Pyrido[1,2-a]benzimidazole-Based Agents Active Against Tuberculosis (TB), Multidrug-Resistant (MDR) TB and Extensively Drug-Resistant (XDR) TB

Article abstract of DOI:10.1002/cmdc.201000490

The struggle against tuberculosis (TB) is still far from over. TB, caused by Mycobacterium tuberculosis, is one of the deadliest infections worldwide. Co-infection with human immunodeficiency virus (HIV) and the emergence of multidrug-resistant tuberculos

Full text of DOI:10.1002/cmdc.201000490

MED
DOI: 10.1002/cmdc.201000490
Pyrido[1,2-a]benzimidazole-Based Agents Active Against
Tuberculosis (TB), Multidrug-Resistant (MDR) TB and
Extensively Drug-Resistant (XDR) TB
Marco Pieroni,[a] Suresh K. Tipparaju,[a] Shichun Lun,[b] Yang Song,[c] A. Willem Sturm,[d]
William R. Bishai,*[b] and Alan P. Kozikowski*[a]
The struggle against tuberculosis (TB) is still far from over. TB,
caused by Mycobacterium tuberculosis, is one of the deadliest
infections worldwide. Co-infection with human immunodefi-
ciency virus (HIV) and the emergence of multidrug-resistant tu-
berculosis (MDR-TB) and extensively drug-resistant tuberculosis
(XDR-TB) strains have further increased the burden for this dis-
ease. Herein, we report the discovery of 2-(4-chlorobenzyl)-3-
methyl-1-oxo-1H,5H-pyrido[1,2-a]benzimidazole-4-carbonitrile
as an effective antitubercular agent and the structural modifi-
cations of this molecule that have led to analogues with im-
proved potency and lower toxicity. A number of these deriva-
tives were also active at sub-micromolar concentrations
against resistant TB strains and devoid of apparent toxicity to
Vero cells, thereby underscoring their value as novel scaffolds
for the development of new anti-TB drugs.
Introduction
The resurgence of tuberculosis (TB) is one of the most serious
public health concerns worldwide, and the causative patho-
gen, Mycobacterium tuberculosis (Mtb), is frequently observed
in immuno-compromised individuals suffering from human im-
munodeficiency virus (HIV).[1] The World Health Organization
(WHO) has reported that Mtb was responsible for 9.2 million
new cases of infection and 1.78 million deaths in 2008, and
that one-third of the world’s population is estimated to be in-
fected by latent TB.[2] The treatment of TB must be continued
for 6–12 months in order to purge both the active bacteria, as
well as the bacteria present in a quiescent state.[3] However, in-
terconnected factors, including the high cost of drugs, the
slow expansion of directly observed treatment, short course
(DOTS), poor patient compliance with the treatment regimen
and physician errors, often result in relapses and the emer-
gence of drug resistance.[4,5] These resistant strains, in turn,
often demand a prolongation of the therapeutic regimen, with
the use of additional drugs that leads to an increase in treat-
ment costs.
introduced into the market over the last half century. Although
a number of novel agents are fueling the TB pipeline,[9,10] RIF,
used to treat TB since 1966,[11] still represents the last novel
class of anti-TB drugs. The foregoing facts strongly highlight
the need for the identification and development of agents ca-
pable of allowing for a shorter treatment regimen while inter-
acting with a unique biological target, thereby avoiding cross
resistance with existing drugs. Many research efforts to identify
new antibiotics have made it apparent that a whole-cell phe-
notypic high-throughput screening (HTS) approach is more
likely to yield lead candidates for further advancement than
the isolated target approach.[12] Indeed, compounds TMC207[13]
[a] Dr. M. Pieroni, Dr. S. K. Tipparaju, Prof. Dr. A. P. Kozikowski
Department of Medicinal Chemistry and Pharmacognosy
College of Pharmacy, University of Illinois at Chicago
833 S. Wood St., Chicago, IL 60612 (USA)
Fax: (+1)312-996-7107
E-mail: kozikowa@uic.edu
[b] Dr. S. Lun, Prof. Dr. W. R. Bishai
Center for Tuberculosis Research
Dept. of Medicine, Division of Infectious Disease
Johns Hopkins School of Medicine
Baltimore, MD 21231-1044 (USA)
Fax: (+1)410-614-8173
The number of new cases of drug-resistant TB has increased
in the recent years, with 440000 cases of multidrug-resistant
TB (MDR-TB) that have led to an estimated 150000 deaths in
2008.[6] By definition, MDR-TB strains lose their susceptibility to
two of the first-line drugs used in therapy, namely isoniazid
(INH) and rifampin (RIF), while extensively drug-resistant tuber-
culosis (XDR-TB) strains are in addition resistant to at least one
fluoroquinolone and one of the injectable anti-TB drugs, such
as amikacin, kanamycin (KAN), or capreomycin.[7] Even more
problematic is the recent isolation of strains resistant to all of
the standard first- and second-line drugs used in TB therapy,[8]
giving rise to the likely threat of a virtually incurable infection.
Despite its global impact on world health, TB is considered a
neglected disease and no new anti-TB therapeutics have been
E-mail: wbishai1@jhmi.edu
[c] Dr. Y. Song
Institute for Tuberculosis Research
College of Pharmacy, University of Illinois at Chicago
833 S. Wood St., Chicago, IL 60612 (USA)
[d] A. W. Sturm
Nelson R. Mandela School of Medicine University of KwaZulu-Natal
Suite Z310 Doris Duke Building 719 Umbilo Road
Durban 4001 Private Bag X7, Congella, Durban 4013, (South Africa)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cphc.201000490.
334
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ChemMedChem 2011, 6, 334 – 342

Pyrido[1,2-a]benzimidazole-Based Antitubercular Agents
and PA824,[14] two of the most promising anti-TB agents in clin-
ical trials, are the result of the improvement of hit compounds
identified after a random anti-TB screening of in-house chemi-
cal libraries at Johnson & Johnson and Ciba-Geigy, respectively.
In our own efforts to discover novel anti-TB chemotypes,[15,16]
phenotypic screening of a library of ~6000 compounds was
carried out affording a number of new hits with anti-TB activity
deemed of interest for further development. Among these
structures, compound 3a, containing a pyrido[1,2-a]benzimid-
azole core, was chosen for further
investigation based on its activity,
relatively low Vero cell toxicity, and
the ease of analogue synthesis.
These structure–activity relation-
ship (SAR) elucidation efforts have
led to the identification of mole-
cules having both an improved
anti-TB potency and selectivity
index. Some of the molecules were
also investigated for their chemical stability in human liver mi-
crosomes, thus providing some insights into the metabolism
of these compounds. Of greatest note is our finding that com-
pound 3h was able to maintain good activity when tested
against a panel of MDR-TB and XDR-TB strains, thus making
these pyrido[1,2-a]benzimidazoles attractive leads for further
development in the quest for improved anti-TB drugs.
Scheme 1. Reagents and conditions: a) (CH3)3SiCl/NaI/CH3CN, CH3CN, RT!
608C, 24 h, 57%; b) tBuOK, tBuOH, THF, 708C, 1–2.5 h, 26–92%; c) Benzimid-
azole-2-acetonitrile, CH3COONH4, neat or toluene, 1508C, 0.5–1.5 h, 51–91%;
d) for 4: TeO2/H2O2, MeOH/H2O, RT, 72 h, 66%; for 5: m-CPBA, acetone, RT,
3 h; e) BBr3, CH2Cl2, 08C!RT, 2 h, 79%. For complete structures see Figure 1.
Results and Discussion
Chemistry
The target compounds (3a–w,
4–6, Figure 1) were synthesized
by employing a straightforward
and efficient protocol described
previously (Scheme 1).[17] Benz-
imidazole-2-acetonitrile was re-
acted with an equimolar amount
of a suitable a-substituted ethyl
acetoacetate (2a–w) in the pres-
ence of ammonium acetate
either under neat conditions or
in anhydrous toluene at 1508C.
