Antimycobacterial activity of substituted isosteres of pyridine- and pyrazinecarboxylic acids. 2

Article abstract of DOI:10.1021/jm000350w

Pyridines and pyrazines substituted with 1,2,4-oxadiazole-5-ones, 1,2,4-oxadiazole-5-thiones, and 1,3,4-oxathiazoline-2-ones were synthesized and tested against Mycobacterium tuberculosis. The two former ring systems were documented in the literature to act as carboxylic acid isosteres. The latter series was synthesized as possible synthetic intermediates to 1,2,4-thiadiazole-3-ones and was included in this study due to their interesting activity. Pivaloyl-oxymethyl derivatives of the isosteres were also prepared in order to increase their lipophilicity and therefore improve their cellular permeability. The derivatized isosteres were expected to be biotransformed by esterases to the active species after penetration of the mycobacterial cell wall. Biological properties of the compounds were compared with the unmodified polar isosteres of pyrazinoic and nicotinic acids. The majority of the compounds exhibited activities ranging from 0.5 to 16 times the potency of pyrazinamide.

Full text of DOI:10.1021/jm000350w

1560
J . Med. Chem. 2001, 44, 1560-1563
An tim ycoba cter ia l Activity of Su bstitu ted Isoster es of P yr id in e- a n d
P yr a zin eca r boxylic Acid s. 2.1
Mikail H. Gezginci, Arnold R. Martin,* and Scott G. Franzblau
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, and
GWL Hansen’s Disease Center, Baton Rouge, Louisiana 70894
Received August 14, 2000
Pyridines and pyrazines substituted with 1,2,4-oxadiazole-5-ones, 1,2,4-oxadiazole-5-thiones,
and 1,3,4-oxathiazoline-2-ones were synthesized and tested against Mycobacterium tuberculosis.
The two former ring systems were documented in the literature to act as carboxylic acid
isosteres. The latter series was synthesized as possible synthetic intermediates to 1,2,4-
thiadiazole-3-ones and was included in this study due to their interesting activity. Pivaloyl-
oxymethyl derivatives of the isosteres were also prepared in order to increase their lipophilicity
and therefore improve their cellular permeability. The derivatized isosteres were expected to
be biotransformed by esterases to the active species after penetration of the mycobacterial cell
wall. Biological properties of the compounds were compared with the unmodified polar isosteres
of pyrazinoic and nicotinic acids. The majority of the compounds exhibited activities ranging
from 0.5 to 16 times the potency of pyrazinamide.
In tr od u ction
In1 the 1940s, Chorine2 and McKenzie3 independently
demonstrated that nicotinamide was effective for the
treatment of murine tuberculosis. The synthesis of
many nicotinamide analogues including the pyrazine
isostere pyrazinamide followed this discovery. It was
Several other acidic heterocycles or functional groups
soon shown that an enzyme called nicotinamidase inside
have been used to replace carboxyl groups or the
the mycobacterium hydrolyzed nicotinamide and pyrazin-
tetrazole ring itself.7-11
amide to the corresponding carboxylic acids, and these
carboxylic acids were the actual active compounds.4
However, nicotinic and pyrazinoic acids did not demon-
strate activity due to their poor penetration into myco-
bacterial cells.
