Design, syntheses, and anti-tuberculosis activities of conjugates of piperazino-1,3-benzothiazin-4-ones (pBTZs) with 2,7-dimethylimidazo [1,2-a]pyridine-3-carboxylic acids and 7-phenylacetyl cephalosporins

Article abstract of DOI:10.1016/j.bmcl.2016.02.076

Tuberculosis (TB) remains one of the most threatening diseases in the world and the need for development of new therapies is dire. Herein we describe the rationale for the design and subsequent syntheses and studies of conjugates between pBTZ and both the imidazopyridine and cephalosporin scaffolds. Overall some compounds exhibited notable anti-TB activity in the range of 2-0.2 μM in the Microplate Alamar Blue (MABA) Assay.

Full text of DOI:10.1016/j.bmcl.2016.02.076

Accepted Manuscript
Design, syntheses, and anti-tuberculosis activities of conjugates of piperazi-
no-1,3-benzothiazin-4-ones (pBTZs) with 2,7-dimethylimidazo [1,2-a]pyri-
dine-3-carboxylic acids and 7-phenylacetyl cephalosporins
Mark W. Majewski, Rohit Tiwari, Patricia A. Miller, Sanghyun Cho, Scott G.
Franzblau, Marvin J. Miller
PII:
S0960-894X(16)30197-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.076
BMCL 23630
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
17 December 2015
23 February 2016
24 February 2016
Please cite this article as: Majewski, M.W., Tiwari, R., Miller, P.A., Cho, S., Franzblau, S.G., Miller, M.J., Design,
syntheses, and anti-tuberculosis activities of conjugates of piperazino-1,3-benzothiazin-4-ones (pBTZs) with 2,7-
dimethylimidazo [1,2-a]pyridine-3-carboxylic acids and 7-phenylacetyl cephalosporins, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.076
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Design, syntheses, and anti-tuberculosis
activities of conjugates of piperazino-1,3-
benzothiazin-4-ones (pBTZs) with 2,7-
dimethylimidazo [1,2-a]pyridine-3-
carboxylic acids and 7-phenylacetyl
cephalosporins.
≠a
≠a,c
a
b
b
Mark W. Majewski, Rohit Tiwari, Patricia A. Miller, Sanghyun Cho, Scott G. Franzblau,
a
and Marvin J. Miller *

Bioorganic & Medicinal Chemistry Letters
Design, syntheses, and anti-tuberculosis activities of conjugates of
piperazino-1,3-benzothiazin-4-ones (pBTZs) with 2,7-dimethylimidazo [1,2-
a]pyridine-3-carboxylic acids and 7-phenylacetyl cephalosporins.
≠
a
≠a,c
a
b
b
Mark W. Majewski, Rohit Tiwari, Patricia A. Miller, Sanghyun Cho, Scott G. Franzblau,
a
and Marvin J. Miller *
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
c-Current Address: Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food & Drug Administration, 10993 New Hampshire
Avenue, Silver Spring, MD 20993.
≠
Both Authors Contributed Equally
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Tuberculosis (TB) remains one of the most threatening diseases in the world and the need for
development of new therapies is dire. Herein we describe the rationale for the design and
subsequent syntheses and studies of conjugates between pBTZ and both the imidazopyridine and
cephalosporin scaffolds. Overall some compounds exhibited notable anti-TB activity in the
range of 2 to 0.2 μM in the microplate alamar blue (MABA) assay.
2
009 Elsevier Ltd. All rights reserved.
Keywords:
Tuberculosis
Benzothiazinones
Imidazopyridine
Cephalosporin
Tuberculosis (TB), caused by Mycobacterium tuberculosis
Mtb), rivals HIV/AIDS as the most notorious single infectious
has been the use of drug cocktails that keep the mycobacterial
population in check by inhibiting not one, but multiple
biochemical processes. Fortunately, a number of promising
(
6
agent, posing a significant risk to global health. In 2013, it was
estimated by the World Health Organization that one-third of the
world population was infected with latent TB, approximately 9
anti-TB agents in development have distinct targets.
7
8
Nitroimidazoles (PA-824), benzothiazinones (BTZ043), and
1
9
million people fell ill with TB, and 1.5 million died of TB. The
imidazopyridines carboxamides all have been reported to be
emergence of multi-drug resistant (MDR) and extensively drug
resistant (XDR) TB have only put further pressure on the
medicinal community, as these new strains pose great challenges
potent anti-TB agents (Figure 1) and do not share target
similarities.
2
to existing treatments. Further, the classic TB drug regimens
have many issues associated with them, such as long treatment
3
duration and adverse drug-drug interactions. In an effort to
expand structure-activity-relationship studies of potential anti-TB
agents, we were interested in designing hybrid scaffolds. Herein,
we describe the synthetic coupling of piperazino-1,3-
4
benzothiazin-4-ones (pBTZs) with 2,7-dimethylimidazo [1,2-
a]pyridine-3-carboxylic acids and cephalosporins and anti-TB
evaluations of the synthesized conjugates.
The current TB treatment regimen for drug sensitive forms of
the disease is long and involves at least 6 months of therapy
involving many of the front line orally active anti-TB agents (e.g.
rifampin, isoniazid). This approach often suffers from poor
patient compliance and can further lead to the progression of
drug resistant TB forms. One potential way of slowing resistance
Figure 1. Promising anti-TB agents currently in development.
Work by Makarov et al. have shown that combination therapy
of pBTZ169, bedaquiline, and pyrazinamide was more effective
5
10
than the frontline TB regimen in murine models. Moreover,

