Functionalized 3-amino-imidazo[1,2-a]pyridines: A novel class of drug-like Mycobacterium tuberculosis glutamine synthetase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.06.045

3-Amino-imidazo[1,2-a]pyridines have been identified as a novel class of Mycobacterium tuberculosis glutamine synthetase inhibitors. Moreover, these compounds represent the first drug-like inhibitors of this enzyme. A series of compounds exploring structural diversity in the pyridine and phenyl rings have been synthesized and biologically evaluated. Compound 4n was found to be the most potent inhibitor (IC₅₀ = 0.38 ± 0.02 μM). This compound was significantly more potent than the known inhibitors, l-methionine-SR-sulfoximine and phosphinothricin.

Full text of DOI:10.1016/j.bmcl.2009.06.045

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4790–4793
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Functionalized 3-amino-imidazo[1,2-a]pyridines: A novel class of drug-like
Mycobacterium tuberculosis glutamine synthetase inhibitors
Luke R. Odell a, Mikael T. Nilsson b, Johan Gising a, Olof Lagerlund a, Daniel Muthas a, Anneli Nordqvist a,
Anders Karlén a, Mats Larhed a,
*
a Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, Biomedical Center, Box 574, SE-751 23 Uppsala, Sweden
b Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Amino-imidazo[1,2-a]pyridines have been identified as a novel class of Mycobacterium tuberculosis glu-
tamine synthetase inhibitors. Moreover, these compounds represent the first drug-like inhibitors of this
enzyme. A series of compounds exploring structural diversity in the pyridine and phenyl rings have been
synthesized and biologically evaluated. Compound 4n was found to be the most potent inhibitor
Received 1 June 2009
Revised 9 June 2009
Accepted 11 June 2009
Available online 14 June 2009
(IC50 = 0.38 0.02
lM). This compound was significantly more potent than the known inhibitors, L-
methionine-SR-sulfoximine and phosphinothricin.
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis
Glutamine synthetase inhibitors
Microwave
3-Aminoimidazo[1,2-a]pyridine
Tuberculosis (TB) is one of the world’s major public health prob-
lems and has been declared a global health emergency by the
World Health Organization.1 In 2006, there were approximately 9
million new cases of TB, resulting in an estimated 1.7 million
deaths.2 In addition, the emergence of multi-drug resistant Myco-
bacterium tuberculosis strains (causing MDR-TB), the growing rate
of TB incidence, the lethal combination represented by HIV co-
infection and the lack of any new antituberculosis agent in the last
40 years, all indicate an urgent need for the development of novel
TB therapies. In particular, new lead structures are required with
novel modes of action.3,4 In the last decade M. tuberculosis gluta-
mine synthetase (MtGS), a key enzyme required for nitrogen
metabolism and mycobacterial cell-wall biosynthesis, has emerged
as a potential target for antibiotics against TB.1,4–6 Exposure of M.
O
O
HOOC
S
HOOC
P
NH
OH
NH2
NH2
1
2
Figure 1. Structure of MSO (1) and PPT (2).
During the course of a high-throughput compound screen and a
subsequent lead validation process, 3-amino-imidazo[1,2-a]pyri-
dines were identified as a novel class of MtGS inhibitors (Fig. 2).
It was quickly recognized that this would provide a scaffold ame-
nable to the rapid exploration of structure–activity relationships.
Indeed, a diverse range of analogues can be readily obtained, in
one step, via an Ugi-type cyclization.14–19 Herein, we describe the
high-speed synthesis and MtGS inhibitory activity of two libraries
of differentially substituted 3-amino-imidazo[1,2-a]pyridines, 3
and 4.
We decided to focus our initial investigations on evaluating
the effect of altering the pyridine ring substituent. Accordingly,
3-amino-imidazo[1,2-a]pyridines (3a–m) were prepared by
microwave-assisted multicomponent reaction (MCR) between
cyclopentylisonitrile, 3-hydroxy-4-methoxy benzaldehyde and an
appropriately substituted 2-aminopyridine.19 All 2-aminopyri-
dines were commercially available with the exception of 5-meth-
oxy-2-aminopyridine, which was prepared by the treatment of
5-iodo-2-aminopyridine with MeOH and CuI under Buchwald’s
tuberculosis to the known GS inhibitor L-methionine-SR-sulfoxim-
ine 1 (MSO) (Fig. 1) has been shown to inhibit both cell wall forma-
tion and mycobacterial growth.1,6 Thus, the development of new
MtGS inhibitors could be useful in developing an effective treat-
ment for MDR-TB. The majority of known GS inhibitors are simple
glutamate analogues and of these, MSO and phosphinothricin 2
(PPT) are the most widely investigated.7 These inhibitors have been
used as lead compounds in several studies,5,7–12 albeit they are po-
lar, flexible, non-selective13 and not particularly drug-like. Accord-
ingly, we were interested in the identification and development of
more drug-like non-amino acid derived MtGS inhibitors.
a
* Corresponding author.
