Synthesis and evaluation of functionalized benzoboroxoles as potential anti-tuberculosis agents Dedicated to Professor Neil K. Garg on the occasion of his receipt of the Tetrahedron Young Investigator Award

Article abstract of DOI:10.1016/j.tet.2016.03.038

Several derivatives of aminobenzoboroxole have been prepared starting from 2-boronobenzaldehyde. All of these derivatives have been evaluated for their anti-mycobacterial activity on Mycobacterium smegmatis and cytotoxicity on breast cancer cell line MCF7. Based on these studies, all the tested molecules have been found to be generally non-toxic and benzoboroxoles with unsubstituted (primary) amines have been found to exhibit good anti-mycobacterial activity. Some of the key compounds have been evaluated for their anti-tubercular activity on Mycobacterium tuberculosis H37Rv using 7H9 and GAST media. 7-Bromo-6-aminobenzoboroxole 4 has been identified as the lead candidate compound for further development.

Full text of DOI:10.1016/j.tet.2016.03.038

Accepted Manuscript
Synthesis and evaluation of functionalized benzoboroxoles as potential anti-
tuberculosis agents
Mohammad A. Alam, Kriti Arora, Shirisha Gurrapu, Sravan K. Jonnalagadda, Grady
L. Nelson, Paul Kiprof, Subash C. Jonnalagadda, Venkatram R. Mereddy
PII:
S0040-4020(16)30170-3
10.1016/j.tet.2016.03.038
TET 27578
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 7 January 2016
Revised Date: 10 March 2016
Accepted Date: 11 March 2016
Please cite this article as: Alam MA, Arora K, Gurrapu S, Jonnalagadda SK, Nelson GL, Kiprof P,
Jonnalagadda SC, Mereddy VR, Synthesis and evaluation of functionalized benzoboroxoles as potential
anti-tuberculosis agents, Tetrahedron (2016), doi: 10.1016/j.tet.2016.03.038.
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Synthesis and evaluation of functionalized
benzoboroxoles as potential anti-tuberculosis
agents
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Mohammad A. Alama, Kriti Arorab, Shirisha Gurrapuc, Sravan K. Jonnalagaddac, Grady L. Nelsonc, Paul
Kiprof,d Subash C. Jonnalagadda,a,e * and Venkatram R. Mereddy.c,d,f *
a Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USA, bTuberculosis Research Section, Laboratory of Clinical
Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892, USA, cIntegrated
Biosciences Graduate Program, University of Minnesota, MN 55812, USA, dDepartment of Chemistry and Biochemistry, University of Minnesota Duluth,
e
f
Duluth, MN 55812, USA, Department of Biomedical and Translational Sciences, Rowan University, Glassboro, NJ 08028, USA, Department of
Pharmacy Practice and Pharmaceutical Sciences, University of Minnesota, Duluth, MN 55812, USA.

1
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Tetrahedron
journal homepage: www.elsevier.com
Synthesis and evaluation of functionalized benzoboroxoles as potential
anti-tuberculosis agents
Mohammad A. Alama, Kriti Arorab, Shirisha Gurrapuc, Sravan K. Jonnalagaddac, Grady L. Nelsonc, Paul
*
Kiprof,d Subash C. Jonnalagadda,a,e and Venkatram R. Mereddy.c,d,f
a Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USA, bTuberculosis Research Section, Laboratory of Clinical Infectious
Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892, USA, cIntegrated Biosciences Graduate
Program, University of Minnesota, MN 55812, USA, dDepartment of Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN 55812, USA,
f
eDepartment of Biomedical and Translational Sciences, Rowan University, Glassboro, NJ 08028, USA, Department of Pharmacy Practice and Pharmaceutical
Sciences, University of Minnesota, Duluth, MN 55812, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Several derivatives of aminobenzoboroxole have been prepared starting from 2-
boronobenzaldehyde. All of these derivatives have been evaluated for their anti-mycobacterial
activity on Mycobacterium smegmatis and cytotoxicity on breast cancer cell line MCF7. Based
on these studies, all the tested molecules have been found to be generally non-toxic and
benzoboroxoles with unsubstituted (primary) amines have been found to exhibit good anti-
mycobacterial activity. Some of the key compounds have been evaluated for their anti-
tubercular activity on Mycobacterium tuberculosis H37Rv using 7H9 and GAST media. 7-
Bromo-6-aminobenzoboroxole 4 has been identified as the lead candidate compound for further
development.
Available online
Keywords:
Benzoxaboroles; nitrobenzoxaborole;
aminobenzoxaborole; bromoaminobenzoxaborole;
anti-tuberculosis agents
2016 Elsevier Ltd. All rights reserved
.
———
Corresponding author. Tel.: +1-856-256-5452; fax: +1- 856-256-4478; e-mail: jonnalagadda@rowan.edu

2
Tetrahedron
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O
Introduction
O
Fuming HNO3
-40oC
O
NaBH4
Tuberculosis, caused by Mycobacterium tuberculosis (M.
