6-Nitro-2,3-dihydroimidazo[2,1-b][1,3]thiazoles: Facile synthesis and comparative appraisal against tuberculosis and neglected tropical diseases

Article abstract of DOI:10.1016/j.bmcl.2017.03.069

As part of a quest for backups to the antitubercular drug pretomanid (PA-824), we investigated the unexplored 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]-thiazoles and related -oxazoles. The nitroimidazothiazoles were prepared in high yield from 2-bromo-4-nitroimidazole via heating with substituted thiiranes and diisopropylethylamine. Equivalent examples of these two structural classes provided broadly comparable MICs, with 2-methyl substitution and extended aryloxymethyl side chains preferred; albeit, S-oxidised thiazoles were ineffective for tuberculosis. Favourable microsomal stability data for a biaryl thiazole (45) led to its assessment in an acute Mycobacterium tuberculosis mouse model, alongside the corresponding oxazole (48), but the latter proved to be more efficacious. In vitro screening against kinetoplastid diseases revealed that nitroimidazothiazoles were inactive versus leishmaniasis but showed interesting activity, superior to that of the nitroimidazooxazoles, against Chagas disease. Overall, “thio-delamanid” (49) is regarded as the best lead.

Full text of DOI:10.1016/j.bmcl.2017.03.069

Accepted Manuscript
6-Nitro-2,3-dihydroimidazo[2,1-b][1,3]thiazoles: facile synthesis and compa-
rative appraisal against tuberculosis and neglected tropical diseases
Andrew M. Thompson, Adrian Blaser, Brian D. Palmer, Robert F. Anderson,
Sujata S. Shinde, Delphine Launay, Eric Chatelain, Louis Maes, Scott G.
Franzblau, Baojie Wan, Yuehong Wang, Zhenkun Ma, William A. Denny
PII:
S0960-894X(17)30318-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.069
BMCL 24821
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 February 2017
21 March 2017
23 March 2017
Please cite this article as: Thompson, A.M., Blaser, A., Palmer, B.D., Anderson, R.F., Shinde, S.S., Launay, D.,
Chatelain, E., Maes, L., Franzblau, S.G., Wan, B., Wang, Y., Ma, Z., Denny, W.A., 6-Nitro-2,3-dihydroimidazo[2,1-
b][1,3]thiazoles: facile synthesis and comparative appraisal against tuberculosis and neglected tropical diseases,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.069
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6-Nitro-2,3-dihydroimidazo[2,1-b][1,3]thiazoles: facile synthesis and
comparative appraisal against tuberculosis and neglected tropical diseases
Andrew M. Thompsona,*, Adrian Blasera, Brian D. Palmera, Robert F. Andersona,b, Sujata S.
Shindea,b, Delphine Launayc, Eric Chatelainc, Louis Maesd, Scott G. Franzblaue, Baojie Wane,
Yuehong Wange, Zhenkun Maf, and William A. Dennya
aAuckland Cancer Society Research Centre, School of Medical Sciences, The University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand; bDepartment of Chemistry, The
University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; cDrugs for
Neglected Diseases initiative, 15 Chemin Louis Dunant, 1202 Geneva, Switzerland;
dLaboratory for Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical,
Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610
Antwerp, Belgium; eInstitute for Tuberculosis Research, College of Pharmacy, University of
Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, USA; fGlobal Alliance
for TB Drug Development, 40 Wall St, New York 10005, USA
*Corresponding author: Dr Andrew M. Thompson, Auckland Cancer Society Research
Centre, School of Medical Sciences, The University of Auckland. Ph: (+649) 923 6145. Fax:
(+649) 373 7502. Email: am.thompson@auckland.ac.nz
Keywords: Tuberculosis, Chagas disease, delamanid, in vivo efficacy, nitroimidazole

As part of a quest for backups to the antitubercular drug pretomanid (PA-824), we
investigated the unexplored 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]-thiazoles and related
-oxazoles. The nitroimidazothiazoles were prepared in high yield from 2-bromo-4-
nitroimidazole via heating with substituted thiiranes and diisopropylethylamine. Equivalent
examples of these two structural classes provided broadly comparable MICs, with 2-methyl
substitution and extended aryloxymethyl side chains preferred; albeit, S-oxidised thiazoles
were ineffective for tuberculosis. Favourable microsomal stability data for a biaryl thiazole
(45) led to its assessment in an acute Mycobacterium tuberculosis mouse model, alongside
the corresponding oxazole (48), but the latter proved to be more efficacious. In vitro
screening against kinetoplastid diseases revealed that nitroimidazothiazoles were inactive
versus leishmaniasis but showed interesting activity, superior to that of the
nitroimidazooxazoles, against Chagas disease. Overall, “thio-delamanid” (49) is regarded as
the best lead.

