Synthesis and antimycobacterial activity of pyrazine and quinoxaline derivatives

Article abstract of DOI:10.1021/jm020310n

A series of pyrazine and quinoxaline derivatives have been synthesized, and their activity against M. tuberculosis (Mtb) and Mycobacterium avium (MAC) are reported. The 4-acetoxy-benzyl ester of pyrazinoic acid and 4′-acetoxybenzyl 2-quinoxalinecarboxylate showed excellent activity against Mtb (MIC ranges of less than 1-6.25 μg/mL) but only modest activity against MAC (MICs of 4-32 μg/mL).

Full text of DOI:10.1021/jm020310n

5
604
J . Med. Chem. 2002, 45, 5604-5606
Brief Articles
Syn th esis a n d An tim ycoba cter ia l Activity of P yr a zin e a n d Qu in oxa lin e
Der iva tives
†
‡
,†
Lainne E. Seitz, William J . Suling, and Robert C. Reynolds*
Organic Chemistry Department and Biochemistry Department, Southern Research Institute, P.O. Box 55305,
Birmingham, Alabama 35255-5305
Received J uly 17, 2002
A series of pyrazine and quinoxaline derivatives have been synthesized, and their activity
against M. tuberculosis (Mtb) and Mycobacterium avium (MAC) are reported. The 4-acetoxy-
benzyl ester of pyrazinoic acid and 4′-acetoxybenzyl 2-quinoxalinecarboxylate showed excellent
activity against Mtb (MIC ranges of less than 1-6.25 µg/mL) but only modest activity against
MAC (MICs of 4-32 µg/mL).
In tr od u ction
Sch em e 1. Preparation of POA Analogues
The worldwide appearance of strains of Mtb that are
resistant to many if not all of the clinically used
antitubercular agents has led to a call for new ap-
1
proaches to treatment of tuberculosis (Tb). In addition,
alteration of clinically used agents that might improve
their initial activity or modify the susceptibility profile
of clinically resistant disease is also a viable approach
to developing more potent antitubercualr drugs. Re-
esterases, thus allowing production of POA even in
clinically resistant forms of Mtb that are not sensitive
to PZA through loss of the amidase activity. In fact,
several POA esters show significant activity against
PZA-susceptible and -resistant Mtb strains as well as
several intrinsically PZA-resistant mycobacteria such
as M. bovis, M. kansasii, and M. avium (MAC).2
2
,3
searchers have reported pyrazinamide (PZA),
niazid, and ethambutol analogues with significant
activity against Tb.
iso-
4
,5
6
PZA is one of the front-line agents prescribed for the
treatment of Mtb. The initial treatment regimen in-
cludes the use of four drugs: PZA, isoniazid, rifampin,
and either ethambutol or streptomycin. Demonstration
of PZA activity against Mtb in vitro requires an acidic
,3
Herein, several pyrazinoic acid ester derivatives are
reported that were designed as potential prodrugs of
1
2
POA through a “self-immolative” process. A “self-
immolative” prodrug is defined as generating an un-
stable intermediate that, following the activation pro-
cess, will extrude the active drug in a number of
7
,8
environment (pH e 5.6). Although PZA has been used
clinically since the 1950s, a proposed mechanism of
action has only recently been reported to be inhibition
of the eukaryotic-like fatty acid synthetase I (FASI) of
1
3
subsequent steps. For example, enzymatic deacylation
of the 4-acetoxy group (3, 8, 10, 17) generates a hydroxy
group. Bioreduction (1, 2, 6, 7, 9, 15, 16) converts the
nitro group into an amino group. Upon activation,
additional electron density in the aromatic π system
would lead to the extrusion of a substituted quinone
methide and POA. Herein, several ester derivatives of
POA, nicotinic acid, 2-quinoxalinecarboxylic acid (QOA),
benzoic acid, and acetic acid were synthesized and
screened for activity against Mtb and MAC in order to
test this approach for inhibitor design.
