Tuning Side Arm Electronics in Unsymmetrical Cyclotriazadisulfonamide (CADA) Endoplasmic Reticulum (ER) Translocation Inhibitors to Improve their Human Cluster of Differentiation 4 (CD4) Receptor Down-Modulating Potencies

Article abstract of DOI:10.1021/acs.jmedchem.5b01832

Cyclotriazadisulfonamide prevents HIV entry into cells by down-modulating surface CD4 receptor expression through binding to the CD4 signal peptide. According to a two-site binding model, 28 new unsymmetrical analogues bearing a benzyl tail group and nine

Full text of DOI:10.1021/acs.jmedchem.5b01832

Article
pubs.acs.org/jmc
Tuning Side Arm Electronics in Unsymmetrical
Cyclotriazadisulfonamide (CADA) Endoplasmic Reticulum (ER)
Translocation Inhibitors to Improve their Human Cluster of
Differentiation 4 (CD4) Receptor Down-Modulating Potencies
Reena Chawla,† Victor Van Puyenbroeck,‡ Nicholas C. Pflug,† Alekhya Sama,† Rameez Ali,†
Dominique Schols,‡ Kurt Vermeire,*,‡,§ and Thomas W. Bell*,†,§
†Department of Chemistry, University of Nevada, 1664 North Virginia Street, Reno, Nevada 89557-0216 United States
‡Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, KU
LeuvenUniversity of Leuven, 3000 Leuven, Belgium
S
* Supporting Information
ABSTRACT: Cyclotriazadisulfonamide prevents HIV entry
into cells by down-modulating surface CD4 receptor
expression through binding to the CD4 signal peptide.
According to a two-site binding model, 28 new unsymmetrical
analogues bearing a benzyl tail group and nine bearing a
cyclohexylmethyl tail have been designed and synthesized. The
most potent new CD4 down-modulator (40 (CK147); IC50 63
nM) has a 4-dimethylaminobenzenesulfonyl side arm. One of
the two side arms was varied with substituents in different
positions. This gave a range of CD4 down-modulation potencies that correlated well with anti-HIV-1 activities. The side arms of
21 of the new benzyl-tailed analogues were modeled by means of quantum mechanical calculations. For CADA analogues with
arenesulfonamide side arms, the pIC50 values for CD4 down-modulation correlated with the component of the electric dipole
moment in the aromatic ring, suggesting that an attractive electronic interaction is a major factor determining the stability of the
complex between the molecule and its target.
CD4 on T cells, and the resulting CD4− cells are
INTRODUCTION
■
immunocompetent.14
Cyclotriazadisulfonamide (CADA) compounds inhibit repli-
cation of various strains of human immunodeficiency virus
(HIV) and simian immunodeficiency virus (SIV) by selectively
down-modulating expression of cell-surface cluster of differ-
Recent studies have shown that CADA (Figure 1a) down-
modulates cell-surface CD4 by a novel mechanism of action.15
Surface expression of most type 1 transmembrane proteins
entiation 4 (CD4).1−6 This type 1 integral transmembrane
requires transport of the nascent protein across the membrane
of the endoplasmic reticulum (ER) during translation, a process
glycoprotein is expressed on immune cells and is the primary
known as cotranslational translocation.16−19 The N-terminal
receptor required by HIV to enter host cells.7,8 Thus, CADA
signal peptide (SP) emerges first from the exit channel of the
compounds are effective HIV entry inhibitors, a class of
ribosome and is recognized by the signal recognition particle,
compounds that are of continuing interest for treatment of HIV
which docks the translating ribosome to the mouth of an ER
infection because of their different resistance profile, compared
to other anti-HIV agents.5,9−11 They are unique among viral
transmembrane channel (a translocon). The SP binds to the
wall of this channel, in some cases (as with CD4)15 initially
entry inhibitors in that they do not simply bind to and block
with the N terminus facing the ER interior (the lumen), then it
accessibility of a cell-surface receptor, they reduce CD4
reorients with the N terminus facing the cytosol,18,20−22
expression levels below the threshold concentration required
for HIV entry.2 Accordingly, CADA compounds are also of
allowing SP cleavage, translocation of the protein into the
lumen, and subsequent transport to the cell membrane. CADA
potential interest for chemotherapy of various immunologically
based diseases involving CD4+ T-cells, including asthma,
inhibits requisite inversion of the SP, blocking translocation of
the protein into the ER lumen, and diverts CD4 to the cytosol,
where it is degraded by proteolysis.15 CADA directly binds the
25-residue signal peptide of human (and apparently, more
rheumatoid arthritis, and diabetes, and also for preventing
organ transplant rejection.12,13 While CD4 down-modulation
may at first sight appear to be a therapeutic strategy that could
compromise the immune system, the African green monkey
avoids progression to acquired immunodeficiency syndrome
(AIDS) when infected by SIV through decreased expression of
Received: November 25, 2015
Published: March 14, 2016
© 2016 American Chemical Society
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Article
generally primate) CD4, but not mouse CD4, which has less
homology with the human CD4 SP. Hence, the significance of
CADA compounds may go far beyond CD4 down-modulation
in that they might unlock a novel approach to suppressing
other cell-surface proteins of therapeutic interest.
To deduce the physicochemical properties of the CADA
binding site, we have performed several structure−activity
relationship (SAR) studies in which the two side arms (Ar1 and
Ar2) and the tail group (R) were varied (Figure 1b).2,6,23,24
A
quantitative structure−activity relationship (QSAR) study
showed the importance of a relatively large, hydrophobic tail
group (R) for high CD4 down-modulation and anti-HIV
potency.24 While CADA was found to interact mainly with the
signal peptide of the nascent CD4 protein, the tail group may
play an important role in anchoring the molecule to the
hydrophobic lateral gate of the translocon. The QSAR study
also showed that electron-donating substitutents in the para
positions of the two benzenesulfonyl side arms increase
potency.24 More recently, this effect was found to be greatest
in unsymmetrical CADA compounds with two different side
arms (Ar1 ≠ Ar2).23 The most potent unsymmetrical analogue
Figure 1. (a) Structure of lead compound CADA. (b) General
structure of previously synthesized CADA compounds with various
side arms (Ar1 and Ar2) and tail groups (R). (c) Structure of potent
analogue VGD020. (d) Proposed unsymmetrical “two-site” binding
model, with the Y-substituted benzenesulfonyl side arm, of interest in
the current study, occupying site 1.
a
Scheme 1
a
*Yield as HCl salt.
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Article
Scheme 2
for CD4 down-modulation in CHO·CD4-YFP cells was found
to be VGD02023 (1, Figure 1c), which was more potent than
either symmetrical analogue with either two p-methylbenzene-
sulfonyl (tosyl) or two p-methoxybenzenesulfonyl side arms.
We also found previously that the 12-membered ring adopts
the same folded conformation in X-ray structures of various
CADA compounds.24 There is a consistent twist about the
isobutylene headgroup, placing the two side arms in two
different orientations. This led to a “two-site” model in which
the two side arm-binding regions are not symmetrically
disposed and have different steric and electronic requirements
(Figure 1d). We hypothesize that when CADA compounds
bind, they stabilize a folded conformation of the CD4 SP, which
prevents further translocation of the nascent CD4 protein
toward the ER lumen. The folded SP may form the CADA
binding site, placing certain amino acid side chains in close
proximity with the two side arms. The purpose of this study
was to vary substituents (Y) and substitution patterns on the
nontosyl side arm to determine the nature of interactions of
this side arm with residues forming its binding site (Figure 1d).
aromatic side arm is introduced by sulfonylation in the fourth
step of the sequence then palladium-catalyzed macrocyclization
produces the target compounds. Many of these macrocycles
were then converted to additional targets by modification of
substituents on the nontosyl aromatic ring, as described later.
The starting material, N-(3-aminopropyl)-p-toluenesulfona-
mide (2), was previously prepared in 48% yield by reaction of
tosyl chloride with excess 1,3-diaminopropane in toluene,
followed by recrystallization to remove the side product, 1,3-
bis[N-(p-toluenesulfonamido)]propane.23 We have found that
the ratio of monotosyl to ditosyl derivatives in the crude
product can be increased by conducting this reaction in
dichloromethane (DCM), rather than toluene, resulting in an
increased yield of 81%. The benzyl or cyclohexylmethyl tail
groups are then installed quantitatively by condensation of 2
with benzaldehyde or cyclohexanecarboxaldehyde, followed by
reducing the resulting Schiff base with sodium borohydride
(Scheme 1).23 Three-carbon chain elongation was achieved by
reaction of 3 or 4 with commercially available N-(3-
bromopropyl)phthalimide in acetonitrile (AN) in the presence
of sodium carbonate and catalytic lithium iodide. Cyclo-
hexylmethylamine 6 was purified by chromatography, as
described previously,23 while benzyl analogue 5 was converted
to the HCl salt, which was purified by trituration with diethyl
ether. Both phthalimide intermediates were deprotected
quantitatively to the corresponding primary amines, 7 and 8,
by hydrazinolysis in ethanol at room temperature.
RESULTS AND DISCUSSION
■
Synthesis. A series of target CADA analogues was designed,
based on the structure of compound 1, which has a
cyclohexylmethyl tail group, one tosyl side arm, and one p-
methoxybenzenesulfonyl side arm.23 The objective was to
explore the steric and electronic requirements of the binding
region for the nontosyl side arm. Most of the new analogues
were prepared by means of the five-step synthesis shown in
Scheme 1, which had been developed previously for the
synthesis of unsymmetrical CADA analogues.23 A majority of
the target compounds were designed with a benzyl tail group,
which generally gives better yields and improves the ease of
chromatographic purification during the later synthetic steps. A
smaller number of cyclohexylmethyl tail analogues were also
prepared, particularly to test the increase in potency relative to
some of the more potent benzyl tail analogues. The substituted
Reaction of benzylamine intermediate 7 with 14 different
sulfonyl chlorides under Schotten−Baumann conditions gave
the 14 different open-chain disulfonamides (9−22) listed in the
table in Scheme 1. Most of these sulfonyl chlorides were
commercially available, but 3-(dimethylamino)benzenesulfonyl
chloride,25 p-(dimethylamino)benzenesulfonyl chloride,26,27
benzo[d][1,3]dioxole-5-sulfonyl chloride,28 and N-methylpyr-
role-3-sulfonyl chloride29 were prepared by literature proce-
dures. Intermediate 19 with a N,N-dimethylaminosulfonyl side
arm was prepared to test the effect of an electron-donating
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Scheme 3
group lacking an aromatic ring. Two cyclohexylmethyl tail
intermediates, 23 and 24, were prepared by sulfonylation of
cyclohexylmethylamine 8. All 16 of the open-chain inter-
mediates were cyclized by palladium catalyzed reaction with 2-
methylene-1,3-propanebis(t-butylcarbonate), as described pre-
viously.23 The yields for this macrocyclization reaction after
rigorous purification of each target compound varied from 15
to 57%. In most cases, byproducts were observed, likely arising
from differences in acidity between the two sulfonamide NH
groups. The t-butoxide anion generated by decomposition of t-
butylcarbonate in this reaction serves as the base to
deprotonate the sulfonamide NH groups. Sodium carbonate
is added to scavenge acidic protons that can quench t-butoxide.
Selective deprotonation of more acidic sulfonamide NH groups
can lead to bimolecular substitution reactions competing more
effectively with macrocyclization of the intermediate. Finally,
Scheme 1 also shows the conversion of p-fluorobenezenesulfo-
namide CK201 (37) to methylthio analogue CK204 (41) via
ipso substitution (SNAr)30 in low, but useful, yield.
Previous SAR studies showed that the CD4 down-
modulation potency of CADA analogues is enhanced by
electron-donating groups on benzenesulfonamide side arms,
particularly in the para position.23,24 Hence, the three nitro
compounds with benzyl tail groups listed in Scheme 1 (27, 28,
and 34) and previously prepared VGD02323 (42), the p-nitro
analogue with a cyclohexylmethyl tail, were reduced with
sodium borohydride and cupric acetate in ethanol/water31 to
the corresponding primary amino compounds 43−46 in good
to excellent yields (Scheme 2). Tertiary heterocyclic amines
were also of interest, so the p-amino analogue with a
cyclohexylmethyl tail (46) was condensed with 2,5-dimethox-
ytetrahydrofuran in a modified Paal−Knorr synthesis32,33 to
produce pyrrole 47 (Scheme 2). Also, 46 was treated with
bis(2-chloroethyl) ether34 to give morpholine analogue 48 in
low yield. Also shown in Scheme 2 is the reaction of o-nitro
benzyl tail analogue 27 with mercaptoethanol and DBU to give
one-armed analogue 49 by ipso attack and decomposition of
the resulting Meisenheimer complex to SO2 and 2-(o-
nitrophenylthio)ethanol.35 This compound was mainly of
interest for the synthesis of additional unsymmetrical analogues
with one benzamide side arm, as described later.
