Design, synthesis and investigation on the structure-activity relationships of N-substituted 2-aminothiazole derivatives as antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2013.11.007

Tuberculosis (TB) is one of the deadliest infectious diseases of all times, and its recent resurgence is a supreme matter of concern. Co-infection with HIV and, in particular, the continuous isolation of new resistant strains, makes the discovery of novel anti-TB agents a strategic priority. The research of novel agents should be driven by the accessibility of the synthetic procedure and, in particular, by the lack of cross-resistance with the drugs already marketed. Moreover, in order to shorten the duration of the therapy, and therefore decrease the rate of resistance, these molecules should be active also against the nonreplicating persistent form (NRP-TB) of the infection. The availability of an in-house small library of compounds prompted us to investigate their anti-TB activity. Two compounds, embodying a 2-aminothiazole scaffold, were found to possess a certain inhibitory activity toward Mycobacterium tuberculosis H₃₇Rv, and therefore a medicinal chemistry campaign was initiated in order to increase the activity of the hit compounds and, especially, construct a plausible body of structure-activity relationships. The potency of the hit compound was successfully improved, and, much more importantly, some of the molecules synthesized were found to be active toward the persistent phenotype, and, also, toward a panel of resistant strains. These findings encourage further investigations around this interesting antitubercular chemotype.

Full text of DOI:10.1016/j.ejmech.2013.11.007

European Journal of Medicinal Chemistry 72 (2014) 26e34
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and investigation on the structureeactivity
relationships of N-substituted 2-aminothiazole derivatives as
antitubercular agents
,
b
b
b
a
Marco Pieroni a , Baojie Wan , Sanghyun Cho , Scott G. Franzblau , Gabriele Costantino
*
a Dipartimento di Farmacia, University of Parma, Parco Area delle Scienze 27/A, Parma 43124, Italy
b Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Tuberculosis (TB) is one of the deadliest infectious diseases of all times, and its recent resurgence is a
supreme matter of concern. Co-infection with HIV and, in particular, the continuous isolation of new
resistant strains, makes the discovery of novel anti-TB agents a strategic priority. The research of novel
agents should be driven by the accessibility of the synthetic procedure and, in particular, by the lack of
cross-resistance with the drugs already marketed. Moreover, in order to shorten the duration of the
therapy, and therefore decrease the rate of resistance, these molecules should be active also against the
nonreplicating persistent form (NRP-TB) of the infection. The availability of an in-house small library of
compounds prompted us to investigate their anti-TB activity. Two compounds, embodying a 2-
aminothiazole scaffold, were found to possess a certain inhibitory activity toward Mycobacterium
tuberculosis H37Rv, and therefore a medicinal chemistry campaign was initiated in order to increase the
activity of the hit compounds and, especially, construct a plausible body of structureeactivity relation-
ships. The potency of the hit compound was successfully improved, and, much more importantly, some
of the molecules synthesized were found to be active toward the persistent phenotype, and, also, toward
a panel of resistant strains. These findings encourage further investigations around this interesting
antitubercular chemotype.
Received 20 August 2013
Received in revised form
14 October 2013
Accepted 5 November 2013
Available online 13 November 2013
Keywords:
Tuberculosis
Medicinal chemistry
Resistance
Drug discovery
2-Aminothiazoles
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
moreover, nearly one-third of the world’s population is estimated
to be latently infected by Mtb [2]. The majority of new cases and
Tuberculosis (TBa), a lung infection mostly caused by Mycobac-
terium tuberculosis (Mtb), is considered one of the most threatening
diseases for public health [1]. Despite the information about TB and
Mtb has become more thorough over the years, the most recent
reports disclosed by the World Health Organization (WHO)
describe a scenario that is still frightful: in 2011, there were an
estimated 8.7 million new cases of TB (13% of which co-infected
with HIV) and 1.4 million people died from the disease;
deaths occur in Asia and Africa: in particular, India and China
together account for almost 40% of the world’s TB new cases,
whereas in the African regions there are the highest rates of cases
and deaths per capita [2]. The current recommended therapeutic
strategy, termed DOTS (Directly Observed Therapy, Short-course)
[3], is based on the co-administration of isoniazid (INH), rifampin
(RMP), ethambutol (EMB), and pyrazinamide (PZA) for the first two
months, followed by a prolonged treatment with INH and RMP for
additional 4e7 months [4,5]. The reason for this exceptionally long
period of therapy resides in the peculiar ability of Mtb to modify his
metabolism in such a way to slow down replication and survive in a
dormant state (NRP-TB, nonreplicating persistent TB) [6], therefore
hiding from the chemotherapeutics. The overall duration of the
treatment, coupled to the undesired side effects, often leads to poor
patient compliance which, in turn, contributes to the emergence of
multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB
(XDR-TB) [7e9]. By definition, MDR-TB is resistant to at least two of
the first-line drugs, principally INH and RMP, whereas XDR-TB
additionally fails to respond to any fluoroquinolone and at least
Abbreviations: CAP, capreomycin; DMF, N,N-dimethyl formamide; DOTS, directly
observed therapy short-course; EDG, electron-donating group; EWG, electron-
withdrawing group; INH, isoniazid; LORA, low oxygen recovery assay; MABA,
microplate Alamar Blue assay; MIC, minimum inhibitory concentration; MDR-TB,
multidrug-resistant tuberculosis; MOX, moxifloxacin; Mtb, Mycobacterium tuber-
culosis; NRP-TB, nonreplicating persistent tuberculosis; R-TB, replicating tubercu-
losis; RMP, rifampin; SDR-TB, single drug resistant tuberculosis; SI, supporting
information; SM, streptomycin; TB, tuberculosis; XDR-TB, extensively drug-
resistant tuberculosis.
* Corresponding author. Tel.: þ39 0521 905054.
