Synthesis, biological evaluation, and structure-activity relationships of novel substituted N-phenyl ureidobenzenesulfonate derivatives blocking cell cycle progression in s-phase and inducing DNA double-strand breaks

Article abstract of DOI:10.1021/jm3006492

Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-

Full text of DOI:10.1021/jm3006492

Article
pubs.acs.org/jmc
Synthesis, Biological Evaluation, and Structure−Activity
Relationships of Novel Substituted N-Phenyl
Ureidobenzenesulfonate Derivatives Blocking Cell Cycle Progression
in S-Phase and Inducing DNA Double-Strand Breaks
Vanessa Turcotte,†,‡ Sebastien Fortin,*,†,‡ Florence Vevey,† Yan Coulombe,§ Jacques Lacroix,†
́
Marie-France Cote,† Jean-Yves Masson,§ and Rene C.-Gaudreault*,†,∥
̂
́
́
†Unite
3L5, Canada
‡Faculte
de Pharmacie, Universite
§Laboratoire de la Stabilite
du Gen
∥Faculte
de Medecine, Universite Laval, Pavillon Vandry, Queb
́ ́ ̂ ́
des Biotechnologies et de Bioingenierie, Centre de Recherche, C.H.U.Q., Hopital Saint-Franco̧ is d’Assise, Quebec, QC, G1L
́
́
Laval, Pavillon Vandry, Queb
ome, Centre de Recherche en Cancer
ec, QC, G1V 0A6, Canada
́
ec, QC, G1V 0A6, Canada
́
́
́
ologie de l'Universite Laval, Quebec, QC, G1R 2J6, Canada
́
́
́
́
́
́
ABSTRACT: Twenty-eight new substituted N-phenyl ureido-
benzenesulfonate (PUB-SO) and 18 N-phenylureidobenzene-
sulfonamide (PUB-SA) derivatives were prepared. Several PUB-
SOs exhibited antiproliferative activity at the micromolar level
against the HT-29, M21, and MCF-7 cell lines and blocked cell
cycle progression in S-phase similarly to cisplatin. In addition,
PUB-SOs induced histone H2AX (γH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks.
In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and
46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was
similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are
members of a promising new class of anticancer agents.
either G2/M or G0/G1 phase. By use of matrix-assisted laser
desorption ionization and electrospray mass spectrometry, N-
phenyl-N′-(2-chloroethyl)ureas exhibiting antimicrotubule ac-
tivity were shown to bind covalently to microtubules via a
unique mechanism of nucleophilic addition involving the
esterification of a Glu residue at position 198 of human β-
tubulin (Gluβ198).13 Of interest, Gluβ198, which is located in a
small pocket adjacent to the colchicine-binding site, is involved
in microtubule stability and dynamics and is also associated
with a mechanism of resistance to Taxotere (docetaxel).14,15
With the objective of developing anticancer agents with
optimal biopharmaceutical properties and lower toxicity, we
recently modified the structure of the N-phenyl-N′-(2-
chloroethyl)urea scaffold by the addition of a benzenesulfonate
group and cyclization of the 2-chloroethylurea (CEU) moiety
into a 1-phenylimidazolidin-2-one heterocycle. The latter
modifications led to a novel class of potent antimicrotubule
agents designated as phenyl 4-(2-oxoimidazolidin-1-yl)-
benzenesulfonates (PIB-SOs, 2).16 PIB-SOs, molecules con-
taining an imidazolidonyl ring, exhibited antiproliferative
activities in the low nanomolar range, blocked cell cycle
progression in G2/M phase, and bound to the colchicine-
binding site, leading to cytoskeleton disruption and apoptosis.
INTRODUCTION
■
N-Phenyl-N′-(2-chloroethyl)ureas (1) are members of a class of
potent antiproliferative agents acting across a large panel of
tumor cell lines and in several animal cancer models (Figure 1).
Several subsets of these monoalkylating agents were shown to
bind covalently to proteins such as βII-tubulin,1−7 thioredoxin-
1,8−10 prohibitin-1,11 and mitochondrial voltage-dependent
anion channel,12 leading to arrest of cell cycle progression in
Figure 1. Structure of N-phenyl-N′-(2-chloroethyl)ureas (1), PIB-SOs
(2), CA-4 (3), and PUB-SOs (4−10).
Received: May 9, 2012
Published: June 13, 2012
© 2012 American Chemical Society
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Table 1. Antiproliferative Activity (IC50) and Effect of PUB-SOs, PUB-SAs, and cDDP on Cell Cycle Progression
a
b
IC50 is expressed as the concentration of drug inhibiting cell proliferation by 50%. For flow cytometry experiments, Jurkat cells were incubated for
24 h in the presence of the selected PUB-SOs and PUB-SAs at a concentration inducing an optimal arrest of cell cycle progression in S-phase. cDDP
was used as positive control; n.e., not evaluated.
Finally, PIB-SOs inhibit angiogenesis and tumor growth in the
chick chorioallantoic membrane (CAM) assay at levels
comparable to combretastatin A-4 (CA-4, 3) and exhibit low
to very low toxicity toward chick embryos.16
The assessment of the antiproliferative activity and the effect
on cell cycle progression of the subset (compounds 4−10) of
novel substituted N-phenyl-N′-(2-chloroethyl)ureas either
rationally designed as antimicrotubule agents or produced as
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a
Scheme 1
a
Reagents: (i) relevant phenol, TEA/DCM or relevant aniline, DMAP/CH3CN; (ii) SnCl2·2H2O/EtOH or Fe, HCl/EtOH; (iii) relevant isocyanate
and appropriate method; (iv) 1 M TBAF/THF.
intermediates in the synthesis of PIB-SOs revealed an unusual
arrest of cell cycle progression in S-phase (compounds 4−6 and
10, Table 1) instead of the G2/M phase, as observed with their
known antimicrotubule counterparts. That unexpected S phase
arrest induced by this new subset of N-phenyl-N′-(2-
chloroethyl)ureas prompted us to determine their structure−
activity relationships and to investigate their mechanism of
action. o-Tolyl 4-[3-(2-chloroethyl)ureido]benzenesulfonate
(4) and 4-hydroxyphenyl 4-[3-(2-chloroethyl)ureido]-
benzenesulfonate (10) were selected as molecular templates
to initiate the structure−activity relationship study. We first
assessed the role and the substitution pattern of the
electrophilic CEU group on ring A (Figure 1) via its
substitution with a 3-chloropropylurea (CPU) or an ethylurea
(EU) moiety, leading to N-phenyl ureidobenzenesulfonate
derivatives (PUB-SOs), molecules containing alkylurea moi-
eties. We subsequently replaced the sulfonyl group bridging the
phenyl rings A and B of PUB-SOs with a bioisosteric
sulfonamide bridge, thereby leading to N-phenylureidobenze-
nesulfonamides (PUB-SAs). Moreover, we studied the effects
of replacing the methyl substituent with an ethyl or propyl
group at the C2 position of the B-ring. We also studied the
effect of a hydroxyl moiety at the C4 position of the B-ring. The
potential antiproliferative activity and effect on cell cycle
progression of these novel compounds were assessed in M21
human skin melanoma, estrogen-dependent MCF-7 breast
adenocarcinoma, and HT-29 colon adenocarcinoma cell lines.
The most potent inhibitors of S-phase progression among these
derivatives were assessed for their potential induction of H2AX
phosphorylation, which parallels the induction of DNA double-
strand breaks, and for their antitumoral activity in HT-1080
human fibrosarcoma cells grafted onto the CAM assay.
RESULTS AND DISCUSSION
■
Chemistry. Scheme 1 depicts the synthetic pathways used
for the preparation of substituted PUB-SO and PUB-SA
analogues. These compounds were prepared by nucleophilic
addition of the appropriate phenols or anilines to nitrobenzene-
1-sulfonyl chloride. Nitrophenyl sulfonates 56−67 and nitro-
phenylsulfonamides 68−73 were reduced to the corresponding
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Figure 2. Effect of PUB-SOs 4, 16, 17, 29−31, 33, 40, 41, 45−47, 49, and cDDP on the phosphorylation of histone H2AX into γH2AX.
anilines 74−91 using iron powder in the presence of HCl or
SnCl2·2H2O to obtain compound 59. PUB-SO and PUB-SA
derivatives substituted with a CEU (4−23), a CPU (24−39),
or a EU (40−55) moiety were prepared by nucleophilic
addition of 2-chloroethyl isocyanate, 3-chloropropyl isocyanate,
or ethyl isocyanate, respectively, to the corresponding anilines.
The different nucleophilicity or electrophilicity of the anilines
and isocyanates used as starting materials led us to use various
bases (4-dimethylaminopyridine and pyridine), solvents (THF,
acetonitrile, and methylene chloride), and reaction conditions
(temperature, microwave heating, etc.) to optimize reaction
yields. Removal of the tert-butyldimethylsilyl protecting group
on compounds 9, 14, 27, 32, 43, and 48 into their
corresponding phenols was performed in the presence of
tetra-n-butylammonium fluoride (TBAF).
Of note, the addition of the isocyanate to aniline provides
low to moderate yields in the synthesis of PUB-SO and PUB-
SA derivatives. The yields for the nucleophilic addition are even
lower when PUB-SAs are involved or when the phenyl ring B is
substituted at position 4 by a tert-butyldimethylsilyl group.
Antiproliferative Activity. The antiproliferative activity of
PUB-SOs and PUB-SAs was assessed in three human cancer
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cell lines, namely, HT-29 colon carcinoma, M21 skin
melanoma, and MCF-7 breast carcinoma cells. These cell
lines were selected as representatives of tumors originating
from the three germ layers (i.e., endoderm (HT-29), mesoderm
(M21), and ectoderm (MCF-7)). Antiproliferative activity was
evaluated using the sulforhodamine B method according to the
NCI/NIH Developmental Therapeutics Program.17 The results
are summarized in Table 1 and are expressed as the IC50. The
antiproliferative activity of several PUB-SOs (compounds 4−7,
10, 12, 13, 16, 17, 26, 30, 31, 45−47, and 49) was equivalent
to or better than with cisplatin (cis-diamminedichloroplatinum-
(II), cDDP). In contrast, the antiproliferative activity of PUB-
SAs was lower than for PUB-SOs and cDDP. Only two PUB-
SAs (compounds 20 and 36) showed antiproliferative activity
comparable to that observed with cDDP.
Effect on Cell Cycle Progression. Table 1 shows the
percentage of Jurkat cells in G0/G1, S, and G2/M phases,
respectively, after treatment with PUB-SOs, PUB-SAs, and
cDDP for 24 h at optimal concentrations regarding the arrest of
cell cycle progression in S-phase. Cell cycle distribution
observed for control cells treated with 0.5% DMSO was 43%,
40%, and 17% in G0/G1, S, and G2/M phases, respectively.
PUB-SOs 5, 6, 10, 30, 31, 33, 40, 41, 45, and 49 caused an S-
phase arrest, thereby increasing the percentage of S-phase cells
by 12−27%. PUB-SOs 4, 16, 17, 29, 46, and 47 strongly
blocked cell cycle progression and this, to a more efficient
extent than cDDP, as measured by an increase in the S-phase
fraction of 30−41%. A concentration of 19 μM cDDP blocked
69% of the Jurkat cell population in S-phase. In contrast, PUB-
SAs did not induce an S-phase block.
Structure−Activity Relationships. As depicted in Table 1
and as previously mentioned, replacing the sulfonyl group
bridging phenyl rings A and B with a bioisosteric sulfonamide
bridge significantly lowered antiproliferative activity and
reduced the effect on cell cycle progression. Consequently,
the spatial conformations of the two phenyl rings conferred by
the bridge between the two phenyl rings are important for the
activity. Moreover, our structure−activity relationship study
shows that the substitution pattern of the pharmacophoric
moiety on the A-ring is an important factor in the
antiproliferative activity and cell cycle arrest caused by a
given derivative. Transposition of the pharmacophoric CEU,
CPU, and EU moieties from C4 to C3 on the A-ring
significantly decreased antiproliferative activity and abolished
the effect on cell cycle progression. Thus, derivatives whose A-
rings have steric hindrance at C3 position with CEU, CPU, or
EU in general did not entail S-phase arrest.
Structure−activity relationship studies revealed that the
nature of the pharmacophoric substituting group is also
important. Derivatives bearing EU and CEU moieties at C4
position of A-ring exhibited antiproliferative activities in the
same range and were more potent than their counterparts
bearing a CPU moiety. Consequently, steric hindrance at this
specific position does not seem to affect the biological activity.
Interestingly, compounds 40, 41, 45−47, 49, and 53 bearing an
EU moiety were potent antiproliferative agents and arrested cell
cycle progression in S-phase. These compounds lack an
electrophilic chlorine substituent, which is involved in the
mechanism of nucleophilic esterification of acidic peptide
residues such as glutamic and aspartic acids.11,13 Thus, the
presence of a chlorine atom and dipole−dipole interactions are
not prerequisites for the biological activity of this group of
compounds, unlike the G2/M or G0/G1 block that is specifically
observed with the N-phenyl-N′-(2-chloroethyl)urea deriva-
tives.13 This suggests that the mechanism of action of
compounds 40, 41, 45−47, 49, and 53 does not likely proceed
via nucleophilic protein alkylation. Another most interesting
feature of PUB-SOs lies in the fact that the B-ring can
accommodate substitution with either a hydroxyl group at C4
or an alkyl (methyl, ethyl, propyl) group at C2 without
significant alteration of their cytocidal activity, and therefore,
steric hindrances do not affect the C2 position. Thus, we
obtained a new class of antiproliferative agents that block the S-
phase, with several of its members exhibiting IC50 values that
are similar to or better than in the case of cDDP, used here as a
positive control.
Phosphorylation of H2AX. Since the major event
occurring in S-phase is DNA replication, we next assessed
whether DNA double-strand breaks are involved in the
mechanism of action of PUB-SOs. According to current
literature,18−21 phosphorylation of Ser-139 at the C-terminus
of histone H2AX (thus yielding γH2AX) occurs upon induction
of DNA double-strand breaks. To address the mechanism of
action of the novel S-phase inhibitors, we evaluated their ability
to induce γH2AX formation. H2AX phosphorylation induced
by compounds 4, 16, 17, 29−31, 33, 40, 41, 45−47, and 49,
which had displayed the highest antiproliferative activity (IC50
< 55 μM) and the ability to block >60% of the S-phase fraction
was assessed by immunofluorescence.22,23 As depicted in Figure
2, the latter group of compounds, when tested at their
respective IC50 value, induced H2AX phosphorylation in M21
cells. Indeed, γH2AX was detected as nuclear red spots in
nuclei (stained in blue using 4′,6-diamidino-2-phenylindole
(DAPI)) of cells treated with 4, 16, 17, 29−31, 33, 40, 41, 45−
47, as well as with cDDP, but was absent from control cells.
The latter data support the notion that the active PUB-SOs act
via the induction of DNA double-strand breaks, which in turn
may account for the S-phase cell arrest induced by these
compounds. Research is in progress to determine the molecular
mechanism responsible for the induction of DNA double-
strand breaks and γH2AX by this category of derivatives.
Antitumoral Activity As Measured with CAM Assays.
