Synthesis and biological evaluation of selective tubulin inhibitors as anti-trypanosomal agents

Article abstract of DOI:10.1016/j.bmc.2017.04.009

African trypanosomiasis is still a threat to human health due to the severe side-effects of current drugs. We identified selective tubulin inhibitors that showed the promise to the treatment of this disease, which was based on the tubulin protein structur

Full text of DOI:10.1016/j.bmc.2017.04.009

Accepted Manuscript
Synthesis and biological evaluation of selective tubulin inhibitors as anti-try-
panosomal agents
Viharika Bobba, Vishal Nanavaty, Nethrie D. Idippily, Anran Zhao, Bibo Li,
Bin Su
PII:
S0968-0896(17)30437-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.009
BMC 13679
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 March 2017
30 March 2017
5 April 2017
Please cite this article as: Bobba, V., Nanavaty, V., Idippily, N.D., Zhao, A., Li, B., Su, B., Synthesis and biological
evaluation of selective tubulin inhibitors as anti-trypanosomal agents, Bioorganic & Medicinal Chemistry (2017),
doi: http://dx.doi.org/10.1016/j.bmc.2017.04.009
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Revision: BMC_2017_354
Synthesis and biological evaluation of selective tubulin inhibitors as
anti-trypanosomal agents
Viharika Bobbaa, Vishal Nanavatyb, Nethrie D. Idippilya, Anran Zhaoa, 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
TITLE RUNNING: Anti-trypanosomal agents
*To whom correspondence should be addressed:
Bin Su, Ph.D.
Bibo Li, Ph.D.
Center for Gene Regulation in Health and Disease Center for Gene Regulation in Health and Disease
Department of Chemistry
College of Sciences and Health Professions
Cleveland State University
2121 Euclid Ave., Cleveland, OH 44115, USA
Phone: 216-687-9219
Fax: 216-687-9298
Department of Biological, Geological
Environmental Sciences
College of Sciences and Health Professions
Cleveland State University
2121 Euclid Ave., Cleveland, OH 44115, USA
Phone: 216-687-2444
&
Email: B.su@csuohio.edu
Fax:
216-687-6972
Email: b.li37@csuohio.edu

Abstract
African trypanosomiasis is still a threat to human health due to the severe side-effects of current
drugs. We identified selective tubulin inhibitors that showed the promise to the treatment of this disease,
which was based on the tubulin protein structural difference between mammalian and trypanosome cells.
Further lead optimization was performed in the current study to improve the efficiency of the drug
candidates. We used Trypanosoma brucei brucei cells as the parasite model, and human normal kidney
cells and mouse macrophage cells as the host model to evaluate the compounds. One new analog
showed great potency with an IC50 of 70 nM to inhibit the growth of trypanosome cells and did not
affect the viability of mammalian cells. Western blot analyses reveal that the compound decreased
tubulin polymerization in T. brucei cells. A detailed structure activity relationship (SAR) was
summarized that will be used to guide future lead optimization.
Key words: trypanosomiasis; tubulin inhibitor; selective index; drug development

1. Introduction
Human African trypanosomiasis, also known as African sleeping sickness, is a vector-borne
parasitic disease in sub-Saharan Africa where medical resources are limited.1-3 Currently there are four
drugs available for the treatment of human African trypanosomiasis, including Suramin, Pentamidine,
Melarsoprol, and Eflornithine.4 However, these drugs have many limitations: 1) high toxicity to the host;
2) only available as intramuscular or intravenous injections; 3) narrow anti-trypanosomiasis spectrum;
and 4) high cost to administer the drugs to the patients. Overall, these drugs are not effective in the
treatment of the disease, and it is urgently needed to develop more effective and inexpensive
chemotherapeutic agents for the treatment of human African trypanosomiasis. 4-6
Tubulin is a well-known target in anti-cancer drug discovery field, and several tubulin interfering
7
agents are the first line chemotherapeutic drugs for cancer treatment in clinic. Tubulin also plays an
essential role during trypanosome cell division. The fast growing rate of trypanosome cells makes the
parasites heavily depend on tubulin polymerization/depolymerization for proliferation.8 More
importantly, tubulin is important for the locomotion of trypanosome cells, which is an essential function
for them to survive. Therefore, tubulin inhibitors have the potential activity to block the T. brucei cell
division and decrease the locomotion function of the flagellum as well, which will lead to cell death.9
These factors suggest that there are multiple advantages of tubulin inhibitors as a novel treatment to
human African trypanosomiasis.
Tubulin is a highly conserved protein within different species. However, different susceptibility to
antimitotic agents are known to exist among different organisms, indicating there are differences of
tubulin structures among different species.10 Based on the differences of the colchicine binding pocket
between mammalian and T. brucei tubulins, selective tubulin inhibitors were developed that showed
great in vitro potency to inhibit T. brucei cell growth without harming mammalian cells at the similar