E-mail address: marco.pieroni@unipr.it (M. Pieroni).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.007

M. Pieroni et al. / European Journal of Medicinal Chemistry 72 (2014) 26e34
27
Fig. 1. 2-aminothiazole derivatives preliminary tested as anti-TB agents.
one of three injectable second line drugs (amikacin, capreomycin or
kanamycin) [8]. The treatment of resistant strains requires a pro-
longation of the therapy, needs more toxic drugs, and increases the
financial burden, thus making TB a vicious cycle; moreover, the
recent isolation of a pan-resistant Mtb strain (TDR-TB, totally drug
resistant tuberculosis) threatens the rise of a virtually incurable
pathogen [10]. These facts highlight the need for novel anti-TB
agents, for which the ability of treating Mtb in a dormant state
and the activity toward resistant strains, coupled to a certain degree
of chemical feasibility, should represent the most important fea-
tures [11e13]. Although a considerable number of anti-TB agents
has been reported over the years, to which, among the others, some
of us has given his personal contribution [14e21], only a handful of
compounds have reached the clinical trials, and bedaquiline
(SirturoÒ), former TMC207 [22], is the only new chemical entity
developed for the treatment of tuberculosis that has reached the
market since the introduction of RMP (1967) [23]. With regard to
anti-TB agents, and to antibacterials more in general, the whole cell
phenotypic assay has demonstrated to be the most fruitful method
to produce lead candidates for further development, especially if
compared to the target-based approaches. For instance, bedaqui-
line, mentioned above, and PA-824 [24], an antimycobacterial
nitrofuran in phase III clinical trials, were discovered following a
cell phenotypic approach. The availability of an in-house chemical
library prompted us to carry out a phenotypic screening against
Mtb in order to obtain novel anti-TB chemotypes. Within the small
set taken under consideration, a number of molecules embodying a
2-aminothiazole scaffold were found to have a certain activity, with
derivative 4a being considered the hit compound (Fig. 1). The
feasibility of the synthesis, and the very preliminary structuree
activity relationships (SAR) stemmed from the analysis of these few
derivatives, encouraged us to perform the synthesis of a number of
structurally related derivatives, to thereby elucidate the anti-TB
potential of these aminothiazole-based compounds. Although the
inhibitory activity toward Mtb of some aminothiazoles and ben-
zothiazoles has already been reported [25], up to now there has
been no attempt to establish a reliable SAR for aminothiazole an-
alogs as anti-TB agents. Hence, keeping intact the aminothiazole
core, which is suggested to act as the pharmacophore, we investi-
gated whether the appropriate substitution at the aromatic rings of
the hit compound 4a could lead to an improvement in the anti-TB
activity and, also, in a reduction of the cytotoxicity (Fig. 2). The
functional groups used as substituents were chosen based on their
size, lipophilicity, and physico-chemical properties, and attached at
various positions of the aromatic ring. Once assessed which sub-
stituents afforded the best activity, attempts to constrain the
structure were made, in order to assess whether a more rigid
structure could better fit the binding site of the target, that is as yet
unknown. Finally, a number of substitutions at the amino moiety
and the C-5 of the 2-aminothiazole were explored so as to delineate
a more detailed SAR. For the most promising compounds additional
assays were carried out: in particular, some of the derivatives were
tested against single drug resistant strains (SDR-TB) and, in addi-
tion, the inhibitory activity of these compounds toward a model of
NRP-TB was evaluated, as will be described below.
1.1. Chemistry
The majority of the target compounds (4aen, 6e11, 15, 22a, 6,
11e14, 17e19h, and 17k) were synthetized in a range of variable
yields by employing an established Hantzsch protocol [26],
refluxing the appropriate bromoacetophenone with an equimolar
amount of thiourea in absolute ethanol (Schemes 1e4). Bromoa-
cetophenones 4, 6e15, 17e19, were readily prepared from the
corresponding ketones after treatment with Br2 at room tempera-
ture in CH2Cl2; thioureas aem, when not commercially available,
were prepared by reacting the corresponding anilines with
ammonium thiocyanate in
1 N HCl at reflux. Only cyclo-
hexylthiourea n was obtained starting from cyclohexyl isothiocy-
anate, by reaction with a solution of 0.5 M ammonia in dioxane at
room temperature, as reported before [27] (Scheme 3). Compound
16a was obtained, although in poor yield, by treating 6a with an
excess of pyridinium chloride, while reaction of 4a with methyl
iodide and 4h with benzyl bromide using NaH as the base afforded
compound 5a and 20h, respectively. In these experimental condi-
tions, the alkylation occurs at 2-amino moiety, and not at the
endocyclic nitrogen, as demonstrated by NOESY experiments (see
Supporting information). The synthesis of compounds 4oer was
accomplished starting from the common precursor 2-bromo-4-
phenylthiazole 23, obtained in a two-steps procedure from the
corresponding bromoacetophenone 4 by treatment with ammo-
nium thiocyanate in ethanol at reflux, followed by cyclization
promoted by a 40% acetic solution of HBr in CH2Cl2 (Scheme 5).
Displacement of the bromine atom with piperidine (4p) or N-ace-
tylpiperazine (4q) was carried out in a microwave reactor at 100 ꢀC
using ethanol as the solvent and Et3N as the base. When the reac-
tion was carried out using DMF as the solvent, compound 4o was
obtained in 47% yield. The synthesis of the indole derivative 4r was
conveniently accomplished by employing a BuchwaldeHartwig
amination protocol in a microwave reactor, using Pd(Ac)2 as the
Fig. 2. Sites for modifications of 2-aminothiazole derivatives.

28
M. Pieroni et al. / European Journal of Medicinal Chemistry 72 (2014) 26e34
Scheme 1. Reagents and conditions: (a) anhydrous ethanol, 70 ꢀC, 10e18 h, (26e98%); (b) NaH, anhydrous DMF, MeI (5a) or benzyl bromide (20h), 0 ꢀC / rt, 18 h, (68e77%); (c)
pyridine hydrochloride, MW, 5 min, 190 ꢀC, 46%. For complete structures, see Table 1.
catalyst, BINAP as the ligand and Cs2CO3 as the base [28], whereas
general Ullman-type conditions led only to several inseparable
decomposition products.