More than 2 billion people are latently infected with Mycobacterium tuberculosis (M. tb) and
up to 10% of these will develop active tuberculosis (TB).1 While renewed efforts over the last
two decades have reduced the TB mortality rate in 1990 by an impressive 45%, an enormous
global disease burden remains (10.4 million new cases in 2015, with 1.4 million deaths).2
Synergy with HIV/AIDS and the ongoing spread of often undiagnosed multidrug-resistant
(MDR) and extensively drug-resistant (XDR) TB undergird this major epidemic.2-4 Current
treatment regimens are complex, lengthy and toxic, yet for almost half a century no new
therapies were developed.4-6 Very recently though, two new highly lipophilic drugs have been
approved for treating MDR-TB: the diarylquinoline bedaquiline and delamanid (1, see Fig.
1).6,7 Pretomanid (PA-824, 2) has also progressed to phase II/III clinical trials within various
new drug regimens having the potential to shorten and simplify therapy for all forms of TB.2,7
N
F
F
N
N
N
N
N
N
F
F
N
N
N
N
DNDI L
N
N
N
N
N
N
N
N
N
N
N
N
Figure 1. Nitro drugs/leads for TB, leishmaniasis and Chagas disease
In collaboration with the TB Alliance, we initially investigated new nitroheterobicyclic ring
analogues of 2, seeking a novel active scaffold upon which to develop a backup series.8
However, amongst the fused 5/6 ring systems examined, only nitroimidazothiazines (e.g., 3)
retained significant potency, pointing to the critical importance of the nitroimidazole moiety
and its interesting ring reduction chemistry.8,9 While 3 was metabolised into inactive oxides
too rapidly to be a useful lead (vide infra), we postulated that novel nitroimidazothiazole
analogues of the known nitroimidazooxazole class10 (cf. 1) might be significantly more stable
due to steric factors, and therefore worthy of assessment, as described herein. For
comparative purposes, we also elected to evaluate corresponding nitroimidazooxazoles. Our
broad strategy was to explore both monoaryl and biaryl side chains, due to the markedly
better potency and in vivo efficacy of biaryl analogues of 2 (which ultimately became our
major focus for backup studies).11,12 Since this work was performed, Mugunthan13 reported
the synthesis of two sugar-derived 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]thiazoles and their
oxazole counterparts as anti-TB agents, but these compounds had poor activities in
comparison to 2, and no further examples have been described to date.
Neglected tropical diseases (NTDs) caused by pathogens such as viruses, bacteria, protozoa
and helminths affect one-sixth of the world’s population, resulting in enormous suffering and
more than half a million deaths annually, yet there is an extreme paucity of safe, effective and

affordable medications for their treatment, and the drug pipelines are almost empty.14,15 We
have recently recounted16 that phenotypic screening of selected nitroimidazole derivatives
(above) against kinetoplastid diseases by the Drugs for Neglected Diseases initiative (DNDi)
unexpectedly led to discovery of the outstanding activity of 4 in mouse and hamster models
of visceral leishmaniasis (VL) and then to its selection as a preclinical candidate. As part of
our backup studies with DNDi, we elucidated SARs for nitroimidazooxazoles against 3
NTDs, identifying efficacious new leads for VL, as well as novel hits for Chagas disease.16
Current therapeutic options for Chagas disease are limited to two drugs (benznidazole 5 and
nifurtimox 6) that can cause serious side effects and have reduced efficacy in chronic cases.17
A phase II clinical study of fexinidazole (7: an investigational drug for human African
trypanosomiasis18) in chronic Chagas patients was interrupted due to safety and tolerability
issues,19 reinforcing the urgent need for new treatments.15 Therefore, as an extension to the
present study, we tested our new analogues in several anti-parasitic assays, seeking
innovative leads for the development of oral agents to combat these NTDs.