The most potent PUB-SOs in each series of CEU, CPU, and
EU that induce an S-phase block (compounds 4, 10, 16, 17, 30,
45, 46, and 47) were tested in ovo using the CAM assay. HT-
1080 human fibrosarcoma cells were selected because they
produce solid tumors that are sensitive to antiangiogenic and
antitumoral agents.24−29 cDDP and CA-4 were used as positive
controls. A mixture of Cremophor EL, 99% ethanol, and PBS
(1/1/14 v/v) was used as an excipient to inject cDDP, CA-4,
and PUB-SOs. cDDP (10 μg/egg) and CA-4 (1 μg/egg)
respectively inhibited tumor growth by 46% and 49% and
exhibited toxicity in 6% and 21% of the chick embryos. As
shown in Figure 3, compounds 4, 10, 16, 17, 30, and 45−47
administered at 30 μg/egg (except for 4 which was used at 10
μg/egg) significantly inhibited tumor growth. Thus, com-
pounds 10, 30, 45, and 47 respectively inhibited tumor growth
by 69%, 68%, 68%, and 65% and exhibited lethality in 15%, 0%,
9%, and 10% of the chick embryos. Compounds 16 and 17
reduced tumor growth by 49%, i.e., to an extent comparable to
that of cDDP and CA-4, but were rather toxic toward chick
embryos (causing death in 33% and 36% of embryos,
respectively). On the other hand, compounds 4 and 46
inhibited tumor growth by 60% and 45%, respectively, while
showing low toxicity in chick embryos (with a 15% and 9%
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diluted in fresh DMEM, and 75 μL aliquots containing serially diluted
concentrations of the drug were added. Final drug concentrations
ranged from 200 μM to 780 nM. DMSO was maintained at a
concentration of <0.5% (v/v) to avoid any related toxicity. Plates were
incubated for 48 h, after which growth was stopped by the addition of
cold trichloroacetic acid to the wells (10% w/v, final concentration),
followed by a 1 h incubation at 4 °C. Plates were then washed 5 times
with water. An amount of 75 μL of a sulforhodamine B solution (0.1%
w/v) in 1% acetic acid was added to each well, and the plates were
incubated for 15 min at room temperature. After staining, unbound
dye was removed by washing 5 times with 1% acetic acid. Bound dye
was solubilized in 20 mM Tris base, and the absorbance was read using
an optimal bandwidth (530−568 nm) with a μQuant Universal
microplate spectrophotometer (Biotek, Winooski, VT). Readings
obtained from treated cells were compared with measurements from
control cell plates fixed on treatment day, and the percentage of cell
growth inhibition was thus calculated for each drug. The experiments
were performed at least twice in triplicate. The assays were considered
valid when the coefficient of variation for a given set of conditions and
within the same experiment was <10%.
Cell Cycle Analysis. Jurkat E6 human leukemic T-cell
lymphoblasts were purchased from the American Type Culture
Collection. Cells were cultured in RPMI 1640 medium supplemented
with 10% (v/v) fetal bovine serum. Cells were maintained at 37 °C in
a water-saturated 5% CO2 atmosphere. PUB-SOs, PUB-SAs, cDDP,
and DMSO were serially diluted in culture medium in a 12-well plate,
starting at a concentration 50% above their respective IC50 toward
M21 cells. Next, 4.0 × 105 Jurkat cells suspended in culture medium
were added to each well and incubated with the drugs for 24 h. Cells
were then harvested, washed with PBS, and resuspended in 250 μL of
PBS containing 3.0 × 105 chicken red blood cells as an internal
standard. Cells were fixed by the addition of 750 μL of ice-cold EtOH
and stored at −20 °C until analysis. Prior to fluorometry, cells were
washed with PBS and resuspended in 1 mL of PBS containing 1 μg/
mL DAPI. Fixed cell suspensions were incubated on ice for 1 h, and
cell cycle distribution was then analyzed using an LSR II flow
cytometer (BD Biosciences, Franklin Lakes, NJ).
Immunofluorescence. Cover slides (22 mm × 22 mm) sterilized
with 70% (v/v) EtOH were placed in six-well plates. To promote cell
adhesion, cover slides were treated with 1.5 mL of a fibronectin
solution in PBS (10 μg/mL) for 1 h at 37 °C. Slides were then rinsed
twice with PBS. M21 cells (1 × 105) were seeded onto the plates and
incubated for 24 h. Cells were then incubated with the test compound
at its IC50 for 24 h at 37 °C. The control solution consisted of DMSO
dissolved in culture medium (0.5%, v/v). Cells were fixed using 1 mL
of formaldehyde at 3.7% and permeabilized by addition of a saponin
solution (0.1% in PBS) containing 3% (w/v) BSA (saponin−BSA).
Cells were incubated with mouse anti-H2AX pS139 antibody
(Millipore, Billerica, MA). Cover slides were next incubated for 3 h
at room temperature and then washed twice with PBS supplemented
with 0.05% (v/v) Tween 20 (PBS-T). Saponin−BSA containing goat
anti-mouse IgG conjugated to AlexaFluor 594 (Invitrogen, Burlington,
Ontario, Canada), and DAPI (Sigma, Oakville, Ontario, Canada) (0.3
μg/mL) was then added. The cover slides were incubated for 2 h at
room temperature and then washed twice with PBS-T and twice with
PBS. The cover slides were mounted with polyvinyl alcohol−1,4,-
diazobicyclo[2.2.2]octane (10−2.5%, v/v) in buffer (5% (v/v) glycerol
and 25 mM Tris buffer, pH 8.7) (Sigma, Oakville, Ontario, Canada).
Cells were visualized using an epifluorescence microscope (Olympus
BX51, Center Valley, PA) with a Qimaging RETIGA EXi camera
(Qimaging, Surrey, British Columbia, Canada).
Figure 3. Effect of PUB-SOs 4, 10, 16, 17, 30, 45−47, CA-4, and
cDDP on the growth of HT-1080 tumors and their toxicity on chick
embryos in the CAM assay. Gray bars represent the percentage of wet
weight of tumors treated with or without excipient. Black bars
represent the percentage of chick embryo mortality.
death rate, respectively), in a manner similar to that of cDDP
and CA-4.
CONCLUSION
■
We have identified and characterized PUB-SOs as a novel class
of anticancer agents that block cell cycle progression in S-phase.
Structure−activity relationships of PUB-SOs indicate that
modification of their sulfonyl group by a bioisosteric
sulfonamide moiety, yielding PUB-SAs, abolishes both their
antiproliferative and cell cycle blocking activities. The
pharmacophoric EU and CEU moieties with a substitution at
C4 on aromatic ring A are required to achieve optimal
antiproliferative activity and S-phase arrest, whereas substitu-
tions with alkyl groups at C2 or a hydroxyl group at C4 on the
B-ring do not significantly affect cytocidal activity. In the series
of PUB-SOs herein synthesized, we have identified compounds
with an antiproliferative activity and ability to cause S-phase
arrest comparable to those of cDDP. Moreover, compounds 4,
16, 17, 29−31, 33, 40, 41, 45−47, and 49 induce H2AX
phosphorylation, in support for a mechanism of action that
involves DNA double-strand breaks, although the molecular
details have yet to be identified. Finally, compounds 4 and 46
are at least as active as cDDP and CA-4 in the CAM assay while
displaying little or no toxic effect on chick embryos, suggesting
that these compounds might represent a promising new class of
anticancer agents.
EXPERIMENTAL SECTION
■
Biological Methods. Antiproliferative Activity. HT-29 colon
carcinoma cells, M21 skin melanoma cells, and MCF-7 breast
carcinoma cells (all of human origin) were purchased from the
American Type Culture Collection (Manassas, VA). Cells were
cultured in high-glucose Dulbecco’s minimal essential medium
(DMEM) supplemented with 5% (v/v) fetal bovine serum (Hyclone,
Logan, UT). The cell lines were maintained at 37 °C in a water-
saturated atmosphere containing 5% CO2. The growth inhibition
potency of all compounds was assessed using the procedure
recommended by the National Cancer Institute for its drug screening
program.17 Briefly, 96-well microtiter plates were seeded with 75 μL of
a suspension of HT-29 (4 × 103), M21 (3.5 × 103), or MCF-7 (3 ×
103) cells per well in DMEM. Plates were incubated at 37 °C and 5%
CO2 for 24 h. Drugs freshly solubilized in DMSO (40 mM) were
CAM Assay. Human HT-1080 fibrosarcoma cells were cultured in
Dulbecco’s minimal essential medium containing 58 mM NaHCO3 25
mM D-glucose, 4 mM L-glutamine, and 0.11 mM sodium pyruvate
supplemented with 5% (v/v) fetal bovine serum. Cells were
maintained at 37 °C in a water-saturated, 5% CO2 atmosphere. HT-
1080 cells were used to assess the antitumoral activity of candidate
drugs in the CAM assay. Briefly, on day 0, freshly fertilized chicken
eggs were purchased from Couvoirs Victoriaville (Victoriaville,
Quebec, Canada). The eggs were incubated for 10 days in a Pro-FI
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egg incubator (Lyon Electric, Chula Vista, CA) fitted with an
automatic egg turner before being transferred to a Roll-X static
incubator for the rest of the incubation period. Eggs were kept at 37
°C in a 60% relative humidity atmosphere for the entire incubation
period. By use of a hobby drill (Dremel, Racine, WI), a hole was
drilled on the side of the egg, and negative pressure was applied to
create a new air sac. A window was opened in this new air sac and was
covered with transparent adhesive tape to prevent contamination. A
freshly prepared HT-1080 cell suspension (40 μL, 3.5 × 105 cells/egg)
was applied directly on the freshly exposed CAM tissue. On day 11,
drugs dissolved in DMSO (40 μM) were extemporaneously diluted at
the required concentrations in the excipient (Cremophor EL/99%
ethanol/PBS, 1/1/14 v/v). The drug solution (100 μL) was injected
into a vein under the CAM. Each experimental group contained 10−
12 eggs that were incubated until day 17. Embryos were euthanized by
cooling at 4 °C for at least 4 h. Tumors were collected, and tumor wet
weight was recorded. The number of dead embryos and signs of
toxicity from the different groups were also recorded.
Chemical Procedures. General. Proton NMR spectra were
recorded on a Bruker AM-300 spectrometer (Bruker, Germany).
Chemical shifts (δ) are reported in parts per million. Reactions using
microwave heating were performed with an Initiator system (Biotage,
Charlottesville, VA). IR spectra were recorded with a Magna FT-IR
spectrometer (Nicolet Instrument Inc., Madison, WI). Uncorrected
melting points were determined on an electrothermal melting point
apparatus. HPLC analyses of compounds 4−8 and 10 were performed
using an Acquity UPLC sample with binary solvent manager equipped
with a Quattro Premier XE tandem quadrupole mass spectrometer
(Waters, Milford, MA). Compounds were analyzed with a Waters
BECH C18 reversed-phase column (1.7 μm, 2.1 mm × 50 mm, 50 °C)
and eluted within 7 min with a MeOH/H2O linear gradient containing
0.1% TFA at 0.6 mL/min. HPLC analysis of other end compounds
was performed using a Prominence LCMS-2020 system with binary
solvent equipped with a UV/vis photodiode array (Shimadzu,
Columbia, MD). Compounds were eluted in 30 min on an Alltech
Alltima C18 reversed-phase column (5 μm, 250 mm × 4.6 mm)
equipped with an Alltech Alltima C18 precolumn (5 μm, 7.5 mm × 4.6
mm) with a MeOH/H2O linear gradient at 1.0 mL/min. Purity of the
final compounds was >95%. All reactions were performed under a
dried Ar atmosphere. All chemicals were supplied by Aldrich
Chemicals (Milwaukee, WI) or VWR International (Mont-Royal,
Quebec, Canada) and used as received unless specified otherwise.
Liquid flash chromatography was performed on silica gel F60, 60A,
acetate. The solution was washed with hydrochloric acid (1 N) and
brine, dried over anhydrous Na2SO4, filtered, and evaporated to
dryness.
Method D. The appropriate isocyanate (1.2 mmol) was added
dropwise to the appropriate aniline (1.0 mmol) in dry acetonitrile (10
mL) under an Ar atmosphere. Pyridine (1.0 mmol) was added to the
solution. The reaction mixture was stirred at room temperature for 7
days. The solvent was evaporated under reduced pressure, and the
compound was purified by flash chromatography.
Method E. The appropriate compound (9, 14, 27, 32, 43, or 48 (0.1
mmol)) was dissolved in dry tetrahydrofuran (5 mL). Tetrabuty-
lammonium fluoride (1M) in dry THF was added dropwise. The
mixture was stirred at room temperature for 24 h. The solvent was
evaporated and the residue dissolved with ethyl acetate (40 mL). The
solution was washed with 40 mL of HCL (1 N), brine, dried over
Na2SO4, filtered, and evaporated to dryness. The crude product was
purified by flash chromatography.
2-Tolyl 4-[3-(2-Chloroethyl)ureido]benzenesulfonate (4).
Method A in dry DCM was used. The crude product was purified
by flash chromatography (silica gel, methylene chloride to methylene
chloride/ethyl acetate (90:10)). Yield, 57%; yellow solid; mp, 101 °C.
1
IR ν: 3369 (NH), 1592 (CO) cm−1. H NMR (CDCl3): δ 8.18 (s,
1H, NH), 7.69−7.67 (m, 2H, Ar), 7.53−7.51 (m, 2H, Ar), 7.12−7.04
(m, 3H, Ar), 6.98−6.95 (m, 1H, Ar), 6.12 (t, 1H, J = 4.8 Hz, NH),
3.58 (brs, 4H, 2 × CH2), 2.05 (s, 3H, CH3). 13C NMR (CDCl3): δ
155.1, 148.1, 145.3, 131.8, 131.5, 129.8, 127.9, 127.3, 127.1, 122.2,
118.1, 44.2, 41.9, 16.3. MS (ESI+) m/z found 368.9; C16H18ClN2O4S
(M+ + H) requires 369.1.
3-Tolyl 4-[3-(2-Chloroethyl)ureido]benzenesulfonate (5).
Method C in dry THF under microwave at 100 °C for 15 min
without washing with HCl (1 N) was used. The crude product was
purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (90:10)). Yield, 29%; orange oil. IR
ν: 3348 (NH), 1594 (CO) cm−1. 1H NMR (CDCl3): δ 8.05 (s, 1H,
NH), 7.67−7.64 (m, 2H, Ar), 7.54−7.51 (m, 2H, Ar), 7.13−7.00 (m,
2H, Ar), 6.84 (s, 1H, Ar), 6.70−6.67 (m, 1H, Ar), 6.06 (t, 1H, J = 5.4
Hz, NH), 3.62−3.56 (m, 4H, 2 × CH2), 2.26 (s, 3H, CH3). 13C NMR
(CDCl3): δ 155.0, 149.4, 145.2, 140.2, 129.9, 129.3, 128.1, 127.3,
122.9, 119.0, 117.9, 44.3, 41.9, 21.2. MS (ESI+) m/z found 368.9;
C16H18ClN2O4S (M+ + H) requires 369.1.
4-Tolyl 4-[3-(2-Chloroethyl)ureido]benzenesulfonate (6).
Method A in dry DCM was used. The crude product was purified
by flash chromatography (silica gel, hexanes/ethyl acetate (80:20) to
hexanes/ethyl acetate (60:40)). Yield, 33%; colorless oil. IR ν: 3369
́
40−63 μm supplied by Silicycle (Quebec, Canada) using a FPX flash
1
(NH), 1539 (CO) cm−1. H NMR (CDCl3): δ 8.17 (s, 1H, NH),
purification system (Biotage, Charlottesville, VA) and using solvent
mixtures expressed as v/v ratios. Solvents and reagents were used
without purification unless specified otherwise. The progress of all
reactions was monitored by TLC on precoated silica gel 60 F254 TLC
plates (VWR). The chromatograms were viewed under UV light at
254 and/or 265 nm.
