4-Substituted Thioquinolines and Thiazoloquinolines: Potent, Selective, and Tween-80 invitro Dependent Families of Antitubercular Agents with Moderate invivo Activity

Article abstract of DOI:10.1002/cmdc.201100309

Two new families of closely related selective, non-cytotoxic, and potent antitubercular agents were discovered: thioquinolines and thiazoloquinolines. The compounds were found to possess potent antitubercular properties invitro, an activity that is dependent on experimental conditions of MIC determination (resazurin test and the presence or absence of Tween-80). To clarify the therapeutic potential of these compound families, a medicinal chemistry effort was undertaken to generate a lead-like structure that would enable murine efficacy studies and help elucidate the invivo implications of the invitro observations. Although the final compounds showed only limited levels of systemic exposure in mice, modest levels of efficacy invivo at nontoxic doses were observed. Two new families: In this work we identified two new classes of potent antitubercular agents. Synthesis and analysis of the pharmacological properties of several analogues led to the discovery of potent and selective derivatives invitro, with moderate invivo activity.

Full text of DOI:10.1002/cmdc.201100309

MED
DOI: 10.1002/cmdc.201100309
4-Substituted Thioquinolines and Thiazoloquinolines:
Potent, Selective, and Tween-80 in vitro Dependent
Families of Antitubercular Agents with Moderate in vivo
Activity
Jaime Escribano, Cristina Rivero-Hernꢀndez, Hilda Rivera, David Barros, Julia Castro-Pichel,
Esther Pꢁrez-Herrꢀn, Alfonso Mendoza-Losana, ꢂÇigo Angulo-Barturen, Santiago Ferrer-
Bazaga, Elena Jimꢁnez-Navarro, and Lluꢃs Ballell*[a]
Two new families of closely related selective, non-cytotoxic,
and potent antitubercular agents were discovered: thioquino-
lines and thiazoloquinolines. The compounds were found to
possess potent antitubercular properties in vitro, an activity
that is dependent on experimental conditions of MIC determi-
nation (resazurin test and the presence or absence of Tween-
80). To clarify the therapeutic potential of these compound
families, a medicinal chemistry effort was undertaken to gener-
ate a lead-like structure that would enable murine efficacy
studies and help elucidate the in vivo implications of the in vi-
tro observations. Although the final compounds showed only
limited levels of systemic exposure in mice, modest levels of
efficacy in vivo at nontoxic doses were observed.
Introduction
Despite the availability of treatments for tuberculosis (TB), the
threat this disease represents is still a painful reality for the ten
million people infected and the two million that die each
year.[1] TB also represents an escalating threat to global health,
with an increased prevalence of multidrug-resistant (MDR) TB
strains, which are resistant to at least the two main first-line TB
drugs isoniazid and rifampicin, and extremely drug-resistant
(XDR) TB strains, which are also resistant to three or more of
the six classes of second-line drugs.
mode of action), and 4) be competitive in terms of cost with
current drugs.
We and others have previously reported the antitubercular
activity of various purine,[4] pyrazolopyrimidine,[5] and benzimi-
dazole thiol ethers[6] (Figure 1). Herein we report our expansion
into a similar and as yet unreported new family of quinoline
thiol ethers endowed with potent antitubercular activity, pro-
nounced selectivity for Mycobacterium tuberculosis H37Rv versus
In some eastern European/central Asian countries
(EECAC) such as Azerbaijan, MDR/XDR strains can ac-
count for up to 22% of infections,[2] with mortality
rates for XDR reaching up to 100% of those infect-
ed.[3] While these numbers are partially attributable
to the misuse of current antitubercular agents, they
are also a direct consequence of the nature of the
treatment: a combination of at least three different
drugs that must be taken for six months or longer.
Due to side effects and the length of treatment, pa-
tients often discontinue therapy, with a consequent
Figure 1. Previously reported thiol ethers with antitubercular activity.
[a] Dr. J. Escribano, C. Rivero-Hernꢀndez, H. Rivera, Dr. D. Barros,
rise in drug-resistant strains and infection relapse
cases. This has lead to a call by the World Health Or-
ganization (WHO) for the widespread implementation
of directly observed treatment short-course (DOTS), in which
treatment compliance is monitored by healthcare workers. Al-
though this approach has been successful where appropriately
implemented (cure rates of >90%), the development of a new
drug for the treatment of TB could still be the most cost-effec-
tive way of tackling the potential pandemic. Specifically, any
new drug should be able to: 1) shorten the duration of treat-
ment, 2) avoid any significant drug–drug interactions with cur-
rent regimens, 3) treat MDR as well as XDR TB patients (new
Dr. J. Castro-Pichel, Dr. E. Pꢁrez-Herrꢀn, Dr. A. Mendoza-Losana,
Dr. ꢂ. Angulo-Barturen, Dr. S. Ferrer-Bazaga, Dr. E. Jimꢁnez-Navarro,
Dr. L. Ballell
Diseases of the Developing World (DDW)
Tres Cantos Medicines Development Campus (TCMDC)
GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos Madrid (Spain)
Fax: (+34)918070550
E-mail: lluis.p.ballell@gsk.com
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100309.
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4-Substituted Thioquinolines and Thiazoloquinolines
other bacteria, and ample safety windows in terms of
cytotoxic potential (compounds 5, 6, and 7).
Results and Discussion
As part of our continuous anti-mycobacterial screen-
ing activities, three new antitubercular compounds
were identified (Table 1). The antibacterial profiles of
these compounds were established by determining
the minimum inhibitory concentration (MIC80) against
M. tuberculosis H37Rv, other members of the Mycobac-
terium genus (M. bovis BCG and M. smegmatis), as well
as a panel of other bacterial species (Staphylococcus
aureus Oxford, Enterococcus faecalis, Haemophilus influ-
enzae, Moraxella catarrhalis, Streptococcus pneumo-
Scheme 1. Synthetic sequence to 4-substituted thioquinolines. Reagents and conditions:
a) AcOH, toluene, reflux, 2 h, then Ph2O, 2408C, 1 h; b) P4S10, pyridine, reflux, 5 h; c) R2Br,
K2CO3, DMF, RT, 2 h.
niae, and Escherichia coli). Additionally, both the cytotoxicity
potential of the compounds in HepG2, MDCK, L6, and CHO cell
lines as well as the possible genotoxicity liabilities toward S. ty-
phimurium were determined.
The acid-catalyzed cyclization–condensation between ani-
lines 9a–c and acetoacetate 8 yielded a range of 2-methyl 6-
substituted quinolones 10–16. Phosphorus pentasulfide medi-
ated thionation of the cyclized compounds yielded thioquino-
lones 17–23. Further S-alkylation with a range of alkyl halides
yielded the final 4-thioquinoline products (Table 2). The choice
of R2 group was directly related to our previous work on
thiopyrazolo[3,4-d]pyrimidines,[5] while only R groups that tol-
erate the harsh cyclization conditions were introduced. SAR
analysis showed how a range of lipophilic para-substituted
benzylic groups were tolerated, with a preference for methyl
(25), tert-butyl (5), trifluoromethyl (24), and chloro (26) substi-
tution patterns. With regard to the R1 position, only methoxy
The compounds were shown to be selective anti-mycobacte-
rials and were found to be endowed with the capacity to dif-
ferentiate between fast-growing (M. smegmatis) and slow-
growing (M. tuberculosis H37Rv and M. bovis BCG) mycobacteria.
This evidence prompted us to initiate a medicinal chemistry
effort to further explore and optimize the antitubercular po-
tential of the hit structures. With this aim in mind and a focus
on compound 5, a straightforward synthetic sequence was
devised to gain access to a number of analogues (Scheme 1).
Table 1. New antitubercular quinoline thiol ether screening hits.[a,b]
Isolate
Amoxicilin
Mupirocin
5
6
7
Anti-mycobacterial activity [MIC80, mgmLꢀ1]:
M. tuberculosis H37Rv
M. bovis BCG
M. smegmatis
Cytotoxicity [IC50, mgmLꢀ1]:[c]
–
–
–
–
–
–
<0.1
<0.1
>32
0.6
0.3
>32
1.2
0.6
>32
HepG2
MDCK
L6
–
–
–
–
–
–
–
–
>31
>31
>31
>31
>64
>64
>64
>64
>31
>31
>31
>31
CHO
Antibacterial activity [MIC90, mgmLꢀ1]:
S. aureus Oxford
E. faecalis
H. influenzae
M. catarrhalis
S. pneumoniae
E. coli
0.125
0.125
32
ꢁ0.06
0.25
0.125
1
>64
>64
>64
16
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
>64
4
>64
>64
0.25
0.25
ꢁ0.06
2
4
Genotoxicity:
S. typhimurium
plus S9 preparation
None at 16 mm
None at 16 mm
None at 16 mm
None at 16 mm
None at 16 mm
None at 16 mm
None at 16 mm
None at 16 mm
None at 16 mm
None at 16 mm
[a] Data represent the mean values of at least three independent experiments. [b] MIC determinations for reference compounds rifampicin and isoniazid
were performed. [c] Selected cell lines: HepG2, human caucasian hepatocyte carcinoma; MDCK, canine kidney; L6, rat skeletal muscle myoblast; CHO, Chi-
nese hamster ovary.
