Lead optimization of selective tubulin inhibitors as anti-trypanosomal agents

Article abstract of DOI:10.1016/j.bmc.2019.02.049

Previously synthesized tubulin inhibitors showed promising in vitro selectivity and activity against Human African Trypanosomiasis. Current aim is to improve the ligand efficiency and reduce overall hydrophobicity of the compounds, by lead optimization. V

Full text of DOI:10.1016/j.bmc.2019.02.049

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lead optimization of selective tubulin inhibitors as anti-trypanosomal agents
Anran Zhaoa,#, Yaxin Lia,#, Cody M. Orahoskea, Brittny Schnurb, Abboud Sabbagha,
⁎
⁎
Wenjing Zhanga, Bibo Lib, , Bin Sua,
a Department of Chemistry, Center for Gene Regulation in Health and Disease, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Ave.,
Cleveland, OH 44115, USA
b Department of Biology, Geo. & Env. Sciences, Center for Gene Regulation in Health and Disease, College of Sciences and Health Professions, Cleveland State University,
2121 Euclid Ave., Cleveland, OH 44115, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Previously synthesized tubulin inhibitors showed promising in vitro selectivity and activity against Human
African Trypanosomiasis. Current aim is to improve the ligand efficiency and reduce overall hydrophobicity of
the compounds, by lead optimization. Via combinatorial chemistry, 60 new analogs were synthesized. For
biological assay Trypanosoma brucei brucei Lister 427 cell line were used as the parasite model and for the host
model human embryonic kidney cell line HEK-293 and mouse macrophage cell line RAW 264.7 were used to test
efficacy. Of the newly synthesized compounds 5, 39, 40, and 57 exhibited IC50s below 5 µM inhibiting the
growth of trypanosome cells and not harming the mammalian cells at equipotent concentration. Comparably, the
newly synthesized compounds have a reduced amount of aromatic moieties resulting in a decrease in molecular
weight. Due to importance of tubulin polymerization during protozoan life cycle its activity was assessed by
western blot analyses. Our results indicated that compound 5 had a profound effect on tubulin function. A
detailed structure activity relationship (SAR) was summarized that will be used to guide future lead optimiza-
tion.
Trypanosomiasis
Tubulin inhibitor
Lead optimization
Drug development
1. Introduction
trypanosome cells, which is an essential function for them to survive.9
Therefore, tubulin inhibitors are expected to be able to block the T.
African sleeping sickness (Human African trypanosomiasis, HAT) is a
vector-borne parasitic disease that is still life threatening to the residents
in sub-Saharan Africa.1 Currently there are five drugs available for the
treatment of human African trypanosomiasis, including Suramin, Penta-
midine, Melarsoprol, Eflornithine, and Nifurtimox.2 However, these drugs
have many limitations: 1) highly toxic to the infected hosts; 2) Parental
administration via intramuscular or intravenous injections, which is dif-
ficult to use in the epidemic area with limited medical resources; 3)
narrow anti-trypanosomiasis spectrum; and 4) high cost to the patients.
Overall, these drugs are not effective in the treatment of the disease, and
there is an urgent need to develop more effective and inexpensive che-
motherapeutic agents for the treatment of HAT.
brucei cell division and decrease the locomotion function of the fla-
gellum as well, which will lead to cell death.10 Furthermore, two dif-
ferent tubulin inhibitors benzimidazoles and dinitroanilies also have
been evaluated for HAT activity. Benzimidazoles general are used as
anthelmintics and antifungal agents.11,12 Although benzimidazoles
have demonstrated selective toxicity by binding with protozoa tubulin
instead of mammalian tubulin.13 Whereas, dinitroanilines are classified
as herbicides that inhibit microtubulin, and these compounds are also
potent anti-protozoal agents which are effective against microtubules of
T. brucei. cells and other parasitic cells.14,15 Hence, these factors suggest
that there are multiple advantages of tubulin inhibitor as a novel drug
for the treatment of HAT.
Tubulin interfering agents have been the first line chemotherapeutic
drugs for decades in cancer treatment.3,4 Tubulin is also a critical
protein of trypanosomal cells and plays an essential role during trypa-
nosome cell division.5,6 The fast growth rate of trypanosome cells in-
dicates tubulin polymerization/depolymerization is essential for pro-
liferation.7,8 In addition, tubulin is the key protein for the locomotion of
Tubulin is a highly conserved protein within different species.
However, different susceptibility to antimitotic agents are known to
exist among different organisms, indicating differences in tubulin
structures among different species.16,17 Based on the differences of the
colchicine binding pockets of mammalian and T. brucei tubulins, se-
lective tubulin inhibitors were developed that showed great in vitro
⁎ Corresponding authors.
E-mail addresses: b.li37@csuohio.edu (B. Li), b.su@csuohio.edu (B. Su).
# Equal contribution.
https://doi.org/10.1016/j.bmc.2019.02.049
Received 8 November 2018; Received in revised form 15 February 2019; Accepted 22 February 2019
0968-0896/PublishedbyElsevierLtd.
Pleasecitethisarticleas:AnranZhao,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2019.02.049

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Figure 1. Core structure of the derivatives.
potency to inhibit T. brucei cell growth without harming mammalian
cells at the similar concentrations.18,19 Some compounds exhibited very
specific inhibitory effect on T. brucei cell growth, with a selectivity
index (IC50 inhibiting human cell growth/IC50 inhibiting T. brucei cell
growth) greater than 100.20 In addition, these compounds showed in
vivo activity to decease T. brucei cell growth in the infected mice.
However, they were not potent enough to clear the infection.20 Further
lead optimization based on the summarized structure activity re-
lationship (SAR) resulted in compound 15′ with better potency and
selectivity.21 Following similar testing it resulted in an IC50 of 70 nM to
inhibit trypanosomal cell proliferation with a selectivity index around
700021. Unfortunately, this compound has four aromatic moieties
(Figure 1), which significantly decreased the solubility and even limited
the in vivo testing. The new analogs contain less hydrophobic moieties
which should result in a reduced Log P value. To validate this as-
sumption, two methods were employed: a classical slow stir method
and computational modeling performed by CHM DRAW.22 Only three
compounds where tested including previous compound 15′ and the
current compounds 5 and 57. Based on the structural similarities it is
not necessary to test all the new compounds but the ones with the
highest selectivity index. Herein, in this lead optimization, we use dif-
ferent strategies and try to reduce the bulkiness of the compounds.
the parasite model. Human normal kidney HEK293 cells and mouse
macrophage RAW264.7 cells were used as the mammalian host
model.21 Results of the cell growth inhibition by the compounds are
listed in Table 1. The selectivity index is calculated by dividing the IC50
s
of the mammalian cell growth inhibition with the IC50s of the T. brucei
cell growth inhibition. Compared to the compounds with IC50 below
1 μM in previous study (Figure 1), the new compounds with reduced
aromatic moieties showed reduced activity against T. brucei cells.
However, the study opened up new scaffold for optimization. The
compounds with less aromatic moieties are more drug-like and have
better potential for the future in vivo study.
For the five moieties (R1 – R5) of the compound scaffold (Figure 1),
various functional groups were introduced to enhance the anti-trypa-
nosomal activity and decrease the cytotoxicity to mammalian cells.
Most compounds have IC50 values for mammalian cells above 50 µM,
indicting the low toxicity of the compounds. Therefore, the structure
activity relationship (SAR) to the mammalian cells of these compounds
is not discussed further. For the anti-trypanosomal activity of these
compounds, the SAR is summarized in detail. For R1 domain, there are
four different groups (methyl, methoxyl, chloro, and fluoro) in this
moiety. Overall, methoxyl group has the lowest activity regardless of
the other moieties, and most compounds in this category have IC50s
above 10 µM against trypanosomal cell growth. For methyl group as R1
and R2, when R3 and R5 are trifluoromethyl groups, the resulted com-
pound 5 showed an IC50 of 2.93 µM to inhibit trypanosomal cell pro-
liferation, and selectivity index of 56 and 85 to two mammalian cell
lines. Whereas R1 is still methyl, but R2 is occupied by ethyl or tri-
fluoromethyl groups, activity of these compounds dropped. When R1 is
chloro group and R2 is methyl group, the compounds lost activity
against trypanosomal cells regardless what groups are in R3-R5 moiety.
When R2 is changed to ethyl group, the compounds gained some ac-
tivity, particularly for compound 39 (R3 = H, R4 = Cl, R5 = NO2) with
an IC50 of 2.21 µM and compound 40 (R3 = CF3, R4 = H, R5 = CF3)
with an IC50 of 1.73 µM. Unfortunately, the selectivity index of these
two compounds are below 50 that is our cutoff to select compounds for
further investigation. When R1 is chloro and R2 is trifluoromethyl
group, the compounds overall are not active as indicated by compounds
41–45. The combination of fluoro group at R1 and methyl group at R2
harms the activity as exhibited by compounds 46–50. When R2 is
changed to ethyl group such as compounds 54 and 55, they obtained
some activity with IC50s around 8 µM. When R2 is changed to tri-
fluoromethyl group, the compounds even gained more activity. Parti-
cularly for compound 57, it shows an IC50 of 2.1 µM and selectivity
index above 119. Considering both the activity and selectivity index,
compounds 5 and 57 showed the most promising activity against T.
brucei cells and also great selectivity with a selectivity index above 50.
Further biological investigation was performed with these two com-
pounds.
2. Results and discussion
2.1. Synthesis of the new tubulin inhibitors
In the previous study, the best compound (Figure 1) showed great
potency and selectivity to inhibit T. brucei cell proliferation. But the
congested four aromatic moieties significantly reduce solubility.21 In
order to increase the hydrophilicity, some aromatic moieties should be
eliminated. Based on the SAR summarized before, moieties A and B are
likely not critical for the anti-trypanosomal activity.18,20,21 Therefore,
in the new design, we used small substituent such as halogens, methyl,
and methoxyl groups to occupy the B moiety, and methyl sulfonamide,
trifluromethyl sulfonamide and ethyl sulfonamide group to occupy the
A moiety as illustrated in Figure 1. For the benzamide moiety, we used
different substituents on the aromatic ring to explore the new SAR.
A total of 60 compounds were synthesized using combinatorial
chemistry strategy. We modified the R1, R2, R3, R4 and R5 moieties of
the core structure with different substituents systematically (Figure 1).
In these new compounds, the methyl, methoxyl, chloro, and fluoro
groups were introduced from the starting materials. Next, the R2 moiety
of the scaffold was modified with different substituted sulfonyl chlor-
ides in order to generate different sulfonamide groups. Then, the nitro
group was reduced to amino group in order to introduce the benzamide
moiety. The synthesis of these new compounds is illustrated in Schemes
1 and 2.