When not commercially avail-
able, a-substituted ethyl acetoa-
cetate derivatives 2 were synthe-
sized by reacting ethyl acetoace-
tate with a substituted bromo-
methyl- or bromoethylbenzene,
using potassium tert-butoxide as
a base in anhydrous tetrahydro-
furan (THF) at 708C.[18] Com-
pound 2u was synthesized by
reacting 4-(2-pyridinyl)benzalde-
hyde with ethyl acetoacetate in
the presence of the Me3SiCl/NaI/
CH3CN reagent from room tem- ment.
Figure 1. Structures of intermediates 2a–w and target compounds 3a–w, 4–6. indicates the point of attach-
*
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335

W. R. Bishai, A. P. Kozikowski et al.
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perature to 608C.[19] Oxidation of 3j with tellurium dioxide/hy-
drogen peroxide resulted in the sulfoxide 4 in good yields,
while the sulfone 5 was obtained using meta-chloroperoxyben-
zoic acid (mCPBA) as an oxidizing agent in acetone. Demethy-
lation of 3h with boron tribromide in anhydrous dichlorome-
thane at 08C to room temperature produced the hydroxy de-
rivative 6 in good yield. Product 3b was further examined
through 2D-NMR spectroscopy in order to assess which one of
the possible tautomeric forms (figure S1 in the Supporting In-
formation) predominates in solution.
obtained from the initial HTS study. Improvements were thus
needed, and the first round of modifications aimed at improv-
ing the activity and toxicity profile were made at the site of
the benzyl ring, as the pyrido[1,2-a]benzimidazole nucleus
along with the keto moiety at C1 and the nitrile group at C4
were believed to be important determinants of activity.
We first evaluated the effect of removal of the benzyl ap-
pendage. As it is apparent from the activity of compound 3w
(MICMABA >128 mgmLꢀ1) that the benzyl ring plays an important
role in conferring the desired anti-TB activity. We next investi-
gated the role of the chlorine atom and its position on the
benzyl ring. Moving the chlorine from the para to the meta
and ortho positions (3b, 3c, MICMABA =128 mgmLꢀ1) also result-
ed in a loss of the anti-TB activity, while the removal of the hal-
ogen atom led to a smaller drop in potency, with 3v having
an MICMABA value of 8.0 mgmLꢀ1. In the majority of cases, intro-
duction of various substituents on the benzyl ring at positions
other than the para position resulted in an increase in the
MICMABA value. In particular, meta substitution with electron-
In vitro biological studies
All of the target compounds synthesized were first tested
against the Mtb H37Rv strain to obtain the minimum inhibitory
concentration (MIC) as detected using the microplate Alamar
Blue assay (MABA), that is, the lowest concentration leading to
at least a 90% reduction of bacterial growth (MICMABA). In addi-
tion, for compounds with low MICMABA values, the MIC was
measured using a broth dilution (BD) method, that is the
lowest concentration of drug preventing visible bacterial
growth in a 5 mL Tween-free Middlebrook broth dilution cul-
ture (MICBD). The apparent cytotoxicity was obtained using
Vero cells. Several derivatives were found to be highly active in
inhibiting the growth of actively replicating Mtb with MICMABA
values of less than 1 mgmLꢀ1 (Table 1). For these compounds,
the MICBD values were the same or slightly higher than those
of the corresponding MICMABA, thereby indicating a cidal effect
for this class of derivatives.
withdrawing groups, such as CF3 (3d, MICMABA =128 mgmLꢀ1
CCF3 =3.18)[20] and OCF3 (3 f, MICMABA =64 mgmLꢀ1, COCF3
,
=
3.53),[20] was less tolerated than small electron-donor groups,
such as CH3 (3g, MICMABA =8 mgmLꢀ1, CCH3 =2.47).[20] On the
other hand, modifications at the para position of the benzyl
group played a key role in enhancing the anti-TB potency with
a variety of substituents being tolerated. Among the analogues
synthesized, the para methoxy-substituted compound 3h ex-
hibited one of the best activities in the series with an MICMABA
value of 0.25 mgmLꢀ1, only fivefold higher than that of INH,
the most effective drug against actively replicating Mtb.
More importantly, 3h also had a favorable selectivity index
with an IC50 value against Vero cells of greater than
128 mgmLꢀ1 and improved physicochemical parameters
(table S2 in the Supporting Information) compared to 3a such
as the ClogP (4.0 vs 4.8), topological polar surface area (TPSA;
70.3 ꢁ2 vs 61.1 ꢁ2), and the pre-
From the HTS studies, compound 3a had an MICMABA value
of 0.12 mgmLꢀ1 versus a toxicity toward Vero cells of >100 mm.
However, upon repeat testing, 3a exhibited a higher MICMABA
value of 0.5 mgmLꢀ1 and showed a sharp decrease in the selec-
tivity index (SI=16) with an IC50 value of only 8 mgmLꢀ1
against the Vero cells, which is not consistent with the data
dicted
aqueous
solubility
(AlogpS; ꢀ3.72 [65 mgLꢀ1] vs
ꢀ4.08 [29 mgLꢀ1]). Analogue 3j,
bearing a para methylthioether
group, retained good potency
(MICMABA =0.5 mgmLꢀ1), almost
comparable with that of the cor-
responding methoxy derivative.
Oxidation of the sulfur to the
Table 1. Anti-TB activity of compounds 3a–w, 4–6 against M. tuberculosis strain H37Rv.
[a]
[b]
Compd
MICMABA
MICBD
IC50 Vero cells[c]
Compd
MICMABA
[mgmLꢀ1
MICBD
[mgmLꢀ1
IC50 Vero cells
[mgmLꢀ1
]
[mgmLꢀ1
]
[mgmLꢀ1
]
]
]
[mgmLꢀ1
]
3a
3b
3c
3d
3e
3 f
3g
3h
3i
3j
3k
3l
3m
INH[e]
0.5
>128
>128
>128
>128
64
1.0
>128
>128
nt
8.0
nt[d]
nt
nt
nt
nt
nt
>64
16
nt
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
4
>128
4.0
nt
nt
nt
nt
1.0
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
>64
nt
nt
nt
nt
nt
4.0
0.5
0.5
8.0
nt
nt
nt
0.25
2.0
nt
nt
1.0
0.5
nt
sulfoxide
4
(MICMABA =
8.0
0.25
2.0
0.5
0.5
0.25
0.25
0.04
>128
4.0
8.0
>128
2.0
2.0 mgmLꢀ1) and the sulfone 5
(MICMABA =1.0 mgmLꢀ1) caused a
small reduction in anti-TB activi-
ty. Interestingly, the introduction
nt
nt
nt
nt
8.0
4.0
nt
5
6
1.0
>128
of
a polar p-hydroxy group
caused a loss of activity, the
MICMABA value of 6 being greater
[a] The lowest concentration of drug leading to at least a 90% reduction of bacterial growth signal by micro-
plate Alamar Blue assay (MABA); values reported are the average of three individual measurements; [b] The
lowest concentration of drug preventing visible bacterial growth in a 5 mL Tween-free Middlebrook broth dilu-
tion (BD) culture; values reported are the average of three individual measurements; [c] Cytotoxicity against
Vero cells; [d] Not tested; [e] Isoniazid.
than 128 mgmLꢀ1
.
