Syntheses and evaluation of substituted aromatic hydroxamates and hydroxamic acids that target Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2015.04.099

Tuberculosis (TB) continues to remain one of the most threatening diseases in the world. With the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains, the need to develop new therapies is dire. The syntheses of a focused library of hydroxamates and hydroxamic acids is described, as well as anti-TB activity in the microplate alamar blue assay (MABA). A number of compounds exhibited good activity against Mtb, with notable compounds exhibiting MIC values in the range of 20-0.71 μM. This work suggests that both hydroxamates and their free acids may be incorporated into more complex scaffolds and serve as potential leads for the development of anti-TB agents.

Full text of DOI:10.1016/j.bmcl.2015.04.099

Accepted Manuscript
Syntheses and evaluation of substituted aromatic hydroxamates and hydroxamic
acids that target mycobacterium tuberculosis
Mark W. Majewski, Sanghyun Cho, Patricia A. Miller, Scott G. Franzblau,
Marvin J. Miller
PII:
S0960-894X(15)00444-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.04.099
BMCL 22684
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
20 February 2015
24 April 2015
30 April 2015
Please cite this article as: Majewski, M.W., Cho, S., Miller, P.A., Franzblau, S.G., Miller, M.J., Syntheses and
evaluation of substituted aromatic hydroxamates and hydroxamic acids that target mycobacterium tuberculosis,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.04.099
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Graphical Abstract
Syntheses and evaluation of substituted
aromatic hydroxamates and hydroxamic
acids that target Mycobacterium tuberculosis.
Leave this area blank for abstract info.
Mark W. Majewski,a Sanghyun Cho,b Patricia A. Miller,a Scott G. Franzblau,b and Marvin J. Millera,*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Syntheses and evaluation of substituted aromatic hydroxamates and hydroxamic
acids that target mycobacterium tuberculosis.
Mark W. Majewski,a Sanghyun Cho,b Patricia A. Miller,a Scott G. Franzblau,b and Marvin J. Millera,*
a Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, 46556, USA
b Institute for Tuberculosis Research, College of Pharmacy, MIC 964, Rm. 412, University of Illinois at Chicago, IL, 60612, USA
*phone: 574 631 7571 Faz: 574 631 6652 email: mmiller1@nd.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Tuberculosis (TB) continues to remain one of the most threatening diseases in the world. With
the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains, the
need to develop new therapies is dire. The syntheses of a focused library of hydroxamates and
hydroxamic acids is described, as well as anti-TB activity in the microplate alamar blue assay
(MABA). A number of compounds exhibited good activity against Mtb, with notable
compounds exhibiting MIC values in the range of 20-0.71 µM. This work suggests that both
hydroxamates and their free acids may be incorporated into more complex scaffolds and serve as
potential leads for the development of anti-TB agents.
Revised
Accepted
Available online
Keywords:
Tuberculosis
Hydroxamate
Hydroxamic Acid
2014 Elsevier Ltd. All rights reserved.
Tuberculosis (TB), caused by Mycobacterium tuberculosis
(Mtb), is a harsh disease that infects approximately one-third of
the global population. TB is a highly contagious disease, the
second major cause of death as the result of a bacterial infection
worldwide.1 TB targets the lungs in most cases and can spread
quite rapidly, a result of the ease in which this infection can be
transmitted among individuals. On average, there is a new case
of TB each second2 and, although most are latent infections, the
actual cases of active TB continue to grow, approaching 10
million as of 2010.3 TB exhibits a high mortality rate, with up to
2 million deaths associated with this pathogen every year.4 Over
the last 25 years, TB has seen a resurgence in both the number of
cases and severity of infection.5 The emergence of multi-drug
resistant (MDR), extensively drug resistant (XDR), and totally
drug resistant (TDR) strains has further exacerbated this
situation.6 TB drug discovery is an arduous process primarily
because of the obstacles that need to be circumvented such as the
thick, lipophilic cell wall characterized by the presence of
mycolic acids and arabinogalactan.7 Prior to December 31st 2012,
there was a 40 year gap between an FDA approved anti-TB
agent, with bedaquiline recently approved.8 With this in mind, a
current interest in our lab is in the development of new anti-TB
agents9 based on a scaffold simplification strategy.10 Herein we
report on the syntheses of such compounds and their anti-
tubercular activities.
promising new compounds have been reported that have
demonstrated potent activity against TB. Although the majority
of these are still in pre-clinical investigations, they may serve as
welcome additions in the fight against TB.