2.2. Biological evaluation of the new derivatives
2.3. Solubility testing for pharmacokinetic profile
The biological activity of the synthesized analogs was determined
The water solubility of compounds 5 and 57 were compared to
with cell proliferation assays. T. b. brucei Lister 427 cells were used as
previously synthesized compound 15′21 by measuring the LogP
2

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 1. Synthesis of derivatives (Compounds 1–30).
value.22,23 In addition, the calculated cLogP values were also obtained
with CHEMDRAW. In Table 2, the data are reported and shown that
newly synthesized compounds 5 and 57 have a reduced hydro-
phobicity. It was found that compound 5 has an experimental Log P
2.4. Biological evaluation of the tubulin targeting effect of compound 5 and
compound 57
Our previous study reveals that the lead compound interferes with
the microtubule dynamics.20 However, we removed two aromatic
moieties in the new compounds and that is a major structural mod-
ification. Although the new analogs still showed activity to selectively
inhibit the proliferation of T. brucei cells, it is not clear if the tubulin
targeting effect still remains after the dramatic structural change, which
is necessary to be examined. To test if the new compound is still a tu-
bulin inhibitor, we determined the amount of the polymerized and non-
polymerized tubulin after treating T. brucei cells with compounds 5 and
57. We found that the compounds increased the amount of non-poly-
merized tubulin and decreased the amount of polymerized tubulin in a
value of 1.957
0.134 compared with its computational prediction of
3.64 for cLog P. Compound 57 had an experimental log P value of
0.998 0.304 compared with its computational predication cLogP of
3.31. Compound 15′ resulted in an experimental Log of
P
2.902
0.199 and the computational prediction cLogP is 5.44. Com-
paring these values with the predicted values, there are major differ-
ences between the predicted and the experimental results. But the hy-
drophobicity trend of these compounds is consistent from prediction to
experiment, and the newly synthesized compounds 5 and 57 have lower
log P values, indicating that they are more hydrophilic.
Scheme 2. Synthesis of derivatives (Compounds 31–60).
3

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 1
Comparison of the growth inhibitory effects of the tubulin inhibitors on mammalian and T. brucei cells.
No.
IC50 value(μM; Mean
SD)
Selectivity
T. brucei
HEK293
RAW264.7
HEK293/T. brucei
RAW264.7/T. brucei
Chemical Structure
1
R
1 = CH3, R2 = CH3 R3 = H, R4 = Cl, R5 = H
R1 = CH3, R2 = CH3 R3 = H, R4 = CN, R5 = H
1 = CH3, R2 = CH3 R3 = H, R4 = H, R5 = CF3
R1 = CH3, R2 = CH3 R3 = H, R4 = Cl, R5 = NO2
1 = CH3, R2 = CH3 R3 = CF3, R4 = H, R5 = CF3
> 62.5
> 62.5
50.26
13.57
8.54
69.05
171.12
109.90
107.90
> 250
68.71
108.76
113.91
140.60
124.20
35.36
135.4
8.72
20.12
< 1
< 1
12
< 2
< 3
7
2
43.53
68.07
39.79
50.76
3
R
16.74
7.57
10.91
3.61
1.43
194.3
112.60
32.60
109.62
27.20
4
73.13
10
14
5
R
2.93
163.1
56
> 85
< 2
< 2
3
6
R1 = CH3, R2 = CH2CH3 R3 = H, R4 = Cl, R5 = H
> 62.5
> 62.5
35.26
19.42
12.47
37.21
> 62.5
21.71
> 62.5
20.65
> 62.5
> 62.5
> 62.5
> 62.5
> 62.5
> 62.5
> 62.5
35.74
> 62.5
11.55
45.55
9.48
125.43
> 250
209.63
144.2
11.88
16.77
28.44
80.06
59.80
14.04
24.58
< 3
ND
6
7
R
R
1 = CH3, R2 = CH2CH3 R3 = H, R4 = CN, R5 = H
1 = CH3, R2 = CH2CH3 R3 = H, R4 = H, R5 = CF3
8
20.21
56.26
84.16
64.97
62.06
9
R1 = CH3, R2 = CH2CH3 R3 = H, R4 = Cl, R5 = NO2
9.66
9.04
23.83
7
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R
R
1 = CH3, R2 = CH2CH3 R3 = CF3, R4 = H, R5 = CF3
1 = CH3, R2 = CF3 R3 = H, R4 = Cl, R5 = H
126.1
10
10
200.50
> 250
130.60
> 250
181.65
> 250
> 250
> 250
164.41
41.97
5
1
R1 = CH3, R2 = CF3 R3 = H, R4 = CN, R5 = H
ND
6
< 3
< 1
< 2
2
R
1 = CH3, R2 = CF3 R3 = H, R4 = H, R5 = CF3
R1 = CH3, R2 = CF3 R3 = H, R4 = Cl, R5 = NO2
1 = CH3, R2 = CF3 R3 = CF3, R4 = H, R5 = CF3
14.46
8.02
32.20
89.65
6.18
89.78
40.66
70.00
> 250
84.77
> 250
> 250
60.09
66.44
84.83
5.66
24.65
15.77
16.32
ND
9
R
R1 = OCH3, R2 = CH3 R3 = H, R4 = Cl, R5 = H
ND
ND
ND
< 3
< 1
ND
ND
> 7
< 2
> 22
> 5
> 26
> 6
> 18
ND
< 3
ND
ND
< 4
18
< 2
ND
< 2
ND
ND
< 1
< 2
2
R
R
1 = OCH3, R2 = CH3 R3 = H, R4 = CN, R5 = H
1 = OCH3, R2 = CH3 R3 = H, R4 = H, R5 = CF3
27.70
R1 = OCH3, R2 = CH3 R3 = H, R4 = Cl, R5 = NO2
37.51
17.57
R
1 = OCH3, R2 = CH3 R3 = CF3, R4 = H, R5 = CF3
R1 = OCH3, R2 = CH2CH3 R3 = H, R4 = Cl, R5 = H
> 250
> 250
> 250
63.65
31.85
29.82
41.66
R
R
1 = OCH3, R2 = CH2CH3 R3 = H, R4 = CN, R5 = H
1 = OCH3, R2 = CH2CH3 R3 = H, R4 = H, R5 = CF3
24.93
R1 = OCH3, R2 = CH2CH3 R3 = H, R4 = Cl, R5 = NO2
18.13
2.95
< 1
> 22
2
R
R
1 = OCH3, R2 = CH2CH3 R3 = CF3, R4 = H, R5 = CF3
1 = OCH3, R2 = CF3 R3 = H, R4 = Cl, R5 = H
6.21
> 250
> 250
> 250
> 250
> 250
> 250
167.45
> 250
> 250
203.57
184.56
> 250
> 250
158.54
63.80
> 250
108.48
> 250
75.23
> 250
> 250
79.94
> 250
> 250
43.60
69.55
> 250
226.08
62.43
125.40
64.07
47.15
> 250
55.82
31.68
136.06
22.25
> 250
32.29
101.79
124.94
151.92
107.38
119.17
198.43
> 250
3.27
24.55
46.66
14.75
R1 = OCH3, R2 = CF3 R3 = H, R4 = CN, R5 = H
R1 = OCH3, R2 = CF3 R3 = H, R4 = H, R5 = CF3
3.91
> 26
2
43.04
14.08
> 62.5
> 62.5
> 62.5
> 62.5
> 62.5
10.08
19.12
37.95
7.81
23.44
6.89
R
R
1 = OCH3, R2 = CF3 R3 = H, R4 = Cl, R5 = NO2
1 = OCH3, R2 = CF3 R3 = CF3, R4 = H, R5 = CF3
> 18
ND
< 2
ND
ND
< 1
7
R1 = Cl, R2 = CH3 R3 = H, R4 = Cl, R5 = H
30.03
49.27
R
R
1 = Cl, R2 = CH3 R3 = H, R4 = CN, R5 = H
1 = Cl, R2 = CH3 R3 = H, R4 = H, R5 = CF3
R1 = Cl, R2 = CH3 R3 = H, R4 = Cl, R5 = NO2
R1 = Cl, R2 = CH3 R3 = CF3, R4 = H, R5 = CF3
29.44
84.83
22.23
29.89
6.58
R
R
1 = Cl, R2 = CH2CH3 R3 = H, R4 = Cl, R5 = H
1 = Cl, R2 = CH2CH3 R3 = H, R4 = CN, R5 = H
13.52
20.73
> 13
> 7
20
> 13
6
114.5
19.90
62.97
53.30
6.39
R1 = Cl, R2 = CH2CH3 R3 = H, R4 = H, R5 = CF3
4.93
72.35
32.72
14.30
63.12
8
R
R
1 = Cl, R2 = CH2CH3 R3 = H, R4 = Cl, R5 = NO2
1 = Cl, R2 = CH2CH3 R3 = CF3, R4 = H, R5 = CF3
2.21
1.08
0.71
29
57
1.73
32.17
19
37
R1 = Cl, R2 = CF3 R3 = H, R4 = Cl, R5 = H
> 62.5
11.00
> 62.5
47.63
47.36
> 62.5
> 62.5
> 62.5
> 62.5
23.68
> 62.5
> 62.5
33.91
8.39
190.90
> 250
209.85
228.48
> 250
216.21
> 250
> 250
23.08
< 4
> 23
< 4
5
< 1
> 23
< 1
1
R
1 = Cl, R2 = CF3 R3 = H, R4 = CN, R5 = H
8.06
R1 = Cl, R2 = CF3 R3 = H, R4 = H, R5 = CF3
39.02
44.82
7.79
7.26
51.81
6.64
R
R
1 = Cl, R2 = CF3 R3 = H, R4 = Cl, R5 = NO2
1 = Cl, R2 = CF3 R3 = CF3, R4 = H, R5 = CF3
26.11
27.31
> 5
< 4
ND
ND
< 1
> 11
< 1
< 1
3
3
R1 = F, R2 = CH3 R3 = H, R4 = Cl, R5 = H
99.88
< 1
ND
< 1
< 2
5
R
1 = F, R2 = CH3 R3 = H, R4 = CN, R5 = H
R1 = F, R2 = CH3 R3 = H, R4 = H, R5 = CF3
1 = F, R2 = CH3 R3 = H, R4 = Cl, R5 = NO2
15.69
45.41
70.28
73.77
49.13
43.96
58.95
R
3.13
R1 = F, R2 = CH3 R3 = CF3, R4 = H, R5 = CF3
16.82
19.71
> 250
53.37
R
R
1 = F, R2 = CH2CH3 R3 = H, R4 = Cl, R5 = H
1 = F, R2 = CH2CH3 R3 = H, R4 = CN, R5 = H
4.83
< 3
< 2
4
26.36
6.79
R1 = F, R2 = CH2CH3 R3 = H, R4 = H, R5 = CF3
94.84
29.38
41.60
R
1 = F, R2 = CH2CH3 R3 = H, R4 = Cl, R5 = NO2
5.68
83.66
10
24
R1 = F, R2 = CH2CH3 R3 = CF3, R4 = H, R5 = CF3
8.78
5.87
1.01
> 250
78.98
> 28
< 2
> 119
3
> 28
< 1
> 119
1
R
R
1 = F, R2 = CF3 R3 = H, R4 = Cl, R5 = H
1 = F, R2 = CF3 R3 = H, R4 = CN, R5 = H
> 62.5
2.10
41.12
1.78
> 250
151.72
193.67
163.76
> 250
34.44
187.75
112.41
R1 = F, R2 = CF3 R3 = H, R4 = H, R5 = CF3
47.35
16.79
9.386
29.86
31.83
97.12
90.18
20.47
93.75
55.98
R
1 = F, R2 = CF3 R3 = H, R4 = Cl, R5 = NO2
12.71
5.49
12
11
R1 = F, R2 = CF3 R3 = CF3, R4 = H, R5 = CF3
17
12
IC50 values represent the Mean
“ND” = not determined.
Standard Deviation (S.D.) of four parallel measurements.
dose-dependent manner (Figure 2), indicating that after the treatment,
the tubulin polymerization in the cells was inhibited by the compounds.
Compound 5 showed much better activity than compound 57 to inhibit
tubulin polymerization. However, the IC50s against trypanosomal cell
proliferation of the two compounds are similar. It suggests that com-
pound 57 may also target the cell proliferation via other mechanisms,
which compensates the relative lower tubulin targeting effect in com-
parison to compound 5. Overall, the results demonstrate that the new
4

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 2
Lop P values assay of compound 5, compound 57 and compound 15′
Compound 5 (λ = 290 nm)
Compound 57 (λ = 241 nm)
Compound 15′ (λ = 212 nm)
experimental Log P
Predicted cLogP
1.957
3.64
0.134
0.998
3.31
0.304
2.902
5.44
0.199
The measurement was repeated three times with water and 1-octanol as the two solvents, and the results are presented as Mean
ChemDraw Ultra 12.0.2.1076.