B
B
O2N
OH
THF:H2O
(9:1)
tuberculosis) is a highly contagious chronic bacterial infection.1
Nearly one third of world’s population has been affected by
latent tuberculosis.1a This infection spreads through the air and
causes millions of deaths every year. The standard treatment of
B
OH
OH
1, 87%
2, 68%
OH
HCOONH4
Pd-C
tuberculosis includes
a four-drug regimen of isoniazid,
Br2-CH3COOH
O
O
B
OH
B
rifampicin, ethambutol, and pyrazinamide for six to nine months.
The emergence of multi-drug resistant (MDR-TB) and
extensively drug resistant strains of this bacterium is a cause of
great concern worldwide (eg. WHO estimates indicate that nearly
half million people globally have developed MDR-TB).1a Hence,
new and highly effective therapeutic strategies are urgently
required for the better treatment of this epidemic. Owing to the
global importance of this disease, there has been an explosive
growth of publications in the area of tuberculosis research.2
Despite this progress, discovery of biologically active small
molecules with novel chemical entities is extremely critical to
combat this disease in future.
H2N
H2N
OH
Br
3, 74%
4, 55%
Br2-CH3COOH
(excess)
Br
O
B
OH
H2N
Br
5, 53%
Scheme 1: Preparation of brominated aminobenzoboroxoles
O
O
O
O
NaBH4
Benzoboroxoles are cyclic boronic acids that are highly stable
under strongly acidic and basic conditions.3 These compounds
have been found to have valuable applications in materials4 and
medicinal3,5 chemistry. While this work was in progress, a patent
application has been recently published on the usage of tricyclic
benzoboroxoles as anti-mycobacterial agents.6 Our long-standing
interest7 in developing novel functionalized boronic acids and
benzoboroxoles as therapeutic agents prompted us to explore the
utility of these scaffolds as potential anti-tubercular agents.
R
H
B
B
B
H2N
R
N
R
N
H
OH
OH
OH
O
O
B
OH
O
B
OH
B
N
N
N
H
H
H
OH
F
Cl
6, 82%
7, 79%
8, 80%
O
O
B
O
N
B
N
H
OH
H
OH
10, 55%
Br
9, 75%
Results and Discussion
Scheme 2: Reductive amination of 6-aminobenzoboroxoles
Based on the literature reports as well as our previous
experience, it was found that the biological activity of
benzoboroxoles greatly diminished upon substitution at the
benzylic carbon in the oxaborole ring. Hence, we envisioned the
preparation of aromatic ring-substituted benzoboroxoles while
leaving the oxaborole methylene group unsubstituted. In this
regard, we chose 6-aminobenzoboroxole 3 as the common
synthon for the preparation of various functionalized derivatives.
The amine 3 was prepared in three steps starting from 2-
boronobenzaldehyde (Scheme 1).7a,8
The 6-aminobenzoboroxole 3 was further derivatized via
alkylation of the amine with alkyl and benzyl halides. Amine 3
was treated with allyl bromide, propargyl bromide, 1,4-
dibromobutane, benzyl bromide9 and p-xylyl bromide in the
presence of potassium carbonate and DMSO to obtain the N,N-
dialkylated aminobenzoboroxoles 11-15 respectively (Scheme
3).
O
O
RBr, K2CO3
DMSO
B
B
H2N
R2N
The reaction of 2-boronobenzaldehyde with sodium
borohydride in THF and water provided benzoboroxoles 1 in
90% yield. Nitration8a of 1 with fuming nitric acid followed by
reduction of nitro group with ammonium formate in the presence
of palladium carbon resulted in the formation of 6-
aminobenzoboroxole 3. Electrophilic aromatic bromination of 3
with bromine in acetic acid provided 7-bromo-6-
aminobenzoboroxole 4 in 55% yield along with ~20% of 5,7-
dibromo-6-aminobenzoboroxole 5. Pure 4 could be readily
obtained upon simple silica gel column chromatography using
hexanes and ethyl acetate as eluents. Addition of excess bromine
(2.5 eq) to 3 yielded 53% of 5 (Scheme 1).
OH
OH
O
O
O
B
N
B
B
N
N
OH
OH
OH
11, 76%
12, 69%
13, 60%
O
B
O
N
B
N
OH
OH
H3C
CH3
14, 70%
15, 75%
The 6-aminobenzoboroxole 3 was then subjected to reductive
amination to prepare monosubstituted derivatives of the amino
group.7a 6-Aminobenzoboroxole was treated with benzaldehyde,
Scheme 3: Dialkylation of 6-aminobenzoboroxoles
The 6-aminobenzoboroxole 3 was also functionalized as the
corresponding amides 16-20 upon reaction with acetic
anhydride10, butyric anhydride, trifloroacetic anhydride, succinic
anhydride11, and phthalic anhydride respectively (Scheme 4).