9
Mtb. The results of a more recent study, however,
suggest that neither PZA nor pyrazinoic acid (POA)
directly inhibit FASI.10 PZA is considered to be a
prodrug of POA, which is believed to be the active
2
inhibitor of Mtb. Activation of PZA to POA is regulated
by a pyrazinamidase present in all PZA-sensitive strains
of Mtb. PZA-resistant strains of Mtb and intrinsically
resistant strains of M. bovis are defective in this
amidase activity because of mutations in the associated
gene and are therefore unaffected by PZA.11
In an effort to circumvent this mechanism of clinical
resistance while maintaining the activity of this class
of agent, pyrazinoic acid ester derivatives have been
prepared and screened in vitro.2,3 It was reasoned that
the ester forms of POA might be activated by bacterial
Ch em istr y
The POA esters (1-10) were synthesized according
to reported procedures (Scheme 1). Esterification of
14
2
-pyrazinecarboxylic acid in the presence of DCC/DMAP
and the appropriate alcohol gave the desired product
*
To whom correspondence should be addressed. Phone: 205-581-
upon purification. The QOA ester derivatives (11-17)
2
454. Fax: 205-581-2877. E-mail: reynolds@sri.org.
†
were prepared by a method adapted from Cynamon et
Organic Chemistry Department.
Biochemistry Department.
‡
al. for the preparation of POA esters (Scheme 2).2
1
0.1021/jm020310n CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/07/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5605
Ta ble 2. Activity of Quinoxaline Derivativesa
Sch em e 2. Preparation of QOA Analogues
MIC (µg/mL)
Mtb
Mtb
MAC
compd
R2
H37Rv H37Ra NJ 211 MlogP
1
12
1
methoxy
n-propoxy
n-octyloxy
2-octyloxy
4-nitrobenzyloxy
2-nitrobenzyloxy
4-acetoxybenzyloxy
NH2
>6.25 >128
>128
128
128
0.84
1.41
2.61
2.70
2.27
2.27
2.15
0.13
0.16
ND
Ta ble 1. Activity of Pyrazine and Acetic Acid Estersa
MIC (µg/mL)
6.25
>6.25
>6.25
64
128
64
1
1
1
1
1
1
1
3
4
5
6
7
8
9
128
Mtb
Mtb
MAC
>6.25 >128
>128
>128
16
>128
>128
>128
compd
X
Y
R
1
H37Rv H37Ra NJ 211 MlogP
>6.25 >128
6.25
0.5
>6.25 >128
HNNH2
>6.25
ND
64
>128
QOA OH
a
Mtb H37Rv (TAACF using BACTEC 12B broth), Mtb H37Ra,
and MAC were screened using Middlebrook 7H9 with glycerol and
OADC enrichment, pH 6.6.
1
2
3
4
5
6
7
8
N
N
2-nitrobenzyloxy
4-nitrobenzyloxy
4-acetoxybenzyloxy
benzyloxy
>6.25
6.25
6.25
>6.25
>6.25
>6.25
>6.25
64
128
<0.25
16
128
128
ND
16
>128
>128
16
>128
>128
>128
ND
0.99
0.99
0.92
0.94
1.87
2.29
3.45
3.38
N
N
N
N
N
N
N
N
2
reported by Cynamon et al. for a series of POA ester
4-CF
3
-benzyloxy
derivatives. Also, medium with OADC at pH 6.6 lowered
the MIC for 3 and 17, but not for 2 and PZA, by 4- and
CH N 4-nitrobenzyloxy
CH CH 4-nitrobenzyloxy
CH CH 4-acetoxybenzyloxy >6.25
N
128
>128 ND
8
-fold, respectively, compared to medium with ADC
POA
N
OH
ND
64
(data not shown). Ethambutol activity was not affected
by the type of enrichment. The MIC of ethambutol for
Mtb H37Ra and MAC was in the range 2-8 µg/mL. The
presence of OADC or ADC in the medium did not alter
the MICs of the derivatives for MAC (data not shown).
Tables 1 and 2 present the MICs for Mtb H37Ra and
MAC determined at pH 6.6 and with OADC enrichment
and for Mtb H37Rv assayed in BACTEC 12B medium.