2. Results and discussion
A total of 38 compounds were synthesized and tested for their
ability to inhibit the growth of R-TB strain H37Rv in a microplate
Alamar Blue assay (MABA) [29]. Compounds showing an encour-
aging MIC in MABA were also tested in a low oxygen recovery assay
Scheme 2. Reagents and conditions: (a) anhydrous ethanol, 70 ꢀC, 10e18 h, (6e64%).
For complete structures, see Table 1.
(LORA) [30], that is an in vitro model for the preliminary assessment
of the activity against the persistent Mtb phenotype. Some of the
Scheme 3. Reagents and conditions: (a) NH3 (0.5M in dioxane), rt, 24 h (53%); (b) anhydrous ethanol, 70 ꢀC, 10e18 h, (3%).
most representative compounds were also tested toward Vero cells
to ascertain the cytotoxicity profile. To further analyze the biolog-
ical profile of these compounds, and rule out the possibility of
cross-resistance with the commonly used chemotherapeutics,
assessment of activity toward SDR-TB strains was carried out as
well. 16 compounds resulted inactive at 128
mM concentration,
whereas 21 derivatives were found to inhibit the growth of R-TB in
the MABA assay at variable concentrations. These modifications led
to a variable range of activities and allowed us to construct a
plausible SAR as will be described below.
Scheme 4. Reagents and conditions: (a) anhydrous ethanol, 70 ꢀC, 10e18 h, (3%).
Scheme 5. Reagents and conditions: (a) 1. NH4SCN, anhydrous ethanol, 75 ꢀC, 1 h; 2. 40% HBr in acetic acid, anhydrous CH2Cl2, rt, 18 h. (b) for 4o, DMF, MW, 120 ꢀC, 25 min (40%);
4peq, piperidine or N-acethylpiperazine, absolute ethanol, MW, 120 ꢀC, 25 min (57e96%); (c) 4r, 5-methyl-1H-indole, anhydrous ethanol, BINAP, Pd(OAc)2, Cs2CO3, MW, 125 ꢀC,
25 min (51%).

M. Pieroni et al. / European Journal of Medicinal Chemistry 72 (2014) 26e34
29
Table 1
Anti-TB activity of compounds 4aen, 5e11, 15, 16, 22a, 6, 11e14, 17e20h and 17k against M. Tuberculosis strain H37Rv.
R1
R2
R3
R4
MIC (
m
M)
IC50 (mM)
a
Compd
MABA
LORA
4a
5a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
6a
6h
7a
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
2-F, 5-CF3
4-OCH3
4-OCH3
4-CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
Ph
Bn
H
H
CH3
H
H
H
H
H
H
H
H
H
H
4-CH3
4-CH3
3-CH3
2-CH3
4-Cl
27.5
31.7
29.8
35.1
19.2
30.1
>128
>128
15.0
30.1
>128
59.5
30.8
>128
13.0
92.4
>128
31.5
15.2
46.6
3-Cl
2-Cl
2,4-Cl
3,5-Cl
3,5-CH3
2-Cl, 4-CH3
4-F
50
H
2,4-CH3
3,5-Cl
4-CH3
3,5-Cl
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
3,5-Cl
-CH2-CH3
H
H
H
H
H
H
H
H
H
H
H
H
Bn
31.8
>128
15.6
>128
>128
>128
>128
63.8
>128
28.6
28.9
8a
9a
4-Cl
2-Cl
4-CH3
e
10a
11a
16a
11h
12h
13h
14h
20h
4-OH
e
e
e
>128
20.0
>128
e
82.7
2-F, 5-CF3
17h
17k
18h
19h
H
H
H
H
3,5-Cl
4-F
>128
>128
>128
43.3
3,5-Cl
3,5-Cl
4n
30.3
15a
22a
>128
>128
INH
RMP
CAP
SM
PA824
0.4
0.1
1.9
0.3
0.8
*Indicates the point of attachment.
a
Cytotoxicity to Vero cells; MIC values determined by MABA are the mean of replicated experiments (SD < 15%); LORA MIC values represent single measurements.

30
M. Pieroni et al. / European Journal of Medicinal Chemistry 72 (2014) 26e34
2.1. Substitutions at the aromatic rings: the aniline portion
results are consistent with the notion that at this position the ac-
tivity is somehow dependent on the size of the substituent and its
lipophilicity. In particular, the bigger and more lipophilic is the
substituent, the higher is the activity (4d > 4a > 4k). This may be
rationalized by the presence of a hydrophobic pocket at the target
biding site, located within the vicinity of the aniline, able to
accommodate a variety of hydrophobic groups. Further insights on
the characteristics of this pocket are given by the activity of 4n,
which is on the same range as for the hit compound 4a (4n,
The first round of modifications aimed at improving the activity
was made at the aniline site. Therefore, keeping intact the 4-(2-
fluoro-5-(trifluoromethyl)phenyl)thiazol-2-amine scaffold, small
functional groups were attached at different positions of this aro-
matic ring. We reasoned that these substituents could affect the
hydrogen-bond donor/acceptor ability of the amino moiety,
thereby playing a role in the interactions with the target binding
site. First we investigated the role of the methyl group and its po-
sition on the aniline ring. Moving the methyl from the para to the
MIC ¼ 30.3
mM and 4a, MIC ¼ 27.5
mM). Moreover, the complete
lack of activity of compound 3 (Fig.1), bearing a 4-pyridine attached
to the amino group of the 2-aminothiazole, might serve as an
additional evidence for this assumption. Then we investigated the
disubstitution at the aniline ring in positions other than the ortho,
both with an electron-donating and an electron-withdrawing
group. While the disubstitution in the meta positions with two
methyl groups did not affect the activity compare to the mono-
meta and the ortho positions (4b, MIC ¼ 29.8
respectively) did not lead to any considerable variation in potency
M) as shown in
mM, 4c, MIC ¼ 35.1
mM,
compared with the hit compound 4a (MIC ¼ 27.5
m
Table 1. Next, at the same positions, we investigated the effect of the
substitution with an electron-withdrawing group as the chlorine.