This list is
highlighted by PA-82412 and BTZ-04313 (Figure 1). A common
feature to both of these classes of anti-TB agent is the presence of
an electron deficient aromatic ring. This moiety continues to be
reported as crucial for activity, especially for the BTZ class of
compounds.14
Figure 1. Two Promising Anti-TB Compounds in Development.
With these findings in mind, we were interested in combining
electron deficient systems with more lipophilic hydroxamates
and hydroxamic acids and testing them against Mtb. Both the
hydroxamate and hydroxamic acid functional group have proven
to be very important in a multitude of medicinally useful
agents.15 The ability of hydroxamic acids to readily chelate metal
ions is well established in the literature,16 thus making them
useful moieties for interactions with metalloproteases. Notably,
this family of iron chelators has been incorporated in
siderophores and has been found to inhibit lipoxygenase, matrix
Although there are a number of classic treatments for TB (i.e.
isoniazid, rifampin), there remain many issues with them,
including drug toxicity, long treatment duration, and adverse
drug-drug interactions. In cases where the TB infection is
treatable, the regimen consists of administration of multiple
drugs for at least six months.11 Fortunately, a number of

metalloproteinases, and histone deacetylases.17 Beyond just the
consideration of metal chelation, we were interested to determine
if the combination of the hydroxamate and hydroxamic acid
functionalities with aromatic rings of varying electron densities
would generate an anti-mycobacterial agent. Generally, the
utilization of both hydroxamates and hydroxamic acids in TB
therapy is scarce.18 Here we report on the syntheses of these
compounds and evaluation of their anti-TB and anti-microbial
activities.
the absence of the nitro group would affect TB activity, so we
coupled 3-bromo-5-(trifluoromethyl)benzoic acid, 13, to give
hydroxamate 14.
First, simple unsubstituted hydroxamates of interest were
generated (Scheme 1). Here, benzoic acid was coupled to both
O-benzylhydroxylamine
allylhydroxylamine
hydrochloride
hydrochloride
and
using
O-
N-(3-
hydrochloride
dimethylaminopropyl)-N′-ethylcarbodiimide
(EDC•HCl) under aqueous conditions to give hydroxamates 2a-
b. To enhance lipophilicity, silylated hydroxamate 2c was
synthesized in 54% yield from 1 and tert-butyldimethylsilyl
hydroxylamine and EDC•HCl in DCM. Next, hydroxamates
with para-substituted aromatic rings were generated (Scheme 2).
First, the appropriately substituted benzoic acid, 3, and
substituted hydroxylamine was coupled with EDC•HCl to give
hydroxamates 4a-e in varying yields. Intermediates 4c-e were
then subjected to hydrogenolysis conditions to give hydroxamic
acids 5a-c. Lastly, the para-nitrohydroxamic acid 7 was made
via the direct reaction of ethyl 4-nitrobenzoate, 6, with
hydroxylamine hydrochloride.
Scheme 3. Syntheses of 3,5-Substituted Compounds.
Lastly, Scheme 4 shows the syntheses of hydroxamates from
simple malonate monoacids. As an extension of our structure-
activity relationship studies, we were interested in further varying
substrate polarity and see if malonate based hydroxamates and
their corresponding acids demonstrated anti-TB activity. Here
Meldrum’s acid, 15, was opened to give malonates 16a-b which
were then coupled to the appropriate substituted hydroxylamine
to give hydroxamates 17a-c. Finally, silyl deprotection with
tetrabutylammonium fluoride (TBAF) afforded 18b-c.
Scheme 1. Syntheses of Hydroxamates 2a-c.
Scheme 4. Syntheses of Hydroxamate Malonates.
All hydroxamates and hydroxamic acids were then subjected
to anti-microbial evaluations. Minimum inhibitory concentration
(MIC) data for select compounds against Mtb in the microplate
alamar blue assay (MABA)19 are displayed in Table 1. From this
data, we observed that some compounds exhibited good media-
dependent activity against Mtb. Often, differences in MIC values
between the two media are attributed to factors such as
compound solubility and media age.20 Among the compounds
tested, both the hydroxamate protecting group and electronics of
the aromatic system did affect activity. The data showed that the
benzyl protected hydroxamates had, in general, better activity
than the allyl hydroxamates. This is possibly due to the fact that
the allyl group represents a decrease in lipophilicity and, as a
result, diminished the compound’s ability to pass through the
thick cell wall of Mtb.