11
concentrations. Some compounds exhibited very specific inhibitory effect on T. brucei cell growth,
with a selective index (IC50 inhibiting human cell growth/IC50 inhibiting T. brucei cell growth) beyond
100. 12 To our surprise, the pharmacophore of these compounds enhancing the mammalian cell growth
inhibition is different to the pharmacophore improving the T. brucei cell growth inhibition. In addition,
these compounds showed in vivo activity to decease T. brucei cell growth in the infected mice. However,
12
the compounds were not potent enough to totally clear the infection yet. Herein, we further lead
optimized the compound based on the summarized structure activity relationship, and identified one
compound with better potency and selectivity than previous ones.
2. Results and Discussion
2.1. Synthesis of the new tubulin inhibitors
In our previous studies, the developed selective sulfonamide tubulin inhibitors showed great in vitro
activity to inhibit the growth of T. brucei cells. However, the potency of the compounds was not good
12
enough to clear up the infections in the animals completely.
In the present study, further lead
optimization was performed to develop more effective analogs. A total of 18 compounds were
synthesized using combinatorial chemistry strategy to increase the selectivity and anti-parasite potency
of the compounds. We modified the R1, R2, and R3 moieties of the core structure with different
substituents systematically (Figure 1).
Figure 1. Core structure of the new derivatives
For these new compounds, we explored different structures at R1 domain including aromatic rings
and aliphatic groups. Next, the R2 moiety of the scaffold was modified with different substituted

sulfonyl chlorides in order to generate different sulfonamide groups. Then, the nitro group was reduced
to amino group in order to introduce R3 group. Last, acylation of the amino group with different
aromatic substituents resulted in different R3 moiety. The synthesis is illustrated in Scheme 1.
O2N
O
R1
O2N
O
R1
O2N
OH
K2CO3, DMF
NaH,
NH
O S O
R2
DMF,R2SO2Cl
substituted
benzyl chloride
or Hexyl Iodine
NH2
NH2
B
A
C
2a. R1: 3-(trifluoromethyl)benzyl
2b. R1: 4-(trifluoromethyl)benzyl
2c. R1: 2-(trifluoromethyl)benzyl
3a. R1: 3-(trifluoromethyl)benzyl;
R2: Methanesulfonyl
3b. R1: 2-(trifluoromethyl)benzyl;
R2: Methanesulfonyl
3c. R1: 2-methyl, 5-
2d. R1: 2-methyl, 5-(trifluoromethyl)benzyl
2e. R1: 1-hexyl
2f. R1: 4-cyano benzyl
(trifluoromethyl)benzyl
R2: Methanesulfonyl
3d. R1: 1-hexyl; R2: Methanesulfonyl
3e. R1: 4-cyano benzyl;
R2: Ethanesulfonyl
3f. R1: 3-(trifluoromethyl)benzyl;
R2: Alpha-Toluene sulfonyl
3g. R1: 4-(trifluoromethyl)benzyl;
R2: Alpha-Toluene sulfonyl
3h. R1: 2-(trifluoromethyl)benzyl;
R2: Alpha-Toluene sulfonyl
3i. R1: 1-hexyl;
R2: Alpha-Toluene sulfonyl
H
O2N
O
R1
H2N
O
R1
O
N
O
R1
CH3I,
NaH, DMF
FeCl3-Zn,
DMF-H2O
K2CO3, 1,4-Dioxane,
substituted acetyl
chloride
R3
N
O S O
R2
N
O S O
R2
N
O S O
R2
F
E
D
R3: 3,5-bis(trifluoromethyl)benzoyl
R3: 2,4-bis(trifluoromethyl)benzoyl
R3: benzo[d][1,3]dioxole-5-carbonyl
R3: 3-cyano benzoyl
Compounds 1-16 are from
different R1, R2, R3 combination
Scheme 1: Synthesis of sulfonamide derivatives
T. brucei cells salvage adenosine from their mammalian host via two transporters, P1 and P2. P1 is
specific for adenosine and inosine, whereas P2 transports adenosine, adenine, melaminophenyl
arsenicals, and diamidines. 13-16 These two proteins are responsible for the active uptake of Pentamidine

and Melasoprol by T. brucei, which contributes to the higher trypanocidal activity of the compounds. 16
Based on this information, we also introduced adenosine or diamidine like structures to our analogs to
examine if the drug uptake could be improved. Two new compounds with such moieties were
synthesized based on the following scheme (Scheme 2).
Scheme 2. Synthesis of analogs containing adenosine or diamidine like structures
2.2. Biological evaluation of the new derivatives
The biological activity of the synthesized analogs was determined with cell proliferation assays. T. b.
brucei Lister 427 cells, which are the bloodstream form of the cells, were used as the parasite model,
and human normal kidney HEK293 cells and mouse macrophage RAW267.4 cells were used as the
mammalian host model. Results of the cell growth inhibition by the compounds are listed in Table 1.
The selective index is calculated by dividing the IC50s of the mammalian cell growth inhibition with the
IC50s of the T. brucei cell growth inhibition. Overall, these compounds show higher activity to inhibit
the growth of parasite cells than mammalian cells.