7.66−7.63 (m, 2H, Ar), 7.53−7.50 (m, 2H, Ar), 7.02 (d, 2H, J = 8.4
Hz, Ar), 6.81 (d, 2H, J = 8.4 Hz, Ar), 6.13 (brs, 1H, NH), 3.58 (brs,
4H, 2 × CH2), 2.25 (s, 3H, CH3). 13C NMR (CDCl3): δ 155.2, 147.2,
145.2, 137.3, 130.3, 129.9, 127.1, 122.0, 118.0, 44.2, 41.9, 20.9. MS
(ESI+) m/z found 368.9; C16H18ClN2O4S (M+ + H) requires 369.1.
4-Methoxyphenyl 4-[3-(2-Chloroethyl)ureido]-
benzenesulfonate (7). Method A in THF was used. The crude
product was purified by flash chromatography (silica gel, methylene
chloride to methylene chloride/ethyl acetate (80:20)). Yield, 46%;
General Procedure for the Synthesis of Compounds 4−55.
Method A. The appropriate isocyanate (1.2 mmol) was added
dropwise to the appropriate aniline (1.0 mmol) in dry methylene
chloride or dry tetrahydrofuran (10 mL) under an Ar atmosphere. The
reaction mixture was stirred at room temperature for 7 days. The
solvent was evaporated under reduced pressure, and the compound
was purified by flash chromatography.
Method B. 2-Chloroethyl isocyanate (1.2 mmol) and 4-
dimethylaminopyridine were added dropwise to a solution of the
appropriate aniline (1.0 mmol) in dry tetrahydrofuran (10 mL) under
an Ar atmosphere. The reaction mixture was heated to reflux and
stirred for 7 days. After the mixture was cooled to room temperature,
the solvent was evaporated under reduced pressure and the crude
compound was purified by flash chromatography.
Method C. The appropriate isocyanate (2.0 mmol) was added
dropwise to appropriate aniline (1.0 mmol) in dry acetonitrile or dry
tetrahydrofuran (10 mL). The reaction was performed either in the
absence or presence of pyridine (1 mmol). The reaction mixture was
stirred from 60 to 130 °C under microwave heating (100 W) for 15−
50 min. The solvent was evaporated and the residue dissolved in ethyl
1
colorless oil. IR ν: 1500 (CO) cm−1. H NMR (CDCl3): δ 7.92 (s,
1H, NH), 7.64−7.62 (m, 2H, Ar), 7.50−7.48 (m, 2H, Ar), 6.86−6.83
(m, 2H, Ar), 6.75−6.72 (m, 2H, Ar), 5.97 (t, 1H, J = 5.2 Hz, NH),
3.72 (s, 3H, CH3), 3.62−3.57 (m, 4H, 2 × CH2). 13C NMR (CDCl3):
δ 158.4, 154.9, 145.1, 142.8, 130.0, 127.1, 123.3, 118.0, 114.6, 55.6,
44.3, 42.0. MS (ESI+) m/z found 385.0; C16H18ClN2O5S (M+ + H)
requires 385.1.
4-(Dimethylamino)phenyl 4-[3-(2-Chloroethyl)ureido]-
benzenesulfonate (8). Method C in dry THF under microwave at
60 °C for 15 min without washing with HCl (1 N) was used. The
crude product was purified by flash chromatography (silica gel,
methylene chloride to methylene chloride/ethyl acetate (95:5)). Yield,
1
22%; white sticky solid. IR ν: 3355 (NH), 1569 (CO) cm−1. H
NMR (CDCl3): δ 7.95 (s, 1H, NH), 7.65−7.63 (m, 2H, Ar), 7.51−
7.49 (m, 2H, Ar), 6.78−6.76 (m, 2H, Ar), 6.50−6.48 (m, 2H, Ar), 5.98
(t, 1H, J = 5.3 Hz, NH), 3.63−3.57 (m, 4H, 2 × CH2), 2.87 (s, 6H, 2
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× CH3). 13C NMR (CDCl3): δ 154.9, 149.4, 145.1, 139.9, 129.9,
127.4, 122.8, 117.9, 112.5, 44.3, 41.9, 40.5. MS (ESI+) m/z found
397.9; C17H21ClN3O4S (M+ + H) requires 398.1.
4-Hydroxyphenyl 3-[3-(2-Chloroethyl)ureido]-
benzenesulfonate (15). Method E was used. The crude product
was purified by flash chromatography (silica gel, ethyl acetate to ethyl
acetate/methanol (90:10)). Yield, 78%; white solid; mp, 156 °C. IR ν:
3500−3100 (OH), 1688 (CO), 1330 (OH) cm−1. 1H NMR
(acetone-d6): δ 8.60 (s, 1H, NH), 8.29 (s, 1H, Ar), 7.93−7.90 (m, 1H,
Ar), 7.57−7.52 (m, 1H, Ar), 7.40−7.37 (m, 1H, Ar), 6.90−6.86 (m,
2H, Ar), 6.80−6.76 (m, 2H, Ar), 6.27 (brs, 1H, NH), 4.00 (t, 2H, J =
7.9 Hz, CH2), 3.63−3.58 (m, 2H, CH2), 2.87 (brs, 1H, OH). 13C
NMR (DMSO-d6): δ 158.9, 155.6, 155.6, 142.4, 141.0, 135.7, 129.4,
123.2, 123.1, 121.9, 115.9, 44.8, 37.0. MS (APSI+) m/z found 371.1;
C15H16ClN2O5S (M+ + H) requires 371.0.
2-Ethylphenyl 4-[3-(2-Chloroethyl)ureido]benzenesulfonate
(16). Method C in dry MeCN under microwave at 130 °C for 40 min
with washing with HCl (1 N) was used. The crude product was
purified by flash chromatography (silica gel, hexanes/ethyl acetate
(75:25) to hexanes/ethyl acetate (50:50)). Yield, 8%; white solid; mp,
127−128 °C. IR ν: 3338 (NH), 1685 (CO) cm−1. 1H NMR
(CDCl3): δ 8.44 (s, 1H, NH), 7.86−7.83 (m, 2H, Ar), 7.73−7.70 (m,
2H, Ar), 7.27−6.97 (m, 4H, Ar), 4.03−3.95 (m, 4H, 2 × CH2), 2.53−
2.45 (m, 2H, CH2), 1.12−1.07 (m, 3H, CH3). 13C NMR (CDCl3): δ
154.5, 147.8, 143.9, 137.2, 130.5, 129.9, 129.6, 127.2, 126.9, 122.0,
117.7, 43.6, 41.9, 22.8, 14.1. MS (APSI+) m/z found 383.1;
C17H20ClN2O4S (M+ + H) requires 383.1.
4-(tert-Butyldimethylsilyloxy)phenyl 4-[3-(2-Chloroethyl)-
ureido]benzenesulfonate (9). Method D was used. The crude
product was purified by flash chromatography (silica gel, methylene
chloride to methylene chloride/ethyl acetate (80:20)). Yield, 99%;
yellow oil. IR ν: 3321 (NH), 1670 (CO) cm−1. 1H NMR (CDCl3):
δ 7.96 (s, 1H, NH), 7.65−7.51 (m, 4H, Ar), 6.82−6.68 (m, 4H, Ar),
5.97 (brs, 1H, NH), 3.61 (brs, 4H, 2 × CH2), 0.94 (s, 9H, 3 × CH3),
0.15 (s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 154.8, 154.7, 145.2,
143.3, 130.0, 127.1, 123.3, 120.8, 118.0, 44.3, 42.0, 25.6, 18.2, −4.5.
4-Hydroxyphenyl 4-[3-(2-Chloroethyl)ureido]-
benzenesulfonate (10). Method E was used. The crude product
was purified by flash chromatography (silica gel, ethyl acetate to ethyl
acetate/methanol (90:10)). Yield, 80%; white solid; mp, 247 °C. IR ν:
3625−3050 (OH), 1686 (CO), 1334 (OH) cm−1. 1H NMR
(acetone-d6): δ 8.60 (s, 1H, NH), 7.89−7.86 (m, 2H, Ar), 7.73−7.70
(m, 2H, Ar), 6.85−6.82 (m, 2H, Ar), 6.78−6.75 (m, 2H, Ar), 6.37
(brs, 1H, NH), 4.04 (t, 2H, J = 7.9 Hz, CH2), 3.65−3.60 (m, 2H,
CH2), 3.31 (brs, 1H, OH). 13C NMR (CDCl3/DMSO-d6): δ 158.5,
156.1, 145.6, 141.6, 129.2, 126.3, 122.9, 116.1, 115.7, 44.4, 36.6. MS
(ESI−) m/z found 369.0; C15H14ClN2O5S (M− − H) requires 369.0.
2-Tolyl 3-[3-(2-Chloroethyl)ureido]benzenesulfonate (11).
Method A in dry DCM was used. The crude product was purified
by flash chromatography (silica gel, methylene chloride to methylene
chloride/ethyl acetate (90:10)). Yield, 57%; sticky solid. IR ν: 3330
2 - P r o p y l p h e n y l 4 - [ 3 - ( 2 - C h l o r o e t h y l ) u re i d o ] -
benzenesulfonate (17). Method C in dry MeCN under microwave
at 130 °C for 50 min with washing with HCl (1 N) was used. The
crude product was purified by flash chromatography (silica gel,
hexanes/ethyl acetate (75:25) to hexanes/ethyl acetate (50:50)).
Yield, 75%; yellow solid; mp, 95 °C. IR ν: 3326 (NH), 1669 (CO)
1
(NH), 1658 (CO) cm−1. H NMR (CDCl3): δ 8.26 (s, 1H, NH),
7.97 (s, 1H, Ar), 7.68−7.65 (m, 1H, Ar), 7.83−7.30 (m, 2H, Ar),
7.14−7.02 (m, 3H, Ar), 6.93−6.90 (m, 1H, Ar), 6.18 (brs, 1H, NH),
3.57 (s, 4H, 2 × CH2), 2.07 (s, 3H, CH3). 13C NMR (CDCl3): δ
155.7, 148.2, 140.4, 136.3, 131.7, 131.5, 129.9, 127.2, 127.0, 124.6,
122.1, 122.0, 118.0, 44.2, 41.9, 16.3. MS (APSI+) m/z found 369.1;
C16H18ClN2O4S (M+ + H) requires 369.1.
1
cm−1. H NMR (CDCl3): δ 7.76−7.73 (m, 2H, Ar), 7.54−7.52 (m,
2H, Ar), 7.32 (brs, 1H, NH), 7.29−6.99 (m, 4H, Ar), 5.58 (brs, 1H,
NH), 3.65−3.64 (m, 4H, 2 × CH2), 2.43 (t, 2H, J = 7.7 Hz, CH2),
1.57−1.51 (m, 2H, CH2), 0.86 (t, 3H, J = 7.3 Hz, CH3). 13C NMR
(CDCl3): δ 154.3, 148.0, 144.7, 135.8, 130.7, 129.8, 128.7, 127.1,
127.0, 122.0, 118.1, 44.4, 42.0, 31.9, 23.0, 13.9. MS (APSI+) m/z
found 397.1; C18H22ClN2O4S (M+ + H) requires 397.1.
2-Ethylphenyl 3-[3-(2-Chloroethyl)ureido]benzenesulfonate
(12). Method A in dry DCM was used. The crude product was
purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (90:10)). Yield, 53%; colorless oil. IR
ν: 3343 (NH), 1658 (CO) cm−1. 1H NMR (CDCl3): δ 8.17 (s, 1H,
NH), 7.95 (s, 1H, Ar), 7.70−7.68 (m, 1H, Ar), 7.41−7.34 (m, 2H, Ar),
7.19−7.03 (m, 3H, Ar), 6.92−6.90 (m, 1H, Ar), 6.08 (brs, 1H, NH),
3.56 (s, 4H, 2 × CH2), 2.50 (q, 2H, J = 7.5 Hz, CH2), 1.07 (t, 3H, J =
7.5 Hz, CH3). 13C NMR (CDCl3): δ 155.8, 147.7, 140.4, 137.2, 136.4,
130.0, 127.4, 127.0, 124.6, 121.9, 121.9, 118.0, 102.7, 44.1, 41.9, 22.8,
14.1. MS (APSI+) m/z found 383.1; C17H20ClN2O4S (M+ + H)
requires 383.1.
3-[3-(2-Chloroethyl)ureido]-N-2-tolylbenzenesulfonamide
(18). Method B was used. The crude product was purified by flash
chromatography (silica gel, methylene chloride to methylene chloride/
ethyl acetate (90:10)) and was crystallized with methylene chloride
and filtered. Yield, 45%; white solid; mp, 164−165 °C. IR ν: 3267
1
(NH), 1642 (CO) cm−1. H NMR (DMSO-d6): δ 9.58 (s, 1H,
NH), 9.06 (s, 1H, NH), 7.94 (s, 1H, Ar), 7.62−7.01 (m, 7H, Ar), 6.51
(brs, 1H, NH), 3.71−3.68 (m, 2H, CH2), 3.48−3.45 (m, 2H, CH2),
2.05 (s, 3H, CH3). 13C NMR (DMSO-d6): δ 154.8, 141.3, 141.0,
134.9, 134.3, 130.7, 129.5, 126.5, 126.4, 126.3, 121.3, 119.2, 115.4,
44.3, 41.3, 17.7. MS (APSI−) m/z found 366.0; C16H17ClN3O3S (M−
− H) requires 366.1.
2 - P r o p y l p h e n y l 3 - [ 3 - ( 2 - C h l o r o e t h y l ) u r e i d o ] -
benzenesulfonate (13). Method A in dry DCM was used. The
crude product was purified by flash chromatography (silica gel,
methylene chloride to methylene chloride/ethyl acetate (90:10)).
3-[3-(2-Chloroethyl)ureido]-N-(2-ethylphenyl)-
benzenesulfonamide (19). Method B was used. The crude product
was purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (90:10)) and was crystallized with
methylene chloride and filtered. Yield, 35%; white solid; mp, 170−171
°C. IR ν: 3274 (NH), 1643 (CO). 1H NMR (DMSO-d6): δ 9.58 (s,
1H, NH), 9.06 (s, 1H, NH), 7.95 (s, 1H, Ar), 7.63−6.91 (m, 7H, Ar),
6.50 (brs, 1H, NH), 3.72−3.68 (m, 2H, CH2), 3.47−3.45 (m, 2H,
CH2), 2.58−2.54 (m, 2H, CH2), 1.01 (t, 3H, J = 7.5 Hz, CH3). 13C
NMR (DMSO-d6): δ 154.9, 141.3, 141.0, 140.5, 134.2, 129.5, 129.0,
126.8, 126.6, 126.2, 121.2, 119.2, 115.4, 44.3, 41.3, 23.2, 14.4. MS
(APCI−) m/z found 379.9; C17H19ClN3O3S (M− − H) requires
380.1.