Scheme 2. Syntheses of para-Substituted Compounds.
The syntheses of hydroxamates with the 3-nitro-5-
(trifluoromethyl) aromatic group are shown in Scheme 3. This is
the same moiety that is featured in BTZ-043 and shown to be
crucial for activity (Figure 1). Thus, we were curious if
combining simple hydroxamates and hydroxamic acids with this
key functionality would affect anti-TB activity. EDC coupling of
3-nitro-5-(trifluoromethyl)benzoic acid, 8, with the appropriate
substituted hydroxylamine hydrochloride gave 9a-b and 10 in
varying yields. For the synthesis of 12, a number of different
paths were tested; however, the best involved esterification to
give methyl ester 11 followed by treatment with KOH and
hydroxylamine hydrochloride (HA•HCl) in MeOH to give 12.
As a result of challenges in the purification of 12, a crude sample
was tested (80-85% pure). We were also interested to see how
Interesting results were also obtained when the electronics of
the aromatic ring were varied. When comparing activities among
the para-substituted hydroxamates and their corresponding acids,

Table 2. Antibacterial Assay Results
MIC
(µM)
Agar Diffusion Zones (mm)
B.
S.
M.
M.
P. aeruginosa
E. coli
M. vaccae
subtilis
ATCC
6633
aureus
luteus
ATCC
10240
vaccae
IMET
10670
Compound
K799
/wt
IMET
10670
SG511
K799/61
DC2
2c
0
19* ppt
0
0
19 ppt
0
0
26/30P
15 ppt
15.5V
0
14/18P
0
0
12.5
nt
4d
19P* ppt
14P ppt
14 ppt
0
5b
0
0
0
18P
14P
0
16
nt
5c
20
17
0
0
0
nt
7
0
0
30/33P
45
13
0
0
0
6.25
3.13
nt
9a
16
20
0
14/20V
0
10
14
20
0
16
0
14
0
0
17P
10/15P
0
17.5
0
20
0
26/32P
0
0
0
12.5
nt
DMSO/MeOH
0
0
1.66
µg/mL
19
1.66 µg/mL
19/26P
Ciprofloxicin
18
0
20
23
21
nt
*All solutions were prepared by first making a 20 mM solution in DMSO and diluting 10-fold with MeOH to give 2 mM solutions. Plates were incubated at 37
ºC for 21 h. M. vaccae was incubated 37 ºC for 44 h. All zones reported are in mm.
KEY:
P:
*:
nt:
Unclear inhibition zone
Misshapen zone of inhibition
Not tested
V:
ppt:
Very unclear inhibition zone
Compound precipitated in media
we were surprised to see only small changes in anti-TB activity
among compounds as both the number and nature of the
With regards to hydroxamate-malonate compounds,
compound 17b was our most potent agent. This hydroxamate,
like compounds 4d and 9a, exhibited both a very lipophilic group
(TBS) and an electron deficient ring (PNB). Anticipating that
activity could be further optimized, compounds 17a, 17c, and
free acid 18 were made and tested. These compounds were
inferior in overall activity. Overall, these results helped further
solidify the notion that electron deficiency, specifically the
incorporation of nitro functionality, may transform simple
scaffolds into anti-TB agents. Further, our data illustrated the
point that greater lipophilicity may assist in potentiating activity
(e.g. 2c, 4a, and 9); however, is not necessarily a requirement for
the anti-TB activity of this library.
substituents were varied.
Anticipating that activity may be
enhanced as the aromatic ring became increasingly electron
deficient, we were intrigued by the small and non-linear changes
in TB activity among compounds 4c-e and 5a-c. Thus, it was
especially interesting that the most polar hydroxamate, free acid
7, demonstrated the best overall anti-TB activity in our library,
while other para-substituted hydroxamates, acids, and even
benzohydroxamic acid (not shown in Table 1) were substantially
less active.
Hypothesizing that activity may be further
potentiated by making the aromatic ring increasingly electron
deficient, we then studied systems featuring 3-nitro-5-
(trifluoromethyl) substitution. Here we saw that 9a was the most
active in this series; however, the activity was again inferior to
para-nitro hydroxamic acid 7.