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Table 2. Antitubercular activity of a 4-thioquinoline substituted array.[a]
MIC80 [mgmLꢀ1
]
Cli [mLminꢀ1 gꢀ1
]
Compd
R1
R2
M. tub. H37Rv
M. smegmatis
Mouse
Human
CHI logD[b]
Albumin binding [%]
98.5
5
OMe
0.01
>32
>30
1.3
5.1
24
25
26
OMe
OMe
OMe
0.06
0.03
0.1
>5
>5
>5
>30
>30
>30
–
–
–
4.3
4.3
4.3
–
97.7
97.8
27
OMe
0.1
>5
>30
–
3.7
97.3
28
29
30
OMe
OMe
OMe
0.3
0.6
>5
>5
>5
>30
>30
>30
–
–
–
3.8
4.1
–
97.5
97.7
–
>5
31
OMe
1.25
>5
>30
14.7
–
–
32
33
34
OCF3
OCF3
Cl
>5
2.5
0.1
>5
>5
>5
11
23.9
>30
–
–
3
5.3
98.7
98.2
98.5
5.1
5.94
35
36
37
38
F
0.3
>5
2.5
>5
>5
>5
>5
>30
1.1
–
5.3
5.8
–
98.3
98.7
–
OBn
OEt
CN
–
–
–
–
>5
–
–
–
INH[c]
RIF[d]
–
–
0.25
0.016
–
–
–
–
–
–
–
–
–
–
[a] Data represent mean values of at least three different experiments. [b] At pH 7.4. [c] Isoniazid. [d] Rifampicin.
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4-Substituted Thioquinolines and Thiazoloquinolines
and chloro groups were tolerated, with dramatic differences in
terms of antitubercular activity stemming from subtle modifi-
cations (compounds 5 versus 32; Table 2).
dependence on the presence of Tween-80, with shifts in MIC
values from <0.1 mgmLꢀ1 to >8 mgmLꢀ1 in both M. tuberculo-
sis and M. bovis BCG (Figure 2). Although the simplest possible
explanation for this variation in MIC values could be attributed
to the permeability-enhancing properties of Tween-80,[9] only
modest increases have been shown previously,[10] and this
would be the first report in which a shift of two orders of mag-
nitude was found in comparing MIC values in the absence and
presence of Tween-80. Additionally, compound solubility was
shown to be independent of the presence of Tween-80 (data
not shown).
While the exquisite anti-mycobacterial selectivity profile of
the compounds was maintained (MIC against M. smegmatis:
>5 mgmLꢀ1), and slow clearance was observed in human mi-
crosomal fractions, the nature of the available in vivo murine
infectious TB model[7] made us focus our efforts toward im-
proving the rapid clearance observed in mouse microsomal
fractions (Cli>30 mLminꢀ1 gꢀ1, Table 2). Mapping the possible
structural sources of the instability indicated that the 6-me-
thoxy substitution makes an important contribution
to fast microsomal clearance (compound 32 versus
5). Compound 30 shows how the hypothetical alky-
lating potential of the a-S-benzylic position does not
appear to contribute greatly to the instability, owing
to the protective nature of the a-S-methyl substitu-
tion toward nucleophilic attack; compound 31 con-
firms this observation. Similarly, some contribution
from the para-benzylic position was envisaged when
comparing compounds 33 and 32.
The presence of the thioether 4-quinoline substitu-
tion pattern proved to be a prerequisite for anti-my-
cobacterial activity. Both ether 39, prepared from 10
by reaction with 4-tert-butylbenzyl bromide, and sulf-
Figure 2. M. bovis BCG MIC values for compound 5 in solid media with or without
Tween-80, with isoniazid (INH) and kanamycin (Kan) as references.
oxide 40, prepared by meta-chloroperoxybenzoic
acid (MCPBA)-mediated oxidation of thioether 5,
were devoid of antitubercular properties (Table 3).
Further evaluation of other important drug discov-
ery parameters such as lipophilicity and protein binding indi-
cated that the molecules lie in what is generally considered to
be borderline drug-like space.[8] This highlights the need to
direct medicinal chemistry activities toward decreasing the lip-
ophilic character of lead molecules in order to avoid the al-
ready known associated poor bioavailability and solubility im-
plications of working with drug leads with high clogP values.
As part of the lead confirmation process, antitubercular ac-
tivity was evaluated under a variety of conditions, including
growth inhibition studies in solid H37Rv 7H10-OADC media in
the absence and presence of Tween-80, a surfactant commonly
employed in M. tuberculosis H37Rv cultures to avoid bacterial
aggregation, which affects estimation of bacterial growth. MIC
Alternatively, recent reports have shown how Mycobacter-
ium spp. can degrade Tween-80, the by-products of which can
be used downstream in lipid metabolism as carbon sources to
support growth of actively growing and non-replicating myco-
bacteria.[11] In fact, a number of lipases and cutinases have
been found in the M. tuberculosis genome that could potential-
ly employ Tween-80 as a substrate. This hypothesis has already
been confirmed for a number of cutinases and mycobacterial
phospholipases.[12] This inherent adaptability of M. tuberculosis
to a variety of carbon sources could therefore be related to
the observed impact of Tween-80 on MIC determination. More-
over, on closer inspection and after having carefully analyzed
the growth inhibition curves (Figure 3), the compounds
showed a concentration-dependent biphasic growth inhibition
behavior in the presence of
determinations with compound
5 showed a remarkable
Tween-80. Although effective
bacterial inhibition at low con-
centrations (0.05 mgmLꢀ1) of
Table 3. Antitubercular activity of O, S, and S=O 4-substituted quinolines.
compound 5 was observed up
to ~80% inhibition, much higher
concentrations
(>1 mgmLꢀ1
)
were required to achieve >95%
growth inhibition. This observa-
tion is a consequence of the
method employed for MIC deter-
mination (resazurin reduction,
see Supporting Information), in
which an erratic color change
Isolate
5
39
40
Anti-mycobacterial activity [MIC80, mgmLꢀ1]:[a]
M. tuberculosis H37Rv 0.01
3.7
>10
[a] Data represent the mean values of at least three independent experiments.
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L. Ballell et al.
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Figure 3. Grown inhibition curve for compound 5.
Scheme 2. Synthetic sequence to thiazoloquinoline analogues 45–67 and
oxazoloquinoline 77. Reagents and conditions: a) HNO3, propionic acid,
110 8C; b) Fe, NH4Cl, EtOH/H2O, reflux; c) R2CO2H, DIPEA, PyBOP, DMF, then
P4S10, pyridine, 808C; d) R2CO2H, DIPEA, PyBOP, DMF, then P2O5, pyridine,
reflux.
indicating 80–90% inhibition can occur over a number of in-
hibitor dilutions, masking the MIC determination.
Although this inhibitor-concentration-dependent phenotype
has been previously shown for fluconazole (also known as the
trailing effect),[13] this is the first report in which such behavior
was observed for a novel family of antitubercular agents, and
it remains unclear what the implications might be for the ther-
apeutic value of such an observation for the compound family
involved. To address this issue, compound 5 was progressed to
in vivo murine pharmacokinetic (PK) evaluation (Table 4). The
compound showed high clearance and insufficient exposure
levels, making it unsuitable for an in vivo proof-of-concept
evaluation.
same dependence on Tween-80. With regard to the R1 posi-
tion, chloro substitution was slightly preferred over methoxy
substitution (compounds 45 versus 46). An exploration of the
chemical space around R2 seems to indicate the preference of
benzyl groups (45–55) over other functional groups such as
pyridinylmethyl (57–59), pyrimidinylmethyl (60), indolylmethyl
(61), 3-oxo-1,2,3,4-tetrahydroquinoxalinylmethyl (62), benzofur-
an (64), and phenoxymethyl (65–66) groups. In addition, SAR
analysis of the para position of the benzyl ring confirms the
preference toward more lipophilic substituents such as methyl,
tert-butyl, trifluoromethyl, and chloro groups (46–51) in con-
trast to more hydrophilic moieties, such as acetamido, methyl-
sulfonyl, and amino functional groups (52–54). The presence
of a methylene group spacer in R2 seems to be essential for
anti-mycobacterial activity (compounds 47 versus 63). Further-
more, compound 56, which has a hydroxy group at the benzyl-
ic position, and compound 68, in which in the methylene is re-
placed by an amino group, lack the activity found in their pre-
vious analogous, 49 and 51, respectively.