SD, cLogP is determined using
Figure 2. Tubulin inhibitor compound 5 and compound 57 inhibit the tubulin polymerization in T. brucei cells. After treatment, the level of polymerized tubulin is
significantly decreased. The band intensities of HEXIM1 and β-actin were quantified using ImageJ (NIH) to generate the figure. The figures are representative of 3
experiments. *p < 0.05 vs. DMSO, **p < 0.01 vs DMSO, *** p < 0.001 by unpaired t-test.
compounds are still tubulin inhibitors and selectively target the tubulin
dynamic regulation in T. brucei cells. They are potent and selective
tubulin inhibitors against trypanosomiasis, and could be the new lead
for further optimization.
plates with a fluorescence indicator at 254 nm (Fluka). Flash column
chromatography was performed using silica gel 60 Å (BDH, 40–63 µm).
Mass spectra were obtained on a Bruker Ion-Trap Mass Spectrometer at
Cleveland State University MS facility Center. All the NMR spectra were
recorded on a Bruker 400 MHz spectrometer using CDCl3 or DMSO‑d6
as the solvent.
3. Conclusion
Reversed-phase HPLC analysis of compounds was conducted on a
Beckman HPLC system with an Auto Sampler. The chromatographic
separation was performed on a C18 column (2.0 mm × 150 mm, 5 μm)
from Phenomenex (Torrance, CA). The mobile phase was employed for
isocratic elution with a flow rate of 0.2 mL/min. The injection volume
was 20 µL and the UV detector was set up at 260 nm.
Due to limitations of current anti-trypanosomiasis drugs, the de-
velopment of new medication for the treatment of African trypanoso-
miasis is still urgently needed. In the present study we focused on the
development of selective tubulin inhibitors for the treatment of this
disease. Based on the inhibitory effects of the compounds on T. brucei
cell proliferation and mammalian cell growth, the selectivity index is
determined in order to identify more potent and selective drug candi-
dates. This resulted in compounds with an IC50s around 2 μM to inhibit
the growth of T. brucei cells, and not affecting the viability of mam-
malian cells even with a concentration that is a hundred folds higher.
Compared to previous studies, the new compounds have only two
aromatic moieties, which increased the solubility and ligand efficacy.
Furthermore, the selective tubulin inhibitor interferes with tubulin
polymerization in T. brucei cells and significantly increased the non-
polymerized tubulin and decreased the polymerized tubulin.
The synthesis procedures of compounds 1–60 are listed below.
4.1.1. General method for the preparation of 2a-2d
Sulfonamides 2a-2d were prepared from arylsubstituted 2-amino-5-
nitrophenols 1a-1b. Dissolve arylsubstituted 2-amino-5-nitrophenol
(15.0 mM) in 40 mL anhydrous DMF add NaH (95% powder, 1.325 g,
52.5 mM, 3.5 equiv) into the solution stir at room temperature for
30 min. After stir at room temperature for 30 min corresponding sul-
fonyl chloride (45 mM, 3 equiv) was added to the mixture the reaction
continued overnight at room temperature. Quench the reaction with
water and the mixture was neutralized with 6 N HCl until pH = 1–2,
yellow intermediate precipitated. Intermediate was collected by filtra-
tion and wash with water, which was used to the next reaction without
further purification
4. Experimental section
4.1. Chemistry
The intermediate was dissolved in 100 mL methanol and 50 mL 4 N
NaOH aq solution was added into the solution, stir at room temperature
for 2 h. After reaction completed neutralized the solution with 6 N HCl
until pH = 1–2. The precipitate was collected by filtration and was with
water and cold ether to provide desire product.
Chemicals were commercially available and used as received
without further purification unless otherwise noted. Moisture sensitive
reactions were carried out under a dry argon atmosphere in flame-dried
glassware. Thin layer chromatography was performed on silica gel TLC
5

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.1.2. General method for the preparation of 2g-2j
4.1.7. N-(4-(Methylsulfonamido)-3-Methylphenyl)-3-(Trifluoromethyl)
Sulfonamides 2g-2j were prepared from aryl-substituted 2-amino-5-
nitrophenols 1c-1d. Dissolve arylsubstituted 2-amino-5-nitrophenol
(15.0 mM) in 150 mL anhydrous DCM, TEA was added into solution
(105.0 mM, 7 equiv). After TEA added corresponding sulfonyl chloride
(45 mM, 3 equiv) added into solution, reacted at room temperature
overnight. After reaction completed DCM evaporated under vacuum
added 200 mL water added into the flask neutralized with 6 N HCl until
pH = 1–2. Collect the solid intermediate by filtration. Wash the inter-
mediate with water, which was used to the next reaction without fur-
ther purification.
Benzamide (3).
Yield 62.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.45 (s, 1H), 9.01 (s,
1H), 8.30 (s, 1H), 8.27 (d, 1H, J = 8.0), 7.98 (d, 1H, J = 8.0) 7.80 (t,
1H, J = 8.0), 7.68 (s, 1H), 7.63 (d, 1H, J = 8.8), 7.28 (d, 1H, J = 8.8),
2.98 (s, 3H), 2.33 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 164.39,
137.52, 136.18, 135.47, 132.29, 131.71, 130.22, 130.16, 129.84,
129.52, 129.21 (q, 1C), 128.64 (m, 1C), 127.52, 124.73, 124.69,
124.66, 124.62 (q, 1C), 123.14, 119.16, 31.14, 18.84. DUIS-MS calcu-
lated for C16H15F3N2O3S, [M−H]-: m/z 371.07, found m/z 370.9.
Purity: 97.8%.
The intermediate was dissolved in 100 mL methanol and 50 mL 4 N
NaOH aq solution was added into the solution, stir at room temperature
for 2 h. After reaction completed neutralized the solution with 6 N HCl
until pH = 1–2. The precipitate was collected by filtration and was with
water and cold ether to provide desire product
4.1.8. 4-Chloro-N-(4-(Methylsulfonamido)-3-Methylphenyl)-3-
Nitrobenzamide (4).
Yield 61.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.73 (s, 1H), 8.69 (s,
1H), 8.38 (s, 1H), 8.32 (d, 1H, J = 8.4), 7.79 (d, 1H, J = 8.4), 7.76 (s,
1H), 7.75 (d, 1H, J = 8.8), 7.35 (d, 1H, J = 8.8), 3.27 (s, 3H), 2.36 (s,
3H). 13C NMR (100 MHz, DMSO‑d6) δ: 162.98, 156.41, 147.96, 138.50,
135.08, 133.60, 133.37, 132.47, 128.69, 125.77, 125.37, 123.15,
119.44, 56.50(solvent) 43.61, 37.52, 18.23(solvent). DUIS-MS calcu-
lated for C15H14ClN3O2S, [M−H]-: m/z 382.03, found m/z 381.9.
Purity: 98.2%.
4.1.3. General method for the preparation of 2e-2f and 2k-2 l
Trifluoroumethylsulfonamides 2e-2f and 2k-2l were prepared from
aryl-substituted 2-amino-5-nitrophenols 1a-1d. Dissolve arylsubstituted
2-amino-5-nitrophenol (15.0 mM) in 150 mL anhydrous DCM and
K2CO3 (75 mM) was added to solution, then cool to 0 °C.
Trifluoromethanesulfonic anhydrous (45 mM) was added into the so-
lution dropwise. The resulting mixture was continuously stirred for 3 h
at 0–5 °C. Water (30 mL) was added to quench the reaction. DCM was
evaporated under vacuum, and then 6 N HCl (10 mL) was added to
acidify the residue. The product was collected by filtration and washed
with water and cool ether, then used for the next reaction without
further purification.
4.1.9. N-(4-(Methylsulfonamido)-3-Methylphenyl)-3,5-Bis
(Trifluoromethyl)Benzamide (5).
Yield 55.4%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.59 (s, 1H), 9.57 (s,
1H), 8.62 (s, 2H), 8.38 (s, 1H), 7.64 (s, 1H), 7.74 (d, 1H, J = 8.6), 7.58 (d,
1H, J = 8.6), 3.33 (s, 3H) ,2.26 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ:
162.70(solvent), 160.21, 137.56, 135.46, 132.52, 131.14, 130.81, 130.43
(q, 1C), 128.97 (m, 1C), 125.56 (m, 1C), 124.97, 123.78, 123.08, 122.26,
119.56, 119.02, 18.51(solvent). DUIS-MS calculated for C17H14F6N2O3S,
[M−H]-: m/z 439.06, found m/z 439.0. Purity: 98.8%.
4.1.4. General method for the preparation of product 1–60
Dissolve intermediates 2a-2 l 1 mM in acetone 10 mL and water
1 mL. After intermediates dissolve into solvent Zn (10 mM, 10 equiv)
and FeCl3 (4 mM, 4 equiv) were added into solution, the result mixture
was stirred at room temperature for 1 h. After the reaction completed
the mixture was filtrate through celite to remove inorganic precipitate.
The eluent was evaporated under vacuum(3a-3 l).
4.1.10. 4-Chloro-N-(4-(Ethylsulfonamido)-3-Methylphenyl)Benzamide
(6).
Yield 68.9%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.30 (s, 1H), 8.99 (s,
1H), 7.99 (d, 2H, J = 8.8), 7.66 (d, 1H, J = 2.4), 7.62 (d, 2H, J = 8.8),
7.58 (dd, 1H, J1 = 2.4, J2 = 8.8), 7.23 (d, 1H, J = 8.8), 3.08 (q, 2H,
J = 8.0), 2.32 (s, 3H), 1.28 (t, 3H, J = 8.0). 13C NMR (100 MHz,
DMSO‑d6) δ: 164.80, 137.55, 136.90, 135.29, 134.01, 131.54, 130.06
(2C), 128.94 (2C), 127.30, 122.99, 119.04, 46.67, 18.92, 8.56. DUIS-
MS calculated for C16H17ClN2O3S, [M−H]-: m/z 351.06, found m/z
351.0; Purity: 96.7%.
Redissolve the intermediate into 10 mL acetone, the corresponding
benzoyl chloride (1.1 mM, 1.1 equiv) was added then 10 mL brine and
10 mL saturated Na2CO3 solution. The result mixture was stirred at
room temperature for 1 h. After the reaction completed neutralized
with 6 N HCl until pH = 1–2, acetone was evaporated under vacuum.
The product was collected by filtration and purify by recrystallization in
ethanol/ water (3:1).
4.1.11. 4-Cyano-N-(4-(Ethylsulfonamido)-3-Methylphenyl)Benzamide
(7).
Yield 60.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.45 (s, 1H), 9.00 (s,
1H), 8.11 (d, 2H, J = 8.4), 8.04 (d, 2H, J = 8.4), 7.67 (d, 1H, J = 2.4),
7.59 (dd, 1H, J1 = 2.4, J2 = 8.4,), 7.25 (d, 1H, J = 8.4), 3.08 (q, 2H,
J = 7.6), 2.33 (s, 3H), 1.27 (t, 3H, J = 7.6). 13C NMR (100 MHz,
DMSO‑d6) δ: 164.49, 139.34, 137.28, 135.29, 132.95, 131.82, 128.87,
127.27, 123.03, 119.09, 118.78, 114.34, 46.70, 18.91, 8.56. DUIS-MS
calculated for C17H17N3O3S, [M−H]-: m/z 342.09, found m/z 341.8.