Now we are reporting the synthesis and antituber-
cular activity of 1,2,4-oxadiazole-5-one and 1,2,4-oxa-
diazole-5-thione isosteres of pyridine- and pyrazinecar-
boxylic acids. In addition to those, three 1,3,4-oxathia-
zoline-2-ones were synthesized (as potential synthetic
intermediates in an unsuccessful attempt to prepare
1,2,4-thiadiazole-5-ones) and tested for activity. All of
the synthesized compounds, except for the nonacidic
oxathiazolinones, demonstrated very polar character-
istics and poor solubility in organic solvents. It was
reported that tetrazole derivatives that contain ad-
ditional basic groups elsewhere in the structure existed
in zwitterionic form and therefore exhibited poor ab-
sorption properties.12 Although the heterocycles syn-
thesized by us have not been studied in this way, it can
be assumed that they also act as zwitterions due to their
similar pKa values to tetrazoles.13 To overcome this
problem, a general prodrug approach was adopted to
improve the biomembrane transport. Thus, the hetero-
cycles were alkylated in an attempt to bioreversibly
modify them by an agent with a metabolic weak point
for easy breakdown into the parent compound by
enzymatic means under physiological conditions. Chloro-
methyl pivalate was chosen as the alkylating agent to
serve this purpose. The resulting pivaloyloxymethyl
(POM) derivatives are expected to be hydrolyzed by
esterases to the hydroxymethyl derivatives, which
spontaneously decompose to the parent compounds
releasing a formaldehyde molecule.12 The in vitro anti-
tubercular activity of the resulting compounds are
One of the effective methods that can lead to new drug
discoveries is the bioisosteric replacement of a functional
group. Numerous functional groups have been reported
as bioisosteric replacements for the carboxylic acid
functionality.5 Among those, the most commonly en-
countered one is probably the tetrazole ring. In the area
of nonpeptidic angiotensin II (AII) receptor antagonists,
especially successful results were obtained when the
carboxylic acid group was replaced by a tetrazole ring.6
These findings led our group to synthesize tetrazole
isosteres of pyridine- and pyrazinecarboxylic acids.1 The
most active compounds in this series included 1 and its
nicotinyl analogue, 3-(2-pivaloyloxymethy-5-tetrazolyl)-
pyridine, and they had the MIC value of 50 µM (Table
2).
* Address correspondence to this author at the Department of
Pharmacology and Toxicology, College of Pharmacy, The University
of Arizona, Tucson, AZ 85721. Phone: (520) 626-2515 Fax: (520) 626-
4063. E-mail: amartin@pharmacy.arizona.edu.
10.1021/jm000350w CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/06/2001

Isosteres of Pyridine- and Pyrazinecarboxylic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1561
Ta ble 2. MIC Values of Compounds 1-16
Ta ble 1. Oxadiazolones 2-7, Oxadiazolethiones 8-13, and
Oxathiazolinones 14-16
MICa
(µM) (µg/mL)
MIC
MICa
(µM)
MIC
(µg/mL)
formulaa
compd
compd
compd
X
R
mp (°C)
pyrazinamideb
400
50
>1600
>1600
800
50
100
200
>1600
49
13
>261
>261
131
14
28
56
>286
isoniazidb
9
0.15
0.018
>286
288
59
59
118
9
2
3
C
CH
N
C
CH
N
C
CH
N
C
CH
N
H
H
H
POM
POM
POM
253-5
233-5
229-31
64-6
88-90
99-101
228-9
223-5
220-2
73-6
45-8
61-3
191-3
115-7
130-2
C7H5N3O2
C7H5N3O2
C6H4N4O2
C13H15N3O4
C13H15N3O4
C12H14N4O4
C7H5N3OS
C7H5N3OS
C6H4N4OS
11
2
>1600
1600
200
200
400
50
10
11
12
13
14
15
16
4b
5
3
4
5
6
7
8
6
7
8
9
10
11
12
13
14
15
16
25
25
4.5
4.5
a
Determined in BACTEC 6A; see the Experimental Section for
POM
POM
POM
C
C
C
13H15N3O3S
13H15N3O3S
12H14N4O3S
b
a description of the MIC determination. For comparison.
C
CH
N
C7H4N2O2S
C7H4N2O2S
C6H3N3O2S
Resu lts a n d Discu ssion
Compounds 2-16 were tested against Mycobacterium
tuberculosis H37Rv (ATCC 27294) with the results
shown in Table 2. The MIC value in BACTEC 6A19
medium of the oxadiazole derivative 4 was 800 µM.
