Synthesis and in vitro activity of novel 1,2,4-triazolo[4,3-a]pyrimidine oxazolidinone antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2011.03.075

The synthesis and antibacterial activity of 3-(4-([1,2,4]triazolo[4,3-a] pyrimidin-3-yl)phenyl)oxazolidin-2-ones is reported. Thiocarbonyl derivatives were found to be potent inhibitors of Gram-positive pathogens and compound 4l was two to fourfold more p

Full text of DOI:10.1016/j.bmcl.2011.03.075

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2887–2889
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro activity of novel 1,2,4-triazolo[4,3-a]pyrimidine
oxazolidinone antibacterial agents
Manoj K. Khera a, , Ian A. Cliffe a, Tarun Mathur b, Om Prakash c
⇑
a Department of Medicinal Chemistry, New Drug Discovery Research, Ranbaxy Laboratories Limited, R&D III, Plot No. 20, Sector-18, Udyog Vihar Industrial Area,
Gurgaon 122 001, India
b Department of Microbiology, New Drug Discovery Research, Ranbaxy Laboratories Limited, R&D III, Plot No. 20, Sector-18, Udyog Vihar Industrial Area, Gurgaon 122 001, India
c Department of Chemistry, Kurukshetra University, Kurukshetra 136 119, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and antibacterial activity of 3-(4-([1,2,4]triazolo[4,3-a]pyrimidin-3-yl)phenyl)oxazolidin-
2-ones is reported. Thiocarbonyl derivatives were found to be potent inhibitors of Gram-positive patho-
gens and compound 4l was two to fourfold more potent than Linezolid.
Received 21 January 2011
Revised 21 March 2011
Accepted 22 March 2011
Available online 29 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Oxazolidinones
Antibacterial
The emergence of bacterial resistance to antibiotics has posed
an increasingly serious concern for medical professionals over
the last two decades1 and multi-drug-resistant Gram-positive bac-
teria,2 including methicillin-resistant Staphylococcus aureus
(MRSA),3 Staphylococcus epidermidis and vancomycin-resistant
Enterococci (VRE), have now become a major problem.4 Oxazolid-
inones are a new class of totally synthetic antibacterial agents
which are active against Gram-positive bacteria and anaerobic bac-
teria.5,6 Linezolid 1 and Eperezolid 2 (Fig. 1) were the initial lead
compounds from the oxazolidinone class and Linezolid 1, devel-
oped by Pharmacia and Upjohn, was the first compound to be ap-
proved in 2000.7 Oxazolidinones inhibit protein synthesis prior to
the chain initiation step by binding to the 23S rRNA of the 50S ribo-
somal subunit, and thereby interfere with the initiator fMet-tRNA
binding to the P-site of the ribosomal peptidyltransferase centre.8,9
This novel mechanism of action combined with potent antibac-
terial activity against resistant Gram-positive target pathogens
stimulated the pharmaceutical industry to further explore the
chemistry of the oxazolidinone class of compounds.
In the present investigation, we disclose a series of 5-amino-
methyl-3-(4-([1,2,4]triazolo[4,3-a]pyrimidin-3-yl)phenyl)oxazoli-
din-2-ones4a–vasantibacterialagentsinwhichthe5-aminomethyl
substituent is attached to a range of functional groups.
The synthesis of compounds 4a–v (Scheme 1) was initiated by
the nucleophilic displacement of 2-chloropyrimidine 5 with hydra-
zine.12 The 2-hydrazinopyrimidine 6 was treated with 4-nitro-
benzaldehyde to obtain the Schiff base 7, which was cyclized
using iodobenzene diacetate to obtain the triazolopyrimidine
derivative 8.13 At this stage, the nitro containing compound 8
was converted to the corresponding aniline 9 using stannous
chloride and further converted to the carbamate derivative 10.