Purity: 96.8%.
4.1.5. 4-Chloro-N-(4-(Methylsulfonamido)-3-Methylphenyl)Benzamide
(1).
Yield 56.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.43 (s, 1H), 8.36 (s,
1H), 8.01 (d, 2H, J = 8.0), 7.75 (s, 1H), 7.74 (d, 1H, J = 8.0), 7.63 (d,
2H, J = 8.0), 7.33 (d, 1H, J = 8.0), 3.25 (s, 3H), 2.34 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.95, 156.41, 138.95, 137.07, 133.81,
133.46, 130.12 (2C), 128.99 (2C), 128.95, 125.69, 123.00, 119.31,
43.62, 37.37, 18.17(solvent). DUIS-MS calculated for C15H15ClN2O3S,
[M−H]-: m/z 337.04, found m/z 336.9. Purity: 95.3%.
4.1.12. N-(4-(Ethylsulfonamido)-3-Methylphenyl)-3-(Trifluoromethyl)
Benzamide (8).
4.1.6. 4-Cyano-N-(4-(Methylsulfonamido)-3-Methylphenyl)Benzamide
(2).
Yield 61.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.43 (s, 1H), 8.99 (s,
1H), 8.30 (s, 1H), 8.27 (d, 1H, J = 7.6), 7.98 (d, 1H, J = 7.6), 7.80 (t,
1H, J = 7.6), 7.67 (s, 1H), 7.61 (d, 1H, J = 8.4), 7.26 (d, 1H, J = 8.4),
3.09 (q, 2H, J = 7.2), 2.34 (s, 3H), 1.28 (t, 3H, J = 7.2). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.37, 137.35, 136.19, 135.29, 132.29,
131.76, 130.21, 130.16, 129.84, 129.52, 129.18 (q, 1C), 128.63 (m,
1C), 127.27, 124.69 (m, 1C), 123.14, 119.17, 46.73, 19.00, 8.56. DUIS-
MS calculated for C17H17F3N2O3S, [M−H]-: m/z 385.08, found m/z
384.8. Purity: 97.5%.
Yield 60.5%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.61 (s, 1H), 8.36 (s,
1H), 8.14 (d, 2H, J = 8.4), 8.05 (d, 2H, J = 8.4), 7.76 (s, 1H), 7.74 (d,
1H, J = 8.0), 7.34 (d, 1H, J = 8.0), 3.25 (s, 3H), 2.35 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.66, 139.13, 133.49, 132.98 (2C), 132.95,
129.03 (2C), 128.96, 125.62, 123.07, 119.39, 118.75, 114.48, 43.59,
18.19. DUIS-MS calculated for C16H15N3O3S, [M−H]-: m/z 328.08,
found m/z 328.0. Purity: 97.3%.
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4.1.13. 4-Chloro-N-(4-(Ethylsulfonamido)-3-Methylphenyl)-3-
Nitrobenzamide (9).
(d, 1H, J = 8.8), 2.34 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 163.07,
138.65, 137.41, 136.42, 131.50, 131.17, 130.84, 130.51 (q, 2C),
129.02 (m, 2C), 128.97, 125.72 (m, 1C), 127.66, 124.95, 122.23,
119.52 (q, 2CF3), 123.16, 121.90, 119.41, 18.32. DUIS-MS calculated
for C17H11F9N2O3S, [M−H]-: m/z 493.03, found m/z 492.8, Purity:
98.1%.
Yield 64.3%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.52 (s, 1H), 9.02 (s,
1H), 8.63 (d, 1H, J = 2.0), 8.26 (dd, 1H, J1 = 8.4, J2 = 2.0), 7.98 (d,
1H, J = 8.4), 7.65 (d, 1H, J = 2.0), 7.59 (dd, 1H, J1 = 2.0, J2 = 8.6),
7.26 (d, 1H, J = 8.6), 3.08 (q, 2H, J = 7.3), 2.33 (s, 3H), 1.27 (t, 3H,
J = 7.3). 13C NMR (100 MHz, DMSO‑d6) δ: 162.91, 137.06, 135.32,
133.29 (2C), 132.45, 131.96, 128.56, 127.28, 125.27 (2C), 123.09,
119.14, 46.74, 18.89, 8.55. DUIS-MS calculated for C16H16ClN3O5S,
[M−H]-: m/z 396.04, found m/z 396.0. Purity: 98.8%.
4.1.20. 4-Chloro-N-(4-(Methylsulfonamido)-3-Methoxylphenyl)
Benzamide (16).
Yield 69.8%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.34 (s, 1H), 8.84 (s,
1H), 8.00 (d, 2H, J = 8.4), 7.63 (d, 2H, J = 8.4), 7.62 (s, 1H), 7.35 (d,
1H, J = 8.8), 7.22 (d, 1H, J = 8.8), 3.84 (s, 3H), 2.93 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.83, 153.56, 139.44, 136.60, 134.00,
130.06 (2C), 129.92 (2C), 127.37, 121.53, 112.76, 104.90, 56.15,
40.06. DUIS-MS calculated for C15H15ClN2O4S, [M−H]-: m/z 353.04,
found m/z 352.9. Purity: 96.7%.
4.1.14. N-(4-(Ethylsulfonamido)-3-Methylphenyl)-3,5-Bis
(Trifluoromethyl)Benzamide (10).
Yield 54.6%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.63 (s, 1H), 9.02 (s,
1H), 8.62 (s, 2H), 8.38 (s, 1H), 7.66 (s, 1H), 7.63 (d, 1H, J = 8.4), 7.29
(d, 1H, J = 8.4), 3.08 (q, 2H, J = 7.2), 2.35 (s, 3H), 1.29, (t, 3H,
J = 7.2). 13C NMR (100 MHz, DMSO‑d6) δ: 162.95, 137.51, 136.98,
135.29, 132.09, 131.49, 131.16, 130.82, 130.49 (q, 1C), 129.02 (m,
2C), 127.24, 125.56 (m, 1C), 123.26, 119.29, 46.79, 18.88, 8.56. DUIS-
MS calculated for C18H16F6N2O3S, [M−H]-: m/z 453.07, found m/z
453.0. Purity: 98.8%.
4.1.21. 4-Cyano-N-(4-(Methylsulfonamido)-3-Methoxylphenyl)Benzamide
(17).
Yield 65.3%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.51 (s, 1H), 8.87 (s,
1H), 8.12 (d, 2H, J = 8.4), 8.05 (d, 2H, J = 8.4), 7.62 (d, 1H, J = 2.0),
7.34 (dd, 1H, J1 = 2.0, J2 = 8.8), 7.23 (d, 1H, J = 8.8), 3.84 (s, 3H),
2.93 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ:164.56, 153.53, 139.31,
138.29, 132.96, 128.96, 127.33, 121.99, 118.77, 114.40, 112.81,
104.91, 56.16, 40.07. DUIS-MS calculated for C16H15N3O4S, [M−H]-:
m/z 344.07, found m/z 344.0. Purity: 98.0%.
4.1.15. 4-Chloro-N-(3-Methyl-4-Trifluoromethanesulfonylamino-Phenyl)
Benzamide (11).
Yield 59.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 11.34 (s, 1H), 10.37 (s,
1H), 7.99 (d, 2H, J = 8.4), 7.74 (s, 1H), 7.64 (m, 3H), 7.30 (d, 1H,
J = 8.8), 2.32 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 164.98, 139.21,
137.02, 136.34, 133.89, 130.10 (2C), 128.96 (2C), 128.91, 128.38,
4.1.22. N-(4-(Methylsulfonamido)-3-Methoxylphenyl)-3-
122.90, 121.80, 119.19. DUIS-MS calculated for
C15H12ClF3N2O3S,
(Trifluoromethyl)Benzamide (18).
[M−H]-: m/z 391.01, found m/z 390.9. Purity: 95.7%.
Yield 65.3%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.50 (s, 1H), 8.86 (s,
1H), 8.30 (s, 1H), 8.28 (d, 1H, J = 7.6), 7.99 (d, 1H, J = 7.6), 7.81 (t,
1H, J = 7.6), 7.62 (s, 1H), 7.37 (d, 1H, J = 8.8), 7.24 (d, 1H, J = 8.8),
3.85 (s, 3H), 2.94 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 164.46,
153.54, 138.35, 136.18, 132.28, 130.23, 130.18, 129.86, 129.54,
129.22 (q, 1C), 128.66 (m, 1C), 127.29, 124.69 (m, 1C), 121.96,
112.92, 105.05, 56.19, 40.07. DUIS-MS calculated for C16H15F3N2O4S,
[M−H]-: 387.06, found 386.9. Purity: 97.0%.
4.1.16. 4-Cyano-N-(3-Methyl-4-Trifluoromethanesulfonylamino-Phenyl)
Benzamide (12).
Yield 52.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 11.40 (s, 1H), 10.54
(s, 1H), 8.11 (d, 2H, J = 8.0), 8.04 (d, 2H, J = 8.0), 7.75 (s, 1H), 7.65
(d, 2H, J = 8.8), 7.25 (d, 1H, J = 8.8), 2.33 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.720, 139.211, 138.969, 136.400, 132.867
(2C), 129.013 (2C), 128.965, 128.636, 122.942, 119.426, 118.75,
114.45, 18.34. DUIS-MS calculated for C16H12F3N3O3S, [M−H]-: m/z
382.05, found m/z 381.9. Purity: 97.9%.
4.1.23. 4-Chloro-N-(4-(Methylsulfonamido)-3-Methoxylphenyl)-3-
Nitrobenzamide (19).
Yield 70.1%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.57 (s, 1H), 8.88 (s,
1H), 8.65 (s, 1H), 8.28 (d, 1H, J = 8.4), 7.99 (d, 1H, J = 8.4), 7.59 (s,
1H), 7.36 (d, 1H, J = 8.4), 7.25 (d, 1H, J = 8.4), 3.84 (s, 3H), 2.94 (s,
3H). 13C NMR (100 MHz, DMSO‑d6) δ: 162.97, 153.50, 147.97, 138.04,
135.27, 133.28, 132.47, 128.60, 127.26, 125.26, 122.20, 112.92,
106.01, 56.20, 40.06. DUIS-MS calculated for C15H14ClN3O6S, [M−H]-:
m/z 398.02, found m/z 397.8. Purity: 98.3%.
4.1.17. N-(3-Methyl-4-Trifluoromethanesulfonylamino-Phenyl)-3-
Trifluoromethyl-Benzamide (13).
Yield 55.4%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.55 (s, 1H), 8.27 (s,
1H), 8.25 (d, 1H, J = 8.0), 7.97 (d, 1H, J = 8.0), 7.79 (t, 1H, J = 8.0),
7.72 (s, 1H), 7.66 (d, 1H, J = 8.4), 7.24 (d, 1H, J = 8.4), 2.32 (s, 3H).
13C NMR (100 MHz, DMSO‑d6) δ: 164.72, 139.00, 136.50, 135.98,
132.28, 130.29, 130.20, 129.88, 129.56, 129.24 (q, 1C), 129.00,
128.77 (m, 1C), 128.51, 124.70 (m, 1C), 123.11, 119.37, 18.29. DUIS-
MS calculated for C16H12F6N2O3S, [M−H]-: m/z 425.04, found m/z
424.8. Purity: 98.7%.
4.1.24. N-(4-(Methylsulfonamido)-3-Methoxylphenyl)-3,5-Bis
(trifluoromethyl)Benzamide (20).
