Benzenesulfonamides: A Unique Class of Chemokine Receptor Type4 Inhibitors

Article abstract of DOI:10.1002/cmdc.201200582

The interaction of CXCR4 with CXCL12 (SDF-1) plays a critical role in cancer metastasis by facilitating the homing of tumor cells to metastatic sites. Based on our previously published work on CXCR4 antagonists, we have synthesized a series of aryl sulfonamides that inhibit the CXCR4/CXCL12 interaction. Analogue bioactivities were assessed with binding affinity and Matrigel invasion assays. Computer modeling was employed to evaluate a selection of the new analogues docked into the CXCR4 X-ray structure and to rationalize discrepancies between the affinity and Matrigel invitro assays. A lead compound displays nanomolar potency in the binding affinity assay (IC₅₀=8.0nM) and the Matrigel invasion assay (100% blockade of invasion at 10nM). These data demonstrate that benzenesulfonamides are a unique class of CXCR4 inhibitors with high potency.

Full text of DOI:10.1002/cmdc.201200582

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DOI: 10.1002/cmdc.201200582
Benzenesulfonamides: A Unique Class of Chemokine
Receptor Type 4 Inhibitors
Suazette Reid Mooring,*[a, b] Jin Liu,[c] Zhongxing Liang,[a, d] Jeffrey Ahn,[a] Samuel Hong,[a]
Younghyoun Yoon,[a] James P. Snyder,*[c] and Hyunsuk Shim*[a, d]
The interaction of CXCR4 with CXCL12 (SDF-1) plays a critical
role in cancer metastasis by facilitating the homing of tumor
cells to metastatic sites. Based on our previously published
work on CXCR4 antagonists, we have synthesized a series of
aryl sulfonamides that inhibit the CXCR4/CXCL12 interaction.
Analogue bioactivities were assessed with binding affinity and
Matrigel invasion assays. Computer modeling was employed to
evaluate a selection of the new analogues docked into the
CXCR4 X-ray structure and to rationalize discrepancies be-
tween the affinity and Matrigel in vitro assays. A lead com-
pound displays nanomolar potency in the binding affinity
assay (IC50 =8.0 nm) and the Matrigel invasion assay (100%
blockade of invasion at 10 nm). These data demonstrate that
benzenesulfonamides are a unique class of CXCR4 inhibitors
with high potency.
Introduction
The G-protein-coupled receptor (GPCR) CXCR4 and
the chemokine stromal cell-derived factor-1 (SDF-1,
also known as CXCL12) play a crucial role in physio-
logical processes such as leukocyte migration/traf-
ficking and hematopoiesis.[1] The interaction of
CXCL12 with CXCR4 has implications in cancer meta-
stasis,[2] and CXCR4 is a co-receptor for HIV-1 infec-
tion.[3] CXCR4 is frequently overexpressed in solid
tumors as compared with normal tissue.[4] Activation
of the CXCR4/CXCL12 pathway can lead to the re-
cruitment of distal stroma by tumor cells to facilitate
tumor growth and metastasis, and can promote
homing of tumor cells to metastatic sites,[5] angio-
genesis,[6] cancer cell survival, and invasion.[4,7] Therefore, dis-
ruption of the interaction between CXCL12 and CXCR4 could
block or delay metastasis.
Figure 1. Structures of CXCR4 blockers IT1t, 1, and peptidic antagonists 2 and 12.
onists[9] that led to a potent dipyridine: WZ811 (1; Figure 1).
Potent pyridine and dipyrimidine analogues have been synthe-
sized by our research group[10] and by others.[11] Exploration of
tunable areas around the dipyridine pharmacophore led us to
Thus far, the most explored non-peptidic anti-CXCR4 agents
are bicyclams such as AMD3100 and its derivatives.[8] However,
their metal-chelating properties may be the cause of cardiotox-
icity and therefore may well limit the clinical use of these com-
pounds.[8] We recently identified a novel class of CXCR4 antag-
prepare
a new class of benzenesulfonamide analogues
(Figure 2). The central phenyl ring was retained because it has
been shown to be important for inhibitory activity.[9a] A binding
affinity assay against the potent CXCR4 antagonist TN14003 (2;
Figure 1) was employed as a primary screening method for the
new analogues.[9a,12] Some of these compounds were further
[a] Dr. S. R. Mooring, Dr. Z. Liang, J. Ahn, S. Hong, Dr. Y. Yoon, Dr. H. Shim
Department of Radiology and Imaging Sciences
Emory University, Atlanta, GA (USA)
E-mail: hshim@emory.edu
[b] Dr. S. R. Mooring
Current address: Department of Chemistry
Georgia State University, Atlanta, GA (USA)
E-mail: smooring@gsu.edu
[c] Dr. J. Liu, Dr. J. P. Snyder
Department of Chemistry and Emory Institute for Drug Discovery
Emory University, Atlanta, GA (USA)
E-mail: jsnyder@emory.edu
[d] Dr. Z. Liang, Dr. H. Shim
Figure 2. Design of new sulfonamide analogues based on the scaffold of
compound 1 (R1, R2, and R3: alkyl or aryl substituents).
Winship Cancer Institute, Emory University, Atlanta, GA (USA)
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analyzed in the Matrigel invasion assay by using full-length
CXCL12.
Chemistry
A small series of mono- and disulfonamide analogues of
1 (Figure 2) were prepared with a selection of substituents to
test the viability of this structural class as CXCR4 antagonists.
Compounds 3a and 3b were synthesized in one step by the
reaction of xylylenediamine with the corresponding sulfonyl
chlorides (Scheme 1). Compounds 5a–n were prepared by the
treatment of 4-(bromomethyl)benzenesulfonyl chloride with
the corresponding secondary amine to give compounds 4a–d,
which were treated with the appropriate amine to give the
final products (Scheme 2). Compound 7 was obtained similarly
by treating 4-(bromomethyl)benzenesulfonyl chloride with N-
methyl-2-pyridinemethanamine to give 6, which was subse-
quently coupled with pyrrolidine in the presence of potassium
carbonate to give the final target (Scheme 2). As shown in
Scheme 3, N-Boc-4-hydroxyani-
Scheme 1. Synthesis of 3a and 3b. Reagents and conditions: a) DIPEA,
CH2Cl2, RT, 3 h.
Therefore, this screen is a semi-quantitative first-pass measure
of the level of activity, and should not be confused with IC50
determination.
Initially, five compounds—3a, 3b, 5a, 5b, and 11 a—were
synthesized and analyzed for binding. As shown in Table 1, the
disulfonamides 3a and 3b were found to be relatively inactive.
However, the monosubstituted sulfonamides 5a, 5b, and 11 a
were more effective, with respective EC values of 10, 1, and
10 nm. Hence, we pursued analogues with the monosubstitut-
line was treated with 2-chloro-
N,N-diethylethanamine to yield
8 and subsequently deprotected
with hydrochloric acid in diox-
ane to give 9 as the hydrochlo-
ride salt. Compound 9 was then
combined with 4-(bromome-
thyl)benzenesulfonyl chloride to
give 10, which yielded 11 a–d
upon treatment with the corre-
sponding amines.