E-mail address: mats@orgfarm.uu.se (M. Larhed).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.045

L. R. Odell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4790–4793
4791
H
O
C
N
NH
N
R1
N
R1
R2
NH2
R2
N
3 and 4
Figure 2. 3-Amino-imidazo[1,2-a]pyridines and retrosynthetic analysis showing assembly via a one-pot three-component condensation reaction.
Table 1
modified Ullman reaction conditions (1,10-phenanthroline,
Cs2CO3, 110 °C)20 providing 3c. The MCR reactions were typically
irradiated, in a sealed vessel, for 20–30 min at 160 °C in the pres-
ence of MgCl2 and the products were isolated either by simple fil-
tration, recrystallization or flash chromatography.21 The potency of
the compounds 3a–m from this first series against MtGS was eval-
uated22 and these results are reported in Table 1.
Synthesis and biological evaluation of 3-amino-imidazo[1,2-a]pyridines (3a–m)
O
H
NH
2
5
6' 5'
2'
6
MgCl2
N
4'
N
R1
OMe
R1
N
C
EtOH, 160 °C
MW, 20 or 30 min
N
3'
OH
NH2
HO
7
8
3a-m
OMe
From Table 1 it is clear that a pyridine ring substituent is ben-
eficial for MtGS inhibition and that a number of synthesized ana-
logues were significantly more active than MSO (i.e., 3c, 3e–g,
3k, 3m). The most active compounds contained large halogen
b
Entry
Producta
R1
IC50
(
lM)
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
—
—
H
>50
>50
6-Me
6-OMe
6-F
6-Cl
6-Br
31.3 0.7
>50
11.2 1.5
8.8 0.4
4.8 0.5
>50
atoms (Cl, Br or I) in the 6-position (i.e., 3f, IC50 = 8.8 0.4
and 3g IC50 = 4.8 0.5 M). Interestingly, the 6-fluorine, 6-trifluo-
romethyl and 6-nitrile containing compounds were essentially
inactive (3d, 3h and 3i IC50 >50 M), suggesting that the potency
lM
l
6-I
l
6-CF3
6-CN
5-Br
8-Br
6-Br, 8-Br
6-Br, 8-Me
—
—
of 3e, 3f and 3g is not related purely to their electron withdrawing
nature. The corresponding 5- and 8-bromine substituted analogues
9
>50
>50
10
11
12
13
MSO
PPT
displayed considerably weaker inhibitory activity (3j, IC50 >50
lM
21.3 1.6
>50
12.7 0.7
51 6c
1.9 0.4c
and 3k, IC50 = 21.3 1.6 M) compared to 3f, indicating that there
l
may be some specific hydrophobic or van der Waals interactions
accessible only by a substituent in the 6-position. The introduction
of an additional 8-bromine or 8-methyl substituent did not im-
Purity >95% by HPLC or 1H NMR.
Values are means of three experiments standard error.
See Ref. 10.
a
prove compound potency (3l, IC50 >50
lM and 3m, IC50 = 12.7
b
c
0.7
lM, respectively).
Table 2
Synthesis and biological evaluation of 6-substituted 3-amino-imidazo[1,2-a]pyridines (3n–s)
NH
NH
R1
X
[Pd]
I,II or III
N
N
OMe
OMe
OH
N
N
OH
3n-s
b
Entry
1
Starting material (X)
Br–
Method
Producta
R1
IC50
>50
(lM)
I
I
3n
2
Br–
3o
>50
N
O
B
O
3
4
II
3p
3q
>50
>50
O
—
As per 3c
5
Br–
I
3r
3s
>50
>50
6
3r
III
a
Purity >95% by HPLC or 1H NMR.
b
Values are means of three experiments. I; Boronic acid (3 equiv), Pd(PPh3)4 (7%), Cs2CO3 (3.5 equiv), DMF, MW, 120 °C, 20 min. II; benzyl bromide (0.8 equiv), Pd(dppf)Cl2
(10%), K2CO3 (3 equiv), EtOH, H2O, MW, 130 °C, 30 min. III; 3r 10% Pd/C (10%), H2 (1 atm.), DMF, 25 °C, 18 h.