The sharp difference in poten-
cy between 6 and the other
pyrido[1,2-a]benzimidazole ana-
336
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ChemMedChem 2011, 6, 334 – 342

Pyrido[1,2-a]benzimidazole-Based Antitubercular Agents
logues may be rationalized by the existence of a hydrophobic
pocket in the target binding site, located within the vicinity of
the para position of the benzyl moiety. This notion is to some
extent further supported by the activity of a small series of de-
rivatives for which the benzyl moiety has been substituted
with a methylpyridinyl group. When the nitrogen of the pyri-
dine ring is in the para position in relation to the linker (3n,
MICMABA >128 mgmLꢀ1), activity is lost, whereas the anti-TB ac-
tivities are somehow restored when the nitrogen atom of the
pyridine ring is moved around the ring as in derivatives 3o,
3p (MICMABA =4.0 mgmLꢀ1), and 3q (MICMABA =0.5 mgmLꢀ1). Re-
placement of the chlorine atom with a smaller fluorine atom
(3i, MICMABA =2.0 mgmLꢀ1) did not significantly affect the po-
tency. However, compounds containing two or three halogen
atoms, namely the fluorine containing analogues 3k
(MICMABA =0.5 mgmLꢀ1) and 3l (MICMABA =0.25 mgmLꢀ1), and
the dichloro derivative 3m (MICMABA =0.25 mgmLꢀ1), were
found to be more potent than the corresponding monosubsti-
tuted derivatives. Interestingly, the biological data of com-
pounds 3a, 3b and 3m support the notion that the activity is
dependent upon the presence of a lipophilic substituent at the
para position of the benzyl ring.
fails to provide any insights into the mechanism of action of
the hits. Some of the pyrido[1,2-a]benzimidazoles that we
have indentified here have previously been reported to inhibit
the biosynthesis of b-1,6-glucan, a key component of the my-
cetes cell wall.[21] This fact, along with the bactericidal nature
of these compounds, leads to the hypothesis that a mycobac-
terial orthologue of the fungal b-1,6-glucan synthases might
be the target for these molecules. Although the presence of
potential b-glucan synthase orthologue(s) in Mtb cannot be ex-
cluded, it is known that the mycobacterial genome does not
appear to contain genes encoding the enzymes traditionally
associated with b-glucan synthesis,[22] and, in addition, it is de-
batable whether or not glucans are crucial components of the
mycobacterial cell wall, thus leaving open the question, what
is the actual biosynthetic pathway inhibited by these com-
pounds?
The most promising compound of the series, i.e., 3h, was
also tested for its ability to inhibit the growth of selected
MDR-TB and XDR-TB strains, namely KZN494, V2475[23] (MDR-
TB), TF-274,[23] and R-506[23] (XDR-TB). It was extremely encour-
aging to find that 3h maintained the same activity, as found
for the susceptible strain H37rv, in three of the mutated Mtb
strains (KZN494, TF-274, and R-506), whereas it failed to show
appreciable activity against the MDR strain V2475 (Table 2). Al-
though this may be considered a weakness for the further ad-
vancement of 3h and related analogues, this information can
be used to better understand the molecular target(s) of these
molecules, which is(are) as yet undetermined. However, the
data at present would appear to suggest that the activity of
this novel class of antimycobacterial agents might depend on
a novel mechanism of action, different from those of the most
commonly used drugs.
Next, bulkier substituents were introduced at the same at-
tachment point so as to obtain further insights regarding the
hypothesized lipophilic pocket and its size. The tert-butyl deriv-
ative 3r retained excellent activity, with an MICMABA value of
0.5 mgmLꢀ1, whereas compound 3u, bearing a 2-pyridyl ring
as the substituent, was found to be less active with an MICMABA
value of 4.0 mgmLꢀ1. These findings give further support to the
hypothesis of the presence of a hydrophobic pocket in the
target binding site in proximity to the para position of the
benzyl ring. Likely, this pocket is important for the interaction
with the present molecules and is able to accommodate a
range of substituents mainly of a nonpolar character. Finally,
we investigated the effect of the methylene bridge on the
Table 2. Comparative anti-TB activity of compounds 3h and selected an-
titubercular drugs against susceptible, MDR and XDR strains of M. tuber-
culosis.
anti-TB activity. Removal of the methylene linker (3s, MICMABA
=
8.0 mgmLꢀ1) did not have any apparent effect on the potency
(3v vs 3s), whereas the elongation of the linker with an addi-
tional methylene group caused the complete loss of the anti-
TB activity (3t, MICMABA >128 mgmLꢀ1).
Compd
V4207
(DS)[a]
TF274
(XDR)[b]
R506
(XDR)[b]
KZN494
(MDR)[c]
V2475
(MDR)[c]
[d]
MICMABA [mgmLꢀ1
]
For selected compounds, MICBD values were determined,
which further confirmed the promising activity of these mole-
cules with SAR consistent with that of the MICMABA values.
Some basic considerations regarding the structure–toxicity re-
lationships for this class of derivatives can be surmised from
the small set of compounds synthesized. Compounds such as
3a, 3i, 3l and 3m, bearing halogen substituents on the benzyl
ring, although exhibiting high anti-TB potency, were found to
be toxic to Vero cells. Compounds such as 3r and the lead 3h,
bearing a tert-butyl and a methoxy group, respectively, did not
show any apparent toxicity in the same cell line (Table 1). Thus,
it might be hypothesized that the electron-withdrawing nature
of the substituent on the benzyl ring is responsible for the cy-
totoxicity, while electron-donating groups afford compounds
with potent anti-TB activity but lacking toxicity to Vero cells.
Although phenotypic screening is a powerful tool in the dis-
covery of new antibacterial leads, in the majority of cases it
3h
0.5–2
0.04
0.063
0.125
0.5
0.5
4–8
>128
1
4–8
>128
0.5
8
>128
0.5
16
128
nt[l]
0.5
2
16
16
2–4
nt
0.5
2
INH[e]
RIF[f]
LEV[g]
OFL[h]
KAN[i]
2
4
2–4
>128
[a] A drug-susceptible strain of Mtb; [b] An extensively drug-resistant
strain of Mtb; [c] A multidrug resistant strain of Mtb; [d] The lowest con-
centration of drug leading to at least a 90% reduction of bacterial
growth signal by microplate Alamar Blue assay (MABA); values reported
are the average of three individual measurements; [e] Isoniazid; [f] Ri-
fampin; [g] Levofloxacin; [h] Ofloxacin; [i] Kanamycin; [l] Not tested.
Selected compounds, namely 3a, 3d, 3e, 3v and 3w, were
also tested for their metabolic stability. After 30 min incubation
in the presence of mouse liver microsomes, the half life and
the ratio of predicted clearance to plasma flow were derived
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W. R. Bishai, A. P. Kozikowski et al.
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1 mgmLꢀ1 and MICBD values similar or slightly higher than the
corresponding MICMABA. Modifications of the benzyl moiety led
to a range of activities and allowed us to draft a preliminary
SAR. Removal of the benzyl group caused a loss in activity,
whereas substitution at its para position led to improved anti-
TB activity, with a variety of substituents being tolerated. Halo-
gens, a methoxy group, an alkyl appendage, and a heteroaro-
matic ring were found to be suitable substituents, whereas the
more hydrophilic hydroxy group caused a loss in activity. The
biological results may be consistent with the existence of a hy-
drophobic region in the target binding site within the proximi-
ty of the benzyl ring. However, the introduction of halogens re-
sulted in compounds that are toxic to Vero cells, whereas elec-
tron-donating substituents coupled strong activities with an
apparent lack of cytotoxicity. Removal of the linker methylene
unit did not affect the potency, while elongation of the linker
with an additional methylene group (3t) led to an inactive
compound. More importantly, against the two XDR-TB and one
MDR-TB strains tested in this study, 3h showed equal in vitro
activities as found against the susceptible control strain, there-
by suggesting potential for use in MDR-TB and XDR-TB chemo-
therapy. The molecular target, mechanism of action, and the in
vivo efficacy of such compounds are currently under investiga-
tion. Overall, the high bactericidal potency against drug-sus-
ceptible and drug-resistant Mtb strains establishes these
pyrido[1,2-a]benzimidazole derivatives as a novel promising
anti-TB chemotype.
Table 3. Metabolic stability of selected compounds in mouse liver micro-
somes.