Results and Discussion
Binding affinity assays
The compounds were initially
screened with a binding affinity
assay involving competition of 2
with the putative antagonists, as
Scheme 2. Synthesis of 5a–n and 7. Reagents and conditions: a) CH2Cl2, RT, 12 h; b) amine, Et3N or K2CO3, CH2Cl2
described in our previous publi-
cations.[12,9a] MDA-MB-231 cells
were pre-incubated with com-
pounds at concentrations of 1,
10, 100, and 1000 nm, then incu-
bated with biotinylated 2 and
streptavidin-conjugated rhoda-
mine to determine the binding
efficiency of the new analogues
to the CXCL12 binding domain
of CXCR4. The effective concen-
tration (EC) is defined as the
lowest concentration at which
a significant decrease in rhoda-
mine fluorescence is observed
relative to control, reflecting
or CH3CN, RT, 12 h; c) CH2Cl2, RT, 12 h; d) pyrrolidine, K2CO3, CH3CN, RT, 12 h.
Scheme 3. Synthesis of 11 a–d. Reagents and conditions: a) CH2Cl2, RT, 3 h; b) HCl/dioxane, dioxane, RT, 12 h;
competitive displacement by 2. c) DIPEA, THF/DMF, RT, 12 h; d) amine, CH2Cl2, RT, 12 h.
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ed sulfonamide motif as potential CXCR4 blockers
using the preliminary and less time-consuming EC
binding assay. Thus, the molecular sectors to the left
(region A) and to the right (region B) of the mono-
substituted sulfonamide structure 5b (Figure 3) were
Table 1. Sulfonamide analogues 3 and 5 evaluated by binding affinity and Matrigel
invasion assays.
Compound
EC [nm] Invasion inhib. [%][a]
10 nm
100 nm
3a
1000
NA
NA
Figure 3. Two regions (A and B) modified for preliminary struc-
ture–activity pattern.
3b
1000
95
100
explored by substitution and binding affinity screen-
ing against 2. The central phenyl ring was left un-
touched, as previous work in our research group has
shown that this ring is critical for activity.[9a,10a] Sever-
al cyclic and acyclic amines were introduced to re-
gion A of the benzenesulfonamide motif (Scheme 2).
The morpholine derivative 5c and the pyrrole deriv-
ative 5 f were not well tolerated, and showed EC
values >1000 nm. However, the potencies of diethyl
(5d) and piperazine (5e) derivatives were similar to
the initially synthesized compounds 5a and 5b.
Small non-aromatic rings appear to be favored in re-
gion A.
5a
10
100
100
5b
5c
5d
5e
5 f
1
1000
10
94
0
100
76
Next, region B was explored by adding substituted
aromatic rings (fluoro, tert-butyl, or methoxy groups)
or a pyridine ring, while retaining the piperidine,
pyrrolidine, or diethylamine groups in region A. Sur-
prisingly, addition of fluoro or methoxy groups at
the para position (5h, 5i, and 5l), as well as ortho-
and meta-fluorine (5m and 5n, respectively), result-
ed in a significant decrease in binding relative to 5b.
However, addition of a tert-butyl moiety to the para
position produced the potent compounds 5j and
5k. Replacement of the phenyl ring of region B with
a pyridine ring (compound 7) likewise enhanced po-
tency. Due to the success of 11 a, additional ana-
logues were prepared that retained the diethylamine
fragment in region B, but varied region A with cyclic,
acyclic, or aromatic substituents. In this case, the
five- and six-membered rings were well tolerated in
region A (11 c and 11 d). However, the morpholine-
substituted 11 b showed poor activity, as was the
case for the other morpholine analogue 5c.
71
78
32
84
1
76
>1000
61
5g
5h
5i
100
1000
1000
48
61
64
72
82
99
Because we used only four concentrations (1, 10,
100, and 1000 nm) for the TN14003 binding assay to
determine EC values, we performed seven-point
evaluations for several compounds, with EC values
ranging from 1 to 1000 nm (Figure 4). The compara-
tive results (Table 2) illustrate that the rhodamine
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assay is semi-quantitative and able to demonstrate
binding at a minimum within a factor of 5–10.
Table 1. (Continued)
Compound
EC [nm] Invasion inhib. [%][a]
10 nm
100 nm
5j
1
40
88
Matrigel invasion assays
The Matrigel invasion assay, a functional probe using
full-length CXCL12, was employed as a secondary
functional assay to test whether the compounds can
block CXCR4/CXCL12-mediated chemotaxis and in-
vasion at two concentrations (10 and 100 nm).[12] The
reason for selecting these two concentrations was to
identify potent compounds that are reasonably
active at least at 100 nm and to show concentration
dependence. The results of both binding affinity and
Matrigel invasion for all analogues are listed in
Table 1 except for compounds showing a binding af-
finity >1000 nm. We previously demonstrated that
the dominant effects of our Matrigel invasion arise
from a CXCR4/CXCL12-mediated process.[10b,12] The
results show that 5a and 5b perform well in both
the affinity binding assay and invasion assay at 95–
100% inhibition of invasion at 100 nm as compared
with antagonist 2 as the positive control (set as
100% inhibition of CXCR4/CXCL12-mediated inva-
sion). Compound 5d also delivered a reasonable
result of 71 and 84% at 10 and 100 nm, respectively.
5k
5l
1
20
27
79
82
>1000
5m
5n
7
1000
1000
1
54
54
39
98
88
44
Compound 5g exhibited
a binding affinity of
11 a
11 b
11 c
10
1000
100
10
77
77
37
25
82
100 nm, although the results in the Matrigel invasion
were moderate at 48% (10 nm) and 72% (100 nm).
Compound 5k showed an EC value of 1 nm in the
binding affinity assay and also gave moderate results
in the Matrigel invasion assay. For the series 11 a–d,
11 a delivered a satisfying result in both the binding
affinity assay (EC=10 nm) and the Matrigel invasion
assays (77% at 10 and 100 nm). Compound 11 d
gave a binding affinity EC value of 10 nm and per-
formed moderately in the Matrigel invasion assay,
with 82% inhibition of invasion at 100 nm.
One striking feature of the data as it pertains to
results across both binding and Matrigel assays is
the behavior of six compounds: 3b, 5c, 5h, 5i, 5m,
and 5n. Each substance furnishes a binding assay EC
value of 1000 nm (Table 1), but a contrary Matrigel
invasion decrease of >75%. A consistent proposal
for the behavior of this subset (29% of the com-
pounds prepared) is provided below in the context
of antagonist orientation in the binding pocket of
CXCR4. One final unusual and unique compound is
7, which evidences a binding concentration of 1 nm,
but a Matrigel invasion outcome of 44%. We regard
this compound as an outlier that needs further ex-
amination.
0
17
34
11 d
[a] NA: not applicable.