The novel 6-nitro-2,3-dihydroimidazo[2,1-b][1,3]thiazoles (“thiazoles”) were readily
accessible in a single step, via the base-assisted solventless reaction (DIPEA, 108 °C) of 2-
bromo-4-nitroimidazole with appropriately substituted thiiranes (Scheme 1). The latter were
conveniently obtained in generally high yield from the corresponding epoxides, following
treatment with thiourea on silica gel.20 Hence, compounds with benzyl ether side chains (8, 9,
and 12, cf. 2) were initially achieved by starting from TBS-protected glycidol (60), or its 2-
methyl derivative (64)21 and elaborating to the thiazole alcohols 63 and 51 (Scheme 1A).
Scheme 1. Reagents and conditions: (i) thiourea/silica gel, CH2Cl2, 20 °C, 2.5-3 h (37-94%);
(ii) 2-bromo-4-nitroimidazole, DIPEA, 108-109 °C, 12-21 h (73-95%); (iii) 1% HCl in 95%
EtOH, 20 °C, 7 h (100%); (iv) 4-OCF3BnBr or 4-BnOBnI or 4-BnOBnCl or 4-IBnBr, NaH,
DMF, 0-20 °C, 0.7-16 h (32-93%); (v) TBAF, THF, 20 °C, 4 h (96%); (vi) (R)-(-)-MTPA-Cl,
DMAP, pyridine, 20 °C, 6 h (99%); (vii) preparative chiral HPLC (CHIRALCEL OD, 40%
iPrOH/hexane) (40-42%); (viii) K2CO3, aq MeOH, 20 °C, 3-3.5 h (100%); (ix) NaH, DMF, 0
°C, 80 min (88-93%); (x) m-CPBA, Na2HPO4, CH2Cl2, 0-20 °C, 2.5-22 h (10-97%); (xi)
ArB(OH)2, toluene, EtOH, 2 M Na2CO3, Pd(dppf)Cl2 under N2, 90 °C, 1 h (67-74%).

The enantiomers of 12 were also regarded as useful early TB targets, but we were not
persuaded that any of the reported methods for thiirane synthesis could ensure products of
sufficiently high optical purity.20 Therefore, racemic alcohol 51 was derivatized to 67 (using
Mosher’s acid chloride, R form), and then the diastereomeric esters (68 and 69) were
separated by chiral preparative HPLC and hydrolysed, leading to 13 and 14. The absolute
configurations of these were deduced from their assay results against M. tb in comparison to
data for known nitroimidazooxazoles,16 as assignment based on the 1H NMR spectra of
Mosher esters derived from β-chiral primary alcohols is not regarded as definitive.22 For the
larger scale preparation of 12 and 15, benzyl ether substituted thiiranes 80 and 81 were
synthesised (Scheme 1B). Reaction of these with 2-bromo-4-nitroimidazole also gave small
amounts (2%) of 5-nitro isomers 24 and 25, while reaction of 2-bromo-4-nitroimidazole with
related epoxides, followed by NaH-assisted ring closure of the alcohol intermediates at 0
°C,16 provided the nitroimidazooxazoles (10, 11, 16, and 17). S-Oxidation of 12 and 15 with
m-CPBA (3-4 equiv) yielded mixtures of the sulfoxide and sulfone derivatives, which were
separated by chromatography on silica gel to give 18-23. Finally, analogues with a biaryl side
chain (26-28) were readily obtained via Suzuki couplings on the iodobenzyl ether 82.
Scheme 2. Reagents and conditions: (i) RPhOH, K2CO3, acetone, 58-60 °C, 24-41 h (68-
95%); (ii) thiourea/silica gel, CH2Cl2, 20 °C, 2-5 h (64-89%); (iii) 2-bromo-4-nitroimidazole,
DIPEA, 105-108 °C, 3-16 h (65-91%); (iv) NaH, DMF, 0 °C, 40-50 min (92-94%); (v) m-
CPBA, Na2HPO4, CH2Cl2, 0-20 °C, 3.5-70 h (19-93%); (vi) RPhOH, K2CO3, NaI, DMF, 72-
82 °C, 1-2.5 d (49-73%); (vii) ArB(OH)2, toluene, EtOH, 2 M Na2CO3, Pd(dppf)Cl2 under
N2, 90 °C, 1.3-3 h (52-67%); (viii) 92, DIPEA, 105 °C, 14 h (17-50%).