O
F
O
F
HO
O
O
O
N
N
N
H
N
N
H
N
N
O
O
O
1
2
1,2,4-Triazolo[4,3-a]pyrimidines have been reported by our
group as potent antibacterial agents,10,11 and compound 3 was also
found to be active against Gram-negative strains (E. coli). This
encouraged us to evaluate the impact on antibacterial activity of
this heterocycle when coupled to the oxazolidinone heterocycle
of Linezolid (Fig. 2).
Figure 1. Structures of Linezolid 1 and Eperezolid 2.
N
N
O
N
N
N
N
N
O
N
N
N
HN
R
3
4a-v
⇑
Corresponding author. Tel.: +91 124 2848718; fax: +91 124 2397546.
E-mail address: manoj.khera.c4@dsin.co.in (M.K. Khera).
Figure 2. Modifications in the series.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.075

2888
M. K. Khera et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2887–2889
NO2
NH2
HN
N
Cl
N
N
N
a
HN
c
d
b
N
N
N
N
N
N
NO2
N
5
6
7
8
N
N
N
O
O
N
N
N
e
O
f
N
O
N
N
N
N
N
N
N
NH2
H
OH
11
9
10
N
O
N
N
N
O
N
O
N
j
g, h
i
N
N
O
N
N
N
O
N
N
O
N
N
H
N
R3
X
NH2
4a-k
X: 12 = OMs
X: 13 = N3
14
O
k
m
l
N
O
N
N
O
N
N
O
N
N
N
O
N
N
N
O
N
N
N
O
H
N
H
N
H
N
H
N
N
O
R1
R3
R2
O
X
S
4l-m
4n-p
X=O, 4q-s
X=S, 4t-v
Scheme 1. Reagents and conditions: (a) N2H4ÁH2O, pyridine/DCM (1:1), 0 °C–rt; (b) 4-nitrobenzaldehyde, ethanol, acetic acid (catalytic), reflux; (c) PhI(OAc)2, DCM, rt; (d)
SnCl2ÁH2O, EtOAc, reflux; (e) benzyl chloroformate, aqueous NaHCO3, acetone, 0 °C; (f) n-BuLi, (R)-glycidyl butyrate, THF, À78 °C–rt; (g) MsCl, Et3N, DCM/DMF (1:1), 0 °C–rt;
(h) NaN3, DMF, 70 °C; (i) Ph3P, THF, H2O, rt–50 °C; (j) R3COCl, Et3N, DCM; (k) R1OCOCl, Et3N, DCM; (l) R2NCO or R2NCS, Et3N, THF; (m) Lawesson’s reagent, dioxane, reflux.
Table 1
In vitro MIC values (
lg/ml) of compounds with variation in the acetamide portion
N
N
O
N
N
O
N
H
N
R
4a-v
Compound
R
E. faecalis ATCC 29212
S. aureus MRSA 43300
S. aureus ATCC 25923
S. epidermedis ATCC 12228
E. coli ATCC 25922
4a
4b
4c
4d
4e
4f
–COCH3
16
16
16
16
16
16
16
16
4
16
16
8
>16
>16
>16
>16
>16
>16
>16
>16
–COCH2CH3
–CO(CH2)2CH3
–CO(CH2)3CH3
–COCH2CH(CH3)2
–COCH(CH3)2
–COCH2Cl
16
16
16
16
>16
4
>16
>16
>16
>16
16
>16
>16
>16
>16
4
4g
4h
–COCHCl2
8
8
4
4
O
4i
16
8
16
8
>16
O
4j
>16
16
>16
16
>16
4k
4l
–COPh
–CSCH3
–CSCH2CH3
–COOCH3
–COOCH2CH3
–COOPh
–CONHCH(CH3)2
–CONHC(CH3)3
–CONH(CH2)3CH3
–CSNHCH(CH3)2
–CSNH(CH2)3CH3
–CSNH(CH2)2OCH3
Linezolid
>16
1
4
>16
0.5
2
>16
1
4
16
16
>16
>16
>16
>16
16
16
>16
2
>16
0.5
2
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
4m
4n
4o
4p
4q
4r
4s
4t
>16
>16
>16
>16
>16
>16
>16
16
16
16
>16
>16
>16
>16
16
8
16
>16
>16
>16
>16
>16
16
8
4u
4v
1
>16
2
16
2
8
1

M. K. Khera et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2887–2889
2889
Table 2
CYP profile and metabolic stability of 4l
Compound
% CYP inhibition (at 10
lM)
Metabolic stability
ml/min/g liver
HLM
1A2
0
2C9
15
2C19
30
2D6
13
3A4
28
4l
0.3
derivatives (4l and 4m) were found to have values closer to that
of Linezolid.