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Prediction of benzenesulfona-
mide analogue binding poses
To explore the possible structur-
al basis behind the binding/Ma-
trigel discrepancy for certain
benzenesulfonamides, we arbi-
trarily classified the analogues
of Table 1 as active (ECꢀ
100 nm) or inactive (ECꢁ
1000 nm). In general, we found
that the unscaled binding free
energies from Prime MM-GBSA
calculations correlate with the
two categories of effective con-
centrations. For compounds re-
garded as active, the unscaled
energies are > j30jkcalmolꢂ1
(Table 3). For example, the best
Glide docking pose of 5a (Fig-
ure 6a, EC=10 nm) is protonat-
ed on the pyrrolidine nitrogen
atom, forms a salt bridge (2.8 ꢁ)
to Asp97, and delivers a predict-
Figure 4. IC50 determination curves for selected compounds a) 5b, b) 5h, and c) 11 a.
ed binding free energy of
ꢂ36.4 kcalmolꢂ1. One oxygen
Computational modeling
atom of the sulfonamide engages in a hydrogen bond with
Arg188. Both the pyrrolidine and phenyl rings fit into small
sub-pockets and make hydrophobic contacts with CXCR4 (Fig-
ure 6b).
Mapping CXCR4 antagonists and CXCL12 N-terminal binding
sites
Computational docking of the synthesized ligands into the
binding pocket of the recently disclosed CXCR4 X-ray struc-
ture[13] was performed to furnish insight into the discrepancies
between the binding affinities (EC values) and Matrigel inva-
sion assay results for the subset of six analogues mentioned
above. Compared with previously solved GPCR X-ray struc-
tures, the binding cavity of CXCR4 is larger and more open
with a cavity volume of 3322 ꢁ3.[13] The small-molecule antago-
nist IT1t only occupies part of the pocket. Several functional
studies of mutant CXCR4 revealed that Asp97, Asp187, Glu288,
Phe87, Asp171, and Phe292 are required for CXCL12 binding,
while the first three residue mutants impair CXCL12 signal-
ing.[14] The cyclic peptides 2 and CVX15 (12) are CXCR4 antago-
nists of known structure. CXCR4 alanine scanning for mutants
identified residues required for the binding of 2 to be Asp171,
Arg188, Tyr190, Gly207, and Asp262.[15] By combining the mu-
tational outcomes with crystal structure analysis, we can map
the binding sites of the CXCR4 antagonists and the CXCL12
N terminus. In this way, antagonist peptides 2 and 12 are
shown to occupy similar sectors of the CXCR4 binding cavity,
as most of the key residues sensitive to the binding of 2 are in
close contact with 12. However, mutational analysis also shows
that the CXCL12 N terminus binds in another sector of the
binding pocket, leading to only partial overlap between pep-
tide antagonist and CXCL12 N-terminal binding (Figure 5).
Superposition of all the docked active analogues suggests
that they reside in a similar location and form either a salt
bridge or a hydrogen bond with Asp97 or Glu288, which play
key roles in CXCL12 binding and signal transduction[14a] (Fig-
ure 6c). For compounds classified as binding inactive (ECꢁ
1000 nm), the unscaled binding free energies are in the range
of ꢂ10 to ꢂ20 kcalmolꢂ1, except for 5h, 5i, and 5l, which fall
between ꢂ40 and ꢂ42 kcalmolꢂ1 (Table 3). No polar interac-
tions or hydrogen bonds are formed between the two key
CXCR4 residues Asp187 or Glu288 for the latter agents as well
as inactive 7. For selected compounds the IC50 values versus
docking scores (Table 4) show a trend similar to the EC values
versus docking scores (Table 3). Superposition of 5h, 5i, and
5l, all with predicted binding free energies in the highly active
range (ꢂ40 to ꢂ42 kcalmolꢂ1), suggests that these agents as-
sociate with CXCR4 in poses that place the structures in a dis-
tinct part of the binding pocket near Asp97. The nitrogen
atoms of the pyrrolidine groups are protonated and form salt
bridges (2.7 ꢁ) to Asp97 (Figure 7). None show overlap with
the peptide 12 binding geometry (Figure 5a,d), unlike the ac-
tives displayed in Figure 6. Thus, it is suggested that these
three compounds bind efficiently to the CXCR4–12 complex,
but are unable to compete with antagonist 2. This explains the
inconsistency between the high experimental 1000 nm concen-
trations and the substantial calculated binding free energies
for 5h, 5i, and 5l, as peptidic antagonist 2 and 12 occupy es-
sentially the same but different sector of the binding pocket
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binding affinities. In addition, the SO2 moiety engages in a bi-
furcated hydrogen bond with Ser285, one face of the fluoro-
phenyl group resides in a hydrophobic environment, and the
para-fluorine atom is closely associated with cationic Lys38
(Figure 7d). It is well known that organic fluorides enjoy
a strong electrostatic interaction with cationic amines sufficient
to alter the classic axial/equatorial rules of conformational anal-
ysis.[16] While the corresponding para-methoxy analogue 5l can
be expected to experience a similar productive interaction
with Lys38, the tert-butyl analogues 5j and 5k do not do so
for both steric and electrostatic reasons, adopting the poses
shown in Figure 6 instead. In sum, this analysis suggests that
the polar para substituents provide an unexpected anchor site,
altering the binding pose while directing the compounds away
from bound 2.
Table 2. Selected sulfonamide analogues evaluated by both single-point
binding affinity (EC) and seven-point IC50 values.
Compound
EC [nm]
IC50 [nm]
5a
10
8.0
5b
5c
1
8.9
1000
5300
As a test of the hypothesis regarding the role of aromatic
fluorine in reshaping the binding pose, we prepared 5m and
5n (Table 1), analogues of 5i in which the para-fluoro substitu-
ent was separately moved to the ortho and meta positions. Ac-
cording to the model of Figure 7d, Arg183 hydrogen bonded
to Asp97 is perfectly poised to anchor the aromatic fluoride at
these centers, analogous to the action of Lys38 at the para po-
sition of 5h. Accordingly, when 5m and 5n were subjected to
the Matrigel assay at 100 nm, they blocked invasion at 98 and
88%, respectively. This compares with 82 and 99% at the same
concentration for 5h and 5i, respectively. Such stabilization is
not unprecedented. As part of a mechanistic enzyme study,
a conformational partition of a CF3-methionine between two
closely spaced arginines in bacteriophage l lysozyme has been
observed by NMR spectroscopy.[17]
5g
5h
5i
100
1000
113
11850
7300
1000
5l
>1000
64900
Conclusions
We synthesized a novel class of benzenesulfonamides that in-
hibit CXCR4 as evidenced by the displacement of antagonist
TN14003 (2) from the receptor. Compounds 5a and 5b were
among the most potent compounds, and exhibit IC50 values of
<10 nm in the binding affinity assay and show >90% inhibi-
tion of invasion in the Matrigel invasion assay relative to the
control. Computer modeling reveals that the potent analogues
interact with key residues Asp97 and Arg188 in the CXCR4
binding pocket, mutations of which interfere with receptor
action. In addition, the modeling provides a satisfying explana-
tion for compounds that perform poorly in the binding affinity
assay against 2, but deliver favorable blockade of Matrigel in-
vasion and MM-GBSA binding free energies reflecting good to
excellent potency. The binding/Matrigel inconsistency is re-
solved by the observation that these compounds are predicted
to dock in the CXCL12 binding region, but, in the expansive
binding site, do not overlap with 2 and thus do not compete
with it. It is unusual for two drug-sized molecules to bind si-
multaneously to the same binding site. However, the muscarin-
ic M2 receptor[18] and the sweet receptor[19] may be two exam-
ples of the phenomenon exemplified by Figure 7b. Unfortu-
nately, these results suggest that the utility of the EC binding
site assay would appear to be compromised as a primary
11 a
10
79
21
11 c
1000
(Figure 5c). It may also explain the activities of 3b, 5h, and 5i
in the Matrigel invasion assay, which incorporates full-length
CXCL12 and overlaps with both sectors of CXCR4 (Figure 5).