4792
L. R. Odell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4790–4793
To further explore the effect of large hydrophobic groups in the
(4m) compounds were twofold less potent than 4e (IC50 = 10.1
1.1 M and 13.2 1.5 M vs 3.3 0.6 M, respectively) demon-
strating a preference for a phenol group in the 30-position. Rewar-
dingly, the introduction of carboxylic acid rendered the
nanomolar potent inhibitor, 4n (IC50 = 0.38 0.02 M).
In summary, three small series of trisubstituted 3-amino-imi-
dazo[1,2-a]pyridines have been investigated as MtGS inhibitors.
The compounds represent the first non-amino acid derived inhibi-
tors of this enzyme. The most effective compounds possessed low
6-position, we decided to prepare a series of compounds where the
halogen was exchanged for various aryl moieties. The target com-
pounds were designed to explore the available chemical space and,
in the case of 3o increase solubility and H-bond potential. These
molecules were smoothly prepared via microwave-assisted Suzuki
cross-coupling reactions23–25 utilizing either aryl bromide 3f or the
corresponding 6-boronic acid pinacol ester derivative, which was
prepared from 2-aminopyridine-5-boronic acid pinacol ester
according to Table 1. Compound 3q was synthesized as per 3c,
however MeOH was replaced by phenol in the Ullman reaction.
In addition, treatment of 3r under catalytic hydrogenation condi-
tions (Pd/C, H2) afforded the saturated phenylethylene derivative
3s. These compounds were then assessed for their ability to inhibit
MtGS and the results are reported in Table 2.
l
l
l
a
l
micromolar (3f, IC50 = 8.8 0.4
IC50 = 3.3 0.6 M) or nanomolar potency (4n IC50 = 0.38
0.02 M). Compound 4n was significantly more active than both
MSO (IC50 = 51 M) and PPT (IC50 = 1.9 0.4 M) the most po-
lM, 3g, IC50 = 4.8 0.5 lM, 4e,
l
l
6
l
l
tent known MtGS inhibitors. Given their drug-like nature, we antic-
ipate they will serve as important lead compounds in the search for
new anti-tuberculosis agents. The chemistry established can easily
be used to smoothly produce additional inhibitors. Work is currently
underway within our laboratory utilizing these structures to expand
the SAR developed herein.
Unfortunately, the compounds in this series (3n–s) failed to
show any significant MtGS inhibitory activity, highlighting a lack
of tolerance towards the introduction of large aryl substituents in
the 6-position of the 3-amino-imidazo[1,2-a]pyridines.
Finally, utilizing compound 3f as the lead structure, we decided
to investigate the effect of altering the C-2 aryl substituent (R) on
MtGS inhibition. Thus, a series of compounds were synthesized
from cyclopentylisonitrile, 5-bromo-2-aminopyridine and an
appropriately substituted aldehyde as per Table 1. Compounds
4a–n were evaluated for their MtGS inhibitory activity and the re-
sults are presented in Table 3.
Table 3 shows that a C-2 phenyl ring with a hydrogen bond do-
nor in the 30-position (i.e., 4e, OH or 4l, NH2) is a clear requisite for
enzyme inhibition. Placement of the –OH in the 20- or 40-positions
(4f and 4b, respectively) results in complete loss in activity. Re-
moval of the hydrogen bond donor capacity through the introduc-
tion of a methoxy group rendered the inactive compound 4g.
Interestingly, 4e displays a twofold increase in activity compared
to 3f suggesting that the 40-methoxy group is not required for inhi-
bition. The introduction of electron withdrawing substituents (4i
and 4j) was also detrimental to activity. However, these substitu-
ents may also disrupt the orientation of the phenyl ring (4i) or
intramolecularly bind the 30-OH (4j) giving rise to the loss in po-
tency. The corresponding 30-aniline (4l) and 3-methylalcohol
Acknowledgments
Our tuberculosis-related work is supported by funding from the
Foundation for Strategic Research (SSF), the Swedish Research
Council (VR), the EU Sixth Framework Program NM4TB
CT:018923, Knut and Alice Wallenberg’s Foundation and Uppsala
University. We also thank Professor Sherry L. Mowbray, Dr. Wojci-
ech W. Krajewski and Dr. Francesco Russo for useful discussions
regarding this Letter.