Several derivatives (2, 3, 12, 17) appeared to have good
activity (MICs, 6.25 µg/mL) against Mtb H37Rv. Two
derivatives, the 4-acetoxybenzyl esters of POA (3) and
9
1
4-nitrobenzyl
4-acetoxybenzyl
>6.25 >128
ND
>128
2.02
2.03
0
8
128
a
Mtb H37Rv (TAACF using BACTEC 12B broth), Mtb H37Ra,
and MAC were screened using Middlebrook 7H9 with glycerol and
OADC enrichment, pH 6.6.
Nucleophilic displacement of 2-quinoxalinecarbonyl chlo-
ride by the appropriate alcohol afforded the desired
ester. Ammonolysis of 2-methoxycarbonylquinoxaline
2
-quinoxaline carboxylate (17), exhibited excellent ac-
tivity against Mtb H37Ra with MICs of 0.25-0.5 µg/mL.
Derivatives 3 and 17 also had moderate activity (MIC,
(
11) by standard methods15 using 7 N methanolic
ammonia produced2-quinoxalinecarboxamide(18)(Scheme
). 2-Quinoxalinecarbohydrazide (19) was also prepared
1
6 µg/mL) against MAC NJ 211. The MIC for two
2
additional strains of MAC, NJ 168 and NJ 3404, was 32
µg/mL for 3, and for 17, the MIC was 4 and 8 µg/mL,
respectively. Another derivative, 4-acetoxybenzyl ac-
etate (10), had good activity against Mtb H37Ra but not
against MAC (Table 1). None of the starting materials
(4-acetoxybenzyl alcohol, 2-nitrobenzyl alcohol, 4-ni-
trobenzyl alcohol) were active at 12.8 µg/mL, the highest
amount tested (data not shown).
from methyl 2-quinoxalinecarboxylate (11) as shown in
Scheme 2 through reaction with hydrazine.
Resu lts a n d Discu ssion
We synthesized several POA ester derivatives, one
nicotinic acid ester derivative, two benzoic acid ester
derivatives, and two acetic acid ester derivatives as
putative inhibitors of Mtb and MAC (Table 1). The
activities of three of the starting materials (4-acetoxy-
benzyl alcohol, 2-nitrobenzyl alcohol, and 4-nitrobenzyl
alcohol) were determined to rule out the possibility of
their participation in inhibition as ester hydrolysis
products. Several novel QOA ester derivatives were also
prepared and evaluated as PZA analogues (Table 2).
The derivatives were tested initially against Mtb H37-
Rv through the Tuberculosis Acquisition and Coordinat-
ing Facility (TAACF: NIH, NIAID Contract AI45246).
They were also assayed in our laboratory against Mtb
H37Ra and MAC. The results are presented in Tables 1
and 2. We also determined the effect of pH (6.6 vs 5.8)
and medium enrichment (ADC vs OADC; see Support-
ing Information) on the activity of derivatives 2, 3, 17,
and PZA against Mtb. The lower pH reduced the MIC
for the derivatives, as well as for PZA, similar to results
Derivatives 3 and 17 were the most active of the
esters tested, and it is noteworthy that both of these
derivatives contain the 4-acetoxybenzyl substituent
discussed previously as a potential “self-immolative”
prodrug. The promising in vitro activity of these two
compounds gives credibility to the “self-immolative”
mechanism. Although we cannot definitively state that
this mechanism is operative, our data are suggestive
that it may play a role in the activity of certain
analogues. The reason for making prodrug forms of POA
is to enhance the intracellular concentration of POA by
passive diffusion of an uncharged species that is then
hydrolyzed to yield the active drug. The relationship
between hydrophobicity and activity in antitubercular
1
6
drugs is well established. POA shows only modest
activity, but ester forms of the acid are known to
significantly enhance antibacterial activity likely through

5
606 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Brief Articles
increased uptake and bioactivation. Theoretically, if
passive diffusion and hydrolysis at the pyrazine (or
quinoxaline) carboxylic acid ester were the primary
mode of delivery and activation, presumably all of the
benzyl esters (1-5 and 11-17) would show similar
activity that positively correlates with the calculated
log P (i.e., the more hydrophobic esters should more
rapidly reach higher intracellular concentrations). Tables
(4) Maccari, R.; Ottana, R.; Monforte, F.; Vigorita, M. G. In vitro
Antimycobacterial Activities of 2′-Monosubstituted Isonicoti-
noylhydrazides and Their Cyanoborane Adducts. Antimicrob.
