Synthesis and in vitro activity towards Mycobacterium tuberculosis of L-serinyl ester and amino derivatives of pyrazinoic acid

Article abstract of DOI:10.3184/030823407X200001

Reactions between either L-serine methyl ester hydrochloride (1), or the cbz derivative, methyl (S)-(+)-2-(benzyloxycarbonylamino)-3-hydroxypropanoate (2), and pyrazinoyl chloride (3), have been studied. Methyl (S)-(+)-2- benzyloxycarbonylamino-3-[(pyrazinecarbonyl)oxy]propionate (4), methyl (S)-(+)-3-hydroxy-2-[(pyrazine-2-carbonyl) aminolpropionoate (7), methyl 2-[(pyrazinecarbonyl)amino]acrylate (8) were obtained. Additional products, methyl (S)-(+)-2-benzyloxycarbonylamino-3-formyloxypropionoate (5) and methyl (R)-(+)-2-benzyloxycarbonylamino-3-chloropropionoate (6), were isolated from reaction of 2 with 3, in the presence of DMF remaining from the preparation of 3, from pyrazinecarboxylic acid. The coupling of pyrazinecarboxylic acid with 1, in the presence of DCC was prevented by the formation of the unreactive adduct between DCC and pyrazinoic acid. The compounds were tested against M. tuberculosis: compounds (8) and (6) exhibited a MIC (μg/ml) value of 50 and 100, respectively, compared to the MIC value of 100 for the first line TB drug, pyrazinamide. The confirmation of the structure of (8) was obtained via X-ray crystallography.

Full text of DOI:10.3184/030823407X200001

180 RESEARCH PAPER
MARCH, 180–184
JOURNAL OF CHEMICAL RESEARCH 2007
Synthesis and in vitro activity towards Mycobacterium tuberculosis of
l-serinyl ester and amino derivatives of pyrazinoic acid
Alessandra C. Pinheiroa,b, Carlos R. Kaiserb, Maria C.S. Lourençoc, Marcus V.N. de Souzaa*,
Solange M.S.V. Wardella* and James L. Wardelld,e
aInstituto de Tecnologia em Fármacos, Far-Manguinhos, FioCruz, CEP 21041-250 Rio de Janeiro, RJ, Brazil
bInstituto de Química, Departamento de Química Orgânica, Universidade Federal do Rio de Janeiro, CEP 68534, 21941-972
Rio de Janeiro, RJ, Brazil
cInstituto de Pesquisas Clínica Evandro Chagas – IPEC – Av. Brasil, 4365 - Manguinhos, FioCruz, CEP 21041-250 Rio de Janeiro,
RJ, Brazil
dInstituto de Química, Departamento de Química Inorgânica, Universidade Federal do Rio de Janeiro, CEP 68563, 21945-970
Rio de Janeiro, RJ, Brazil
eDepartment of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen AB24 3UE, UK
Reactions between either L-serine methyl ester hydrochloride (1), or the cbz derivative, methyl (S)-(+)-2-
(benzyloxycarbonylamino)-3-hydroxypropanoate (2), and pyrazinoyl chloride (3), have been studied. Methyl (S)-(+)-2-
benzyloxycarbonylamino-3-[(pyrazinecarbonyl)oxy]propionate (4), methyl (S)-(+)-3-hydroxy-2-[(pyrazine-2-carbonyl)
amino]propionoate (7), methyl 2-[(pyrazinecarbonyl)amino]acrylate (8) were obtained. Additional products, methyl
(S)-(+)-2-benzyloxycarbonylamino-3-formyloxypropionoate (5) and methyl (R)-(+)-2-benzyloxycarbonylamino-3-
chloropropionoate (6), were isolated from reaction of 2 with 3, in the presence of DMF remaining from the preparation
of 3, from pyrazinecarboxylic acid. The coupling of pyrazinecarboxylic acid with 1, in the presence of DCC was
prevented by the formation of the unreactive adduct between DCC and pyrazinoic acid. The compounds were tested
against M. tuberculosis: compounds (8) and (6) exhibited a MIC (mg/ml) value of 50 and 100, respectively, compared
to the MIC value of 100 for the first line TB drug, pyrazinamide. The confirmation of the structure of (8) was obtained
via X-ray crystallography.