Introduction of the chlorine atom at the para position of the aniline
ring, led to a slight improvement of the activity compared to the
substitution (4i, MIC ¼ 30.1
m
M vs 4b, MIC ¼ 29.8
mM), we were
para methyl substitution (4d, MIC ¼ 19.2
Switching from the para to the meta position, an almost 2-fold
reduction in activity is noticed (4d, MIC ¼ 19.2 M vs 4e,
MIC ¼ 30.1 M), whereas the activity is completely lost when the
M). A
mM vs 4a, MIC ¼ 27.5
mM).
pleased to notice that two chlorines at the same positions yielded a
compound about 2-fold more active than the mono-substituted
m
counterpart (4h, MIC ¼ 15.0
mM vs 4e, MIC ¼ 30.1
mM). While it is
m
somehow surprising the capability of the methyl groups not to
affect the potency of the molecules, it is reasonable to assume that
the introduction of an additional chlorine atom in 4e leads to an
increase of the hydrophobicity, a characteristic likely to be impor-
tant for the molecule in order to better penetrate the thick myco-
bacterial cell wall. Reassuming, with regard to this portion of the
molecule, small lipophilic substituents are well tolerated, especially
in the para and meta positions, whereas in the ortho position sub-
stituents like the chlorine led to loss of activity. Disubstitution at
the meta positions, in particular with chlorine, led to lower MIC
values, likely for the improvement of hydrophobicity that they
confer. All these data lead to hypothesize the presence of a quite
large hydrophobic pocket at the target binding site.
chlorine is introduced in the ortho position (4f, MIC ¼ >128
m
plausible explanation of this result might encompass the steric
hindrance produced by the chlorine atom on the amino group of
the thiazole, preventing it from establishing interactions with the
surrounding amino acids at the target binding site. This notion is to
some extent further corroborated by the lack of activity of 4g and 4j
(MIC ¼ >128
mM), in which a characteristic deemed favorable for
the activity such as the methyl or the chlorine in the para position,
was coupled to the detrimental chlorine atom in the ortho position.
Likely, this effect is not observed with the methyl group because of
its smaller size. Indeed, compound 4l (MIC ¼ 30.8
mM), bearing an
ortho, para dimethyl substitution, showed an activity comparable to
that of the hit compound 4a. Interestingly, the introduction of a
fluorine atom in the para position of the aniline led to a 2-fold
decrease in activity compared to the para-methyl analog (4a,
2.2. Substitutions at the aromatic rings: the 4-phenyl ring
MIC ¼ 27.5
m
M vs 4k, MIC ¼ 59.5
m
M) and 3-fold compared to the
The phenyl ring at the C-4 of the 2-aminothiazole is crucial for
activity, as both its removal and its shift tothe C-5 position led to loss
para-chlorine (4d, MIC ¼ 19.2
m
M vs 4k, MIC ¼ 59.5 M). These
m
of activity (15a [31], 22a, MICs ¼ >128
mM). We wanted then to
Table 2
investigate which pattern of functional groups could confer the best
activity. Removal of both the substituents present in the hit com-
pound led to a roughly 2-fold decrease in activity with respect to the
Anti-TB activity of compounds 4oer against M. Tuberculosis strain H37Rv.
substituted analogs (11a, MIC ¼ 63.8
11h, MIC ¼ 28.6 M vs 4h, MIC ¼ 15.0
ring, number of different electron-donating and electron-
m
M vs 4a, MIC ¼ 27.5
mM and
m
mM). Then, as for the aniline
a
withdrawing groups were attached at various position of the
phenyl ring keeping intact, for comparative purpose, the para-
methyl substitution at the aniline ring. Quite surprisingly, small
electron-donating groups such as the methyl and the hydroxyl, or
electron-withdrawing groups such as the chlorine and the tri-
fluoromethyl, failed to show any activity (7a, 8a [32], 9a, 10a, 16a,
Comp
R
MABA MIC (mM)
4o
*N-(CH3)2
>128
4p
4q
4r
56.5
MIC ¼ >128
methoxy substitution, was found to be the most active compound of
M). So we tried to couple the more favorable
mM). On the other hand, compound 6a [33], bearing a 4-
117.8
>128
the series (MIC ¼ 13.0
m
substitution pattern at the aniline ring (the 3,5 dichloro substitu-
tion) to that at the phenyl ring (the 4-methoxy group), but, although
still one of the most active compounds of the series, we did not
INH
RMP
CAP
SM
PA824
0.4
0.1
1.9
0.3
0.8
notice any evident improvement in the activity (6h, MIC 15.6 mM).
Although the small set of compounds synthesized, we might assume
that at the position para of the phenyl ring, slightly more polar
substituents guarantee a more favorable interaction with the target
binding site, likely due to the presence of polar amino acids in the
close proximity. On the other hand, the lack of activity for the 4-
hydroxy derivative 16a might be due to the excessive polar
*Indicates the point of attachment.
MIC values determined by MABA are the mean of replicated experiments
(SD < 15%); LORA MIC values represent single measurements.

M. Pieroni et al. / European Journal of Medicinal Chemistry 72 (2014) 26e34
31
character of this functional group that leads to penetration issues as
thiazole can be alsotri-substituted (see 5a, MIC ¼ 31.7
m
M), the indole
already reported elsewhere. In conclusion, the phenyl ring at the C-4
of the 2-aminothiazole is important for the activity, and a polar
substituent at the para position of this ring confers the best activity.
derivative 4r (MIC ¼ >128
mM) was synthesized, resulting inactive.
Apparently, flexibility of the aromatic ring attached at the 2-amino
moiety is required to confer the desired anti-TB activity.