Lechartler et. al. recently demonstrated that the combination of
clofazimine, a frontline anti-leprosy agent with pBTZ169 was
found to be synergistic against both replicating and non-
replicating Mtb. Intuitively, agents can be designed in a way
that they might have multiple targets. Therefore, we envisioned
chemical conjugation of 1,3-benzothiazin-4-ones, DprE1
inhibitors, with 7-acylamino cephalosporins and 2,7-
The primary scaffold, pBTZ (Figure 3) for synthetic and
biological activity studies, was synthesized as a TFA salt by
removing the Boc group of its N-Boc-protected precursor (Boc-
pBTZ-Boc, Figure 3), which in turn was synthesized according
11
1
7
to the published procedure.
pBTZ-imidazopyridine conjugate,
procedure was used to generate the necessary carboxylic acid.
For the syntheses of our first
a
previously published
18
dimethylimidazo[1,2-a]pyridine-3-carboxylic
acids.
This intermediate was then reacted with pBTZ to give conjugate
Cephalosporins and BTZs target the peptidoglycan and
arabinogalactan component of the bacterial cell wall respectively,
whereas the imidazo[1,2-a]pyridine-3-carboxamides have been
4 (Scheme 1).
12
shown to inhibit the cellular energy dependent process.
The β-lactam family continues to be a hallmark of medicinal
chemistry, having been discovered over 80 years ago, yet still
making up the majority of the antibiotic market. Currently, the
cephalosporin class of β-lactam antibiotics is the most widely
used type of β-lactam, with sales estimated at 11.9 billion dollars,
Scheme 1. Synthesis of pBTZ-imidazopyridine conjugate (4) without any
linker.
13
topping the list of antibacterial agents in 2009. As shown in
Figure 2, one of the reasons the cephalosporins have endured the
test of time is because a multitude of different functionalities
have been successfully incorporated at the C-3’ position of the
cephalosporin core (1). To date, a myriad of different agents,
In order to explore the effect of a linker between pBTZ and
the imidazopyridine scaffold, we chose to incorporate 4-
aminomethyl benzoic acid as a representative amino acid linker.
As shown in Scheme 2, the Boc group of benzyl 4-(((tert-
butoxycarbonyl)amino)methyl)benzoate (5) was deprotected and
the subsequent amine (6) was coupled with 3 to obtain 2,7-
dimethylimidazo[1,2-a]pyridine-3-carboxamide, 7. Compound 7
was then subjected to hydrogenolysis to give carboxylic acid 8
which was used without further purification for coupling with
pBTZ in the presence of tetramethylfluoroformamidinium
hexafluorophosphate (TFFH) and DIPEA to obtain the final
conjugate 9 in 36% yield.
1
4
such as quinolone antibiotics (1) and pyridyl N-oxide toxins
15
(
2) have been conjugated to the cephalosporin core. With the
above precedence in mind, we focused on the syntheses and anti-
TB evaluations of conjugates of pBTZs and cephalosporins.
Figure 2. Two examples of cephalosporin conjugates previously reported
The major scaffolds considered for this study are illustrated in
Figure 3. Our synthetic strategy involved the synthesis of the
1
,3-benzothiazin-4-one scaffold, wherein we opted to utilize the
16
core of the recently reported analog of BTZ043, pBTZ, as one
can introduce appropriate substituents at the terminal piperazinyl
nitrogen potentially without affecting the target interaction
(
DprE1) with the nitro aromatic functionality. The next part
involved syntheses of imidazopyridine-pBTZ conjugate without
Scheme 2) and with (Scheme 3) different linkers. Lastly, the
(
syntheses of appropriately functionalized cephalosporins were
performed (Schemes 4 and 5).
Scheme 2. Syntheses of pBTZ-imidazopyridine conjugate 9.
For our syntheses of pBTZ-cephalosporin conjugates, we
began with the construction of two activated cephalosporins for
eventual pBTZ conjugation. As shown in Scheme 3, starting
Figure 3. Scaffolds of interest in this study