Keywords: synthesis, tuberculosis, l-serine derivatives, pyrazinoic acid derivatives, crystal structure, didehydroamino acid
Tuberculosis (TB) has once again become a major worldwide
public health problem and was declared a global health
emergency in 1993 by the World Health Organisation
(WHO).1
Among second-line TB drugs are the L-serine derivatives,
cycloserine and terizidone, shown in Fig. 2.
As part of our research for new compounds with anti-TB
activities, we initiated a study of compounds containing
moieties of both first-line and second-line TB drugs, namely
pyrazinoic acid and serine derivatives. We now wish to report
our findings, including in vitro test results.
Together with rifampicin and isoniazid, pyrazinamide
(Fig. 1) is a currently used first-line drug. Its inclusion in TB
treatment regimen decreases the duration of the treatment
from twelve to six months. Pyrazinamide is a pro-drug,
only active in acidic media on hydrolysis to pyrazinoic
acid, the active metabolite, produced by the mycobacterial
enzyme pyrazinamidase. The inhibition of the growth of
Mycobacterium tuberculosis by pyrazinoic acid is based on
blocking the synthesis of fatty acids by deactivating the fatty
acid synthetase I enzyme.2
Results and discussion
Routes to serinyl-pyrazine derivatives were attempted using
either L-serine methyl ester hydrochloride (1), or the cbz
derivative,3 methyl (S)-(+)-2-(benzyloxycarbonylamino)-
3-hydroxypropanoate (2) formed from L-serine as shown in
CH3
CH3
CH3
CONHNH2
N
CONH2
AcO
H3CO
OH
OH
OH
OH
CH3
N
H
N
Pyrazinamide
N
H3C
Isoniazide
CH3
O
O
O
OH
N
N
O
CH3
NCH3
Rifampicin
Fig. 1
* Correspondent. E-mail: swardell@far.fiocruz.br; marcos_souza@far.fiocruz.br
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JOURNAL OF CHEMICAL RESEARCH 2007 181
O
NH
O
H2N
OH
N
O
O
N
HN
NH
H2N
CO2Me
Terizidone
O
O
L-Serine
Cycloserine
Fig. 2
Scheme 1, and pyrazinoyl chloride or methyl pyrazinoate.
propionoate (6), in 15% yield. Characterisations of the
products were generally achieved using NMR and IR
spectroscopies. The three distinct carbonyl groups in 4 exhibit
three C=O peaks in the IR spectrum at 1760 (CO2Me), 1725
(CO2R) and 1690 (CONH) cm-1and three C=O absorptions
at 169.8 (CO2Me), 163.0 (CO2R) and 156.1 (CONH) ppm in
the 13C NMR spectrum. Product 5 is formed from reaction
of the hydroxyl in 2 with the residual DMF left amongst the
pyrazinoyl chloride, while compound 6 is formally obtained
by chloride substitution of the OH group in 2.
Pyrazinoyl chloride (3), was generated from pyrazine-
carboxylic acid on treatment with excess SOCl2 in
dichloromethane, in the presence of a catalytic amount of DMF.
However, attempts to isolate pyrazinoyl chloride completely
from the reaction mixture on removal of all volatiles only led
to decomposition and tar formation. Hence, evaporation was
only used to concentrate the reaction mixture to 1–2 ml in
volume, which consequently left some DMF and maybe also
some SOCl2 or HCl, admixed with the acid chloride.