Because p-methoxy side arm analogue 1 was the most potent
CADA compound at the beginning of the current study,
variations with p-oxygen substituents having different steric and
electronic properties were of interest (Scheme 3). In the
cyclohexylmethyl tail series, 1 was demethylated with boron
tribromide to give phenol 51, which was alkylated quantitatively
to produce p-ethoxy analogue 52. To further probe steric
tolerance of the binding site in this region and produce
analogues that might potentially be conjugated to imaging tags
or solid supports, phenol 51 was alkylated with N-(3-
bromopropyl)phthalimide to give 53, which was converted to
primary amine 54 by hydrazinolysis. In the benzyl tail series,
BBr3 demethylation of 50 (VGD02723) gave phenol 55, which
was alkylated with propargyl bromide to give p-propargyloxy
analogue 56. Also shown in Scheme 3 is the conversion of 55 to
the N,N-dimethylthiocarbamate ester 57. This was intended as
an intermediate in the synthesis of the p-thiol analogue via a
reported rearrangement,36,37 which did not succeed in this case.
The final planned series of analogues has one tosyl side arm
and also various benzamide side arms, as shown in Scheme 4.
This series was designed to determine if two arenesulfonamide
Scheme 4
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side arms are required for high potency and if the trends of
substituent effects in arenesulfonamides are similar in
benzamides. One-armed analogue 49 was accordingly acylated
with benzoyl chloride and various para substituted benzoyl
chlorides in good yields, as shown in Scheme 4. By the same
method used in the arenesulfonamide side arm series, p-nitro
analogue 60 was also reduced to p-amino analogue 62. Finally,
49 was also treated with N-morpholinosulfonyl chloride38 to
prepare sulfamate 63, in which the nontosyl side arm contains
an electron-donating tertiary amino group but lacks an aromatic
ring (Scheme 4).
Table 1. CD4 Down-Modulating and Anti-HIV Potencies
and Cytotoxicities of Benzyl-Tailed CADA Compounds
c
e
IC50 CD4 (μM) IC50 HIV (μM)
a
b
f
compd
struct
mean SD
mean SD
CC50 (μM)
25
26
27
28
29
30
31
32
33
34
35
36
37
38
41
43
44
45
49
50
55
56
57
58
59
60
61
62
63
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 2
Scheme 2
Scheme 2
Scheme 2
Scheme 3
Scheme 3
Scheme 3
Scheme 3
Scheme 4
Scheme 4
Scheme 4
Scheme 4
Scheme 4
Scheme 4
1.46 0.05
0.55 0.17
5.25 1.10
0.70 0.09
1.20 0.01
1.35 0.30
0.31 0.06
0.17 0.08
1.06 0.12
2.02 0.29
1.58 0.20
3.17 0.64
0.92 0.11
0.79 0.10
0.23 0.05
2.12 0.39
1.02 0.09
2.42 0.42
>50
>150
>150
>150
>150
>150
>150
>150
>150
>150
>150
>150
>150
>150
>150
>150
70.3
1.35 0.20
35.9 16.7
27.7 7.20
3.26 1.06
7.00 1.99
0.86 0.24
0.62 0.13
14.2 1.70
>50
Potency. A total of 37 new CADA compounds were
synthesized, 28 with benzyl tail groups and nine with
cyclohexylmethyl tails. Samples of each of the new compounds
were tested for CD4 down-modulation, either in their
analytically pure (>95%) free base forms or as hydrochloride
salts, as indicated in the Experimental Section or Supporting
Information for compositions determined by combustion
microanalysis. As for previous CADA compounds,23,24 most
of the samples tested were stoichiometric hydrates of the
monohydrochloride salts (or the dihydrochloride salts when a
basic site was present in a side arm). The CD4 down-
modulation potencies are described here as IC50 values
(concentration producing a 50% decrease in CD4 expression,
measured in Chinese hamster ovary/CD4/yellow fluorescent
protein (CHO·CD4-YFP) cells after 24 h of drug treatment).
The IC50 values for all 28 new benzyl-tailed analogues are given
in Table 1, along with that of previously reported reference
compound 50, which has a p-methoxybenzenesulfonyl side arm.
The structure−activity relationships for benzyl-tailed com-
pounds displayed in Table 1 show a number of interesting
trends. As observed previously for symmetrical and unsym-
metrical CADA compounds, electron-donating groups (EDGs)
in the para position of the benzenesulfonamide side arm
contribute to high potency. The most potent compounds have
the best EDGs in the para position, dimethylamino in 32 (IC50
0.17 μM), methylthio in 41 (IC50 0.23 μM), and methoxy in 50
(IC50 0.29 μM), while 27 with para-nitro, a strong electron-
withdrawing group (EWG), is only weakly active (IC50 5.25
μM). Even in the absence of a phenyl ring on the sulfonyl
group, dimethylamino and N-morpholino groups in 35 and 63
give significant potencies (IC50 1.58 and 1.08 μM, respectively).
The benzamide side arm compounds 58−62 are decidedly less
potent than the corresponding sulfonamides, 50, CADA24
(IC50 0.40 μM), 27, 25, and 45, suggesting either that the
aromatic rings are oriented differently because of the nearly
planar amide group or that the sulfonyl group is important as a
double hydrogen-bond acceptor (HBA).
In the sulfonamide series, when an additional methoxy group
is placed in the meta position of the p-methoxybenzensulfonyl
side arm of 50 (IC50 0.29 μM), potency is unchanged (IC50
0.31 μM for 31), within experimental error, suggesting that the
total electron density of the benzene ring is not important.
When the second methoxy group is moved to the ortho
position in 30, potency is reduced (IC50 1.35 μM), suggesting
that an ortho group could disrupt the binding conformation of
this side arm. Replacement of the p-methoxy group of 50 with
trifluoromethoxy in 29 increases the IC50 value by almost 1 μM,
while replacement of methoxy by propargyl in 56 increases it by
only about 0.1−0.2 μM, suggesting that electronics are more
important than sterics in this position. Finally, replacing OMe
or NMe2 with OH or NH2, respectively, greatly decreases
potency, despite the similar electron donating abilities of OR vs
12.77 3.17
>50
4.66 0.06
>50
1.44 0.14
6.93 1.41
6.22 1.05
7.72 2.00
nd
>150
63.4
d
>50 (20%)
0.29 0.09
3.58 0.54
0.46 0.07
6.28 2.04
4.22 0.84
3.78 2.38
> 150
>150
13.6
0.55 0.09
2.84 0.73
0.55 0.07
>50
>150
106
3.86 0.81
3.95 0.68
>50
23.6
19.9
d
>50 (39%)
>150
15.8
7.32 1.70
>10
d
>50 (15%)
>10
13.7
1.08 0.10
8.99 3.26
>150
a
b
Compounds were used as HCl salts. Location of structural diagram.
c
IC50: inhibitory concentration 50%; concentration at which 50%
down-modulation of CD4 expression measured in CD4-YFP trans-
fected CHO cells after 24 h of drug treatment. Values are the mean
d
standard deviation with n ≥ 3. Activity of compound is too weak to
calculate IC50 value. Activity is represented as percentage CD4 down-
modulation measured at highest (50 μM) compound concentration
e
used. IC50: inhibitory concentration 50%; concentration at which 50%
reduction of HIV-1 NL4.3 (X4) replication in MT-4 cells was
measured. Values are mean standard deviation with n = 2 or 3; nd,
f
not determined. CC50: cytotoxic concentration 50%; concentration
required to reduce viability of MT-4 cells by 50%.
OH and NR2 vs NH2. The increases in IC50 value for these
changes are ca. 3.3 μM (50 to 55) and 2.2 μM (32 to 45),
respectively. This suggests that the introduction of hydrogen-
bond donating (HBD) groups on the benzene ring may greatly
change the manner in which the molecule binds and is oriented
within the binding site.
The IC50 values for the nine new cyclohexylmethyl-tailed
analogues are given in Table 2, along with that of previously
reported reference compound 1, which has a p-methoxybenze-
nesulfonyl side arm. All of the new cyclohexylmethyl tail
compounds also have a single substituent in the para position of
the nontosyl side arm. Replacing the benzyl tail with the more
hydrophobic cyclohexylmethyl group increases the potency of
the reference compound 50 by a factor of 2 for 1 (IC50 0.15
μM). For the compounds with the best EDG (dimethylamino),
the cyclohexylmethyl tail group increases potency by a factor of
3: IC50 0.17 μM for 32 to 0.063 μM for 40, which is now the
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Table 2. CD4 Down-Modulating and Anti-HIV Potencies
and Cytotoxicities of Cyclohexylmethyl-Tailed Compounds
c
d
IC50 CD4 (μM) IC50 HIV (μM)
a
b
e
compd
struct
mean SD
mean SD
CC50 (μM)
1
Figure 1
0.15 0.01
1.10 0.13
0.063 0.005
1.00 0.15
0.18 0.01
0.13 0.01
1.14 0.02
0.19 0.01
0.75 0.06
>50
0.49 0.06
1.95 0.24
0.18 0.03
>10
99
13.2
44
39
40
46
47
48
51
52
53
54
Scheme 1
Scheme 1
Scheme 2
Scheme 2
Scheme 2
Scheme 3
Scheme 3
Scheme 3
Scheme 3
10.6
48
1.25 0.40
0.36 0.02
nd
>150
>150
19.6
32.2
11.6
Figure 2. (a) Correlation between anti-HIV-1 (NL4.3) activities in
MT-4 cells and CD4 expression/down-modulation in CHO·CD4-YFP
cells for 27 compounds (1, 26−33, 35, 37−41, 43−45, 47, 48, 50, 52,
55, 56, 58, 59, and 63). Mean IC50 values from Tables 1 and 2 were
used, with pIC50 = −log10(IC50). (b) Antiviral activities in TZM-bl
cells infected with X4- (NL4.3) and R5-tropic (BaL) HIV-1 strains
correlate well for 14 compounds (CADA, 1, 26−33, 43, 45, 58, and
59). The pIC50 values are plotted as calculated from mean IC50 values
of three independent experiments (see Table S1 in Supporting
Information).
0.62 0.19
nd
nd
a
b
Compounds were used as HCl salts. Location of structural diagram.
c
IC50: inhibitory concentration 50%, i.e., concentration at which 50%
down-modulation of CD4 expression was measured in stably CD4-
YFP transfected CHO cells after 24 h of drug treatment. Values are the
d
mean + standard deviation with n = 3. IC50: inhibitory concentration
50%; concentration at which 50% reduction of HIV-1 NL4.3 (X4)
replication in MT-4 cells was measured. Values are mean standard
deviation with n = 2 or 3; nd, not determined. CC50: cytotoxic
concentration 50%; concentration required to reduce viability of MT-4
cells by 50%.
e
(Figure 2b), indicating that the new unsymmetrical compounds
exert equal anti-HIV activities, irrespective of the coreceptor
used by the viral strain.
Molecular Modeling. The SAR study just discussed has
revealed three important lessons about the effects of nontosyl
side arm substituents on CD4 down-modulation activity: (1)
potency is enhanced by strong EDGs, particularly in the para
position of the ring, (2) steric bulk in the para position only
weakly decreases potency, and (3) HBD groups, such as OH
and NH2 greatly decrease potency. How can these effects be
understood in the context of the hypothesis that CADA
compounds inhibit cotranslational translocation of CD4 by
stabilizing a folded conformation of the CD4 signal peptide in
the transmembrane channel (translocon)? The effect of
introducing a HBD group is interesting because a new
hydrogen bond should, in principle, stabilize a complex
between a small molecule and a peptide or protein target.
Adding a HBD group should increase water solubility and may
affect distribution within the cell, but CADA compounds are
otherwise quite hydrophobic and even an analogue with an OH
or NH2 group should easily cross the cell membrane.