3-[3-(2-Chloroethyl)ureido]-N-(2-propylphenyl)-
benzenesulfonamide (20). Method D was used. The crude product
was purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (80:20)). Yield, 12%; yellow solid;
mp, 207 °C. IR ν: 3318 (NH), 1633 (CO) cm−1. 1H NMR
(acetone-d6): δ 8.55 (s, 1H, NH), 8.01−7.98 (m, 1H, Ar), 7.81 (s, 1H,
Ar), 7.52−6.92 (m, 6H, Ar), 6.41 (brs, 1H, NH), 3.74−3.70 (m, 2H,
CH2), 3.61−3.57 (m, 2H, CH2), 2.48−2.42 (m, 2H, CH2), 1.63−1.53
1
Yield, 12%; yellow oil. IR ν: 3300 (NH), 1657 (CO) cm−1. H
NMR (CDCl3): δ 8.42 (s, 1H, NH), 8.08 (s, 1H, Ar) 7.57−7.55 (m,
1H, Ar), 7.38−7.32 (m, 2H, Ar), 7.14−7.05 (m, 3H, Ar), 6.93−6.91
(m, 1H, Ar), 6.29 (brs, 1H, NH), 3.55 (s, 4H, 2 × CH2), 2.45−2.41
(m, 2H, CH2), 1.49−1.45 (m, 2H, CH2), 0.84−0.80 (m, 3H, CH3).
13C NMR (CDCl3): δ 155.9, 147.9, 140.4, 136.5, 135.8, 130.7, 129.9,
127.2, 127.0, 124.6, 121.9, 118.0, 102.7, 44.1, 41.9, 31.8, 23.0, 13.9. MS
(APSI+) m/z found 397.1; C18H22ClN2O4S (M+ + H) requires 397.1.
4-(tert-Butyldimethylsilyloxy)phenyl 3-[3-(2-Chloroethyl)-
ureido]benzenesulfonate (14). Method D was used. The crude
product was purified by flash chromatography (silica gel, methylene
chloride to methylene chloride/ethyl acetate (90:10)). Yield, 85%;
1
white sticky solid. IR ν: 3004 (NH), 1710 (CO) cm−1. H NMR
(CDCl3): δ 8.16 (s, 1H, NH), 7.90 (s, 1H, Ar), 7.75−7.72 (m, 1H,
Ar), 7.33−7.24 (m, 2H, Ar), 6.82−6.79 (m, 2H, Ar), 6.69−6.66 (m,
2H, Ar), 6.09 (brs, 1H, NH), 3.61 (s, 4H, 2 × CH2), 0.93 (s, 9H, 3 ×
CH3), 0.14 (s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 155.3, 154.7,
143.3, 140.4, 135.3, 129.8, 124.6, 123.2, 122.3, 120.8, 118.0, 44.3, 42.0,
25.6, 18.2, −4.5.
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(m, 2H, CH2), 0.87 (t, 3H, J = 7.3 Hz, CH3). 13C NMR (acetone-d6):
δ 160.1, 150.4, 146.4, 144.8, 138.3, 137.0, 135.5, 134.7, 134.6, 131.4,
129.0, 126.9, 123.1, 49.1, 46.9, 37.9, 27.9, 19.0. MS (APSI−) m/z
found 393.9; C18H21ClN3O3S (M− − H) requires 394.1.
crude product was purified by flash chromatography (silica gel,
chloroform to chloroform/ethyl acetate (80:20)). Yield, 55%; white
1
solid; mp, 100 °C. IR ν: 1634 (CO) cm−1. H NMR (CDCl3): δ
8.29 (s, 1H, NH), 8.02 (s, 1H, Ar), 7.64−7.61 (m, 1H, Ar), 7.41−7.31
(m, 2H, Ar), 7.18−7.03 (m, 3H, Ar), 6.93−6.91 (m, 1H, Ar), 6.00
(brs, 1H, NH), 3.54 (t, 2H, J = 6.2 Hz, CH2), 3.40−3.36 (m, 2H,
CH2), 2.44 (t, 2H, J = 7.7 Hz, CH2), 1.97−1.89 (m, 2H, CH2), 1.55−
1.42 (m, 2H, CH2), 0.86−0.81 (t, 3H, J = 7.3, CH3). 13C NMR
(CDCl3): δ 156.1, 148.0, 140.6, 136.5, 135.8, 130.7, 129.9, 127.2,
127.0, 124.5, 121.9, 121.8, 117.9, 42.4, 37.5, 32.5, 31.8, 23.0, 13.9. MS
(APSI+) m/z found 411.2; C19H24ClN2O4S (M+ + H) requires 411.1.
4-(tert-Butyldimethylsilyloxy)phenyl 3-[3-(3-Chloropropyl)-
ureido]benzenesulfonate (27). Method D was used. The crude
product was purified by flash chromatography (silica gel, methylene
chloride to methylene chloride/ethyl acetate (95:5)). Yield, 83%;
yellowish oil. IR ν: 1662 (CO) cm−1. 1H NMR (CDCl3): δ 8.24 (s,
1H, NH), 8.06 (s, 1H, Ar), 7.57−7.55 (m, 1H, Ar), 7.28−7.21 (m, 2H,
Ar), 6.81−6.78 (m, 2H, Ar), 6.69−6.67 (m, 2H, Ar), 6.05 (brs, 1H,
NH), 3.59−3.55 (m, 2H, CH2), 3.44−3.40 (m, 2H, CH2), 1.98−1.95
(m, 2H, CH2), 0.93 (s, 9H, 3 × CH3), 0.14 (s, 6H, 2 × CH3). 13C
NMR (CDCl3): δ 156.1, 154.6, 143.3, 140.6, 135.4, 129.6, 124.6,
123.2, 122.2, 120.8, 118.0, 42.4, 37.5, 32.6, 25.6, 18.1, −4.5.
4-[3-(2-Chloroethyl)ureido]-N-2-tolylbenzenesulfonamide
(21). Method A in dry DCM was used. The crude product was
purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (90:10)) and was recrystallized with
MeOH and filtered. Yield, 40%; yellowish solid; mp, 134−135 °C. IR
1
ν: 3074 (NH), 1683 (CO) cm−1. H NMR (CDCl3/DMSO-d6): δ
8.97 (s, 2H, 2 × NH), 7.79−7.64 (m, 4H, Ar), 7.38−7.26 (m, 4H, Ar),
6.31 (brs, 1H, NH), 3.87−3.75 (m, 4H, 2 × CH2), 2.24 (s, 3H, CH3).
13C NMR (DMSO-d6): δ 154.7, 144.3, 135.1, 134.1, 132.7, 130.7,
127.9, 126.4, 126.3, 117.0, 44.3, 41.3, 17.7. MS (APSI+) m/z found
368.1; C16H19ClN3O3S (M+ + H) requires 368.1.
4-[3-(2-Chloroethyl)ureido]-N-(2-ethylphenyl)-
benzenesulfonamide (22). Method A in dry DCM was used. The
crude product was purified by flash chromatography (silica gel,
methylene chloride to methylene chloride/ethyl acetate (90:10)) and
was recrystallized with MeOH and filtered. Yield, 10%; white solid;
1
mp, 214−215 °C. IR ν: 3100 (NH), 1682 (CO) cm−1. H NMR
(CDCl3/DMSO-d6): δ 7.53−7.40 (m, 4H, Ar), 7.13−7.01 (m, 4H,
Ar), 3.60−3.49 (m, 4H, 2 × CH2), 2.38 (q, 2H, J = 7.6 Hz, CH2), 0.99
(t, 3H, J = 7.6, CH3). 13C NMR (DMSO-d6): δ 154.7, 144.2, 140.3,
134.4, 132.3, 128.9, 128.0, 126.6, 126.5, 126.1, 117.0, 44.3, 41.2, 23.1,
14.4. MS (APSI+) m/z found 382.1; C17H21ClN3O3S (M+ + H)
requires 382.1.
4-Hydroxyphenyl 3-[3-(3-Chloropropyl)ureido]-
benzenesulfonate (28). Method E was used. The crude product
was purified by flash chromatography (silica gel, ethyl acetate to ethyl
acetate/methanol (90:10)). Yield, 60%; yellowish oil. IR ν: 3450−
3050 (OH), 1666 (CO), 1364 (OH) cm−1. 1H NMR (acetone-d6):
δ 8.12−8.11 (m, 1H, Ar), 7.78−7.63 (m, 1H, Ar), 7.44−7.28 (m, 2H,
Ar), 6.84−6.68 (m, 4H, Ar), 3.67−3.62 (m, 2H, CH2), 3.38−3.34 (m,
2H, CH2), 2.02−1.94 (m, 2H, CH2). 13C NMR (acetone-d6): δ 157.0,
155.8, 143.1, 142.5, 136.6, 130.3, 124.0, 124.0, 121.7, 117.9, 116.6,
43.3, 37.8, 33.7. MS (APSI+) m/z found 385.1; C16H18ClN2O5S (M+
+ H) requires 385.1.
4-[3-(2-Chloroethyl)ureido]-N-(2-propylphenyl)-
benzenesulfonamide (23). Method B was used. The crude product
was purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (90:10)) and was crystallized with
methylene chloride and filtered. Yield, 21%; white solid; mp, 178−180
1
°C. IR ν: 3376 (NH), 1684 (CO) cm−1. H NMR (DMSO-d6): δ
9.36 (s, 1H, NH), 9.15 (s, 1H, NH), 7.64−7.57 (m, 4H, Ar), 7.21−
6.91 (m, 4H, Ar), 6.62 (brs, 1H, NH), 3.73−3.69 (m, 2H, CH2),
3.48−3.46 (m, 2H, CH2), 2.55−2.47 (m, 2H, CH2), 1.44−1.39 (m,
2H, CH2), 0.85 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (DMSO-d6): δ
154.7, 144.2, 138.8, 134.6, 132.4, 129.6, 128.0, 126.4, 126.4, 126.1,
117.0, 44.3, 41.3, 32.3, 22.9, 14.0. MS (APSI−) m/z found 393.9;
C18H21ClN3O3S (M− − H) requires 394.1.
2-Tolyl 4-[3-(3-Chloropropyl)ureido]benzenesulfonate (29).
Method C in dry MeCN under microwave at 130 °C for 45 min with
washing with HCl (1 N) was used. The crude product was purified by
flash chromatography (silica gel, methylene chloride to methylene
chloride/ethyl acetate (90:10)). Yield, 41%; sticky solid. IR ν: 3395
1
(NH), 1675 (CO) cm−1. H NMR (CDCl3): δ 7.90 (s, 1H, NH),
7.70−6.67 (m, 2H, Ar), 7.53−7.50 (m, 2H, Ar), 7.14−6.96 (m, 4H,
Ar), 3.58−3.54 (m, 2H, CH2), 3.42−3.38 (m, 2H, CH2), 2.06 (s, 3H,
CH3), 2.00−1.92 (m, 2H, CH2). 13C NMR (CDCl3): δ 155.2, 148.1,
145.3, 131.8, 131.5, 129.8, 127.8, 127.3, 127.1, 122.2, 118.0, 42.3, 37.5,
32.3, 16.3. MS (APSI+) m/z found 383.1; C17H20ClN2O4S (M+ + H)
requires 383.1.
2-Tolyl 3-[3-(3-Chloropropyl)ureido]benzenesulfonate (24).
Method C in dry MeCN under microwave at 130 °C for 45 min with
washing with HCl (1 N) was used. The crude product was purified by
flash chromatography (silica gel, methylene chloride to methylene
chloride/ethyl acetate (90:10)). Yield, 45%; white solid; mp, 129 °C.
1
IR ν: 3327 (NH), 1626 (CO) cm−1. H NMR (DMSO-d6): δ 9.05
2 - E t h yl p h e n y l 4 - [ 3 - ( 3 - C h l o r o p r o p y l ) u r e i d o ] -
benzenesulfonate (30). Method A in dry DCM was used. The
crude product was purified by flash chromatography (silica gel,
hexanes/ethyl acetate (75:25) to hexanes/ethyl acetate (25:75)).
Yield, 85%; yellowish sticky solid. IR ν: 3363 (NH), 1664 (NH) cm−1.
1H NMR (CDCl3): δ 8.07 (s, 1H, NH), 7.71−7.68 (m, 2H, Ar), 7.54−
7.51 (m, 2H, Ar), 7.19−7.07 (m, 3H, Ar), 6.98−6.96 (m, 1H, Ar), 5.88
(brs, 1H, NH), 3.56−3.52 (m, 2H, CH2), 3.41−3.36 (m, 2H, CH2),
2.48 (q, 2H, J = 7.6 Hz, CH2), 1.96−1.92 (m, 2H, CH2), 1.08 (t, 3H, J
= 7.6 Hz, CH3). 13C NMR (CDCl3): δ 155.4, 147.7, 145.4, 137.2,
130.0, 129.7, 127.8, 127.4, 127.0, 121.9, 118.0, 42.3, 37.5, 32.4, 22.8,
14.1. MS (APSI+) m/z found 397.1; C18H22ClN2O4S (M+ + H)
requires 397.1.
(s, 1H, NH), 8.19 (s, 1H, Ar), 7.70−6.67 (m, 1H, Ar), 7.54−7.52 (m,
1H, Ar), 7.37−7.21 (m, 4H, Ar), 6.99−6.97 (m, 1H, Ar), 6.45 (brs,
1H, NH), 3.69−3.67 (m, 2H, CH2), 3.25−3.23 (m, 2H, CH2), 2.06 (s,
3H, CH3), 1.93−1.91 (m, 2H, CH2). 13C NMR (DMSO-d6): δ 160.2,
153.0, 147.0, 140.8, 137.0, 136.2, 135.3, 132.5, 128.5, 127.1, 125.3,
121.3, 48.3, 41.9, 37.8, 21.0. MS (APSI+) m/z found 383.1;
C17H20ClN2O4S (M+ + H) requires 383.1.
2 - E t h y l p h e n y l 3 - [ 3- ( 3 - C h l o r o p r o p y l ) u r e i d o ] -
benzenesulfonate (25). Method C in dry MeCN under microwave
at 130 °C for 45 min with washing with HCl (1 N) was used. The
crude product was purified by flash chromatography (silica gel,
hexanes/ethyl acetate (75:25) to hexanes/ethyl acetate (25:75)).
Yield, 36%; white solid; mp, 102 °C. IR ν: 3326 (NH), 1633 (CO)
1
cm−1. H NMR (DMSO-d6): δ 9.04 (s, 1H, NH), 8.21 (s, 1H, Ar),
2-Propylphenyl 4-[3-(3-Chloropropyl)ureido]-
benzenesulfonate (31). Method C in dry MeCN under microwave
at 130 °C for 45 min with washing with HCl (1 N) was used. The
crude product was purified by flash chromatography (silica gel,
hexanes/ethyl acetate (75:25) to hexanes/ethyl acetate (25:75)).
7.69−7.67 (m, 1H, Ar), 7.56−7.51 (m, 1H, Ar), 7.40−7.23 (m, 4H,
Ar), 6.99−6.67 (m, 1H, Ar), 6.45 (t, 1H, J = 5.6 Hz, NH), 3.71−3.67
(m, 2H, CH2), 3.27−3.20 (m, 2H, CH2), 2.52−2.45 (m, 2H, CH2),
1.94−1.89 (m, 2H, CH2), 1.06 (t, 3H, J = 7.6 Hz, CH3). 13C NMR
(DMSO-d6): δ 160.2, 152.5, 147.0, 141.8, 140.8, 135.3, 132.7, 132.5,
128.5, 126.9, 125.2, 121.2, 48.2, 1.9, 37.8, 27.4, 19.3. MS (APSI+) m/z
found 397.1; C18H22ClN2O4S (M+ + H) requires 397.1.