The amide with phthalic anhydride cyclized intramolecularly to
provide the cylic imide 20. Similarly, a sulfonamide derivative
21 was also prepared by treating the amine 3 with 4-
nitrobenzenesulfonyl chloride.12
4-flourobenzaldehyde,
4-chlorobenzaldehyde,
4-
bromobenzaldehyde, and 2-furaldehyde in methanol to form the
intermediate imines, followed by reduction of the imines with
sodium borohydride to obtain N-monoalkylated 6-aminobenzo
boroxoles 6-10 respectively (Scheme 2).

3
O
as the negative control. We also evaluated the effectiveness of
ACCEPTED MANUSCRIPT
O
anhydride
O
B
B
the hydrochloride salt of 4 under these conditions. This
compound as well as its hydrochloride salt were found to be
bacteriostatic at lower concentrations but at higher concentration
of >40µM, they were found to exhibit good bactericidal
properties (Table 2).
H2N
R
N
H
OH
OH
O
O
O
O
O
O
B
B
B
NH
N
H
F3C
N
H
OH
OH
OH
16, 67%
17, 76%
18, 80%
Table 2: Minimum Bactericidal Concentration
O
O
O
O
O
O
O
S
B
CFU*/mL Conc.
CFU*/mL
1.06 x 108
165
HO
B
N
N
B
Compound Conc.
DMSO
Rifampicin 0.1 µM
NH
H
OH
OH
OH
3.8 x 108
-
O
19, 67%
O2N
20, 75%
O
21, 40%
-
Scheme 4: Synthesis of 6-amidobenzoboroxoles
290
1.0 µM
4
4 µM
4 µM
4.59 x 105 40 µM
5.51 x 104 40 µM
750
After synthesizing various functionalized benzoboroxoles,
these molecules 1-21 were evaluated for their antimycobacterial
activity. Before testing their antimycobacterial potential, all the
molecules were evaluated for their general cytotoxicity against
breast cancer cell line MCF-7. All of the tested compounds did
not exhibit any significant cytotoxicity even at 50µM.
Encouraged by their lack of cytotoxicity, we screened them for
their biological activity against Mycobacterium smegmatis (M.
smegmatis). This strain is especially useful in screening large
chemical libraries because of the microbe’s speed of growth and
its non-pathogenic nature that allows its use in biosafety level II
hoods. Several laboratories have reported on the effectiveness of
M. smegmatis as a surrogate screen for compounds that inhibit
the growth of M. tuberculosis.13 We employed Kirby-Bauer disk
method for the identification of lead compounds. The compounds
that exhibited >3 cm zone of inhibition were further tested for
their efficacy against M. tuberculosis. Unfortunately, most of the
amine substituted derivatives exhibited weak or no inhibitory
properties in the Kirby-Bauer disk method. Of all the derivatives,
the 6-aminobenzoxaborole 3, 7-bromo-6-aminobenzoboroxole 4
and 5,7-dibromo-6-aminobenzoboroxole 5 were found to be the
most active with zone of inhibition values >5 cm. However, 5
was not selected for further studies due to its high bromine
content which may preclude its suitability as a pharmaceutical
agent. Consequently, the two aminobenzoboroxoles 3 and 4 were
further subjected to detailed biological evaluation against M.
tuberculosis.
4**
1475
*CFU = colony forming units **Hydrochloride salt of bromo-
aminobenzoboroxole 4 was used.
We then carried out the minimum anaerobicidal concentration
study to determine if the candidate compounds have activity
under conditions of dormancy. There is very little metabolic
activity under these conditions hence most compounds including
clinically used isoniazid fail to provide therapeutic benefit.
Metronidazole was used as the positive control and DMSO was
used as the negative control in this study. Unfortunately,
aminobenzoboroxole 4 and its salt did not exhibit any significant
activity at lower or higher concentrations (10 and 100µM) under
these conditions (Table 3).
Table 3: Minimum Anaerobicidal Concentration
Conc.
(µM)
Conc.
(µM)
-
-
Compound
DMSO
Metronidazole 100
Isoniazid
4
4**
CFU*/mL
CFU*/mL
1.32 x 107
1.73 x105
1.36 x107
7.13 x 106
6.55 x 106
-
-
-
10
10
10
-
100
100
4.81 x 106
4.30 x 106
*CFU = colony forming units **Hydrochloride salt of 7-
bromo-6-aminobenzoboroxole 4 was used.
Finally, we also carried out the intracellular macrophage-
killing assay, which provides information about the ability of the
test compounds to kill in macrophages, which are the natural
hosts of Mtb. If a compound shows killing of Mtb in
macrophages then it has better chances of showing efficacy in an
animal model of disease. The candidate benzoboroxole 4 and its
salt showed some cidality in the macrophages but not as much as
the positive control INH (Table 4).