For the three moieties (R1 – R3) of the compound scaffold (Figure 1), various functional groups
were introduced to enhance the anti-trypanosomal activity and decrease the cytotoxicity to mammalian
cells. For R1 domain, the benzyl moiety with the ortho and meta trifluoride methyl groups overall
decreases the activity to inhibit mammalian cell growth (compounds 1-6). However, the inhibition of the
parasite cell growth was not promising. Most compounds have IC50 values above 5 µM, which is a
concentration difficult to reach in the blood circulation system. The alkyl group hexane at the R1 moiety
(compounds 7-8) significantly decreases the inhibition to mammalian cell growth, but their activity
against T. brucei cell proliferation is not promising either.
For R2 moiety, it seems that a bulky aromatic group promotes the activity to inhibit the growth of T.
brucei cells. Compounds 9, 10, 11 and 15 all have IC50s below 5 µM. In addition, these compounds
show weaker activity to affect mammalian cell growth, which significantly improve the selectivity of
the compounds.
For the R3 moiety, we mainly focused on the electron withdrawing groups, which was based on the
12,17
structure activity relationship summarized in our previous studies.
When electron donating group
was introduced (compound 16), the inhibition to T. brucei cell growth was decreased. We did not
observe any improvement on the activity of compounds 17 and 18, even the adenosine or diamine like
structures were introduced to enhance their cell uptake. It is possible that the uptake of the compounds
has been increased, but their targeting effect has been diminished due to this structural modification.
Compound 15 so far shows the best activity against T. brucei cells with an IC50 of 70 nM. It also shows
great selectivity with a selective index more than 7000, which is far better than the compounds from
previous studies. 12
Table 1. Comparison of the growth inhibitory effects of the tubulin inhibitors on mammalian and T.
brucei cells

Entry
Structure
IC50 against T. IC50
against IC50
cells macrophage macrophage HEK293 cells HEK293
267.4 cells/IC50 of (µM) cells/IC50
of IC50
against IC50
of
brucei
(µM)
RAW
cells (µM)
T. brucei
of T. brucei
1
8.58 3.82
8.42 4.13
>500
>58
53.95 6.81
80.63 3.05
6
2
3
4
>500
>59
>51
>38
10
>51
3
9.85 4.83
>500
>500
>500
13.33 6.64
34.77 7.65
5
6
15.58 7.17
13.01 6.75
>500
>500
>32
>38
>500
>32
8
102.1 22.95
7
8
23.04 11.76 >500
30.13 15.52 >500
>22
>17
>500
>500
>22
>17

9
2.08 1.01
1.78 0.72
3.39 1.22
59.81 1.53 29
175.57 2.04 99
293.1 18.23 86
69.54 4.63
20.51 1.77
>500
33
10
11
12
>147
>70
12
13
14
7.13 3.27
10.46 5.23
7.81 3.83
>500
>70
>500
>500
>48
158.1 39.91 15
69.85 0.9
9
13.26 2.88
2

CF3
15
16
0.07 0.02
>500
>7143
>21
>500
>500
>7143
>21
H
N
O
O
N
S
O
O
CN
23.47 12.88 >500
17
18
>50
>50
>500
>500
>10
>10
>500
>500
>10
>10
2.3. Biological evaluation of the tubulin targeting effect of compound 15
Our previous study reveals that these compounds interfere with the microtubule dynamics. 12 To test
if the new compound is still a tubulin inhibitor, we determined the amount of the polymerized and the
un-polymerized tubulin after treating T. brucei cells with compound 15. We found that the compound
decreased the amount of polymerized tubulin in a dose-dependent manner (Figure 2), indicating that
after the treatment, the tubulin polymerization in the cells was inhibited by the compound. Therefore,
the new compound is still a tubulin inhibitor and selectively targets the tubulin dynamic regulation in T.
brucei cells. It is a potent and selective tubulin inhibitor to against trypanosomiasis.