Representative antibacterial evaluations performed using the
Kirby-Bauer agar diffusion assay,21 are shown in Table 2. As
shown, some compounds that were active against TB were also
quite active against M. vaccae. Based on the aforementioned
anti-TB activity, this result was not completely unexpected since
both bacterial strains are from the mycobacteria genus.
Additionally, for compounds that showed notable zones of
inhibition against M. vaccae (2c, 7, 9a, and 14) minimum
inhibitory concentrations (MICs) were determined. As can be
seen 9a was the most active against M. vaccae, with a MIC of
3.13 µM.
In conclusion, a number of compounds synthesized exhibited
notable anti-TB activity (MIC values 15.54 to 0.71 µM) and
specific antimicrobial activity. Although at this stage of our
studies the exact target and/or general mode of action remain
unknown, these compounds may serve as potential scaffolds for
the generation of new anti-TB agents.

Riccardi, G.; Mikusova, K.; Johnsson, K. J. Am. Chem. Soc. 2012,
1345, 912-915.
Acknowledgments
14. Tiwari, R.; Moraski, G. C.; Krchnak, V.; Miller, P. A.; Colon-
Martinez, M.; Herrero, E.; Oliver, A. G.; Miller, M. J. J. Am.
Chem. Soc. 2013, 135, 3539-3549.
We acknowledge Nonka Sevova (Mass Spectrometry and
Proteomics Facility, UND) for mass spectroscopic analyses. We
acknowledge the University of Notre Dame and the NIH (NIH-
2R01-AI054193-05A2) for support of this work. M.W.M
acknowledges an ECK Institute Global Fellowship (2014-2015,
UND).
15. a) Flipo, M.; Beghyn, T.; Charton, J.; Leroux, V. A.; Deprez, B.
P.; Deprez-Poulain, R. F. Bioorg. Med. Chem. 2007, 15, 63-76. b)
Irwin, J. J.; Raushel, F. M.; Shoichet, B. K. Biochem. 2005, 44,
12316-12328. c) Lin, Y. M.; Miller, M. J. J. Org. Chem. 1999, 64,
7451-7458.
16. Day, J. A.; Cohen, S. M. J. Med. Chem. 2013, 56, 7997-8007.
17. Antonello, M.; “Hydroxamic acids: Biological properties and
potential uses as therapeutic agents.” In The Chemistry of
hydroxylamines, oximes, and hydroxamic acids, Rappoport, Z;
Liebman, J. F., Ed.; Wiley: New York, 2011; Vol. 2; p 731.
18. a) Kohn, H.; Kim, M. G.; Krause, K. L.; Briggs, J. M.; Benedik,
M.; Strych, U. “Hydroxamate and hydroxylamine compounds
inhibiting alanine racemase and having antimycobacterial
activity.” WO 2005020973 A2, March 10, 2005. b) Burgos, E.;
Roos, A. K.; Mowbray, S. L.; Salmon, L. Tetrahedron Lett. 2005,
46, 3691-3694.
References and notes
1. Chitre, T. S.; Bothara, K. G. J. Chem. Pharm. Res. 2011, 3, 479-
488.
2. Tuberculosis
World
Health
Organization.
2007.
http://who.int/mediacentre/factsheets/fs104/en/index.html.
Retrieved 12 November 2009. Fact sheet No. 104
3. Dye, C.; Williams, B. G. Science, 2010, 328, 856-861.
4. Kaithamanakallam, R. P.; Karunakaran, R.; Srikumar, P. S. Int. J.
Pharm. Pharm. Sci. 2013, 5, 662-664.
19. a) Collins, L.; Franzblau, S. G. Antimicrob. Agents. Chemother.
1997, 41, 1004-1009. b) De Voss, J. J.; Rutter, K.; Schroeder, B.
Su, H.; Zhu, Y.; Barry, C. E. 3rd Proc. Natl. Acad. Sci. USA.
2000, 97, 1252-1257. c) Cho, S. H.; Warit, S.; Wan, B.; Hwang,
C. H.; Pauli, G. F.; Franzblau, S. G. Antimicrob. Agents
Chemother. 2007, 51, 1380-1385.
5. a) Mays, E. E. J. Natl. Med. Assoc. 1990, 82, 829-831. b)
Kempker, R. R.; Rabin, A. S.; Nikolaishvili, K.; Kalandadze, I.;
Gogishvili, S.; Blumberg, H. M.; Vashakidze, S. Clin. Infect. Dis.