A further observation provides additional support for this in-
terpretation, while taking into account a unique structural
factor for 5h, 5i, and 5l in terms of the para-fluoro or para-
methoxy groups located within the benzyl substituent. That is,
5h and its congeners are predicted to adopt alternative poses,
as depicted in Figure 7b and Figure 7c. The protonated amine
forms a salt bridge with Asp97, consistent with the same inter-
action for all the active analogues as monitored by the EC
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in an eight-well slide chamber for two days. The cells
were pre-incubated with the test compounds for
15 min, and then the cells were fixed with 4% formalde-
hyde. The fixed cells were subsequently incubated for
45 min with biotinylated 2 (50 ngmLꢂ1).[10b,12] Cells were
incubated for 30 min in streptavidin–rhodamine at
a 1:150 dilution (Jackson Immuno Research Laborato-
ries, West Grove, PA, USA) after washing three times
with PBS. Finally, the slides were washed with PBS and
mounted in an anti-fade mounting solution (Molecular
Probes, Eugene, OR, USA). Five pictures of stained cells
for each treatment were taken on a Nikon Eclipse E800
microscope. Pictures were analyzed quantitatively with
ImageJ, and IC50 values for each compound were fitted
with GraphPad Prism 4.
Matrigel cell invasion assays
Matrigel invasion chambers from BD Biocoat Cellware
(San Jose, CA, USA) were used for invasion assays. MDA-
MB-231 cells were cultured on a layer of Matrigel in the
upper chamber with test compounds at 10 or 100 nm,
while 200 ngmLꢂ1 CXCL12 was added in the lower
chamber as a chemoattractant. Detailed procedures for
the binding affinity and invasion assays have been de-
scribed in previous publications.[2c,10b,12]
Figure 5. Mapping the location of peptide ligands onto the CXCR4 binding cavity based
on X-ray structure and point mutations sensitive to ligand binding. a) CXCL12 N terminus
(yellow circle); b) peptidic antagonist 2 (orange circle); c) CVX15 (12) (cyan circle); d) su-
perposition of CXCL12, 2, and 12 binding sites.
Table 3. Effective concentration and predicted relative binding free ener-
gies for active and inactive compounds.
Active
Compd
EC [nm]
MM-GBSA
[kcalmolꢂ1
Inactive
Compd
EC [nm]
MM-GBSA
[kcalmolꢂ1
]
]
5b
5j
5k
7
5e
5a
11 a
5d
1
1
1
1
1
10
10
10
ꢂ37.4
ꢂ31.4
ꢂ29.7
ꢂ40.3
ꢂ31.7
ꢂ36.4
ꢂ34.5
ꢂ33.5
3a
3b
3c
5 f
5h
5i
1000
1000
1000
1000
1000
1000
1000
ꢂ21.5
ꢂ20.7
ꢂ13.4
ꢂ15.0
ꢂ42.1
ꢂ40.0
ꢂ40.3
5l
screen for CXCR4 antagonists, and, at the very least, needs to
be complemented by a second functional assay.
Experimental Section
Initial screening of anti-CXCR4 small molecules based on
a binding affinity assay
Binding affinity and cell invasion assays are basic tools that apply
to the initial screening. MDA-MB-231 cells cultured in an eight-well
slide chamber were pre-incubated with the test compounds at 1,
10, 100, and 1000 nm. The cells were fixed with 4% formaldehyde
and incubated with 50 nm biotinylated 2 followed by rhodamine
staining.
IC50 measurement for select sulfonamide analogues: IC50 values of se-
lected compounds were tested at 1, 4, 10, 40, 100, 400, and
1000 nm, or at 0.1, 0.4, 1, 4, 10, 40, and 100 mm, based on the re-
sults of initial screening. MDA-MB-231 cells (2ꢂ104) were cultured
Figure 6. a) Structure of 5a; b) best docking pose for 5a in the CXCR4 bind-
ing pocket; c) superposition of the Glide/MM-GBSA best binding poses in
CXCR4 for all active analogues.
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Computational protein–ligand docking
Table 4. IC50 (binding affinity assay) determination of selected com-
pounds and the corresponding docking score.
The acid dissociation constant (pKa) of the benzenesulfonamide de-
rivatives was predicted by ACD software.[20] Based on these values,
nitrogen sites in these compounds were protonated when the pKa
was estimated to be >7. All prepared benzenesulfonamide deriva-
tives with the appropriate N site protonated were docked flexibly
into the cavity region of the human chemokine receptor CXCR4
crystal structure (PDB code: 3ODU) devoid of the small-molecule
antagonist IT1t using Glide with standard precision (Schrçdinger,
LLC).[21] This methodology regards the protein structure as a rigid
body, but treats the ligand as a conformationally flexible molecule.
The resulting CXCR4–benzenesulfonamide complexes were subse-
quently sorted energetically with the MM-GBSA scoring algorithm,
which provides an estimate of relative binding free energies.[22] The
volume of the CXCR4 cavity was obtained by the web-based
CASTp package.[23]
Compound
IC50
[nm]
MM-GBSA
[kcalmolꢂ1
]
5j
4.6 ꢂ31.4
5a
8.0 ꢂ36.4
Chemistry
11 c
21.1 ꢂ31.4
1
General: H and 13C NMR spectra were recorded on an INOVA-400
(400 MHz) spectrometer. The spectra obtained in CDCl3 or
[D6]DMSO was referenced to the residual solvent peak. Chemical
shifts (d) are reported in parts per million (ppm) relative to residual
non-deuterated solvent as an internal reference. The following ab-
breviations are used to explain the multiplicities: s=singlet, d=
doublet, t=triplet, q=quartet, dd=doublet of doublets, dt=dou-
blet of triplets, m=multiplet, br=broad. Mass spectra were re-
corded on a JEOL spectrometer at Emory University Mass Spec-
trometry Center.
5c
5 f
5285
ꢂ13.4
ꢂ15.0
9849
General procedure for the synthesis of 3: To a solution of xylyle-
nediamine (1 equiv) in CH2Cl2 was added DIPEA (2 equiv) and the
appropriate sulfonyl chloride (2 equiv). The reaction mixture was
stirred at room temperature for 3 h. The white precipitate was fil-
tered and washed with CH2Cl2 to give the product as white crys-
tals.
5i
30359
ꢂ40.0
N,N’-[1,4-Phenylenebis(methylene)]dibenzenesulfonamide (3a):
1
White crystals (527 mg, 34%): H NMR (400 MHz, CDCl3): d=8.22 (s,
2H), 7.84 (d, J=8.0 Hz, 2H), 7.62 (m, 6H), 7.13 (s, 4H), 3.89 ppm (s,
4H); 13C NMR (400 MHz, [D6]DMSO): d=140.7, 136.6, 132.34,
129.19, 127.44, 126.44, 45.8 ppm; HRMS: m/z [M+H]+ calcd for
C20H20N2O4S2: 415.07917, found: 415.07879.