Figure 2. Tubulin inhibitor compound 15 inhibits the tubulin polymerization in T. brucei cells. After
treatment, the level of polymerized tubulin is significantly decreased. The experiment was
independently repeated for three times and the representative one is presented.
3. Conclusion
Due to the limitations of the current anti-trypanosomiasis drugs, the development of new medicines
for the treatment of African trypanosomiasis is still urgently needed. We focus 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 selective index is determined
in order to identify more potent and selective drug candidates. The current study results in a compound
with an IC50 of 70 nM to inhibit the growth of T. brucei cells, and does not affect the viability of
mammalian cells even with a concentration that is more than 7,000 fold higher. Furthermore, the selective
tubulin inhibitor interferes with tubulin polymerization in T. brucei cells and decreases the polymerized
tubulin in cytosol. However, the solubility of this compound limits the formulation for the in vivo study.
We are currently working on further structural modification to increase the solubility of the compound and
expect to remain or improve its potency and selectivity.
4. Experimental Section
4.1. Chemistry
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 plates with a
fluorescence indicator at 254 nm (Fluka). Flash column chromatography was performed using silica gel
60Å (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.
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.
The synthesis of compounds 1-16 followed the procedure described in previous studies. 18-20
The reaction procedure is illustrated in Scheme 1.
The final step of the reaction and the product characterization are listed below.
N-(4-(N-methylmethylsulfonamido)-3-((3-(trifluoromethyl)benzyl)oxy)phenyl)-3,5-bis(trifluoromethyl)
benzamide (1). 3,5-bis(trifluoromethyl)benzoyl chloride was used and stirred for 2 hrs. Yield 80.3%,
1
Pale Yellow solid. H NMR (400MHz, CDCl3) δH 8.36 (s, 3H), 8.09 (s, 1H), 7.93 (d, 1H), 7.74 (s, 1H),
7.66 (d, 2H), 7.59 (t, 1H), 7.32 (d, 1H), 6.97 (q, 1H), 5.19 (s, 2H), 3.26 (s, 3H), 2.88 (s, 3H); 13C NMR
(100MHz, CDCl3) δC 162.97,155.23, 138.78, 136.73, 136.47, 132.62, 132.50, 132.28, 130.95, 129.42,
127.58, 125.68, 125.39, 124.46, 124.22, 113.01, 105.51, 69.84, 38.33, 37.76, 29.71. ESI-MS calculated
for (C25H19N2SO4F9) [M - H]-: 613.0; Molecular Weight (calculated from structure): 614.48. HPLC
mobile phase: 80% ACN with 0.5% Formic acid, retention time: 2.31 mins, purity: >99%
N-(4-(N-methylmethylsulfonamido)-3-((3-(trifluoromethyl)benzyl)oxy)phenyl)-2,4-bis(trifluoromethyl)
benzamide (2). 2,4-bis(trifluoromethyl)benzoyl chloride was used and stirred for 2 hrs. Yield 92.6%,

Pale Yellow solid. 1H NMR (400MHz, DMSO) δH 10.84 (s, 1H), 8.23 (d, 2H), 7.98 (d, 1H), 7.84 (d, 1H),
7.73 (d, 1H), 7.63 (d, 2H), 7.32 (d, 1H), 7.23 (d, 1H), 7.21 (d, 1H), 5.27 (s, 2H), 3.14 (s, 3H), 2.92 (s,
3H); 13C NMR (100 MHz, DMSO) δC 164.83, 155.54, 139.94, 138.38, 132.29, 131.97, 130.50, 130.06,
129.15, 129.75, 126.15, 125.98, 125.73, 125.22, 124.87, 112.46, 105.34, 69.33, 38.21, 37.96. ESI-MS
calculated for (C25H19N2SO4F9) [M - H]-: 613.02; Molecular Weight (calculated from structure): 614.48.
HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention time: 2.14 mins, purity: >99%
N-(4-(N-methylmethylsulfonamido)-3-((2-(trifluoromethyl)benzyl)oxy)phenyl)-3,5-bis(trifluoromethyl)
benzamide (3). 3,5-bis(trifluoromethyl)benzoyl chloride was used and stirred at room temperature for 3
hrs. Yield 78.1 %, Pale Yellow solid. 1H NMR (400MHz, DMSO) δH 10.78 (s, 1H), 8.60 (s, 2H), 8.40 (d,
1H), 7.88 (d, 1H), 7.83 (d, 1H), 7.77 (t, 1H), 7.69 (s, 1H), 7.62 (t, 1H), 7.43 (d, 1H), 7.35 (d, 1H), 5.32
13
(s, 2H), 3.12 (s, 3H), 2.87 (s, 3H); C NMR (100 MHz, DMSO) δC 163.24, 155.34, 140.03, 137.40,
134,71, 133.49, 131.54, 130.84, 129.40, 129.10, 126.59, 125.82, 124.93, 122.22, 113.69, 106.12, 66.85,
38.17, 37.95. ESI-MS calculated for (C25H19N2SO4F9) [M - H]-: 612.92; Molecular Weight (calculated
from structure): 614.48. HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention time: 2.40
mins, purity: >99%
N-(4-(N-methylmethylsulfonamido)-3-((2-(trifluoromethyl)benzyl)oxy)phenyl)-2,4-bis(trifluoromethyl)
benzamide (4). 2,4-bis(trifluoromethyl)benzoyl chloride was used and stirred for 2.5 hrs. Yield 67.1%,
1
pale yellow solid. H NMR (400 MHz, DMSO) δH 10.83 (s, 1H), 8.23 (d, 2H), 7.98 (d, 1H), 7.87 (q,
13
2H), 7.76 (t, 1H), 7.62 (t, 1H), 7.51 (s, 1H), 7.34 (s, 2H), 5.30 (s, 2H), 3.13 (s, 3H), 2.88 (s, 3H); C
NMR (100 MHz, DMSO) δC 164.81, 155.45, 140.01, 134.76, 133.46, 132.20, 130.63, 130.53, 129.30,