Compd
Half life [min]
Q
p,h [%][a]
3a
3d
3e
3v
3w
6.50
14.96
9.43
7.82
51.21
95.4
90.0
93.5
94.5
72.5
[a] Percentage ratio of predicted clearance over plasma flow.
and calculated based on the well-stirred model (Table 3; see
also table S4 in the Supporting Information). Although the set
of derivatives tested is modest, basic conclusions about the
metabolism of these compounds can be made. The presence
of electron-withdrawing groups in the para position leads to a
shorter half life, as does the unsubstituted benzyl group. Sub-
stituents in the meta position are somehow able to enhance
the stability of these compounds, especially in the case of a
strong electron-withdrawing group. The methyl derivative 3w
was found to be the most stable member of the set of com-
pounds tested, leading to the conclusion that the benzyl
moiety is crucial for the anti-TB activity, but also constitutes
the metabolic “softspot” for this series of compounds.
Although the synthesis of these pyrido[1,2-a]benzimidazoles
has been described earlier, the question as to which one of the
possible tautomers is present in solution has not been ad-
dressed. Therefore, compound 3b was investigated in order to
assign the exchangeable proton at dH 13.47 ppm and the
neighboring protons thereby determining the structure of the
molecule (figure S2 in the Supporting Information). We first
used a combination of correlation spectroscopy (COSY) and
heteronuclear multiple bond correlation (HMBC) to assign each
proton’s resonance signal to the corresponding carbon. Once
each proton of the structure was characterized (table S3 in the
Supporting Information), selective 1D-nuclear Overhauser
effect spectroscopy (NOESY) was used to determine each pro-
ton’s proximity to dH 13.47 ppm. Briefly, the resonance signal
at dH 13.47 ppm was irradiated with a selective shaped pulse,
so as to detect only those protons that are proximal to it. Only
one peak, at dH 7.52 ppm, was revealed in the experiment,
thus establishing that the exchangeable proton was attached
at the nitrogen of the benzimidazole ring (figure S2 in the Sup-
porting Information), and that the pyridone form is the major
tautomer in solution. However, as is well known, the tautomer-
ic equilibrium can be shifted upon binding of the compound
to its molecular target.
Experimental Section
Biology: The minimum inhibitory concentration (MIC) values were
determined using M. tuberculosis H37Rv and the Kwa-Zulu Natal
strains. Initial MIC screening was done with the microplate Alamar
Blue assay (MABA) according to published procedures.[23b] MICBD
determinations were made by using broth dilution methods. Re-
ported MIC values are an average of three individual measure-
ments. For a description of the biological assays, see the Support-
ing Information.
Chemistry: 1H NMR and 13C NMR spectra were recorded on a
Bruker spectrometer at 400 MHz and 100 MHz, respectively, with
tetramethylsilane (TMS) as an internal standard. 19F NMR spectra
were recorded on a Bruker spectrometer at 376 MHz with tri-
fluoroacetic acid (TFA) as an external standard. Standard abbrevia-
tions indicating multiplicity were used as follows: s=singlet, d=
doublet, dd=doublet of doublets, t=triplet, q=quadruplet, m=
multiplet and br=broad. High-resolution mass spectrometry
(HRMS) was performed on Q-TOF-2TM (Micromass). TLC was per-
formed with Merck 60 F254 silica gel plates. Column chromatogra-
phy was performed using CombiFlash Rf system with RediSep col-
umns or alternatively using Merck silica gel (40–60 mesh). Prepara-
tive HPLC was carried out on a Shimadzu SCL-10A VP instrument
with an ACE 5-AQ (21.2 mmꢂ150 mm) column. Analytical HPLC
was carried out on Agilent 1100 HPLC system with a Synergi 4 mm
Hydro-RP 80 A column, on a variable wavelength detector G1314A.
Method 1: Flow rate=1.4 mLminꢀ1; gradient elution over 20 min,
from 30% CH3CN/H2O to 100% CH3CN with 0.05% TFA. For 2D-
NMR experiments a spectrometer Bruker at 900 MHz was used:
3b was dissolved in 0.75 mL of [D6]DMSO (99.96% deuterium) and
transferred into a 5 mm NMR tube. A Bruker AV900 spectrometer
Conclusions
Based on data from a phenotypic screening assay of about
6000 compounds, which identified compound 3a as a good
lead candidate, we constructed a small set of analogous
pyrido[1,2-a]benzimidazoles. A number of the synthesized
compounds were found to exhibit excellent bactericidal activi-
ties, with several of them having MICMABA values lower than
338
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 334 – 342

Pyrido[1,2-a]benzimidazole-Based Antitubercular Agents
1
equipped with a 5 mm ATM BBO probe was used to collect 1H,
DEPTQ, COSY, HSQC, HMBC and sel 1D-NOESY data used in reso-
nance assignment. Spectral information was referenced to the cor-
responding residual solvent peak.
oil); H NMR (400 MHz, CDCl3): d=1.22 (t, J=7.2 Hz, 3H), 2.23 (s,
3H), 3.05–3.20 (m, 2H), 3.73 (t, J=7.6 Hz, 1H), 4.16 (q, J=7.2 Hz,
2H), 7.03 (dd, J1 =2.0 HzJ2 =8.0 Hz, 1H), 7.25–7.30 (m, 1H), 7.30–
7.35 ppm (m, 1H).
The purity of the target compounds was determined to be >98%
Ethyl 2-(pyridin-2-yl-methyl)-3-oxobutanoate (2p): Purified by
by analytical HPLC (table S1 in the Supporting Information).
column chromatography (EtOAc-hexane 1:4). Yield 92% (colorless
oil); H NMR (400 MHz, CDCl3): d=1.21 (t, J=7.2 Hz, 3H), 2.32 (s,
3H), 3.25–3.45 (m, 2H), 4.16 (q, J=7.2 Hz, 2H), 4.29 (t, J=7.6 Hz,
1H), 7.10 (m, 1H), 7.18 (d, J=7.6 Hz, 1H), 7.57 (m, 1H), 8.46 ppm
(m, 1H).
1
General procedure for the synthesis of 2d, f, g, i, j, k, l, m, p, t:
To
a suspension of tBuOK (1.2 equiv) in anhydrous THF
(2 mLmmolꢀ1), ethyl acetoacetate (1.1 equiv) and tBuOH (0.1 equiv)
were added at 08C under argon. The resulting clear solution was
stirred for 30 min, and then the appropriate benzyl bromide
(1 equiv) was added dropwise at 08C. The reaction mixture was
stirred at 708C until consumption of the starting material as deter-
mined by TLC, and then the reaction mixture was quenched with
H2O and saturated aq. NaHCO3. The aqueous layer was extracted
with Et2O (3ꢂ10 mL) and the combined organic extracts were
washed with brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by flash chromatography to obtain the
desired product. Compounds 2a, b, c, e, h, o, q, r, s, v, w were
commercially available, while analytical data for compounds 2d,[24]
f,[24] i,[25] m,[24] n,[26] matched the data previously published.
Ethyl 2-[(4-chlorophenyl)ethyl]-3-oxobutanoate (2t): Purified by
column chromatography (EtOAc-hexane 1:4). Yield 26% (colorless
1
oil); H NMR (400 MHz, CDCl3): d=1.28 (t, J=7.2 Hz, 3H), 2.10–2.20
(m, 2H) 2.21 (s, 3H), 2.55–2.65 (m, 2H) 3.38 (t, J=7.2 Hz, 1H), 4.20
(q, J=7.2 Hz, 2H), 7.10 (d, J=8.4 Hz, 2H), 7.24 ppm (d, J=8.4 Hz,
2H).