with commercially available 7-aminocephalosporanic acid (7-
Interestingly, however, one of the imidazopyridine intermediates
(7) for the syntheses of conjugate 9 was quite active with an MIC
of 0.21 μM in 7H12 media and 4.1 μM in GAS media. Often,
differences in activity in different media occur as the result of
factors such as compound solubility, different carbon sources,
19
ACA), tert-butyl esterification followed by acylation and
20
enzymatic deprotection gave 10. The hydroxyl group was then
converted to an activated carbonate by reaction with 1,2,2,2-
tetrachloroethylchloroformate to give 11. Cephalosporin 11 was
then coupled to pBTZ to give 12 and deprotection with TFA gave
conjugate 13.
22
and media age.
Scheme 4 . Synthesis of pBTZ-cephalosporin 16.
SAR studies of the cephalosporin-pBTZ conjugates also
revealed some media dependent activity against Mtb. Conjugate
Scheme 3. Synthesis of pBTZ-Cephalosporin 13.
1
5a was the most active, with MIC values of 2.03 and 1.51 μM in
the 7H12 and GAS media, respectively. The difference in
activity between conjugates 13 and 16; however, was somewhat
surprising. Since both conjugates possessed the free carboxylate
normally associated with potentiating β-lactams for activity, it
was anticipated that activity might have been enhanced. Instead,
the protected conjugates, (e.g. 12 and 15a) were more potent
agents in general. This may be due to the greater lipophilicity of
these intermediates relative to their free acid counterparts, thus
allowing entry into Mtb. Interestingly, however, intermediate
As with our pBTZ-imidazopyridine syntheses, we were also
interested in exploring the effect of a linker on the anti-TB
activities of the cephalosporin-pBTZ conjugates. As a result of
the lipophilic cell wall (thick layer of mycolic acids)
characteristic of Mtb, we tested various protected cephalosporin-
pBTZ conjugates to see whether these more lipophilic protected
cephalosporins would exhibit anti-TB activity. As shown in
Scheme 4, we started with cephalosporin 10. Reaction with
thionyl chloride gave 14a in good yield and subsequent reactions
of 14a and 14b (commercially available) with NaI and pBTZ
gave intermediates 15a-b. Deprotection of 15b with TFA gave
conjugate 16.
1
5b was completely devoid of activity. Thus, it seemed that the
choice of protecting group might significantly activate or
deactivate these compounds.
Table 1. MIC Determinations (μM) for Select Compounds Against
Mycobacterium Tuberculosis (H37Rv)in the Microplate Alamar Blue
Assay (MABA).
All compounds were then subjected to anti-TB evaluations
21
against Mtb in the Microplate Alamar Blue assay (MABA). As
shown in Table 1, a number of compounds exhibited good
activity against Mtb including the broad activity that conjugates 4
and 15a exhibited against the H37Rv strain of Mtb in two
different mycobacterial growth media (7H12 and GAS).
Although notable, the observed activities of all highlighted
conjugates were still significantly lower than either the prototype
imidazopyridine analogs or pBTZ 169, BTZ043 or the precursor
pBTZ-Boc. The anti-TB evaluations of conjugate 9 indicated
that the introduction of a linker between the imidazopyridine
scaffold and pBTZ was detrimental for anti-TB activity. We
additionally explored the syntheses and anti-TB evaluations of
amino acid linkers such as β-alanine and γ-aminobutyric acid
a
b
a
b
Cmpd.
7H12
GAS
Cmpd.
7H12
GAS
4
7
9
2.10
0.21
>10
1.43
4.10
>10
16
86.8
48.9
Rifampin
BTZ-043
pBTZ-Boc
0.05
0.04
<0.004
0.28
<0.004
<0.20
1
1
1
1
2
3
>50
22.22
63.45
11.96
1.51
1.49
4.63
2.03
>100
15a
1
5b
>100
a
7
H12 = 7H9 medium + casitone, palmitric acid, albumin, and
(see Supporting Information). Nevertheless, the anti-TB
b
catalase; GAS = glycerol-alanine salts medium
evaluations of these conjugates again indicated that the presence
of a linker had a deleterious effect on the anti-TB activity.