Table 4. PK parameters determined for compound 5.[a]
t1/2
[h]
cmax
[mgmLꢀ1
clast
[mgmLꢀ1
AUC(0ꢀt h)
Vd
[Lkgꢀ1
Cli
[b]
[c]
]
]
[mghmLꢀ1
]
]
[mLminꢀ1 gꢀ1
]
0.90
0.35
0.004
0.26
9.66
0.124
[a] Data represent the mean values of at least four independent experi-
ments after intravenous administration to C57BL/6 mice at a dose of
2 mgkgꢀ1 as a solution in encapsine 20%. [b] tmax =15 min. [c] tlast =4 h.
A small subset of N-substituted 8-aminomethyl thiazoloqui-
nolines was then prepared by cleavage of N-Boc-protected
thiazoloquinoline 67 and consecutive reductive amination with
several aromatic and heteroaromatic aldehydes (Scheme 3).
None of these secondary amines 70–76, or the parent com-
pound 69 showed any significant activity against M. tuberculo-
sis H37Rv (Table 5).
In light of this evidence, further medicinal chemistry efforts
led to the discovery of the 4-methyl[1,3]thiazolo[4,5-c]quinoline
core structure as an opportunity to overcome the rapid clear-
ance observed in mice microsomal fractions. The thiazoloqui-
noline potentially stabilizes the most obvious metabolic liabili-
ty, that is, the thioether functionality, in theory rendering an
improved PK profile over the previous scaffold.
Compounds were prepared by following the synthetic se-
quence shown in Scheme 2. Nitration and subsequent iron/am-
monium chloride reduction of 2-methyl-6-substituted quino-
lones 10 and 12 yielded the corresponding 3-aminoquinolones
43–44. PyBOP-assisted coupling of 3-aminoquinolones with an
array of carboxylic acids followed by thionation/dehydration
with phosphorus pentasulfide led to assembly of the thiazole
ring and formation of the required substituted thiazoloquino-
lines 45–67.
Scheme 3. Synthetic sequence to 71–78. Reagents and conditions: a) HCl
(4m), dioxane, RT; b) aldehyde, NaBH(OAc)3, RT.
Next, we focused our attention on the thiazoloquinoline
ring. Several modifications in the core structure were envis-
aged to discern if this particular configuration is essential for
The anti-mycobacterial activities of these compounds were
evaluated and are listed in Table 5; the MIC values showed the
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4-Substituted Thioquinolines and Thiazoloquinolines
Table 5. Antitubercular activity of a 4-methyl[1,3]thiazolo[4,5-c]quinoline substituted array.
[a]
[a]
Compd
R1
R2
MIC80 [mgmLꢀ1
]
Compd
R1
Cl
R2
MIC80 [mgmLꢀ1
]
45
OMe
0.024
61
>4
46
47
Cl
Cl
0.035
0.055
62
63
Cl
Cl
>4
>30
48
Cl
0.035
64
Cl
>4
49
50
51
Cl
Cl
Cl
0.035
0.035
0.022
65
66
67
Cl
Cl
Cl
>4
>4
>2
52
Cl
>4
68
Cl
>2
53
54
55
56
57
58
59
60
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
>4
3.4
0.1
>4
0.2
2
69
70
71
72
73
74
75
76
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
>1
>2
>4
3.5
2.9
>4
>4
>4
1.6
>4
[a] Determined toward M. tuberculosis H37Rv; data represent the mean values of at least three independent experiments.
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anti-mycobacterial activity. Thus, oxazoloquinoline 77 was pre-
pared from 44 through a similar approach as its parent com-
pound 49, but in this case with phosphorus pentoxide as the
cyclization/dehydration agent (Scheme 2). Moreover, thiazolo-
quinolinone 80 was prepared from commercially available 6-
chloro-2-methyl-4H-3,1-benzoxazin-4-one in three consecutive
steps (Scheme 4).
Table 6. Antitubercular activity of compounds 77, 80, and 83–86.
[a]
Compd
X
Y
R1
R2
R3
MIC80 [mgmLꢀ1
]
77
80
83
84
85
86
O
S
S
S
S
S
CH
CH
N
N
N
Cl
Cl
MeO
Me
O
Me
Me
Me
Me
Me
Cl
Cl
Cl
Cl
>4
>4
0.9
>4
>4
>4
O
(MeO)2OPO
Cl
N
Cl
[a] Determined toward M. tuberculosis H37Rv; data represent the mean
values of at least three independent experiments.
mycobacterial activity. Thus, sulfur atom replacement for
oxygen in oxazoloquinoline 77 led to a significant decrease in
potency (compound 77 versus 47) as previously observed for
the thioquinoline family, or the presence of naphthyridine in-
stead of quinoline ring (compound 83 versus 49).
Scheme 4. Synthetic sequence to thiazoloquinolinone 80. Reagents and con-
ditions: a) DBU, THF, RT, overnight, then HCl, MeOH, reflux, 3 h; b) 4-
ClPhCH2CO2H, DIPEA, PyBOP, DMF, RT, 2 h; c) KOH, Me2NCSCl, MeOH, RT, 1 h,
then BF3·OEt, CH2Cl2, MW, 1108C, 10 min.
Compound 48 was selected as a lead candidate for in vivo
murine efficacy studies. Although compound 45, with a me-
thoxy group, was found to have higher potency than com-
pound 46, the murine microsomal fraction clearance value
strongly favors the chloro substitution pattern found in com-
pound 48 (data not shown). Under our experimental condi-
tions, the oral administration of 48 does not exhibit acute tox-
icity >1000 mgkgꢀ1 for 1 day in mice. No significant variations
in body weight, necropsy, clinical chemistry, or hematology
values were observed. With this data in hand, the oral PK pro-
file of the compound at 100 and 300 mgkgꢀ1 was then estab-
lished. Despite the poor absorption (Figure 4), an event most
likely linked to low solubility in aqueous media, the compound
was further progressed to evaluation for in vivo murine anti-TB
efficacy.[7] The compound showed a 1.5-log decrease in myco-
bacterial burden (Figure 5) with respect to untreated
Several thiazolonaphthyridines were then synthesized by the
sequence illustrated in Scheme 5. Briefly, compound 83 was
prepared by cyclization–condensation between 5-amino-2-me-
thoxypyridine (9d) and acetoacetate, subsequent nitration and
reduction with iron/ammonium chloride, coupling with the
corresponding carboxylic acid, and thionation/cyclization with
phosphorus pentasulfide. Treatment with hydrogen bromide
gave thiazolonaphthyridinone 84, which could be then phos-
phorylated to 85 or halogenated to 86 with phosphoryl chlo-
ride using different conditions. The antitubercular activity of
these compounds is listed in Table 6.
As is the case for 4-thioquinolines, subtle substitutions in
the core structure translate into dramatic changes in anti-
controls. We believe this result, when put in the con-
text of the very modest exposure levels achieved in
mice and of the fact that AUC values at 100 and
300 mgkgꢀ1 were found to be similar, leaves room
for further optimization. Future synthetic efforts will
be focused on the optimization of physicochemical
parameters (clogP and solubility).
Conclusions
In summary, two new families of closely related com-
pounds (thioquinolines and thiazoloquinolines) with
potent antitubercular activity have been presented,
and further work is necessary to optimize the physi-
cochemical properties of the compounds in order to
obtain leads with improved in vivo DMPK properties.
Scheme 5. Synthetic sequence to thiazolonaphthyridines 83–86. Reagents and conditions:
a) 8, AcOH, toluene, reflux, 2 h, then Ph2O, 2408C; b) HNO3, propionic acid, 1108C; c) Fe,
NH4Cl, EtOH/H2O, reflux, then 4-ClPhCH2CO2H, DIPEA, PyBOP, DMF, then P4S10, pyridine,
808C; d) HBr, AcOH, 1008C; e) POCl3, 908C, then MeOH; f) POCl3, MW, 1608C.