References and notes
1. Harth, G.; Horwitz, M. A. Infect. Immun. 2003, 71, 456.
2. Global Tuberculosis Control: Surveillance, Planning, Financing. WHO Report
2009. Geneva, World Health Organization (WHO/HTM/TB/2009.411).
3. Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.
4. Duncan, K. Curr. Pharm. Des. 2004, 10, 3185.
5. Berlicki, L.; Obojska, A.; Forlani, G.; Kafarski, P. J. Med. Chem. 2005, 48, 6340.
6. Harth, G.; Horwitz, M. A. J. Exp. Med. 1999, 189, 1425.
7. Berlicki, L.; Kafarski, P. Bioorg. Med. Chem. 2006, 14, 4578.
8. Logusch, E. W.; Walker, D. M.; McDonald, J. F.; Franz, J. E. Biochemistry 1989, 28,
3043.
9. Forlani, G.; Obojska, A.; Berlicki, L.; Kafarski, P. J. Agric. Food Chem. 2006, 54,
796.
10. Nordqvist, A.; Nilsson, M. T.; Röttger, S.; Odell, L. R.; Krajewski, W. W.;
Andersson, E.-L.; Larhed, M.; Mowbray, S. L.; Karlén, A. Bioorg. Med. Chem. 2008,
16, 5501.
Table 3
Synthesis and biological evaluation of 3-amino-imidazo[1,2-a]pyridines (4a–n)
11. Lagerlund, O.; Odell, L. R.; Mowbray, S. L.; Nilsson, M. T.; Krajewski, W. W.;
Nordqvist, A.; Karlén, A.; Larhed, M. Comb. Chem. High Throughput Screening
2007, 10, 783.
12. Selvam, C.; Goudet, C.; Oueslati, N.; Pin, J. P.; Acher, F. C. J. Med. Chem. 2007, 50,
4656.
13. Griffith, O. W.; Horwitz, M.; Harth, G. WO045539, 2004.
14. DiMauro, E. F.; Kennedy, J. M. J. Org. Chem. 2007, 72, 1013.
15. Blackburn, C.; Guan, B.; Fleming, P.; Shiosaki, K.; Tsai, S. Tetrahedron Lett. 1998,
39, 3635.
NH
Br
Br
MgCl2
O
N
N
R
N
C
EtOH, 160 °C
MW, 20 or 30 min
R
H
N
NH2
4a-n
Entry
Producta
R
IC50
(lM)
b
16. Blackburn, C. Tetrahedron Lett. 1998, 39, 5469.
17. Groebke, K.; Weber, L.; Mehlin, F. Synlett 1998, 661.
18. Blackburn, C.; Guan, B. Tetrahedron Lett. 2000, 41, 1495.
19. Yimin Lu, W. Z. QSAR Comb. Sci. 2004, 23, 827.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
H
>50
>50
>50
ndc
3.3 0.6
>50
>50
>50
>50
>50
30-OMe,40-OHC6H3
C6H5
20. Wolter, M.; Nordmann, G.; Job, G. E.; Buchwald, S. L. Org. Lett. 2002, 4, 973.
21. Example synthesis: Compound 3f. To a microwave transparent vial (2–5 mL)
with a teflon coated stirring bar was added cyclopentylisonitrile (0.192 g,
2 mmol), 3-hydroxy-4-methoxybenzaldehyde (0.304 g, 2 mmol), 2-amino-5-
bromopyridine (0.346 g, 2 mmol), MgCl2 (0.019 g, 0.1 mmol) and EtOH (2 mL).
The vial was then sealed under air and heated at 160 °C by microwave
irradiation for 20 min using a fixed hold time. After cooling, the mixture was
diluted with ethyl acetate and brine (20 mL each) and the two layers separated.