2-Propylphenyl 3-[3-(3-Chloropropyl)ureido]-
benzenesulfonate (26). Method C in dry MeCN under microwave
at 130 °C for 45 min with washing with HCl (1 N) was used. The
1
Yield, 27%; yellow oil. IR ν: 3352 (NH), 1672 (CO) cm−1. H
NMR (CDCl3): δ 7.72−7.50 (m, 4H, Ar), 7.16−6.96 (m, 4H, Ar),
3.58−3.54 (m, 2H, CH2), 3.41−3.37 (m, 2H, CH2), 2.43−3.39 (m,
2H, CH2), 1.99−1.94 (m, 2H, CH2), 1.55−1.45 (m, 2H, CH2), 0.86−
0.81 (m, 3H, CH3). 13C NMR (CDCl3): δ 155.1, 147.9, 145.1, 135.7,
130.8, 129.7, 128.1, 127.2, 127.0, 121.9, 118.1, 42.3, 37.6, 32.3, 31.8,
6202
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23.0, 13.9. MS (APSI+) m/z found 411.2; C19H24ClN2O4S (M+ + H)
requires 411.1.
3079 (NH), 1678 (CO) cm−1. 1H NMR (DMSO-d6): δ 9.35 (s, 1H,
NH), 8.95 (s, 1H, NH), 7.56−7.49 (m, 4H, Ar), 7.13−7.00 (m, 4H,
Ar), 6.45 (brs, 1H, NH), 3.70−3.66 (m, 2H, CH2), 3.27−3.21 (m, 2H,
CH2), 2.02 (s, 3H, CH3), 1.93−1.89 (m, 2H, CH2). 13C NMR
(DMSO-d6): δ 154.8, 144.5, 135.1, 134.0, 132.1, 130.7, 127.9, 126.4,
126.3, 126.2, 116.9, 43.0, 36.6, 32.5, 17.7. MS (ESI−) m/z found
380.1; C17H19ClN3O3S (M− − H) requires 380.1.
4-(tert-Butyldimethylsilyloxy)phenyl 4-[3-(3-Chloropropyl)-
ureido]benzenesulfonate (32). Method D was used. The crude
product was purified by flash chromatography (silica gel, methylene
chloride to methylene chloride/ethyl acetate (90:10)). Yield, 60%;
yellowish oil. IR ν: 3303 (NH), 1672 (CO) cm−1. 1H NMR
(CDCl3): δ 7.89 (s, 1H, NH), 7.65−7.62 (m, 2H, Ar), 7.52−7.50 (m,
2H, Ar), 6.82−6.79 (m, 2H, Ar), 6.72−6.68 (m, 2H, Ar), 5.78 (brs,
1H, NH), 3.58−3.55 (m, 2H, CH2), 3.41−3.37 (m, 2H, CH2), 1.98−
1.94 (m, 2H, CH2), 0.95−0.91 (s, 9H, 3 × CH3), 0.15 (s, 6H, 2 ×
CH3). 13C NMR (CDCl3): δ 155.2, 154.7, 145.4, 143.3, 130.0, 126.9,
123.3, 120.8, 117.9, 42.3, 37.5, 32.4, 25.6, 18.1, −4.5.
4-Hydroxyphenyl 4-[3-(3-Chloropropyl)ureido]-
benzenesulfonate (33). Method E was used. The crude product
was purified by flash chromatography (silica gel, ethyl acetate to ethyl
acetate/methanol (90:10)). Yield, 63%; Yellowish oil. IR ν: 3620−
3375 (OH), 1678 (CO), 1359 (OH). 1H NMR (DMSO-d6): δ 9.67
(s, 1H, OH), 9.17 (s, 1H, NH), 7.64 (s, 4H, Ar), 6.80−6.68 (m, 4H,
Ar), 6.54 (t, 1H, J = 5.6, NH), 3.72−3.67 (m, 2H, CH2), 3.28−3.22
(m, 2H, CH2), 1.97−1.88 (m, 2H, CH2). 13C NMR (DMSO-d6): δ
156.2, 154.7, 146.3, 141.4, 129.7, 125.1, 123.1, 117.1, 115.9, 43.0, 36.7,
32.5. MS (APSI+) m/z found 385.1; C16H18ClN2O5S (M+ + H)
requires 385.1.
4-[3-(3-Chloropropyl)ureido]-N-(2-ethylphenyl)-
benzenesulfonamide (38). Method A in dry DCM was used. The
crude product was purified by flash chromatography (silica gel,
methylene chloride to methylene chloride/ethyl acetate (90:10)) and
was recrystallized with MeOH and filtered. Yield, 13%; white solid;
mp, 192 °C. IR ν: 3076 (NH), 1679 (CO) cm−1. 1H NMR
(CDCl3/DMSO-d6): δ 7.54−7.41 (m, 4H, Ar), 7.18−7.03 (m, 4H,
Ar), 3.58 (t, 2H, J = 6.3 Hz, CH2), 3.36−3.31 (m, 2H, CH2), 2.38 (q,
2H, J = 7.5 Hz, CH2), 1.99−1.91 (m, 2H, CH2), 1.01 (t, 3H, J = 7.5
Hz, CH3). 13C NMR (DMSO-d6): δ 155.5, 145.6, 140.4, 135.5, 133.3,
129.7, 129.0, 127.2, 126.9, 126.8, 117.8, 43.2, 37.8, 33.7, 24.1, 14.7. MS
(ESI−) m/z found 394.1; C18H21ClN3O3S (M− − H) requires 394.1.
4-[3-(3-Chloropropyl)ureido]-N-(2-propylphenyl)-
benzenesulfonamide (39). Method A in dry DCM was used. The
crude product was purified by flash chromatography (silica gel,
methylene chloride to methylene chloride/ethyl acetate (95:15)) and
was recrystallized with MeOH and filtered. Yield, 11%; white solid;
1
mp, 219−220 °C. IR ν: 3277 (NH), 1657 (CO) cm−1. H NMR
3-[3-(3-Chloropropyl)ureido]-N-2-tolylbenzenesulfonamide
(34). Method D was used. The crude product was purified by flash
chromatography (silica gel, methylene chloride to methylene chloride/
ethyl acetate (70:30)) and recrystallized with MeOH and filtered.
Yield, 40%; yellowish solid; mp, 150 °C. IR ν: 3353 (NH), 1648 (C
(DMSO-d6): δ 9.11 (s, 1H, NH), 7.80−7.60 (m, 4H, Ar), 7.44−7.08
(m, 4H, Ar), 6.54−6.48 (m, 2H, 2 × NH), 3.51−3.47 (m, 2H, CH2),
3.26−3.22 (m, 2H, CH2), 1.95−1.90 (m, 2H, CH2), 1.78−1.73 (m,
2H, CH2), 1.61−1.59 (m, 2H, CH2), 0.91 (t, 3H, J = 7.2 Hz, CH3).
13C NMR (DMSO-d6): δ 154.8, 152.7, 145.4, 142.9, 134.1, 130.9,
130.5, 129.9, 129.6, 127.1, 116.6, 43.0, 37.8, 36.7, 32.6, 22.5, 14.1. MS
(APSI−) m/z found 408.0; C19H23ClN3O3S (M− − H) requires 408.1.
2-Tolyl 3-(3-Ethylureido)benzenesulfonate (40). Method A in
dry DCM was used. The crude product was purified by flash
chromatography (silica gel, methylene chloride/ethyl acetate (92:8) to
methylene chloride/ethyl acetate (88:12)). Yield, 80%; yellowish
sticky solid. IR ν: 3317 (NH), 1655 (CO) cm−1. 1H NMR
(CDCl3): δ 8.27 (s, 1H, NH), 8.00 (s, 1H, Ar), 7.63−7.61 (m, 1H,
Ar), 7.35−7.27 (m, 2H, Ar), 7.09−7.03 (m, 3H, Ar), 6.93−6.90 (m,
1H, Ar), 5.89 (brs, 1H, NH), 3.26−3.19 (m, 2H, CH2), 2.07 (s, 3H,
CH3), 1.07 (t, 3H, J = 7.1 Hz, CH3). 13C NMR (CDCl3): δ 156.1,
148.2, 140.8, 136.4, 131.7, 131.5, 129.8, 127.2, 127.0, 124.4, 122.1,
121.6, 117.9, 35.0, 16.3, 15.1. MS (APSI+) m/z found 335.1;
C16H19N2O4S (M+ + H) requires 335.1.
1
O) cm−1. H NMR (DMSO-d6): δ 9.55 (s, 1H, NH), 8.85 (s, 1H,
NH), 7.91 (s, 1H, Ar), 7.58−7.56 (m, 1H, Ar), 7.40−7.35 (m, 1H, Ar),
7.16−7.08 (m, 4H, Ar), 6.70−6.67 (m, 1H, Ar), 6.33 (t, 1H, J = 5.5
Hz, NH), 3.70−3.66 (m, 2H, CH2), 3.25−3.19 (m, 2H, CH2), 2.03 (s,
3H, CH3), 1.95−1.88 (m, 2H, CH2). 13C NMR (DMSO-d6): δ 155.0,
141.2, 134.9, 134.2, 130.7, 129.4, 126.4, 126.3, 121.2, 118.9, 115.3,
113.1, 43.1, 36.6, 32.6, 17.7. MS (APSI+) m/z found 382.1;
C17H21ClN3O3S (M+ + H) requires 382.1.
3-[3-(3-Chloropropyl)ureido]-N-(2-ethylphenyl)-
benzenesulfonamide (35). Method D was used. The crude product
was purified by flash chromatography (silica gel, hexanes/ethyl acetate
(40:60) to hexanes/ethyl acetate (10:90)) and recrystallized with
methanol and filtered. Yield, 14%; yellowish solid; mp, 125 °C. IR ν:
3316 (NH), 1641 (CO) cm−1. 1H NMR (DMSO-d6): δ 9.05 (s, 1H,
NH), 7.97 (s, 1H, Ar), 7.77−7.75 (m, 1H, Ar), 7.53−7.43 (m, 2H, Ar),
7.27−7.22 (m, 3H, Ar), 6.97−6.95 (m, 1H, Ar), 6.42 (t, 1H, J = 5.5,
NH), 3.69 (t, 2H, J = 6.4 Hz, CH2), 3.25−3.21 (m, 2H, CH2), 2.26 (q,
2H, J = 7.3 Hz, CH2), 1.96−1.87 (m, 2H, CH2), 0.98 (t, 3H, J = 7.3
Hz, CH3). 13C NMR (acetone-d6/CDCl3): δ 156.2, 147.4, 141.9,
140.0, 132.1, 131.1, 130.1, 129.9, 126.9, 124.9, 122.5, 118.8, 117.4,
43.2, 37.8, 33.7, 24.2, 14.3. MS (APSI−) m/z found 393.9;
C18H21ClN3O3S (M− − H) requires 394.1.
3-[3-(3-Chloropropyl)ureido]-N-(2-propylphenyl)-
benzenesulfonamide (36). Method D was used. The crude product
was purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (80:20)) and recrystallized with
MeOH and filtered. Yield, 6%; yellowish solid; mp, 112 °C. IR ν: 3319
(NH), 1642 (CO). 1H NMR (DMSO-d6): δ 9.55 (s, 1H, NH), 8.85
(s, 1H, NH), 7.93 (s, 1H, Ar), 7.58−756 (m, 1H, Ar), 7.41−7.36 (m,
1H, Ar), 7.20−7.05 (m, 4H, Ar), 6.93−6.91 (m, 1H, Ar), 6.33 (brs,
1H, NH), 3.70−3.66 (m, 2H, CH2), 3.25−3.19 (m, 2H, CH2), 2.48−
2.43 (m, 2H, CH2), 1.94−1.86 (m, 2H, CH2), 1.41−1.28 (m, 2H,
CH2), 0.81 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (DMSO-d6): δ 155.0,
141.2, 139.0, 138.8, 134.4, 129.6, 129.4, 126.6, 126.5, 126.2, 121.1,
119.0, 115.3, 43.1, 36.7, 32.6, 32.3, 22.9, 14.0. MS (ESI−) m/z found
408.1; C19H23ClN3O3S (M− − H) requires 408.1.
2-Ethylphenyl 3-(3-Ethylureido)benzenesulfonate (41).
Method A in dry DCM was used. The crude product was purified
by flash chromatography (silica gel, methylene chloride/ethyl acetate
(92:8) to methylene chloride/ethyl acetate (88:12)). Yield, 78%; white
solid; mp, 76−78 °C. IR ν: 3297 (NH), 1655 (CO) cm−1. 1H NMR
(CDCl3): δ 8.27 (s, 1H, NH), 7.99 (s, 1H, Ar), 7.66−7.63 (m, 1H,
Ar), 7.39−7.32 (m, 2H, Ar), 7.17−7.01 (m, 3H, Ar), 6.94−9.91 (m,
1H, Ar), 5.88 (brs, 1H, NH), 3.25−3.19 (m, 2H, CH2), 2.51 (q, 2H, J
= 7.5, CH2), 1.14−1.04 (m, 6H, 2 × CH3). 13C NMR (CDCl3): δ
156.0, 147.8, 140.8, 137.2, 136.5, 129.9, 129.8, 127.3, 126.9, 124.3,
121.9, 121.5, 117.9, 35.0, 22.8, 15.1, 14.0. MS (APSI+) m/z found
349.1; C17H21N2O4S (M+ + H) requires 349.1.
2-Propylphenyl 3-(3-Ethylureido)benzenesulfonate (42).
Method A in dry DCM was used. The crude product was purified
by flash chromatography (silica gel, methylene chloride/ethyl acetate
(92:8) to methylene chloride/ethyl acetate (88:12)). Yield, 99%;
sticky solid. IR ν: 3343 (NH), 1655 (CO) cm−1. 1H NMR
(CDCl3): δ 8.28 (s, 1H, NH), 8.03 (s, 1H, Ar), 7.62−7.60 (m, 1H,
Ar), 7.41−7.29 (m, 2H, Ar), 7.16−7.02 (m, 3H, Ar), 6.95−6.92 (m,
1H, Ar), 5.90 (brs, 1H, NH), 3.25−3.18 (m, 2H, CH2), 2.44 (t, 2H, J
= 7.7 Hz, CH2), 1.52−1.44 (m, 2H, CH2), 1.06 (t, 3H, J = 7.1 Hz,
CH3), 0.82 (t, 3H, J = 7.3 Hz, CH3). 13C NMR (CDCl3): δ 156.1,
148.0, 140.8, 136.6, 135.8, 130.7, 129.8, 127.1, 127.0, 124.4, 121.9,
121.5, 117.9, 35.0, 31.8, 23.0, 15.1, 13.8. MS (APSI+) m/z found
363.1; C18H23N2O4S (M+ + H) requires 363.1.
4-[3-(3-Chloropropyl)ureido]-N-2-tolylbenzenesulfonamide
(37). Method A in dry DCM was used. The crude product was
purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (80:20)) and was recrystallized with
MeOH and filtered. Yield, 20%; white solid; mp, 204−205 °C. IR:
6203
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4-(tert-Butyldimethylsilyloxy)phenyl 3-(3-Ethylureido)-
benzenesulfonate (43). Method D was used. The crude product
was purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (75:25)). Yield, 48%; yellowish oil.
7.73−7.63 (m, 4H, Ar), 6.84−6.75 (m, 4H, Ar), 6.02 (brs, 1H, NH),
3.27−3.21 (m, 2H, CH2), 1.12 (t, 3H, J = 7.1 Hz, CH3), 3.08 (brs, 1H,
OH). 13C NMR (acetone-d6): δ 156.9, 155.2, 147.3, 143.2, 130.5,
127.2, 124.1, 117.9, 116.5, 35.2, 15.5. MS (APSI+) m/z found 337.1;
C15H17N2O5S (M+ + H) requires 337.1.