The compounds are endowed with Tween-80 in vitro
dependent anti-mycobacterial activity, an observation
that does not translate into a complete lack of anti-
2258
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ChemMedChem 2011, 6, 2252 – 2263

4-Substituted Thioquinolines and Thiazoloquinolines
sured. 1H NMR spectra were re-
corded at 300 MHz on a Varian
spectrometer. Chemical shifts (d)
are given in ppm relative to the
solvent reference as an internal
standard (CDCl3, d=7.24 ppm;
[D6]DMSO, d=2.50 ppm; CD3OD,
d=3.31 ppm). Data are reported
as follows: chemical shift (multi-
plicity (s for singlet, d for doublet,
t for triplet, q for quartet, sept for
septet, m for multiplet, br broad),
coupling constant [Hz], integra-
tion). HPLC–MS analyses were con-
ducted on an Agilent 1100 instru-
ment equipped with a Sunfire C18
column (30 mm
ꢅ 2.1 mm i.d.,
Figure 4. Peripheral blood levels of compound 48 after oral administration to C57BL/6 mice at doses of 100 and
300 mgkgꢀ1, as a suspension in 1% methyl cellulose (n=4); tlast =24 h.
3.5 mm packing diameter) at 408C
coupled with a Waters ZMD2000
mass spectrometer; the method of
ionization was alternate-scan posi-
tive and negative electrospray.
Most of the compounds used in biological testing had
purity of >98%, as determined by HPLC analysis. Meth-
od A: flow: 1.5 mLminꢀ1, eluents: 1% aq. HCO2H/CH3CN,
ramp 0–0.2 min 90:10, 0.2–3.5 min 90:10!10:90, 3.5–
4.0 min 10:90, 4.0–5.0 min 90:10; Method B: flow:
1.5 mLminꢀ1, eluents: 1% aq. HCO2H/CH3CN, ramp 0–
0.2 min 70:30, 0.2–3.5 min 70:30!10:90, 3.5–4.0 min
10:90, 4.0–5.0 min 70:30; Method C: flow: 1.5 or
1.0 mLminꢀ1, eluents: aq. 0.05m NH4OAc/CH3CN, ramp
0–0.2 min 90:10, 0.2–3.5 min 90:10!10:90, 3.5–4.0 min
10:90, 4.0–5.0 min 90:10. 6-Benzyloxy-2-methyl-4(1H)-qui-
nolinone (13), 6-fluoro-2-methyl-4(1H)-quinolinone (14),
6-ethoxy-2-methyl-4(1H)-quinolinone (15), and 2-methyl-
4-oxo-1,4-dihydro-6-quinolinecarbonitrile (16) were pur-
chased from UkrOrgSynthesis Building Blocks; 2-[(2-
methyl-6-methoxy-4-quinolinyl)thio]-1-(4-methoxypheny-
l)ethanone (31) was purchased from Asinex. All commer-
cially available compounds were used as received, with-
out further purification. Representative procedures and
physical properties for examples of characterized ana-
logues are described. Characterization data for all com-
pounds are detailed in the Supporting Information.
Figure 5. Antitubercular activity of 48 in a murine model of acute infection, with isonia-
zid (INH) as reference. Mice were infected with 105 CFU, and lung homogenates were ob-
tained 9 days after infection (n=5 mice per group). Data were analyzed by one-factor
ANOVA and Tukey’s HSD post hoc multiple comparison test; p<0.05 was considered sig-
nificant. The boxes extend from the 25th to the 75th percentile, with a line at the
median; error bars indicate the highest and lowest values observed.
General procedure for the preparation of 2-methyl-4-
quinolinone derivatives 10–12 and 81: AcOH (6 mL)
was added to a suspension of aniline derivative (9a–c;
80 mmol) and ethyl acetylacetate (9.63 g, 94 mmol) in
toluene (150 mL). The reaction mixture was held at reflux for 2 h
with azeotropic removal of H2O by means of a Dean–Stark appara-
tus. Solvent was evaporated under vacuum, the formed solid was
suspended in Ph2O (20 mL) and heated at 2408C for 1 h. The reac-
tion mixture was cooled to 508C, and warm hexanes (100 mL) was
added. The formed precipitate was filtered off, washed with hex-
anes (3ꢅ5 mL) and dried under vacuum to yield 10–12, which
were used without further purification.
tubercular efficacy in vivo; the compounds present an in vitro
biphasic concentration-dependent antitubercular behavior. Fur-
ther studies are currently ongoing that will help elucidate the
therapeutic potential of this compound family.
Experimental Section
Chemistry
General: All commercially available reagents and solvents were
used without further purification unless otherwise stated. Automat-
ed flash chromatography was performed on a Biotage FlashMaster
II system with peak detection at 254 nm. All products were ob-
tained as amorphous solids, and melting points were not mea-
6-Methoxy-2-methyl-4(1H)-quinolinone (10): Yield: 12.5 g, 83%;
1H NMR (300 MHz, CD3OD): d=7.61 (d, J=2.8 Hz, 1H, ArH), 7.48
(d, J=9.1 Hz, 1H, ArH), 7.29 (dd, J=9.1 and 2.8 Hz, 1H, ArH), 6.19
(s, 1H, ArH), 3.88 (s, 3H, CH3), 2.44 ppm (s, 3H, CH3); MS (ES+): m/z
(%): 109.1 (100) [M+H]+.
ChemMedChem 2011, 6, 2252 – 2263
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2259

L. Ballell et al.
MED
General procedure for the thionation of 2-methyl-4-quinolinone
derivatives 10–16: P4S10 (888 mg, 2 mmol) was added in one por-
tion to a suspension of 2-methyl-4-quinolinone (10–16; 2 mmol) in
dry pyridine (25 mL). The reaction mixture was heated for 5 h at
reflux with stirring. The resulting solution was cooled in an ice
bath, and H2O (5 mL) was added. The mixture was concentrated to
10 mL, and H2O (10 mL) was added to the resulting crude. The
formed precipitate was filtered off, washed with H2O (3ꢅ3 mL),
and dried under vacuum to yield pure products 17–19, 22, and 23.
4-{[(4-tert-butyl)phenyl]methoxy}-6-methoxy-2-methylquinoline
(39). Yield: 60 mg, 17%; H NMR (300 MHz, CDCl3): d=8.74 (d, J=
8.9 Hz, 1H, ArH), 7.55–7.46 (m, 4H, ArH), 7.45–7.39 (m, 2H, ArH),
6.85 (s, 1H, ArH), 5.42 (s, 2H, CH2), 3.94 (s, 3H, CH3), 3.04 (s, 3H,
CH3), 1.36 ppm (s, 9H, 3ꢅCH3); MS (ES+), m/z (%): 336.2 (74)
[M+H]+, 147.1 (100); LC–MS, tR =2.61 min, Method A.
1
Procedure for the preparation of sulfoxide 40: MCPBA (16 mg,
0.07 mmol) was added to a stirred suspension of compound 5
(50 mg, 0.14 mmol) in CH2Cl2 (5 mL), and the reaction was stirred
for 1 h at 08C. The reaction mixture was concentrated to dryness.
The solid was purified by column chromatography to obtain 4-{[(4-
tert-butyl)phenyl]methylsulfinyl}-6-methoxy-2-methylquinoline (40).
Yield: 12 mg, 46%; 1H NMR (300 MHz, CDCl3): d=8.03 (d, J=
9.1 Hz, 1H, ArH), 7.49 (s, 1H, ArH), 7.36 (d, J=9.2 and 2.8 Hz, 1H,
ArH), 7.17 (d, J=8.3 Hz, 2H, ArH), 6.84 (d, J=2.8 Hz, 1H, ArH), 6.76
(d, J=8.3 Hz, 2H, ArH), 4.21 (d, J=12.7 Hz, 1H, CHH), 4.13 (d, J=
12.7 Hz, 1H, CHH), 3.84 (s, 3H, CH3), 2.68 (s, 3H, CH3), 1.25 ppm (s,
9H, 3ꢅCH3); MS (ES+), m/z (%): 368.2 (100) [M+H]+; LC–MS, tR =
2.90 min, Method A.
6-Methoxy-2-methyl-4(1H)-quinolinethione (17): Yield: 400 mg,
1
98%; H NMR (300 MHz, [D6]DMSO): d=12.03 (brs, 1H, NH), 7.40
(d, J=2.7 Hz, 1H, ArH), 6.75 (d, J=9.1 Hz, 1H, ArH), 6.63 (s, 1H,
ArH), 6.57 (dd, J=9.1 and 2.8 Hz, 1H, ArH), 3.13 (s, 3H, CH3),
1.68 ppm (s, 3H, CH3); MS (ES+): m/z (%): 205.1 (100) [M+H]+.