Phenyl ether substituted thiazoles (and oxazole congeners) were similarly accessed from
appropriately substituted epoxides16,23,24 (Scheme 2A). The latter approach became necessary
when attempts to form 29 and 33 from the alcohols 63 and 51 via Mitsunobu reactions (or
from the mesylate derivative of 51 by reaction with the phenol in K2CO3/DMF) proved too
low-yielding, due to either facile elimination to an exocyclic alkene (in the case of 63) or the
competitive formation of other close-running side products (for 51). Of note, a more direct
route to the epoxides 98 and 101 (Scheme 2B), starting from commercial 2-(chloromethyl)-2-
methyloxirane (97), allowed a critical non-oxidative synthesis of the latter (which contained a

sensitive tertiary amine). Unexpectedly, Suzuki couplings on thiazole 100 (to give 43-45)
proved to be particularly difficult, requiring higher catalyst loadings and longer reaction
times for completion (consistent with catalyst poisoning by sulfur); however, it is unclear
why this complication was more evident with this shorter side chain length. Lastly, an
alternative base-assisted reaction of thiirane 92 with 5-bromo-3-nitro-1,2,4-triazole (103)
(DIPEA, 105 °C) resulted in a separable 3:1 mixture of nitro and bromo products (53 and 58)
and oxidation of 53 with m-CPBA (1.8 equiv) provided the remaining targets (55-57). All
new compounds were characterised by 1H NMR, MS, melting point, and combustion analysis
(or HRMS and HPLC); full synthetic procedures and characterisation data for several
representative examples have been provided in the Supporting Information.
Table 1. In vitro antitubercular and antiparasitic activities of benzyloxymethyl-substituted
nitroimidazothiazoles and nitroimidazooxazoles
MICa,b (µM)
MABA LORA VERO L. inf T. cruzi T. bruc MRC-5
IC50c,b (µM)
compd Fm
X
Y
S
S
O
O
S
R
A
A
A
A
A
H
H
H
H
Me
OCF3
OBn
OCF3
OBn
4.4
4.4
14
15
3.4
43
63
7.9
5.8
51
12
34
50
>128
>128
>128 0.11
>128
NT
24
64
48
0.87
0.62
0.60
6.9
0.67
0.57
1.6
0.28
0.42
3.2
>64
6.2
>64
61
>64
>64
>64
>64
>64
43
8
9
10d
11
12
13
14
15
16d
17
18
19
20
21
22
23
24
25
26
27
28
11
1.5
57
>64
>64
56
OCF3 0.24
OCF3 0.20
56
Ae Me
Af Me
A
A
A
S
>64
>64
31
S
S
OCF3
OBn
6.1
3.2
52
NT
>64
64
Me
Me
Me
O
O
OCF3 0.24
OBn 0.30
>128 0.28
60
60
60
>128
NT
NT
NT
NT
NT
NT
45
NT
NT
NT
NT
7.3
>64
>64
56
>64
>64
56
>64
>64
>64
>64
>64
>64
62
Ag Me
Ah Me
Ag Me
Ah Me
SO OCF3 >128 >128
SO OCF3 >128 >128
1.6
3.0
>64
>64
0.33
2.3
>64
0.98
22
SO OBn
SO OBn
>128 >128
>128 >128
0.08
0.49
0.97
0.09
7.3
A
A
B
B
C
C
C
Me SO2 OCF3 >128
Me SO2 OBn >128 >128
55 59
>128 >128
21
Me
Me
Me
Me
Me
S
S
S
S
S
OCF3
OBn
F
CF3
OCF3
>64
55
47
19
53
>64
>64
>64
>64
1.5
1.1
1.4
>64
>64
>64
>64
56
56
0.15
0.04
0.14
>64
1.1
2.8
aMinimum inhibitory concentration against M. tb, measured under aerobic (MABA)26 or
hypoxic (LORA)27 conditions. bEach value (except the single test VERO data) is the mean of
≥2 independent determinations (NT means not tested). For complete results (mean SD)
please refer to the Supporting Information. cIC50 values for inhibition of growth of the
parasites Leishmania infantum, Trypanosoma cruzi, and Trypanosoma brucei, or for
cytotoxicity toward human lung fibroblasts (MRC-5 cells) and VERO cells. dRef. 16. e(R)-
Enantiomer. f(S)-Enantiomer. gLess polar diastereoisomer (TLC). hMore polar
diastereoisomer (TLC).