Lastly, cephalosporin-pBTZ conjugates were also screened
for antibacterial activity. As shown in Table 2, the conjugates
targeted Gram-positive bacteria, exhibiting both potent zones of
inhibition (see Supporting Information) and MIC values. Of
outstanding interest was both cephalosporin-pBTZ conjugates 13
and 16, demonstrating notable inhibition against M. vaccae and
B. subtilis, with MICs of 0.2 μM and <0.003 μM, respectively.
3.
Zumla, A.; Nahid, P.; Cole, S.T. Nat. Rev. Drug Dis. 2013, 12,
88-404.
3
4
5
.
.
Trefzer, C.; Skovierova, H.; Buroni, S.; Bobovska, A.; Nenci,
S.; Molteni, E.; Pojer, F.; Pasca, M. R.; Makarov, V.; Cole, S. T.;
Riccardi, G.; Mikusova, K.; Johnsson, K. J. Am. Chem. Soc.
2012, 134, 912-915.
a) Minarini, P. R. R.; de Souza, A. O.; Soares, E. G.; Barata, L. E.
S.; Silva, C. L.; Bentley, M. V. Maria Vitoria, L. B. Drug. Dev.
Ind.
Pharm.
2012,
38,
259-263.
b)
http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5211a1.htm c)
Zumla, A.; Raviglione, M.; Hafner, R.; von Reyn, C. F. N. Engl. J.
Med . 2013, 368, 745-755.
Table 2. Minimum Inhibitory Determinations (μM) for Select Compounds.
B. subtilis
S. aureus
SG511
0.2
M. luteus
M. vaccae
6.
7.
Zumla, A.; Raviglione, M.; Hafner, R.; von Reyn, C. F. N. Engl.
J. Med. 2013, 368, 745-755.
Cmpd.
ATCC
ATCC
10240
IMET 10670
6
633
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.
1
, X = OAc
<0.1
nt
3.13
>200
nt
nt
0.03
0.20
0.8
nt
BTZ043
>200
12.5
12.5
1.56
0.4
8
.
Makarov, V.; Manina, G.; Mikusova, K.; Mollmann, U.; Ryabova,
O.; Saint-Joanis, B.; Dhar, N.; Pasca, M. R.; Buroni, S.; Lucarelli,
A. P.; Milano, A.; De Rossi, E.; Belanova, M.; Bobovska, A.;
Dianiskova, P.; Kordulakova, J.; Sala, C.; Fullam, E.; Schneider,
P.; McKinney, J. D.; Brodin, P.; Christophe, T.; Waddell, S.;
Butcher, P.; Albrethsen, J.; Rosenkrands, I.; Brosch, R.; Nandi,
V.; Bharath, S.; Gaonkar, S.; Shandil, R. K.; Balasubramanian, V.;
Balganesh, T.; Tyagi, S.; Grosset, J.; Riccardi, G.; Cole, S. T.
Science 2009, 324, 801-804.
1
3
0.80-1.60
1.56
1
5a
5b
nt
1
0.1-0.2
<0.003
0.15-0.32
nt
1
6
1.56
nt
12.5
nt
9
1
.
Cheng, Y.; Moraski, G. C.; Cramer, J.; Miller, M. J.; Schorey J. S.
PLoS One. 2014, 9, e87483.
Ciprofloxicin
nt
0.
Makarov, V.; Lechartier, B.; Zhang, M.; Neres, J.; van der Sar, A.
M.; Raadsen, S. A.; Hartkoorn, R. C.; Ryabova, O. B.; Vocat, A.;
Decosterd, L. A.; Widmer, N.; Buclin, T.; Bitter, W.; Andries, K.;
Pojer, F.; Dyson, P. J.; Cole, S. T. EMBO Mol. Med. 2014, 6, 372-
Compounds were dissolved in MeOH/DMSO
KEY: B. subtilis = Bacillus subtilis, S. aureus. = Staphylococcus aureus, M.
luteus = Micrococcus luteus, M. vaccae = Mycobacterium vaccae.
nt = not tested
3
83.
To summarize, we have synthesized a focused set of
conjugates between pBTZ and both imidazopyridines and 7-
phenylacetamido cephalosporins and tested them for anti-TB
activity in the MABA assay. The product of direct conjugation