General procedure for the alkylation of 2-methyl-4-quinoline-
thione derivatives 17–23: The appropriate alkyl bromide
(1.5 equiv) was added to a suspension of 2-methyl-4(1H)-quinoline-
thione (17–23; 1 equiv) and K2CO3 (5 equiv) in DMF (2 mLmmolꢀ1),
and the reaction was stirred for 2 h at room temperature. The reac-
tion mixture was quenched by the addition of aqueous NH3 (28%,
3 mL). An aqueous solution of NH4Cl (1n, 75 mL) was added, and
the formed precipitate was filtered off and washed with H2O (3ꢅ
10 mL). The solid was purified by column chromatography to
obtain pure products 5, 24–30, 32–34, and 37–38.
General procedure for the nitration of 2-methyl-6-substituted 4-
quinolones 10, 12, and 81: A suspension of 2-methyl-4-quinolinol
derivative (10, 12, 81; 50 mmol) in propionic acid (100 mL) was
heated at 1108C with stirring. HNO3 (6.1 mL) was then added drop-
wise over 20 min. The reaction mixture was heated for 2 h at
110 8C with vigorous stirring. The resulting suspension was cooled
to room temperature and filtered off, the solids were washed with
cold EtOH (3ꢅ5 mL) and dried under vacuum to yield the products
41–42 and 82.
4-{[(4-tert-Butyl)phenyl]methylthio}-6-methoxy-2-methylquino-
line (5): Yield: 1.1 g, 68%; 1H NMR (300 MHz, CDCl3): d=7.88 (d,
J=8.8 Hz, 1H, ArH), 7.41–7.27 (m, 6H, ArH), 7.12 (s, 1H, ArH), 4.28
(s, 2H, CH2), 3.89 (s, 3H, CH3), 2.66 (s, 3H, CH3), 1.32 ppm (s, 9H, 3ꢅ
CH3); MS (ES+): m/z (%): 352.1 (100) [M+H]+; LC–MS, tR =2.78 min,
Method A.
6-Methoxy-2-methyl-3-nitro-4-quinolinol (41): Yield: 8.53 g, 73%;
1H NMR (300 MHz, [D6]DMSO): d=12.42 (brs, 1H, NH), 7.58 (d, J=
9.1 Hz, 1H, ArH), 7.53 (d, J=2.3 Hz, 1H, ArH), 7.39 (dd, J=9.1 and
2.9 Hz, 1H, ArH), 3.85 (s, 3H, CH3), 2.49 ppm (s, 3H, CH3); MS (ES+),
m/z (%): 234.1 (100) [M+H]+.
Procedure for the synthesis of 2-methyl-4-thioquinoline deriva-
tives 35–36: P4S10 (222 mg, 0.5 mmol) was added in one portion to
a suspension of 2-methyl-4-quinolinone (13, 14; 0.5 mmol) in dry
pyridine (5 mL). The reaction mixture was heated for 5 h at reflux
with stirring. The solvent was removed, and the remaining residue
was poured into H2O (5 mL) and extracted with CH2Cl2 (3ꢅ10 mL).
The combined organic layers were dried over MgSO4. The solvent
was evaporated, and LC–MS analysis of the crude was completed,
confirming the presence of quinolinethiones (20, 21). The resulting
solid was suspended in DMF (3 mL), and K2CO3 (345 mg, 2.5 mmol)
and the corresponding alkyl bromide (0.75 mmol) were added, and
the reaction was stirred for 2 h at room temperature. The reaction
mixture was quenched by the addition of aqueous NH3 (28%,
1 mL). An aqueous solution of NH4Cl (1n, 10 mL) was added, and
the formed precipitate was filtered off. The solid was purified by
column chromatography to obtain pure products 35 and 36.
Procedure for the reduction of 2-methyl-3-nitro-4-quinolinol de-
rivatives 41–42: A suspension of 2-methyl-3-nitro-4-quinolinol
(41–42; 15 mmol) in EtOH (200 mL) was heated at reflux. Fe0
(8.37 g, 150 mmol) and an aqueous solution of NH4Cl (60 mL,
150 mmol, 2.5m) were added, and the reaction was stirred at
reflux to completeness. The warm mixture was filtered through a
Celite patch, and the remaining solids were washed with warm
EtOH (60 mL). The filtrate was concentrated to dryness and was re-
suspended with trituration in H2O (40 mL). The formed precipitate
was filtered off and washed with H2O (2ꢅ10 mL) and hexanes (2ꢅ
10 mL) to yield 3-amino-2-methyl-4-quinolinol derivatives 43–44.
3-Amino-6-methoxy-2-methyl-4-quinolinol (43): Yield: 1.76 g,
1
58%; H NMR (300 MHz, CD3OD): d=7.65–7.43 (m, 2H, ArH), 7.28–
4-[(4-tert-Butylphenyl)methylthio]-6-fluoro-2-methylquinoline
(35): Yield: 48 mg, 28%; 1H NMR (300 MHz, CDCl3): d=7.97 (dd,
J=9.2 and 5.4 Hz, 1H, ArH), 7.70 (dd, J=10.0 and 2.8 Hz, 1H, ArH),
7.44 (ddd, J=9.1, 2.8 and 1.2 Hz, 1H, ArH), 7.41–7.31 (m, 4H, ArH),
7.14 (s, 1H, ArH), 4.28 (s, 2H, CH2), 2.67 (s, 3H, CH3), 1.32 ppm (s,
9H, 3ꢅCH3); MS (ES+): m/z (%): 340.1 (100) [M+H]+; LC–MS, tR =
3.13 min, Method A.
7.18 (m, 1H, ArH), 3.90 (s, 3H, CH3), 2.50 ppm (s, 3H, CH3); MS
(ES+), m/z (%): 204.1 (100) [M+H]+.
General procedure for the formation of thiazoloquinoline deriv-
atives 45–67: 3-Amino-2-methyl-4-quinolinol (43–44) and the cor-
responding carboxylic acid (1 equiv) were suspended in DMF
(2 mL), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate (PyBOP; 1.5 equiv) and ethyldiisopropylamine
(DIPEA; 3 equiv) were added. The reaction mixture was stirred for
2 h at room temperature and left to cool at 48C for 1 h. The result-
ing suspension was filtered off and washed with H2O (3ꢅ2 mL)
and hexanes (2ꢅ2 mL). The dried solid was suspended in pyridine
(2 mL), and P4S10 (1 equiv) was added in one portion. The mixture
was heated at 808C for 2 h. H2O (4 mL) was then added, and the
mixture was allowed to cool to room temperature. The white pre-
Procedure for the preparation of quinoline 39: 4-tert-Butylbenzyl
bromide (340 mg, 1.5 mmol) was added to a suspension of 6-me-
thoxy-2-methyl-4(1H)-quinolinethione (10; 200 mg, 1.05 mmol) and
K2CO3 (724 mg, 5.25 mmol) in DMF (5 mL), and the reaction was
stirred for 5 h at room temperature. The reaction mixture was fil-
tered off, and the organic solution was concentrated to dryness.
The solid was purified by column chromatography to obtain
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4-Substituted Thioquinolines and Thiazoloquinolines
cipitate was filtered off, washed with H2O (2ꢅ2 mL) and hexane
(2ꢅ2 mL) to yield the corresponding tiazoloquinoline derivatives
45–67.
quenched by the addition of satd. NaHCO3 (10 mL) and the aque-
ous layer was extracted with CH2Cl2/MeOH (4:1; 3ꢅ15 mL). The or-
ganic fraction was dried over Na2SO4 and concentrated to dryness.
The crude was dissolved in MeOH and purified by HPLC to give
the thiazoloquinolinone derivatives 70–76.
2-[(4-tert-Butylphenyl)methyl]-4-methyl-8-methoxy[1,3]thiazolo-
1
[4,5-c]quinoline (45): Yield: 2.52 g, 59%; H NMR (300 MHz, CDCl3):
d=8.03 (d, J=9.1 Hz, 1H, ArH), 7.45–7.34 (m, 4H, ArH), 7.31 (dd,
J=9.1 and 2.8 Hz, 1H, ArH), 7.06 (d, J=2.8 Hz, 1H, ArH), 4.52 (s,
2H, CH2), 3.92 (s, 3H, CH3), 3.08 (s, 3H, CH3), 1.35 ppm (s, 9H, 3ꢅ
CH3); MS (ES+), m/z (%): 377.2 (100) [M+H]+; LC–MS, tR =3.47 min,
Method B.