The structures and in vitro biological assay data for the 41 new and 11 known16,25 target
compounds are summarised in Tables 1 and 3. For the TB studies, MIC values describing
growth inhibitions of ≥90% against M. tb (strain H37Rv) were measured under both aerobic
and hypoxic conditions (MABA26 and LORA27 assays, respectively). This recognised the
finding that 2 displays unique modes of action under these differing oxygenation states,7 and
that testing compounds against bacteria exposed to one or more host-relevant stresses is
postulated to assist the discovery of drugs with greater sterilizing ability against persistent
bacteria.28 Cytotoxicity against mammalian cells (VERO) was also tested in a 72 h assay.26 In
the NTD work, 50% inhibitory concentrations provided quantification of the levels of activity
against three protozoan parasites (L. infantum, T. cruzi, and T. brucei) or cytotoxic effects on
human lung fibroblasts (MRC-5 cells).29 In all cases (except for VERO), recorded data are
mean values derived from two or more independent experiments.
In line with our original studies on fused 5/6 ring nitroheterobicycles,8 our first side chain
targets in the new nitroimidazothiazole series were simple benzyl ethers having either a 4-
trifluoromethoxy or 4-benzyloxy substituent (8, 9, 12, and 15; Table 1). This choice reflected
both the excellent in vivo antitubercular effects of 2 and the superior in vitro potency of its 4-
benzyloxybenzyl analogue (an order of magnitude better than 2,30 as also shown for the
racemates8). Although the latter compound was reportedly inferior to 2 in vivo (due to less
optimal pharmacokinetic properties),31 we had elected to employ this 4-benzyloxyaryl side
chain in several studies8,30 as an SAR tool to probe the potential to enhance in vitro potency
by increasing lipophilicity.11 Such effects may be derived from hydrophobic binding to
specific aromatic residues within the N terminus of the known nitroreductase Ddn.32
Table 2. Reduction potentials, solubility values, microsomal stabilities, and in vivo
antitubercular efficacy data for selected analogues
Solubilityb
(µg/mL)
pH 7 pH 1
Microsomesc
(% remaining at 1 h)
In vivo efficacy
against M. tb
(ratio vs 2)d
68
E(1)a
(mV)
compd
1
H
M
87
94
0.7
0.31
19
116
71
82
42
1.00
2
-534
7
5
3e
-534
4f
2.4
2.4
9.3
12
13
16
19
22
45
48
49
51
52
-537
9
29
68
0
30
11
0.025
-492
-519
6
7
0.25
0.41
0.18
84
82
75
81
5.7
115
13
-527
-526
5
6
aOne-electron reduction potentials, determined8 by pulse radiolysis. bSolubility in water (pH
7) or 0.1 M HCl (pH 1) at 20 °C, measured16 by HPLC. cPooled human (H) or CD-1 mouse
(M) liver microsomes. dFold reduction in lung colony forming units (CFUs) for compound
compared with the fold CFU reduction for 2 in a mouse model26 of acute TB infection. eRef.
8. fRef. 16.