Yield 63.4%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.69 (s, 1H), 8.90 (s,
1H), 8.62 (s, 2H), 8.39 (s, 1H), 7.59 (d, 1H, J = 2.0), 7.37 (dd, 1H,
4.1.18. 4-Chloro-N-(3-Methyl-4-Trifluoromethanesulfonylamino-Phenyl)-
3-Mitro-Benzamide (14).
J1 = 2.0, J2 = 8.8), 7.27 (d, 1H, J = 8.8), 3.85 (s, 3H), 2.95 (s, 3H). 13
C
NMR (100 MHz, DMSO‑d6) δ: 162.95, 153.48, 137.92, 137.49, 131.49,
131.16, 130.83, 130.50 (q, 2C), 129.02 (m, 2C), 127.21, 125.63 (m,
1C), 127.68, 124.96, 122.25, 199.53 (q, 2CF3), 122.31, 113.08, 105.15,
56.21, 40.04. DUIS-MS calculated for C17H14F6N2O4S, [M−H]-: m/z
455.05, found m/z 454.9. Purity: 96.1%.
Yield 52.9%. 1H NMR (400 MHz, DMSO‑d6) δ: 11.38 (s, 1H), 10.63
(s, 1H), 8.65 (s, 1H), 8.27 (d, 1H, J = 8.4), 7.99 (d, 1H, J = 8.4), 7.74
(s, 1H), 7.66 (d, 1H, J = 8.6), 7.26 (d, 1H, J = 8.6), 2.33 (s, 3H). 13C
NMR (100 MHz, DMSO‑d6) δ: 163.03, 147.89, 138.81, 136.46, 135.15,
133.34, 132.47, 129.01, 128.72, 128.67, 125.34, 123.01, 119.30,
18.34. DUIS-MS calculated for C15H11ClF3N3O5S, [M−H]-: m/z 436.00,
found m/z 435.9. Purity: 96.8%.
4.1.25. 4-Chloro-N-(4-(Ethylsulfonamido)-3-Methoxylphenyl)Benzamide
(21).
Yield 69.9%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.35 (s, 1H), 8.84 (s,
1H), 7.99 (d, 2H, J = 8.4), 7.63 (d, 2H, J = 8.4), 7.61 (d, 1H, J = 2.0),
7.34 (dd, 1H, J1 = 2.0, J2 = 8.4) ,7.23 (d, 1H, J = 8.4), 3.82 (s, 3H),
2.99 (q, 2H, J = 7.2), 1.26 (t, 3H, J = 7.2). 13C NMR (100 MHz,
DMSO‑d6) δ: 164.93, 153.38, 138.44, 136.95, 134.00, 130.06, 128.96,
4.1.19. N-(3-Methyl-4-Trifluoromethanesulfonylamino-Phenyl)-3,5-Bis-
Trifluoromethyl-Benzamide (15).
Yield 53.6%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.69 (s, 1H), 8.61 (s,
2H), 8.46 (s, 1H), 8.39 (s, 1H), 7.72 (s, 1H), 7.67 (d, 1H, J = 8.8), 7.28
7

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
127.25, 121.73, 112.74, 104.80, 56.11, 46.61, 8.48. DUIS-MS calcu-
lated for C16H17ClN2O4S, [M−H]-: m/z 367.05, found m/z 366.9,
Purity: 97.5%.
139.19, 132.99 (2C), 129.78, 129.01 (2C), 118.75, 114.51, 112.66,
107.67, 104.79, 56.25. DUIS-MS calculated for C16H12F3N3O4S,
[M−H]-: m/z 398.04, found m/z 397.9. Purity: 96.2%.
4.1.26. 4-Cyano-N-(4-Ethanesulfonylamino-3-Methoxyphenyl)Benzamide
(22).
4.1.32. N-(3-Methoxy-4-Trifluoromethanesulfonylamino-Phenyl)-3-
Trifluoromethyl-Benzamide (28).
Yield 65.8%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.50 (s, 1H), 8.86 (s,
1H), 8.11 (d, 2H, J = 8.4), 8.05 (d, 2H, J = 8.4), 7.61 (d, 1H, J = 2.0),
7.34 (dd, 1H, J1 = 2.0, J2 = 8.4), 7.24 (d, 1H, J = 8.4), 3.83 (s, 3H),
3.00 (q, 2H, J = 7.2), 1.26 (t, 3H, J = 7.2). 13C NMR (100 MHz,
DMSO‑d6) δ: 164.54, 153.32, 139.32, 138.13, 132.95, 128.96, 127.11,
122.07, 118.76, 114.40, 112.83, 104.87, 56.14, 46.65, 8.47. DUIS-MS
calculated for C17H17N3O4S, [M−H]-: m/z 358.09, found m/z 357.9,
Purity: 96.9%.
Yield 45.8%. 1H NMR (400 MHz, DMSO‑d6) δ: 11.21 (s, 1H), 10.58
(s, 1H), 8.30 (s, 1H), 8.28 (d, 1H, J = 7.6), 8.00 (d, 1H, J = 7.6), 7.82
(t, 1H, J = 7.6), 7.65 (s, 1H), 7.42 (d, 1H, J = 8.4), 7.25 (d, 1H,
J = 8.4), 3.34 (s, 1H). 13C NMR (100 MHz, DMSO‑d6) δ: 164.69,
155.47, 140.49, 138.52, 136.07, 132.03, 131.07, 130.78, 130.27,
129.74, 129.24 (q, 1C), 128.85, 128.47 (m, 1C), 124.74 (m, 1C),
112.75, 104.91, 56.27. DUIS-MS calculated for
C16H12F6N2O4S,
[M−H]-: m/z 441.03, found m/z 440.9. Purity: 98.7%.
4.1.27. N-(4-(Ethylsulfonamido)-3-Methoxylphenyl)-3-(Trifluoromethyl)-
Benzamide (23).
4.1.33. 4-Chloro-N-(3-Methoxy-4-Trifluoromethanesulfonylamino-
Phenyl)-3-Nitro-Benzamide (29).
Yield 62.3%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.50 (s, 1H), 8.85 (s,
1H), 8.30 (s, 1H), 7.28 (d, 1H, J = 7.6), 7.99 (d, 1H, J = 7.6), 7.81 (t,
1H, J = 7.6), 7.60 (s, 1H), 7.36 (d, 1H, J = 8.8), 7.25 (d, 1H, J = 8.8),
3.84 (s, 3H), 3.01 (q, 2H, J = 7.2), 1.26 (t, 3H, J = 7.2). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.44, 153.34, 138.20, 136.18, 132.28,
130.23, 130.18, 129.86, 129.53, 129.22 (q, 1C), 128.68 (m, 1C),
127.14, 128.52, 125.81, 123.10, 120.38 (q, CF3), 124.65 (m, 1C),
122.00, 112.91, 104.97, 56.15, 46.65, 8.48. DUIS-MS calculated for
C17H17F3N2O4S, [M−H]-: m/z 401.08, found m/z 400.9. Purity: 97.8%.
Yield 50.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 11.22 (s, 1H), 10.66
(s, 1H), 8.65 (s, 1H), 8.28 (d, 1H, J = 8.0), 8.00 (d, 1H, J = 8.0), 7.63
(s, 1H), 7.41 (d, 1H, J = 8.4), 7.26 (d, 1H, J = 8.4), 56.29 (s, 3H). 13C
NMR (100 MHz, DMSO‑d6) δ:163.11, 155.47, 147.88, 140.19, 135.16,
133.32, 132.49, 129.76, 128.72, 125.32, 118.48, 112.76, 104.90,
56.29. DUIS-MS calculated for C15H11ClF3N3O6S, [M−H]-: m/z 452.01,
found m/z 451.9. Purity: 97.8%.
4.1.34. N-(3-Methoxy-4-Trifluoromethanesulfonylamino-Phenyl)-3,5-Bis-
Trifluoromethyl-Benzamide (30).
4.1.28. 4-Chloro-N-(4-(ethylsulfonamido)-3-methoxylphenyl)-3-
nitrobenzamide (24).
Yield 52.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 11.29 (s, 1H), 10.77
(s, 1H), 8.62 (s, 2H), 8.40 (s, 1H), 7.63 (s, 1H), 7.42 (d, 1H, J = 8.6),
7.28 (d, 1H, J = 8.6), 3.86 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ:
163.19, 155.47, 140.14, 137.40, 131.51, 131.18, 130.95, 130.52 (q,
2C), 129.79, 129.07 (m, 2C), 125.70 (m, 1C), 127.66, 124.94, 122.23,
119.52 (q, 2CF3), 118.53, 112.90, 105.02, 56.30. DUIS-MS calculated
for C17H11F9N2O4S, [M−H]-: m/z 509.03, found m/z 508.9, Purity:
97.7%.
Yield 69.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.56 (s, 1H), 8.86 (s,
1H), 8.64 (d, 1H, J = 2.0), 8.27 (dd, 1H, J1 = 8.4, J2 = 2.0), 7.99 (d,
1H, J = 8.4), 7.57 (d, 1H, J = 1.6), 7.34 (dd, 1H, J1 = 1.6, J2 = 8.8),
7.26 (s, 1H, J = 8.8), 3.83 (s, 3H), 3.01 (q, 2H, J = 7.2), 1.26 (t, 3H,
J = 7.2). 13C NMR (100 MHz, DMSO‑d6) δ: 162.86, 152.33, 147.87,
137.90, 135.27, 133.28, 132.47, 128.60, 127.15, 125.27, 122.20,
112.90, 104.90, 56.15, 45.67, 8.48. DUIS-MS calculated for
C
16H16ClN3O6S, [M−H]-: m/z 412.04, found m/z 411.9. Purity: 96.7%.
4.1.35. 4-Chloro-N-(3-Chloro-4-(MethylsulfonamidoPhenyl)Benzamide
(31).
4.1.29. N-(4-(Ethylsulfonamido)-3-Methoxylphenyl)-3,5-Bis
(Trifluoromethyl)Benzamide (25).
Yield 56.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.49 (s, 1H), 9.41 (s,
1H), 8.06 (s, 1H), 7.99 (d, 2H, J = 8.4), 7.71 (d, 1H, J = 8.4), 7.64 (d,
2H, J = 8.4), 7.44 (d, 1H, J = 8.4), 3.03 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) δ: 165.10, 138.75, 137.20, 133.62, 130.14 (2C), 130.09,
129.77, 129.22, 129.03 (2C), 212.42, 119.97, 41.31. DUIS-MS calcu-
lated for C14H12Cl2N2O3S, [M−H]-:356.99, found 356.8. Purity: 98.9%.
Yield 62.4%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.68 (s, 1H), 8.88 (s,
1H), 8.62 (s, 2H), 8.40 (s, 1H), 7.57 (d, 1H, J = 2.0), 7.35 (q, 1H,
J1 = 2.0, J2 = 8.6), 7.28 (d, 1H, J = 8.6), 3.84 (s, 3H), 3.01 (q, 2H,
J = 7.3), 1.26 (t, 3H, J = 7.3). 13C NMR (100 MHz, DMSO‑d6) δ:
162.93, 153.30, 137.77, 137.51, 131.50, 131.17, 130.84, 130.51 (q,
2C), 129.01 (m, 2C), 127.68, 124.96, 122.25, 119.53 (q, 2CF3), 127.03,
125.67 (m, 1C), 122.38, 113.09, 105.11, 56.20, 46.72, 8.48. DUIS-MS
calculated for C18H16F6N2O4S, [M−H]-: m/z 469.07, found m/z 468.9.
Purity: 98.2%.
4.1.36. 4-Cyano-N-(3-Chloro-4-(MethylsulfonamidoPhenyl)Benzamide
(32).