The major product, albeit in a low 34% yield, from
reaction of such a concentrated impure solution of pyrazinoyl
chloride (3), with 2 was the ester, methyl (S)-(+)-2-
benzyloxycarbonylamino-3-[(pyrazinecarbonyl)oxy]propionate
(4),4 see Scheme 2, via reaction at the free hydroxyl group
of 2. Other products isolated from the reaction mixture by
column chromato-graphy were methyl (S)-(+)-2-(benzyloxy-
carbonylamino)-3-formyloxy-propionoate (5), in 17% yield,
and methyl (R)-(+)-2-benzyloxycarbonylamino-3-chloro-
The reaction of another sample of pyrazinecarbonyl
chloride, obtained in a similar manner as indicated above,
with L-serine methyl ester hydrochloride (1), produced
methyl
(S)-(+)-3-hydroxy-2-[(pyrazine-2-carbonyl)amino]
propionoate (7), methyl 2-[(pyrazinecarbonyl)amino]acrylate
(8), in yields of 35 and 30%, respectively, (Scheme 2). The
two distinct carbonyl groups in 7 exhibited two C=O peaks
in the IR spectrum at 1743 (CO2CH3) and 1674 (CONH)
cm-1 and two C=O absorptions at 171.2 (CO2Me) and 164.0
OH
OH
OH
a
b
Cbz
+
N
H
CO2Me
H3N
Cl-
CO2Me
H2N
CO2H
1
2
L-Serine
100%
80%
Scheme 1 (a) SOCl2, MeOH, RT, 24 h; (b) NaHCO3, H2O, PhCH2OCOCl, 0°C, 3 h.
N
OCHO
Cl
O
N
Cbz
Cbz
Cbz
O
+
N
H
CO2Me
+
N
H
CO2Me
N
H
CO2Me
6
5
17%
4
34%
15%
b
N
COCl
N
N
CO2H
a
OH
O
O
CH2
CO2Me
c
N
N
N
N
N
N
H
3
CO2Me
N
H
+
7
35%
8
30%
Scheme 2 (a) SOCl2, DMF, DCM, RT, 3 h; (b) DMF, Et3N, THF, 2, RT, 5 h; (c) DMF, Et3N, THF, 1, RT, 5 h.
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182 JOURNAL OF CHEMICAL RESEARCH 2007
Fig. 3 (a). Atom arrangements and numbering scheme for one, molecule A, of the two independent molecules of 8.
(b) Intramolecular H-bonding in 8, for both the independent molecules. Probability elipsoids drawn at the 50% level.
(CONH) ppm in the 13C NMR spectrum. The OH group is
apparent from the stretching frequency at 3384 cm-1 in the IR
spectrum and the absorption at 2.37 ppm exchangeable with
in an one-pot synthesis from pyrazinecarboxylic acid, SOCl2
and MeOH.13 However, methyl pyrazinecarboxylate was found
to be unreactive towards L-serine methyl ester hydrochloride,
1, under a number of conditions, including refluxing THF in the
presence of Et3N, and under 50 bar pressure.
1
D2O in the H NMR spectrum. As in 6, the NH proton in 7
1
appears as a doublet at 8.62 ppm in the H NMR spectrum.
The coupling reaction of pyrazinecarboxylic acid with
L-serine methyl ester hydrochloride in the presence of
dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole
(HOBt) was also attempted, the target being 7 in a higher yield
than obtained from the acid chloride. DCC, which activates the
acid on forming an adduct, has proved especially successful in
aiding amide synthesis from aminoacid derivatives in peptide
and nucleotide chemistry. However, alternative products have
been frequently reported in attempted DCC assisted coupling
reactions between other carboxylic acids, RCO2H, and an
amine or an alcohol. These alternative products for amine
reactions include anhydrides [RCO2OCR], ureas [cyclohexyl-
NHCONH-cyclohexyl] and acylureas [cyclohexyl-NHCON
(COR)NH-cyclohexyl].14-15 From the DCC, HOBt, pyrazine-
carboxylic acid and L-serine methyl ester hydrochloride
reaction mixture in CH2Cl2, the major product isolated in
~70% yield was the acylurea derivative, 9, obtained by
rearrangement of the initial pyrazinecarboxylic acid/DCC
adduct (10), and a little cyclohexyl-NHCONH-cyclohexyl:
no amide 7 was detected. The acylurea derivatives, such as 9,
unlike the initial adducts, such as 10, are unreactive towards
amines and alcohols.