Ethyl 2-[4-(pyridin-2-yl)-benzyl]-3-oxobutanoate (2u): To a stirred
mixture of (CH3)3SiCl (3.44 mL, 27.3 mmol), NaI (4.1 g, 27.3 mmol),
ethyl acetoacetate (0.689 mL, 5.45 mmol) and CH3CN (1 mLmmolꢀ1
of (CH3)3SiCl), 4-(pyridin-2-yl)benzaldehyde (1 g, 5.45 mmol) was
added at 08C. The mixture was stirred for 6 h at RT, and then over-
night at 608C. After cooling, H2O was added and the organic layer
was extracted with EtOAc, washed with aq. Na2S2O3, brine, dried
over MgSO4, filtered, and concentrated in vacuo. The resulted
yellow powder was used for the next reaction step without further
Ethyl 2-[3-(trifluoromethyl)benzyl]-3-oxobutanoate (2d):[24] Puri-
fied by column chromatography (EtOAc-hexane 1:4). Yield 67%
1
(colorless oil); H NMR (400 MHz, CDCl3): d=1.21 (t, J=7.2 Hz, 3H),
2.23 (s, 3H), 3.15–3.25 (m, 2H), 3.78 (t, J=8.0 Hz, 1H), 4.47 (q, J=
7.2 Hz, 2H), 7.30–7.55 ppm (m, 4H).
1
purification (0.88 g, yield 57%); H NMR (400 MHz, CDCl3): d=1.27
(t, J=7.2 Hz, 3H), 2.27 (s, 3H), 3.25–3.45 (m, 2H), 4.16 (q, J=7.2 Hz,
2H), 4.36 (t, J=7.6 Hz, 1H), 7.30–7.40 (m, 1H), 7.72 (d, J=8.0 Hz,
2H), 8.00 (d, J=8.4 Hz, 2H), 8.18 (d, J=8.4 Hz, 2H), 8.75 ppm (d,
J=7.6 Hz, 1H).
Ethyl 2-[3-(trifluoromethoxy)benzyl]-3-oxobutanoate (2 f):[24] Puri-
fied by column chromatography (EtOAc-hexane 1:4). Yield 87%
1
(colorless oil); H NMR (400 MHz, CDCl3): d=1.24 (t, J=7.2 Hz, 3H),
2.20 (s, 3H), 2.32 (s, 3H), 3.12–3.20 (m, 2H), 3.78 (t, J=7.6 Hz, 1H),
4.16 (q, J=7.2 Hz, 2H), 6.80–7.00 (m, 3H), 7.15–7.25 ppm (m, 1H).
General procedure for the synthesis of 3a–w: Benzimidazole-2-
acetonitrile (1 equiv), ammonium acetate (2.2 equiv) and the ap-
propriate substituted ethyl acetoacetate 2a–w (1.2 equiv) were re-
acted either in neat or in anhydrous toluene at 1508C for 0.5–1.5 h.
After cooling, the solid was washed with a mixture of EtOH/Et2O
(1:1, 3ꢂ10 mL), collected by filtration and purified either through
HPLC or recrystallized from EtOH/DMF. In the cases of the reactions
in toluene, after cooling the solvent was evaporated, and the re-
sulting solid washed with a mixture of EtOH/Et2O (1:1, 3ꢂ10 mL),
collected by filtration and purified through HPLC. Analytical data
for compounds 3s[22] and 3v[17] matched the data published previ-
ously.
Ethyl 2-(3-methylbenzyl)-3-oxobutanoate (2g): Purified by
column chromatography (EtOAc-hexane 1:4). Yield 69% (colorless
1
oil); H NMR (400 MHz, CDCl3): d=1.22 (t, J=7.2 Hz, 3H), 2.23 (s,
3H), 3.15–3.25 (m, 2H), 3.78 (t, J=8.0 Hz, 1H), 4.47 (q, J=7.2 Hz,
2H), 7.30–7.55 ppm (m, 4H).
Ethyl 2-(4-fluorobenzyl)-3-oxobutanoate (2i):[25] Purified by
column chromatography (EtOAc-hexane 1:4). Yield 65% (colorless
1
oil); H NMR (400 MHz, CDCl3): d=1.27 (t, J=7.2 Hz, 3H), 2.26 (s,
3H), 2.55–2.65 (m, 2H), 3.46 (t, J=7.2 Hz, 1H), 4.28 (q, J=7.2 Hz,
2H), 7.12 (d, J=8.4 Hz, 2H), 7.22 ppm (d, J=8.4 Hz, 2H).
Ethyl 2-[4-(methylthio)benzyl]-3-oxobutanoate (2j): Purified by
2-(4-Chlorobenzyl)-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]benzimid-
azole-4-carbonitrile (3a): Neat; purified by preparative HPLC. Yield
73% (white powder); 1H NMR (400 MHz, [D6]DMSO): d=2.32 (s,
3H), 3.91 (s, 2H), 7.22 (d, J=8.4 Hz, 2H); 7.26 (d, J=8.4 Hz, 2H),
7.33 (m, 1H), 7.45–7.55 (m, 2H), 8.56 (d, J=8.0 Hz, 1H), 13.47 ppm
(s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=18.3, 30.6, 69.8, 111.3,
114.0, 116.3, 116.8, 122.2, 126.6, 127.8, 128.2, 129.9, 130.4, 131.7,
139.5, 145.5, 147.4, 158.7 ppm; HRMS (ESI) calcd for C20H14ClN3O:
348.0898 [M+H]+, found: 348.0896.
column chromatography (EtOAc-hexane 1:4). Yield 62% (colorless
1
oil); H NMR (400 MHz, CDCl3): d=1.23 (t, J=7.2 Hz, 3H), 2.20 (s,
3H), 3.09–3.20 (m, 2H), 3.75 (t, J=7.6 Hz, 1H), 4.47 (q, J=7.2 Hz,
2H), 7.11 (d, J=8.4 Hz, 2H), 7.22 ppm (d, J=8.4 Hz, 2H).
Ethyl 2-(2,3,4-trifluorobenzyl)-3-oxobutanoate (2k): Purified by
column chromatography (EtOAc-hexane 1:4). Yield 81% (white
1
solid); H NMR (400 MHz, CDCl3): d=1.23 (t, J=7.2 Hz, 3H), 2.25 (s,
3H), 3.10–3.22 (m, 2H), 3.80 (t, J=8.0 Hz, 1H), 4.20 (q, J=7.2 Hz,
2H), 6.85–6.95 ppm (m, 2H).
2-(3-Chlorobenzyl)-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]benzimid-
azole-4-carbonitrile (3b): Neat; purified by preparative HPLC. Yield
62% (white powder); 1H NMR (400 MHz, [D6]DMSO): d=2.33 (s,
3H), 3.95 (s, 2H), 7.17–7.26 (m, 4H); 7.34 (m, 1H), 7.51 (m, 2H),
8.58 (d, J=8.0 Hz, 1H), 13.47 ppm (s, 1H); 13C NMR (400 MHz,
[D6]DMSO): d=18.3, 30.8, 69.8, 111.2, 113.5, 116.2, 116.8, 122.1,
125.8, 126.5, 126.7, 127.7, 127.7, 130.0, 131.6, 132.9, 143.1, 145.5,
147.3, 158.7 ppm; HRMS (ESI) calcd for C20H14ClN3O: 348.0898 [M+
H]+, found: 348.0900.
Ethyl 2-(2,4-difluorobenzyl)-3-oxobutanoate (2l): Purified by
column chromatography (EtOAc-hexane 1:4). Yield 61% (colorless
1
oil); H NMR (400 MHz, CDCl3): d=1.22 (t, J=7.2 Hz, 3H), 2.23 (s,
3H), 3.05–3.15 (m, 2H), 3.81 (t, J=8.0 Hz, 1H), 4.15 (q, J=7.2 Hz,
2H), 6.70–6.80 (m, 2H), 7.05–7.20 ppm (m, 1H).