Remembering that the SP is a relatively small target (only 25
amino acids), the binding site is likely to be poorly defined and
may be conformationally stabilized by interactions between the
SP and the protein subunits of the translocon. While the SP has
been identified as the principal target of CADA compounds,
part of the molecule, especially the hydrophobic tail, may also
interact with the translocon. Introducing a good HBD group
might disrupt the folded SP conformation needed to block
translocation, either by changing the way the molecule interacts
with the SP, or by weakening its hold on the SP because of a
new interaction between the molecule and a hydrogen-bond
acceptor on the translocon.
most potent CADA analogue synthesized to date. As observed
in the benzyl-tailed series, replacing NMe2 with HBD group
NH2 greatly decreases potency (IC50 1.0 μM for 46), and the
same effect is observed for replacement of OMe by OH (IC50
0.15 for 1 vs 1.14 μM for 51). Replacing NMe2 in 40 with
heterocyclic substituents N-pyrrolyl or N-morpholino decreases
potency by factors of 3 (IC50 0.18 μM for 47) or 2 (IC50 0.13
μM for 48), respectively, again suggesting a steric constraint for
nonlinear substituents in the binding site at this ring position.
Compounds 52 (p-ethoxy, IC50 0.19 μM) and 53 (p-(3-(N-
phthalimido)propyloxy), IC50 0.75 μM) further probe sterics in
this area and show a very small decrease in potency for a change
from OMe to OEt and a more significant drop with the much
larger substituent in 53. Finally, replacement of the phthalimide
group on this chain with NH2 (compound 54) makes CD4
down-modulation undetectable, again showing the deleterious
effect of hydrogen bond donors on potency.
As a consequence of reduced CD4 cell surface levels caused
by treatment with active compounds, HIV is prevented from
infecting its target cells. Thus, we tested the activities of the
compounds against the infection of the T-cell line MT-4 with
the CXCR4-using HIV-1 laboratory strain NL4.3 (Tables 1 and
2). As observed for previously reported analogues,2,24 for the
new unsymmetrical compounds a good correlation was seen
between potencies for CD4 receptor down-modulation and for
inhibition of HIV entry into CD4+ T-helper lymphocytes
(Figure 2a). Only the most potent 27 new compounds were
included in this correlation because the CD4 down-modulation
assay is more sensitive than the anti-HIV assay, as can be
gleaned from Figure 2a, and the 10 least potent compounds did
not give exact IC50 values for inhibition of HIV replication in
vitro. In addition, the antiviral potencies of a representative set
of analogues were determined against a CCR5-using and a
CXCR4-using HIV-1 strain (BaL and NL4.3, respectively;
Table S1 in Supporting Information). Comparison of the
antiviral pIC50 values of these compounds between both
representative HIV-1 strains revealed a remarkable correlation
We propose that non-HBD polar substituents on the
nontosyl side arm directly affect the strength of the interaction
between the small molecule and the CD4 SP. Our initial
hypothesis was that the nontosyl side armmight act as a π−
electron donor, so EDGs enhance this interaction with the SP
by increasing the electron density of this aromatic ring.
Returning to Table 1, the significant activity of o-nitro analogue
28 (IC50 0.70 μM) contradicts this hypothesis because NO2 is
well-known as a powerful EWG. We then noticed that when the
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NO2 group is moved from the ortho to the meta position in 34,
the IC50 increases to 2.02 μM, and then to 5.25 μM for p-nitro
analogue 27. This strongly suggested that the important
parameter is not the electron density of the aromatic ring but
its dipole moment. The sulfonyl group itself is a strong EWG
and produces an electric dipole in the benzene ring in the
absence of other substituents, as shown in Figure 3a.
enesulfonamides modeling the nontosyl side arms of 21 new
benzyl-tailed CADA analogues shown in Schemes 1−4, plus
CADA and 50. The initial study group omitted dimethylami-
nosulfamide 35, which lacks an arene ring, but included N-
morpholinosulfamide 63, in which the morpholine ring may
bind in an orientation that is similar to the benzene rings of
other analogues. The geometries were optimized for each
molecule and aligned with the coordinate system shown in
Figure 4a. Then the dipole moments were calculated, resulting
Figure 3. Orientations and relative sizes of in-plane dipole moment
components in arenesulfonamide side arms, represented by N,N-
dimethylbenzenesulfonamides. (a) Dipole induced by sulfonyl group.
(b) Enhanced dipole produced by p-EDG. (c) Dipole produced by p-
EWG stronger than sulfonyl. (d) Dipole produced by o-EWG.
Conjugating EDGs in the para position set up a push−pull
situation, increasing the electric dipole component in the plane
of the aromatic ring, with its positively charged end toward the
para ring carbon atom (Figure 3b). A strong EWG in the para
position competes with the sulfonyl group and would produce a
small dipole in the opposite direction if it is a better EWG (e.g.,
nitro) than sulfonyl (Figure 3c). Vector addition of the dipole
produced by the sulfonyl group and the dipole generated by a
strong EWG in the ortho position would produce a sizable
dipole moment oriented as shown in Figure 3d.
The activities of the dimethoxy analogues 30 and 31 are also
consistent with this dipole moment theory. The 3,4-
dimethoxybenzenesulfonyl analogue 31 has the same potency
(IC50 0.31 μM) as the p-dimethoxy reference compound 50
(IC50 0.29 μM), within experimental error. The second
methoxy group should increase the net dipole, but change its
orientation and possibly also produce steric hindrance. The 2,4-
dimethoxy analogue 30 is significantly less potent (IC50 1.35
μM). In this case, the two EDGs compete for orientation of the
dipole, so its component along the axis between the sulfonyl
group and the para position will be much smaller.
Figure 4. (a) Alignment of model N,N-dimethylsulfonamides with the
Cartesian coordinate system. The ring is in the xy plane, the S−C
bond is along the x axis, and the S−N bond is in the xz plane. (b)
Energy-minimized structure of side arm model of 25 (R = H) and
orientation of the calculated dipole (μtot: large red arrow pointing from
the negative charge to the positive charge).
The dipole moment of a specific aromatic ring has not often
been recognized as a dominant factor in small molecule−
protein interactions, although two QSAR studies of sulfona-
mide carbonic anhydrase inhibitors have found dipole moments
to be a dominant factor in modeling inhibition activity, one
using the dipole component along the S−N bond39 and the
other using the total molecular dipole moment.40 A more
recent study of antileishmanial benzenesulfonamides also used
the molecular dipole moment as a prominent factor in the
QSAR model.41 No specific interaction between the
sulfonamide dipole and amino acid residues was proposed in
any of these publications, but in these cases in the literature,
and also in the binding of CADA compounds to the CD4 SP,
the arenesulfonamide may π-stack with an aromatic side chain
or with a planar functional group of the peptide/protein such as
an amide linkage or a polar side chain group.
in a total dipole moment (μtot) and the x, y, and z components
of the dipole for each molecule (μx, μy, and μz, respectively),
which are listed in Table 3. Chemists conventionally draw
dipole vectors pointing from the positive charge to the negative
charge, as in Figure 3, while the true mathematical sign of a
dipole moment is opposite.42 Hence, the x component of the
dipole produced by the sulfonamide substituent shown in
Figure 3a is positive along the x-axis defined in Figure 4a, rather
than negative. The energy-minimized structure of the side arm
model for compound 25 lacking an R substituent on the
benzenesulfonamide ring is shown in Figure 4b, along with the
orientation of the calculated dipole.
The total dipole moments and the x, y, and z components of
the dipole moments for each model compound were plotted
separately against the CD4 down-modulation pIC50 values of
each CADA analogue. The use of pIC50 values in these plots
was based on the assumption that the IC50 value for CD4
down-modulation is primarily determined by the affinity of the
To test the theory that the arenesulfonamide dipole is a
major contributor to the strength of the interaction between
CADA compounds and the CD4-SP, we performed quantum
mechanical calculations on a series of 23 N,N-dimethylar-
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Table 3. Dipole Moments of 23 N,N-Dimethylsulfonamides
Modeling Side Arms of Benzyl-Tailed CADA Compounds
side arm dipole moment (Debye)
a
b
c
compd
struct
pIC50
μx
μy
μz
μtot
CADA*
50*
25*
26*
27*
28*
29*
30*
31*
32*
33*
34*
36
Figure 1
6.40
6.54
5.84
6.26
5.28
6.15
5.92
5.87
6.51
6.77
5.97
5.69
5.50
6.04
6.10
6.64
5.66
5.99
5.62
5.45
6.34
5.20
5.97
3.47
3.92
2.81
3.21
−1.24
3.20
0.23
3.68
3.56
6.09
3.52
−0.16
−5.20
1.26
4.19
3.35
2.22
3.67
5.03
3.19
3.41
5.02
−0.97
0.90
−0.29
−0.90
−0.60
0.00
3.37
3.30
3.40
3.35
3.95
3.64
3.89
2.08
3.25
3.19
3.15
3.47
3.13
3.42
3.39
3.31
2.66
4.20
4.14
3.32
3.32
6.01
3.19
4.91
5.13
4.50
4.68
4.14
5.63
4.05
5.69
4.78
6.93
4.87
4.30
6.26
3.76
5.48
4.73
3.82
5.58
6.56
4.62
4.77
7.90
3.36
Scheme 3
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 1
Scheme 2
Scheme 2
Scheme 2
Scheme 3
Scheme 3
Scheme 3
Scheme 4
−2.86
−1.10
−3.80
0.55
Figure 5. Plot of the x component of dipole moments calculated for
16 model arenesulfonamides vs pIC50 values for CD4 down-
modulation by the corresponding CADA compounds (CADA, 25−
34, 37, 38, 41, 50, and 56).
0.85
−1.17
2.54
contains the C−S bond of the arenesulfonamide group. The
sulfonamide EWG establishes a positive dipole along the x-axis
(μx = 2.81 D for 25), and this is increased to 6.09 D by the
strong EDG (p-NMe2) of 32, the most potent compound in
the series. Accordingly, the weakest CD4 down-modulator in
the correlation displayed in Figure 5 is p-nitro analogue 27, and
in this case μx is actually reversed (−1.24 D). Moving the nitro
group to the ortho position in 28 restores the effective
orientation of μx and increases it relative to the unsubstituted
25. Accordingly, 28 (μx = 3.20 D) is more potent than 25.
There are many possible reasons why the correlation shown
in Figure 5 is significant but not more perfect. First, compounds
with different physiochemical properties may distribute differ-
ently between the aqueous medium and the cells, and also
within the cells, resulting in different concentrations at the CD4
SP target within the translocon. Second, the positions of
substituents on the aromatic ring alter the conformation of the
arenesulfonamide group, especially when the substituent is in
the ortho position. This could change van der Waals, hydrogen
bonding, and polar interactions between the sulfonamide group
and the binding site. Additionally, substituent changes do not
simply alter molecular dipoles, they also change sterics and can
produce new interactions within the binding site. Considering
all this, it is remarkable that a significant correlation is observed
between potency and a single molecular variable (μx). This
indicates that the distribution of electrons in the nontosyl side
arm has a strong influence on affinity of the compound for the
CD4 SP and that this aromatic ring likely forms a strong polar
interaction with one or more key amino acid residues or other
functional groups within the binding site.
−1.51
−0.91
1.01
37*
38*
41*
43
0.39
1.61
44
0.17
45
−0.79
0.40
55
56*
57
0.25
−1.04
−0.35
63
a
Compounds were used as HCl salts; asterisks indicate compounds
included in the final correlation (Figure 4). Location of structural
diagram. IC50: inhibitory concentration 50%, i.e., concentration (μM)
at which 50% down-modulation of CD4 expression was measured in
stably CD4-YFP transfected CHO cells after 24 h of drug treatment;
pIC50 = −log(IC50) based on the IC50 values for CD4 down-
modulation as listed in Table 1.
b
c
compound for its target, the CD4 SP, in which case the pIC50
value should be roughly proportional to the negative enthalpy
of binding (−ΔH). The best correlation in these initial plots
(not shown) was observed for the plot of the x component vs
pIC50, but this correlation was poor (R2 = 0.173). Considering
that for this correlation to be meaningful, the ring attached to
the sulfonyl group of all the compounds has to rest in the same
position in the binding site, 36 and 63 lacking carbocyclic
aromatic rings were excluded from the study. Also considering
that potency-reducing HBD groups may alter the overall
binding configuration, phenol 55 and anilines 43−45 were also
excluded. Finally, thiocarbamate 57 was removed from the
study group because the bulky thiocarbamate substituent would
not likely be coplanar with the benzene ring and would have its
own dipole outside the xy plane; this could confound the
correlation. A plot of the x-component of the dipole moment of
the remaining 16 model compounds against the CD4 down-
modulation pIC50 values of the corresponding CADA
compounds (Figure 5) shows a significant correlation (R2 =
0.625).