Table 1: Minimum Inhibitory Concentration
MIC99* (7H9)
MIC99* (GAST)
Week 2
Compound
Week 1
Week 2
62.5-125
31.25
7.8
1
62.5
15.6
3.9
62.5
3
15.6
1.9
4
Isoniazid
0.7
0.7
0.15
*MIC99 expressed in µM
Table 4: Intracellular Macrophage Killing Assay
Initially, parent benzoboroxole 1, amines 3 and 4 were
evaluated for their minimum inhibitory concentration (MIC99)
under aerobic conditions. Isoniazid was used as the positive
control. Compared to 1, the amine derivatives 3 and 4 exhibited
superior inhibitory properties. The amine 3 was found to be ~2-4
times more potent than 1 whereas the bromo derivative 4 was
found to be ~8-16 times more potent than 1 in 7H9 medium. The
MIC99 values of 1, 3 and 4 were also determined in more
sensitive glycerol-alanine-salts-tween (GAST) medium.14 In this
study, 3 was found to be ~4 times more potent than 1 and 4 was
found to be ~30 times more potent than 1 (Table 1).
Conc.
Conc.
Day 1
Day 6
Compound
(
µM
)
CFU*/mL
(
-
-
µM)
CFU*/mL
Untreated
Isoniazid
4
-
2.16 x 105
1.80 x 105
4
4
4
1.34 x103
4.31 x104
6.09 x 104
-
40
40
2.28 x 104
2.44 x 104
4**
*CFU = colony forming units, **Hydrochloride salt of 7-
bromo-6-aminobenzoboroxole 4 was used.
Based on the results, complete loss in biological activity was
observed upon structural changes on the 6-position primary
amino group in benzoboroxole. We observed that the conversion
of primary amine to secondary amines (eg. compounds 6-10,
Scheme 2), tertiary amines (eg. compounds 11-15, Scheme 3), as
well as amides and sulphonamides (eg. compounds 16-21,
We then carried out the minimum bactericidal concentration
of 4 under aerobic conditions to evaluate whether these
compounds were bacteriostatic or bactericidal. In this case,
rifampicin was used as the positive control and DMSO was used

4
Tetrahedron
Scheme 4) resulted in significant loss in biological acvitiy.
CHN Analysis: (Found: C, 27.12%; H, 2.05%; N, 4.34%;
ACCEPTED MANUSCRIPT
However, some of the compounds such as 6-
aminobenzoboroxole 3, 7-bromo-6-aminobenzoboroxole 4, and
5,7-dibromo-6-aminobenzoboroxole 5 exhibit good biological
activity, and efforts are on going towards their development as
anti-tuberculosis agents.
C7H6BBr2NO2 requires: C, 27.41%; H, 1.97%; N, 4.57%)
6-(benzylamino)benzo[c][1,2]oxaborol-1(3H)-ol (6): 1H NMR
(500 MHz, DMSO-d6): δ 8.91 (s, 1H), 7.37-7.24 (m, 4H), 7.22 (t,
J = 7.0 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.87 (s, 1H), 6.76 (d, J
= 8.0 Hz, 1H), 6.23 (br s, 1H), 4.82 (s, 2H), 4.28 (s, 2H); 13C
NMR (125 MHz, DMSO-d6): δ 148.3, 142.0, 140.8, 128.7, 127.6,
127.0, 121.9, 117.0, 112.7, 70.0, 47.1; CHN Analysis: (Found: C,
70.56%; H, 5.78%; N, 5.62%; C14H14BNO2 requires: C, 70.33%;
H, 5.90%; N, 5.86%)
Conclusion
In conclusion, we have synthesized several derivatives of 6-
aminobenzoboroxole starting from 2-boronobenzaldehyde. Some
of the protocols employed in the synthesis include electrophilic
aromatic bromination, reductive amination, dialkylation, and
amide formation. All the synthesized derivatives have been
evaluated for their zone of inhibition against M. smegmatis and
cytotoxic properties against MCF7 cell line. From this study, two
lead derivatives 3 and 4 have been identified for further anti-
mycobacterial activity against M. tuberculosis H37Rv using 7H9
and GAST media. 3 and 4 have been found to be several times
more potent than their parent benzoboroxole 1 in both these
assays. Some of the other biology studies on 3 and 4 include
minimum bactericidal concentration under aerobic and anaerobic
conditions. Based on all these studies, 7-bromo-6-
aminobenzoboroxole 4 has been identified as the lead candidate
compound for further structure-activity studies to identify a
derivative that exhibits much superior properties than the existing
clinically used drugs.
6-((4-fluorobenzyl)amino)benzo[c][1,2]oxaborol-1(3H)-ol (7):
1H NMR (500MHz, DMSO-d6): δ 8.91 (s, 1H), 7.41-7.38 (m,
2H), 7.16-7.07 (m, 3H), 6.86 (s, 1H), 6.76 (d, J = 8.5 Hz, 1H),
6.24 (t, J = 6.0 Hz, 1H), 4.82 (s, 2H), 4.27 (d, J = 6.0 Hz, 2H);
13C NMR (125 MHz, DMSO-d6): δ 161.6 (d, JF = 300.0 Hz),
148.1, 142.1, 136.9, 129.4, 122.0, 117.1, 115.4 (d, JF = 26.2 Hz),
112.8, 70.0, 46.4; CHN Analysis: (Found: C, 65.24%; H, 4.97%;
N, 5.60%; C14H13BFNO2 requires: C, 65.41%; H, 5.10%; N,
5.45%).