[(8-Chloro-4-methyl[1,3]thiazolo[4,5-c]quinolin-2-yl)methyl][(6-
trifluoromethyl-3-pyridinyl)methyl]amine (70): Yield: 51 mg,
64%; H NMR (300 MHz, [D6]DMSO): d=8.89 (s, 1H, ArH), 8.35 (d,
J=2.3 Hz, 1H, ArH), 8.29 (s, 1H, NH), 8.23 (d, J=7.9 Hz, 1H, ArH),
8.12 (d, J=8.9 Hz, 1H, ArH), 7.98 (d, J=8.0 Hz, 1H, ArH), 7.80 (dd,
J=8.9 and 2.3 Hz, 1H, ArH), 4.84 (s, 2H, CH2), 4.46 (s, 2H, CH2),
3.00 ppm (s, 3H, CH3); MS (ES+), m/z (%): 425.0 (42) [M+2H]+,
423.0 (100) [M+H]+; LC–MS, tR =3.71 min, Method C.
1
8-Chloro-4-methyl-2-[(4-trifluoromethylphenyl)methyl][1,3]thi-
1
azolo[4,5-c]quinoline (48): Yield: 372 mg, 65%; H NMR (300 MHz,
CDCl3): d=8.08 (d, J=8.6 Hz, 1H, ArH), 7.81 (d, J=2.0 Hz, 1H, ArH),
7.70–7.58 (m, 3H, ArH), 7.57–7.50 (m, 2H, ArH), 4.60 (s, 2H, CH2),
3.09 ppm (s, 3H, CH3); 13C NMR (100.6 MHz, [D6]DMSO): d=191.9
(C), 171.1(C), 154.5 (C), 147.0 (C), 142.1 (C), 141.9 (C), 138.9 (C),
131.1 (CH), 131.1 (C), 130.2 (CH), 129.3 (CH), 125.7 (CH), 124.0 (CH),
123.1 (C), 67.7 (CH2), 22.1 ppm (CH3);MS (ES+), m/z (%): 394.9 (43)
[M+2H]+, 393.0 (100) [M+H]+; HRMS-ESI: m/z [M+H]+ calcd for
C19H12ClF3N2S: 393.0435, found: 393.0436; LC–MS, tR =3.29 min,
Method C.
Procedure for the formation of oxazoloquinoline 77: 3-Amino-6-
chloro-2-methyl-4-quinolinol (44; 208 mg, 1 mmol) and 4-methyl-
phenylacetic acid (150 mg, 1 mmol) were suspended in DMF
(2 mL), and PyBOP (520 mg, 1 mmol) and DIPEA (0.53 mL, 2 mmol)
were added. The reaction mixture was stirred for 2 h at room tem-
perature. The resulting suspension was filtered off and washed
with DMF (2ꢅ3 mL), H2O (3ꢅ3 mL), and hexanes (2ꢅ3 mL). The
solid was dried, suspended in pyridine (10 mL), and P2O5 (142 mg,
1 mmol) was added in one portion. The mixture was heated at
reflux for 5 h. H2O (4 mL) was then added, and the mixture was al-
lowed to cool to room temperature. The white precipitate was fil-
tered off, washed with H2O (2ꢅ2 mL) and hexane (2ꢅ2 mL) to
Procedure for the synthesis of thiazoloquinoline derivative 68:
1-Isocyanate-4-methylbenzene (96 mg, 0.71 mmol) and Et3N
(0.134 mL, 0.96 mmol) were added to a solution of 3-amino-6-
chloro-2-methyl-4-quinolinol (44; 100 mg, 0.48 mmol) in EtOH
(6 mL). The reaction mixture was stirred for 2 days at 708C. The sol-
vent was partially evaporated (1 mL), and the resulting suspension
was filtered off and washed with hexanes (3ꢅ4 mL). The dried
solid was suspended in pyridine (4 mL), and P4S10 (197 mg,
0.44 mmol) was added in one portion. The mixture was heated at
reflux for 2 h. H2O (4 mL) was then added, and the mixture was al-
lowed to cool to room temperature. The white precipitate was fil-
tered off, washed with H2O (2ꢅ2 mL) and hexane (2ꢅ2 mL). The
solid was purified by preparative HPLC to yield 8-chloro-4-methyl-
N-(4-methylphenyl)[1,3]thiazolo[4,5-c]quinolin-2-amine (68; 10 mg,
yield
8-chloro-4-methyl-2-[(4-methylphenyl)methyl][1,3]oxazolo
1
[4,5-c]quinoline (77; 129 mg, 40%); H NMR (300 MHz, CDCl3): d=
8.08–8.03 (m, 2H, ArH), 7.60 (dd, J=9.1 and 2.3 Hz, 1H, ArH), 7.34–
7.28 (m, 2H, ArH), 7.20–7.15 (m, 2H, ArH), 4.52 (s, 2H, CH2), 4.05 (s,
3H, CH3), 3.09 ppm (s, 3H, CH3); MS (ES+), m/z (%): 325.1 (37)
[M+2H]+, 323.1 (100) [M+H]+; LC–MS, tR =3.09 min, Method C.
Synthesis of quinolinone 78: 6-Chloro-2-methyl-4H-3,1-benzoxa-
zin-4-one (8 g, 40.9 mmol) in THF (60 mL) was added dropwise to a
solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 6.23 g,
40.9 mmol) and ethyl isocyanoacetate (4.63 g, 40.9 mmol) in THF
(60 mL) at room temperature. The solution was stirred overnight.
AcOH (20% in H2O; 50 mL) was then added with ice bath cooling.
H2O (140 mL) was then added, and the organic solvent was re-
moved under vacuum. The resulting suspension was filtered off,
the solids were suspended in MeOH (70 mL), and HCl (16 mL, 37%)
was added to the mixture. The suspension was heated at reflux for
3 h with formation of a new white precipitate. The mixture was al-
lowed to cool to room temperature, and the solids were filtered
off, washed with Et2O (50 mL) and dried under vacuum to give 3-
amino-6-chloro-4-hydroxy-2(1H)-quinolinone (78; 5.72 g, 66%);
1H NMR (300 MHz, [D6]DMSO): d=12.05 (s, 1H, NH), 8.02 (d, J=
2.2 Hz, 1H, ArH), 7.53 (dd, J=8.8 and 2.2 Hz, 1H, ArH), 7.34 (d, J=
8.8 Hz, 1H, ArH), 7.05 ppm (brs, 2H, NH2); MS (ES+), m/z (%): 213.0
(37) [M+2H]+, 211.0 (100) [M+H]+.
1
6%); H NMR (300 MHz, CD3OD): d=10.87 (s, 1H, NH), 8.07 (d, J=
2.0 Hz, 1H, ArH), 8.00 (d, J=8.9 Hz, 1H, ArH), 7.70 (d, J=8.3 Hz, 2H,
ArH), 7.65 (dd, J=9.0 and 2.2 Hz, 1H, ArH), 7.22 (d, J=8.3 Hz, 1H,
ArH), 2.89 (s, 3H, CH3), 2.30 ppm (s, 3H, CH3); MS (ES+), m/z (%):
342.0 (48) [M+2H]+, 340.0 (100) [M+H]+; LC–MS, tR =3.53 min,
Method A.
Preparation of compound 69: 1-tert-Butyl [(8-chloro-4-methyl[1,3]
thiazolo[4,5-c]quinolin-2-yl)methyl]carbamate (970 mg, 2.67 mmol)
was suspended in 4m HCl (6.67 mL, 26.7 mmol) in dioxane, and
the mixture was stirred for 2 h at room temperature. The mixture
was concentrated to dryness and resuspended in satd. Na2CO3
(10 mL). The aqueous mixture was extracted with CH2Cl2/MeOH 4:1
(4ꢅ15 mL). The organic layer was dried over MgSO4, filtered off,
concentrated to dryness, and purified by column chromatography
to give [(8-chloro-4-methyl[1,3]thiazolo[4,5-c]quinolin-2-yl)methyl]-
Synthesis of acetamide 79: PyBOP (2.47 g, 4.75 mmol) and DIPEA
(1.658 mL, 9.50 mmol) were added to a suspension of 3-amino-6-
chloro-4-hydroxy-2(1H)-quinolinone (78; 1 g, 4.75 mmol) and 4-
chlorophenylacetic acid (0.810 g, 4.75 mmol) in DMF (4 mL). The
clear solution was allowed to react for 2 h. H2O (5 mL) was then
added, and the reaction was evaporated to near dryness under
vacuum. The resulting crude was resuspended in H2O (20 mL) to
give a white precipitate that was filtered off and purified by
column chromatography to give N-(6-chloro-4-hydroxy-2-oxo-1,2-
dihydro-3-quinolinyl)-2-(4-chlorophenyl)acetamide (79; 430 mg,
1
amine (69; 420 mg, 60%); H NMR (300 MHz, [D6]DMSO): d=8.46
(d, J=2.2 Hz, 1H, ArH), 8.23 (d, J=8.9 Hz, 1H, ArH), 7.89 (dd, J=8.9
and 2.3 Hz, 1H, ArH), 4.76 (s, 2H, CH2), 4.56 (brs, 2H, NH2),
3.07 ppm (s, 3H, CH3); MS (ES+), m/z (%): 266.0 (45)
[M+2H]+, 264.0 (100) [M+H]+; LC–MS, tR =2.45 min, Method C.