In summary, we have described initial results from a study of 3-
(4-([1,2,4]triazolo[4,3-a]pyrimidin-3-yl)phenyl)oxazolidin-2-ones.
The thioamide derivatives 4l and 4m were found to display a good
antibacterial activity profile against all the Gram-positive patho-
gens tested. Compound 4l was also found to be metabolically sta-
ble and devoid of any CYP liability up to a concentration of 10 lM.
Further modifications in this series of compounds including the
impact of additional fluorine atom substitution in the phenyl ring
and substitutions on the triazolopyrimidine ring will be the subject
of further communications.
Figure 3. MIC values of 4l and Linezolid against Gram-positive strains.
Deprotonation followed by reaction with (R)-glycidyl butyrate
yielded the oxazolidinone alcohol 11. This was converted to the
corresponding azide 13 by a series of nucleophilic reactions. The
azide derivative was converted to the corresponding amine 14
with triphenyl phosphine in aqueous THF with heating.14 It should
be noted that hydride-containing reagents (e.g. LAH) or hydrogena-
tion methods were not suitable for this transformation owing to
the reactivity of the triazolopyrimidine ring. The amine derivative
14 was converted to the corresponding amides 4a–k, carbamates
4n–p, ureas 4q–s and thioureas 4t–v using standard procedures.
The amides 4a–b were also converted to the corresponding thio-
amides 4l–m using Lawesson’s reagent.15
Acknowledgements
We thank the analytical chemistry department of New Drug
Discovery Research, Ranbaxy Research Laboratories for support.
We also thank the metabolism and pharmacokinetics department
for ADME related support.
References and notes
1. Service, R. F. Science 1995, 270, 724.
2. Swartz, M. N. Proc. Natl. Sci. U.S.A. 1994, 91, 2420.
Final compounds 4a–v were screened16 against target patho-
gens including both Gram-positive (E. faecalis ATCC 29212, S. aur-
eus MRSA 43300, S. aureus ATCC 25923 S. epidermidis ATCC
12228) and Gram-negative (E. Coli ATCC 25922) strains (Table 1).