3-(3-Ethylureido)-N-2-tolylbenzenesulfonamide (50). Method
D was used. The crude product was purified by flash chromatography
(silica gel, hexanes/ethyl acetate (50:50) to ethyl acetate) and was
recrystallized with MeOH and filtered. Yield, 15%; white solid; mp,
199−200 °C. IR ν: 3333 (NH), 1685 (CO) cm−1. 1H NMR
(CDCl3/DMSO-d6): δ 9.55 (s, 1H, NH), 8.81 (s, 1H, NH), 7.90 (s,
1H, Ar), 7.59−7.57 (m, 1H, Ar), 7.40−7.35 (m, 1H, Ar), 7.16−7.08
(m, 4H, Ar), 7.00−6.97 (m, 1H, Ar), 6.17 (brs, 1H, NH), 3.16−3.07
(m, 2H, CH2), 2.03 (s, 3H, CH3), 1.06 (t, 3H, J = 7.2 Hz, CH3). 13C
NMR (DMSO-d6): δ 154.9, 141.3, 141.2, 135.0, 134.2, 130.7, 129.4,
126.4, 126.4, 126.3, 121.1, 118.8, 115.2, 34.0, 17.7, 15.4. MS (APSI+)
m/z found 334.2; C16H20N3O3S (M+ + H) requires 334.1.
1
IR ν: 3370 (NH), 1659 (CO) cm−1. H NMR (CDCl3): δ 7.98 (s,
1H, NH), 7.83 (s, 1H, Ar), 7.79−7.76 (m, 1H, Ar), 7.30−7.26 (m, 2H,
Ar), 6.82−6.79 (m, 2H, Ar), 6.68−6.65 (m, 2H, Ar), 5.60 (brs, 1H,
NH), 3.29−3.22 (m, 2H, CH2), 1.13−1.08 (m, 2H, CH2), 0.93 (s, 9H,
3 × CH3), 0.13 (s, 6H, 2 × CH3). 13C NMR (DMSO-d6): δ 155.8,
154.6, 143.4, 140.7, 135.4, 129.7, 124.5, 123.2, 122.0, 120.7, 117.9,
35.1, 25.6, 18.1, 15.2, −4.5.
4-Hydroxyphenyl 3-(3-Ethylureido)benzenesulfonate (44).
Method E was used. The crude product was purified by flash
chromatography (silica gel, ethyl acetate to ethyl acetate/methanol
(90:10)). Yield, 50%; white sticky solid. IR ν: 1649 (CO) cm−1. 1H
NMR (DMSO-d6): δ 9.67 (s, 1H, OH), 8.95 (s, 1H, NH), 8.09 (s, 1H,
Ar), 7.66−7.26 (m, 3H, Ar), 6.82−6.68 (m, 4H, Ar), 6.27 (t, 1H, J =
5.1 Hz, NH), 3.14−3.09 (m, 2H, CH2), 1.06 (t, 3H, J = 7.0 Hz, CH3).
13C NMR (DMSO-d6): δ 156.3, 154.8, 141.8, 141.3, 134.9, 129.9,
123.0, 123.0, 120.2, 116.2, 116.0, 34.1, 15.3. MS (APSI+) m/z found
337.1; C15H17N2O5S (M+ + H) requires 337.1.
N-(2-Ethylphenyl)-3-(3-ethylureido)benzenesulfonamide
(51). Method A in dry DCM was used. The crude product was
purified by flash chromatography (silica gel, hexanes/ethyl acetate
(40:60) to hexanes/ethyl acetate (80:20)) and was recrystallized with
MeOH and filtered. Yield, 4%; yellowish solid; mp, 229−230 °C. IR ν:
3312 (NH), 1643 (CO) cm−1. 1H NMR (DMSO-d6): δ 8.97 (s, 1H,
NH), 7.96 (s, 1H, NH), 7.78−7.76 (m, 1H, Ar), 7.53−7.24 (m, 6H,
Ar), 6.97−6.95 (m, 1H, Ar), 6.23 (brs, 1H, NH), 3.18−3.11 (m, 2H,
CH2), 2.28−2.24 (m, 2H, CH2), 1.22−0.95 (m, 6H, 2 × CH3). 13C
NMR (DMSO-d6): δ 154.8, 145.7, 141.6, 139.0, 132.1, 131.6, 130.6,
129.7, 129.2, 126.5, 123.0, 120.2, 116.7, 34.1, 22.8, 15.4, 13.8. MS
(APSI−) m/z found 346.0; C17H20N3O3S (M− − H) requires 346.1.
3-(3-Ethylureido)-N-(2-propylphenyl)benzenesulfonamide
(52). Method D was used. The crude product was purified by flash
chromatography (silica gel, methylene chloride to methylene chloride/
ethyl acetate (75:25)) and was recrystallized with MeOH and filtered.
Yield, 11%; yellowish solid; mp, 147 °C. IR ν: 3288 (NH), 1649 (C
2-Tolyl 4-(3-Ethylureido)benzenesulfonate (45). Method A in
dry DCM was used. The crude product was purified by flash
chromatography (silica gel, methylene chloride to methylene chloride/
ethyl acetate 90:10)). Yield, 70%; white solid; mp, 137−138 °C. IR ν:
1
3363 (NH), 1663 (CO) cm−1. H NMR (CDCl3): δ 8.08 (s, 1H,
NH), 7.66−7.63 (m, 2H, Ar), 7.55−7.52 (m, 2H, Ar), 7.11−7.06 (m,
3H, Ar), 6.96−6.93 (m, 1H, Ar), 5.69 (brs, 1H, NH), 3.27−3.19 (m,
2H, CH2), 2.05 (s, 3H, CH3), 1.09 (t, 3H, J = 7.1 Hz, CH3). 13C NMR
(CDCl3): δ 155.5, 148.2, 145.8, 131.7, 131.5, 129.7, 127.4, 127.2,
126.9, 122.2, 117.8, 34.9, 16.3, 15.2. MS (APSI+) m/z found 335.1;
C16H19N2O4S (M+ + H) requires 335.1.
2-Ethylphenyl 4-(3-Ethylureido)benzenesulfonate (46).
Method A in dry DCM was used. The crude product was purified
by flash chromatography (silica gel, hexanes/ethyl acetate (65:35) to
hexanes/ethyl acetate (55:45)). Yield, 83%; orange solid; mp, 123 °C.
1
O) cm−1. H NMR (DMSO-d6): δ 9.54 (s, 1H, NH), 8.81 (s, 1H,
NH), 7.93 (s, 1H, Ar), 7.60−7.57 (m, 1H, Ar), 7.41−6.91 (m, 6H, Ar),
3.17−3.10 (m, 2H, CH2), 2.47 (m, 2H, J = 7.8 Hz, CH2), 1.41−1.32
(m, 2H, CH2), 1.06 (t, 3H, J = 7.1 Hz, CH3), 0.82 (t, 3H, J = 7.2 Hz,
CH3). 13C NMR (DMSO-d6): δ 154.8, 141.3, 141.2, 138.8, 134.4,
129.6, 129.3, 126.6, 126.5, 126.1, 121.0, 118.8, 115.3, 34.0, 32.3, 22.9,
15.4, 14.0. MS (APSI+) m/z found 362.2; C18H24N3O3S (M+ + H)
requires 362.2.
1
IR ν: 3389 (NH), 1671 (CO) cm−1. H NMR (CDCl3): δ 8.19 (s,
1H, NH), 7.72−769 (m, 2H, Ar), 7.56−7.52 (m, 2H, Ar), 7.18−7.07
(m, 3H, Ar), 6.99−6.96 (m, 1H, Ar), 5.81 (brs, 1H, NH), 3.28−3.22
(m, 2H, CH2), 2.49 (q, 2H, J = 7.5 Hz, CH2), 1.12−1.05 (m, 6H, 2 ×
CH3). 13C NMR (CDCl3): δ 155.5, 147.7, 145.6, 137.2, 130.0, 129.7,
127.4, 126.9, 121.9, 117.9, 102.6, 35.0, 22.8, 15.2, 14.0. MS (APSI+)
m/z found 349.1; C17H21N2O4S (M+ + H) requires 349.1.
4-(3-Ethylureido)-N-2-tolylbenzenesulfonamide (53). Method
A in dry DCM was used. The crude product was purified by flash
chromatography (silica gel, methylene chloride to methylene chloride/
ethyl acetate (75:25)) and was recrystallized with MeOH and filtered.
Yield, 15%; white solid; mp, 246−247 °C. IR ν: 3051 (NH), 1679
2-Propylphenyl 4-(3-Ethylureido)benzenesulfonate (47).
Method A in dry DCM was used. The crude product was purified
by flash chromatography (silica gel, hexanes/ethyl acetate (65:35) to
hexanes/ethyl acetate (55:45)). Yield, 73%; yellowish solid; mp, 108
°C. IR ν: 3383 (NH), 1666 (CO) cm−1. 1H NMR (CDCl3): δ 8.08
(s, 1H, NH), 7.72−7.69 (m, 2H, Ar), 7.55−7.52 (m, 2H, Ar), 7.16−
7.06 (m, 3H, Ar), 6.99−6.97 (m, 1H, Ar), 5.67 (brs, 1H, NH), 3.26−
3.22 (m, 2H, CH2), 2.42 (t, 2H, J = 7.7 Hz, CH2), 1.55−1.43 (m, 2H,
1
(CO) cm−1. H NMR (CDCl3/DMSO-d6): δ 7.46−7.22 (m, 4H,
Ar), 7.12−6.91 (m, 4H, Ar), 3.15 (q, 2H, J = 7.2 Hz, CH2), 1.92 (s,
3H, CH3), 1.04 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (DMSO-d6): δ
154.7, 144.6, 135.1, 134.0, 131.9, 130.7, 127.8, 126.4, 126.3, 126.2,
116.8, 34.0, 17.7, 15.3. MS (APSI−) m/z found 331.9; C16H18N3O3S
(M− − H) requires 332.1.
CH2), 1.10 (t, 3H, J = 7.2 Hz, CH3), 0.83 (t, 3H, J = 7.3 Hz, CH3). 13
C
NMR (CDCl3): δ 155.3, 147.9, 145.5, 135.7, 130.7, 129.7, 127.8,
127.2, 127.0, 122.0, 117.8, 35.1, 31.8, 23.0, 15.2, 13.9. MS (APSI+) m/
z found 363.1; C18H23N2O4S (M+ + H) requires 363.1.
N-(2-Ethylphenyl)-4-(3-ethylureido)benzenesulfonamide
(54). Method A in dry DCM was used. The crude product was
purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (75:25)) and was recrystallized with
MeOH and filtered. Yield, 53%; white solid; mp, 223−224 °C. IR ν:
3107 (NH), 1683 (CO) cm−1. 1H NMR (DMSO-d6): δ 9.35 (s, 1H,
NH), 8.91 (s, 1H, NH), 7.55−7.49 (m, 4H, Ar), 7.21−7.05 (m, 4H,
Ar), 6.89 (t, 1H, J = 5.4 Hz, NH), 3.17−3.08 (m, 2H, CH2), 2.55−2.48
(m, 2H, CH2), 1.22−1.05 (m, 6H, 3 × CH3). 13C NMR (DMSO-d6):
δ 154.5, 144.5, 140.2, 134.4, 131.9, 128.9, 127.9, 126.5, 126.4, 126.1,
116.8, 34.0, 23.1, 15.3, 14.4. MS (APSI+) m/z found 346.0;
C17H20N3O3S (M+ + H) requires 346.1.
4-(tert-Butyldimethylsilyloxy)phenyl 4-(3-Ethylureido)-
benzenesulfonate (48). Method D was used. The crude product
was purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (75:25)). Yield, 53%; yellowish oil.
1
IR ν: 3357 (NH), 1665 (CO) cm−1. H NMR (CDCl3): δ 7.93 (s,
1H, NH), 7.66−7.60 (m, 2H, Ar), 7.49−7.42 (m, 2H, Ar), 7.86−7.78
(m, 2H, Ar), 6.72−6.66 (m, 2H, Ar), 5.50 (brs, 1H, NH), 3.28−3.20
(m, 2H, CH2), 1.15−1.05 (m, 3H, CH3), 0.92 (s, 9H, 3 × CH3), 0.14
(s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 155.1, 154.6, 145.5, 143.4,
131.0, 129.9, 123.3, 120.7, 117.7, 35.7, 25.6, 18.1, 15.2, −4.5.
4-Hydroxyphenyl 4-(3-Ethylureido)benzenesulfonate (49).
Method E was used. The crude product was purified by flash
chromatography (silica gel, ethyl acetate to ethyl acetate/methanol
(90:10)). Yield, 66%; white oil. IR ν: 3450−3075 (OH), 1666 (C
4-(3-Ethylureido)-N-(2-propylphenyl)benzenesulfonamide
(55). Method A in dry DCM was used. The crude product was
purified by flash chromatography (silica gel, methylene chloride to
methylene chloride/ethyl acetate (75:25)) and was recrystallized with
MeOH and filtered. Yield, 21%; yellowish solid; mp, 203 °C. IR ν:
1
O), 1357 (OH) cm−1. H NMR (acetone-d6): δ 8.48 (s, 1H, NH),
6204
dx.doi.org/10.1021/jm3006492 | J. Med. Chem. 2012, 55, 6194−6208

Journal of Medicinal Chemistry
Article
1
3098 (NH), 1678 (CO) cm−1. H NMR (CDCl3/DMSO-d6): δ
7.21−7.14 (m, 3H, Ar), 6.98 (d, 1H, J = 7.3 Hz, Ar), 2.12 (s, 3H,
CH3). 13C NMR (CDCl3): δ 148.2, 148.0, 138.1, 133.8, 132.0, 131.3,
130.9, 128.7, 127.7, 127.3, 123.5, 122.0, 16.3.
9.02 (s, 1H, NH), 8.77 (s, 1H, NH), 7.51−7.44 (m, 4H, Ar), 7.10−
6.88 (m, 4H, Ar), 6.14 (brs, 1H, NH), 3.19−3.11 (m, 2H, CH2), 2.46
(t, 2H, J = 7.9 Hz, CH2), 1.45−1.32 (m, 2H, CH2), 1.08 (t, 3H, J = 7.1
Hz, CH3), 0.82 (t, 3H, J = 7.2 Hz, CH3). 13C NMR (DMSO-d6): δ
154.8, 144.4, 138.5, 134.5, 131.9, 129.3, 127.7, 126.0, 125.9, 125.7,
116.5, 34.0, 33.3, 22.9, 15.2, 13.8. MS (APSI−) m/z found 360.0;
C18H22N3O3S (M− − H) requires 360.1.
General Procedure for the Synthesis of Compounds 56−73.
For method F, the 3-nitrobenzene-1-sulfonyl chloride or 4-nitro-
benzene-1-sulfonyl chloride (7.5 mmol) was dissolved in dry
methylene chloride (20 mL) under a dry Ar atmosphere. The selected
phenol or aniline (7.5 mmol) and trietylamine were then added
dropwise to the solution. The reaction mixture was stirred for 24 h at
room temperature. The solvent was evaporated and the residue
dissolved in ethyl acetate. The solution was washed with 1 N HCl, 1 N
NaOH, brine, dried over anhydrous Na2SO4, filtered, and evaporated
to dryness.
2-Ethylphenyl 3-Nitrobenzenesulfonate (63). Method F was
used. Yield, 78%; colorless oil. IR ν: 1533 (NO2), 1381 (NO2) cm−1.