2.5. Substitution at the C-5 of the 2-aminothiazole
2.3. Substitution at the 2-amino moiety
We finally wanted to investigate whether substitutions at the C-
5 of the 2-aminothiazole were tolerated, and, if so, what charac-
teristics should these substituents possess. Moreover, it is known
that 2-aminothiazoles without blocking substituents at C-5 can
undergo oxidation in vivo to generate potentially toxic reactive
epoxide metabolites, therefore the presence of a bulky substituent
at the C-5 might be useful in terms of improving some pharma-
cokinetic characteristics [34]. A small group such as the methyl
doesn’t affect the activity compared to the counterpart (12h,
It is known that in the majority of cases the polar portions of the
molecule allow for the most specific interactions, and therefore are
seldom modified. We wanted to explore whether the hydrogen of
the 2-aminothiazole was important for the activity. Compound 5a
(MIC ¼ 31.7
m
M) still maintains an activity comparable to that of the
hit compound 4a (MIC ¼ 27.5
m
M), thereby indicating that the
capability of being a hydrogen-bond donor is not crucial for the
activity. This prompted us to gain further insights into what degree
of hindrance was tolerated in regard to the substitution at this
position; unfortunately, slightly larger group, such as an ethyl (4m,
MIC ¼ 28.9
abolished when
MIC ¼ >128 M). Surprisingly, the activity is restored when a
benzyl moiety is used as the substituent (14h, MIC ¼ 20.0 M).
m
M, vs 11h, MIC ¼ 28.6
mM), whereas the activity is
phenyl group is introduced (13h,
a
MIC ¼ >128
m
M) or more hindered substituent, such as the benzyl
m
group (20h, MIC ¼ >128
mM) failed to yield active compounds.
m
Encouraged by the activity of compounds such 5a and 4n, we also
investigated if the amino moiety could be embodied into a cyclo-
aliphatic ring. The piperidine derivative 4p had a MIC 2-fold higher
Assessment of the collocation of these data within the SAR is
difficult, but we might speculate that the benzyl ring, by virtue of its
higher flexibility compared to the phenyl ring, can accommodate
into the target binding site in a more favorable manner that is,
presumably, distant from the phenyl ring at the C-4 of the 2-
aminothiazole. To reassume, several substituents, from the small
methyl to the larger benzyl ring, but not the phenyl, can be intro-
duced at the C-5 position of the 2-aminothiazole.
than that of the hit compound 4a (4p, MIC ¼ 56.5
MIC ¼ 27.5 M), but the lack of a lipophilic appendage, as for 4o
(MIC ¼ >128 M), or the introduction of a more polar moiety, as in
M),
mM vs 4a,
m
m
the case of the N-acetylpiperazine derivative 4q (MIC ¼ 117.6
m
impaired the anti-TB activity (Table 2). Briefly, we can state that a
small group such as the methyl can substitute the hydrogen of the
2-aminothiazole, but larger moieties lead to loss of activity.
2.6. Activity toward resistant strains and NRP-TB
2.4. Constrained structures
Some of the compounds were also tested in LORA, a plausible
surrogate for NRP-TB. In the majority of the cases, the LORA MIC
values are reported to be several fold higher than those of MABA
MIC [14e16,19e21], so it was very remarkable to notice that com-
pound 4h had exactly the same activity in LORA as toward the
Our investigation was also directed to explore whether a more
rigid structure could affect the activity. As a matter of fact, it is known
that if the less flexible structure mimics the bound conformation at
the target binding site, animprovementof activity mightbeobserved.
We therefore synthesized a small set of derivatives in which the 2-
aminothiazole lays on the same plan of the phenyl ring attached at
actively replicating strain (4h, MABA MIC ¼ 15.0
mM vs LORA
MIC ¼ 15.2
m
M). Compound 4d and 6a still retained appreciable
activity in the LORA assay, although the LORA MIC value being
C-4 via either
MIC ¼ >128 M) or an ethylene bridge (19h, MIC ¼ 43.3
estingly, compound 19h had an MIC value only slightly higher than
that of the counterpart (19h, MIC ¼ 43.3 M, vs 11h, MIC ¼ 28.6 M).
a
methylene (17h, MIC
¼
>128
m
M, 17k,
almost 2-fold higher than that of the MABA MIC (4d, MABA
m
mM). Inter-
MIC ¼ 19.2
m
M and LORA MIC ¼ 31.5
m
M, 6a, MABA MIC ¼ 13.0
mM
and LORA MIC ¼ 31.8
m
M), following the trend noticed in our pre-
m
m
viously reported works. Among the current TB drugs, only RMP and
PZA have been reported to show good activity toward the dormant
bacteria, and it is generally accepted that targeting the NRP-TB
plays a crucial role in shortening the TB treatment. These two
compounds were finally evaluated against Mtb strains that are
resistant to two first-line and 2 s-line TB drugs (Table 3). Both 4d
and 4h were found to maintain their anti-TB activity against RMP,
INH, MOX, and CAP resistant strains, suggesting a different mode of
action and indicating that this compound class holds promise for
the treatment of resistant TB as well. Although it is a matter of
concern the fact that RMP and INH are likely more active than our
best derivatives against the respective resistant strains, however, in
a preliminary study such as our work is supposed to be, the lack of
cross resistance toward a biological target always represents an
encouraging starting point, from which further improvements are
expected.
These results bring over further insights about the interaction be-
tween the molecule and the target: a) likely, we may suppose that the
thiazoleand thephenyl ring, atthetargetbindingsite, arecoplanar; b)
however, the phenyl ring must be positioned at a suitable distance
with respect to the thiazole. Derivative 18h, that can be considered
the constrained analog of 22a, is inactive as well. Following a similar
investigational approach on the other portion of the molecule, and
taking into consideration the fact that the 2-amino group of the
Table 3
Activity of compounds 4d, h against SDR M. Tuberculosis strains.
Comp
MABA MIC (
r-RMPa
mM)
r-INHb
r-MOXc
r-CAPd
4d
4h
10.9
9.7
10.4
7.1
0.06
>8
3.21
0.43
15.5
12.0
0.14
3.60
3.68
0.88
17.4
12.0
0.05
0.81
>16
1.26
RMP
INH
CAP
PA-824
>4
0.85
1.92
0.51
2.7. Cytotoxicity
Some of the derivatives synthesized were tested to assess their
apparent cytotoxicity toward Vero cells. In general, the selectivity
index, that in this case is the ratio between IC50 toward Vero cells
and the MABA MIC toward Mtb, for a compound to be considered a
a
Rifampin (RMP) resistant strain.
Isoniazid (INH) resistant strain.
Moxifloxacin (SM) resistant strain.
b
c
d
Capreomycin (KN) resistant strain.