126.57, 125.70, 112.64, 105.18, 66.76, 38.15, 37.95. ESI-MS calculated for (C25H19N2SO4F9) [M - H]-:
612.86; Molecular Weight (calculated from structure): 614.48. HPLC mobile phase: 80% ACN with
0.5% Formic acid, retention time: 2.18 mins, purity: >99%
N-(3-((2-methyl-5-(trifluoromethyl)benzyl)oxy)-4-(N-methylmethylsulfonamido)phenyl)-3,5-bis(trifluo
romethyl)benzamide (5). 3,5-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield
1
71.3%, white solid. H NMR (400 MHz, DMSO) δH 10.80 (s, 1H), 8.60 (s, 2H), 8.39 (s, 1H), 7.90-7.36
(m, 6H), 5.25 (s, 2H), 3.51 (s, 3H), 3.13 (s, 3H), 2.91 (s, 3H); 13C NMR (100 MHz, DMSO) δC 163.21,
141.76, 139.89, 137.31, 136.39, 131.49, 131.18, 129.03, 126.08, 125.15, 124.92, 113.60, 106.46, 67.81,
66.81, 38.18, 38.05. ESI-MS calculated for (C26H21N2SO4F9) [M - H]-: 626.86; Molecular Weight
(calculated from structure): 628.51. HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention
time: 2.63 mins, purity: >99%
N-(3-((2-methyl-5-trifluoromethyl)benzyl)oxy)-4-(N-methylmethylsulfonamido)phenyl)-2,4-bis(trifluor
omethyl)benzamide (6). 2,4-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield
59.3%, white solid. 1H NMR (400 MHz, DMSO) δH 10.86 (s, 1H), 8.23 (d, 2H), 7.98-7.27 (m, 7H), 5.23
(s, 2H), 3.12 (s, 3H), 2.89 (s, 3H), 2.44 (s, 3H); 13C NMR (100 MHz, DMSO) δC 164.85, 155.67, 149.50,
148.15, 141.82, 139.94, 136.35, 131.69, 131.47, 130.49, 125.88, 125.15, 112.49, 105.36, 67.75, 66.81,
38.13, 38.04. ESI-MS calculated for (C26H21N2SO4F9) [M - H]-: 626.91; Molecular Weight (calculated
from structure): 628.51. HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention time: 2.21
mins, purity: >99%

N-(3-(hexyloxy)-4-N-methylmethylsulfonamido)phenyl)-3,5-bis(trifluoromethyl)benzamide
(7).
3,5-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield 80.2%, pale yellow solid.
1H NMR (400 MHz, CDCl3) δH 8.53 (s, 1H), 8.38 (s, 2H), 8.07 (s, 1H), 7.80 (d, 1H), 7.20 (d, 1H), 6.79
(q, 1H), 4.06 (t, 2H), 3.26 (s, 3H), 2.97 (s, 3H), 1.82 (m, 2H), 1.47 (m, 2H), 1.36 (m, 4H), 0.93 (m, 3H);
13C NMR (100 MHz, CDCl3) δC 163.03, 155.76, 138.81, 136.57, 132.59, 132.25, 127.69, 125.47,
125.07, 124.26, 121.55, 112.48, 105.27, 68.55, 38.11, 37.67, 31.45, 29.19, 25.77, 22.55, 13.96. ESI-MS
calculated for (C23H26N2SO4F6) [M - H]-: 539.00; Molecular Weight (calculated from structure): 540.52.
HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention time: 2.79 mins, purity: >99%
N-(3-(hexyloxy)-4-N-methylmethylsulfonamido)phenyl)-2,4-bis(trifluoromethyl)benzamide
(8).
2,4-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield 74.0%, pale yellow solid.
1H NMR (400 MHz, CDCl3) δH 7.95 (s, 1H), 7.92 (t, 2H), 7.78 (d, 2H), 7.28 (t, 1H), 6.67 (q, 1H), 4.09
13
(t, 2H), 3.26 (s, 3H), 2.90 (s, 3H), 1.85 (q, 2H), 1.61-1.35 (m, 6H), 0.94 (t, 3H); C NMR (100 MHz,
CDCl3) δC 164.44, 155.92, 138.60, 132.86, 132.82, 132.53, 129.43, 129.25, 128.58, 125.23, 124.16,
123.90, 121.44, 111.91, 104.88, 68.55, 37.97, 37.59, 31.45, 29.19, 25.78, 22.55, 13.97. ESI-MS
calculated for (C23H26N2SO4F6) [M - H]-: 539.01; Molecular Weight (calculated from structure): 540.52.
HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention time: 2.37 mins, purity: >99%
N-(4-(N-methyl-1-phenylmethylsulfonamido)-3-((3-(trifluoromethyl)benzyl)oxy)phenyl)-3,5-bis(trifluor
omethyl)benzamide (9). 3,5-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield
71.2%, white solid. 1H NMR (400 MHz, CDCl3) δH 8.35 (d, 3H), 8.07 (s, 1H), 7.69-7.28 (m, 10H), 6.96
(q, 1H), 6.88 (d, 1H), 5.00 (s, 2H), 4.32 (s, 2H), 3.11 (s, 3H); 13C NMR (100 MHz, CDCl3) δC 155.49,

