv) was
added to the reaction mixture and stirred for 15 min. The reaction
mixture was cooled to 0 °C and methanesulfonyl chloride (0.9 mL,
11 mmol, 1.1 equiv) was added dropwise to the solution. The reac-
tion was warmed to room temperature and stirred for 10 h. The
reaction was quenched with water and extracted with DCM (3ꢃ).
The organic layers were combined and washed with brine and
water, dried over MgSO4, and concentrated in vacuo. The remain-
ing brown residue was purified using silica gel chromatography
(1:1 EtOAc/hexanes) to yield an off-white solid (0.5 g, 15%). 1H
NMR (300 MHz, CD3OD) d 7.17 (d, J = 6.9 Hz, 2H), 6.89 (d,
J = 6.9 Hz, 2H), 3.96 (t, J = 5.4 Hz, 2H), 3.39 (t, J = 3.5 Hz, 2H), 2.85
(s, 3H), 1.42 (s, 9H). 13C NMR (75 MHz, CD3OD) d 123.96, 115.07,
67.00, 41.73, 39.77, 37.44, 27.54. HRMS calcd for C14H22N2O5S,
330.1249; found, 331.13245 [M+H]+.
6.2.41. N-(3-(3,4-Dichlorophenylamino)propyl)-2-(4-(methyl-
sulfonamido)phenoxy)acetamide (59)
Compound 59 was obtained using carboxylic acid 29 and amine
34. The crude material was purified using silica gel chromatogra-
phy (2 EtOAc/1 hexanes to 100% EtOAc gradient) to give a white
foam. Trituration with MeOH give a white solid obtained by filtra-
tion (0.177 g, 64%). 1H NMR (600 MHz, CD3OD) d 7.22 (d, J = 9.1 Hz,
2H), 7.14 (d, J = 9.1 Hz, 1H), 6.97 (d, J = 9.1 Hz, 2H), 6.68 (d,
J = 2.9 Hz, 1H), 6.48 (dd, J1 = 9.1 Hz, J2 = 2.9 Hz, 1H), 4.50 (s, 2H),
3.38 (mult, 2H), 3.05 (t, J = 6.7 Hz, 2H), 2.87 (s, 3H), 1.81 (quint,
J = 6.7 Hz, 2H). 13C NMR (150 MHz, CD3OD) d 171.3, 156.9, 150.3,
133.6, 133.3C, 125.0, 119.4, 116.8, 114.5, 113.7, 68.6, 42.0, 38.9,
38.0, 29.8. HRMS calcd for C18H21Cl2N3O4S, 445.0630; found,
446.07031 [M+H]+. Anal. (C18H21Cl2N3O4S) C, H, N.
6.2.46. N-(4-(2-Aminoethoxy)phenyl)methanesulfonamide
hydrochloride (65)
Sulfonamide 64 (0.5 g, 2 mmol, 1.0 equiv), was dissolved in
DCM (10 mL). Trifluoroacetic acid (5 mL) was added to the solution
and the reaction mixture was stirred for 3 h at room temperature.
The reaction was quenched with water and extracted with DCM.
The organics were washed with brine and then dried over MgSO4.
Volatiles were removed in vacuo to yield 0.17 g of a brown oil. The
free base was converted to the HCl salt by bubbling HCl (g) through
a solution of the amine dissolved in EtOAc. The resulting precipi-
tate was filtered to yield a purple solid (0.2 g, 49%). 1H NMR
(300 MHz, CD3OD) d 7.13 (d, J = 9 Hz, 2H), 6.91 (d, J = 9 Hz, 2H),
4.12 (t, J = 4.8 Hz, 2H), 3.28 (t, J = 3.6 Hz, 2H).
6.2.42. N-(3-(3,4-Dichlorophenylamino)propyl)-3-(4-
(methylsulfonamido)phenyl)propanamide (60)
Compound 60 was obtained using carboxylic acid 31 and amine
34. The crude material was purified using silica gel chromatogra-
phy (2 EtOAc/1 hexanes to 100% EtOAc gradient) to give a white
foam which solidified in vacuo (0.182 g, 40%). 1H NMR (300 MHz,
CD3OD) d 7.99 (br s, 1H), 7.21–7.14 (mult, 5H), 6.68 (d, J = 2.3 Hz,
1H), 6.48 (dd, J1 = 8.8 Hz, J2 = 2.6 Hz, 1H), 3.23–3.19 (mult, 2H),
2.95 (t, J = 6.7 Hz, 2H), 2.89 (s, 3H), 2.47 (t, J = 7.6 Hz, 2H), 1.68
(quint, J = 6.7 Hz, 2H). 13C NMR (150 MHz, CD3OD) d 174.0, 148.9,
137.5, 136.4, 132.3, 130.4, 129.2, 120.9, 118.0, 113.1, 112.3, 40.5,
37.8, 37.6, 36.7, 31.0, 28.5. HRMS calcd for C19H23Cl2N3O3S,
443.0837; found, 444.09059 [M+H]+. Anal. (C19H23Cl2N3O3S) C, H,
N.