Compounds showed varying degrees of activity against Gram-posi-
tive pathogens; however, all the compounds were found to be inac-
tive against E. coli. A weak enhancement of activity was observed
among the alkyl amide derivatives 4a–k when the alkyl group
was relatively small in size (compare acetamide 4a and cyclopro-
pyl amide 4i with the larger alkylamides 4b–f, 4j–k). The surpris-
ingly moderate to good activity of relatively large chloroacetamide
derivatives 4g–h may arise from electronic effects such as
enhancement of double bond character within the carbonyl group
of the amide. Such enhancement of electron density in this region
would be expected to occur when the carbonyl oxygen atom is re-
placed by sulphur and it was gratifying to see that the thioamide
derivatives 4l–m displayed excellent potency against all Gram-
positive target pathogens tested. Indeed, thioacetamide 4l was
found to be two- to four- fold more potent than Linezolid. As ex-
pected, the sterically demanding and less electron rich carbamate,
urea and thiourea systems 4n–v had moderate to low activity
against Gram-positive pathogens. In order to establish the spec-
trum of activity, the most potent compound 4l was tested against
other Gram-positive strains including Streptococcus pneumoniae
and Streptococcus pyogenes ( Fig. 3). Compound 4l, being active
against these strains, indicates that it could be useful in treating
upper respiratory tract bacterial infections. This compound was
also profiled for its CYP liability17 and metabolic stability18 and
found to be stable in human liver microsome and to have no
3. Tomasz, A. N. Eng. J. Med. 1994, 330, 1247.
4. Selvekumar, N.; Srinivas, D.; Khera, M. K.; Kumar, M. S.; Mamidi, N. V. S. R.;
Sarnaik, H.; Chandershekhar, C.; Rao, B. S.; Raheem, M. A.; Das, J.; Iqbal, J.;
Rajagopalan, R. J. Med. Chem. 2002, 45, 3953.
5. Brickner, S. J. Curr. Pharm. Des. 1996, 2, 175.
6. Phillips, O. A. Curr. Opin. Invest. Drugs 2003, 4, 117.
7. Brickner, S. J.; Hutchinson, D. K.; Barbachyn, M. R.; Manninen, P. R.; Ulanowicz,
D. A.; Garmon, S. A.; Grega, K. C.; Henges, S. K.; Toops, D. S.; Ford, C. W.;
Zurenko, G. E. J. Med. Chem. 1996, 39, 673.
8. Swaney, S. M.; Aoki, H.; Ganoza, M. C.; Shinabarger, D. L. Antimicrob. Agents
Chemother. 1998, 42, 3251.
9. Aoki, H.; Ke, L.; Poppe, S. M.; Poel, T. J.; Weaver, E. A.; Gadwood, R. C.; Thomas,
R. C.; Shinabarger, D. L.; Ganoza, M. C. Antimicrob. Agents Chemother. 2002, 46,
1080.
10. Prakash, O.; Bhardwaj, V.; Kumar, R.; Tyagi, P.; Aneja, K. R. Eur. J. Med. Chem.
2004, 39, 1073.
11. Kumar, R.; Nair, R. R.; Dhiman, S. S.; Sharma, J.; Prakash, O. Eur. J. Med. Chem.
2009, 44, 2260.
12. Srinivasan, V.; Jebaratnam, D. J.; Budil, D. E. J. Org. Chem. 1999, 64, 5644.
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14. Vaultier, A.; Knouzi, N.; Carrie, R. Tetrahedron Lett. 1983, 24, 763.
15. Cava, M. P.; Levinson, M. I. Tetrahedron 1985, 41, 5061.
16. Minimum inhibitory concentrations (MICs) were determined as per the CLSI
guidelines 2009, 29(2), M07-A8.
17. CYP inhibition was evaluated in vitro using a standard commercial kit (BD-
Gentest) comprising recombinant CYPs, fluorogenic substrates, standard
inhibitors, buffers and stop reagents. The recombinant CYP metabolizes the
(non-fluorescent) fluorogenic substrate into
a fluorescent product, whose
fluorescence is measured in a fluorescent plate reader. The concentration-
dependent ability of compounds to reduce this fluorescence for each individual
CYP is measured and reported as CYP inhibition.
18. Compound 4l (10 lM concentration) was incubated with liver microsomes
(1 mg/mL), obtained from BD Gentest, at 37 °C in phosphate buffer (pH 7.4)
with UDPGA and an NADPH regenerating system. Samples were withdrawn at
specific time points up to 30 min. Reaction was immediately quenched and the
parent compound quantified by LCMS. The data was analyzed using GRAPHPAD
PRISM software.
19. Apparent Caco-2 cell permeability was calculated using an in-silico ADME
prediction tool (Qik Prop from Schrodinger molecular modeling suite). The
predicted permeability values for compounds 4a, 4b, 4l, 4m and Linezolid were
found to be 55, 81, 277, 311 and 520 nm/s, respectively.
CYP-related liabilities up to a concentration of 10 lM (Table 2).
Interestingly, the predicted permeability19 of amide derivatives
(4a and 4b) was low; however, the corresponding thioamide