Ethyl 2-(3,4-dichlorobenzyl)-3-oxobutanoate (2m):[24] Purified by
column chromatography (EtOAc-hexane 1:4). Yield 70% (colorless
ChemMedChem 2011, 6, 334 – 342
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
339

W. R. Bishai, A. P. Kozikowski et al.
MED
2-(2-Chlorobenzyl)-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]benzimid-
azole-4-carbonitrile (3c): Neat; recrystallized from EtOH/DMF. Yield
81% (white powder); 1H NMR (400 MHz, [D6]DMSO): d=2.26 (s,
3H), 3.98 (s, 2H), 6.91 (d, J=7.2 Hz, 1H), 7.14 (t, J=7.2 Hz, 1H),
7.21 (t, J=7.2 Hz, 1H), 7.35 (m, 1H), 7.45 (d, J=8.0 Hz, 1H), 7.46–
7.55 (m, 2H), 8.55 (d, J=8.0 Hz, 1H), 13.57 ppm (s, 1H); 13C NMR
(400 MHz, [D6]DMSO) d=18.3, 29.0, 70.0, 111.3, 112.0, 116.3, 116.9,
122.3, 126.7, 127.3, 127.7, 127.8, 128.5, 129.1, 131.8, 133.2, 137.1,
145.8, 148.1, 158.7 ppm; HRMS (ESI) calcd for C20H14ClN3O:
348.0898 [M+H]+, found: 348.0898.
7.45–7.55 (m, 2H), 8.58 (d, J=8.0 Hz, 1H), 13.50 ppm (s, 1H);
19F NMR (376 MHz, [D6]DMSO): d=ꢀ116.2; HRMS (ESI) calcd for
C20H14FN3O: 332.1194 [M+H]+, found 332.1203.
3-Methyl-1-oxo-2-[4-(thiomethyl)benzyl]-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3j): Neat; purified by preparative HPLC.
1
Yield 81% (white powder); H NMR (400 MHz, [D6]DMSO): d=2.32
(s, 3H), 2.39 (s, 3H), 3.89 (s, 2H), 7.12 (d, J=8.4 Hz, 2H); 7.15 (d, J=
8.4 Hz, 2H), 7.35 (m, 1H), 7.45–7.53 (m, 2H), 8.57 (d, J=8.0 Hz, 1H),
13.67 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=15.1, 18.4,
30.6, 69.7, 111.3, 114.4, 116.3, 116.9, 122.3, 126.4, 126.7, 127.9,
128.7, 131.8, 134.9, 137.4, 145.5, 147.1, 158.8 ppm; HRMS (ESI)
calcd for C21H17N3OS: 360.1165 [M+H]+, found 360.1153.
3-Methyl-1-oxo-2-[3-(trifluoromethyl)benzyl]-1H,5H-pyrido[1,2-a]-
benzimidazole-4-carbonitrile (3d): Neat; recrystallized from EtOH/
DMF. Yield 83% (white powder); 1H NMR (400 MHz, [D6]DMSO):
d=2.35 (s, 3H), 4.05 (s, 2H), 7.30–7.40 (m, 1H); 7.40–7.55 (m, 5H),
7.60 (s, 1H), 8.58 (d, J=8.4 Hz, 1H), 13.51 ppm (s, 1H); 13C NMR
(400 MHz, [D6]DMSO): d=18.4, 31.1, 69.9, 99.6, 111.4, 113.5, 116.3,
122.3, 123.7 (q, J=270 Hz), 124.6 (q, J=3.2 Hz), 126.7, 127.9, 128.9,
129.2, 129.4 (q, J=40 Hz), 131.8, 132.1, 142.1, 145.7, 147.5,
158.8 ppm; HRMS (ESI) calcd for C21H14F3N3O: 380.1016 [M+H]+,
found: 380.1035.
3-Methyl-1-oxo-2-(2,3,4-trifluorobenzyl)-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3k): Neat; purified by preparative HPLC.
1
Yield 50% (white powder); H NMR (400 MHz, [D6]DMSO): d=2.33
(s, 3H), 3.93 (s, 2H), 6.90 (m, 1H), 7.11 (m, 1H); 7.33 (m, 1H), 7.45–
7.55 (m, 2H), 8.53 (d, J=8.0 Hz, 1H), 13.54 ppm (s, 1H); 19F NMR
(376 MHz, [D6]DMSO): d=ꢀ161.0 (app t, J=24 Hz), ꢀ137.5 (dd,
J1 =4.0 Hz, J2 =16.0 Hz), ꢀ137.2 ppm (dd, J1 =4.0 Hz, J2 =16.0 Hz);
HRMS (ESI) calcd for C20H12F3N3O: 366.0884 [M+H]+, found
366.0874.
3-Methyl-2-(3-methoxybenzyl)-1-oxo-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3e): Neat; purified by preparative HPLC.
2-(2,4-Difluorobenzyl)-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3l): Neat; recrystallized from EtOH/DMF.
1
Yield 83% (white powder); H NMR (400 MHz, [D6]DMSO): d=2.32
(s, 3H), 3.68 (s, 3H), 3.92 (s, 2H), 6.70–6.85 (m, 3H); 7.10–7.15 (m,
1H), 7.33 (m, 1H), 7.45–7.55 (m, 2H), 8.58 (d, J=8.4 Hz, 1H),
13.60 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=18.3, 31.2,
54.9, 69.8, 110.9, 111.3, 114.0, 114.3, 116.2, 116.9, 120.2, 122.4, 126.5,
127.9, 129.3, 131.8, 142.1, 145.6, 147.2, 158.8, 159.4 ppm; HRMS
(ESI) calcd for C21H17N3O2: 344.1394 [M+H]+, found 344.1395.
1
Yield 75% (white powder); H NMR (400 MHz, [D6]DMSO): d=2.33
(s, 3H), 3.90 (s, 2H), 7.10–7.20 (m, 2H), 7.33 (m, 1H), 7.45–7.55 (m,
2H), 8.55 (d, J=8.4 Hz, 1H), 13.54 ppm (s, 1H); 19F NMR (376 MHz,
[D6]DMSO): d=ꢀ112.4 (d, J=4.0 Hz), ꢀ112.1 ppm (d, J=8.0 Hz);
HRMS (ESI) calcd for C20H13F2N3O: 350.1088 [M+H]+, found
350.1092.
3-Methyl-1-oxo-2-[3-(trifluoromethoxy)benzyl]-1H,5H-pyrido[1,2-a]-
benzimidazole-4-carbonitrile (3 f): Neat; purified by preparative
HPLC. Yield 63% (grey powder); H NMR (400 MHz, [D6]DMSO): d=
2-(3,4-Dichlorobenzyl)-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3m): Neat; purified by preparative HPLC.
Yield 51% (white powder); H NMR (400 MHz, [D6]DMSO): d=2.36
1
1
2.34 (s, 3H), 4.01 (s, 2H), 7.15–7.40 (m, 5H); 7.45–7.55 (m, 2H), 8.58
(d, J=8.4 Hz, 1H), 13.66 ppm (s, 1H); 13C NMR (400 MHz,
[D6]DMSO): d=18.4, 30.9, 69.9, 111.3, 113.5, 116.3, 116.9, 118.2,
120.5, 121.4, 122.3, 126.7, 127.0, 127.9, 130.1, 131.8, 143.5, 145.7,
147.5, 148.5, 158.8 ppm; HRMS (ESI) calcd for C21H14F3N3O2:
398.1088 [M+H]+, found 398.1099.
(s, 3H), 3.98 (s, 2H), 7.20–7.25 (m, 1H), 7.36 (m, 1H), 7.45–7.55 (m,
4H), 8.59 (d, J=8.0 Hz, 1H), 13.61 ppm (s, 1H); 13C NMR (400 MHz,
[D6]DMSO): d=18.4, 30.4, 69.9, 99.6, 111.3, 113.3, 116.3, 116.9,
122.3, 126.7, 127.8, 128.5, 129.9, 130.4, 130.8, 131.7, 141.9, 145.7,
147.6, 158.8 ppm; HRMS (ESI) calcd for C20H13Cl2N3O: 380.0363
[MꢀH]+, found 380.0375.
3-Methyl-2-(3-methylbenzyl)-1-oxo-1H,5H-pyrido[1,2-a]benzimid-
azole-4-carbonitrile (3g): Neat; recrystallized from EtOH/DMF.
Yield 91% (white powder); H NMR (400 MHz, [D6]DMSO): d=2.22
3-Methyl-1-oxo-2-[(pyridin-4-yl)methyl]-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3n): Neat; recrystallized from EtOH/DMF.