MICs of the POM derivatives of the oxadiazoles 5-7
were 50, 100, and 200 µM, respectively. The POM
derivatives of the oxadiazolethiones 11-13 exhibited
MIC values of 200-400 µM, and the oxathiazoline-2-
ones 14-16 exhibited MIC values of 25-50 µM. The
MIC values of all the other compounds tested were
greater than or equal to 1600 µM, limited solubility
prohibiting an accurate determination. The unprotected
polar heterocycles exhibited only minimal activity with
the exception of 3-pyrazinyl-1,2,4-oxadiazole-5(4H)-one
(4), which was half as potent as pyrazinamide. The
oxadiazolethione series (8-10) was devoid of activity at
the concentrations tested except for the 3-pyrazinyl-
1,2,4-oxadiazole-5(4H)-thione (10), which was 4 times
less potent than pyrazinamide.
As expected, the polar, unprotected isosteres of pyri-
dine- and pyrazinecarboxylic acids 2-4 and 8-10
showed little or no inhibition of M. tuberculosis. We
believe that this phenomenon is due to their structural
similarity to nicotinic, isonicotinic, and pyrazinoic acids,
which themselves are devoid of antitubercular activity,
while derivatives thereof such as pyrazinamide are
effective inhibitors. Pyrazinamide and nicotinamide are
known to be converted by a nicotinamidase into pyrazi-
noic and nicotinic acids, which are thought to be the
actual active species in the mycobacteria.3 However, the
highly polar nature of the acids prevents their efficient
penetration of the mycobacterial cell wall, rendering the
compounds inactive. Polarity appeared to be the only
determinant of the activity at this stage of the experi-
ments as demonstrated by the fact that slightly more
lipophilic pyrazine derivatives were the only compounds
with any activity.
a
Analyses were within 0.4% of the calculated values except
b
where noted (footnote b). N: calcd, 34.14; found, 33.57.
discussed and compared with the unmodified polar
isosteres of nicotinic and pyrazinoic acids.
Ch em istr y
Pyridine-3-14 and 4-carboxamidoximes15 that served
as starting materials were synthesized according to a
published procedure with some improvement.16 Pyra-
zine carboxamidoxime was synthesized using a similar
procedure.17
1,2,4-Oxadiazole-5(4H)-ones 2-4 (Table 1) were syn-
thesized by reacting the corresponding amidoxime with
methyl chloroformate and then thermally cyclizing the
resulting acylated intermediates. The heterocycles were
alkylated with chloromethyl pivalate in the presence of
KI to give POM derivatives 5-7. 1,2,4-Oxadiazole-
5(4H)-thiones 8-10 were synthesized using two differ-
ent methodologies. In the first, the acetylated carbox-
amidoximes were reacted with carbon disulfide in the
presence of a base to yield 8 and 9. However, the
pyrazine derivate 10 decomposed during workup. There-
fore, another methodology was applied to synthesize all
three compounds, which involved thiocarbonyldiimida-
zole (TCDI) and a base. The alkylation of the thione
derivatives 8-10 went smoothly to afford the corre-
sponding POM derivatives 11-13. 1,3,4-Oxathiazoline-
2-ones 14-1618 were prepared by the action of chloro-
carbonylsulfenyl chloride on the corresponding carbox-
amides. Attempted (3 + 2) cycloadditions of 14-16 with
tosyl cyanide failed to give the desired 1,2,4-thiadiazole-
3-ones, yielding elemental sulfur and the corresponding
nitriles instead.