138.56, 136.90, 136.47, 131.55, 130.77, 130.68, 129.29, 129.02, 128.70, 127.61, 126.26, 125.53, 125.10,
112.96, 105.63, 69.60, 58.12, 38.54. ESI-MS calculated for (C31H23N2SO4F9) [M - H]-: 689.01;
Molecular Weight (calculated from structure): 690.58. HPLC mobile phase: 80% ACN with 0.5%
Formic acid, retention time: 2.73 mins, purity: >99%
N-(4-(N-methyl-1-phenylmethylsulfonamido)-3-((3-(trifluoromethyl)benzyl)oxy)phenyl)-2,4-bis(trifluor
omethyl)benzamide (10). 2,4-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield
1
67.5%, white solid. H NMR (400 MHz, DMSO) δH 7.88 (s, 1H), 7.80-7.63 (m, 6H), 7.56 (t, 2H), 7.35
(d, 5H), 7.08 (d, 1H), 6.76 (q, 1H), 5.28 (s, 2H), 4.26 (s, 2H), 3.16 (s, 3H); 13C NMR (100 MHz, DMSO)
δC 155.70, 139.87, 139.15, 138.75, 138.41, 132.15, 131.66, 131.36, 130.24, 130.00, 129.15, 128.75,
128.50, 127.75, 126.15, 125.05, 124.95, 112.40, 69.32, 57.08, 38.46. ESI-MS calculated for
(C31H23N2SO4F9) [M - H]-: 688.94; Molecular Weight (calculated from structure): 690.58. HPLC mobile
phase: 80% ACN with 0.5% Formic acid, retention time: 2.36 mins, purity: >99%
N-(4-(N-methyl-1-phenylmethylsulfonamido)-3-((4-(trifluoromethyl)benzyl)oxy)phenyl)-3,5-bis(trifluor
omethyl)benzamide (11). 3,5-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield
1
98.3%, white solid. H NMR (400 MHz, DMSO) δH 10.74 (s, 1H), 8.59 (s, 2H), 8.40 (s, 1H), 7.69 (q,
5H), 7.37 (q, 6H), 7.08 (d, 1H), 5.32 (s, 2H), 4.46 (s, 2H), 3.12 (s, 3H); 13C NMR (100 MHz, DMSO) δ
C
155.57, 141.80, 139.83, 137.32, 131.40, 131.17, 130.84, 130.24, 129.05, 128.76, 128.58, 128.54, 126.15,
125.87, 125.83, 113.45, 106.30, 69.42, 57.12, 38.54. ESI-MS calculated for (C31H23N2SO4F9) [M - H]-:
688.87; Molecular Weight (calculated from structure): 690.58. HPLC mobile phase: 80% ACN with
0.5% Formic acid, retention time: 3.01 mins, purity: >99%

N-(4-(N-methyl-1-phenylmethylsulfonamido)-3-((2-(trifluoromethyl)benzyl)oxy)phenyl)-3,5-bis(trifluor
omethyl)benzamide (12). 3,5-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield
1
99%, white solid. H NMR (400 MHz, DMSO) δ 10.76 (s, 1H), 8.60 (s, 2H), 8.39 (s, 1H), 7.96-7.12
H
13
(m, 12H), 5.34 (s, 2H), 4.39 (s, 2H), 3.08 (s, 3H); C NMR (100 MHz, DMSO) δ 163.21, 155.48,
C
139.98, 137.40, 134.78, 133.44, 131.67, 131.34, 131.14, 130.81, 130.78, 130.20, 129.34, 129.08, 128.74,
128.55, 127.18, 126.88, 126.55, 126.49, 126.18, 126.05, 125.76, 124.93, 122.22, 113.62, 106.02, 66.82,
57.11, 38.50. ESI-MS calculated for (C31H23N2SO4F9) [M - H]-: 688.96; Molecular Weight (calculated
from structure): 690.58. HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention time: 2.92
mins, purity: >99%
N-(4-(N-methyl-1-phenylmethylsulfonamido)-3-((2-(trifluoromethyl)benzyl)oxy)phenyl)-2,4-bis(trifluor
omethyl)benzamide (13). 2,4-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield
1
98%, white solid. H NMR (400 MHz, CDCl3) δ 7.57-7.28 (m, 14H), 7.05 (d, 1H), 6.96 (q, 1H), 5.31
H
13
(s, 2H), 4.18 (s, 2H), 3.10 (s, 3H); C NMR (100 MHz, CDCl3) δ 164.41, 155.53, 138.45, 134.10,
C
132.46, 132.05, 130.75, 130.05, 129.42, 129.00, 128.62, 128.58, 128.53, 127.88, 126.21, 112.90, 105.29,
66.85, 57.87, 38.42. ESI-MS calculated for (C31H23N2SO4F9) [M - H]-: 688.92; Molecular Weight
(calculated from structure): 690.58. HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention
time: 2.34 mins, purity: >99%
N-(3-(hexyloxy)-4-(N-methyl-1-phenylmethylsulfonamido)phenyl)-3,5-bis(trifluoromethyl)benzamide
1
(14). 3,5-bis(trifluoromethyl)benzoyl chloride was used and stirred for 3 hrs. Yield 99%, white solid. H
NMR (400 MHz, CDCl3) δ 8.10 (s, 2H), 7.89 (s, 1H), 7.70 (s, 1H), 7.60-7.22 (m, 4H), 6.95 (q, 1H),
H