CONCLUSIONS
■
A total of 28 new unsymmetrical analogues bearing a benzyl tail
group and nine new unsymmetrical analogues bearing a
cyclohexylmethyl tail have been designed, synthesized, and
tested for CD4 down-modulation and suppression of HIV-1
replication in vitro. This resulted in a wide range of CD4 down-
modulation potencies, which correlated well with anti-HIV
potencies. The most potent new CD4 down-modulator is
compound 40 (IC50 63 nM), with a cyclohexylmethyl tail, a
tosyl side arm, and a second 4-dimethylaminobenzenesulfonyl
side arm. For a series of 16 CADA analogues with
arenesulfonamide side arms, a significant correlation was
observed between the pIC50 values of the compounds for
CD4 down-modulation and the component of the electric
dipole moment in the aromatic ring and along the C−S axis.
This indicates that one side arm may π-stack with a key
functional group in the CD4 SP and that the resulting attractive
The roughly linear correlation observed between the CD4
down-modulation pIC50 values of 16 CADA compounds and
the x-components of the dipole moments calculated for the
nontosyl side arms of each compound suggests that the
electron distribution in this side arm plays a major role in
stabilizing the complex with the CD4 SP. The x-component of
the side arm dipole lies in the plane of the aromatic ring and
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Computational Modeling. Model N,N-dimethylsulfonamide
molecules, including N,N-dimethylbenzenesulfonamides (Figure 3a),
representing the side arms of 21 new unsymmetrical disulfonamide
CADA compounds with a benzyl tail, plus CADA and compound 50,
were built and visualized using the open-source molecular editor
Avogadro.43,44 Geometry optimizations were then performed using
Gaussian 03 revision C.02545 accessed via the web browser-based
graphical user interface (GUI) WebMO (version 12.1).46 Geometry
optimizations of the model molecules were done using a B3LYP47−50
functional with a 6-31G(d)51−54 standard basis set. The benzene rings
of the geometrically optimized molecular structures were then aligned
approximately in the xy plane, keeping the S−C1−C4 atoms aligned
long the x axis using the program Avogadro.43,44 To obtain accurate
results, single-point calculations of the geometrically optimized and
aligned molecular structures of the model molecules were performed
at B3LYP47−50 with def2-TZVP55,56 basis set levels in the program
ORCA 2.9.1.57
interaction is a major factor determining the stability of the
complex between the molecule and the CD4 SP target. The
results of this study should enable design of more potent
CADA analogues and help determine molecular details of the
interaction between CADA compounds and the CD4 SP, which
will lead to a more complete understanding of this unique
mechanism of action.
EXPERIMENTAL SECTION
■
Biological Evaluation: CD4 Down-Modulation. To study the
effect of the CADA analogues on CD4 expression, CHO cells, stably
expressing CD4-YFP (human CD4 fused at its COOH-terminus to the
yellow fluorescent protein), were treated for 24 h with serial dilutions
(1:5) of the compounds at 37 °C. Cells were then washed, fixed in 1%
formaldehyde, and analyzed immediately. Data were acquired with a
FACSCalibur flow cytometer (BD Biosciences) using the 488 nm laser
line and CellQuest software (BD Biosciences). YFP was measured
with a FL-1 detector, and data were analyzed with FLOWJO software
(Tree Star, San Carlos, CA). Down-modulation of CD4 was evaluated
by the decrease in fluorescence intensity on CADA-treated cells
relative to matched, untreated cells. To calculate the efficiency of CD4
down-modulation, the median fluorescence intensity (MFI) for YFP
for each sample was expressed as a percentage of the MFI of control
cells (after subtracting the background MFI of the nontransfected
control cells).
Chemistry: General Methods. All reactions were performed
under an atmosphere of dry nitrogen. Reagents and solvents purchased
from Aldrich Chemical Co., Acros Organics, or Fisher Scientific were
of ACS reagent grade or better and were used without purification,
unless indicated otherwise. Anhydrous AN was distilled from CaH2.
HCl (2 N) in methanol/water was prepared from 42 mL of concd aq
HCl (12.1 N) and 210 mL of methanol. For macrocyclization
reactions, the disulfonamide intermediate (previously washed with aq
NaOH, as described for deprotonation of HCl salts) and 2-methylene-
1,3-propanebis(tert-butylcarbonate) were dried in vacuo for at least 16
h then dissolved in anhydrous AN with dppb and Pd2(dba)3 added
last. All the equipment required for macrocyclization reactions,
including magnetic stir bars, spatulas, syringes, and needles, were
dried overnight at 110 °C. Trituration of HCl salts was done by
sonication in anhydrous diethyl ether (5−15 mL, unless indicated
otherwise) for 5 min and filtration, repeating the process two more
times. “Overnight” periods are ca. 16 h. Organic solutions were dried
with anhydrous Na2SO4, and sample drying in vacuo was performed at
0.1 mm and room temperature. Column chromatography was
performed with Sorbent Technologies neutral alumina (50−200
μm) or standard grade silica (32−63 μm), unless noted otherwise.
Chromatotron chromatography was performed with Sorbent Tech-
nologies neutral alumina (UV254 with gypsum). Melting points were
measured on a Thomas−Hoover or Mel-Temp apparatus and are
uncorrected. 1H NMR (400 or 500 MHz) and 13C NMR (100 or 125
MHz) spectra were acquired on a Varian 400 or a Varian Unity+ 500
spectrometer. All chemical shifts (δ) are reported in ppm units relative
Viral Replication. MT-4 cells (obtained from the American Type
Culture Collection) were seeded in 96-well plates at 3.75 × 105 cells/
mL (150 μL/well) filled with culture medium (Roswell Park Memorial
Institute 1640, 10% (v/v) fetal bovine serum, 2 mM L-glutamine) and
test compounds. Cells were preincubated with the compounds for 15
min before addition of the laboratory HIV-1 strain NL4.3 (obtained
from the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH) at (50 μL; 30 pg/well) and incubation at 37 °C.
Four days after infection, cell viability was quantified by the MTS/
phenazine ethosulfate (PES) method. Absorbance was measured at
490 and 700 nm and used to calculate the 50% inhibitory
concentration (IC50) of each compound for viral replication.
To compare the antiviral effect of CADA analogues on X4-tropic
and R5-tropic HIV-1 strains, we used CD4+, CXCR4+, and CCR5+
TZM-bl cells expressing firefly luciferase and Escherichia coli β-
galactosidase (a kind gift from Dr. G. Vanham, ITG, Antwerp,
Belgium). Cells (50 μL; 2 × 105 cells/mL) were resuspended in cell
culture medium supplemented with 15 μg/mL diethylaminoethyl-
dextran (DEAE-dextran; Sigma-Aldrich, Diegem, Belgium) and
preincubated for 30 min at 37 °C in 96-well plates with test
compounds diluted in 100 μL of cell culture medium. Next, HIV-1 X4
strain NL4.3 or HIV-1 R5 strain BaL (also obtained from the AIDS
Research and Reference Reagent Program, Division of AIDS, NIAID)
were added (50 μL) according to the TCID50 of the viral stocks. Two
days postinfection, viral replication was measured by luminescence.
Steadylite Plus reagent (PerkinElmer, Zaventem, Belgium) was mixed
with lyophilized substrate according to manufacturer’s guidelines.
Supernatant (120 μL) was removed, and 75 μL of Steadylite plus
substrate solution was added to the 96-well plates. Next, the plates
were incubated in dark for 10 min in a closed plate shaker (PHMP,
Grant, Shepreth, Cambridgeshire, UK). Finally, cell lysis was scored
microscopically, and 100 μL was transferred to white Lumitrac 96-well
plates (Greiner Bio-One, Frickenhausen, Germany) to measure the
relative luminescence units (RLUs) using a SpectraMax L microplate
reader and Softmax Pro software (Molecular Devices, Sunnyvale, CA,
USA) with an integration time of 0.6 s and a dark adapt of 5 min.
Cytotoxicity. MT-4 cells were seeded in transparent 96-well plates
at 104 cells per well in Dulbecco’s Modified Eagle Medium (Life
Technologies, Waltham, MA, USA) with 10% (v/v) fetal bovine serum
and 10 mM HEPES. Subsequently, compounds were added and the
cell/compound mixture was incubated at 37 °C for 48 h. The 50%
cytotoxic concentration (CC50) of each compound was determined
from the reduction of viability of cells exposed to the compound, as
measured by the MTS/phenazine ethosulfate (PES) method.
1
to solvent resonances, as follows: H, CDCl3/TMS = 0.00, DMSO-d6
= 2.50, CD3OD = 3.31; 13C, CDCl3 = 77.23, DMSO-d6 = 39.7,
CD3OD = 49.15 ppm. Infrared spectra (IR) were recorded on a
Nicolet 6700 FTIR spectrometer. Low-resolution mass spectra (MS)
were acquired on a Waters Micromass ZQ electrospray ionization
quadrupole mass spectrometer with positive ion detection (capillary
voltage = 3.5 kV). High-resolution mass spectra (HRMS) were
acquired on an Agilent 6230 TOF mass spectrometer. Samples for
elemental analysis were dried at 78 °C (0.1 mm) for 2 days, unless
stated otherwise, and microanalysis was performed by NuMega
Resonance Laboratories, Inc. Samples for biological testing were
greater than 95% pure, as shown by combustion microanalysis.
N-(3-Aminopropyl)-p-toluenesulfonamide (2). A solution of 40 g
(0.21 mol) of TsCl in 200 mL of DCM was added dropwise to 156 g
(2.10 mol) of 1,3-diaminopropane, which was stirred vigorously at 0
°C. The resulting solution was stirred overnight and allowed to
gradually warm to room temperature. The solvent was then removed
by rotary evaporation, then a mixture of the residue and 800 mL of 1:1
(v/v) methanol/water was stirred for 30 min at room temperature.
Cooling overnight in a refrigerator gave white crystals that were
collected by vacuum filtration, washed with cold water, and dried in
vacuo, yielding 0.45 g of 1,3-bis(p-toluenesulfonamido)propane side
product. The filtrate was concentrated to dryness by rotary
evaporation. The residue was dried in vacuo and recrystallized by
dissolving in minimal boiling water and storage in a refrigerator for 2 d.
The resulting white solid was collected by vacuum filtration, washed
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with cold water, and dried in vacuo, yielding 39 g (81%) of the desired
product, mp 112−115 °C (lit.23,58,59 113−115, 112−114, 114−117
°C).
temperature for 24 h, then the layers were separated. The aqueous
layer was extracted with DCM (2 × 10 mL). The combined organic
solutions were dried, filtered, and concentrated by rotary evaporation.
The residue was dried in vacuo, then stirred with 13 mL of 2 N HCl in
MeOH/H2O for 5 h. The solvent was removed by rotary evaporation,
and the residue was dried in vacuo. The resulting brown glassy solid
was triturated with ether then dried in vacuo, yielding 0.84 g (85%) of
11·HCl as a brown solid. A mixture of 0.84 g of 11·HCl, 20 mL of
DCM, 20 mL of 2 N aq NaOH, and 20 mL of satd aq NaCl at was
stirred room temperature for 4 h, then the layers were separated. The
aqueous layer was extracted with DCM (2 × 15 mL). The combined
organic solutions were dried, filtered, and concentrated by rotary
evaporation. The residue was dried in vacuo, yielding 0.75 g (81%) of
N-(3-(N′-Phthalimido)propyl)-N-(3-(p-toluenesulfonamido)-
propyl)benzylamine (5). A mixture of 4.98 g (15.6 mmol) of 3,23 1.58
g (14.9 mmol) of Na2CO3, 0.57 g (4.2 mmol) of LiI, and 95 mL of AN
was stirred at room temperature as a solution of 8.44 g (31.5 mmol) of
N-(3-bromopropyl)phthalimide in 15 mL of AN was added dropwise
over 30 min. The resulting mixture was stirred and boiled under reflux
for 24 h, cooled to room temperature, then filtered through a fine
porosity sintered glass funnel. The residue washed with 30 mL of AN,
then the combined filtrates were concentrated by rotary evaporation.