The results also showed that derivatization of these
compounds to more lipophilic precursors, which made
ionization impossible and presumably facilitated pen-
etration of the cell wall, was a successful method to
improve their physicochemical properties. Modification
of inactive oxadiazole 2 with the POM group produced
compound 5, which was 8 times as potent as the
currently used drug pyrazinamide. Likewise, protection
of inactive 3 resulted in 6, which is 4 times as potent
as pyrazinamide. Derivatization of compound 4 in-
creased 4 times its already existing potency. The
protected derivatives of the oxadiazolethiones 8 and 9
(11 and 12) were twice as potent as pyrazinamide,

1562 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10
Gezginci et al.
dropwise to a solution of 5 mmol of the respective oxadiazolone
and 5 mmol of AcOK in 10 mL of DMF. NaI (5 mmol) was
added, and the mixture was stirred at room temperature for
72 h. The reaction mixture was diluted with 20 mL of H2O
and extracted with 3 × 40 mL of EtOAc. The organic layers
were combined and washed with 120 mL of 1% Na2SO3
solution, 120 mL of H2O, and 120 mL of brine. The solution
was dried over Na2SO4, and the solvent was evaporated. A
yellow oil was obtained, which was chromatographed on silica
gel using hexanes/EtOAc (7:3 for 5 and 6, and 8:2 for 7) as
eluent. Colorless to yellow oils were obtained, which slowly
crystallized. Recrystallization from a mixture of hexanes and
whereas 10, which was 4 times less potent than pyrazi-
namide, achieved equal potency with the drug when it
was protected. To our surprise, the 1,3,4-oxathiazoline-
2-ones 14-16 were the most active compounds in the
series, having activities ranging from 8 to 16 times as
potent as pyrazinamide. Interestingly enough, a mem-
ber of this series (15) was recently reported as a
microbicide in a patent application.18
In summary, the results show that bioreversible
modification of pyridine- and pyrazinecarboxylic acid
bioisosteres to more lipophilic precursors can increase
their activity. Although the compounds were not tested
against pyrazinamide-resistant strains of M. tubercu-
losis, they can be expected to be effective against most
of these strains because of the knowledge that most
pyrazinamide resistance results from lack of pyrazina-
midase (nicotinamidase) enzyme in the bacteria.20 Since
the compounds do not contain an amide bond that has
to be hydrolyzed for activity, pyrazinamidase would not
be necessary for the activation of the synthesized
compounds. In the case of the 1,3,4-oxathiazoline-2-ones
14-16, the species responsible for the antimycobacterial
activity is unknown, so it is uncertain if these com-
pounds would be active against pyrazinamide-resistant
strains.
1
EtOAc gave 5-7. 5: yield 33%. H NMR (CDCl3): δ 8.88 (d,
1
2H), 7.59 (d, 2H), 5.65 (s, 2H), 1.18 (s, 9H). 6: yield 37%. H
NMR (CDCl3): δ 8.90 (m, 2H), 7.99 (m, 1H), 7.55 (m, 1H), 5.63
1
(s, 2H), 1.19 (s, 9H). 7: yield 51%. H NMR (CDCl3): δ 9.29
(d, 1H), 8.81 (d, 1H), 8.66 (d, 1H), 6.09 (s, 2H), 1.08 (s, 9H).
Gen er a l P r oced u r es for th e P r ep a r a tion of th e Oxa -
d ia zoleth ion es 8-10. Meth od 1 for 8 a n d 9. Acetic anhy-
dride (7.3 mmol) was added dropwise to a mixture of 7.3 mmol
of the respective amidoxime and 7.3 mmol of Et3N in 30 mL
of CH2Cl2. The mixture was stirred at room temperature for 4
h. The solids were filtered, washed with CH2Cl2 and H2O, and
air-dried. The filtrate was washed with 30 mL of H2O and
evaporated. The solids were combined with the ones obtained
from the mother liquor and dissolved in 40 mL of DMF. The
solution was cooled to 0 °C in an ice-water bath, and 25.5
mmol of CS2 and 19.2 mmol of a 50% dispersion of NaH in
mineral oil were added. The mixture was stirred for 1 h. A 1
N HCl solution (80 mL) was added very carefully, and the
mixture was stirred for another hour and refrigerated over-
night. The crystals were collected by filtration, washed with
H2O, and recrystallized twice from EtOH to give 8 or 9.