The aqueous layer was washed twice with ethyl acetate (20 mL) and the
combined organic phases were concentrated in vacuo. The crude product was
thereafter purified by recrystallization from ethyl acetate. Yield: 0.530 g, 66%;
1H NMR (400 MHz, CDCl3): d 1.39–1.48 (m, 2H), 1.53–1.58 (m, 2H), 1.67–1.77
(m, 4H), 3.05 (d, J = 4.4 Hz, 1H), 3.61–3.66 (m, 1H), 3.92 (s, 3H), 6.02 (br s, 1H),
6.93 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 2.0, 9.2 Hz, 1H), 7.40 (d, J = 9.2 Hz, 1H), 7.50
(dd, J = 2.0, 8.4 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 8.22 (d, J = 2.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3): d 23.8, 33.7, 56.2, 59.3, 106.6, 111.0, 113.8, 118.2, 119.6,
40-OHC6H4
30-OHC6H4
20-OHC6H4
30-OMeC6H4
20,30-DiOMeC6H3
20-Cl,30-OHC6H3
30-OH,40-NO2C6H3
30-NO2C6H4
>50
30-NH2C6H4
30-(CH2OH)C6H4
30-(COOH)C6H4
10.1 1.1
13.2 1.5
0.38 0.02
a
Purity >95% by HPLC or 1H NMR.
Values are means of three experiments standard error.
IC50 could not be determined due to poor solubility.
b
c

L. R. Odell et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4790–4793
4793
122.9, 125.5, 127.3, 127.7, 137.9, 140.0, 146.0, 146.6. LC–ESI-MS: m/z 402
(M+1). Calcd for C19H20BrN3O2: C, 56.73; H, 5.01; N, 10.45. Found: C, 56.44; H,
5.01; N, 10.27.
under air and heated at 120 °C by microwave irradiation for 30 min using a
fixed hold time. After cooling, the mixture was diluted with ethyl acetate and
brine (20 mL each) and the two layers separated. The aqueous layer was
washed twice with ethyl acetate (20 mL) and the combined organic phases
were concentrated in vacuo. The crude product was thereafter purified by flash
chromatography eluting with ethyl acetate/hexane (3:2). Yield: 0.064 g, 65%;
1H NMR (400 MHz, DMSO-d6) d 1.45–1.50 (m, 4H), 1.58–1.62 (m, 2H), 1.70–
1.74 (m, 2H), 3.55–3.58 (m, 1H), 3.80 (s, 3H), 4.26 (d, J = 4.6 Hz, 1H), 6.85 (d,
J = 8.5 Hz, 1H), 7.04 (d, J = 16.4 Hz, 1H), 7.18 (d, J = 16.4 Hz, 1H), 7.19–7.22 (m,
1H), 7.28–7.37 (m, 3H), 7.45 (dd, J = 1.7, 9.5 Hz, 1H) 7.51 (d, J = 7.5, 2H) 7.55
(dd, J = 2.2, 8.2 Hz, 1H), 7.65 (d, J = 2.4 Hz, 1H), 8.25 (br s, 1H), 8.69 (s, 1H). 13C
NMR (100 MHz, CDCl3): d 24.0, 33.5, 56.2, 59.0, 112.7, 114.9, 117.0, 118.6,
121.5, 122.3, 122.3, 125.7, 126.3, 126.8, 128.0, 128.2, 128.4, 129.1, 136.5, 137.5,
140.5, 146.7, 147.4. LC–ESI-MS: m/z 426 (M+1) Calcd for C27H27N3O2: C, 76.21;
H, 6.40; N, 9.87. Found: C, 76.02; H, 6.39; N, 9.79.
22. The assay was performed essentially as previously described.10 However,
25 mM of MgCl2, 1 mM of ATP and 30 mM of monosodium
used and stock solutions of the compounds (10 mM) were prepared in DMSO. A
concentration of 50 M was chosen as the upper limit for IC50 determinations
L-glutamate were
l
due to solubility problems encountered with some compounds at higher
concentrations.
23. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
24. (a) Alonso, F.; Beletskaya, I. P.; Yus, M. Tetrahedron 2008, 64, 3047; (b) Larhed,
M.; Moberg, C.; Hallberg, A. Acc. Chem. Res. 2002, 35, 717.
25. Example synthesis: Compound 3r. To a microwave transparent vial (2–5 mL)
with a teflon coated stirring bar was added 3f (0.080 g, 0.2 mmol), trans-2-
phenylvinylboronic acid (0.088 g, 0.6 mmol), Cs2CO3 (0.234 g, 0.7 mmol),
Pd(PPh3)4 (0.016 g, 0.013 mmol) and DMF (2 mL). The vial was then sealed