Yield 66.8%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.66 (s, 1H), 9.43 (s,
1H), 8.12 (d, 2H, J = 8.4), 8.07 (s, 1H), 8.06 (d, 2H, J = 8.4), 7.71 (d,
1H, J = 8.4), 7.46 (d, 1H, J = 8.4), 3.04 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) δ: 164.82, 138.94, 138.46, 133.01 (2C), 130.05, 129.19,
129.04 (2C), 121.51, 120.05, 118.72, 114.59, 99.99, 41.32. DUIS-MS
calculated for C15H12ClN3O3S, [M−H]-: m/z 348.02, found m/z 347.9,
Purity: 98.7%.
4.1.30. 4-Chloro-N-(3-Methoxy-4-Trifluoromethanesulfonylamino-
Phenyl)-B enzamide (26).
Yield 45.5%. 1H NMR (400 MHz, DMSO‑d6) δ: 11.14 (s, 1H), 10.44
(s, 1H), 8.00 (d, 2H, J = 8.4), 7.66 (s, 1H), 7.64 (d, 2H, J = 8.4), 7.40
(d, 1H, J = 8.4), 7.22 (d, 1H, J = 8.4), 3.84 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ: 165.06, 155.45, 140.66, 137.09, 133.86,
130.11 (2C), 129.72, 128.99 (2C), 118.03, 112.58, 104.76, 56.23.
DUIS-MS calculated for C15H12ClF3N2O4S, [M−H]-: m/z 407.01, found
m/z 406.9. Purity: 97.4%.
4.1.37. N-(3-Chloro-4-(MethylsulfonamidoPhenyl)-3-(Trifluoromethyl)
Benzamide (33).
Yield 69.8%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.84 (s, 1H), 9.45 (s,
1H), 8.34 (s, 1H), 8.11 (s, 1H), 7.99 (d, 1H, J = 6.8), 7.80 (m, 3H), 7.46
(d, 1H, J = 8.4), 3.04 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 164.71,
138.72, 135.71, 132.55, 130.19, 130.16, 129.84, 129.52, 129.21 (q,
1C), 130.13, 129.93, 129.11, 128.85 (m, 1C), 125.00 (m, 1H), 121.76,
120.24, 41.29. DUIS-MS calculated for C15H12ClF3N2O3S, [M−H]-: m/z
391.01, found m/z 390.9. Purity: 98.7%.
4.1.31. 4-Cyano-N-(3-Methoxy-4-Trifluoromethanesulfonylamino-Phenyl)
Benzamide (27).
Yield 45.0%. 1H NMR (400 MHz, DMSO‑d6) δ: 11.22 (s, 1H), 10.60
(s, 1H), 8.12 (d, 2H, J = 8.4), 8.05 (d, 2H, J = 8.4), 7.66 (d, 1H,
J = 1.6), 7.40 (dd, 1H, J1 = 1.6, J2 = 8.4), 7.24 (d, 1H, J = 8.4), 3.84
(s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 164.80, 155.46, 140.41,
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Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.1.38. 4-Chloro-N-(3-Chloro-4-(methylsulfonamido)Phenyl)-3-
Nitrobenzamide (34).
4.1.44. N-(3-Chloro-4-(Ethylsulfonamido)Phenyl)-3,5-Bis
(Trifluoromethyl)Benzamide (40).
Yield 68.8%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.72 (s, 1H), 9.45 (s,
1H), 8.65 (s, 1H), 8.27 (d, 1H, J = 8.4), 8.05 (s, 1H), 8.00 (d, 1H,
J = 8.4), 7.70 (d, 1H, J = 8.8), 7.47 (d, 1H, J = 8.8), 3.04 (s, 3H). 13C
NMR (100 MHz, DMSO‑d6) δ: 163.13, 147.88, 138.24, 134.91, 133.35,
132.53, 130.21, 130.03, 129.16, 128.93, 125.35, 121.60, 120.12,
41.34. DUIS-MS calculated for C14H11Cl2N3O5S, [M−H]-: m/z 401.97,
found m/z 401.8. Purity: 98.6%.
Yield 54.1%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.81 (s, 1H), 9.43 (s,
1H), 8.61 (s, 2H), 8.38 (s, 1H), 8.03 (s, 1H), 7.70 (d, 1H, J = 8.6), 7.49
(d, 1H, J = 8.6), 3.14 (q, 2H, J = 7.2), 1.30 (t, 3H, J = 7.2). 13C NMR
(100 MHz, DMSO‑d6) δ: 163.18, 137.95, 137.16, 131.54, 131.20,
130.87, 130.54 (q, 2C), 130.45, 129.73, 129.06 (m,2C), 128.89, 125.78
(m,1C), 127.62, 124.91, 122.20, 119.48 (q, 2CF3), 121.75, 120.24,
47.67, 8.53. DUIS-MS calculated for C17H13ClF6N2O3S, [M−H]-: m/z
473.02, found m/z 472.9. Purity: 97.2%.
4.1.39. N-(3-Chloro-4-(Methylsulfonamido)Phenyl)-3,5-Bis
4.1.45. 4-Chloro-N-(3-Chloro-4-(Trifluoromethylsulfonamido)-Phenyl)-
Benzamide (41).
(Trifluoromethyl)Benzamide (35).
Yield 51.2%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.80 (s, 1H), 9.45 (s,
1H), 8.62 (s, 2H), 8.40 (s, 1H), 8.05 (s, 1H), 7.73 (d, 1H, J = 8.8), 7.50
(d, 1H, J = 8.8), 3.05 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 163.21,
138.14, 137.20, 131.54, 131.21, 130.88, 130.55 (q, 2C) 130.33,
130.00, 129.11, 129.07 (m, 2C), 127.64, 124.93, 122.23, 119.51 (q,
2CF3), 125.83 (m, 1C), 121.80, 120.26, 40.08. DUIS-MS calculated for
Yield 48.2%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.72 (s, 1H), 8.11
(d, 2H, J = 8.4), 8.10 (s, 1H), 8.06 (d, 2H, J = 8.4), 7.75 (d, 1H,
J = 8.8), 7.46 (d, 1H, J = 8.8). 13C NMR (100 MHz, DMSO‑d6)
δ:164.96, 139.66, 138.87, 133.02 (2C), 131.73, 130.44, 129.08 (2C),
121.50, 120.08, 118.71, 114.66. DUIS-MS calculated for
C
14H9Cl2F3N2O3S, [M−H]-: m/z 410.96, found m/z 410.8. Purity:
C
16H11ClF6N2O3S, [M−H]-: m/z 459.00, found m/z 458.8. Purity:
98.2%.
99.2%.
4.1.46. 4-Cyano-N-(3-Chloro-4-(Trifluoromethylsulfonamido)-Phenyl)-
Benzamide (42).
4.1.40. 4-Chloro-N-(3-Chloro-4-(Ethylsulfonamido)Phenyl)Benzamide
(36).
Yield 50.5%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.70 (s, 1H), 8.11
(d, 2H, J = 8.4), 8.08 (d, 1H, J = 2.0), 8.06 (d, 2H, J = 8.4), 7.73 (dd,
1H, J1 = 8.8, J2 = 2.0), 7.45 (d, 1H, J = 8.8). 13C NMR (100 MHz,
DMSO‑d6) δ: 164.92, 138.89, 137.63, 133.02 (2C), 131.55, 130.21,
125.35, 129.07 (2C), 121.49, 120.06, 118.71, 114.63. DUIS-MS calcu-
lated for C15H9ClF3N3O3S, [M−H]-: m/z 401.99, found m/z 401.8.
Purity: 97.2%.
Yield 66.3%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.48 (s, 1H), 9.38 (s,
1H), 8.05 (s, 1H), 7.99 (d, 2H, J = 8.0), 7.69 (d, 1H, J = 8.6), 7.63 (d,
2H, J = 8.0), 7.45 (d, 1H, J = 8.6), 3.12 (q, 2H, J = 7.2), 1.30 (t, 3H,
J = 7.2). 13C NMR (100 MHz, DMSO‑d6) δ: 165.07, 138.57, 137.19,
133.63, 130.14, 129.87, 129.83, 129.02, 121.40, 119.96, 99.99, 47.61,
8.56. DUIS-MS calculated for C15H14Cl2N2O3S, [M−H]-: m/z 371.00,
found m/z 370.9. Purity: 98.1%.
4.1.47. N-(3-Chloro-4-(Trifluoromethylsulfonamido)-Phenyl)-3-
(Trifluoromethyl)-Benzamide (43).
Yield 49.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.73 (s, 1H), 8.31 (s,
1H), 8.28 (d, 1H, J = 7.6), 8.11 (s, 1H), 8.00 (d, 1H, J = 7.6), 7.81 (t,
1H, J = 7.6), 7.78 (d, 1H, J = 8.8), 7.46 (d, 1H, J = 8.8). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.85, 139.87, 135.73, 132.42, 131.80,
130.54, 130.31, 130.20, 129.89, 129.58, 129.27 (q, 1C), 128.96 (m,
1C), 124.83 (m, 1C), 128.47, 125.76, 123.05, 120.34 (q, CF3), 121.58,
120.13, 118.66. DUIS-MS calculated for C15H9ClF6N2O3S, [M−H]-: m/z
444.98, found m/z 444.8. Purity: 98.6%.
4.1.41. 4-Cyano-N-(3-Chloro-4-(Ethylsulfonamido)Phenyl)Benzamide
(37).
Yield 62.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.65 (s, 1H), 9.41 (s,
1H), 8.06 (s, 1H), 8.11 (d, 2H, J = 8.0), 8.05 (d, 2H, J = 8.0), 7.69 (d,
1H, J = 8.6), 7.46 (d, 1H, J = 8.6), 3.13 (q, 2H, J = 7.2), 1.29 (t, 3H,
J = 7.2). 13C NMR (100 MHz, DMSO‑d6) δ: 164.80, 138.94, 138.28,
133.00 (2C), 130.16, 129.79, 129.04 (2C), 128.98, 121.48, 120.05,
118.72, 114.60, 47.63, 8.56. DUIS-MS calculated for C16H14ClN3O3S,
[M−H]-: m/z 362.04, found m/z 361.9. Purity: 97.8%.
4.1.48. 4-Chloro-N-(3-Chloro-4-(Trifluoromethylsulfonamido)Phenyl)-3-
Nitrobenzamide (44).
4.1.42. N-(3-Chloro-4-(Ethylsulfonamido)Phenyl)-3-(Trifluoromethyl)
Benzamide (38).
Yield 49.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.79 (s, 1H), 8.645
(s, 1H), 8.27 (d, 1H, J = 8.4), 8.08 (s, 1H), 8.10 (d, 1H, J = 8.4), 7.74
(d, 1H, J = 8.8), 7.47 (d, 1H, J = 8.8). 13C NMR (100 MHz, DMSO‑d6)
δ: 163.29, 147.87, 139.51, 134.83, 133.39, 132.54, 131.78, 130.54,
128.91, 125.41, 121.89, 121.58, 120.14. DUIS-MS calculated for
Yield 62.5%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.66 (s, 1H), 9.41 (s,
1H), 8.31 (s, 1H), 8.28 (d, 1H, J = 7.4), 8.06 (s, 1H), 8.00 (d, 1H,
J = 7.4), 7.81 (t, 1H, J = 7.4), 7.72 (d, 1H, J = 8.6), 7.47 (d, 1H,
J = 8.6), 3.13 (q, 2H, J = 7.2), 1.30 (t, 3H ,J = 7.2). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.68, 138.38, 135.82, 132.38, 130.29,
130.08, 130.21, 129.89, 129.57, 129.25 (q, 1C), 129.80, 128.97,
128.88 (m, 1C), 128.49, 125.78, 123.07, 120.36 (q, CF3), 124.79 (m),
121.58, 120.11, 47.63, 8.56. DUIS-MS calculated for C16H14ClF3N2O3S,
[M−H]-: m/z 405.03, found m/z 404.9. Purity: 99.1%.