N,N’-[1,4-Phenylenebis(methylene)]bis(4-methoxybenzenesulfo-
namide) (3b): White crystals (562 mg 60%): 1H NMR (400 MHz,
[D6]DMSO): d=7.94 (brs, 2H), 7.73 (d, J=8.8 Hz, 4H), 7.14 (s, 4H),
7.10 (d, J=9.2 Hz, 4H), 3.88 (s, 4H), 3.83 ppm (s, 6H); 13C NMR
(400 MHz, [D6]DMSO): d=162.1, 136.6, 132.3, 128.7, 127.4, 114.3,
55.62, 45.8 ppm; HRMS: m/z [M+H]+ calcd for C22H24N2O6S2:
477.11486, found: 477.11457.
General procedure for the synthesis of 4: To a solution of 4-(bro-
momethyl)benzene-1-sulfonyl chloride (1 equiv) in CH2Cl2 (0.1m)
was added the amine (2 equiv). The reaction mixture was allowed
to stir at room temperature for 2 h to overnight. The reaction mix-
ture was washed with H2O and brine, and the combined organic
layers were dried over MgSO4 and concentrated to give the final
compound.
Figure 7. a) Structure of 5h; b) best docking pose of 5i (cyan) in CXCR4
N-Benzyl-4-(bromomethyl)-N-methylbenzenesulfonamide (4a):
Off-white solid, (116 mg, 20%): H NMR (400 MHz, CDCl3): d=7.84
(d, J=8.0 Hz, 2H), 7.59 (d, J=8.0 Hz, 2H), 7.34–7.31 (m, 5H), 4.65
(s, 2H), 4.16 (s, 2H), 2.61 ppm (s, 3H); 13C NMR (400 MHz, CDCl3):
d=142.7, 137.6, 135.6, 130.0, 129.4, 128.9, 128.5, 128.2, 128.1, 54.3,
with X-ray position of 12 (yellow); c) superposition of best binding poses of
protonated 5h (cyan), 5i (magenta), and 5l (yellow) in CXCR4; d) close-up
view of protonated 5h showing key interactions with residues on adjacent
helices in the binding site. The conformation of Lys38 was established by
conformational searching of its side chain following docking of 5h.
1
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34.5, 31.8 ppm; HRMS: m/z [M+H]+ calcd for C15H15BrN2O2SNa:
376.9930, found: 376.9932.
4.13 (s, 2H), 3.70 (s, 2H), 2.59 (m, 7H), 1.09 ppm (t, J=7.0 Hz, 6H);
13C NMR (400 MHz, CDCl3): d=144.3, 136.2, 135.7, 129.8, 128.8,
128.5, 128.0, 127.6, 57.0, 54.2, 47.0, 34.5, 11.4 ppm; HRMS: m/z
[M+H]+ calcd for C19H27N2O2S 347.1788, found: 347.1787.
4-(Bromomethyl)-N-(4-fluorobenzyl)-N-methylbenzenesulfona-
mide (4b): White powder (235 mg, 32%): 1H NMR (400 MHz,
CDCl3): d=7.81 (d, J=8.4 Hz, 2H), 7.58 (d, J=8.4 Hz, 2H), 7.27 (d,
J=8.4 Hz, 2H), 7.03 (d, J=8.4 Hz, 2H), 4.53 (s, 2H), 4.13 (s, 2H),
2.61 ppm (s, 3H); HRMS: m/z [M+H]+ calcd for C15H17N2O2F1S1:
372.0065, found: 372.0064.
N-Benzyl-N-methyl-4-(piperazin-1-ylmethyl)benzenesulfonamide
(5e): White solid, (141 mg, 46%): 1H NMR (400 MHz, CDCl3): d=
7.72 (d, J=8.4 Hz, 2H), 7.46 (d, J=8.8 Hz, 2H), 7.28–7.19 (m, 5H),
4.08 (s, 2H), 3.51 (s, 2H), 2.86 (t, J=4.8 Hz, 4H), 2.54 (s, 3H),
2.39 ppm (brs, 4H); 13C NMR (400 MHz, CDCl3): d=141.3, 136.2,
135.4, 128.2, 128.8, 128.1, 127.6, 126.9, 64.2, 57.0, 54.7, 46.3,
34.9 ppm; HRMS: m/z [M+H]+ calcd for C19H26N3O2S: 360.1740,
found: 360.1741 [M+H]+.
4-(Bromomethyl)-N-(4-(tert-butyl)benzyl)-N-methylbenzenesulfo-
namide (4c): White powder (686 mg, 90%): 1H NMR (400 MHz,
CDCl3): d=7.81 (d, J=8.4 Hz, 2H), 7.57 (d, J=8.4 Hz, 2H), 7.35 (d,
J=8.8 Hz, 2H), 7.21 (d, J=8.4 Hz, 2H), 4.53 (s, 2H), 4.13 (s, 2H),
2.62 (s, 3H), 1.31 ppm (s, 9H); 13C NMR (400 MHz, CDCl3): d=151.2,
142.7, 137.7, 132.5, 129.9, 128.3, 128.2, 125.8, 53.9, 34.7, 34.5, 31.8,
31.5, 31.36 ppm; HRMS: m/z [M+H]+ calcd for C19H24NO2BrNaS:
432.0603, found: 432.0607.
4-[(1H-Pyrrol-1-yl)methyl]-N-benzyl-N-methylbenzenesulfona-
1
mide (5 f): White powder (103 mg, 53%): H NMR (400 MHz, CDCl3):
d=7.77 (dd, J=2.0, 8.0 Hz, 2H), 7.32–7.21 (m, 7H), 6.70 (t, J=
2.0 Hz, 2H), 6.23 (t, J=2.4 Hz, 2H), 5.16 (s, 2H), 4.23 (s, 2H),
2.56 ppm (s, 3H); 13C NMR (400 MHz, CDCl3): d=143.6, 136.9, 135.7,
128.9, 128.5, 128.1, 127.5, 121.4, 109.4, 54.3, 52.9, 34.5 ppm; HRMS:
m/z [M+H]+ calcd for C19H21N2O2S: 341.1318, found: 341.1316.
4-(Bromomethyl)-N-(4-methoxybenzyl)-N-methylbenzenesulfona-
mide (4d): Off-white powder (581 mg, 81%): 1H NMR (400 MHz,
CDCl3): d=7.81 (d, J=8.4 Hz, 2H), 7.57 (d, J=8.8 Hz, 2H), 7.20 (d,
J=8.8 Hz, 2H), 6.86 (d, J=8.4 Hz, 2H), 4.53 (s, 2H), 4.10 (s, 2H),
3.80 (s, 3H), 2.59 ppm (s, 3H); 13C NMR (400 MHz, CDCl3): d=159.6,
142.7, 137.6, 132.1, 129.9, 129.4, 128.1, 127.5, 114.7, 114.3, 55.5,
53.7, 34.3, 31.8 ppm; HRMS: m/z [M+H]+ calcd for
C16H18NO3BrNaS: 406.0083, found: 406.0093.