6-((4-chlorobenzyl)amino)benzo[c][1,2]oxaborol-1(3H)-ol (8):
1H NMR (500MHz, DMSO-d6): δ 8.92 (s, 1H), 7.37-7.22 (m,
4H), 7.07 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H), 6.74 (d, J = 8.5 Hz,
1H), 6.28 (t, J = 6.2 Hz, 1H), 4.82 (s, 2H), 4.28 (d, J = 6.0 Hz,
2H); 13C NMR (125 MHz, DMSO-d6): δ 148.0, 142.2, 140.0,
131.5, 129.4, 128.7, 122.0, 117.0, 112.8, 70.0, 46.4; CHN
Analysis: (Found: C, 61.39%; H, 4.85%; N, 4.95%;
C14H13BClNO2 requires: C, 61.48%; H, 4.79%; N, 5.12%).
Experimental Section
General Methods: All operations were carried out under an inert
atmosphere of nitrogen. Glassware for all reactions was oven
dried at 125 °C and cooled under nitrogen prior to use. Liquid
reagents and solvents were introduced by oven-dried syringes or
6-((4-bromobenzyl)amino)benzo[c][1,2]oxaborol-1(3H)-ol (9):
1H NMR (500MHz, DMSO-d6): δ 8.92 (s, 1H), 7.51 (d, J = 8.0
Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.5 Hz, 1H), 6.83
(s, 1H), 6.74 (d, J = 8.5 Hz, 1H), 6.28 (br s, 1H), 4.82 (s, 2H),
4.26 (s, 2H); 13C NMR (125 MHz, DMSO-d6): δ 148.0, 142.2,
140.4, 131.6, 129.8, 122.0, 119.9, 117.1, 112.8, 70.0, 46.5; CHN
Analysis: (Found: C, 52.72%; H, 4.02%; N, 4.49%;
C14H13BBrNO2 requires: C, 52.88%; H, 4.12%; N, 4.41%).
cannulas through septa sealed flasks under
a nitrogen
atmosphere. THF was distilled from sodium benzophenone ketyl.
All other solvents and reagents were purchased and used without
further purification. The 1H and 13C NMR spectra were plotted on
a Bruker-500 spectrometer fitted with a Quad probe. Elemental
analysis was performed on Perkin Elmer PE2400 CHN analyzer.
6-((furan-2-ylmethyl)amino)benzo[c][1,2]oxaborol-1(3H)-ol
(10): 1H NMR (500MHz, DMSO-d6) δ 8.94 (s, 1H), 7.57 (d, J =
2.0 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 6.95 (s, 1H), 6.81-6.84 (m,
1H), 6.39-6.28 (m, 2H), 6.06 (t, J = 6.2 Hz, 1H), 4.84 (s, 2H),
4.25 (d, J = 6.0 Hz, 2H); 13C NMR (125 MHz, DMSO-d6) δ
153.9, 147.9, 142.3, 121.9, 117.1, 112.9, 110.8, 107.3, 70.0, 31.2;
CHN Analysis: (Found: C, 62.79%; H, 5.37%; N, 6.20%;
C12H12BNO3 requires: C, 62.93%; H, 5.28%; N, 6.12%).
6-amino-7-bromobenzo[c][1,2]oxaborol-1(3H)-ol (4): To
a
stirred solution of aminobenzoboroxole 3 (1mmol) in acetic acid
(4 mL) was added bromine (1 mmol) at 0oC and stirred for 5h.
Upon completion via thin layer chromatography (TLC), reaction
mixture was quenched by the addition of saturated NaHCO3
followed by extraction with ethyl acetate (3x15mL). The
combined organic layers were dried over anhydrous MgSO4,
concentrated under vacuum and purified via silica gel column
chromatography (hexanes: ethyl acetate) to obtain pure
6-(diallylamino)benzo[c][1,2]oxaborol-1(3H)-ol (11): To
a
1
stirred solution of aminobenzoboroxole 3 (1mmol) in DMSO (5
mL) was added allyl bromide (6 mmol) and K2CO3 (3.0 mmol).
The reaction mixture was stirred for 12h. Upon completion
(TLC), reaction mixture was extracted with ethyl acetate and
water. The combined organic layers were dried over anhydrous
MgSO4, concentrated under vacuum and purified via silica gel
column chromatography (hexanes: ethyl acetate) to obtain pure
compound 4 (55%). H NMR (500 MHz, DMSO-d6): δ 8.83 (s,
1H), 7.07 (d, J = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 5.12 (s,
2H), 4.81 (s, 2H); 13C NMR (125 MHz, DMSO-d6): δ 145.2,
143.7, 121.4, 119.5, 109.5, 69.1; CHN Analysis: (Found: C,
36.85%; H, 3.25%; N, 6.25%; C7H7BBrNO2 requires: C, 36.90%;
H, 3.10%; N, 6.15%).
1
6-amino-5,7-dibromobenzo[c][1,2]oxaborol-1(3H)-ol (5): To a
stirred solution of 6-aminobenzoboroxole 3 (1mmol) in acetic
acid (5 mL) was added bromine (2.5 mmol) and stirred for 5h.