General synthesis for the formation of thiazoloquinolines 70–
76:
[(8-Chloro-4-methyl[1,3]thiazolo[4,5-c]quinolin-2-yl)methyl]-
amine (69; 50 mg, 0.19 mmol) and the corresponding aldehyde
(0.38 mmol) were dissolved in a mixture of CH2Cl2/MeOH 4:1
(5 mL) and NaBH(OAc)3 (121 mg, 0.57 mmol) was added portion-
wise every hour with stirring (8 h, eight portions). The mixture was
1
26%) as a clear white-blue powder. H NMR (300 MHz, [D6]DMSO):
d=11.93 (s, 1H, NH), 9.92 (s, 1H, OH), 7.79 (d, J=2.3 Hz, 1H, ArH),
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L. Ballell et al.
MED
7.55 (dd, J=8.8 and 2.3 Hz, 1H, ArH), 7.44–7.38 (m, 4H, ArH), 7.30
(d, J=8.7 Hz, 1H, ArH), 3.92 ppm (s, 2H, CH2); MS (ES+), m/z (%):
367 (11) [M+4H]+, 365.0 (67) [M+2H]+, 363.0 (100) [M+H]+.
Preparation of compound 85: 2-[(4-Chlorophenyl)methyl]-4-
methyl[1,3]thiazolo[4,5-c]-1,5-naphthyridin-8(9H)-one (84; 20 mg,
0.058 mmol) was suspended in POCl3 (1 g, 6.52 mmol), and the
mixture was heated at 908C for 30 min. The mixture was concen-
trated to dryness and dissolved in MeOH for loading onto HPLC for
purification to give 2-[(4-chlorophenyl)methyl]-4-methyl[1,3]thi-
azolo[4,5-c]-1,5-naphthyridin-8-yl dimethyl phosphate (85; 7 mg,
27%); 1H NMR (300 MHz, CDCl3): d=8.55 (d, J=8.8 Hz, 1H, ArH),
7.40–7.30 (m, 5H, ArH), 4.52 (s, 2H, CH2), 4.05 (s, 3H, CH3), 4.01 (s,
3H, CH3), 3.14 ppm (s, 3H, CH3); MS (ES+), m/z (%): 452.1 (43)
[M+2H]+, 450.1 (100) [M+H]+; LC–MS, tR =2.82 min, Method C.
Preparation of compound 80: KOH (15.45 mg, 0.275 mmol) was
suspended in MeOH (5 mL), and N-(6-chloro-4-hydroxy-2-oxo-1,2-
dihydro-3-quinolinyl)-2-(4-chlorophenyl)acetamide (79; 100 mg,
0.275 mmol) was added to the mixture. After 5 min stirring, 2-
chloro-N,N-dimethylethanethioamide (34.0 mg, 0.275 mmol) was
added in one portion. The mixture was allowed to react for 1 h,
and the white precipitate formed was filtered off, washed with H2O
(5 mL), and dried under vacuum. The white solid was suspended in
CH2Cl2 (5 mL), and BF3·OEt (117 mg, 0.826 mmol) was added to the
mixture in one portion. The mixture was sealed and heated with
stirring under microwave irradiation (10 min, 1108C). The mixture
was concentrated to dryness and purified by preparative HPLC to
give 8-chloro-2-[(4-chlorophenyl)methyl][1,3]thiazolo[4,5-c]quinolin-
4(5H)-one (80; 20 mg, 20%); 1H NMR (300 MHz, [D6]DMSO): d=
12.05 (s, 1H, NH), 7.88 (d, J=2.3 Hz, 1H, ArH), 7.54 (dd, J=8.8 and
2.3 Hz, 1H, ArH), 7.46–7.44 (m, 4H, ArH), 7.42 (d, J=8.8 Hz, 1H,
ArH), 4.52 ppm (s, 2H, CH2); MS (ES+), m/z (%): 365.0 (12)
[M+4H]+, 363.0 (74) [M+2H]+, 361.0 (100) [M+H]+; LC–MS, tR =
2.96 min, Method B.
Synthesis of naphthyridine 86: 2-[(4-Chlorophenyl)methyl]-4-
methyl[1,3]thiazolo[4,5-c]-1,5-naphthyridin-8(9H)-one (84; 20 mg,
0.058 mmol) was suspended in POCl3 (1 g, 6.52 mmol), and the
mixture was heated at 1608C for 2 min under microwave irradia-
tion. The mixture was concentrated to dryness and dissolved in
MeOH for loading onto HPLC for purification to give 8-chloro-2-[(4-
chlorophenyl)methyl]-4-methyl[1,3]thiazolo[4,5-c]-1,5-naphthyridine
1
(86; 6 mg, 29%); H NMR (300 MHz, CDCl3): d=8.39 (d, J=8.8 Hz,
1H, ArH), 8.39 (d, J=8.8 Hz, 1H, ArH), 7.38–7.33 (m, 4H, ArH), 4.53
(s, 2H, CH2), 3.12 ppm (s, 3H, CH3); MS (ES+), m/z (%): 364.0 (9)
[M+4H]+, 362.0 (65) [M+2H]+, 360.0 (100) [M+H]+, LC–MS, tR =
3.53 min, Method C.
Synthesis of naphthyridine 83: 2-Methyl-6-methoxy-3-nitro-1,5-
naphthyridin-4-ol (82; 580 mg, 2.46 mmol) was suspended in EtOH
(40 mL), and the mixture was heated at reflux. Fe0 (1.377 g,
24.6 mmol) and an aqueous solution of NH4Cl (2.5m, 9.86 mL,
24.6 mmol) were added, and the mixture was heated at reflux for
2 h. The warm mixture was filtered through a Celite patch, and the
remaining solids were washed with warm EtOH (5 mL). The result-
ing liquids were concentrated to dryness to give a dark-brown
powder. The solids were suspended DMF (5 mL), and DIPEA
(1.723 mL, 9.86 mmol) was added to the mixture followed by 4-
chlorophenylacetic acid (547 mg, 3.21 mmol) and PyBOP (1.716 g,
3.3 mmol). The resulting mixture was stirred at room temperature
for 4 h. H2O (25 mL) was then added, and the mixture was allowed
to precipitate for 15 min. The formed solids were filtered off,
washed with H2O, and dried under vacuum. The solid (420 mg)
was suspended in pyridine (5 mL), and P4S10 (522 mg, 1.17 mmol)
was added in one portion. The mixture was heated at reflux for
2 h. H2O (4 mL) was then added, and the mixture was allowed to
cool to room temperature. The white precipitate was filtered off
and purified by column chromatography to give 2-[(4-chloro-
phenyl)methyl]-4-methyl-8-methoxy[1,3]thiazolo[4,5-c]-1,5-naphthy-
ridine (83; 320 mg, 38%). 1H NMR (300 MHz, CDCl3): d=8.41 (d,
J=9.1 Hz, 1H, ArH), 7.41–7.35 (m, 4H, ArH), 7.13 (d, J=9.1 Hz, 1H,
ArH), 4.54 (s, 2H, CH2), 4.07 (s, 3H, CH3), 3.15 ppm (s, 3H, CH3); MS
(ES+), m/z (%): 358.0 (37) [M+2H]+, 356.0 (100) [M+H]+; LC–MS,
tR =3.60 min, Method C.
Biology
All experimental work with animals was performed by following
the 3R principles: replacing, reducing, and refining animal testing,
and took place in our Association for the Assessment of Laboratory
Animal Care International (AAALAC-1)-accredited facilities under
the control and approval of the Tres Cantos GSK local ethics com-
mittee.