32
M. Pieroni et al. / European Journal of Medicinal Chemistry 72 (2014) 26e34
valuable lead has usually to be >10. Therefore, we wanted to
investigate whether the toxicological profile of derivative 4a
Advance spectrometer (600 MHz); 13C NMR spectra were recorded
on a Bruker spectrometer at 100 MHz. Chemical shifts ( scale) are
d
(IC50 ¼ 92.4
m
M, SI 3.4) could be improved in order to yield a better
reported in parts per million (ppm) relative to the central peak of
the solvent. 1H NMR Spectra are reported in order: multiplicity and
number of protons; signals were characterized as s (singlet), dd
(doublet of doublet), t (triplet), m (multiplet), br s (broad signal).
HRMS experiments were performed using a LTQ ORBITRAP XL
Thermo by Thermo-scientific instrument coupled to HPLC
lead compound. Unfortunately, modifications leading to an
enhancement of activity, also resulted in a counterproductive
improvement of cytotoxicity (4d, IC50 ¼ 46.6
mM, SI 2.4, 4h
IC50 ¼ 50.0
m
M, SI 3.3). Then we tried to modify the 2-aminothiazole
scaffold through the introduction of a methyl group at the 2-amino
moiety; we were pleased to notice that the compound was
endowed with a column Alltima C18 5
m 150 mm*4.6 mm, Alltech
apparently not toxic (5a, IC50 > 128
activity comparable to that of the hit compound. Unfortunately, as
reported above, other attempts to substitute the 2-amino moiety
m
M), while maintaining an
Italia Srl. Reactions were monitored by TLC, on Kieselgel 60 F 254
(DC-Alufolien, Merck). All the final compounds were more than 95%
pure by analytical HPLC.
with bulkier groups led to
a
drop of the activity (4m,
MIC ¼ >128
m
M; 20h, MIC ¼ >128
m
M). However, the substitution
4.2. Biology
of the 4-phenyl ring with a more polar EDG such as the methoxy
group at the para position, gave the best compromise between
The MICs were determined using Mtb H37Rv ATCC 27294 in
MABA and LORA assays according to published procedures [29,30].
The reported MICs are an average value from 2 to 3 individual ex-
periments. Similarly, cytotoxicities were determined on Vero cells
according to a published procedure. For a brief description of the
biological assays see the Supporting information.
activity and lack of cytotoxicity (6a, MIC ¼ 13.0
mM, IC50 > 128 mM).
Summarizing, the extended investigation around the SAR for these
antitubercular 2-aminothiazoles, led to the synthesis of more active
compounds, moreover devoid of apparent cytotoxicity.
3. Conclusion
4.3. General procedure for the synthesis of compounds 4aen, 6e11,
15a, 6, 11e14, 17e19h (Hantzsch synthesis)
A total of 38 2-aminothiazole derivatives were synthesized in
this work, allowing to build a reliable SAR with regards to their
anti-TB activity. Most notable compounds had MICs ranging in the
low micromolar values, and, more importantly, selected com-
pounds were found to be active not only toward the actively
replicating mycobacterial strain, but also toward the nonreplicating
persistent phenotype in low oxygen conditions. Moreover, when
tested against a panel of single-drug resistant Mtb strains, de-
rivatives 4d and 4h maintained the same activity as for the wild
type, indicating that these derivatives act with a mechanism of
action different from those of the currently used drugs, although as
yet unknown. The various modifications explored gave a number of
valuable hints for the design of additional analogs. In particular, at
the amino group of the 2-aminothiazole scaffold, an aromatic ring,
preferably adorned with bulky and lipophilic functional groups
such as chlorine, is better tolerated; on the other side of the
molecule, attached at the C-4 position of the 2-aminothiazole, an
aromatic ring is required to confer the desired activity. Lipophilic
substituents of various sizes are tolerated in positions other than
the para, where a more polar group (such as the methoxy moiety)
seems to be necessary in order to maintain a certain anti-TB ac-
tivity, likely for the presence of polar amino acids in the vicinity of
the pocket accommodating the molecule. All of these findings
made possible a preliminary perception of the shape and stereo-
electronic characteristics of the target binding site. Compound 6a,
in which an encouraging improvement of activity is coupled to a
lack of apparent cytotoxicity, also by virtue of its preserved activity
toward NRP-TB, can be considered as a lead chemotype worth of
further investigations toward the synthesis of novel antitubercular
2-aminothiazoles. Studies aimed at improving the activity of these
compounds, and establishing preliminary ADMET properties, are
therefore underway in our research groups.
The properly substituted bromoacetophenone 4, 6e15, 17e19 (1
equiv) and thioureas aen (1 equiv) were solubilized in dry ethanol
(5 mL mmolꢁ1 of bromoacetophenone) and reacted at 70 ꢀC until
consumption of the starting materials as indicated by TLC. After
cooling, the solvent was evaporated and the residue obtained was
washed with diethyl ether (3 ꢂ 10 mL) to give the title compounds
as a powder in good overall yields. In some cases, the crude ma-
terial was purified by flash column chromatography. Yields, puri-
fication methods and purity are reported in details in the SI.
Analytical data for compounds n, 6a, 8a, 15a, matched the data
published previously [27,33,32,31].
4.4. Cyclohexylthiourea (n) [27]
Cyclohexylisocyanate 21 (1 g, 7.12 mmol) was solubilized in a
0.5 M solution of NH3 in dioxane (22.3 mL) and the mixture was
stirred for 24 h at rt. The solvent was then evaporated and the solid
obtained was washed with petroleum ether (40 mL) to give a white
powder (584 mg, 53%) that was used in the next reaction step
without further purification. The analytical data obtained matched
those reported in literature [27].