The residue was dried in vacuo, giving 8.1 g of a viscous brown oil,
which was stirred at room temperature for 5 h with 95 mL of 2 N HCl
in MeOH/H2O. The mixture was concentrated to dryness by rotary
evaporation then dried in vacuo. The residue was triturated with ether
(3 × 50 mL) then dried in vacuo, yielding 8.22 g (97%) of 5·HCl as a
1
pure 11. H NMR (500 MHz, CDCl3) δ 8.24 (d, 9 Hz, 2 H, m-
ArSO2), 7.92 (d, 9 Hz, 2 H, o-ArSO2), 7.62 (d, 8 Hz, 2 H, o-Ts), 7.24
(d, 8 Hz, 2 H, m-Ts), 7.19 (m, 2 H, m-Bn), 7.13 (m, 3 H, o,p-Bn), 5.58
(bs, 2 H, NH), 3.38 (s, 2 H, CH2Ph), 2.95 (t, 6 Hz, 2 H,
CH2NHSO2Ar), 2.87 (t, 6 Hz, 2 H, CH2NHTs), 2.40 (m, 4 H,
CH2NBn), 2.36 (s, 3 H, CH3), 1.63 (m, 4 H, CCH2C). 13C NMR
(125 MHz, CD3OD) δ 146.0, 143.5, 138.0, 136.7, 129.7, 129.1, 128.6,
128.3, 127.5, 127.1, 124.3, 58.9, 52.4, 52.0, 42.4, 26.2, 26.0, 21.5. A
mixture of 0.1 g of 11 and 5 mL of 2 N HCl in MeOH/H2O was
stirred at room temperature for 5 h. The solvent was removed by
rotary evaporation, and the residue was dried in vacuo. The resulting
brown glassy solid was triturated with ether then dried in vacuo,
yielding 85 mg of pure 11·HCl as a brown solid, mp 163−170 °C
(dec). 1H NMR (500 MHz, CD3OD) δ 8.41 (d, 8 Hz, 2 H, m-ArSO2),
8.06 (d, 8 Hz, 2 H, o-ArSO2), 7.73 (d, 8 Hz, 2 H, o-Ts), 7.54 (m, 5 H,
Bn), 7.40 (d, 8 Hz, 2 H, m-Ts), 4.37 (s, 2 H, CH2Ph), 3.24 (m, 4 H,
CH2NBn), 2.99 (t, 7 Hz, 2 H, CH2NHSO2), 2.91 (t, 7 Hz, 2 H,
CH2NHSO2), 2.42 (s, 3 H, CH3), 1.98 (m, 4 H, CCH2C). 13C NMR
(125 MHz, CD3OD) δ 150.2, 145.8, 143.6, 136.9, 130.9, 130.0, 129.5,
129.2, 128.0, 126.7, 124.1, 57.1, 50.0, 49.8, 39.7, 39.6, 23.9, 23.8, 20.0.
IR (neat, cm−1) 3078 (w), 2940 (w), 2557 (w), 1528 (m), 1455 (w),
1325 (m), 1172 (s), 1088 (m), 1072 (m), 913 (w), 854 (w), 812 (w),
738 (s), 685 (s), 658 (s). MS m/z 561 (MH+). Anal. Calcd for
C26H32N4O6S2·HCl: C, 52.30; H, 5.57; N, 9.38. Found: C, 52.20; H,
5.74; N, 9.42.
1
beige solid, mp 197−201 °C (dec). H NMR (500 MHz, CDCl3) δ
11.99 (bs, 1 H, NH+), 7.83 (m, 2 H, Phth), 7.73 (m, 4 H, o-Ts, Phth),
7.56 (m, 2 H, o-Bn), 7.28 (m, 5 H, m-Ts, m,p-Bn), 6.37 (bs, 1 H,
TsNH), 4.34 (m, 2 H, CH2Ph), 3.73 (t, 8 Hz, 2 H, CH2NPhth), 3.26
(bs, 2 H, CH2NBn), 3.13 (bs, 2 H, CH2NBn), 3.06 (t, 8 Hz, 2 H,
CH2NHTs), 2.41 (s, 3 H, CH3), 2.30 (m, 2 H, CCH2C), 2.18 (m, 2 H,
CCH2C). 13C NMR (125 MHz, CD3OD) δ 168.3, 143.6, 137.0, 134.1,
131.9, 130.6, 129.8, 129.5, 126.7, 122.9, 56.8, 50.3, 49.6, 39.6, 34.2,
23.8, 22.8, 20.0. IR (neat, cm−1) 3092 (w), 2493 (w), 1767 (w), 1705
(s), 1398 (m), 1329 (m), 1156 (s), 1094 (m), 1025 (m), 811 (m), 759
(m), 717 (s), 661 (m). MS m/z 506 (MH+). Anal. Calcd for
C28H31N3O4S·HCl: C, 62.04; H, 5.95; N, 7.75. Found: C, 61.70; H,
6.33; N, 7.97. A mixture of 5·HCl, 70 mL of 2 N aq NaOH, 70 mL of
DCM, and 70 mL of satd aq NaCl was stirred at room temperature for
5 h, then the layers were separated. The aqueous layer was extracted
with DCM (2 × 50 mL). The combined organic solutions were
washed with 50 mL of satd aq NaCl, then dried. Filtration, rotary
1
evaporation and drying in vacuo gave 7.35 g (93%) of pure 5. H
NMR (400 MHz, CDCl3/TMS) δ 7.85 (d, 8 Hz, 2 H, Phth), 7.71 (m,
4 H, o-Ts, Phth), 7.24 (m, 7 H, Bn, m-Ts), 5.99 (bs, 1 H, TsNH), 3.59
(t, 6 Hz, 2 H, CH2NPhth), 3.48 (s, 2 H, CH2Ph), 3.04 (m, 2 H,
CH2NHTs), 2.46 (t, 6 Hz, 2 H, CH2NBn), 2.42 (t, 6 Hz, 2 H,
CH2NBn), 2.35 (s, 3 H, CH3), 1.80 (m, 2 H, CCH2C), 1.65 (m, 2 H,
CCH2C). 13C NMR (100 MHz, CDCl3) δ 168.3, 142.9, 137.3, 134.1,
133.9, 132.0, 129.5, 129.1, 128.4, 127.1, 123.3, 123.2, 59.0, 58.5, 51.8,
50.9, 42.3, 36.0, 25.9, 21.4.
N-(3-Aminopropyl)-N-(3-(p-toluenesulfonamido)propyl)-
benzylamine (7). A mixture of 8.32 g (16.5 mmol) of 5, 12.5 mL of
hydrazine monohydrate, and 240 mL of EtOH was stirred at room
temperature overnight. The reaction mixture was filtered, and the
residue was washed with 20 mL of EtOH. The combined filtrates were
concentrated to minimum volume by rotary evaporation, made basic
(pH 10) with aq NaOH, then extracted with DCM (3 × 50 mL). The
combined extracts were dried, filtered, and concentrated by rotary
evaporation. The residue was dried in vacuo to give 6.2 g (100%) of 7
as a light-yellow, viscous oil. 1H NMR (400 MHz, CDCl3) δ 7.70 (d, 8
Hz, 2 H, o-Ts), 7.23 (m, 7 H, Bn, m-Ts), 3.47 (s, 2 H, CH2Ph), 2.98
(t, 6 Hz, 2 H, CH2NHTs), 2.67 (t, 6 Hz, 2 H, CH2NH2), 2.43 (m, 4
H, CH2N), 2.42 (s, 3 H, CH3), 1.61 (m, 4 H, CCH2C). 13C NMR
(100 MHz, CDCl3) δ 142.9, 138.8, 137.4, 129.5, 129.0, 128.4, 127.1,
127.0, 52.7, 51.4, 42.8, 40.3, 30.1, 25.6, 21.5. IR (neat, cm−1) 3384 (w),
2942 (w), 2859 (w), 1696 (w), 1631 (w), 1453 (m), 1319 (m), 1150
(s), 1085 (m), 816 (m), 715 (s), 694 (s). MS m/z 376 (MH+). Anal.
Calcd for C20H29N3O2S·1.25H2O: C, 60.35; H, 7.98; N, 10.56. Found:
C, 60.12; H, 7.88; N, 10.60.
N′-(p-(Dimethylamino)benzenesulfonyl)-N″-(p-toluenesulfonyl)-
[N,N-bis(3-amino-propyl)benzylamine] (16). A mixture of 0.28 g
(0.75 mmol) of 7, 0.17 g (0.77 mmol) of p-(dimethylamino)-
benzenesulfonyl chloride,62,63 5 mL of DCM, 5 mL of satd aq Na2CO3,
and 5 mL of satd aq NaCl was stirred vigorously at room temperature
for 24 h then the layers were separated. The aqueous layer was
extracted with DCM (2 × 10 mL). The combined organic solutions
were dried, filtered, and concentrated by rotary evaporation. The
residue was dried in vacuo then stirred with 10 mL of 2 N HCl in
MeOH/H2O at room temperature for 5 h. The solvent was removed
by rotary evaporation and the residue was dried in vacuo, triturated
with ether, and dried in vacuo, yielding 0.43 g (91%) of pure 16·2HCl,
mp 70−85 °C (dec). 1H NMR (500 MHz, CD3OD) δ 7.86 (d, 8 Hz, 2
H, o-ArSO2), 7.71 (d, 8 Hz, 2 H, o-Ts), 7.53 (m, 2 H, o-Bn), 7.48 (m,
3 H, m,p-Bn), 7.37 (m, 4 H, m-ArSO2, m-Ts), 4.32 (s, 2 H, CH2Ph),
3.18 (m, 10 H, (CH3)2N, CH2NBn), 2.90 (m, 4 H, CH2NHSO2), 2.40
(s, 3 H, TsCH3), 1.95 (m, 4 H, CCH2C). 13C NMR (125 MHz,
CD3OD) δ 143.5, 136.9, 130.9, 129.9, 129.5, 129.1, 129.0, 128.8,
126.7, 57.0, 50.0, 39.6, 23.6, 20.0. IR (neat, cm−1) 2955 (w), 2949 (w),
2579 (m), 1708 (m), 1592 (m), 1451 (m), 1335 (s), 1219 (m), 1158
(s), 1109 (m), 1087 (m), 1023 (m), 974 (w), 941 (m), 870 (m), 812
(m), 800 (m), 736 (s), 690 (s), 650 (s), 586 (m). MS m/z 559
(MH+). Anal. Calcd for C28H38N4O4S2·2HCl·H2O: C, 51.76; H, 6.52;
N, 8.62. Found: C, 52.12; H, 6.93; N, 8.97. A mixture of 0.43 g of 16·
2HCl, 20 mL of DCM, 20 mL of 2 N aq NaOH, and 20 mL of satd aq
NaCl was stirred at room temperature for 4 h, then the layers were
separated. The aqueous layer was extracted with DCM (2 × 25 mL).
The combined organic solutions were dried, filtered, and concentrated
by rotary evaporation. The residue was dried in vacuo, giving 0.37 g
Procedure for Synthesis of 11−15, 17−19, 21, and 22.
Benzo[d][1,3]dioxole-5-sulfonyl chloride60 and 3-(dimethylamino)-
benzenesulfonyl chloride61 were prepared according to literature
procedures. Following is a representative example: N′-(p-nitro-
benzenesulfonyl)-N″-(p-toluenesulfonyl)-[N,N-bis(3-aminopropyl)-
benzylamine] (11). A mixture of 0.62 g (1.7 mmol) of 7, 0.37 g (1.7
mmol) of p-nitrobenzenesulfonyl chloride, 11 mL of DCM, 11 mL of
satd aq Na2CO3, and 11 mL of satd aq NaCl was stirred at room
1
(88%) of 16 as a yellowish-brown, viscous residue. H NMR (500
MHz, CDCl3) δ 7.70 (d, 8 Hz, 2 H, o-Ts), 7.65 (d, 8 Hz, 2 H, o-
ArSO2), 7.25 (m, 7 H, m-Ts, Bn), 6.66 (d, 8 Hz, 2 H, m-ArSO2), 3.32
2642
DOI: 10.1021/acs.jmedchem.5b01832
J. Med. Chem. 2016, 59, 2633−2647

Journal of Medicinal Chemistry
Article
(s, 2 H, CH2Ph), 3.03 (s, 6 H, (CH3)2N), 2.90 (m, 4 H, CH2NHSO2),
2.42 (s, 3 H, TsCH3), 2.40 (m, 4 H, CH2NBn), 1.62 (m, 4 H,
CCH2C). 13C NMR (125 MHz, CDCl3) δ 152.7, 143.1, 138.3, 137.0,
129.6, 129.0, 128.9, 128.4, 127.2, 127.0, 125.0, 110.9, 58.7, 52.1, 52.0,
42.3, 42.2, 40.1, 26.04, 26.01, 21.5.