6.2.47. 2-Chloro-N-(3,4-dichlorophenyl)acetamide (66)
3,4-Dichloroaniline (32, 2.0 g, 12 mmol, 1.0 equiv) and triethyl-
amine (6.8 mL, 49 mmol, 4 equiv) were suspended in DCM (50 mL).
The mixture was cooled to 0 °C and chloroacetyl chloride (0.98 mL,
12 mmol, 1 equiv) was added dropwise. Immediate evolution of
gas occured upon addition of the acid chloride as well as a notable
color change from red-orange to dark brown. The reaction was
warmed to room temperature and stirred for 2 h. The reaction mix-
ture was washed with 1 M HCl. The aqueous layer was reextracted
with DCM. The organics were combined and dried over MgSO4. The
organics were removed and the solid was concentrated in vacuo to
give an off-white solid (2.9 g, 99%). 1H NMR (300 MHz, CDCl3) d
8.28 (br s, 1H), 7.81 (mult, 1H), 7.41 (mult, 2H), 4.21 (s, 2H). 13C
NMR (75 MHz, CDCl3) d 130.8, 121.9, 119.4, 43.0.
6.2.43. tert-Butyl N-2-(4-nitrophenoxy)ethylcarbamate (62)
4-Nitrophenol (61, 1.9 g, 14 mmol, 1.1 equiv), tert-butyl N-2-
hydroxyethylcarbamate (2.0 mL, 13.0 mmol, 1.0 equiv) and tri-
phenylphosphine (4.47 g, 18.0 mmol, 1.4 equiv) were dissolved in
60 mL of dry THF. To this mixture, DIAD (3.6 mL, 18.0 mmol,
1.4 equiv) was added dropwise at 0 °C. The reaction was allowed
to slowly warm to room temperature and stirred for 16 h. The
resulting mixture was concentrated in vacuo and the remaining
yellow-orange residue was dissolved in EtOAc, treated with 1.0 N
NaOH and washed with water and brine. The aqueous layer was

C. A. Mosley et al. / Bioorg. Med. Chem. 17 (2009) 6463–6480
6479
6.2.48. N-(3,4-Dichlorophenyl)-2-(2-(4-(methylsulfonamido)-
phenoxy)ethylamino) acetamide (67)
1.1 equiv) was added, and the mixture was stirred at room temper-
ature for 12 h. The solution was concentrated in vacuo and the res-
idue was taken up in ethyl acetate, washed with brine, dried over
Na2SO4, and concentrated in vacuo to give a colorless oil. The crude
material was purified using silica gel chromatography (100%
EtOAc) to give a white foam (0.070 g, 58%). 1H NMR (400 MHz,
CDCl3) d 7.72 (s, 1H), 7.16 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.6 Hz,
2H), 7.02 (s, 1H), 6.64 (d, J = 2.9 Hz, 1H), 6.41 (dd, J1 = 8.5 Hz,
J2 = 2.9 Hz, 1H), 3.64 (t, J = 6.0 Hz, 2H), 3.40–3.36 (mult, 4H), 2.97
(s, 3H), 2.57 (t, J = 7.3 Hz, 2H), 1.26 (t, J = 7.3 Hz, 2H). 13C
(75 MHz, CDCl3) d 152.6, 147.1, 137.3, 136.8, 135.6, 130.9, 129.5,
121.6, 118.0, 113.6, 112.5. HRMS calcd for C19H21Cl2N3O3S,
441.0681; found, 442.07527 [M+H]+. HPLC (a) 99.1% (b) 99.6%.
Compound 65 (0.26 g, 1 mmol, 1.9 equiv) was suspended in THF
(20.0 mL). Triethylamine (0.2 mL, 1 mmol, 2 equiv) was added to
the mixture and 66 (0.15 g, 0.6 mmol, 1 equiv) was added after
10 min. The reaction mixture was refluxed and stirred for 16 h.