between pBTZ and imidazopyridine (compound 4) exhibited
anti-TB activity albeit not as impressive as its precursors and the
introduction of linkers between the two precursor scaffolds in 4
resulted in dramatic loss of activity. Anti-TB activity was
observed only for the very lipophilic 15a, whereas similarly
lipophilic analog 15b was completely inactive. Potent Gram-
positive antibacterial activity was seen for cephalosporin-pBTZ
conjugates 13 and 15-16.
11.
Lechartier, B.; Hartkoorn, R. C.; Cole, S. T. Antimicrob. Agents
Chemother. 2012, 56, 5790-5793.
1
2.
Moraski, G. C.; Markley, L. D.; Hipskind, P. A.; Boshoff, H; Cho,
S.; Franzblau, S. G.; Miller, M. J. ACS Med. Chem. Lett. 2011,
2, 466-470.
1
1
3.
4.
Hamad, B. Nat. Rev. Drug Dis. 2010, 9, 675-676.
a) Albrecht, H. A.; Beskid, G.; Christenson, J. G.; Deitcher, K. H.;
Georgopapadakou, N. H.; Keith, D. D.; Konzelmann, F. M.;
Pruess, D. L.; Wei, C. C. J. Med. Chem. 1994, 37, 400-407. b)
Albrecht, H. A.; Christenson, J. G. Adv. Med. Chem. 1992, 1,
2
07-234.
Acknowledgments
1
5.
O’Callaghan, C. H.; Sykes, R. B.; Staniworth, S. E.
Antimicrob. Agents Chemother. 1976, 10, 245-248.
We acknowledge the University of Notre Dame, and the NIH
(
NIH-2R01-AI054193-05A2) for support of this work. We
16.
Gao, C.; Ye, T. H.; Wang, N. Y.; Zeng, X. X.; Zhang, L. D.;
Xiong, Y.; You, X. Y.; Xia, Y.; Xu, Y.; Peng, C. T.; Zuo, W. Q.;
Wei, Y.; Yu, L.T. Bioorg. Med. Chem. Lett. 2013, 23, 4919-
acknowledge Nonka Sevova (Mass Spectrometry and Proteomics
Facility, UND) for mass spectroscopic analyses and Viktor
Krchňák for LC/MS and prep LC assistance.
4
922.
M.W.M
acknowledges an ECK Global Health Fellowship (2014-2015,
UND).
1
1
7.
8.
Peng, C. T.; Gao, C.; Wang, N. Y.; You, X. Y.; Zhang, L. D.;
Zhu, Y. X.; Xv, Y.; Zuo, W. Q.; Ran, K.; Deng, H. X.; Lei, Q.;
Xiao, K. J.; Yu, L. T.; Bioorg. Med. Chem. Lett. 2015, 25, 1373-
1
376.
References and notes
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.
1
2
.
.
Global Tuberculosis Report 2014,
http://www.who.int/tb/publications/global_report/en/
Gandhi, N. R.; Nunn, P.; Dheda, K.; Schaaf, H. S.; Zignol, M.;
19.
Glinka, T.; Huie, K.; Cho, A.; Ludwikow, M.; Blais, J.; Griffith,
D.; Hecker, S.; Dudley, M. Bioorg. Med. Chem. 2003, 11, 591-
600.
Van Soolingen D.; Jensen, P.; Bayona, J. Lancet 2010, 375, 1830-
1
843.

2
2
0.
1.
Patterson, L. D.; Miller, M. J. J. Org. Chem. 2010, 75, 1289-
292.
1
a) Collins, L.; Franzblau, S. G. Antimicrob. Agents
Chemother. 1997, 41, 1004-1009. b) DeVoss, J. J.; Rutter, K.;
rd
Schroeder, B.; Su, H.; Zhu, Y.; Barry, C. E. 3 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.
2
2.
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,
4
53-488.
Supplementary Material
Synthetic procedures, compound characterization data, and
MABA assay procedures are available online. This material is
available free of charge via the Internet.