N′-(3-N-Methylpyrrole-1-sulfonyl)-N″-(p-toluenesulfonyl)-[N,N-
bis(3-aminopro-pyl)benzylamine] (20). A mixture of 0.13 g (0.72
mmol) of 3-N-methylpyrrole-1-sulfonyl chloride,64−66 0.30 g (0.80
mmol) of 7, and 5 mL of DCM was stirred vigorously at room
temperature for 24 h. Concentration by rotary evaporation and drying
in vacuo gave 0.41 g (91%) of crude product, which was partially
purified by silica column chromatography and then by chromatotron
(70:30 (v/v) ethyl acetate/hexane), yielding 113 mg (27%) of
product. After many unsuccessful purification attempts, this impure
material was used in the next step.
methylene-5-(p-toluenesulfonyl)-1,5,9-triazacyclo-dodecane (25). A
mixture of 0.11 g (0.21 mmol) of 9, 0.15 g (0.52 mmol) of 2-
methylene-1,3-propanebis(tert-butylcarbonate), 5.4 mg (0.014 mmol)
of 1,4-bis(diphenylphosphinobutane) (dppb), 60 mg (0.007 mmol) of
tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3), 12.5 mg
(0.120 mmol) of Na2CO3, and 11 mL of anhydrous AN was stirred
and heated under reflux for 24 h then allowed to cool to room
temperature. The reaction mixture was concentrated to dryness by
rotary evaporation, and the residue was dissolved in 15 mL of DCM.
The solution was washed with satd aq NaHCO3 (2 × 7 mL) and 7 mL
of satd aq NaCl, dried, filtered, and rotary evaporated to dryness. The
crude product was purified by chromatotron chromatography (25:75
(v/v) ethyl acetate/hexane), giving 62 mg (52%) of pure 25. 1H NMR
(400 MHz, CDCl3/TMS) δ 7.78 (d, 8 Hz, 2 H, o-PhSO2), 7.66 (d, 8
Hz, 2 H, o-Ts), 7.58 (m, 1 H, p-PhSO2), 7.52 (m, 2 H, m-PhSO2), 7.31
(d, 8 Hz, 2 H, m-Ts), 7.23 (m, 3 H, m,p-Bn), 7.14 (m, 2 H, o-Bn), 5.21
(s, 2 H, CCH2), 3.89 (s, 2 H, H2/4), 3.83 (s, 2 H, H4/2), 3.39 (s, 2
H, CH2Ph), 3.16 (t, 7 Hz, 2 H, H6/12), 3.11 (t, 7 Hz, 2 H, H12/6),
2.43 (s, 3 H, CH3), 2.37 (m, 4 H, H8,10), 1.65 (m, 4 H, H7,11). 13C
NMR (100 MHz, CDCl3) δ 143.5, 139.2, 138.8, 138.3, 135.4, 132.6,
129.8, 129.2, 128.8, 128.2, 127.3, 127.2, 127.0, 116.2, 59.0, 51.3, 50.7,
49.6, 49.5, 44.2, 43.9, 24.6, 24.4, 21.5. A mixture of 40 mg of 25 and 10
mL of 2 N HCl in MeOH/H2O was stirred for 5 h, concentrated by
rotary evaporation, and dried in vacuo. The residue was triturated with
ether and dried in vacuo, giving 41.4 mg of pure 25·HCl, mp 122 °C
(dec). 1H NMR (400 MHz, CDCl3) δ 12.37 (bs, 1 H, NH+), 7.75 (d,
8 Hz, 4 H, o-PhSO2, o-Ts), 7.60 (m, 5 H, m,p-PhSO2, o-Bn), 7.45 (m,
3 H, m,p-Bn), 7.34 (d, 8 Hz, 2 H, m-Ts), 5.37 (s, 2 H, CCH2), 4.14
(s, 2 H, CH2Ph), 3.73 (m, 4 H, H2,4), 3.39 (m, 2 H, H8/10), 3.17 (m,
6 H, H8/10,6,12), 2.45 (s, 3 H, CH3), 2.29 (m, 2 H, H7/11), 2.00 (m,
2 H, H7/11). 13C NMR (100 MHz, CDCl3) δ 144.4, 141.5, 136.5,
133.38, 133.35, 130.9, 130.1, 130.0, 129.43, 129.40, 128.8, 127.51,
127.5, 119.5, 58.6, 52.9, 52.7, 48.6, 48.5, 46.2, 21.5, 20.3, 20.2. IR
(neat, cm−1) 3059 (w), 2927 (w), 1598 (w), 1473 (m), 1333 (s), 1157
(s), 1088 (m), 950 (w), 915 (m), 733 (s), 690 (s), 674 (m), 653 (m),
580 (s), 545 (m), 534 (m). MS m/z 568 (MH+). Anal. Calcd for
C30H37N3O4S2·HCl·H2O: C, 57.91; H, 6.48; N, 6.75. Found: C, 57.73;
H, 6.41; N, 6.59.
N′-(p-Butoxybenzenesulfonyl)-N″-(p-toluenesulfonyl)-[N,N-bis(3-
aminopropyl)cy-clohexylmethylamine] (23). Prepared from 823 and
p-butoxybenzenesulfonyl chloride by the method described for 11.
1
Free base: H NMR (500 MHz, CDCl3) δ 7.73 (m, 4 H, o-Ts, o-
ArSO2), 7.29 (d, 8 Hz, 2 H, m-Ts), 6.94 (d, 8 Hz, 2 H, m-ArSO2), 4.01
(t, 6 Hz, 2 H, OCH2), 2.96 (m, 4 H, CH2NHSO2), 2.42 (s, 3 H,
TsCH3), 2.34 (t, 6 Hz, 4 H, CH2NCH2Cy), 2.04 (d, 7 Hz, 2 H,
CH2Cy), 1.79 (quint, 8 Hz, 2 H, CH2Et), 1.63 (m, 10 H, NCCH2CN,
Cy), 1.49 (quint, 7 Hz, 2 H, CH2Me), 1.34 (m, 1 H, CH), 1.15 (m, 2
H, Cy), 0.98 (t, 8 Hz, 3 H, CH2CH3), 0.78 (m, 2 H, Cy). 13C NMR
(125 MHz, CDCl3) δ 162.3, 143.1, 137.0, 131.2, 129.6, 129.1, 127.1,
114.6, 68.1, 62.3, 62.0, 53.0, 42.5, 35.6, 31.9, 31.0, 26.7, 26.6, 26.1,
1
26.0, 25.9, 21.5, 19.1, 13.8. 23·HCl, mp 64−73 °C (dec): H NMR
(500 MHz, CD3OD/TMS) δ 7.78 (d, 8 Hz, 2 H, o-Ts), 7.74 (d, 8 Hz,
2 H, o-ArSO2), 7.40 (d, 8 Hz, 2 H, m-Ts), 7.07 (d, 8 Hz, 2 H, m-
ArSO2), 4.05 (m, 2 H, OCH2), 3.31 (m, 2 H, CH2NCH2Cy), 3.22 (m,
2 H, CH2NCH2Cy), 2.94 (m, 2 H, CH2Cy), 2.44 (m, 4 H,
CH2NHSO2), 2.42 (s, 3 H, TsCH3), 1.90 (quint, 8 Hz, 2 H, CH2Et),
1.79 (m, 10 H, NCCH2CN, Cy), 1.70 (quint, 7 Hz, 2 H, CH2Me),
1.51 (m, 1 H, CH), 1.38 (m, 2 H, Cy), 1.23 (t, 8 Hz, 3 H, CH2CH3),
1.00 (m, 2 H, Cy). 13C NMR (125 MHz, CD3OD) δ 162.7, 143.6,
136.9, 131.1, 129.5, 128.8, 126.7, 114.5, 67.9, 59.6, 51.13, 51.07, 39.6,
33.2, 30.9, 30.3, 25.49, 25.45, 25.0, 23.43, 23.38, 20.1, 18.8, 12.7. IR
(neat, cm−1) 3285 (w), 3074 (w), 2930 (m), 2851 (m), 2594 (w),
1741 (w), 1598 (s), 1500 (m), 1320 (s), 1307 (s), 1256 (s), 1149 (s),
1100 (s), 968 (m), 827 (s), 705 (s), 660 (s). MS m/z 594 (MH+).
Anal. Calcd for C30H47N3O5S2·HCl: C, 57.17; H, 7.68; N, 6.67.
Found: C, 57.19; H, 8.00; N, 6.43.
1-(p-Butoxybenzenesulfonyl)-9-cyclohexylmethyl-3-methylene-5-
(p-toluenesulfonyl)-1,5,9-triazacyclododecane (39). Prepared by the
1
method described for 25. Free base: H NMR (400 MHz, CDCl3) δ
7.70 (d, 8 Hz, 2 H, o-Ts), 7.67 (d, 8 Hz, 2 H, o-ArSO2), 7.33 (d, 8 Hz,
2 H, m-Ts), 6.98 (d, 8 Hz, m-ArSO2), 5.18 (s, 2 H, CCH2), 4.03 (t,
6 Hz, 2 H, OCH2), 3.81 (s, 2 H, H2/4), 3.77 (s, 2 H, H4/2), 3.15 (m,
4 H, H6,12), 2.44 (s, 3 H, TsCH3), 2.26 (m, 4 H, H8,10), 1.96 (d, 7
Hz, 2 H, CH2Cy), 1.80 (m, 2 H, CH2Et), 1.62 (m, 10 H, H7,11, Cy),
1.52 (m, 2 H, CH2Me), 1.25 (m, 1 H, CH), 1.15 (m, 2 H, Cy), 0.98 (t,
8 Hz, 3 H, CH2CH3), 0.70 (m, 2 H, Cy). 13C NMR (100 MHz,
CDCl3) δ 162.5, 143.4, 140.0, 135.7, 129.8, 129.7, 129.3, 127.2, 116.5,
114.7, 68.1, 62.1, 51.1, 50.6, 50.4, 50.3, 44.2, 44.0, 35.9, 31.9, 31.0,
26.8, 26.0, 24.5, 24.3, 21.5, 19.1, 13.8. 39·HCl, mp 92−97 °C (dec):
1NMR (400 MHz, CD3OD) δ 7.64 (d, 8 Hz, 2 H, o-ArSO2), 7.60 (d, 8
Hz, 2 H, o-Ts), 7.34 (d, 8 Hz, 2 H, m-Ts), 7.02 (d, 8 Hz, m-ArSO2),
5.30 (s, 1 H, CCH2), 5.29 (s, 1 H, CCH2), 3.99 (t, 6 Hz, 2 H,
OCH2), 3.64 (s, 4 H, H2,4), 3.35 (m, 2 H, H8/10), 3.23 (m, 2 H, H8/
10), 3.11 (m, 4 H, H6,12), 2.99 (d, 7 Hz, 2 H, CH2Cy), 2.36 (s, 3 H,
TsCH3), 1.92 (m, 2 H, CH2Et), 1.70 (m, 10 H, H7, 11, Cy), 1.42 (m,
2 H, CH2Me), 1.27 (m, 1 H, CH), 1.15 (m, 2 H, Cy), 0.97 (m, 2 H,
Cy), 0.90 (t, 8 Hz, 3 H, CH2CH3). 13C NMR (100 MHz, CD3OD) δ
163.2, 144.4, 142.7, 133.4, 129.7, 129.6, 127.5, 127.4, 118.4, 114.7,
70.0, 60.5, 52.6, 48.5, 33.0, 30.8, 30.2, 25.5, 25.1, 25.0, 20.1, 20.0, 19.4,
18.8, 12.7. IR (neat, cm−1) 3089 (w), 3086 (w), 2921 (m), 2851 (m),
1732 (m), 1595 (m), 1494 (m), 1338 (s), 1252 (s), 1188 (m), 1142
(m), 1109 (m), 1026 (m), 999 (m), 950 (m), 900 (m), 860 (m), 827
(m), 705 (m), 550 (m). MS m/z 646 (MH+). Anal. Calcd for
C34H51N3O5S2·HCl·1/3CH2Cl2: C, 58.03; H, 7.47; N, 5.91. Found: C,
57.85; H, 7.35; N, 5.67.