Yield 78% (white powder); H NMR (400 MHz, [D6]DMSO): d=2.33
1
1
(s, 3H), 2.34 (s, 3H), 3.92 (s, 2H), 6.90–7.10 (m, 3H), 7.11 (m, 1H);
7.36 (m, 1H), 7.45–7.55 (m, 2H), 8.60 (d, J=8.0 Hz, 1H), 13.49 ppm
(s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=18.4, 21.1, 31.1, 69.8,
111.3, 114.5, 116.3, 117.0, 122.2, 125.0, 126.5, 126.6, 127.9, 128.2,
128.6, 131.8, 137.3, 140.4, 147.2, 158.8 ppm; HRMS (ESI) calcd for
C21H17N3O: 328.1444 [M+H]+, found 328.1450.
(s, 3H), 3.98 (s, 2H), 7.22 (m, 2H), 7.34 (m, 1H), 7.45–7.55 (m, 2H),
8.40, (m, 2H), 8.56 (d, J=7.6 Hz, 1H), 13.50 ppm (s, 1H); 13C NMR
(400 MHz, [D6]DMSO): d=18.4, 30.7, 70.0, 99.6, 111.4, 112.5, 116.3,
122.2, 123.6, 126.6, 127.9, 132.0, 145.8, 147.7, 149.4, 149.8,
158.8 ppm; HRMS (ESI) calcd for C19H14N4O: 315.1240 [M+H]+,
found 315.1245.
3-Methyl-2-(4-methoxybenzyl)-1-oxo-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3h): Neat; purified by preparative HPLC.
Yield 74% (white powder); H NMR (400 MHz, [D6]DMSO): d=2.33
(s, 3H), 3.67 (s, 3H), 3.87 (s, 2H), 6.79 (d, J=8.0 Hz, 2H); 7.03 (d, J=
8.0 Hz, 2H), 7.33 (m, 1H), 7.45–7.55 (m, 2H), 8.58 (d, J=8.0 Hz, 1H),
13.61 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=18.3, 30.3,
55.0, 69.6, 111.2, 113.7, 114.9, 116.3, 116.9, 122.2, 126.6, 127.9, 129.0,
131.8, 132.3, 145.5, 146.9, 157.4, 158.8 ppm; HRMS (ESI) calcd for
C21H17N3O2: 344.1394 [M+H]+, found 344.1395.
3-Methyl-1-oxo-2-[(pyridin-3-yl)methyl]-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3o): Toluene; purified by preparative
HPLC. Yield 85% (white powder); 1H NMR (400 MHz, [D6]DMSO):
d=2.39 (s, 3H), 3.99 (s, 2H), 7.27 (m, 1H), 7.36 (m, 1H), 7.45–7.55
(m, 2H), 7.61 (d, J=8.0 Hz, 1H), 8.37, (s, 1H), 8.50 (s, 1H), 8.60 (d,
J=8.0 Hz, 1H), 13.52 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO):
d=18.8, 29.1, 70.3, 111.8, 113.6, 116.6, 117.4, 122.5, 123.9, 127.0,
128.3, 132.6, 135.9, 136.5, 146.2, 147.5, 147.7, 149.8, 159.2 ppm;
HRMS (ESI) calcd for C19H14N4O: 315.1240 [M+H]+, found 315.1254.
1
2-(4-Fluorobenzyl)-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]benzimid-
azole-4-carbonitrile (3i): Neat; recrystallized from EtOH/DMF. Yield
91% (white powder); 1H NMR (400 MHz, [D6]DMSO): d=2.34 (s,
3H), 3.93 (s, 2H), 7.04 (m, 2H); 7.20–7.30 (m, 2H), 7.35 (m, 1H),
3-Methyl-1-oxo-2-[(pyridin-2-yl)methyl]-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3p): Toluene; purified by preparative
HPLC. Yield 71% (white powder); 1H NMR (400 MHz, [D6]DMSO):
d=2.41 (s, 3H), 4.10 (s, 2H), 7.18 (m, 1H), 7.24 (d, J=8.0 Hz, 1H),
340
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Pyrido[1,2-a]benzimidazole-Based Antitubercular Agents
7.35 (m, 1H), 7.45–7.55 (m, 2H), 7.65 (t, J=7.6 Hz, 1H), 8.44, (m,
1H), 8.58 (d, J=8.0 Hz, 1H), 13.52 ppm (s, 1H); 13C NMR (400 MHz,
[D6]DMSO): d=19.0, 34.6, 70.2, 111.8, 113.5, 116.6, 117.4, 121.7,
122.6, 122.8, 127.0, 128.3, 132.4, 137.0, 146.1, 148.1, 149.2, 159.1,
160.0 ppm; HRMS (ESI) calcd for C19H14N4O: 315.1240 [M+H]+,
found 315.1255.
2,3-Dimethyl-1-oxo-1H,5H-pyrido[1,2-a]benzimidazole-4-carbo-
nitrile (3w): Neat; recrystallized from EtOH/DMF. Yield 73% (white
powder); 1H NMR (400 MHz [D6]DMSO): d=2.01 (s, 3H), 2.30 (s,
3H), 7.32 (bd s, 1H), 7.45–7.55 (m, 2H), 8.55 (d, J=6.8 Hz, 1H),
13.33 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=11.7, 18.5,
69.3, 111.0, 111.2, 116.2, 117.2, 122.1, 126.5, 127.8, 131.8, 145.3,
145.9, 158.6 ppm; HRMS (ESI) calcd for C14H11N3O: 238.0975 [M+
H]+, found 238.0973.
2-[(6-Chloropyridin-3-yl)methyl]-3-methyl-1-oxo-1H,5H-pyrido-
[1,2-a]benzimidazole-4-carbonitrile (3q): Toluene; purified by
preparative HPLC. Yield 88% (white powder); 1H NMR (400 MHz,
[D6]DMSO): d=2.37 (s, 3H), 3.94 (s, 2H), 7.35–7.37 (m, 2H), 7.45–
7.55 (m, 2H), 7.67 (d, J=8.0 Hz, 1H), 8.31 (s, 1H), 8.56 (d, J=8.0 Hz,
1H), 13.51 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=18.8,
28.3, 70.3, 111.7, 113.4, 116.6, 117.2, 122.7, 124.3, 127.0, 128.2,
132.1, 136.1, 139.3, 146.0, 147.8, 148.2, 149.8, 159.0 ppm; HRMS
(ESI) calcd for C19H13ClN4O: 349.0851 [M+H]+, found 349.0867.
3-Methyl-2-[4-(methylsulfinyl)benzyl]-1-oxo-1H,5H-pyrido[1,2-a]-
benzimidazole-4-carbonitrile (4): To a flask containing 3j (61 mg,
0.17 mmol) suspended in MeOH (2 mL) were added TeO2 (27 mg,
0.17 mmol), 30% H2O2 (0.1 mL), and a drop of concd HCl. The mix-
ture was stirred at RT for 72 h. Water (10 mL) was then added, and
the resulting precipitate was collected by filtration. The precipitate
was washed with H2O (3ꢂ5 mL) and purified by preparative HPLC
to afford 4 as a white solid (42 mg, 66%); 1H NMR (400 MHz,
[D6]DMSO): d=2.37 (s, 3H), 2.68 (s, 3H), 4.08 (s, 2H), 7.35 (m, 1H),
7.42 (d, J=8.0 Hz, 2H); 7.45–7.53 (m, 2H), 8.59 (d, J=8.0 Hz, 1H),
13.54 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=18.8, 31.5,
43.6, 70.2, 111.7, 114.2, 116.7, 117.3, 122.7, 124.1, 127.1, 127.5,
128.2, 129.2, 132.2, 144.0, 146.0, 147.8, 159.2 ppm; HRMS (ESI)
calcd for C21H17N3O2S: 374.0969 [MꢀH]+, found 374.0983.
3-Methyl-1-oxo-2-[(4-tert-butyl)benzyl]-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (3r): Neat; purified by preparative HPLC.