1H NMR (CDCl3): δ 8.75 (s, 1H, Ar), 8.54 (d, 1H, J = 7.7 Hz, Ar),
8.23 (d, 1H, J = 7.7 Hz, Ar), 8.21−7.80 (m, 1H, Ar), 7.24−7.13 (m,
3H, Ar), 7.02−7.00 (m, 1H, Ar), 2.52 (q, 2H, J = 7.6 Hz, CH2), 1.13
(t, 3H, J = 7.6 Hz, CH3). 13C NMR (CDCl3): δ 148.3, 147.5, 138.3,
137.0, 133.7, 130.7,130.2, 128.6, 127.8, 127.2, 123.6, 121.8, 22.8, 14.04.
2-Propylphenyl 3-Nitrobenzenesulfonate (64). Method F was
used. Yield, 80%; yellowish oil. IR ν: 1533 (NO2), 1381 (NO2) cm−1.
1H NMR (CDCl3): δ 8.75 (s, 1H, Ar), 8.55 (d, 1H, J = 7.9 Hz, Ar),
8.23 (d, 1H, J = 7.9 Hz, Ar), 7.82−7.77 (m, 1H, Ar), 7.23−7.15 (m,
3H, Ar), 7.05−7.03 (m, 1H, Ar), 2.44 (t, 2H, J = 7.8 Hz, CH2), 1.58−
1.47 (m, 2H, CH2), 0.87 (t, 3H, J = 7.4 Hz, CH3). 13C NMR (CDCl3):
δ 148.3, 147.7, 138.4, 135.5, 133.7, 130.9, 130.7, 128.6, 127.6, 127.3,
123.6, 121.8, 31.9, 23.0, 13.9.
For method G, the 3-nitrobenzene-1-sulfonyl chloride or 4-
nitrobenzene-1-sulfonyl chloride (8 mmol) was dissolved in dry
acetonitrile (10 mL) under an Ar atmosphere. The relevant aniline (8
mmol) and 4-dimethylaminopyridine were successively added
dropwise, and the mixture was stirred for 48 h at room temperature.
The solvent was evaporated and the residue dissolved in ethyl acetate.
The solution was washed with hydrochloric acid (1 N), brine, dried
over Na2SO4, filtered, and evaporated to dryness.
2-Tolyl 4-Nitrobenzenesulfonate (56). Method F was used.
Yield, 98%; yellowish solid; mp, 84−85 °C. IR ν: 1533 (NO2), 1191
(SO) cm−1. 1H NMR (CDCl3): δ 8.35 (d, 2H, J = 8.8 Hz, Ar), 8.06
(d, 2H, J = 8.8 Hz, Ar), 7.19−7.10 (m, 3H, Ar), 6.96−6.93 (m, 1H,
Ar), 2.09 (s, 3H, CH3). 13C NMR (CDCl3): δ 151.0, 148.0, 141.7,
132.0, 131.4, 129.8, 127.6, 127.3, 124.5, 121.9, 16.3.
3-Tolyl 4-Nitrobenzenesulfonate (57). Method F was used.
Yield, 97%; yellowish solid; mp, 94−95 °C. IR ν: 1533 (NO2), 1351
(NO2) cm−1. 1H NMR (CDCl3): δ 8.35 (d, 2H, J = 8.8 Hz, Ar), 8.02
(d, 2H, J = 8.8 Hz, Ar), 7.19−7.06 (m, 2H, Ar), 6.86 (s, 1H, Ar),
6.73−6.70 (m, 1H, Ar), 2.29 (s, 3H, CH3). 13C NMR (CDCl3): δ
151.0, 149.2, 141.1, 140.6, 129.9, 129.6, 128.5, 124.3, 122.7, 118.8,
21.2.
4-(tert-Butyldimethylsilyloxy)phenyl 3-Nitrobenzenesulfo-
nate (65). Method F was used. Yield, 95%; yellowish oil. IR ν:
1
1542 (NO2), 1377 (NO2) cm−1. H NMR (CDCl3): δ 8.65 (s, 1H,
Ar), 8.52−8.50 (m, 1H, Ar), 8.14−8.12 (m, 1H, Ar), 7.78−7.73 (m,
1H, Ar), 6.86−6.83 (m, 2H, Ar), 6.75−6.72 (m, 2H, Ar), 0.95 (s, 9H,
3 × CH3), 0.17 (s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 155.0, 148.2,
143.0, 137.4, 133.9, 130.6, 128.6, 123.8, 123.1, 121.0, 25.6, 18.2, −4.5.
2-Ethylphenyl 4-Nitrobenzenesulfonate (66). Method F was
used. Yield, 90%; white solid; mp, 82 °C. IR ν: 1530 (NO2), 1376
(NO2) cm −1. 1H NMR (CDCl3): δ 8.39 (d, 2H, J = 8.7 Hz, Ar), 8.10
(d, 2H, J = 8.7 Hz, Ar), 7.25−7.12 (m, 3H, Ar), 7.00−6.97 (m, 1H,
Ar), 2.50 (q, 2H, J = 7.5 Hz, CH2), 1.12 (t, 3H, J = 7.5 Hz, CH3). 13
C
NMR (CDCl3): δ 150.9, 147.6, 141.8, 137.0, 130.2, 129.7, 127.8,
127.2, 124.4, 121.7, 22.9, 14.04.
2-Propylphenyl 4-Nitrobenzenesulfonate (67). Method F was
used. Yield, 85%; yellowish solid; mp, 58 °C. IR ν: 1525 (NO2), 1375
(NO2) cm−1. 1H NMR (CDCl3): δ 8.40 (d, 2H, J = 8.8 Hz, Ar), 8.10
(d, 2H, J = 8.8 Hz, Ar), 7.23−7.13 (m, 3H, Ar), 7.02−6.99 (m, 1H,
Ar), 2.42 (t, 2H, J = 7.7 Hz, CH2), 1.59−1.46 (m, 2H, CH2), 0.87 (t,
3H, J = 7.4 Hz, CH3). 13C NMR (CDCl3): δ 150.9, 147.8, 141.9,
135.6, 131.0, 129.7, 127.6, 127.2, 124.4, 121.8, 31.9, 23.0, 13.9.
3-Nitro-N-2-tolylbenzenesulfonamide (68). Method G was
used. Yield, 94%; white solid; mp, 156 °C. IR ν: 1529 (NO2), 1354
4-Tolyl 4-Nitrobenzenesulfonate (58). Method F was used.
Yield, 96%; white solid; mp, 94−95 °C. IR ν: 1520 (NO2), 1199 (S
1
O) cm−1. H NMR (CDCl3): δ 8.35 (d, 2H, J = 8.7 Hz, Ar), 8.01 (d,
1
(NO2) cm−1. H NMR (DMSO-d6): δ 10.03 (s, 1H, NH), 8.51−8.44
2H, J = 8.7 Hz, Ar), 7.09 (d, 2H, J = 8.2 Hz, Ar), 6.85 (d, 2H, J = 8.2
Hz, Ar), 2.30 (s, 3H, CH3). 13C NMR (CDCl3) δ: 151.0, 147.1, 141.0,
137.8, 130.5, 129.9, 124.3, 121.8, 20.8.
(m, 2H, Ar), 8.09−8.06 (m, 1H, Ar), 7.91−7.86 (m, 1H, Ar), 7.20−
6.91 (m, 4H, Ar), 2.06 (s, 3H, CH3). 13C NMR (DMSO-d6): δ 147.8,
142.1, 134.7, 134.2, 132.6, 131.4, 131.0, 127.4, 127.0, 126.8, 126.6,
121.4, 17.7.
4-Methoxyphenyl 4-Nitrobenzenesulfonate (59). Method F
was used. Yield, 89%; white solid; mp, 150−151 °C. IR ν: 1540
N-(2-Ethylphenyl)-3-nitrobenzenesulfonamide (69). Method
F was used. The crude product was purified by recrystallized with
MeOH and filtered. Yield, 56%; white solid; mp, 159−160 °C. IR ν:
1
(NO2), 1378 (NO2) cm−1. H NMR (DMSO-d6): δ 8.47 (d, 2H, J =
8.7 Hz, Ar), 8.14 (d, 2H, J = 8.7 Hz, Ar), 7.02−6.91 (m, 4H, Ar), 3.74
(s, 3H, CH3). 13C NMR (DMSO-d6): δ 158.3, 151.1, 142.1, 139.6,
130.1, 125.0, 123.2, 115.1, 55.6.
1
1532 (NO2), 1352 (NO2) cm−1. H NMR (CDCl3): δ 8.74 (s, 1H,
Ar), 8.59−8.56 (m, 1H, Ar), 8.37−8.34 (m, 1H, Ar), 7.87−7.82 (m,
1H, Ar), 7.53−7.42 (m, 2H, Ar), 7.24−7.18 (m, 1H, Ar), 6.87−6.85
(m, 1H, Ar), 2.28 (q, 2H, J = 7.5 Hz, CH2), 1.09 (t, 3H, J = 7.5 Hz,
CH3). 13C NMR (CDCl3): δ 148.2, 145.6, 140.9, 134.5, 131.6, 131.5,
131.4, 130.6, 130.1, 128.8, 126.9, 124.3, 23.6, 14.0.
4-(Dimethylamino)phenyl 4-Nitrobenzenesulfonate (60).
Method F was used. The crude product was purified by flash
chromatography (silica gel, hexanes/ethyl acetate (90:10) to hexanes/
ethyl acetate (70:30)). Yield, 41%; orange solid; mp, 129−130 °C. IR
1
ν: 1513 (NO2), 1187 (SO) cm−1. H NMR (DMSO-d6): δ 8.47−
3-Nitro-N-(2-propylphenyl)benzenesulfonamide (70). Meth-
8.44 (m, 2H, Ar), 8.14−8.11 (m, 2H, Ar), 6.86−6.83 (m, 2H, Ar),
6.64−6.61 (m, 2H, Ar), 2.87 (s, 6H, 2 × CH3). 13C NMR (DMSO-
d6): δ 149.4, 139.0, 130.1, 124.9, 122.4, 112.5, 40.1.
od G was used. Yield, 55%; yellowish solid; mp, 64−65 °C. IR ν: 1529
1
(NO2), 1350 (NO2) cm−1. H NMR (CDCl3): δ 8.60−8.55 (m, 1H,
Ar), 8.40−8.34 (m, 1H, Ar), 8.05−8.02 (m, 1H, Ar), 7.69−7.63 (m,
1H, Ar), 7.16−7.07 (m, 4H, Ar), 2.34 (t, 2H, J = 7.7 Hz, CH2), 1.43−
1.33 (m, 2H, CH2), 0.81 (t, 3H, J = 7.3 Hz, CH3). 13C NMR (CDCl3):
δ 148.2, 141.7, 136.4, 134.6, 132.8, 130.4, 130.2, 127.4, 127.2, 127.1,
124.9, 122.5, 32.7, 23.2, 13.8.
4-(tert-Butyldimethylsilyloxy)phenyl 4-Nitrobenzenesulfo-
nate (61). Method F was used. Yield, 96%; yellowish solid; mp,
94−95 °C. IR ν: 1495 (NO2), 1377 (NO2) cm−1. 1H NMR (CDCl3):
δ 8.33 (d, 2H, J = 8.7 Hz, Ar), 7.98 (d, 2H, J = 8.7 Hz, Ar), 6.84−6.70
(m, 4H, Ar), 0.92 (s, 9H, 3 × CH3), 0.15 (s, 6H, 2 × CH3). 13C NMR
(CDCl3): δ 154.9, 151.0, 143.1, 140.9, 130.0, 124.3, 123.1, 121.0, 25.6,
18.1, −4.5.
4-Nitro-N-2-tolylbenzenesulfonamide (71). Method G was
used. Yield, 86%; orange solid; mp, 158 °C. IR ν: 1528 (NO2), 1343
1
(NO2) cm−1. H NMR (CDCl3/MeOD): δ 7.93−7.90 (m, 2H, Ar),
2-Tolyl 3-Nitrobenzenesulfonate (62). Method F was used.
Yield, 91%; white solid; mp, 63−64 °C. IR ν: 1533 (NO2), 1351
7.52−7.49 (m, 2H, Ar), 6.74−6.62 (m, 4H, Ar), 1.65 (s, 3H, CH3).
13C NMR (DMSO-d6): δ 149.7, 146.1, 134.6, 134.1, 131.0, 128.2,
127.0, 126.8, 126.6, 124.6, 17.7.
1
(NO2) cm−1. H NMR (CDCl3): δ 8.70 (s, 1H, Ar), 8.53 (d, 1H, J =
8.0 Hz, Ar), 8.20 (d, 1H, J = 7.7 Hz, Ar), 7.83−7.77 (m, 1H, Ar),
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N-(2-Ethylphenyl)-4-nitrobenzenesulfonamide (72). Method
G was used. Yield, 84%; orange solid; mp, 149 °C. IR ν: 1531 (NO2),
2-Tolyl 3-Aminobenzenesulfonate (80). Yield, 56%; yellow
1
solid; mp, 86 °C. IR ν: 3463 (NH2), 3364 (NH2) cm−1. H NMR
1
1344 (NO2) cm−1. H NMR (CDCl3): δ 8.19−8.16 (m, 2H, Ar),
(CDCl3): δ 7.27−6.89 (m, 8H, Ar), 4.52 (brs, 2H, NH2), 2.11 (s, 3H,
CH3). 13C NMR (CDCl3): δ 148.4, 145.9, 137.0, 131.7, 131.6, 130.2,
127.0, 126.9, 122.3, 121.0, 118.8, 114.5, 16.3.
7.83−7.77 (m, 2H, Ar), 7.39−6.77 (m, 4H, Ar), 2.34 (q, 2H, J = 7.5
Hz, CH2), 0.92 (t, 3H, J = 7.5 Hz, CH3). 13C NMR (CDCl3): δ 149.7,
146.2, 140.9, 133.4, 129.2, 128.3, 127.3, 126.9, 126.4, 124.6, 23.22,
14.5.
2-Ethylphenyl 3-Aminobenzenesulfonate (81). Yield, 46%;
1
orange solid; mp, 52 °C. IR ν: 3478 (NH2), 3385 (NH2) cm−1. H
4-Nitro-N-(2-propylphenyl)benzenesulfonamide (73). Meth-
NMR (CDCl3): δ 7.31−6.88 (m, 8H, Ar), 4.73 (brs, 2H, NH2), 2.53
(q, 2H, J = 7.5 Hz, CH2), 1.11 (t, 3H, J = 7.5 Hz, CH3). 13C NMR
(CDCl3): δ 147.9, 145.2, 137.3, 137.1, 130.2, 129.9, 127.2, 126.9,
122.1, 121.4, 119.2, 114.9, 22.8, 14.0.
2-Propylphenyl 3-Aminobenzenesulfonate (82). Yield, 8.7%;
orange oil. IR ν: 3489 (NH2), 3397 (NH2) cm−1. 1H NMR (CDCl3):
δ 7.26−7.00 (m, 8H, Ar), 4.87 (brs, 2H, NH2), 2.45 (t, 2H, J = 7.8 Hz,
CH2), 1.56−1.46 (m, 2H, CH2), 0.87 (t, 3H, J = 7.3 Hz, CH3). 13C
NMR (CDCl3): δ 148.1, 144.8, 137.3, 135.8, 130.6, 130.3, 127.0,
126.9, 122.1, 121.6, 119.5, 115.2, 31.8, 23.0, 13.9.
od G was used. Yield, 91%; white solid; mp, 120−121 °C. IR ν: 1530
1
(NO2), 1343 (NO2) cm−1. H NMR (DMSO-d6): δ 9.25−9.22 (m,
2H, Ar), 8.78−8.75 (m, 2H, Ar), 8,26−7.64 (m, 4H, Ar), 3.27 (t, 2H, J
= 7.9 Hz, CH2), 2.21−2.13 (m, 2H, CH2), 1.61 (t, 3H, J = 7.3 Hz,
CH3). 13C NMR (DMSO-d6): δ 151.0, 146.2, 133.6, 130.2, 129.9,
128.3, 127.2, 126.5, 125.0, 124.6, 32.3, 23.0, 13.9.