4.5. 4-(4-Hydroxyphenyl)-N-(4-methylphenyl)thiazol-2-amine
(16a)
4-(4-Methoxyyphenyl)-N-(4-methylphenyl)thiazol-2-amine 6a
[33] (50 mg, 0.17 mmol) and pyridine hydrochloride (97 mg,
0.84 mmol) were reacted for 5 min in a microwave reactor (190 ꢀC,
215 W) under neat conditions. The crude material was solubilized
in CH2Cl2 and washed with water (3 ꢂ 10 mL), brine and dried
(Na2SO4). After filtration, the solvent was removed in vacuo and the
dark red solid was purified by flash chromatography (CH2Cl2e
MeOH 95:5) to give 16a as a light yellow powder (22 mg, 46%): 1H
4. Experimental section
4.1. Chemistry
NMR (400 MHz, MeOD):
d
¼ 2.31 (s, 3H), 6.76 (s, 1H), 6.83 (d,
Microwave assisted synthesis was performed using a CEM Mi-
crowave Synthesizer e Discover ModelÔ. All products were char-
acterized by 1H NMR and 13C NMR. The 1H NMR spectra were
recorded on a Bruker 300 Avance spectrometer (300 MHz), on a
Bruker 400 Avance spectrometer (400 MHz) and an an Agilent 600
J ¼ 8.6 Hz, 2H), 7.14 (d, J ¼ 8.3 Hz, 2H), 7.52 (d, J ¼ 8.3 Hz, 2H),
7.72 ppm (d, J ¼ 8.61 Hz, 2H); 13C NMR (100 MHz, MeOD):
¼ 19.41,
d
98.7, 114.8, 117.42, 127.01, 129.10, 131.1, 129.1, 138.9, 151.1, 156.9,
165.1 ppm; HRMS (ESI) calculated for C16H14N2OS [M þ H]þ
283.0827, found: 283.0903.

M. Pieroni et al. / European Journal of Medicinal Chemistry 72 (2014) 26e34
33
4.6. 4-(2-Fluoro-5-trifluoromethylphenyl)-N-methyl-N-(4-
methylphenyl)thiazol-2-amine (5a)
J ¼ 1.9 Hz, 1H), 8.52 (dd, J1 ¼ 2.2 Hz, J2 ¼ 7.0 Hz, 1H); HRMS
calculated for C10H4BrF4NS: m/z 325.92 (100.0%), 327.92 (97.4%),
[M þ H]þ, found 325.9262, 327.9237.
4a (35 mg, 0.10 mmol) was added to a suspension of NaH (5 mg,
0.10 mmol) in dry DMF (2 mL) at 0 ꢀC. After stirring for 15 min,
methyl iodide (22 mg, 0.13 mmol) was added and the reaction
mixture was stirred at rt overnight. The mixture was then
cautiously poured into ice water (10 mL), extracted with EtOAc
(3 ꢂ 10 mL) and the organic layers were washed with brine and
dried (Na2SO4). After filtration, the solvent was removed in vacuo
and the yellow oil obtained was purified by flash column chro-
matography (EtOAcepetroleum ether 5:95) to give 5a as a white
4.9. 4-(2-Fluoro-5-trifluoromethylphenyl)-N,N-dimethylthiazol-2-
amine (4o)
Compound 23 (100 mg, 0.31 mmol) and Et3N (91.1 mg,
0.93 mmol) were solubilized in dry DMF (2 mL) and the solution
was reacted for 25 min in a microwave reactor (120 ꢀC, 250 W). The
solution was then poured into ice-water, the aqueous layer was
extracted with EtOAc and the combined organic extracts were
washed with brine and dried over Na2SO4. After filtration, the
solvent was removed in vacuo and the white solid obtained was
purified by flash column chromatography (EtOAcepetroleum ether
5:95) to give 4o as a gray solid (35 mg, 47%): 1H NMR (400 MHz,
solid (33 mg, 77%): 1H NMR (600 MHz, CDCl3):
d
¼ 2.38 (s, 3H), 3.59
(s, 3H), 7.02 (s, 1H), 7.18 (t, J ¼ 3.2 Hz, 1H), 7.24 (d, J ¼ 8.8 Hz, 2H),
7.30 (d, J ¼ 8.8 Hz, 2H), 7.49e7.53 (m,1H), 8.52 (d, J ¼ 3.2 Hz,1H). 13
C
NMR (100 MHz, CDCl3):
d
¼ 15.8, 35.1, 103.0,111.2, 118.3,119.9,120.4
(J ¼ 3 Hz), 121.2 (J ¼ 272 Hz), 122.4, 125.2, 131.5, 138.4, 155.8, 157.4,
DMSO-d6):
d
¼ 3.20 (s, 6H), 7.11 (d, J ¼ 2.3 Hz, 1H), 7.21 (app t,
164.0.
J ¼ 9.8 Hz, 1H), 7.49e7.53 (m, 1H), 8.51 ppm (d, J ¼ 5.3 Hz, 1H); 13
C
Following a similar procedure, but using benzyl bromide in
place of methyl iodide, and starting from compound 4h, compound
20h was prepared in 68% yield.
NMR (100 MHz, DMSO-d6):
d
¼ 40.3, 107.4, 107.5, 116.2, 116.4, 123.4
(J ¼ 231 Hz), 125.2 (J ¼ 2.2 Hz), 127.7 (J ¼ 4.2 Hz), 144.1, 160.6, 163.1,
169.8 ppm. HRMS (ESI) calculated for C12H10F4N2S: 291.0501
[M þ H]þ, found: 291.0579.