The formation of 7 indicates the much greater reactivity of the
amino group relative to the hydroxyl group in 1 towards the
acyl chloride.
The two distinct carbonyl groups in 8 exhibited two peaks in
the IR spectrum at 1744 (CO2Me) and 1636 (CONH) cm-1and
two absorptions at 163.6 (CO2Me) and 161.0 (CONH) ppm
1
in the 13C NMR spectrum. The H NMR spectrum showed
the NH proton as a singlet at 10.2 ppm, and the two protons
of the double bond as broad singlet absorptions at 6.85 and
6.07 ppm. J(HA,HB) couplings for HAHBC = R(R') are
generally in the range of 2-4 Hz; clearly these were not
resolved in our spectrum, nor in the reported 1H NMR spectra
of other H2C =C(NHCOR)CO2H compounds [see for example:
ref. 5] The formation of the alkenyl compound 8, in which the
C =C double bond is in conjugation with the CO2Me group, is
derived from dehydration of the alcohol 7, facilitated by the
acidic reaction conditions. Phenyl and picolinyl analogues
of 8, PhCONHC( =CH2)CO2Me and 2-pyridinyl-CONHC
( =CH2)CO2Me were indicated in a patent to be readily obtained
from successive treatment of ArCONHCH(CH2OH)CO2Me
(Ar = Ph or 2-pyridinyl) with SOCl2 and a base.6
Compound 8 is an didehydroamino acid. Didehydroamino
acids, in general have been synthesised by several routes,7
including b-elimination of serine derivatives. They have been
shown to be useful intermediates in amino acid and peptide
synthesis and are constituents of a number of naturally
occurring antibiotic and phytotoxic peptides.7-11
Confirmation of the structure of 8 was obtained via X-ray
crystallography. Figure 3a shows the atom arrangements
and the numbering scheme for one, molecule A, of the two
independent molecules of 8: the other molecule B, which has
B in place of A in the numbering scheme, is very similar as
shown by geometric parameters available from CCDC.
The core atoms of the side chains, C7, N2, C8 and C10
are near co-planar with their attached pyrazine rings, angles
between the best planes for these side chain atoms and the ring
atoms being 3.74(2) and 2.70(1)°, respectively in molecules
Aand B. Extensive strong intramolecular H-bonding, as shown
in Fig. 3b and as indicated by the PLATON analysis, cement
the particular conformation for each independent molecule.
Intermolecular C–H---O H-bonding link the two independent
molecules as shown in Fig. 4. Further weak intermolecular
hydrogen bonding involving the methyl hydrogens, were also
indicated by the PLATON program.
O
O
N
N
O
N
N
N
H
N
N
O
NH
9
10
The lack of high yielding reactions with pyrazinoyl chloride,
led to the attempted use of methyl pyrazinecarboxylate, formed
Fig. 4 Intermolecular C–H---O interactions linking the two
independent molecules of 8.
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JOURNAL OF CHEMICAL RESEARCH 2007 183
Interestingly, Ljungvist and Folkers reported16 the successful
formation of the p-nitrophenyl pyridine-2-carboxylate in 70%
yield from p-nitrophenol and pyridine-2-carboxylic acid in the
presence of DCC in CH2Cl2, but only 16% was obtained in DMF,
along with 30% of 1,4-dicyclohexyl-2-(2-pyridylcarbonyl)
semicarbazide. In contrast, good yields of the p-nitrophenyl
esters of 3- and 4-pyridinecarboxylic acids were obtained even
in DMF. Such results for similar nitrogen heteroaromatic acids
show how small changes effect the outcome of these DCC
assisted reactions.
Reaction between pyrazinecarbonyl chloride (3), and methyl (S)-
(+)-2-(benzyloxycarbonyl)-3-hydroxypropanoate (2). Formation of
methyl (S)(+)-2-benzyloxycarbonylamino-3-(pyrazinecarbonyloxy)
propionate(4),methyl(S)-(+)-2-benzyloxycarbonylamino-3-formyloxy-
propionoate (5), and methyl (R)-(+)-2-benzyloxycarbonylamino-3-
chloro-propionoate (6).