Of these initial targets, the 2-methyl thiazole 12 appeared to be the most promising for TB
(MABA MIC 0.24 µM), being fairly comparable to 2 (MABA MIC 0.50 µM, LORA MIC
2.6 µM)11 and slightly superior to 3 (MABA MIC 1.1 µM, LORA MIC 13 µM).8 Thiazole 12
also had equivalent solubility to 4 and a one-electron reduction potential very similar to 2 and
3 (Table 2). The R enantiomer of 12 (13) was 9- to 31-fold more potent than the S enantiomer
14 in the two MIC assays, consistent with the recorded 63-fold difference in aerobic activity
for the enantiomers of a phenoxy analogue in the nitroimidazooxazole series.33 However, the
S-oxidised derivatives 18-23 were generally devoid of any antitubercular activity (despite 19
and 22 respectively having 16 and 31 mV lower one-electron reduction potentials than
corresponding nitroimidazothiazine oxide analogues;8 Table 2), while the 5-nitro isomer of
12 (24) was more than 200-fold less active in the MABA assay. Moreover, unlike the SAR
for nitroimidazooxazines (e.g., 2),11,12 the formation of biaryl analogues of 12 (26-28)
actually resulted in a decreased level of potency.
These findings led us to investigate the shorter chain length phenyl ethers (29, 30, 33, 34, and
37-42) and their biaryl derivatives (43-45; Table 3). Here, a 2-methyl substituent on the
thiazole ring was again favoured, but 33 (MABA MIC 0.49 µM) was not superior to 12, and
S-oxidation (37-42) still conferred a 29- to >260-fold potency loss (MABA). Nevertheless,
the aerobic potencies of the biaryl derivatives 43-45 were notably improved (up to 8-fold
over 33), although activity in the hypoxic assay was poor. Interestingly, the racemic thiazole
analogue of 1 (49) was equivalent in potency to the biaryl compound 45, while the simple
alcohol 51 and the nitrotriazole analogue 53 were relatively ineffective, in line with SAR for
the oxazine series.8 However, in order to better understand the significance of these data, it
was necessary to consider oxazole congeners having the same side chains. The oxazole
benzyl ethers (10, 11, 16, 17) were all less active than related thiazoles in the LORA assay (3-
to 19-fold), whereas in the MABA assay, 10 and 11 were less active than 8 and 9 but 16 was
equipotent to 12 and 17 was 11-fold better than 15. Similarly, oxazole phenyl ether 35 was
superior to 33 under aerobic conditions only (6-fold better in the MABA assay but 6-fold
worse in the LORA assay). For the biaryl oxazole derivatives (46-48), the fluoro analogue 46
was more effective in both TB assays than thiazole 43 but there was little variance between
the other pairs and racemic 1 (50) was only marginally better than 49. Therefore, apart from a
couple of exceptions, the nitroimidazothiazoles and nitroimidazooxazoles were fairly
comparable in terms of their overall in vitro antitubercular activity.
To determine whether the nitroimidazothiazole class could be developed any further, it was
important to assess the potential for metabolism (e.g., to the inactive oxides), as this was an
irreparable flaw with the nitroimidazothiazine series, based on the microsomal stability data
obtained for 3 (Table 2). The (2R) benzyl ether 13 was indeed metabolised very rapidly,
particularly by mouse liver microsomes, and was clearly less stable than the related oxazole
16. Nevertheless, results for the more lipophilic and shorter linked biaryl derivative 45 were
more encouraging, with an approximately 80% recovery of the parent compound following a
1 h exposure to either mouse or human liver microsomes, similar to data for the equivalent
oxazole 48. Therefore, compounds 45 and 48 were further evaluated head-to-head (together
with 2) in a mouse model of acute TB infection, dosing orally for 3 weeks (daily at 100
mg/kg, for 5 days each week).11,26 Here, in comparison to 2 (Table 2), oxazole 48 gave a 115-
fold greater reduction in colony-forming units (CFUs) in the lung, whereas thiazole 45 was
significantly less efficacious (5.7-fold better than 2, 20-fold less than 48). Both delamanid 1
and the VL lead 4 also demonstrated better activities than 45 in this model, although benzyl
ether 16 was inferior (consistent with its low microsomal stability). Based on these findings,
we elected to discontinue development of the nitroimidazothiazole class for TB.