2012, 54, e51-e54.
6. Velayati, A. A.; Farnia, P.; Masjedi M. R.; Ibrahim, T. A.; Tabarsi
P.; Haroun R. Z.; Kuan H. O.; Ghanavi J.; Farnia P.; Varahram M.
Eur. Respir. J. 2009, 34, 1202-1203.
20. Franzblau, S. G.; DeGroote, M. A.; Cho, S. H.; Andries, K.;
Nuermberger, E.; Orme, I. M.; Mdluli, K.; Angelo-Barturen, I.;
Dick, T.; Dartois, V.; Lenaerts, A. J. Tuberculosis 2012, 92, 453-
488.
7. Zumla, A.; Nahid, P.; Cole, S. T. Nat. Rev. 2013, 12, 388-404.
8. Wong, E. B.; Cohen, K. A.; Bishai, W. R. Trends Microbiol. 2013,
21, 493-501.
21. a) Afonin, S.; Glaser, R. W.; Berditchevskaja, M.; Wadhwani, P.;
Gührs, K.-H.; Möllmann, U.; Perner, A.; Ulrich, A. S. Chem. Bio.
Chem. 2003, 4, 1151-1163. b) Murray, P. R.; Baron, E. J.; Pfaller,
M. A.; Tenover, F. C.; Yolken, R. H. Manual of Clinical
Microbiology, 7th ed.; American Society for Microbiology:
Washington, DC, 1999.
9. a) Moraski, G. C.; Oliver, A. G.; Markley, L. D.; Cho, S.;
Franzblau, S. G.; Miller, M. J. Bioorg. Med. Chem Lett. 2014, 24,
3493-3498. b) Cheng, Y.; Moraski, G. C.; Cramer, J.; Miller, M.
J.; Schorey, J. S. PLoS One. 2014, 9, e87483/1-e87483/8. c)
Moraski, G. C.; Markley, L. D.; Cramer, J.; Hipskind, P. A.;
Boshoff, H.; Bailey, M. A.; Alling, T.; Ollinger, J.; Parish, T.;
Miller, M. J. ACS Med. Chem. Lett. 2013, 4, 675-679. d) Juarez-
Hernandez, R. E.; Franzblau, S. G.; Miller, M. J. Org. Biomol.
Chem. 10, 7584-7593.
Supplementary Material
10. Tiwari, R.; Mӧllmann, U.; Cho, S.; Franzblau, S. G.; Miller, P. A.;
Miller, M. J. ACS Med. Chem. Lett. 2014, 5, 587-591.
Data associated with this article, including experimental
procedures and characterization data for all compounds, is
available with the online version of this article.
11. Morbidoni, H. Cur. Res. Med. Rev. 2009, 5, 190-200.
12. Singh, R.; Manjunatha, U.; Boshoff, H. I. M.; Ha, Y. H.;
Niyomrattanakit, P.; Ledwidge, R.; Dowd, C.S.; Lee, I. Y; Kim,
P.; Zhang, L.; Kang, S.; Keller, T. H.; Jiricek, J.; Barry, C. E. III,
Science, 2008, 322, 1392-1395.
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13. Trefzer, C.; Skovierova, H.; Buroni, S.; Bobovska, A.; Nenci, S.;
Molteni, E.; Pojer, F.; Pasca, M. R.; Makarov, V.; Cole, S. T.;

Table 1. MIC Determinations (µM) for Select Compounds Against
Mycobacterium Tuberculosis in the Microplate Alamar Blue Assay
Compound
7H12a
GASb
Compound
7H12a
GASb
>100
>100
>50
48.45
>100
24.29
13.60
37.61
42.26
15.54
47.31
>100
>100
>100
2a
2b
2c
4a
4b
4c
4d
4e
5a
5b
5c
7
7.79
>100
>100
>100
>100
19.48
>100
86.59
>100
98.65
>100
0.71
10.65
43.17
>100
29.96
>100
80.53
27.11
3.04
9a
9b
90.42
>100
>100
47.31
>100
>100
>100
48.07
10
11
12*
14
17a
17b
17c
18
98.58
>100
a7H12 = 7H9 medium + casitone, palmitric
acid, albumin, and catalase
Rifampin
0.03
<0.016
bGAS = glycerol-alanine salts medium
*Crude Material Screened