Pyrazinecarbonyl chloride (3),was prepared from the reaction of
pyrazinecarboxylic acid (1 g, 8.06 mmol), thionyl chloride (2.4 ml,
32.24 mmol) and DMF (0.06 ml, 0.81 mmol) in dichloromethane
(30 ml) at room temperature, under stirring and nitrogen atmosphere.
After 3–4 hours, the reaction mixture was concentrated by evaporation
to 1–2 ml and the crude mixture was used as such in the next stage.
To the stirred solution of methyl (S)-(+)-2-(benzyloxycarbonyl)-
3-hydroxypropanoate, 2, 7.25 mmol) and triethylamine (1.5 ml,
10.9 mmol) in anhydrous THF (25 ml) at 0°C was added
pyrazinecarbonyl chloride, 3, concentrate. The reaction mixture was
stirred for 5 hours at RT, quenched with water (20 ml), and extracted
with ethyl acetate. The combined organic phases were dried over
anhydrous sodium sulfate and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel,
eluting with hexane: ethyl acetate gradient (up to 20%) to give
methyl (S)-(+)-2-(benzyloxycarbonylamino)-3-(pyrazinecarbonyloxy)
propionate (4), methyl (S)-(+)-2-(benzyloxycarbonylamino)-3-
(formyloxy)-propionoate (5), and methyl (R)-(+)-2-(benzyloxy-
carbonylamino)-3-chloropropionoate (6).
Methyl (S)-(+)-2-(benzyloxycarbonylamino)-3-(pyrazinecarbonyl-
oxy)propionate (4): Yield: 34%, as a colourless oil. 1H NMR
(400 MHz, DMSO-d6) d: 9.22 (d; J = 1.0; 1H; H3); 8.90 (d; J = 2.0;
1H; H6); 8.83 (dd; J = 2.0 and 1.0; 1H; H5); 8.12 (d; J = 7.5; 1H;
NH); 7.32 (m; 5H; Ph); 5.06 (s; 2H; CH2Ph); 4.66 (m; 2H; CH'OCO
and CH); 4.58 (dd; J = 12.5 and 4.5; 1H; CHOCO); 3.69 (s; 3H;
CH3). 13C NMR (100 MHz; DMSO-d6) d: 169.8; 163.0; 156.1; 148.2;
145.9; 144.9; 142.7; 136.7; 128.4; 127.9; 127.8; 65.8; 64.3; 52.9 52.4.
IR (cm-1; KBr): 1760 (C(O)OCH3); 1725 (C(O)OR; 1690 (C(O)NR).
[a]D30: + 38.7 (c = 1.0, CHCl3). HRMS-ESI (m/z): [M + Na]+ calcd
for C17H17N3O6·Na+, 382.1015; found, 382.0982.
Biological activity
The anti-mycobacterial activities of the compounds were
assessed against M. tuberculosis ATTC 27294 using the micro
plate Alamar Blue assay (MABA).17 The results are listed in
Table 1. Compounds 8 and 6 exhibited a MIC (mg/ml) value
of 50 and 100, respectively, which can be compared with the
MIC value of 100 for the first line TB drug, pyrazinamide.
Experimental
General
Melting points were determined on a Buchi apparatus and are
uncorrected. IR spectra were determined using a Thermo Nicolet
Nexus 670 spectrometer in potassium bromide discs. Mass spectra
recorded on a Q-TOF mass spectrometer Typical ESI-MS (Micromass,
Manchester, UK); NMR spectra were obtained at room temperature
with a Bruker Avance 500 spectrometer operating at 500.00 MHz
(1H) and 125.0 MHz (13C), in DMSO-d6 solutions. Optical rotation
was measured with a Polartronic NH8 polarimeter at ~25°C.
Thin layer chromatography was carried out on silica gel plates, using
ethyl acetate/methanol mixtures as eluents, with products detected by
UV light. For column chromatographic purification, column grade
silica gel 60 (0.063–0.200 mm mesh size) was employed.