N′-(p-(Dimethylamino)benzenesulfonyl)-N″-(p-toluenesulfonyl)-
[N,N-bis(3-amino-propyl)cyclohexylmethylamine] (24). Prepared
from 823 and p-(dimethylamino)benzenesulfonyl chloride62,63 by the
1
method described for 11. Free base: H NMR (500 MHz, CDCl3) δ
7.74 (d, 8 Hz, 2 H, o-Ts), 7.68 (d, 8 Hz, 2 H, o-ArSO2), 7.31 (d, 8 Hz,
2 H, m-Ts), 6.68 (d, 8 Hz, 2 H, m-ArSO2), 3.05 (s, 6 H, (CH3)2N),
2.99 (t, 7 Hz, 2 H, CH2NHSO2), 2.95 (t, 7 Hz, 2 H, CH2NHSO2),
2.43 (s, 3 H, CH3), 2.37 (t, 6 Hz, 4 H, CH2NCH2Cy), 2.05 (d, 7 Hz, 2
H, CH2Cy), 1.63 (m, 10 H, CCH2C, Cy), 1.34 (m, 1 H, CH), 1.15
(m, 2 H, Cy), 0.81 (m, 2 H, Cy). 13C NMR (125 MHz, CDCl3) δ
152.8, 143.1, 137.1, 129.6, 128.9, 127.1, 110.9, 53.2, 52.9, 40.1, 31.9,
1
26.6, 26.0, 25.8, 21.5. 24·2HCl, mp 73−93 °C (dec): H NMR (500
MHz, CDCl3) δ 8.08 (d, 8 Hz, 2 H, o-Ts), 7.95 (d, 8 Hz, 2 H, o-
ArSO2), 7.77 (d, 8 Hz, 2 H, m-Ts), 7.29 (d, 8 Hz, 2 H, m-ArSO2), 3.28
(s, 3 H, (CH3)2N), 3.24 (s, 3 H, (CH3)2N), 3.18 (d, 7 Hz, 2 H,
CH2Cy), 2.99 (m, 4 H, CH2NHCH2Cy), 2.41 (m, 4 H, CH2NHSO2),
2.40 (s, 3 H, TsCH3), 2.13 (m, 10 H, CCH2C, Cy), 1.89 (m, 1 H,
CH), 1.69 (m, 2 H, Cy), 1.13 (m, 2 H, Cy). 13C NMR (125 MHz,
CDCl3) δ 143.6, 137.0, 129.5, 128.5, 126.7, 112.3, 105.0, 59.7, 51.2,
51.0, 39.6, 33.2, 30.3, 25.5, 25.0, 23.4, 23.3, 20.1. IR (neat, cm−1) 3270
(w), 3062 (w), 2924 (m), 2851 (m), 2576 (w), 1735 (m), 1595 (s),
1512 (m), 1436 (m), 1369 (s), 1304 (s), 1225 (m), 1145 (s), 1081
(s), 940 (m), 815 (s), 773 (m), 708 (m), 644 (s). MS m/z 565
(MH+). Anal. Calcd for C28H44N4O4S2·2HCl: C, 52.74; H, 7.27; N,
8.79. Found: C, 52.36; H, 6.89; N, 9.00.
9-Cyclohexylmethyl-1-(p-(dimethylamino)benzenesulfonyl)-3-
Procedure for Synthesis of 25−38 by Macrocyclization.
Following is a representative example: 1-(benzenesulfonyl)-9-benzyl-3-
methylene-5-(p-tolu-enesulfonyl)-1,5,9-triazacyclododecane (40).
1
Prepared by the method described for 25. Free base: H NMR (400
2643
DOI: 10.1021/acs.jmedchem.5b01832
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Journal of Medicinal Chemistry
Article
MHz, CDCl3) δ 7.68 (d, 8 Hz, 2 H, o-Ts), 7.60 (d, 8 Hz, 2 H, o-
ArSO2), 7.31 (d, 8 Hz, 2 H, m-Ts), 6.68 (d, 8 Hz, 2 H, m-ArSO2), 5.18
(m, 2 H, CCH2), 3.86 (s, 2 H, H4), 3.70 (s, 4 H, H2), 3.24 (t, 7 Hz,
2 H, H6), 3.06 (s, 6 H, (CH3)2N), 3.04 (m, 2 H, H12), 2.44 (s, 3 H,
TsCH3), 2.28 (t, 5 Hz, 2 H, H8), 2.22 (t, 5 Hz, 2 H, H10), 1.96 (d, 7
Hz, 2 H, CH2Cy), 1.64 (m, 10 H, H7,11, Cy), 1.54 (m, 2 H, Cy), 1.15
(m, 1 H, CH), 0.68 (m, 2 H, Cy). 13C NMR (100 MHz, CDCl3) δ
152.7, 143.2, 138.2, 136.1, 129.7, 129.1, 127.2, 123.2, 116.3, 110.9,
62.2, 52.0, 50.6, 50.1, 49.7, 44.5, 43.6, 40.0, 35.9, 31.8, 26.8, 26.0, 24.9,
23.9, 21.5. 40·HCl, mp 111−113 °C (dec): 1NMR (400 MHz,
CD3OD) δ 7.69 (d, 8 Hz, 2 H, o-Ts), 7.64 (d, 8 Hz, 2 H, o-ArSO2),
7.44 (d, 8 Hz, 2 H, m-Ts), 6.96 (d, 8 Hz, m-ArSO2), 5.37 (m, 2 H,
CCH2), 3.71 (m, 4 H, H2,4), 3.43 (m, 2 H, H8/10), 3.33 (m, 2 H,
H8/10), 3.29 (s, 6 H, (CH3)2N), 3.17 (m, 4 H, H6,12), 2.97 (d, 7 Hz,
2 H, CH2Cy), 2.44 (s, 3 H, TsCH3), 2.00 (m, 4 H, H7,11), 1.79 (m, 8
H, Cy), 1.15 (m, 1 H, CH), 0.68 (m, 2 H, Cy). 13C NMR (100 MHz,
CDCl3) δ 144.4, 142.9, 133.4, 129.7, 129.2, 127.4, 118.2, 112.1, 70.0,
60.5, 52.8, 52.7, 48.6, 39.6, 33.0, 30.6, 30.2, 25.5, 25.1, 25.0, 20.1, 20.0,
19.9. IR (neat, cm-1) 2927 (w), 2845 (w), 1735 (w), 1595 (s), 1515
(w), 1350 (s), 1142 (s), 1148 (m), 944 (w), 913 (w), 870 (w), 782
(s), 711 (w), 647 (s). MS m/z 617 (MH+). Anal. Calcd for
C32H48N4O4S2·2HCl·2H2O: C, 52.95; H, 7.50; N, 7.72. Found: C,
53.30; H, 7.33; N, 7.93.
9-Benzyl-3-methylene-1-(p-thiomethoxybenzenesulfonyl)-5-(p-
toluenesulfonyl)-1,5,9-triazacyclododecane (41). A solution of 0.50
g (0.85 mmol) of 37 and 0.30 g (1.1 mmol) of tetrabutylammonium
chloride in 5 mL of anhydrous THF was added under nitrogen by
cannula to a stirred suspension of 75 mg (1.1 mmol) of sodium
thiomethoxide in 5 mL of anhydrous THF. The mixture was stirred
overnight then 5 mL of MeOH was added. The solvents were removed
by rotary evaporation. A solution of the residue in 50 mL of ethyl
acetate was washed with 5% aq NaOH, dried, and rotary evaporated to
afford 0.6 g of a dark-brown oil. Alumina column chromatography
(25:75 (v/v) ethyl acetate/hexane) gave the desired product, which
was stirred with 50 mL of a solution of 2 N HCl in MeOH/H2O for 4
h. Rotary evaporation gave a residue that was triturated with ether and
dried in vacuo, yielding 0.31 g (59%) of 41·HCl. 1H NMR (500 MHz,
CD3OD) δ 7.77 (d, 8 Hz, 2 H, o-Ts), 7.67 (d, 8 Hz, 2 H, o-ArSO2),
7.56 (m, 5 H, Bn), 7.44 (d, 8 Hz, m-Ts), 7.13 (d, 8 Hz, 2 H, m-
ArSO2), 5.38 (s, 2 H, CCH2), 4.35 (s, 2 H, CH2Ph), 3.88 (s, 3 H,
SCH3), 3.74 (s, 4 H, H2,4), 3.42 (m, 2 H, H8/10), 3.32 (m, 2 H, H8/
10), 3.16 (m, 4 H, H6/12), 2.43 (s, 3 H, TsCH3), 2.02 (m, 4 H,
H7,11). 13C NMR (125 MHz, CD3OD) δ 163.7, 142.7, 133.4, 130.4,
129.9, 129.7, 129.6, 129.5, 129.2, 127.7, 127.4, 125.2, 114.3, 65.4, 58.1,
54.9, 52.3, 23.8, 20.14, 20.06, 14.0, 13.2, 7.8. IR (neat, cm−1) 2924 (s),
2851 (m), 1735 (s), 1598 (s), 1670 (m), 1650 (m), 1347 (s), 1259
(s), 1220 (m), 1158 (s), 1103 (m), 1026 (m), 1002 (w), 980 (w), 941
(m), 901 (m), 837 (m), 770 (s), 702 (m), 647 (m). MS m/z 599
(100%, MH+ − CH3), 614 (30%, MH+) Anal. Calcd for
C31H39N3O4S3·HCl: C, 57.26; H, 6.20; N, 6.46. Found: C, 57.08; H,
6.44; N, 6.84. Free base 41: 1H NMR (500 MHz, CDCl3) δ 7.77 (d, 8
Hz, 2 H, o-Ts), 7.67 (d, 8 Hz, 2 H, o-ArSO2), 7.32 (d, 8 Hz, m-Ts),
7.24 (m, 5 H, Bn), 7.05 (d, 8 Hz, 2 H, m-ArSO2), 5.24 (s, 2 H, C
CH2), 3.88 (s, 3 H, SCH3), 3.82 (s, 4 H, H2,4), 3.45 (s, 2 H, CH2Ph),
3.13 (m, 4 H, H6,12), 2.43 (s, 3 H, TsCH3), 2.10 (m, 4 H, H8,10),
1.67 (m, 4 H, H7, 11). 13C NMR (125 MHz, CDCl3) δ 143.1, 137.0,
131.6, 129.6, 129.4, 129.2, 128.6, 128.2, 127.6, 127.4, 127.1, 125.4,
114.2, 58.5, 55.6, 51.5, 45.7, 41.8, 25.7, 21.5, 14.8, 8.6.
The residue was stirred with 2 N HCl in MeOH/H2O for 30 min, and
then the mixture was concentrated by rotary evaporation and the
residue was dried in vacuo then triturated with ether. A solution of the
residue in 50 mL of DCM was washed with 10% aq Na2CO3 (3 × 20
mL) and then with 20 mL of satd aq NaCl and then dried and
concentrated by rotary evaporation. Silica column chromatography
1
(1:1 (v/v) ethyl acetate/hexane) gave 0.37 g (73%) of pure 49. H
NMR (500 MHz, CDCl3) δ 7.64 (d, 8 Hz, 2 H, o-Ts), 7.27−7.16 (m,
7 H, m-Ts, Bn), 5.10 (s, 1 H, CCH2), 5.08 (s, 1 H, CCH2), 3.95
(s, 2 H, H2), 3.36 (s, 2 H, H4), 3.20 (t, 6 Hz, 2 H, H12), 3.18 (s, 2 H,
CH2Ph), 2.68 (t, 6 Hz, 2 H, H10), 2.47 (t, 6 Hz, 2 H, H8), 2.39 (s, 3
H, CH3), 2.14 (t, 6 Hz, 2 H, H6), 1.55 (m, 2 H, H11), 1.44 (quint, 7
Hz, 2 H, H7). 13C NMR (125 MHz, CDCl3) δ 129.9, 129.8, 129.5,
128.8, 128.7, 128.2, 127.7, 127.2, 126.8, 59.0, 58.6, 58.4, 53.4, 49.2,
1
21.5. 49·2HCl, mp 190 °C (dec): H NMR (500 MHz, CDCl3) δ
+
11.31 (bs, 1 H, NH+), 9.89 (bs, 2 H, NH2 ), 7.71 (m, 2 H, o-Ts), 7.56
(m, 2 H, o-Bn), 7.38 (m, 3 H, m,p-Bn), 7.32 (d, 8 Hz, 2 H, m-Ts), 5.76
(s, 1 H, CCH2), 5.46 (s, 1 H, CCH2), 4.46 (s, 2 H, H4), 4.15−
3.31 (m, 10 H, H2,8,10,12,CH2Ph), 2.41 (s, 3 H, CH3), 2.30 (m, 2 H,
H7), 2.03 (m, 2 H, H11). 13C NMR (125 MHz, CDCl3) δ 144.6,
135.4, 132.6, 131.3, 130.1, 130.0, 129.3, 128.9, 127.6, 123.0, 58.9, 54.8,
48.1, 47.7, 45.6, 44.2, 42.6, 29.7, 21.7, 17.1. IR (neat, cm−1) 3389 (m),
2955 (m), 2585 (m), 1705 (w), 1595 (m), 1454 (s), 1338 (s), 1158
(s), 1106 (m), 1087 (m), 1026 (w), 997 (m), 941 (m), 916 (m), 812
(m), 800 (m), 736 (m), 693 (s), 648 (m). MS m/z 428 (MH+). Anal.