13
6.72 (s, 1H), 4.34 (s, 2H), 4.01 (t, 2H), 1.68 (q, 2H), 1.59 (s, 3H), 1.35 (quin, 8H), 0.92 (t, 3H); C
NMR (100 MHz, CDCl3) δ 134.15, 132.75, 130.68, 128.87, 128.74, 19.15, 105.75, 100.15, 31.48,
C
28.15, 25.60, 22.52, 13.98. ESI-MS calculated for (C29H30N2SO4F6) [M - H]-: 615.56; Molecular Weight
(calculated from structure): 616.61. HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention
time: 2.84mins, purity: >99%
4-cyano-N-(4-(N-methyl-1-phenylmethylsulfonamido)-3-((4-(trifluoromethyl)benzyl)oxy)phenyl)benza
1
mide (15). 4-cyano benzoyl chloride was used and stirred for 2.5 hrs. Yield 96%, Pale brown solid. H
NMR (400 MHz, DMSO) δH 10.57 (S, 1H), 8.06 (t, 4H), 7.73 (d, 5H), 7.36 (d, 6H), 7.07 (d, 1H), 5.31 (s,
2H), 4.46 (s, 2H), 3.12 (s, 3H); 13C NMR (100 MHz, DMSO) δC 206.97, 164.75, 156.25, 142.25, 140.45,
139.15, 132.99, 131.39, 130.27, 129.00, 128.75, 128.56, 125.86, 113.19, 106.07, 69.38, 57.11, 38.25,
31.14. ESI-MS calculated for (C30H24N3SO4F3) [M - H]-: 578.12; Molecular Weight (calculated from
structure): 579.59. HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention time: 3.12 mins,
purity: >99%
N-(3-((3-cyanobenzyl)oxy)-4-(N-methylethylsulfonamido)phenyl)benzo[d][1,3]dioxole-5-carboxamide
(16). Benzo[d][1,3]dioxole-5-carbonyl chloride was used and stirred for 2 hrs. Yield 94%, white solid.
1H NMR (400 MHz, CDCl3) δ 7.56-6.81 (m, 11H), 6.09 (d, 2H), 5.20 (s, 2H), 3.27 (s, 3H), 3.08 (q,
H
2H), 1.32 (t, 3H); 13C NMR (100 MHz, CDCl3) δ 148.38, 139.75, 137.64, 132.14, 132.09, 131.15,
C
129.68, 128.55, 125.25, 121.78, 113.25, 112.34, 108.26, 107.65, 105.11, 101.98, 69.42, 46.38, 38.36,
8.12. ESI-MS calculated for (C25H23N3SO6) [M - H]-: 492.34; Molecular Weight (calculated from
structure): 493.53. HPLC mobile phase: 80% ACN with 0.5% Formic acid, retention time: 1.98 mins,
purity: >99%

Compound 17 was synthesized as described below.
The precursor compound was dissolved in dichloromethane (DCM) and then ethanol (EtOH) was
added. The reaction was bubbled with HCl gas for 15-20 minutes and the mixture was stirring at room
temperature overnight until the precursor was gone. Then the reaction was quenched with water and the
solid product was filtered. This intermediate product was dissolved in 10 ml of methanol (MeOH) and
bubbled with ammonia for 10-15 mins, the mixture was stirring for hours at room temperature until the
intermediate was gone. The formed solid product was filtered, washed, and dried.
N-(3-((3-carbamimidoylbenzyl)oxy)-4-(N-methylmethylsulfonamido)phenyl)-3,5-bis(trifluoromethyl)be
nzamide (17). Yield 65%. Pale yellow solid. 1H NMR (400 MHz, DMSO) δ 9.23 (s, 1H), 8.62 (s, 2H),
H
8.39 (s, 1H), 7.96 (s, 2H), 7.75 (s, 2H), 7.46 (t, 2H), 7.36 (m, 3H), 5.23 (s, 2H), 3.17 (s, 4H), 2.94 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 163.16, 162.90, 155.50, 140.26, 137.56, 136.78, 131.52, 130.63,
C
129.33, 128.82, 126.61, 125.70, 124.95, 122.24, 119.75, 113.40, 106.45, 70.11, 38.20. ESI-MS
calculated for (C25H22N4O4SF6) [M - H]-: 587.61; Molecular Weight (calculated from structure): 588.52.
HPLC mobile phase: 83% ACN with 0.5% Formic acid, retention time: 7.23 mins, purity: >99%
Procedure for the synthesis compound 18:
The precursor amine (1 eq) was refluxed with 2-chloropyrimidin-4-amine (1.5 eq) in the presence of
n-BuOH and a drop of trifluoroacetic acid (TFA) for 4-5 hours. The solid product formed was filtered,
washed, and dried.
N-(4-((4-aminopyrimidin-2-yl)amino)-2-((2,5-dimethylbenzyl)oxy)phenyl)-N-methylmethanesulfonami
1
de (18): 2-chloropyrimidin-4-amine was used and refluxed for few hours. Yield 86%. Yellow solid. H
NMR (400 MHz, DMSO) δ 10.46 (s, 1H), 8.29 (s, 2H), 7.90 (d, 1H), 7.62 (s, 1H), 7.28 (t, 2H), 7.13
H