Upon completion (TLC), the reaction mixture was quenched by
the addition of saturated NaHCO3 and extracted with ethyl
acetate (3x15mL). The combined organic layers were dried over
anhydrous MgSO4 and concentrated under vacuum and purified
via silica gel column chromatography (hexanes: ethyl acetate) to
obtain pure compound 5 (53%). 1H NMR (500 MHz, DMSO-d6):
δ 9.11 (s, 1H), 7.51 (s, 1H), 5.24 (s, 2H), 4.85 (s, 2H); 13C NMR
(125 MHz, DMSO-d6): δ 142.3, 142.0, 125.3, 112.3, 109.9, 68.7;
compound 10 (76%). H NMR (500MHz, DMSO-d6): δ 8.97 (s,
1H), 7.17 (d, J = 8.0 Hz, 1H), 7.03 (s, 1H), 6.84 (d, J = 8.5 Hz,
1H), 5.90-5.84 (m, 2H), 5.18-5.12 (m, 4H), 4.87 (s, 2H), 3.94-
3.91 (m, 4H); 13C NMR (125 MHz, DMSO-d6): δ 147.9, 141.9,
134.9, 128.6, 122.0, 116.3, 113.5, 70.0, 53.1; CHN Analysis:
(Found: C, 68.25%; H, 6.92%; N, 6.02%; C13H16BNO2 requires:
C, 68.16%; H, 7.04%; N, 6.11%).
6-(di(prop-2-yn-1-yl)amino)benzo[c][1,2]oxaborol-1(3H)-ol
1
(12): H NMR (500MHz, DMSO-d6): δ 9.08 (s, 1H), 7.30-7.27
(m, 2H), 7.11 (d, J = 8.5 Hz, 1H), 4.92 (s, 2H), 4.14 (s, 4H), 2.51
(s, 2H); 13C NMR (125 MHz, DMSO-d6): δ 147.1, 145.1, 122.1,

5
119.7, 116.9, 80.3, 75.6, 70.0, 40.8; CHN Analysis: (Found: C,
69.23%; H, 5.29%; N, 6.29%; C13H12BNO2 requires: C, 69.38%;
H, 5.37%; N, 6.22%).
CHN Analysis: (Found: C, 64.38%; H, 3.44%; N, 5.18%;
C15H10BNO4 requires: C, 64.56%; H, 3.61%; N, 5.02%).
ACCEPTED MANUSCRIPT
N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)-4-
6-(pyrrolidin-1-yl)benzo[c][1,2]oxaborol-1(3H)-ol (13): 1H
NMR (500MHz, DMSO-d6): δ 8.94 (s, 1H), 7.18 (d, J = 8.5 Hz,
1H), 6.86 (s, 1H), 6.69 (d, J = 8.5 Hz, 1H), 4.87 (s, 2H), 3.22 (t, J
= 6.0 Hz, 4H), 1.97-1.95 (m, 4H); 13C NMR (125 MHz, DMSO-
d6): δ 147.6, 141.1, 122.0, 115.7, 112.7, 70.0, 48.0, 25.4; CHN
Analysis: (Found: C, 64.96%; H, 7.02%; N, 6.79%; C11H14BNO2
requires: C, 65.07%; H, 6.95%; N, 6.90%).
6-(dibenzylamino)benzo[c][1,2]oxaborol-1(3H)-ol (14): 1H
NMR (500 MHz, DMSO-d6): δ 8.95 (s, 1H), 7.35-7.23 (m, 10H),
7.12 (d, J = 8.5 Hz, 1H), 7.07 (s, 1H), 6.84 (d, J = 8.0 Hz, 1H),
4.84 (s, 2H), 4.71 (s, 4H); 13C NMR (125 MHz, DMSO-d6): δ
148.0, 142.2, 139.4, 128.9, 127.2, 127.1, 122.1, 116.4, 113.6,
70.0, 54.8; CHN Analysis: (Found: C, 76.35%; H, 6.30%; N,
4.16%; C21H20BNO2 requires: C, 76.62%; H, 6.12%; N, 4.25%).
nitrobenzene sulfonamide (21): To a stirred solution of
aminobenzoboroxole 3 (1mmol) in dioxane (5 mL) was added 4-
nitrobenzenesulfonyl chloride (1.1 mmol) and stirred for 1h.
Upon completion (TLC), reaction mixture was quenched by the
addition of dilute HCl followed by extraction with ethyl acetate.
The combined organic layers were dried over anhydrous MgSO4,
concentrated under vacuum and purified by silica gel column
chromatography (hexanes: ethyl acetate) to obtain pure
1
compound 21 (40%). H NMR (500MHz, DMSO-d6): δ 9.34 (s,
1H), 7.99-7.90 (m, 4H), 7.76 (s, 1H), 7.57-7.52 (m, 2H), 5.06 (s,
2H); 13C NMR (125 MHz, DMSO-d6): δ 167.7, 154.1, 135.2,
132.1, 131.1, 130.3, 129.8, 123.9, 122.4, 70.4; CHN Analysis:
(Found: C, 46.64%; H, 3.19%; N, 8.25%; C13H11BN2O6S
requires: C, 46.73%; H, 3.32%; N, 8.38%).