Exp er im en ta l Section
Gen er a l. Melting points were determined with an electro-
thermal capillary melting point apparatus and are uncor-
rected. 1H and 13C nuclear magnetic resonance (NMR) spectra
were obtained using a Varian Gemini 200 spectrometer at 200
and 50 MHz, respectively. Chemical shifts are reported in ppm
downfield (δ) from internal TMS. Elemental analyses were
performed by Desert Analytics of Tucson, Arizona, and were
within 0.4% of the calculated values except where stated
otherwise (Table 1). Column chromatography was performed
using silica gel purchased from Aldrich Chemical Co. (60 A,
70-230 mesh).
Meth od 2 for 8-10. To a stirred suspension of 7.3 mmol
of the respective amidoxime in 60 mL of MeCN was added 10.9
mmol ofTCDI (90%) in one portion. A clear solution formed
for a moment, and then a yellow precipitation followed. To this
mixture, 29.2 mmol of DBU was added dropwise. A clear
solution was obtained again, which was stirred at room
temperature overnight. The mixture was poured into 120 mL
of H2O and neutralized using 6 N HCl. The precipitate was
filtered and washed with H2O. The mother liquor was refriger-
ated overnight, and the solids were filtered, combined with
the first crop, and recrystallized from ethanol to give 8-10.
8: yield method 1: 35%, method 2: 80%. 1H NMR (DMSO-
d6): δ 8.91 (dd, 2H), 8.06 (dd, 2H), 6.19 (br s, 1H). 9: yield
Gen er a l P r oced u r e for th e P r ep a r a tion of Oxa d ia zo-
lon es 2-4. The respective amidoxime14,15,17 (10 mmol) and a
magnetic stirring bar were placed in a 100 mL round-bottomed
flask, and the flask was sealed with a rubber septum. N2 was
introduced, and 15 mL of anhydrous DMF was added via
syringe. After the addition of 12 mmol pyridine, the mixture
was stirred at room temperature until dissolution and then
cooled to 0 °C in an ice-water bath. Methyl chloroformate (11
mmol) was added, and the mixture was stirred for 3 h while
warming to room temperature. The mixture was diluted with
30 mL of H2O, and the resulting solution was extracted with
3 × 30 mL of EtOAc. The organic phases were combined and
dried over anhydrous Na2SO4. Upon filtration and evaporation
of the solvent, a yellow oil (in the case of 2 and 3) was obtained.
The pyrazine derivative 4 gave, upon dilution of the reaction
mixture with H2O, a solid, which was washed with H2O and
air-dried. The oils were dissolved in 60 mL of toluene and
refluxed for 24 h. The solid pyrazine derivative was suspended
in 60 mL of xylenes and refluxed (140 °C) for 24 h. Then the
mixtures were allowed to cool to room temperature, and the
volatiles were removed under vacuum (2 and 3) or let stand
overnight (4). The solids were recrystallized twice from EtOH
(2 and 4) or MeCN (3) to give 2-4. 2: yield 48%. 1H NMR
(DMSO-d6): δ 8.83 (d, 2H), 7.77 (d, 2H), 3.50 (br s, 1H). 3:
1
method 1: 28%, method 2: 79%. H NMR (DMSO-d6): δ 9.08
(d, 1H), 8.84 (dd, 1H), 8.34 (ddd, 1H), 7.70 (dd, 1H). 10: yield
1
89%. H NMR (DMSO-d6): δ 9.25 (d, 1H), 8.90 (m, 2H).