Agents Chemother. 2002, 46 (2), 294-299 and references therein.
(
5) Georgieva, N.; Gadjeva, V. Isonicotinoylhydrazone Analogues of
Isoniazid: Relationship between Superoxide Scavenging and
Tuberculostatic Activities. Biochemistry (Moscow) 2002, 67 (5),
588-591 and references therein.
6) Reynolds, R. C.; Bansal, N.; Rose, J .; Friedrich, J .; Suling, W.
J .; Maddry, J . A. Ethambutol-Sugar Hybrids as Potential
Inhibitors of Mycobacterial Cell-Wall Biosynthesis. Carbohydr.
Res. 1999, 317 (1-4), 164-179.
(7) McDermott, W.; Tompsett, R. Activation of Pyrazinamide and
Nicotinamide in Acidic Environments in Vitro. Am. Rev. Tuberc.
1954, 70, 748-754.
(
1
and 2 both give log P values for the synthetic com-
1
7
pounds as calculated by the method of Morgiuchi et al.
In fact, many of the analogues showed little or no
activity including the more hydrophobic compounds 5
and 13. The benzyl analogue 4 has virtually the same
MlogP value but is at least 50 times less active than
the acetoxybenzyl analogue 3. The in vitro activity of 4
(
8) Heifets, L. B.; Flory, M. A.; Lindholm-Levy, P. J . Does Pyrazinoic
Acid as an Active Moiety of Pyrazinamide Have Specific Activity
Against M. tuberculosis? Antimicrob. Agents Chemother. 1989,
33, 1252-1254.
(
9) Zimhony, O.; Cox, J . S.; Welch, J . T.; Vilcheze, C.; J acobs, W. R.
Pyrazinamide Inhibits the Eukaryotic-like Fatty Acid Synthase
I (FASI) of M. tuberculosis. Nat. Med. 2000, 6, 1043-1047.
3
for Mtb and MAC at pH 5.8 has been reported.
Interestingly, the 2-nitro and the 4-nitrobenzyl ana-
logues (1 and 2; 15 and 16) are much less active than
the acetoxybenzyl analogues (3 and 17), suggesting that
the nitro function does not appear to be bioreduced
under these conditions. This result may not be surpris-
ing considering the wealth of data regarding bioreducing
(10) Boshoff, H. I.; Mizrahi, V.; Barry, C. E. III Effects of Pyrazina-
mide on Fatty Acid Synthesis by Whole Mycobacterial Cells and
Purified Fatty Acid Synthetase I. J . Bacteriol. 2002, 8, 2167-
2172.
(
11) Scorpio, A.; Zhang, Y. Mutations in pncA, a Gene Encoding
Pyrazinamidase/Nicotinamidase, Cause Resistance to the An-
tituberculosis Drug Pyrazinamide in Tubercle bacillus. Nat. Med.
1996, 2, 662-667.
1
8
(12) Reynolds, R. C.; Tiwari, A.; Harwell, J . E.; Gordon, D. G.;
Garrett, B. D.; Gilbert, K. S.; Schmid, S. M.; Waud, W. R.; Struck,
R. S. Synthesis and Evaluation of Several New (2-chloroethyl)-
nitrosocarbamates as Potential Anticancer Agents. J . Med.
Chem. 2000, 43, 1484-1488.
groups as chemotherapeutic prodrugs. However, under
1
9
conditions of hypoxia as in encased tuberculous le-
sions, bioreduction may be more favorable.20 In vitro
2
1
assays under hypoxic conditions are now available,
and we are pursuing screening in these assays.