Sulforhodamine-B assay for cytotoxicity: MCF-7 cells were
cultured in Iscove's Modified Eagle Medium containing 10%
Hyclone-III and 1% antibiotic (500,000 units penicillin-
streptomycin). The cells were seeded at a concentration of
~2x104 cells per well in 48 well plates and incubated for 18-24
hours at 37oC and 5% CO2 atmosphere. The test compounds were
initially diluted in DMSO and further diluted 1000 times in
growth media so that the final DMSO concentration was <0.1%.
Growth media was removed from 48 well plates and test
compound in 400µL of growth media were added. Taxol was
used as the positive control whereas DMSO and growth media
were used as negative controls. All the compounds were tested in
triplicates at 50 and 10µM. The plates were incubated for 72
hours and growth media was removed. The wells were washed
with 1mL of 1% DPBS and the plates were dried. 100µL of 0.5%
SRB (in 1% acetic acid) was added in each well and incubated at
37oC for 45 minutes. The wells were washed 5 times with 1%
acetic acid solution to remove residual SRB and the plates were
dried. The cellular protein was dissolved in 400µL of 10mM tris
base (pH 10.2) and absorbance was recorded at 540nm.
%Survival was calculated using the formula (Absorbance of test
compound/Absorbance of DMSO control) x 100%.
6-(bis(4-methylbenzyl)amino)benzo[c][1,2]oxaborol-1(3H)-ol
(15): H NMR (500MHz, DMSO-d6): δ 8.97 (s, 1H), 7.21-7.07
1
(m, 10H), 6.83-6.85 (m, 1H), 4.85 (s, 2H), 4.62 (s, 4H), 2.28 (s,
6H); 13C NMR (125 MHz, DMSO-d6): δ 148.2, 142.2, 136.3,
129.5, 129.1, 127.1, 122.1, 116.6, 113.8, 70.0, 54.6, 21.2; CHN
Analysis: (Found: C, 77.20%; H, 6.62%; N, 3.83%; C23H24BNO2
requires: C, 77.33%; H, 6.77%; N, 3.92%).
N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)acetamide
(16): To a stirred solution of aminobenzoboroxole 3 (1mmol) in
dioxane (5 mL) was added acetic anhydride (1.2 mmol) and
stirred for 1h. Upon completion (TLC), reaction mixture was
quenched by the addition of dilute HCl followed by extraction
with ethyl acetate. The organic layers were dried under
anhydrous MgSO4, concentrated under vacuum and purified via
silica gel column chromatography (hexanes: ethyl acetate) to
obtain amidobenzoboroxole 16 (67%).
N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6yl)butyramide
1
(17): H NMR (500MHz, DMSO-d6): δ 9.89 (s, 1H), 9.20 (s,
1H), 8.02 (s, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.0 Hz,
1H), 4.94 (s, 2H), 2.31 (t, J = 7.2 Hz, 2H), 1.66-1.61 (m, 2H),
0.94 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ
171.5, 148.9, 138.6, 122.8, 121.9, 121.5, 70.2, 38.8, 19.1, 14.1;
CHN Analysis: (Found: C, 60.13%; H, 6.38%; N, 6.45%;
C11H14BNO3 requires: C, 60.32%; H, 6.44%; N, 6.39%).
Bacterial strains, media and growth conditions: M. tuberculosis
H37Rv was grown in Middlebrook 7H9 broth medium (Difco)
supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) tween 80
and albumin dextrose catalase (ADC): 5g/L bovine serum
albumin (BSA); 2g/L dextrose; and 0.81g/L NaCl. M.
tuberculosis was also cultured in GAST medium consisting of
0.3g/L bacto-casitone, 4g/L dibasic potassium phosphate, 2g/L
citric acid, 1g/L L-alanine, 1.2g/L magnesium chloride
hexahydrate, 0.6g/L potassium sulfate, 2g/L ammonium chloride,
1% glycerol (v/v) and 0.05% tween 80 (v/v) at pH 6.6. For
generating non-replicating persistent cells M. tuberculosis was
cultured in Middlebrook Dubos medium (Difco) supplemented
with 5g/L BSA, 7.5g/L glucose and 0.81g/L NaCl. For
determination of viable cell counts, M. tuberculosis cells were
plated on Middlebrook 7H11 plates supplemented with 0.5%
glycerol (v/v) and oleic albumin dextrose catalase (OADC):
0.06% oleic acid; 4mg/L NaOH; 50g/L BSA; 2g/L dextrose; and
0.81g/L NaCl. All cultures were incubated at 37°C.