The reaction mixture was basified with NaHCO3 and extracted with
DCM (2ꢃ). The organic layers were washed with water and brine,
and then dried over MgSO4, and concentrated in vacuo. The result-
ing free base was converted to the HCl salt by bubbling HCl (g)
through a solution of the substrate in EtOAc. The volatiles were re-
moved in vacuo to yield an orange solid (0.2 g, 76%). 1H NMR
(300 MHz, CD3OD) d 7.85 (d, J = 2.4 Hz, 1H), 7.43 (mult, 1H), 7.38
(mult, 1H), 7.11 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 4.40
(mult, 2H), 3.93 (s, 2H), 3.20 (t, J = 3.6 Hz, 2H) 2.87 (s, 3H). 13C
NMR (100 MHz, CD3OD) d 165.8, 155.9, 137.5, 132.1, 131,8,
130.5, 127.2, 123.8, 123.3, 121.3, 115.2, 67.4, 54.7, 39.1, 37.6.
HRMS calcd for C17H19Cl2N3O4S, 431.0473; found, 432.05423
[M+H]+. Anal. (C17H19Cl2N3O4S) C, H, N.
6.2.52. N-(4-(2-(3-(3,4-Dichlorophenyl)-2-oxoimidazolidin-1-
yl)ethoxy)phenyl)methanesulfonamide (71)
Compound 71 was obtained using amine 69 (white foam,
0.100 g, 56%). 1H NMR (400 MHz, CDCl3) d 7.62 (d, J = 2.5 Hz, 1H),
7.30 (dd, J1 = 8.9 Hz, J2 = 2.5 Hz, 1H), 7.25 (d, J = 8.9 Hz, 1H), 7.12
(d, J = 8.9 Hz, 2H), 6.77 (d, J = 8.9 Hz, 2H), 4.05–4.02 (mult, 2H),
3.70–3.59 (mult, 6H), 2.84 (s, 3H). 13C NMR (100 MHz, CDCl3) d
157.4, 156.8, 140.0, 132.6, 130.4, 130.0, 125.5, 124.6, 118.9,
116.6, 115.4, 67.4, 38.9. HRMS calcd for C18H19Cl2N3O4S,
444.0473; found, 444.05451 [M]+. HPLC (a) 98.3% (b) 97.6%.
General method for preparation of amines 68 and 69, exempli-
fied by 68.
6.2.49. N-(4-(3-(2-(3,4-Dichlorophenylamino)ethylamino)-
propyl)phenyl)methanesulfonamideꢁHCl (68)
Amide 49 (0.500 g, 1.2 mmol) was dissolved in THF (30.0 mL).
After cooling to 0 °C, a solution of lithium aluminum hydride
(2.0 M solution in THF, 2.3 mL, 4.6 mmol, 4.0 equiv) was added
dropwise. After stirring for 10 min at 0 °C, the ice bath was re-
moved and the reaction mixed was warmed to room temperature
and stirred for 12 h. The mixture was diluted with DCM and water
to give an emulsion. Rochelle’s salt (satd) was added and the mix-
ture stirred for 20 min before filtering over a pad of Celite. The
resulting liquid was separated, and the organics were washed with
brine, dried over MgSO4, and concentrated in vacuo to give a white
foam. The free base was converted to the HCl salt by bubbling HCl
(g) through a solution of substrate dissolved in ethanol. The white
powder precipitated out and was collected by filtration (0.358 g,
74%) .1H NMR (300 MHz, CDCl3) d 7.20–7.11 (mult, 5H), 6.69 (d,
J = 2.8 Hz, 1H), 6.46 (dd, J1 = 8.8 Hz, J2 = 2.8 Hz, 1H), 4.36 (br s,
1H), 3.16 (mult, 2H), 3.00 (s, 3H), 2.88 (t, J = 6.2 Hz, 2H), 2.66 (t,
J = 7.1 Hz, 4H), 1.86–1.79 (mult, 3H). 13C NMR (75 MHz, CDCl3) d
148.1, 139.4, 134.8, 132.8, 130.7, 129.7, 121.6, 119.7, 113.9,
112.9, 49.0, 48.2, 43.2, 39.3, 32.9, 31.5. HRMS calcd for
C18H22Cl2N3O2S, 429.0681; found, 431.08143 [M+H]+. Anal.
(C18H23Cl2N3O2Sꢁ0.45 HCl) C, H, N.
Acknowledgements
The authors are grateful to Drs. S. Nakanishi, P. Seeburg, and S.
F. Heinemann for cDNAs encoding the glutamate receptor subunits.
We thank Kimberly Vellano for excellent technical assistance. This
work was supported in part by the NINDS (NS036654, ST;
NS036604, RD).
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6.2.50. N-(4-(2-(2-(3,4-Dichlorophenylamino)ethylamino)-
ethoxy)phenyl)methanesulfonamideꢁHCl (69)
Compound 69 was obtained from amide 52 (0.486 g, 50%). 1H
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6.81 (d, J = 8.8 Hz, 2H), 6.68 (d, J = 2.8 Hz, 1H), 6.46 (dd,
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