Table 3. In vitro antitubercular and antiparasitic activities of (phenoxy or hydroxy)methyl-
substituted nitroimidazothiazoles, nitroimidazooxazoles, and triazole-based analogues
MICa,b (µM)
MABA LORA VERO L. inf T. cruzi T. bruc MRC-5
IC50c,b (µM)
compd Fm
X
Y
S
S
R
A
A
A
A
A
A
A
A
H
H
OCF3
OBn
OCF3
OBn
4.5
>128
2.0
8.2
29
73
>128
>128
115
>64
56
0.85
1.6
>64
11
>64
56
29
30
31d
32
H
H
Me
Me
Me
Me
O
O
S
S
O
O
0.15
3.4
>64
>64
0.33
1.3
>64
>64
55
1.5
3.1
0.38
0.14
1.8
1.9
2.3
1.7
0.19
0.75
1.8
0.46
0.21
0.035
0.085
0.52
1.5
0.89
0.055
0.40
NT
>64
>64
>64
>64
>64
28
>64
19
0.96 >128 >128
OCF3 0.49
OBn 0.76
OCF3 0.077
OBn 0.79
10
5.3
55
24
>128
>128
NT
>64
>64
>64
>64
>64
>64
53
51
>64
45
33
34
35d,e
36
NT
Af Me
Ag Me
Af Me
Ag Me
SO OCF3 >128 >128 >128
>64
41
37
38
SO OCF3
SO OBn
SO OBn
26
>128 >128 >128
>128
14
58
>128
0.36
0.98
>64
3.8
>64
52
39
40
25
15
>128
>128
42
>64
55
A
A
B
B
B
B
B
B
C
C
D
D
E
Me SO2 OCF3
Me SO2 OBn
41
42
>128 >128 >128
43
44
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
S
S
F
CF3
0.21
0.12
>64 >128
46 >128
>64
>64
45
3.3
11
5.3
53
13
>64
>64
>64
>64
>64
>64
>64
>64
NT
>64
>64
>64
1.2
S
OCF3 0.063 >128 >128
>64
9.2
1.0
0.85
59
3.4
NT
>64
10
45
46d
47d
48d
49
O
O
O
S
O
S
O
S
O
F
CF3
0.043
0.081 >128 >128
OCF3 0.088
11
>128
64
71
26
>128
>128
>128
NT
>128
104
0.073
0.051
10
16
21
50d,e
51
>128
116
>128
NT
18
53
>64
16
52d,e
53
52
OCF3
0.49
0.59
0.60
1.2
54d
55
E
OCF3 >128 >128 >128
20
Ef Me
Eg Me
SO OCF3
SO OCF3
Me SO2 OCF3
Me
Me
6.4
9.3
2.2
24
61
90
50
98
46
89
60
7.1
3.0
2.1
34
0.90
1.3
>64
>64
0.74
0.42
42
56
57
E
F
F
27
>128
>128
1.3
6.2
13
S
O
OCF3
OCF3
58
59d
46
60
43
aMinimum inhibitory concentration against M. tb, measured under aerobic (MABA)26 or
hypoxic (LORA)27 conditions. bEach value (except the single test VERO data) is the mean of
≥2 independent determinations (NT means not tested). For complete results (mean SD)
please refer to the Supporting Information. cIC50 values for inhibition of growth of the
parasites Leishmania infantum, Trypanosoma cruzi, and Trypanosoma brucei, or for
cytotoxicity toward human lung fibroblasts (MRC-5 cells) and VERO cells. dRef. 16. eRef.
25. fLess polar diastereoisomer (TLC). gMore polar diastereoisomer (TLC).

Following our discovery that nitroimidazooxazoles such as 4 were highly efficacious against
VL, we screened these thiazole analogues for possible utility against NTDs (Tables 1 and 3).
However, none of the nitroimidazothiazoles showed any significant activity against VL (L.
inf IC50s >40 µM). A novel nitroreductase (NTR2) has recently been identified as the enzyme
responsible for the activation of 4 in Leishmania parasites.34 In our previous SAR study of
4,16 we had shown that both the position of the nitro group (at C-6) and the imidazole ring
itself were essential for the retention of VL potency, broadly mimicking the structural
requirements for TB activity (involving substrate binding to the nitroreductase Ddn32). The
one-electron reduction potential of thiazole alcohol 51 (-527 5 mV; Table 2) was equivalent
to that obtained for the corresponding oxazole alcohol 52 (and similar to data measured for 2,
3, and 12). Therefore, the observed lack of activity against VL for nitroimidazothiazoles
cannot be accounted for by any differences in reduction potential, which may point instead to
a highly critical role for the ring oxygen atom of 4 in binding to NTR2, in order for
successful activation of the nitroimidazole “warhead” to occur.