General antimicrobial activity assay: Whole-cell antimicrobial activity
was determined by broth microdilution using the Clinical and Lab-
oratory Standards Institute (CLSI)-recommended procedure, Docu-
ment M7-A7, Methods for Dilution Susceptibility Tests for Bacteria
that Grow Aerobically. Some compounds were evaluated against a
panel of Gram-positive and Gram-negative organisms, including
Staphylococcus aureus, Enterococcus faecalis, Haemophilus influen-
zae, Moraxella catarrhalis, Streptococcus pneumoniae, and Escheri-
chia coli. Minimum inhibitory concentration (MIC) values were de-
termined as the lowest concentration of compound producing
>80 or 90% decrease in fluorescence observed.
Mycobacterium tuberculosis H37Rv inhibition assay: MIC determina-
tions for each tested compound were performed in 96-well flat-
bottomed polystyrene microtiter plates. Ten twofold drug dilutions
in neat DMSO starting at 400 mm were performed; these drug solu-
tions (5 mL each) were added to 95 mL Middlebrook 7H9 medium
(Difco cat. no. 271310; columns A–H, rows 1–10 of the plate
layout). Isoniazid was used as a positive control; eight twofold dilu-
tions of isoniazid starting at 160 mgmLꢀ1 were prepared, and 5 mL
of this control curve were added to 95 mL Middlebrook 7H9
medium (row 11, columns A–H). Neat DMSO (5 mL) was added to
row 12 (growth and blank controls). The inoculum was standar-
dized to ~1ꢅ107 CFUmLꢀ1 and diluted 1:100 in Middlebrook 7H9
Preparation of naphthyridinone 84: 2-[(4-chlorophenyl)methyl]-4-
methyl-8-methoxy[1,3]thiazolo[4,5-c]-1,5-naphthyridine
(83;
200 mg, 0.562 mmol) was suspended in a 33% solution of HBr in
AcOH (2 g, 4.68 mmol), and the stirred mixture was heated at
1008C for 1 h. The resulting residue was concentrated to dryness
purified by HPLC to give 2-[(4-chlorophenyl)methyl]-4-methyl-
[1,3]thiazolo[4,5-c]-1,5-naphthyridin-8(9H)-one (84; 78 mg, 41%);
1H NMR (300 MHz, [D6]DMSO): d=12.32 (s, 1H, NH), 8.01 (d, J=
9.1 Hz, 1H, ArH), 7.51–7.43 (m, 4H, ArH), 6.70 (d, J=9.1 Hz, 1H,
ArH), 4.61 ppm (s, 2H, CH2), 2.48 ppm (s, 3H, CH3); MS (ES+), m/z
(%): 344.0 (35) [M+2H]+, 342.0 (100) [M+H]+; LC–MS, tR =
2.31 min, Method C.
broth (Middlebrook ADC enrichment,
a dehydrated culture
medium that supports the growth of mycobacterial species;
Becton–Dickinson cat. no. 211887) to produce the final inoculum
of H37Rv strain (ATCC 25618). This inoculum (100 mL) was added to
the entire plate except wells G-12 and H-12 (blank controls). All
plates were placed in a sealed box to prevent drying of the periph-
eral wells, and were incubated at 378C without shaking for 6 days.
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ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 2252 – 2263

4-Substituted Thioquinolines and Thiazoloquinolines
A resazurin solution was prepared by dissolving one tablet of resa-
zurin (Resazurin Tablets for Milk Testing; VWR International Ltd. cat.
no. 330884Y) in 30 mL sterile phosphate-buffered saline (PBS); this
solution was added to each well (25 mL per well). Fluorescence was
20 mLkgꢀ1 vol. admin.; dose level: 100, 300, and 1000 mgkgꢀ1
;
feeding regimen: fed ad libitum; control group: vehicle, 1%
methyl cellulose; analytical method: Irwin test, body weight evolu-
tion, MDwF, clinical chemistry, hematology, reticulocytes, autopsy,
PK study at first day and three points for remain of study; statistics:
Kruskal Wallis and Dunns test (no parametric).
measured (Spectramax M5, Molecular Devices, lex =530 nm, lem
=
590 nm) after 48 h to determine the MIC value.
MIC in solid media: MIC values for each tested compound were
performed in 24-well flat-bottomed polystyrene microtiter plates;
11 twofold drug dilutions were carried out in 7H10-OADC (1 mL,
558C) and pooled in the plate (DMSO was used as positive con-
trol). The inoculum was standardized to 5ꢅ104 CFUmLꢀ1 in Middle-
brook 7H9 broth. This inoculum was placed in each well (10 mL per
well). All plates were placed in a sealed box to prevent drying out
and were incubated at 378C for 15 days. MIC in these assays was
defined as the lower concentration of the compound where <5%
of starting CFUs were observed.
Acknowledgements
The authors are grateful to the TB Alliance for financial support.
We thankfully acknowledge Fꢀtima Ortega for pharmacokinetic
evaluations, Joaquꢃn Rullas for efficacy studies, Antonio Martꢃnez
for essential animal lab upkeep and daily maintenance, Marꢃa
Jesffls Almela for cytotoxicity studies, Marꢃa Teresa Fraile for for-
mulation studies, Josꢁ Fiandor for advice and guidance for me-
dicinal chemistry activities, and Sophie Huss for in vitro clearance
evaluation.
Cytotoxicity assays: The cytotoxic effect of the compounds was
tested on various cell lines. Moreover, two different exposure times
(4 and 48 h) at 378C in a humidified 5% CO2 atmosphere and two
methods for determining cell damage (resazurin reduction and ATP
content) were done. The well-known toxic compound doxorubicin
was used as cytotoxic control. For all assays, the cell culture
medium used was EMEM supplemented with 5% fetal calf serum;
the concentration of DMSO never exceeded 0.5%.[14] Selected cell
lines: HepG2, human caucasian hepatocyte carcinoma; MDCK,
canine kidney; L6, rat skeletal muscle myoblast; CHO, Chinese
hamster ovary.
Keywords: antibiotics
· antitubercular agents · medicinal
chemistry · trailing effect · Tween-80
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Genotoxicity assays: Genotoxicity was measured by the Salmonella
typhimurium SOS/umu-assay,[15] and is based on the ability of DNA-
damaging agents to induce the expression of the umu operon. The
strains used were S. typhimurium TA1535/pSK1002 and NM2009.
The pSK1002 plasmid carries a umuC–lacZ fusion gene that produ-
ces a hybrid protein with b-galactosidase activity, the expression of
which is controlled by the umu regulatory region. The plasmid pre-
pared from E. coli CSH26/pSK1002 was introduced into S. typhimuri-
um TA1535 by DNA to create TA1535/pSK1002. The other strain,
NM2009, possesses elevated O-acetyltransferase levels and was
constructed by subcloning the corresponding gene into a plasmid
vector pACYC184 and introducing this plasmid into the original
strain TA1535/pSK1002. The criterion for positive response is a two-
fold increase in b-galactosidase activity over the mean control
values, showing a clear dose-related response.
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Pharmacokinetic studies: Species: C57BL/6 mouse, female, 18–20 g;
route: p.o., oral gavage; dose level: 100 and 300 mgkgꢀ1
,
20 mLkgꢀ1 vol. admin.; feeding regimen: fed ad libitum; compart-
ment analyzed: peripheral total blood; vehicle p.o. PK (suspension):
1% methyl cellulose (1% MC); sampling scheme: 15, 30 and
45 min, and 1, 1.5, 2, 3, 4, 8 and 24 h; n=4 per time point; analyti-
cal method (sensitivity): LC–MS (LLQ=1–5 ngmLꢀ1 in 25 mL blood);
data analysis: WinNonlin 5.0, non-compartmental analysis (NCA),
supplementary analysis with GraphPad 4 software.
Tolerability studies: Compound 48 was administered at 1000, 300,
and 100 mgkgꢀ1 p.o. to five animals each, by gavage. After admin-
istration, Irwin tests were done to every group, and body weight
was recorded 24 h after administration. All groups were sacrificed
by CO2 overdose, blood samples were taken by intracardiac punc-
ture for further analysis, and necropsy was done. Species: C57BL/6J
mice, 20 g ꢂ10%; sex: female, n=5 per group; protocol: CEEA
nos. 4 and 19; AESOP: AP1937v3; study design: 1 dose, o.d. maxi-
mal dose without findings (MDwF); route: p.o. oral gavage 20 g;
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Received: June 24, 2011
Revised: August 24, 2011
Published online on September 16, 2011
ChemMedChem 2011, 6, 2252 – 2263
ꢄ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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