1
Yield 79% (white powder); H NMR (400 MHz, [D6]DMSO): d=1.22
(s, 9H), 2.36 (s, 3H), 3.91 (s, 2H), 7.13 (d, J=8.0 Hz, 2H); 7.24 (d, J=
8.0 Hz, 2H), 7.34 (m, 1H), 7.44–7.55 (m, 2H), 8.59 (d, J=8.0 Hz, 1H),
13.48 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=18.4, 30.7,
31.2, 34.1, 69.6, 99.6, 111.3, 114.7, 116.3, 117.0, 122.2, 125.0, 126.6,
127.7, 127.9, 131.8, 137.4, 145.5, 146.9, 148.0, 158.8 ppm; HRMS
(ESI) calcd for C24H23N3O: 370.1914 [M+H]+, found 370.1915.
3-Methyl-2-[4-(methylsulfonyl)benzyl]-1-oxo-1H,5H-pyrido[1,2-a]-
benzimidazole-4-carbonitrile (5): To a solution of 3j (50 mg,
0.14 mmol) in acetone (3 mL), a solution of m-CPBA (80%, 36 mg,
0.21 mmol) in acetone (1 mL) was added at 08C, and the reaction
mixture was stirred at RT for 3 h. Water (7 mL) was then added, the
solvent concentrated, and the resulting precipitate was collected
by filtration. The precipitate was washed with H2O (3ꢂ5 mL), Et2O
(3ꢂ5 mL) and purified by preparative HPLC to afford 5 as a white
3-Methyl-1-oxo-2-phenyl-1H,5H-pyrido[1,2-a]benzimidazole-4-
carbonitrile (3s):[25] Neat; purified by preparative HPLC. Yield 81%
(white powder); 1H NMR (400 MHz, [D6]DMSO): d=2.18 (s, 3H),
7.17 (t, J=7.6 Hz, 1H); 7.25–7.35 (m, 3H), 7.35–7.45 (m, 3H), 7.50
(d, J=8.0 Hz, 1H), 8.49 (d, J=8.0 Hz, 1H), 13.48 ppm (s, 1H);
13C NMR (400 MHz, [D6]DMSO): d=19.7, 70.9, 113.5, 115.7, 120.0,
125.2, 126.4, 127.8, 129.4, 131.5, 137.1, 146.5, 158.8, 172.1 ppm;
HRMS (ESI) calcd for C19H13N3O: 300.1131 [M+H]+, found 300.1132.
1
solid; H NMR (400 MHz, [D6]DMSO): d=2.41 (s, 3H), 3.15 (s, 3H),
4.08 (s, 2H), 7.36 (m, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.45–7.55 (m,
4H), 7.79 (d, J=8.4 Hz, 1H), 8.59 (d, J=8.0 Hz, 1H), 13.54 ppm (s,
1H); 13C NMR (400 MHz, [D6]DMSO): d=18.8, 31.9, 43.6, 70.2, 112.9,
114.9, 116.7, 117.3, 123.5, 124.0, 127.1, 127.5, 128.2, 130.2, 132.2,
144.0, 146.1, 147.8, 159.2 ppm; HRMS (ESI) calcd for C21H17N3O3S:
390.0918 [MꢀH]+, found 390.0933.
2-[(4-Chlorophenyl)ethyl]-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]-
benzimidazole-4-carbonitrile (3t): Toluene; purified by preparative
HPLC. Yield 78% (white powder); 1H NMR (400 MHz, [D6]DMSO):
d=2.22 (s, 3H), 2.70 (m, 2H), 2.78 (m, 2H), 7.24 (d, J=8.4 Hz, 2H),
7.25–7.40 (m, 5H), 7.45–7.55 (m, 2H), 8.59 (d, J=8.0 Hz, 1H),
13.57 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=17.8, 28.4,
33.5, 69.3, 111.2, 114.6, 116.2, 117.0, 122.1, 126.5, 127.7, 128.2,
130.3, 130.5, 131.7, 140.8, 145.3, 146.2, 158.3 ppm; HRMS (ESI)
calcd for C21H16ClN3O: 362.1055 [M+H]+, found 362.1039.
2-(4-Hydroxybenzyl)-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]benz-
imidazole-4-carbonitrile (6): To a suspension of 3h (100 mg,
0.29 mmol) in anhydrous CH2Cl2 (8 mL), BBr3 (2.32 mL, 2.33 mmol)
was added dropwise at 08C and the reaction mixture was stirred
for 2 h at RT. Upon completion, the reaction mixture was quenched
with H2O (5 mL), and the organic solvent was evaporated. The
solid resulted was collected and purified by preparative HPLC to
3-Methyl-1-oxo-2-[4-(pyridin-2-yl)benzyl]-1H,5H-pyrido[1,2-a]-
benzimidazole-4-carbonitrile (3u): Toluene; purified by prepara-
tive HPLC. Yield 52% (yellow powder); 1H NMR (400 MHz,
[D6]DMSO): d=2.34 (s, 3H), 4.01 (s, 2H), 7.20–7.40 (m, 4H), 7.45–
7.55 (m, 2H), 7.80–8.00 (m, 4H), 8.10 (m, 1H), 8.50–8.60 (m, 3H),
13.50 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=18.1, 31.0,
69.8, 111.3, 114.1, 116.3, 116.9, 121.0, 122.3, 122.9, 126.7, 126.9,
127.9, 128.6, 131.8, 135.0, 138.9, 142.3, 145.6, 147.4, 148.2, 155.2,
158.9 ppm; HRMS (ESI) calcd for C25H18N4O: 391.1540 [M+H]+,
found 391.1539.
1
give 6 as white solid (75 mg, 79%); H NMR (400 MHz, [D6]DMSO):
d=2.34 (s, 3H), 3.84 (s, 2H), 6.62 (d, J=8.0 Hz, 2H); 7.00 (d, J=
8.0 Hz, 2H), 7.35 (m, 1H), 7.45–7.55 (m, 2H), 8.60 (d, J=8.0 Hz, 1H),
9.10 (s, 1H), 13.46 ppm (s, 1H); 13C NMR (400 MHz, [D6]DMSO): d=
18.3, 30.2, 69.6, 111.3, 115.1, 116.3, 117.0, 122.3, 126.7, 127.8, 128.9,
130.5, 131.7, 145.5, 146.9, 155.3, 158.8 ppm; HRMS (ESI) calcd for
C20H15N3O2: 330.1237 [M+H]+, found 330.1225.
2-Benzyl-3-methyl-1-oxo-1H,5H-pyrido[1,2-a]benzimidazole-4-
carbonitrile (3v):[18] Neat; recrystallized from EtOH/DMF. Yield 90%
(white powder); 1H NMR (400 MHz, [D6]DMSO): d=2.35 (s, 3H),
3.97 (s, 2H), 7.10–7.30 (m, 5H), 7.35 (m, 1H), 7.45–7.55 (m, 2H),
8.60 (d, J=8.0 Hz, 1H), 13.49 ppm (s, 1H); 13C NMR (400 MHz,
[D6]DMSO): d=18.3, 31.1, 69.6, 111.2, 114.4, 116.2, 116.9, 122.2,
125.8, 126.5, 127.8, 128.0, 128.3, 131.7, 140.5, 145.5, 147.0,
158.7 ppm; HRMS (ESI) calcd for C20H15N3O: 314.1288 [M+H]+,
found 314.1301.
Acknowledgements
The support of US National Institutes of Health (grants AI30036
and AI079590) is gratefully acknowledged. We are grateful to
Prof. P. Moodley, W. R. Jacobs, Jr., and M. Larsen for generously
sharing clinical isolates of M. tuberculosis from KwaZulu Natal
and for providing their drug susceptibility phenotypes. Dr. S. C.
Cho and Prof S. G. Franzblau are acknowledged for the pheno-
ChemMedChem 2011, 6, 334 – 342
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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W. R. Bishai, A. P. Kozikowski et al.
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Received: November 16, 2010
Published online on January 21, 2011
342
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ChemMedChem 2011, 6, 334 – 342