N-Benzyl-N-methyl-4-{[(pyridin-2-ylmethyl)amino]methyl}benze-
1
nesulfonamide (5g): Yellow solid (14 mg, 21%): H NMR (400 MHz,
CDCl3): d=8.56 (d, J=4.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 2H), 7.66 (td,
J=8.0, 1.6 Hz, 1H), 7.34–7.24 (m, 6H), 7.20–7.17 (m, 1H), 4.12 (s,
2H), 3.96 (s, 2H), 3.95 (s, 2H), 2.57 ppm (s, 3H); 13C NMR (400 MHz,
CDCl3): d=159.2, 149.4, 145.6, 144.5, 136.7, 135.9, 135.7, 129.4,
129.4, 129.4, 128.9, 128.7, 128.5, 128.0, 127.7, 127.6, 122.6, 122.3,
122.0, 58.1, 54.5, 54.2, 52.9, 34.5, 29.8 ppm; HRMS: m/z [M+H]+
calcd for C21H24N3O2S: 382.1584, found: 382.1582.
General procedure for the synthesis of 5: To a solution of 4
(1 equiv) in CH3CN was added K2CO3 (2 equiv) and the amine
(1 equiv). The reaction mixture was allowed to stir at room temper-
ature overnight. The organic solvent was removed by rotary evap-
oration, and the residue was dissolved in CH2Cl2 and washed with
H2O and brine, dried over MgSO4, and concentrated. The crude
product was purified by column chromatography.
4-[(Diethylamino)methyl]-N-(4-fluorobenzyl)-N-methylbenzene-
sulfonamide (5h): White solid (45 mg, 59%): 1H NMR (400 MHz,
CDCl3): d=7.77 (d, J=8.4 Hz, 2H), 7.60 (d, J=8.4 Hz, 2H), 7.26 (t,
J=7.4 Hz, 2H), 7.02 (t, J=7.0 Hz, 2H), 4.12 (s, 2H), 3.65 (s, 2H),
2.60–2.55 (m, 7H), 1.07 ppm (t, J=7.2 Hz, 6H); 13C NMR (400 MHz,
CDCl3): d=161.0, 135.8, 131.6, 130.3, 130.2, 129.5, 127.6, 115.9,
115.7, 57.4, 53.6, 47.2, 34.5, 12.0 ppm; HRMS: m/z [M+H]+ calcd
for C19H26N3FO2S: 365.1694, found: 365.1690.
N-Benzyl-N-methyl-4-(pyrrolidin-1-ylmethyl)benzenesulfonamide
1
(5a): Off-white solid, (52 mg, 41%): H NMR (400 MHz, CDCl3): d=
7.81 (d, J=8.4 Hz, 2H), 7.58 (d, J=8.4 Hz, 2H), 7.31–7.27 (m, 5H),
4.15 (s, 2H), 3.74 (s, 2H), 2.60 (s, 7H), 1.85 ppm (m, 4H); 13C NMR
(400 MHz, CDCl3): d=136.0, 135.9, 129.6, 128.9, 128.6, 128.1, 127.7,
60.3, 54.5, 54.3, 34.6, 23.7 ppm; 13C NMR (400 MHz, CDCl3): d=
144.6, 135.8, 135.7, 129.3, 128.6, 128.3, 127.9, 127.5, 60.1, 54.3, 54.1,
34.3, 29.7, 23.5 ppm; HRMS: m/z [M+H]+ calcd for C19H25N2O2S:
345.16313, found: 345.16296.
N-(4-Fluorobenzyl)-N-methyl-4-(pyrrolidin-1-ylmethyl)benzene-
sulfonamide (5i): Yellow solid (42 mg, 57%): 1H NMR (400 MHz,
CDCl3): d=7.76 (d, J=8.0 Hz, 2H), 7.52 (d, J=8.0 Hz, 2H), 7.25 (t,
J=8.4, 2H), 7.00 (d, J=8.4 Hz, 2H), 4.09 (s, 2H), 3.69 (s, 2H), 2.57–
2.51 (m, 7H), 1.80 ppm (brs, 4H); 13C NMR (400 MHz, CDCl3): d=
145.0, 136.0, 131.6, 130.3, 130.2, 129.6, 127.7, 115.9, 115.7, 60.3,
54.5, 53.6, 34.5, 23.7 ppm; HRMS: m/z [M+H]+ calcd for
C19H24N2FO2S: 363.1537, found: 363.1534.
N-Benzyl-N-methyl-4-(piperidin-1-ylmethyl)benzenesulfonamide
1
(5b): Off-white solid (40 mg, 30%): H NMR (400 MHz, CDCl3): d=
7.76 (d, J=8.8 Hz, 2H), 7.50 (d, J=8.4 Hz, 2H), 7.29 (m, 5H), 4.13 (s,
2H), 3.53 (s, 2H), 2.58 (s, 3H), 2.38 (s, 4H), 1.58 (q, J=5.4 Hz, 4H),
1.42 ppm (brs, 2H); 13C NMR (400 MHz, CDCl3): d=136.2, 135.8,
131.1, 129.9, 128.8, 128.6, 128.1, 127.6, 63.1, 54.8, 54.3, 38.9, 34.6,
30.5, 29.1, 25.9, 24.2, 23.9, 23.2, 14.2, 11.2 ppm; HRMS: m/z [M+
H]+ calcd for C20H27N2O2S: 359.17878, found: 359.17856.
N-[4-(tert-Butyl)benzyl]-4-[(diethylamino)methyl]-N-methylben-
zenesulfonamide (5j): White solid (54 mg, 56%). NMR (400 MHz,
CDCl3): d=7.80 (d, J=8.4 Hz, 2H), 7.57 (d, J=8.4 Hz, 2H), 7.37 (d,
J=8.0 Hz, 2H), 7.25 (d, J=8.4 Hz, 2H), 4.15 (s, 2H), 3.67 (s, 2H),
2.63 (s, 3H), 2.58 (q, J=7.2 Hz, 4H), 1.34 (s, 9H), 1.09 ppm (t, J=
7.2 Hz, 6H); 13C NMR (400 MHz, CDCl3): d=151.1, 146.1, 135.9,
132.8, 129.4, 128.3, 127.6, 125.7, 57.4, 53.9, 47.2, 34.7, 34.5, 31.5,
29.9, 12.01 ppm; HRMS: m/z [M+H]+ calcd for C23H35N2O2S
403.2414, found: 403.2418.
N-Benzyl-N-methyl-4-(morpholinomethyl)benzenesulfonamide
1
(5c): White solid (86 mg, 57%): H NMR (400 MHz, CDCl3): d=7.78
(d, J=8.4 Hz, 2H), 7.52 (d, J=8.4 Hz, 2H), 7.31–7.24 (m, 5H), 4.13
(s, 2H), 3.71 (t, J=4.4 Hz, 4H), 3.59 (s, 2H), 2.59 (s, 3H), 2.46 ppm
(t, J=4.8 Hz, 4H); 13C NMR (400 MHz, CDCl3): d=143.5, 136.2,
135.6, 129.5, 128.7, 128.4, 127.9, 127.6, 67.0, 62.7, 54.1, 53.7,
34.4 ppm; HRMS: m/z [M+H]+ calcd for C19H25N2O3S: 361.1580,
found: 361.1581.