Gen er a l P r oced u r e for th e Alk yla tion of Oxa d ia zole-
th ion es 8-10 (11-13). Chloromethyl pivalate (7.1 mmol) was
added dropwise to a stirred solution of 5.9 mmol of the
respective oxadiazolethione and 7.1 mmol of Et3N in 10 mL of
DMF. The solution was stirred at room temperature for 7 h,
and another equivalent of chloromethyl pivalate and Et3N was
added. The solution was stirred at room temperature for a total
of 24 h. For the last 1.5 h, it was heated at 50 °C. The mixture
was cooled to room temperature and poured into crushed ice
with vigorous stirring. In the case of 11 and 13, the solids were
collected by filtration, washed with water, and recrystallized
twice from petroleum ether to give 11 and 13. Compound 12
separated as an oil, which was extracted with 3 × 50 mL of
EtOAc. The solution was dried over Na2SO4 and filtered, and
the solvent was evaporated. The residue was dissolved heating
slightly in hexanes and filtered. Hexanes were removed under
vacuum. A tan oil was obtained, which was chromatographed
on silica gel using hexanes/EtOAc (9:1) as eluent. The product
solidified in the freezer and was recrystallized from petroleum
ether in the freezer to give 12. 11: yield 83%. 1H NMR
(CDCl3): δ 8.80 (dd, 2H), 7.94 (dd, 2H), 5.83 (s, 2H), 1.22 (s,
9H). 12: yield 78%. 1H NMR (CDCl3): δ 9.31 (dd, 1H), 8.77
(dd, 1H), 8.34 (m, 1H), 7.44 (m, 1H), 5.83 (s, 2H), 1.22 (s, 9H).
13: yield 84%. 1H NMR (CDCl3): δ 9.37 (d, 1H), 8.77 (m, 2H),
5.86 (s, 2H), 1.22 (s, 9H).
1
yield 52%. H NMR (DMSO-d6): δ 8.94 (br s, 1H), 8.75 (br s,
1H), 8.15 (d, 1H), 7.58 (q, 1H), 3.81 (br s, 1H). 13C NMR
(DMSO-d6): δ 159.92, 155.86, 152.85, 146.91, 134.06, 124.46,
120.01. 4: yield: 57%. 1H NMR (DMSO-d6): δ 9.13 (d, 1H),
8.81 (m, 2H), 3.61 (br s, 1H). 13C NMR (DMSO-d6): δ 159.69,
156.41, 147.57, 144.91, 142.60, 139.11.
Gen er a l P r oced u r e for th e Alk yla tion of Oxa d ia zolo-
n es 2-4 (5-7). Chloromethyl pivalate (6 mmol) was added

Isosteres of Pyridine- and Pyrazinecarboxylic Acids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 10 1563
(10) Kees, K. L.; Caggiano, T. J .; Steiner, K. E.; Fitzgerald, J . J ., J r.;
Kates, M. J .; Christos, T. E.; Kulishoff, J . M., J r.; Moore, R. D.;
McCaleb, M. L. Studies on New Acidic Azoles as Glucose-
Lowering Agents in Obese, Diabetic db/db Mice. J . Med. Chem.
1995, 38, 617-628.
(11) Allan, R. D.; J ohnston, G. A. R. Synthetic Analogs for the Study
of GABA as a Neurotransmitter. Med. Res. Rev. 1983, 3, 91-
118.
(12) The pKa of phenyltetrazole was reported as about 4.5: Alexander,
J .; Renyer, M. L.; Rork, G. S. Investigation of N-[(Acyloxy)alkyl]
Ester as a Prodrug Model for Drugs Containing the Phenyl-
tetrazole Moiety. J . Pharm. Sci. 1994, 83, 893-897
(13) Kohara, Y.; Kubo, K.; Imamiya, E.; Wada, T.; Inada, Y.; Naka,
T. Synthesis and Angiotensin II Receptor Antagonistic Activities
of Benzimidazole Derivatives Bearing Acidic Heterocycles as
Novel Tetrazole Bioisosteres. J . Med. Chem. 1996, 39, 5228-
5235.