4.7. 5-Phenyl-N-(4-methylphenyl)thiazol-2-amine (22a)
4.10. 4-(2-Fluoro-5-trifluoromethylphenyl)-2-(piperidin-1-yl)
thiazole (4p)
To a solution of phenylacetaldehyde 22 (500 mg, 4.16 mmol)
in CH2Cl2 (5 mL), a solution of Br2 (697 mg, 4.36 mmol) in
CH2Cl2 (4 mL) was added dropwise. After consumption of the
starting material according to TLC, the solvent was removed in
vacuo having care not to heat the bath more than 20 ꢀC, so as to
prevent the evaporation of the brominated compound. Without
further purification, the crude red oil obtained was quickly
reacted with (p-tolyl)thiourea a (831 mg, 5.01 mmol) in abso-
lute ethanol (5 mL) at reflux for 2 h. The solvent was evaporated
and the slurry residue was washed with water. The aqueous
layer was extracted with EtOAc (3 ꢂ 7 mL) and the combined
organic layers were washed with brine, dried (Na2SO4) and
concentrated in vacuo. Purification by flash column chroma-
tography (CH2Cl2eMeOH 98:2) gave the title compound (30 mg,
3% overall yield) as a white powder. 1H NMR (400 MHz, CDCl3):
A solution of 23 (62 mg, 0.19 mmol), piperidine (39 mg,
0.46 mmol) and Et3N (102.0 mg, 1.01 mmol) in dry ethanol (3 mL)
was heated for 30 min in a microwave reactor (120 ꢀC, 200 W). The
solvent was evaporated and the residue was solubilized with EtOAc
and washed with water (15 mL). The organic layers were washed
with brine, dried over Na2SO4, filtered and concentrated in vacuo to
give a residue that is purified through flash column chromatog-
raphy (EtOAcepetroleum ether 5:95) to afford 4p as a yellow wax
(60 mg, 96%); 1H NMR (400 MHz, DMSO-d6):
d
¼ 1.70e1.76 (m, 6H),
3.53e3.58 (m, 4H), 7.13 (d, J ¼ 2.5 Hz, 1H), 7.18e7.23 (m, 1H), 7.49e
7.52 (m, 1H), 8.50 ppm (dd, J1 ¼ 2.2 Hz, J2 ¼ 7.0 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6):
d
¼ 24.0, 25.1, 49.5, 108.9, 117.6, 123.0, 123.5
(J ¼ 12 Hz), 126.3 (J ¼ 2.8 Hz), 126.9 (J ¼ 3.2 Hz), 143.0, 160.5, 163.1,
170.2. HRMS (ESI) calculated for C15H14F4N2S [M þ H]þ 331.0814,
found: 331.0892.
d
¼ 2.38 (s, 3H), 7.18e7.40 (m, 7H), 7.47e7.50 ppm (m, 3H), 8.30
(bp, 1H); 13C NMR (100 MHz, CDCl3):
d
¼ 20.8, 119.1, 125.6, 127.0,
127.2, 128.9, 130.1, 132.1, 132.3, 134.0, 137.8, 165.5 ppm; HRMS
(ESI) calculated for C16H14N2S: 267.0878 [M þ H]þ, found:
267.0947.
Following a similar procedure, but using N-acetylpiperazine in
place of piperidine, compound 4q was prepared in 57% yield.
4.11. 4-(2-Fluoro-5-trifluoromethylphenyl)-2-(5-methyl-1H-indol-
1-yl)thiazole (4r)
4.8. 2-Bromo-4-(2-fluoro-5-trifluoromethylphenyl)thiazole (23)
Ammonium thiocyanate (38 mg, 0.52 mmol) was solubilized in
absolute ethanol (5 mL) and the solution was heated at 75 ꢀC. 2-
bromo-1-(2-fluoro-5-(trifluoromethyl)acetophenone 4 (100 mg,
0.35 mmol) was added portion wise and the reaction mixture was
stirred for 1 h at the same temperature. The solvent was removed
in vacuo and the yellow solid obtained was washed with water
(3 ꢂ 10 mL) and dried. The compound obtained was solubilized in
Compound 23 (75 mg, 0.23 mmol) and 5-methyl-1H-indole
(32.8 mg, 0.25 mmol) were solubilized in anhydrous toluene (2 mL),
and Cs2CO3 (91.2 mg, 0.28 mmol), BINAP (3.2 mg, 0.014 mmol) and
Pd(OAc)2 (8.7 mg, 0.014 mmol) were added, and the reaction
mixture was heated for 25 min in a microwave reactor (125 ꢀC,
200 W). The solvent was evaporated and the residue was washed
with water. The aqueous layer was then extracted with EtOAc
(3 ꢂ 7 mL), and the combined organic extracts were washed with
brine, dried over Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by flash column chromatography
(EtOAcepetroleum ether 5:95) to obtain the title compound as a
dry CH2Cl2 (3 mL) and a solution of 40% HBr in acetic acid (100 mL,
1.65 mmol) was added dropwise at 0 ꢀC, and the mixture was
stirred at rt overnight. After 18 h, additional 40% HBr in acetic acid
(100
mL, 1.65 mmol) was added and the reaction mixture was
stirred at the same temperature until consumption of the limiting
reagent. The organic layer was then washed with water
(3 ꢂ 10 mL), sat. NaHCO3 solution (1 ꢂ 10 mL), brine and dried
over Na2SO4. Purification by flash column chromatography
(EtOAcepetroleum ether 10:90) gave the desired compound 23
(63 mg, 56% overall yield) as a white solid. 1H NMR (400 MHz,
white solid (44 mg, 51%). 1H NMR (400 MHz, DMSO-d6):
d
¼ 2.51 (s,
3H), 6.70 (dd, J1 ¼ 0.6 Hz, J2 ¼ 3.5 Hz, 1H), 7.26e7.33 (m, 2H), 7.47 (s,
1H), 7.57 (d, J ¼ 2.2 Hz, 1H), 7.59e7.64 (m, 1H), 7.72 (d, J ¼ 3.5 Hz,
1H), 8.24 (d, J ¼ 8.5 Hz, 1H), 8.63 ppm (dd, J1 ¼ 2.2 Hz, J2 ¼ 7.0 Hz,
1H). 13C NMR (100 MHz, DMSO-d6):
d
¼ 21.3, 107.2, 112.1, 112.8,
116.5, 116.8, 121.5, 123.6 (J ¼ 270 Hz), 125.7, 126.2, 127.2 (J ¼ 32 Hz),
CDCl3):
d
¼ 7.25e7.30 (m, 1H), 7.57e7.63 (m, 1H), 7.78 (d,
127.6 (J ¼ 3.2 Hz),130.5,131.9,133.2, 144.4,159.4,160.6,163.1. HRMS

34
M. Pieroni et al. / European Journal of Medicinal Chemistry 72 (2014) 26e34
(ESI) calculated for C19H12F4N2S [M þ H]þ 377.0657, found:
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Author contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
Acknowledgments
Sara Tanzarella, Elena Ferrari and Giannamaria Annunziato are
kindly acknowledged for the contribution in the synthesis of
derivatives.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.11.007.
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