Calcd For C24H33N3O2S·2HCl·CH2Cl2·H2O: C, 49.76; H, 6.51; N,
6.96. Found: C, 49.68; H, 6.95; N, 7.26.
9-Cyclohexylmethyl-1-(p-hydroxybenzenesulfonyl)-3-methylene-
5-(p-toluenesulfon-yl)-1,5,9-triazacyclododecane (51). A solution of
1.94 g (3.2 mmol) of 1 in 150 mL of anhydrous DCM was stirred at
room temperature under nitrogen as 45 mL of 1.0 M BBr3 in DCM
was added dropwise. The mixture was stirred at room temperature for
21 h then cooled to 0 °C, and 150 mL of NH4OH (30 wt % in H2O)
was added slowly. The resulting mixture was stirred vigorously at room
temperature for 48 h and then the layers were separated. The organic
layer was washed with H2O (2 × 120 mL), dried, filtered, concentrated
by rotary evaporation, and dried in vacuo. Column chromatography on
alumina (9:1 (v/v) EtOAc/MeOH) gave 1.72 g of partially protonated
51. A solution of this crude product in 15 mL of DCM was stirred with
30 mL of satd aq NH4OH for 0.5 h, and then the layers were
separated. The organic layer was washed with water (2 × 20 mL),
dried, filtered, concentrated by rotary evaporation, and dried in vacuo
1
to give 1.4 g (74%) of 51 as a yellow solid. H NMR (500 MHz,
CDCl3) δ 7.67 (d, 8 Hz, 2 H, o-Ts), 7.65 (d, 9 Hz, 2 H, o-ArSO2), 7.33
(d, 8 Hz, 2 H, m-Ts), 6.93 (d, 9 Hz, 2 H, m-ArSO2), 5.18 (s, 2 H, C
CH2), 3.80 (s, 2 H, H2/4), 3.78 (s, 2 H, H4/H2), 3.15 (m, 4 H,
H6,12), 2.44 (s, 3 H, CH3), 2.29 (m, 4 H, H8,10), 1.98 (d, 7 Hz, 2 H,
CH2Cy), 1.63 (m, 8 H, H7,11,Cy), 1.26 (s, 1 H, Cy), 1.12 (m, 4 H,
Cy), 0.70 (m, 2 H, Cy). 13C NMR (125 MHz, CDCl3) δ 160.4, 143.6,
138.3, 135.4, 129.8, 129.6, 127.3, 116.7, 116.2, 62.1, 51.4, 50.7, 50.2,
50.0, 44.7, 44.5, 35.7, 31.9, 26.7, 26.0, 24.2, 24.0, 21.5. IR (neat, cm−1)
3383 (w), 2927 (w), 2857 (w), 2802 (w), 1580 (w), 1453 (w), 1331
(w), 1155 (m), 1090 (w), 908 (s), 729 (s). MS m/z 590 (MH+). Anal.
Calcd for C30H43N3O5S2: C, 61.09; H, 7.35; N, 7.12. Found: C, 59.62;
H, 7.50; N, 7.14.
Synthesis of Compounds 58−61. Following is a representative
example: 9-benzyl-3-methylene-5-(p-methoxybenzoyl)-1-(p-toluene-
sulfonyl)-1,5,9-triazacyclododecane (58). To a cold (0 °C) solution
of 0.12 g (0.29 mmol) of 49 in 5 mL of DCM were added 54 mg (0.68
mmol) of pyridine and 0.14 g (0.82 mmol) of 4-methoxybenzoyl
chloride. The mixture was stirred at room temperature for 48 h and
then concentrated by rotary evaporation. The resulting viscous yellow
oil was shaken with 10 mL of water and 10 mL of CHCl3, and then the
layers were separated. The aqueous layer was extracted with CHCl3 (2
× 10 mL). The combined organic solutions were washed with 10 mL
of 15% aq NaOH, dried, filtered, and concentrated by rotary
evaporation. The residue was dried in vacuo then chromatographed
on silica gel (20:80 (v/v) ethyl acetate/hexane), giving 118 mg (73%)
of 58 as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.61 (d, 8 Hz, 2
H, o-Ts), 7.36 (d, 8 Hz, 2 H, o-ArCO), 7.31 (d, 8 Hz, 2 H, m-Ts), 7.20
9-Benzyl-3-methylene-1-(p-toluenesulfonyl)-1,5,9-triazacyclodo-
decane (49). A mixture of 0.73 g (1.2 mmol) of 27 and 0.55 mL (0.56
g, 3.7 mmol) of DBU in 25 mL of AN was stirred at room temperature
for 15 min, then 0.10 mL (0.11 g, 1.4 mmol) of 2-mercaptoethanol
was added. The mixture was stirred at room temperature for 24 h and
passed through a short silica gel column, eluting with AN, then the
eluent was concentrated by rotary evaporation. A mixture of the
residue, 40 mL of 2-propanol, 40 mL of H2O, and 1.5 g (18 mmol) of
NaHCO3 was stirred for 24 h, diluted with 50 mL of satd aq NaCl and
2 N aq NaOH was added dropwise until the pH reached 14. The
mixture was extracted with DCM (3 × 30 mL), and the combined
extracts were dried (Na2SO4) then concentrated by rotary evaporation.
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Article
(m, 5 H, Bn), 6.85 (d, 8 Hz, 2 H, m-ArCO), 5.22 (s, 2 H, CCH2),
4.24 (s, 2 H, H2/4), 3.81 (s, 3 H, OCH3), 3.58 (s, 2 H, H4/2), 3.45 (s,
2 H, CH2Ph), 2.91 (m, 4 H, H6,12), 2.61 (t, 6 Hz, 2 H, H8/10), 2.43
(s, 3 H, TsCH3), 2.36 (t, 6 Hz, 2 H, H10/8), 1.99 (quint, 2 H, H7/
11), 1.78 (quint, 7 Hz, 2 H, H11/7). 13C NMR (125 MHz, CDCl3) δ
172.3, 160.7, 143.8, 139.3, 129.8, 128.8, 128.4, 128.1, 127.6, 126.9,
113.7, 58.7, 56.1, 55.3, 51.1, 48.6, 46.3, 27.0, 21.5. The free base was
stirred with 30 mL of 2 N HCl in MeOH/H2O, and then solvent was
removed by rotary evaporation. The residue was dried in vacuo,
triturated with ether, and then dried in vacuo to give 126 mg (73%
overall) of pure 58·HCl, mp 149−151 °C (dec). 1H NMR (500 MHz,
CD3OD) δ 7.69 (d, 8 Hz, 2 H, o-Ts), 7.56 (m, 2 H, o-Bn), 7.48 (m, 3
H, m,p-Bn), 7.41 (m, 4 H, m-Ts, o-ArCO), 6.98 (d, 9 Hz, 2 H, m-
ArCO), 5.42 (s, 1 H, CCH2), 5.18 (s, 1 H, CCH2), 4.36 (s, 2 H,
CH2Ph), 3.82 (s, 4 H, H2/4), 3.42 (m, 4 H, H8,10), 3.28 (s, 3 H,
OCH3), 3.22 (m, 4 H, H6,12), 2.42 (s, 3 H, TsCH3), 2.10 (m, 2 H,
H7/11), 1.96 (m, 2 H, H11/7). 13C NMR (125 MHz, CD3OD) δ
173.7, 161.4, 144.3, 134.6, 130.9, 129.9, 129.8, 129.2, 129.1, 128.2,
127.4, 127.1, 116.1, 113.7, 58.9, 55.9, 54.5, 20.2. IR (neat, cm−1) 3343
(w), 3071 (w), 2964 (w), 2918 (w), 1836 (w), 1635 (s), 1610 (m),
1509 (w), 1448 (m), 1408 (m), 1375 (m), 1329 (m), 1292 (m), 1237
(s), 1182 (w), 1152 (m), 1026 (m), 996 (m), 974 (m), 886 (m), 835
(s), 806 (m), 778 (m), 739 (s), 696 (s), 675 (s), 586 (m), 571 (m).
MS m/z 562 (MH+). Anal. Calcd for C32H39N3O4S·HCl·3H2O: C,
58.93; H, 7.11; N, 6.44. Found: C, 58.93; H, 6.71; N, 6.50.
ABBREVIATIONS USED
■
AIDS, acquired immunodeficiency syndrome; AN, acetonitrile;
b, broad; CADA, cyclotriazadisulfonamide; CC50, 50%
cytotoxic concentration; CD4, complex of differentiation 4;
CHO, Chinese hamster ovary; d, doublet; DABCO, 1,4-
diazabicyclo[2.2.2]octane; dba, dibenzylideneacetone; DBU, 8-
diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane;
DEAE-dextran, diethylaminoethyldextran; DMF, N,N′-dime-
thylformamide; dppb, 1,4-bis(diphenylphosphino)butane;
EDG, electron donating group; ER, endoplasmic reticulum;
ESI, electrospray ionization; EWG, electron withdrawing
group; FBS, fetal bovine serum; GUI, graphical user interface;
h, hour(s); HBA, hydrogen bond acceptor; HBD, hydrogen
bond donor; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid; HPLC, high pressure liquid chromatography;
HIV, human immunodeficiency virus; IC50, 50% inhibitory
concentration; IR, infrared spectroscopy; m, medium or
multiplet; MFI, mean fluorescence intensity; min, minute(s);
Morph, morpholine; MS, mass spectrometry; MTS, 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium, inner salt; NMR, nuclear magnetic
resonance; PES, phenazine ethosulfate; PBMC, peripheral
blood mononuclear cell; PBS, phosphate buffered saline;
Phth, phthalimide; Pyrr, pyrrole; q, quartet; QSAR, quantitative
structure−activity relationship; quint, quintet; RLU, relative
luminescence unit; s, singlet or strong; SAR, structure−activity
relationship; SIV, simian immunodeficiency virus; SOD,
superoxide dismutase; SP, signal peptide; t, triplet; TCID50,
50% tissue culture infective dose; THF, tetrahydrofuran; tlc,
thin-layer chromatography; TOF, time-of-flight; w, weak; YFP,
yellow fluorescent protein
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01832.
Detailed synthetic procedures and characterization data
(1H and 13C NMR, IR, MS, and combustion micro-
analysis) for compounds 9, 10, 43−48, 52−57, 62, and
63, characterization data only for compounds 12−15,
17−19, 21, 22, 26−38, and 59−61, and table listing
antiviral activities of CADA compounds on X4-tropic
and R5-tropic HIV-1 strains (PDF)
REFERENCES
■
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AUTHOR INFORMATION
■
Corresponding Authors
*For T.W.B. (Chemistry): phone, 775-784-1842; fax, 775-750-
3391; E-mail, twb@unr.edu.
*For K.V. (Biology): phone, (+32) 16 33 73 71; E-mail, kurt.
vermeire@rega.kuleuven.be.
Author Contributions
§T.W.B. and K.V. contributed equally.
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ACKNOWLEDGMENTS
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This work was supported by the KU Leuven (GOA 10/014 and
PF/10/018), the Foundation of Scientific Research (FWO no.
G-0485-08 and G-0528-12), The Foundation Dormeur, Vaduz
and the CHAARM project (no. 242135) of the European
Commission. We are grateful to S. Claes, R. Provinciael, E. Van
Kerckhove, and E. Fonteyn for excellent technical assistance.
Funding from the National Science Foundation (grant CHE-
0521191) in support of NMR spectrometers in the Department
of Chemistry, University of Nevada, Reno, is also gratefully
acknowledged.
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