(14) 3-Cyanopyridine (6.24 g, 0.06 mol) and 8.34 g (0.12 mol) of NH2-
OH‚HCl were dissolved in 60 mL of H2O. A solution of 12.72 g
(0.12 mol) of Na2CO3 in 45 mL of H2O was cautiously added,
and the resulting solution was stirred and heated at 70 °C
overnight. The solution was cooled to room temperature, satu-
rated with NaCl, and extracted with 4 × 50 mL of EtOAc. The
solution was dried over anhydrous Na2SO4, and the solvent was
evaporated, giving 5.12 g of colorless crystals (62%). Reported
yield was 38%.
(15) 4-Cyanopyridine (6.24 g, 0.06 mol) was dissolved in 9 mL of
EtOH (slightly heated). NH2OH‚HCl (8.34 g, 0.12 mol) was
dissolved in 9 mL of H2O and added to the 4-cyanopyridine
solution. Na2CO3 (12.72 g, 0.12 mol) was dissolved in 75 mL of
H2O and added cautiously to the above solution. The suspension
was heated at 70 °C overnight. The mixture was cooled to room
temperature and kept at 4 °C for a few hours. The crystals were
separated by filtration and washed with H2O, giving 7.40 g of
colorless crystals (90%). Reported yield was 57%.
Gen er a l P r oced u r e for th e P r ep a r a tion of Oxa th ia -
zolin on es 14-16.18 A mixture of 30 mmol of the respective
carboxamide and 10 mmol of chlorocarbonylsulfenyl chloride
in 100 mL of toluene was refluxed for 3 h. The precipitate was
filtered and washed with 50 mL of EtOAc. The filtrate and
the washings were combined and evaporated. The residue was
purified by column chromatography using hexanes/EtOAc
(1:1) as eluent and recrystallized from a mixture of hexanes
and EtOAc to give 14-16. 14: yield 43%. 1H NMR (CDCl3): δ
1
8.83 (dd, 2H), 7.82 (dd, 2H). 15: yield 43%. H NMR (CDCl3):
δ 9.15 (d, 1H), 8.75 (dd, 1H), 8.20 (ddd, 1H), 7.42 (m, 1H). 13C
NMR (CDCl3): δ 172.93, 155.33, 153.04, 148.48, 134.46,
123.64, 122.04. 16: yield 33%. 1H NMR (CDCl3): δ 9.29 (d,
1H), 8.77 (m, 2H). 13C NMR (CDCl3): δ 172.11, 154.42, 147.11,
144.62, 144.14, 140.07.
Deter m in a tion of Biologica l Activity. The compounds
were tested for inhibition of M. tuberculosis H37Rv ATCC 27294
using the BACTEC 460 system as previously described.19
Percent inhibition was calculated as 1 - (growth index of test
sample/growth index of control) × 100. The minimum inhibi-
tory concentration is defined as the minimum concentration
that inhibited 99% of the inoculum.
Ack n ow led gm en t. This work was funded by the
Arizona Disease Control Research Commission, Phoe-
nix, AZ, Contract 9617. The authors thank Anita Biswas
for her reliable technical assistance in the determination
of the bioactivities.
Refer en ces
(1) Part 1: Wachter, G. A.; Davis, M. C.; Martin, A. R.; Franzblau,
S. G. Antimycobacterial Activity of Substituted Isosteres of
Pyridine and Pyrazinecarboxylic Acids. J . Med. Chem. 1998, 41,
2436-2438.
(2) Chorine, M. V. Action of Nicotinamide on Bacilli of the Species
Mycobacterium. C. R. Hebd. Se´ances Acad. Sci. 1945, 220, 150-
156.
(3) McKenzie, D.; Malone, L.; Kushner, S.; Oleson, J . J .; Subbarow,
Y. The Effect of Nicotinic Acid Amide on the Experimental
Tuberculosis of White Mice. J . Lab. Clin. Med. 1948, 33, 1249-
1253.
(4) Konno, K.; Feldmann, F. M.; McDermott, W. Pyrazinamide
Susceptibility and Amidase Activity of Tubercle Bacilli. Am. Rev.
Respir. Dis. 1967, 95, 461-469.