2,2,2-trifluoro-N-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-
1
6-yl)acetamide (18): H NMR (500MHz, DMSO-d6): δ 11.30 (s,
1H), 9.32 (s, 1H), 8.07 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.46 (d,
J = 8.5 Hz, 1H), 4.99 (s, 2H); 13C NMR (125 MHz, DMSO-d6): δ
155.1 (q, JF = 36.0 Hz), 151.6, 135.5, 124.6, 123.6, 122.4, 118.5
(q, JF = 285.0 Hz), 70.2; CHN Analysis: (Found: C, 44.01%; H,
2.99%; N, 5.60%; C9H7BF3NO3 requires: C, 44.13%; H, 2.88%;
N, 5.72%).
4-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-yl)amino)-4-
1
oxobutanoic acid (19): H NMR (500MHz, DMSO-d6): δ 12.13
(br s, 1H), 9.99 (s, 1H), 9.20 (s, 1H), 8.01 (s, 1H), 7.61 (d, J = 8.0
Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 4.94 (s, 2H), 2.55 (m, 4H); 13
C
NMR (125 MHz, DMSO-d6): δ 174.4, 170.5, 148.9, 138.6, 122.6,
121.93, 121.3, 70.2, 31.5, 29.3; CHN Analysis: (Found: C,
53.18%; H, 4.74%; N, 5.49%; C11H12BNO5 requires: C, 53.05%;
H, 4.86%; N, 5.62%).
Minimum inhibitory concentration (MIC99): MIC99 was
determined in 7H9 as well as GAST medium by micro-dilution
broth method. Briefly, M. tuberculosis was grown to an optical
density at 650nm (OD650) of 0.2-0.3 in 7H9 or GAST medium.
Cells were diluted to final OD650 of 0.0002 (1:1000 of parent
culture) in desired medium. Ten-fold serial dilution of the test
compounds was made in triplicate rows of a 96-well plate. The
2-((1-hydroxy-1,3-dihydrobenzo[c][1,2]oxaborol-6-
yl)carbamoyl)benzoic acid (20): 1H NMR (500MHz, DMSO-d6):
δ 9.36 (s, 1H), 8.00-7.93 (m, 4H), 7.78 (s, 1H), 7.59-7.55 (m,
2H), 5.08 (s, 2H); 13C NMR (125 MHz, DMSO-d6): δ 167.7,
154.1, 135.2, 132.1, 131.2, 130.3, 129.8, 123.9, 122.5, 70.4;

6
Tetrahedron
ACCEPTED MANUSCRIPT
above dilution of cells was then added to all the wells of the 96-
on 7H11 plates (supplemented with OADC). After 6 days of
well plate. The assay was performed in duplicate and each
compound dilution was set-up in triplicate rows per plate. The
plates were incubated at 37°C and observations were noted on
day-7, and day-14. MIC99 was noted as the lowest concentration
of test compound that inhibited visible growth after 14 days of
incubation.
treatment, all macrophages were lysed (by adding 0.1% SDS)
and serial dilutions were plated on 7H11 plates (supplemented
with OADC). Isoniazid (35µM) was used as a positive control
and DMSO was used as a negative control.
Acknowledgments
Minimum bactericidal concentration: M. tuberculosis was
grown to OD650 0.2-0.3 in 7H9 medium and cells were then
diluted to OD650 0.002 (1:100 of parent culture). For enumeration
of viable numbers on day 0, serial dilutions of the diluted cultures
were plated in duplicate on 7H11 plates (supplemented with
OADC). 1mL aliquots of the diluted culture were then dispensed
in duplicate wells of 24-well plates. Treatments with chosen test
compounds were carried out at 4µM and 40µM for 7 days. Serial
dilutions were then plated on 7H11 medium (supplemented with
OADC) and colonies were counted after 3-week incubation at
37°C and CFU/ml were determined.
The work was funded, in part by the Intramural Research
Program at NIAID, NIH. We thank Dr. Clifton E. Barry and Dr.
Helena Boshoff (NIAID, NIH) for their guidance with the study.
We thank Dr. John Dahl, Department of Biology, University of
Minnesota Duluth for kindly providing Mycobacterium
smegmatis culture. We thank the Departments of Chemistry and
Biochemistry at Rowan University and University of Minnesota
Duluth as well as the Integrated Biosciences Program.
References
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persistent (NRP) cells of M. tuberculosis were obtained by
culturing the cells in Wayne model system.15 Briefly, M.
tuberculosis was cultured in Dubos medium to an OD650 of 0.2-
0.3. Cells were then diluted to OD650 0.005 and dispensed in glass
bottles leaving a head-space ratio of 0.5. The bottles were sealed
with wax and placed on a magnetic stir plate at 37°C. The cells
were allowed to stir for 21 days at which point the culture
reached NRP2 state. The bottles were removed from the stir plate
and opened in an anaerobic chamber. For determination of day 0
cell numbers, serial dilutions of the culture were plated on 7H11
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concentrations of the candidate compounds for 7 days in the
anaerobic chamber at 37°C. After 7 days, the plates were
removed from the chamber and serial dilutions were then made
and plated on 7H11 plates (supplemented with OADC). Viable
numbers were counted after 4 weeks of incubation at 37°C and
CFU/ml was determined.
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