General Procedure for the Synthesis of Compounds 74−91.
The appropriate nitro compound (2.0 mmol) was dissolved in a
mixture of EtOH and H2O (40 mL, 10:1). Powdered iron (8.0 mmol)
and five drops of hydrochloric acid (12 M) were added. The mixture
was refluxed overnight. After the mixture was cooled at room
temperature, the solvent was evaporated. HCl (1 N, 100 mL) was
added and the mixture was extracted with ethyl acetate (100 mL). The
organic solutions were pooled, washed with brine, dried over
anhydrous Na2SO4, and concentrated under reduced pressure.
2-Tolyl 4-Aminobenzenesulfonate (74). Yield, 88%; yellowish
solid; mp, 66−67 °C. IR ν: 3387 (NH2), 1592 (NH2) cm−1. 1H NMR
(CDCl3): δ 7.55−7.53 (m, 2H, Ar), 7.13−7.10 (m, 3H, Ar), 7.04−7.00
(m, 1H, Ar), 6.62−6.60 (m, 2H, Ar), 4.41 (brs, 2H, NH2), 2.24 (s, 3H,
CH3). 13C NMR (CDCl3/DMSO-d6): δ 153.1, 148.3, 131.5, 131.4,
130.3, 126.7, 122.3, 121.4, 113.5, 16.4.
3-Tolyl 4-Aminobenzenesulfonate (75). Yield, 92%; white
solid; mp, 67−68 °C. IR ν: 3389 (NH2), 1592 (NH2) cm−1. 1H
NMR (CDCl3/MeOD): δ 7.45−7.42 (m, 2H, Ar), 7.05−7.00 (m, 1H,
Ar), 6.94−6.91 (m, 1H, Ar), 6.75 (s, 1H, Ar), 6.66−6.58 (m, 3H, Ar),
4.37 (brs, 2H, NH2), 2.15 (s, 3H, CH3). 13C NMR (CDCl3/MeOD):
δ 152.2, 149.6, 139.9, 130.5, 129.2, 127.8, 123.0, 121.8, 119.2, 114.1,
21.0.
4-(tert-Butyldimethylsilyloxy)phenyl 3-Aminobenzenesulfo-
nate (83). Yield, 73%; yellow solid; mp, 101 °C. IR ν: 3495 (NH2),
1
3391 (NH2) cm−1. H NMR (CDCl3): δ 8.29 (s, 1H, Ar), 7.98−7.96
(m, 1H, Ar), 7.62−7.60 (m, 1H, Ar), 7.54−7.46 (m, 1H, Ar), 6.87−
6.80 (m, 2H, Ar), 6.74−6.68 (m, 2H, Ar), 4.42 (brs, 2H, NH2), 0.95
(s, 9H, 3 × CH3), 0.16 (s, 6H, 2 × CH3). 13C NMR (CDCl3): δ 154.5,
146.1, 143.6, 136.1, 130.0, 123.3, 120.8, 120.7, 118.9, 114.6, 25.6, 18.2,
−4.5.
2-Ethylphenyl 4-Aminobenzenesulfonate (84). Yield, 95%;
1
orange solid; mp, 71 °C. IR ν: 3467 (NH2), 3375 (NH2) cm−1. H
NMR (CDCl3): δ 7.48−7.45 (m, 2H, Ar), 7.30−7.20 (m, 3H, Ar),
7.01−6.98 (m, 1H, Ar), 6.68−6.65 (m, 2H, Ar), 6.41 (brs, 2H, NH2),
2.46 (q, 2H, J = 7.5 Hz, CH2), 1.05 (t, 3H, J = 7.5 Hz, CH3). 13C
NMR (CDCl3): δ 154.7, 147.6, 136.8, 130.3, 129.8, 127.0, 121.9,
118.7, 112.8, 22.2, 14.1.
2-Propylphenyl 4-Aminobenzenesulfonate (85). Yield, 93%;
white solid; mp, 93−94 °C. IR ν: 3473 (NH2), 3378 (NH2) cm−1. 1H
NMR (CDCl3): δ 7.59 (d, 2H, J = 8.4 Hz, Ar), 7.18−7.03 (m, 4H, Ar),
6.66 (d, 2H, J = 8.4 Hz, Ar), 4.51 (brs, 2H, NH2), 2.44 (t, 2H, J = 7.8
Hz, CH2), 1.55−1.48 (m, 2H, CH2), 0.87 (t, 3H, J = 7.3 Hz, CH3).
13C NMR (CDCl3): δ 151.7, 148.2, 135.9, 130.6, 130.5, 126.8, 123.7,
122.3, 122.3, 114.1, 31.8, 23.0, 14.0.
4-Tolyl 4-Aminobenzenesulfonate (76). Yield, 92%; orange
1
solid; mp, 130−132 °C. IR ν: 3394 (NH2), 1596 (NH2) cm−1. H
NMR (CDCl3): δ 7.52 (d, 2H, J = 8.5 Hz, Ar), 7.05 (d, 2H, J = 8.5 Hz,
Ar), 6.85 (d, 2H, J = 8.5 Hz, Ar), 6.61 (d, 2H, J = 8.5 Hz, Ar), 4.36
(brs, 2H, NH2), 2.28 (s, 3H, CH3). 13C NMR (CDCl3): δ 152.1,
147.6, 136.8, 130.7, 130.0, 122.5, 122.2, 113.8, 20.9.
3-Amino-N-2-tolylbenzenesulfonamide (86). Yield, 41%;
brown solid; mp, 102−103 °C. IR ν: 3404 (NH2), 3341 (NH2)
1
cm−1. H NMR (DMSO-d6): δ 9.40 (s, 1H, NH), 7.26−6.76 (m, 8H,
Ar), 5.64 (brs, 2H, NH2), 2.04 (s, 3H, CH3). 13C NMR (DMSO-d6): δ
148.8, 141.3, 135.2, 134.1, 130.6, 129.5, 126.3, 126.2, 126.2, 117.6,
113.7, 111.4, 17.7.
4-Methoxyphenyl 4-Aminobenzenesulfonate (77). To a
solution of the nitro compound 59 (2.0 mmol) in ethanol (40 mL)
was added stannous chloride dihydrate (12.0 mmol), and the mixture
was refluxed for 6 h. After the mixture was cooled at room
temperature, the solvent was evaporated. The residue was then
taken up in 300 mL of 1 N NaOH and extracted with ether (200 mL).
The combined organic extracts were washed with brine, dried over
Na2SO4, and concentrated under reduced pressure. Yield, 97%; orange
3-Amino-N-(2-ethylphenyl)benzenesulfonamide (87). Yield,
52%; yellow solid; mp, 145−147 °C. IR ν: 3453 (NH2), 3371 (NH2)
1
cm−1. H NMR (CDCl3/DMSO-d6): δ 8.06 (s, 1H, NH), 7.40−6.83
(m, 8H, Ar), 5.27 (brs, 2H, NH2) 2.31 (q, 2H, J = 7.4 Hz, CH2), 1.01
(t, 3H, J = 7.4 Hz, CH3). 13C NMR (CDCl3/DMSO-d6): δ 147.8,
146.0, 139.2, 132.4, 131.5, 130.0, 129.3, 128.7, 125.8, 119.8, 116.0,
113.6, 22.7, 13.7.
1
solid; mp, 161−162 °C. IR ν: 1594 (NH2) cm−1. H NMR (DMSO-
d6): δ 7.38 (d, 2H, J = 8.8 Hz, Ar), 6.89 (s, 4H, Ar), 6.62 (d, 2H, J =
8.8 Hz, Ar), 6.37 (brs, 2H, NH2), 3.72 (s, 3H, CH3). 13C NMR
(DMSO-d6): δ 157.7, 154.5, 142.8, 130.4, 123.3, 117.9, 114.6, 112.8,
55.5.
3-Amino-N-(2-propylphenyl)benzenesulfonamide (88).
Yield, 91%; yellow solid; mp, 144−145 °C. IR ν: 3400 (NH2), 3254
1
(NH2) cm−1. H NMR (CDCl3): δ 8.37 (s, 1H, NH), 7.27−6.72 (m,
8H, Ar), 4.81 (brs, 2H, NH2), 2.24−2.19 (m, 2H, CH2), 1.22−1.10
(m, 2H, CH2), 0.61−0.56 (m, 3H, CH3). 13C NMR (CDCl3): δ 147.2,
145.3, 140.0, 132.8, 132.0, 130.3, 129.7, 129.5, 126.1, 120.0, 118.3,
114.4, 32.6, 22.9, 14.4.
4-(Dimethylamino)phenyl 4-Aminobenzenesulfonate (78).
Yield, 98%; white solid; mp, 192−194 °C. IR ν: 1593 (NH2) cm−1.
1H NMR (acetone-d6): δ 7.46−7.43 (m, 2H, Ar), 6.82−6.73 (m, 4H,
Ar), 6.63−6.60 (m, 2H, Ar), 5.76 (brs, 2H, NH2), 2.90 (s, 6H, 2 ×
CH3). 13C NMR (acetone-d6): δ 154.8, 150.1, 141.3, 131.3, 123.6,
121.7, 113.8, 113.1, 40.5.
4-Amino-N-2-tolylbenzenesulfonamide (89). Yield, 89%;
orange solid; mp, 148−149 °C. IR ν: 3478 (NH2), 3380 (NH2)
cm−1. 1H NMR (DMSO-d6): δ 9.04 (s, 1H, NH), 7.28 (d, 2H, J = 8.6
Hz, Ar), 7.13−7.00 (m, 4H, Ar), 6.55 (d, 2H, J = 8.6 Hz, Ar), 5.95
(brs, 2H, NH2), 2.03 (s, 3H, CH3). 13C NMR (DMSO-d6): δ 152.7,
135.6, 133.7, 130.6, 128.6, 126.2, 126.1, 125.9, 125.6, 112.6, 17.7.
4-Amino-N-(2-ethylphenyl)benzenesulfonamide (90). Yield,
57%; orange solid; mp, 171 °C. IR ν: 3479 (NH2), 3380 (NH2) cm−1.
1H NMR (DMSO-d6): δ 9.04 (s, 1H, NH), 7.31 (d, 2H, J = 8.5 Hz,
Ar), 7.19−6.93 (m, 4H, Ar), 6.57 (d, 2H, J = 8.5 Hz, Ar), 5.95 (brs,
2H, NH2), 2,54 (q, 2H, J = 7.5 Hz, CH2), 1.01 (t, 3H, J = 7.5 Hz,
4-(tert-Butyldimethylsilyloxy)phenyl 4-Aminobenzenesulfo-
nate (79). The crude product was purified by flash chromatography
(silica gel, hexanes/ethyl acetate (90:10) to hexanes/ethyl acetate
(70:30)). Yield, 66%; orange solid; mp, 91−93 °C. IR ν: 1644 (NH2)
1
cm−1. H NMR (CDCl3): δ 7.50 (d, 2H, J = 8.7 Hz, Ar), 6.84−6.81
(m, 2H, Ar), 6.71−6.68 (m, 2H, Ar), 6.60 (d, 2H, J = 8.7 Hz, Ar), 4.33
(brs, 2H, NH2), 0.95 (s, 9H, 3 × CH3), 0.19 (s, 6H, 2 × CH3). 13C
NMR (CDCl3): δ 154.3, 152.0, 143.8, 130.8, 123.5, 122.4, 120.6,
113.7, 25.6, 18.2, −4.5.
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CH3). 13C NMR (DMSO-d6): δ 152.7, 139.9, 134.9, 128.8, 128.7,
126.2, 126.0, 125.7, 112.6, 112.3, 23.1, 14.4.
4-Amino-N-(2-propylphenyl)benzenesulfonamide (91).
ships, and biological characterization of N-aryl-N′-(2-chloroethyl)ureas
as new selective alkylating agents. J. Med. Chem. 2001, 44, 694−702.
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generation of N-aryl-N′-(1-alkyl-2-chloroethyl)ureas as microtubule
disrupters: synthesis, antiproliferative activity, and β-tubulin alkylation
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C.-Gaudreault, R. Selective alkylation of βII-tubulin and thioredoxin-1
by structurally related subsets of aryl chloroethylureas leading to either
anti-microtubules or redox modulating agents. Bioorg. Med. Chem.
2008, 16, 7277−7290.
(9) Fortin, J. S.; Cote, M. F.; Lacroix, J.; Patenaude, A.; Petitclerc, E.;
C.-Gaudreault, R. Cycloalkyl-substituted aryl chloroethylureas inhibit-
ing cell cycle progression in G0/G1 phase and thioredoxin-1 nuclear
translocation. Bioorg. Med. Chem. Lett. 2008, 18, 3526−3531.
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Gaudreault, R. Aromatic 2-chloroethyl urea derivatives and bio-
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Med. Chem. 2008, 16, 7477−7488.
Yield, 77%; orange solid; mp, 153−154 °C. IR ν: 3475 (NH2), 3379
1
(NH2) cm−1. H NMR (CDCl3/MeOD): δ 7.05−6.66 (m, 8H, Ar),
6.27 (brs, 2H, NH2), 2.08 (t, 2H, J = 7.8 Hz, CH2), 1.13−1.03 (m, 2H,
CH2), 0.53 (t, 3H, J = 7.3 Hz, CH3). 13C NMR (DMSO-d6): δ 154.2,
152.7, 138.3, 135.1, 130.6, 129.5, 128.6, 126.1, 125.9, 125.7, 112.6,
112.3, 32.3, 22.9, 14.0.
AUTHOR INFORMATION
Corresponding Author
■
*For S.F.: phone, 418-525-4444, extension 52364; fax, 418-525-
4372; e-mail, sebastien.fortin.81@gmail.com. For R.C.-G.:
phone, 418-525-4444, extension 52363; fax, 418-525-4372; e-
mail, rene.c-gaudreault@crsfa.ulaval.ca.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the Canadian Institutes of Health
Research (R.C.-G, Grants MOP-79334 and MOP-89707).
Work in the Masson laboratory was supported by the CIHR.
S.F. is a recipient of a studentship from the Canadian Institutes
of Health Research (Grant CGD-83623). We are indebted to
Dr. Richard Poulin for his dedicated help and insightful
comments of this manuscript.
ABBREVIATIONS USED
■
CEU, 2-chloroethylurea; PIB-SO, phenyl 4-(2-oxoimidazolidin-
1-yl)benzenesulfonate; PUB-SO, N-phenyl ureidobenzenesul-
fonate; PUB-SA, N-phenylureidobenzenesulfonamide; CPU, 3-
chloropropylurea; EU, ethylurea; cDDP, cis-
diamminedichloroplatinum(II) (cisplatin); CA-4, combretasta-
tin A-4; CAM, chick chorioallantoic membrane; DMEM,
Dulbecco’s minimal essential medium; PBS-T, PBS supple-
mented with 0.05% (v/v) Tween 20
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