(5) Thornber, C. W. Isosterism and Molecular Modification in Drug
Design. Chem. Soc. Rev. 1979, 8, 563-580.
(6) Carini, D. J .; Duncia, J . V.; Aldrich, P. E.; Chiu, A. T.; J ohnson,
A. L.; Pierce, M. E.; Price, W. A.; Santella, J . B., III; Wells, G.
J .; Wexler, R. R.; Wong, P. C.; Yoo, S.; Timmermans, P. B. M.
W. M. Nonpeptide Angiotensin II Receptor Antagonists: The
Discovery of a Series of N-(Biphenylmethyl)imidazoles as Potent,
Orally Active Antihypertensives. J . Med. Chem. 1991, 34, 2525-
2547.
(7) Ellingboe, J . W.; Alessi, T. R.; Dolak, T. M.; Nguyen, T. T.; Tomer,
J . D.; Guzzo, F.; Bagli, J . F.; McCaleb, M. L. Antihyperglycemic
Activity of Novel Substituted 3H-1,2,3,5-Oxathiadiazole 2-Ox-
ides. J . Med. Chem. 1992, 35, 1176-1183.
(16) Delaby, R.; Reynaud, P.; Tupin, T. Amidines et Amidoximes
Pyridiques. Bull. Soc. Chim. Fr. 1957, 714-717.
(17) Cyanopyrazine (6.3 g, 0.06 mol) was diluted with 9 mL of EtOH.
Hydroxylamine hydrochloride (8.34 g, 0.12 mol) was dissolved
in 9 mL of H2O and added to the cyanopyrazine solution. Two
phases separated. Na2CO3 (12.72 g, 0.12 mol) was dissolved in
75 mL of H2O and added cautiously to the above mixture. After
the addition of the first few milliliters of the Na2CO3 solution, a
large amount of white substance precipitated, which did not
dissolve upon the addition of the rest of the solution. The whole
mixture was heated at 70 °C for 5 h and left standing at room
temperature overnight. The crystals were filtered and washed
with H2O. The filtrate was saturated with NaCl and extracted
with 3 × 50 mL of EtOAc. Solution was dried over anhydrous
Na2SO4, and the solvent was evaporated. The crystals were
combined with the initial precipitate giving 7.98 g of colorless
needles (97%).
(18) The synthesis of 15 was recently described as a microbicide with
no physical or analytical data: Holzl, W.; Schnyder, M. Micro-
bicidal Active Substances. Int. Pat. Appl., WO 00/07446, 2000.
(19) Collins, L. A.; Franzblau, S. G. Microplate Alamar Blue Assay
versus BACTEC 460 System for High Throughput Screening of
Compounds Against Mycobacterium tuberculosis and Mycobac-
terium avium. Antimicrob. Agents Chemother. 1997, 41, 1004-
1009.
(8) Ellingboe, J . W.; Lombardo, L. J .; Alessi, T. R.; Nguyen, T. T.;
Guzzo, F.; Guinosso, C. J .; Bullington, J .; Browne, E. N. C.; Bagli,
J . F.; Wrenn, J .; Steiner, K.; McCaleb, M. L. Antihyperglycemic
Activity of Novel Naphthalenyl 3H-1,2,3,5-Oxathiadiazole 2-Ox-
ides. J . Med. Chem. 1993, 36, 2485-2493.
(9) Unangst, P. C.; Shrum, G. P.; Connor, D. T.; Dyer, R. D.; Schrier,
D. J . Novel 1,2,4-Oxadiazoles and 1,2,4-Thiadiazoles as Dual
5-Lipoxygenase and Cyclooxygenase, Inhibitors. J . Med. Chem.
1992, 35, 3691-3698.
(20) Hirano, K.; Takahashi, M.; Kazumi, Y.; Fukasawa, Y.; Abe, C.
Mutation in pcnA is
a major mechanism of pyrazinamide
resistance in Mycobacterium tuberculosis. Tuber. Lung Dis. 1998,
78, 117-122.
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