C
14H8Cl2F3N3O5S, [M−H]-: m/z 455.94, found m/z 455.8. Purity:
97.8%.
4.1.49. N-(3-Chloro-4-(Trifluoromethylsulfonamido)Phenyl)-3,5-Bis
(Trifluoromethyl)Benzamide (45).
Yield 48.8%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.85 (s, 1H), 8.61 (s,
2H), 8.40 (s, 1H), 8.07 (s, 1H), 7.75 (d, 1H, J = 8.8), 7.48 (d, 1H,
J = 8.8). 13C NMR (100 MHz, DMSO‑d6) δ: 163.34, 137.16, 131.63,
131.54, 131.21, 130.78, 130.54 (q, 2C), 130.87, 130.31, 129.09 (m,
2C), 125.87 (m, 1C), 127.63, 124.92, 122.21, 119.49 (q, 2CF3), 121.98,
121.75, 120.25. DUIS-MS calculated for C16H8ClF9N2O3S, [M−H]-: m/z
512.97, found m/z 512.8. Purity: 97.3%.
4.1.43. 4-Chloro-N-(3-Chloro-4-(Ethylsulfonamido)-Phenyl)-3-
Nitrobenzamide (39).
Yield 67.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.70 (s, 1H), 9.42 (s,
1H), 8.64 (s, 1H), 8.27 (d, 1H, J = 8.4), 8.03 (s, 1H), 8.00 (d, 1H,
J = 8.4), 7.69 (d, 1H, J = 8.8), 7.48 (d, 1H, J = 8.8), 3.13 (q, 2H,
J = 7.2), 1.30 (t, 3H, J = 7.2). 13C NMR (100 MHz, DMSO‑d6) δ:
163.11, 147.88, 138.06, 134.92, 133.34, 132.53, 130.31, 129.77,
128.97, 128.82, 125.35, 121.55, 120.10, 47.65, 8.56. DUIS-MS calcu-
lated for C15H13Cl2N3O5S, [M−H]-: m/z 415.99, found m/z 415.8.
Purity: 98.8%.
4.1.50. 4-Chloro-N-(3-Fluoro-4-(Methylsulfonamido)Phenyl)Benzamide
(46).
Yield 52.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.57 (s, 1H), 9.48 (s,
1H), 7.99 (d, 2H, J = 8.0), 7.84 (d, 1H, J = 12.8), 7.64 (d, 2H, J = 8.0),
9

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.1.56. 4-Cyano-N-(3-Fluoro-4-(Ethylsulfonamido)Phenyl)Benzamide
7.54, (d, 1H, J = 8.6), 7.37 (t, 1H, J = 8.6), 3.01 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ: 165.09, 157.64, 155.21 (1C), 138.87, 138.77
(1C), 137.17, 133.69, 130.14 (2C), 129.02 (2C), 128.43, 120.47,
120.34 (1C), 116.75, 116.72 (1C), 108.45, 108.20 (1C), 40.07. DUIS-
MS calculated for C14H12ClFN2O3S, [M−H]-: m/z 341.02, found m/z
340.9. Purity: 96.8%.
(52).
Yield 53.2%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.68 (s, 1H), 9.53 (s,
1H), 8.11 (d, 2H, J = 8.4), 8.05 (d, 2H, J = 8.4), 7.84(d, 1H, J = 12.8),
7.53 (d, 2H, J = 8.8), 7.39 (t, 1H, J = 8.8), 3.08 (q, 2H, J = 7.2), 1.28
(t, 3H, J = 7.2) . 13C NMR (100 MHz, DMSO‑d6) δ: 164.80, 157.37,
154.94 (1C), 138.99, 138.39, 138.29 (1C), 133.00 (2C), 129.04 (2C),
128.25, 120.79, 120.65 (1C), 128.73, 116.84, 116.80 (1C), 114.56,
108.48, 108.23 (1C), 46.72, 8.48. DUIS-MS calculated for
4.1.51. 4-Cyano-N-(3-Fluoro-4-(Methylsulfonamido)Phenyl)Benzamide
(47).
Yield 48.9%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.68 (s, 1H), 9.51 (s,
1H), 8.11 (d, 2H, J = 8.4), 8.05 (d, 2H, J = 8.4), 7.85 (d,1H, J = 12.4),
7.54 (d, 1H, J = 8.8), 7.39 (t, 1H, J = 8.8), 3.02 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ: 164.82, 157.56, 155.13 (1C), 139.00, 138.55,
138.45 (1C), 133.00 (2C), 129.04 (2C), 128.38, 120.79, 120.66 (1C),
118.72, 116.84, 116.81 (1C), 114.57, 108.54, 108.29 (1C), 56.50.
DUIS-MS calculated for C15H12FN3O3S, [M−H]-: m/z 332.05, found m/
z 332.0. Purity: 98.8%.
C
16H14FN3O3S, [M−H]-: m/z 346.07, found m/z 345.8. Purity: 97.7%.
4.1.57. N-(3-Fluoro-4-(Ethylsulfonamido)Phenyl)-3-(Trifluoromethyl)
Benzamide (53).
Yield 52.3%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.66 (s, 1H), 9.531
(s, 1H), 8.29 (s, 1H), 8.27 (d, 1H, J = 7.2), 8.00 (d, 1H, J = 7.2), 7.83
(m, 2H), 7.53 (d, 1H, J = 8.0), 7.40 (d, 1H, J = 8.0), 3.09 (q, 2H,
J = 7.0), 1.28 (t, 3H, J = 7.0). 13C NMR (100 MHz, DMSO‑d6) δ:
164.69, 157.39, 154.97 (1C), 138.48, 138.38(1C), 135.87, 132.36,
130.29, 130.21, 129.88, 129.56, 129.24 (q, 1C), 128.87 (m, 1C),
128.26, 124.73 (m, 1C), 120.71, 120.58 (1C), 116.89, 116.85 (1C),
108.57, 108.31 (1C), 46.74, 8.49. DUIS-MS calculated for
4.1.52. N-(3-Fluoro-4-(Methylsulfonamido)Phenyl)-3-(Trifluoromethyl)
Benzamide (48).
Yield 43.5%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.66 (s, 1H), 9.50 (s,
1H), 8.30 (s, 1H), 8.28 (d, 1H, J = 8.0), 8.00 (d, 1H, J = 8.0), 7.85 (d,
1H, J = 12.8), 7.81 (d, 1H, J = 8.0), 7.55 (d, 1H, J = 8.8), 7.40 (d, 1H,
J = 8.8). 3.02 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ: 164.71,
157.59, 155.16 (1C), 138.63, 138.53(1C), 135.88, 132.36, 130.30,
130.21, 129.89, 129.57, 129.90 (q, 1C), 128.44, 125.78, 123.07,
120.36 (q, CF3), 128.38, 124.73 (m, 1C), 123.07, 120.71, 120.58 (1C),
116.90, 116.87 (1C), 108.63, 108.38 (1C), 40.06. DUIS-MS calculated
for C15H12F4N2O3S, [M−H]-: m/z 375.04, found m/z 374.8. Purity:
98.2%.
C
16H14F4N2O3S, [M−H]-: m/z 389.06, found m/z 388.9. Purity: 97.5%.
4.1.58. 4-Chloro-N-(3-Fluoro-4-(Ethylsulfonamido)Phenyl)-3-
Nitrobenzamide (54).
Yield 52.5%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.75 (s, 1H), 9.55 (s,
1H), 8.64 (s, 1H), 8.27 (dd, 1H, J1 = 2.0, J2 = 8.4), 8.00 (d, 1H,
J = 8.4), 7.83 (d, 1H, J = 12.8), 7.52 (dd, 1H, J1 = 8.8, J2 = 2.0), 7.40
(t, 1H, J = 8.8), 3.09 (q, 2H, J = 7.3), 1.28 (t, 3H, J = 7.3). 13C NMR
(100 MHz, DMSO‑d6) δ: 163.12, 157.33, 155.00 (1C), 147.87, 138.17,
138.07 (1C), 134.96, 133.35, 132.50, 128.78, 128.24, 128.23 (1C),
125.37, 120.94, 120.81 (1C), 116.91, 116.88 (1C), 109.58, 109.33
(1C), 46.766, 6.48. DUIS-MS calculated for C15H13ClFN3O5S, [M−H]-:
m/z 400.02, found m/z 399.9. Purity: 98.8%.
4.1.53. 4-Chloro-N-(3-Fluoro-4-(Methylsulfonamido)Phenyl)-3-
Nitrobenzamide (49).
Yield 42.6%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.73 (s, 1H), 9.521
(s, 1H), 8.64 (s, 1H), 8.27 (d, 1H, J = 8.4), 7.999 (d, 1H, J = 8.4), 7.83
(d, 1H, J = 12.8), 7,53 (d, 1H, J = 8.8), 7.40 (t, 1H, J = 8.8, 3.023 (s,
3H). 13C NMR (100 MHz, DMSO‑d6) δ: 163.14, 157.52, 155.10 (1C),
149.89, 138.31, 138.21 (1C), 134.97, 133.34, 132.52, 128.80, 128.37,
125.35, 120.95, 120.92 (1C), 116.92, 116.56 (1C), 108.64, 108.40
(1C), 40.06. DUIS-MS calculated for C14H11ClFN3O5S, [M−H]-: m/z
386.00, found m/z 385.8; Purity: 96.3%.
4.1.59. N-(3-Fluoro-4-(Ethylsulfonamido)Phenyl)-3,5-Bis
(Trifluoromethyl)Benzamide (55).
Yield 47.9%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.87 (s, 1H), 9.56 (s,
1H), 8.62 (s, 2H), 8.40 (s, 1H), 7.84 (d, 1H, J = 12.8), 7.53 (d, 1H,
J = 8.8), 7.42 (t, 1H, J = 8.8), 3.10 (q, 2H, J = 7.2), 1.28 (t, 3H,
J = 7.2). 13C NMR (100 MHz, DMSO‑d6) δ: 163.20, 157.30, 154.87
(1C), 138.07, 137.97 (1C), 137.22, 131.51, 131.18, 130.84, 130.54 (q,
2C), 129.08 (m, 2C), 128.17, 125.77 (m, 1C), 127.65, 124.93, 122.22,
119.50 (2CF3), 121.07, 120.94 (1C), 117.07, 117.03 (1C), 108.76,
108.50 (1C), 46.80, 8.49. DUIS-MS calculated for C17H13F7N2O3S,
[M−H]-: m/z 457.05, found m/z 456.8. Purity: 98.5%.
4.1.54. N-(3-Fluoro-4-(Methylsulfonamido)Phenyl)-3,5-Bis
(Trifluoromethyl)Benzamide (50).
Yield 41.7%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.85 (s, 1H), 9.54 (s,
1H), 8.61 (s, 2H), 8.40 (s, 1H), 7.84 (d, 1H, J = 12.8), 7.54 (d, 1H,
J = 8.8), 7.42 (d, 1H, J = 8.8), 3.03 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6) δ: 163.22, 157.50, 155.06 (1C), 138.21, 138.11 (1C), 137.23,
131.51, 131.19, 130.86, 130.53 (q, 2C), 129.08 (m, 2C), 128.27,
127.65, 124.93, 122.22, 119.50 (q, 2CF3), 125.82 (m, 1C), 121.08,
120.95 (1C), 117.06, 117.04 (1C), 108.81, 108.56 (1C), 40.06. DUIS-
MS calculated for C16H11F7N2O3S, [M−H]-: m/z 443.03, found m/z
442.8. Purity: 97.8%.
4.1.60. 4-Chloro-N-(3-Fluoro-4-(Trifluoromethylsulfonamido)Phenyl)
Benzamide (56).