(m, 3H), 6.19 (d, 1H), 5.13 (s, 2H), 3.10 (s, 3H), 2.85 (s, 3H), 2.32 (s, 3H), 2.27 (s, 3H); 13C NMR (100
MHz, DMSO) δ 164.98, 155.89, 153.75, 139.03, 135.23, 134.63, 133.92, 131.63, 130.31, 129.31,
C
125.46, 113.53, 106.80, 98.51, 68.99, 60.85, 38.27, 38.03, 21.05, 18.46. ESI-MS calculated for
(C21H25N5O3S) [M - H]-: 426.01; Molecular Weight (calculated from structure): 427.52. HPLC mobile
phase: 83% ACN with 0.5% Formic acid, retention time: 8.38 mins, purity: >99%
4.2. Cell culture
HEK293 kidney cells and mouse macrophage RAW267.4 cells were obtained from ATCC
(Rockville, MD) and maintained in RPMI1640 medium supplemented with 10% fetal bovine serum
(FBS), 2 mmol/L L-Glutamine, 1 mmol/L sodium pyruvate, and 100 U/mLpenicillin-streptomycin. FBS
was heat inactivated for 30 min at 56 ºC. Mammalian cells were grown at 37 ºC in a Heraeus
water-jacketed incubator with 5% CO2. Bloodstream form T. b. brucei Lister 427 cells were cultured in
21
HMI-9 medium with 10% FBS at 37 ºC in a Heraeus water-jacketed incubator with 7.5% CO2. The
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS)
reagents 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).
4.3. Mammalian cell viability analysis
The MTT assay was used to examine the effect of tubulin inhibitors on the growth of HEK293 and
RAW267.4 cells in four replications. 3000 cells per well were seeded with RPMI1640 medium in
96-well flat-bottomed plates for 24 hrs and were then exposed to various concentrations of test
compounds dissolved in DMSO (final concentration 0.1%) in medium for 48 hrs. 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 2 hrs. Supernatants were removed from the wells, and

the reduced MTT dye was solubilized with 200 µl/well DMSO. Absorbance at 570 nm was determined
on a SpectraMax Plus384 spectrophotometer (Molecular Devices). Data obtained with quadruplication
were normalized and fitted to a dose–response curve using GraphPad Prism v.5 (GraphPad).
4.4. T. brucei cell viability analysis
The (MTS) assay was used to examine the effect of tubulin inhibitors on T. b. brucei cell viability 11.
5,000 cells of T. brucei were seeded in 96 well plates and treated with 0.1% DMSO and tested agents at
various concentrations for 48 hrs at 37 °C. Subsequently, 20 µL of MTS (5% PMS) from the CellTiter
Cell Proliferation Assay (Promega) was added to 200 µL of T. b. brucei culture in each well and
incubated at 37 °C for 3 hrs. Soluble formazan, produced by viable cells due to reduction of MTS, was
measured at 490 nm with a SpectraMax Plus384 spectrophotometer (Molecular Devices). Data obtained
with quadruplication were normalized and fitted to a dose-response curve using GraphPad Prism v.5
(GraphPad).
4.5. T. brucei cell lysate preparation and western blot assay
T. brucei cells were incubated with 0.5% DMSO and different doses of compound 15 for 7 hrs. Cell
pellets were harvested by centrifugation at 1,500 rpm for 10 mins at 4°C, 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 protease inhibitor (Roche), and lysed with 300 µl of lysis buffer (80 mM Pipes, pH 6.8, 1 mM
EGTA, 1 mM MgCl2, 0.2% Triton X-100, 10% glycerol and protease inhibitor (Roche)) at 30°C for 5
mins. The cell lysate was centrifuged at 12,000 rpm for 10 mins at 4°C, and the supernatant was
transferred into a fresh Eppendorf tube. Pellets were re-suspended in 50 µl of lysis buffer and sonicated.
50 µl of 2 x SDS buffer was added subsequently, and the sample was boiled at 95°C for 5 mins. Protein
lysates from equal number of cells were separated on 10% polyacrylamide gels by electrophoresis.

Proteins were transferred onto nylon hyblot CL membranes. Tubulin antibody (TAT-1, a gift from Dr. K.
Gull) was used in the following western analysis.
5. Acknowledgements
This research was supported by the grant 2R15AI 103889-02 (B. Su) and Center for Gene
Regulation in Health and Disease (GRHD) of Cleveland State University.
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Graphical Abstract
Synthesis and biological evaluation of selective tubulin inhibitors as anti-trypanosome agents
Viharika Bobba, Vishal Nanavaty, Nethrie D.
Idippily, Anran Zhao, Bibo Li, Bin Su

ꢀ A series of sulfonamide tubulin inhibitors were synthesized
ꢀ The compounds were examined with mammalian cells and trypanosomal cells to identify selective
drug candidates
ꢀ A new analog showed high selectivity and potency against the growth of trypanosome cells
ꢀ The compound decreased tubulin polymerization in trypanosome cells