(
13) Niculescu-Duvaz, D.; Niculescu-Duvaz, I.; Friedlos, F.; Martin,
J .; Spooner, R.; Davies, L.; Marais, R.; Springer, C. J . Self-
Immolative Nitrogen Mustard Prodrugs for Suicide Gene Therapy.
J . Med. Chem. 1998, 41, 5297-5309.
Ack n ow led gm en t. This research was funded by the
National Institutes of Health (Grant AI45317 to R.C.R.,
Principal Investigator). We also thank the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility
(
(
(
14) Steglich, W.; Neises, B. Simple Method for the Esterification of
Carboxylic Acids. Angew. Chem., Int. Ed. Engl. 1978, 17, 522-
5
24.
15) Yosioka, I.; Otomasu, H. Studies on Phenazines. XV. The Ring
Cleavage of Phenazine. (1). 2,3-Quinoxalinedicarboxylic Acid.
Chem. Pharm. Bull. 1957, 5, 277-279.
16) Barry, C. E., III; Slayden, R. A.; Sampson, A. E.; Lee, R. E. Use
of Genomics and Combinatorial Chemistry in the Development
of New Antimycobacterial Drugs. Biochem. Pharmacol. 2000,
59, 221-31.
17) Moriguchi, I.; Hirono, S.; Liu, Q.; Nakagome, I.; Hirano, H.
Comparison of Reliability of logP Values for Drugs Calculated
by Several Methods. Chem. Pharm. Bull. 1994, 42, 976-978 and
references therein.
(TAACF) through a research and development contract
with the U.S. National Institute of Allergy and Infec-
tious Diseases. The M. avium strains were provided by
L. Heifets, National J ewish Center for Immunology and
Respiratory Diseases, Denver, Colorado.
(
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the syntheses of compounds 1-19, analytical data,
and procedures for the biological assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
(
18) Denny, W. A. The Role of Hypoxia-Activated Prodrugs in Cancer
Therapy. Lancet Oncol. 2000, 1 (1), 25-29.
(
19) Rosenkrands, I.; Slayden, R. A.; Crawford, J .; Aagaard, C.; Barry,
C. E. III; Andersen, P. Hypoxic Response of Mycobacterium
tuberculosis Studied by Labeling and Proteome Analysis of
Cellular and Extracellular Proteins. J . Bacteriol. 2002, 184,
Refer en ces
3
485-3491.
(
1) Duncan, K. The Impact of Genomics on the Search for Novel
(20) (a) Zhang, Z.; Hillas, P. J .; Ortiz de Montellano, P. R. Reduction
of Peroxides and Dinitrobenzenes by Mycobacterium tuberculosis
Thioredoxin and Thioredoxin Reductase. Arch. Biochem. Bio-
phys. 1999, 363, 19-26. (b) Murugasu-Oei, B.; Dick, T. Bacte-
ricidal Activity of Nitrofurans against Growing and Dormant
Mycobacterium bovis BCG. J . Antimicrob. Chemother. 2000, 46,
917-919.
Tuberculosis Drugs. Novartis Found. Symp. 1998, 217, 228-
2
37.
(
2) Cynamon, M. H.; Klemens, S. P.; Chou, T. S.; Gimi, R. H.; Welch,
J . T. Antimycobacterial Activity of a Series of Pyrazinoic Acid
Esters. J . Med. Chem. 1992, 35, 1212-1215 and references
therein.
(
3) Cynamon, M. H.; Gimi, R.; Gyenes, F.; Sharpe, C. A.; Bergmann,
K. E.; Han, H.-J .; Gregor, L. B.; Rapolu, R.; Luciano, G.; Welch,
J . T. Pyrazinoic Acid Esters with Broad Spectrum in Vitro
Antimycobacterial Activity. J . Med. Chem. 1995, 38, 3902-3907
and references therein.
(21) Flynn, J . L.; Chan, J . Minireview. Tuberculosis: Latency and
Reactivation. Infect. Immun. 2001, 69 (7), 4195-4201 and
references therein.
J M020310N