Yield 45.6%. 1H NMR (400 MHz, DMSO‑d6) δ: 16.20 (s, 1H), 10.59
(s, 1H), 7.99 (d, 2H, J = 8.4), 7.87 (d, 1H, J = 12.8), 7.64 (d, 2H,
J = 8.4), 7.58 (d, 1H, J = 8.4), 7.39 (t, 1H, J = 8.4). 13C NMR
(100 MHz, DMSO‑d6) δ: 165.26, 159.04, 158.57 (1C), 140.28, 140.31,
140.13 (1C), 137.26, 133.59, 130.17 (2C), 129.04 (2C), 119.34, 119.14
(1C), 116.82, 116.79 (1C), 108.32, 108.07(1C). DUIS-MS calculated for
4.1.55. 4-Chloro-N-(3-Fluoro-4-(Ethylsulfonamido)Phenyl)Benzamide
(51).
C
14H9ClF4N2O3S, [M−H]-: m/z 394.99, found m/z 393.8, Purity:
97.8%.
Yield 50.2%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.51 (s, 1H), 9.51 (s,
1H), 7.99 (d, 2H, J = 8.4), 7.83 (dd, 1H, J1 = 2.0, J2 = 12.8), 7.64 (d,
2H, J = 8.4), 7.52 (d, 1H, J = 8.8), 7.37 (t, 1H, J = 8.8), 3.08 (q, 2H,
J = 7.2), 1.28 (t, 3H, J = 7.2). 13C NMR (100 MHz, DMSO‑d6) δ:
165.08, 157.44, 155.01 (1C), 138.70, 138.60 (1C), 137.16, 133.68,
130.13, 129.02, 128.33, 120.46, 120.33 (1C), 116.74, 116.70 (1C),
108.38, 108.13 (1C), 46.70, 8.48. DUIS-MS calculated for
4.1.61. 4-Cyano-N-(3-Fluoro-4-(Trifluoromethylsulfonamido)Phenyl)
Benzamide (57).
Yield 47.6%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.77 (s, 1H), 8.11
(d, 2H, J = 8.0), 8.06 (d, 2H, J = 8.0), 7.89 (d, 1H, J = 12.8), 7.59 (d,
1H, J = 8.8), 7.42 (t, 1H, J = 8.8). 13C NMR (100 MHz, DMSO‑d6) δ:
165.02, 158.67, 156.21 (1C), 138.89, 133.16, 133.14 (1C), 133.01
(2C), 130.31, 129.09 (2C), 121.94, 121.91 (1C), 118.70, 116.93,
116.90 (1C), 114.66, 108.40, 108.15 (1C). DUIS-MS calculated for
C
15H14ClFN2O3S, [M−H]-: m/z 355.03, found m/z 354.9. Purity:
97.5%.
10

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
C15H9F4N3O3S, [M−H]-: m/z 386.02, found m/z 385.8. Purity: 97.9%.
4.3. Cell culture
HEK293 kidney cells and mouse macrophage RAW264.7 cells were
obtained from ATCC (Rockville, MD) and maintained in RPMI1640
medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-
Glutamine, 1 mM sodium pyruvate, and 100 U/mL penicillin-strepto-
mycin. FBS was heat inactivated for 30 min at 56 °C. Mammalian cells
were grown at 37 °C in a Heraeus water-jacketed incubator with 5%
CO2. Bloodstream form T. b. brucei Lister 427 cells were cultured in
HMI-9 medium with 10% FBS at 37 °C in a Heraeus water-jacketed
incubator with 7.5% CO2. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
boxymethoxyphenyl)-2-(4-sulfophenyl)–2H-tetrazolium (MTS) re-
agents were ordered from Promega life science (Madison, WI). 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was
ordered from Sigma-Aldrich (Milwaukie, WI).
4.1.62. N-(3-Fluoro-4-(Trifluoromethylsulfonamido)-Phenyl)-3-
(Trifluoromethyl)Benzamide (58).
Yield 47.5%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.78 (s, 1H), 8.30 (s,
1H), 8.29 (d, 2H, J = 7.6), 8.00 (d, 1H, J = 7.6), 7.91 (d, 1H, J = 12.4),
7.83 (t, 1H, J = 7.6), 7.61 (d, 1H, J = 8.2), 7.42 (t, 1H, J = 8.2) . 13C
NMR (100 MHz, DMSO‑d6) δ: 164.90, 158.68, 156.23 (1C), 140.42,
140.34 (1C), 135.78, 132.42, 132.38, 130.30, 130.27, 130.22, 129.90,
129.58, 129.26 (q, 1C), 128.917 (m, 1C), 124.81 (m, 1C), 123.06,
122.49 (1C), 116.99, 116.96 (1C), 108.50, 108.25 (1C). DUIS-MS cal-
culated for C15H9F7N2O3S, [M−H]-: m/z 429.01, found m/z 428.8.
Purity: 98.3%.
4.1.63. 4-Chloro-N-(3-Fluoro-4-(Trifluoromethylsulfonamido)Phenyl)-3-
Nitrobenzamide (59).
4.4. Mammalian cell viability analysis
Yield 43.5%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.76 (s, 1H), 10.48
(s, 1H), 8.65 (s, 1H), 8.28 (dd, 1H, J1 = 8.4, J2 = 1.2), 8.00 (t, 1H,
J = 8.0), 7.86 (d, 1H, J = 12.8), 7.61 (m, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ: 163.34, 158.67, 156.22 (1C), 147.88, 140.24, 140.14 (1C),
134.87, 133.38, 132.54, 130.37, 128.90, 125.41, 121.89, 121.34 (1C),
117.01, 116.98 (1C), 108.50, 108.25 (1C). DUIS-MS calculated for
The MTT assay was used to examine the effect of tubulin inhibitors
on the growth of HEK 293 and RAW 264.7 cells in four replications.
3000 cells per well were seeded with RPMI1640 medium in 96-well
flat-bottomed plates for 24 h and were then exposed to various con-
centrations of test compounds dissolved in DMSO (final concentra-
tion ≤ 0.1%) in medium for 48 h. Controls received DMSO at a same
concentration as that in drug-treated cells. Cells were incubated in
200 µL of 0.5 mg/mL of MTT reagent diluted in fresh media at 37 °C for
3 h. Supernatants were removed from the wells, and the reduced MTT
dye was solubilized with 200 µL/well DMSO. Absorbance at 570 nm
C
14H8ClF4N3O5S, [M−H]-: m/z 439.97, found m/z 438.8.
Purity:98.1%.
4.1.64. N-(3-Fluoro-4-(Trifluoromethylsulfonamido)Phenyl)-3,5-Bis
(Trifluoromethyl)Benzamide (60).
Yield 40.2%. 1H NMR (400 MHz, DMSO‑d6) δ: 10.94 (s, 1H), 8.61 (s,
2H), 8.40 (s, 1H), 7.90 (d, 1H, J = 12.4), 7.59 (d, 1H, J = 8.6), 7.45 (t,
1H, J = 8.6). 13C NMR (100 MHz, DMSO‑d6) δ: 163.43, 158.67, 156.21
(1C), 140.17, 140.07 (1C), 137.15, 131.56, 131.19, 130.86, 130.53 (q,
2C), 130.35, 129.15, 129.13 (3C), 125.84 (m, 1C), 127.63, 124.91,
122.20, 119.49 (q, 2CF3), 117.13, 117.10 (1C), 108.65, 108.40 (1C).
DUIS-MS calculated for C16H8F10N2O3S, [M−H]-: m/z 497.00, found
m/z 497.0. Purity: 98.2%.
was determined on
a
SpectraMax Plus384 spectrophotometer
(Molecular Devices). Data obtained with quadruplication were nor-
malized and fitted to a dose–response curve using GraphPad Prism v.5
(GraphPad).
4.5. T. brucei cell viability analysis
The (MTS) assay was used to examine the effect of tubulin inhibitors
on T. brucei cell viability.21 5000 cells of T. brucei were seeded in 96
well plates and treated with 0.1% DMSO and tested agents at various
concentrations for 48 h at 37 °C. Subsequently, 20 μL of MTS (5% PMS)
from the CellTiter Cell Proliferation Assay (Promega) was added to
200 μL of T. brucei culture in each well and incubated at 37 °C for 3 h.
Soluble formazan, produced by viable cells due to reduction of MTS,
4.2. Solubility assay
Compounds 5, 57 and 15′ were used in the determination of their
log P values. This method follows the OECD test guidelines for the slow
stir method, with slight modification due to the data collection
23
method.22,
The data was collected on a UV–Vis spectrophotometer
was measured at 490 nm with
a SpectraMax Plus 384 spectro-
Shimadzu UV-1280 and standard UV Quartz 10 mm cuvette was used.
The chemical was purchased from VWR brand EMPLURA 1-octanol
99% purity. The Flask where equipped with Teflon coated magnetic
stirrers and Coring PC420D stir plates where used. The 1-octanol and
DDH2O where placed into two separate containers containing both
phases to become mutual saturated by allowing them to stir for 48 h.
Then the two phases where separated by a separation funnel. Com-
pounds 5, 57, and 15′ now were dissolved in 1-octanol pre-saturated
with water. These stock solutions are then centrifuged at 5000 rpm for
5 min. The supernatant was taken to be the working solution. Take 5 mL
working solution of each into new clear flask, and add identical volume
water saturated with 1-octanol, then stir at room temperature for 48 h.
After that, stop stirring; take two-phase solution out, centrifuge at
5000 rpm for 5 min to separate this two-phase system. Removal of the
1-octanol phase from the solution then transferred for UV spectro-
photometry to achieve absorbance at the correct wavelength. According
photometer (Molecular Devices). Data obtained with quadruplication
were normalized and fitted to a dose-response curve using GraphPad
Prism v.5 (GraphPad).
4.6. T. brucei cell lysate preparation and western blot assay
T. brucei cells were incubated with 0.5% DMSO and different doses
of compound 5 and compound 57 for 12 h. Cell pellets were harvested
by centrifugation at 1500 rpm for 10 min at room temperature, washed
twice with 1X TDB buffer (5 mM KCl, 80 mM NaCl, 1 mM MgSO4,
20 mM Na2HPO4, 2 mM NaH2PO4, 20 mM glucose, pH 7.4) with pro-
tease inhibitor (Roche) and PMSF (1 mM, Amresco), and lysed with
30 µL of lysis buffer (80 mM Pipes, pH 6.8, 2 mM EGTA, 1 mM MgCl2,
0.15% Triton X-100, 10% glycerol, protease inhibitor (Roche) and
PMSF) at 4 °C for 5 min. The cell lysate was centrifuged at 15,000 g for
30 min at 4 °C, and the supernatant was transferred into a fresh
Eppendorf tube. Pellets were re-suspended in 40 µL of lysis buffer
(RIPA, Thermo), and the sample was boiled at 95 °C for 15 min. Protein
lysates from equal number of cells (1.83 × 107, split into tubulin non-
polymerized and polymerized fractions) were separated on 10% poly-
acrylamide gels by electrophoresis. Proteins were transferred onto
PVDF membranes. Tubulin antibody (Prod # MA1-19162, Life tech-
nology) was used in the following western analysis.
to the procedure, concentrations were determined by UV measurement
23
so, equation written as
:
Ax
Log P= log
A0 Ax
where A0 and Ax are the initial and final absorbance values of organic
layer.
11

A. Zhao, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Acknowledgements
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