N-[4-(tert-Butyl)benzyl]-N-methyl-4-(pyrrolidin-1-ylmethyl)benze-
nesulfonamide (5k): White powder (51 mg, 55%): 1H NMR
(400 MHz, CDCl3): d=7.77 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz,
2H), 7.32 (s, J=8.4 Hz, 2H), 7.19 (d, J=8.0 Hz, 2H), 4.10 (s, 2H),
3.72 (s, 2H), 2.58 (s, 7H), 1.83 (s, 4H), 1.24 ppm (s, 9H); 13C NMR
(400 MHz, CDCl3): d=151.1, 136.3, 132.7, 129.7, 128.3, 127.8, 125.7,
N-Benzyl-4-[(diethylamino)methyl]-N-methylbenzenesulfona-
mide (5d): White solid (117 mg, 60%): 1H NMR (400 MHz, CDCl3):
d=7.77 (d, J=8.4 Hz, 2H), 7.58 (d, J=8.4 Hz, 2H), 7.28 (m, 5H),
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60.1, 54.5, 53.9, 34.7, 34.5, 31.5, 29.9, 23.7 ppm; HRMS: m/z [M+
4-[2-(Diethylamino)ethoxy]benzenamine (9): To a solution of 8
(1.99 g, 6.46 mmol) in dioxane (10 mL) in an ice bath was added
4m HCl in dioxane (3 mL). The reaction was allowed to stir over-
night and warmed to room temperature. Et2O was added to the re-
action mixture, and the white precipitate was filtered to give prod-
uct (1.49 g, 51%): H NMR (400 MHz, CDCl3): d=6.74 (d, J=8.8 Hz,
2H), 6.63 (d, J=8.8 Hz, 2H), 4.00 (t, J=6.4 Hz, 2H), 2.87 (t, J=
6.4 Hz, 2H), 2.66 (q, J=7.2 Hz, 4H), 1.08 ppm (t, J=6.8 Hz, 6H).
H]+ calcd for C23H33N2O2S: 403.2257, found: 403.2261.
N-(4-Methoxybenzyl)-N-methyl-4-(pyrrolidin-1-ylmethyl)benze-
nesulfonamide (5l): Yellow powder (142 mg, 77%): 1H NMR
(400 MHz, CDCl3): d=7.81 (d, J=8.4 Hz, 2H), 7.53 (d, J=8.4 Hz,
2H), 7.20 (d, J=8.4 Hz, 2H), 6.85 (d, J=8.4 Hz, 2H), 4.07 (s, 2H),
3.79 (s, 2H), 3.70 (s, 3H), 2.56–2.52 (m, 7H), 1.83–1.80 ppm (m, 4H);
13C NMR (400 MHz, CDCl3): d=159.5, 145.0, 136.0, 129.9, 129.5,
127.7, 114.2, 60.3, 55.5, 54.5, 53.8, 34.3, 29.9, 23.7 ppm; HRMS: m/z
[M+H]+ calcd for C20H27N2O3S: 375.1737, found: 375.1737.
1
4-(Bromomethyl)-N-{4-[2-(diethylamino)ethoxy]phenyl}benzene-
sulfonamide (10): To a solution of 4-(bromomethyl)benzene-1-sul-
fonyl chloride (823 mg, 3.0 mmol) in DMF (15 mL) was added 9
(635 mg, 3.0 mmol); the mixture was allowed to stir at room tem-
perature overnight. The reaction mixture was diluted with CH2Cl2
(20 mL), washed with H2O (2ꢂ100 mL) and brine (1ꢂ100 mL),
dried over MgSO4, and concentrated. The crude product was puri-
fied by column chromatography, silica gel, CH2Cl2/MeOH (15:1 !
10:1 ! 5:1) to give an off-white solid (1.27 g, 90%): 1H NMR
(400 MHz, CDCl3): d=7.70 (d, J=8.4 Hz, 2H), 7.41 (d, J=8.0 Hz,
2H), 6.98 (d, J=8.8 Hz, 2H), 6.65 (d, J=9.2 Hz, 2H), 4.55 (d, J=
6.0 Hz, 2H), 4.36 (t, J=4.6 Hz, 2H), 3.47 (t, J=4.4 Hz, 2H), 3.26 (brs,
4H), 1.39 ppm (t, J=7.2 Hz, 6H); HRMS: m/z [M+H]+ calcd for
C19H26N2O3SBr: 441.0842, found: 441.0847.
N-(2-Fluorobenzyl)-N-methyl-4-(pyrrolidin-1-ylmethyl)benzene-
1
sulfonamide (5m): White powder (84 mg, 63%): H NMR (400 MHz,
CDCl3): d=7.71 (d, J=8.0 Hz, 2H), 7.52 (d, J=8.4 Hz, 2H), 7.41 (td,
J=8.0, 1.6 Hz, 1H), 7.22–7.19 (m, 1H), 7.09 (t, J=7.2 Hz, 1H), 6.95
(t, J=10 Hz, 1H), 4.18 (s, 2H), 3.71 (s, 2H), 2.60–2.57 (m, 7H),
1.79 ppm (s, 4H); 13C NMR (400 MHz, CDCl3): d=160.1, 139.8, 136.2,
130.9, 129.9, 127.8, 124.7, 122.8, 115.7, 59.9, 54.4, 47.2, 34.9,
23.7 ppm; HRMS: m/z [M+H]+ calcd for C19H23O2N2F: 363.1537,
found: 363.1536.
N-(3-Fluorobenzyl)-N-methyl-4-(pyrrolidin-1-ylmethyl)benzene-
sulfonamide (5n): White powder (113 mg, 85%): 1H NMR
(400 MHz, CDCl3): d=7.71 (d, J=6.8 Hz, 2H), 7.49 (d, J=8.4 Hz,
2H), 7.23–7.20 (m, 1H), 7.02–6.91 (m, 3H), 4.07 (s, 2H), 3.66 (s, 2H),
2.55 (s, 3H), 2.51 (brs, 4H), 1.76 ppm (t, J=6.8 Hz, 4H); 13C NMR
(400 MHz, CDCl3): d=162.5, 139.8, 137.0, 128.9, 127.8, 127.0, 123.2,
118.8, 113.7, 60.0, 54.3, 59.2, 34.8, 23.7 ppm; HRMS: m/z [M+H]+
calcd for C19H23O2N2F: 363.1537, found: 363.1536 [M+H]+.
General procedure for the synthesis of 11: A solution of 10
(1 equiv) and secondary amine (1 equiv) in CH2Cl2 was stirred at
room temperature overnight. The solvent was removed by rotary
evaporation, and the crude product was purified by column chro-
matography (silica gel 9:1 ! 6:1 CH2Cl2/MeOH).