In other screening results, the thiazoles were generally ineffective against human African
trypanosomiasis (T. brucei IC50s mostly >10 µM), except for two sulfoxide derivatives (20
and 39), which displayed moderate potencies (IC50s ~0.35 µM). However, while the latter
results were intriguing, the lack of activity shown by the parent thiazoles (15 and 34) and the
(3- to 11-fold) weaker potencies observed for the more polar sulfoxide diastereomer forms
(21 and 40) and their sulfone analogues (23 and 42) discouraged further assessment of these
hits. In contrast, many thiazole compounds, including 15 and 34 and (notably) their oxidised
derivatives (20, 21, 23, 39, 40, and 42), as well as biaryls such as 26-28 and 43-45, and the
thiazole analogue of 1 (49), all showed interesting activity (IC50s of 0.035-0.8 µM) against T.
cruzi, the protozoan parasite responsible for Chagas disease. For the 6-nitroimidazothiazoles,
potency tended to increase with lipophilicity (Fig. 2; CLogP data from ChemDraw v. 15.0).
Interestingly, the nitrotriazole-based congener 53 also showed reasonable potency in the same
assay (IC50 0.49 µM) but its S-oxidised derivatives (55-57) were cytotoxic. Overall, where
oxazole comparators were available, the thiazoles were superior in 11/13 cases (and by at
least 5-fold for most of these), suggesting a general advantage for this latter class. Previous
studies directed at finding a more stable backup to 2 had verified the faster metabolism of its
4-benzyloxybenzyl analogue and the improved stability achieved by removal of benzylic
methylenes.30,35 Therefore, we were unsurprised by results from a first assessment of sulfone
23 revealing a fairly short (25 min) half-life in human liver microsomes. Taken together,
these data point to biaryls 43-45 and especially the delamanid counterpart 49 (T. cruzi IC50
0.055 µM; IC90 0.19 µM) as the most useful new antitrypanosomal leads.
7.5
7.25
7
6.75
6.5
6.25
6
5.75
3
3.5
4
4.5
5
5.5
6
CLogP
Figure 2. Effect of lipophilicity on potency against T. cruzi for 15 6-nitroimidazothiazoles

To summarise, we first set out to synthesise and assess the utility of novel
nitroimidazothiazole analogues of the known nitroimidazooxazole class as potential
antitubercular agents. The ring system was easily constructed in one high yielding step,
providing simple access to the target compounds. Based on their promising MIC data,
comparable to those of the corresponding oxazoles, we then examined the microsomal
stabilities of two of the most potent examples, benzyl ether 13 and biaryl 45. However, while
the latter compound demonstrated a low susceptibility toward in vitro metabolism, equivalent
to results for the oxazole congener 48, it was found to be 20-fold less effective than 48 in a
mouse model of acute TB infection. The unexpected identification of racemic oxazole 35,
then 4, as potential development candidates for VL triggered more extensive SAR studies and
the discovery of novel leads for Chagas disease, including the interesting triazole 54. Despite
their close structural similarity to nitroimidazooxazoles and equivalent reduction potentials,
nitroimidazothiazoles surprisingly lacked any utility for VL but showed superior activity
against Chagas disease. Importantly, the latter activity was not ablated by oxidation of the
thiazole sulfur, indicating that the potent “thio-delamanid” analogue 49 may represent a
useful new lead that is worthy of further assessment for this highly neglected disease.
Acknowledgments
The authors thank the Global Alliance for Tuberculosis Drug Development and the Drugs for
Neglected Diseases initiative [specific funding from UK Department for International
Development, Dutch Ministry of Foreign Affairs, Federal Ministry of Education and
Research (BMBF) through KfW/GERMANY, the Bill & Melinda Gates Foundation,
Médecins Sans Frontières and an anonymous donor] for financial support through
collaborative research agreements, and Sisira Kumara (ACSRC) for the solubility
measurements.
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