N-{4-[2-(Diethylamino)ethoxy]benzyl}-4-(piperazin-1-ylmethyl)-
benzenesulfonamide (11a): Brown oil (245 mg, 20%): 1H NMR
(400 MHz, CDCl3): d=7.61 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.0 Hz,
2H), 6.94 (d, J=8.8 Hz, 2H), 6.72 (d, J=9.2 Hz, 2H), 3.97 (t, J=
6.0 Hz, 2H), 3.49 (s, 2H), 2.92 (q, J=4.8 Hz, 4H), 2.84 (t, J=6.0 Hz,
2H), 2.64 (m, 4H), 2.42 (brs, 4H), 1.07 ppm (t, J=7.2 Hz, 6H);
13C NMR (400 MHz, CDCl3): d=143.9, 136.2, 135.8, 129.7, 129.4,
128.8, 128.6, 128.1, 127.7, 115.2, 63.1, 54.3, 51.8, 48.0, 46.0, 34.5,
29.9,14.3, 11.9 ppm; HRMS: m/z [M+H]+ calcd for C23H34N4O3S:
447.2424, found: 447.2420.
4-(Bromomethyl)-N-methyl-N-(pyridin-2-ylmethyl)benzenesulfo-
namide (6): Follows the same general procedure as for the synthe-
sis of 4. The crude product was used without further purification
1
(1.24 g, 94%): H NMR (400 MHz, [D6]DMSO): d=8.52 (d, J=4.4 Hz,
1H), 7.90–7.82 (m, 3H), 7.71 (d, J=8.4 Hz, 2H), 7.45 (d, J=8.0 Hz,
1H), 7.38–7.34 (m, 1H), 4.86 (s, 2H), 4.30 (s, 2H), 2.70 ppm (s, 3H);
13C NMR (400 MHz, CDCl3): d=155.5, 148.2, 142.7, 128.3, 136.7,
129.7, 127.6, 123.2, 122.6, 54.5, 44.9, 35.5 ppm; HRMS: m/z [M+
Na]+ calcd for C14H15N2O2BrNaS: 376.9930, found: 376.9928.
N-Methyl-N-(pyridin-2-ylmethyl)-4-(pyrrolidin-1-ylmethyl)benze-
nesulfonamide (7): Follows the general procedure as for the syn-
thesis of 5. The crude product was purified by column chromatog-
raphy (20:1 CH2Cl2/MeOH) to give the final compound as a brown
N-{4-[2-(Diethylamino)ethoxy]phenyl}-4-(morpholinomethyl)ben-
zenesulfonamide (11b): Yellow solid (25 mg, 10%): 1H NMR
(400 MHz, CDCl3): d=7.68 (d, J=7.6 Hz, 2H), 7.39 (d, J=7.6 Hz,
2H), 7.00 (d, J=8.0 Hz, 2H), 6.71 (d, J=8.4 Hz, 2H), 4.37 (brs, 2H),
3.70 (t, J=4.8 Hz, 4H), 3.51 (s, 2H), 3.20 (brs, 2H), 2.94 (q, J=
7.6 Hz, 4H), 2.42 (t, J=4.4 Hz, 4H), 1.23 ppm (t, J=8.8 Hz, 6H);
HRMS: m/z [M+H]+ calcd for C23H34N3O4S: 448.2265, found:
448.2261 [M+H]+.
1
oil (72.5 mg, 50%): H NMR (400 MHz, CDCl3): d=8.47 (m, 1H), 7.77
(d, J=8.4 Hz, 2H), 7.71 (td, J=8.0, 2.0 Hz, 2H), 7.56–7.52 (m, 2H),
7.20–7.18 (m, 2H), 4.29 (s, 2H), 3.73 (s, 2H), 2.69 (s, 3H), 2.57 (s,
4H), 1.84 ppm (s, 4H); 13C NMR (400 MHz, CDCl3): d=156.6, 149.3,
145.1, 137.3, 135.8, 129.6, 127.7, 122.9, 122.6, 60.2, 56.3, 54.4, 35.5,
23.7 ppm; HRMS: m/z [M+H]+ calcd for C18H24N3O2S: 346.158,
found: 346.1584.
N-{4-[2-(Diethylamino)ethoxy]phenyl}-4-(piperidin-1-ylmethyl)-
benzenesulfonamide (11c): Yellow oil (20 mg, 9%): 1H NMR
(400 MHz, CDCl3): d=7.64 (d, J=8.4 Hz, 2H), 7.39 (d, J=8.0 Hz,
2H), 6.98 (dt, J=8.8, 3.6 Hz, 2H), 6.72 (dt, J=9.2, 3.6, 2H), 4.15 (t,
J=5.2 Hz, 2H), 3.55 (s, 2H), 2.99 (t, J=8.0 Hz, 2H), 2.79 (q, J=
6.8 Hz, 4H), 2.40 (brs, 4H), 1.60 (m, 4H), 1.41 (brs, 2H), 1.10 ppm
(t, J=8.4 Hz, 6H); 13C NMR (400 MHz, CDCl3): d=156.8, 143.9,
137.8, 129.6, 127.4, 125.4, 115.1, 66.0, 63.1, 54.6, 51.6, 47.8, 29.8,
25.9, 24.2, 11.2 ppm; HRMS: m/z [M+H]+ calcd for C35H36N3O3S:
448.2265, found: 448.2261.
tert-Butyl 4-[2-(diethylamino)ethoxy]phenylcarbamate 8: To a so-
lution of tert-butyl-4-hydroxyphenylcarbamate (2 g, 9.56 mmol) in
DMF (20 mL) was added 2-chloro-N,N-diethylethanamine (1.97 g,
11.48 mmol) and NaOH (0.96 g, 23.9 mmol). The reaction mixture
was stirred at room temperature for 3 h. The reaction mixture was
filtered, and the filtrate was diluted with CH2Cl2 (20 mL). The organ-
ic layer was washed with brine (2ꢂ100 mL), dried over MgSO4, and
concentrated. The crude product was purified by column chroma-
tography, silica gel, CH2Cl2/MeOH (9:1) to give a brown solid
N-(4-(2-(Diethylamino)ethoxy)phenyl)-4-(pyrrolidin-1-ylmethyl)-
benzenesulfonamide (11d): Yellow semi-solid (40 mg, 19%):
1H NMR (400 MHz, CDCl3): d=7.65 (d, J=8.4 Hz, 2H), 7.37 (d, J=
8.4 Hz, 2H), 7.00 (d, J=9.2 Hz, 2H), 6.72 (d, J=8.8 Hz, 2H), 3.96 (t,
J=6.4 Hz, 2H), 3.61 (s, 2H), 3.25 (s, 2H), 2.83 (t, J=6.0 Hz, 2H), 2.61
1
(1.99 g, 60%): H NMR (400 MHz, CDCl3): d=7.22 (d, J=8.9 Hz, 2H),
6.75 (d, J=9.2 Hz, 2H), 4.14 (t, J=5.6 Hz, 2H), 3.09 (t, J=5.2 Hz,
2H), 2.88 (q, J=7.2 Hz, 4H), 1.44 (s, 9H), 6.08 ppm (t, J=7.2 Hz,
6H).
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 622 – 632 631

CHEMMEDCHEM
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We are grateful to Dr. Haipeng Hu for assistance in the prepara-
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(S.R.M.) and NIH R01CA165306 (H.S.).
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Published online on March 6, 2013
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ChemMedChem 2013, 8, 622 – 632 632