Methyl
(S)-(+)-2-(benzyloxycarbonylamino)-3-(formyloxy)-
Synthesis
l-Serine methyl ester hydrochloride salt (1): To a stirred solution of
thionyl chloride (69.5 ml, 0.95 mol) in methanol (400 ml) at 0°C was
added l-serine (20 g, 0.19 mol). The reaction mixture was stirred
for 24 h at room temperature, the solvent removed to give l-serine
methyl ester hydrochloride salt, 1 (147.2 g) in quantitative yield. m.p.
163.1°C (Lit.: 163°C26). 1H NMR (400 MHz, DMSO-d6) d: 8.60
(s, 3H, H3N+), 5.64 (s, 1H, OH), 4.08 (t, J = 4 Hz, 1H, CH), 3.82
(d, J = 4 Hz, 2H, CH2), 3.73 (s, 3H, CH3).13C NMR (100 MHz,
DMSO-d6) d: 168.5, 59.5, 54.4, 52.8.IR (cm-1; KBr): 3362 (O-H);
1743 (CO2Me).
propionoate (5): Yield: 17%, as a colourless oil. 1H NMR (400 MHz,
DMSO-d6) d: 8.21 (s; 1H; OCOH); 7.97 (d; J = 8; 1H; NH); 7.34
(m; 5H; Ph); 5.06 (s; 2H; CH2Ph); 4.46 (m; 2H; CH'OCOH and CH);
4.26 (dd; J = 11.2 and 6.4; 1H; CHOCOH); 3.67(s; 3H; CH3). 13C
NMR (100 MHz; DMSO-d6) d: 170.3; 162.2; 156.6; 137.0; 128.9;
128.4; 128.1; 66.3; 62.6; 53.3; 52.8. IR (cm-1; KBr): 1750 (C(O)H);
1719 (C(O)OCH3); 1692 (C(O)NR). [a]D30: + 10.0 (c = 1.0, CHCl3).
HRMS-ESI (m/z): [M + Na]+ calcd for C13H15NO6· Na+, 304.0797;
found, 304.0790.
Methyl (R)-(+)-2-(benzyloxycarbonylamino)-3-chloropropionoate
(6): Yield: 15%. m.p. 53.0°C. 1H NMR (400 MHz, DMSO-d6) d: 7.98
(d; J = 8; 1H; NH); 7.33 (m; 5H; Ph); 5.07 (s; 2H; CH2Ph); 4.49
(m, 1H; CH); 3.90 (dd; J = 11.5 and 4.5; 1H; CH'Cl); 3.83 (dd;
J = 11.5 and 7.5; 1H; CHCl); 3.36 (s; 3H; CH3). 13C NMR (100 MHz;
DMSO-d6) d: 169.9; 156.4; 137.0; 128.7; 128.3; 128.0; 66.1; 55.6;
52.8; 44.1. IR (cm-1; KBr): 1746 (C(O)OCH3); 1686 (C(O)NR).
[a]D30: + 39.3 (c = 1.0, CHCl3). HRMS-ESI: [M + Na]+ calcd for
C12H14ClNO4·Na+, 294.0509; found, 294.0520.
Methyl (S)-(+)-2-(benzyloxycarbonylamino)-3-hydroxypropanoate
(2): To
a reaction mixture containing l-serine methyl ester
hydrochloride salt (1), (13.2 g, 0.11 mol), water (100 ml), diethyl
ether (75 ml) and sodium bicarbonate (48 g, 0.55 mol) at 0°C was
added dropwise benzyl chloroformate (21 ml, 0.15 mol). After 2 h
at 0°C and 1 hour at room temperature, the reaction was quenched
with pyridine, and water was added to solubilise all salts. The organic
layer was washed with HCl (2.5N), dried ( MgSO4), filtered and
concentrated. The residue was chromatographed (10%→30% EtOAc/
hexanes) affording 2 as a colourless oil (22.4 g, 80% yield. 1H NMR
(400 MHz, CDCl3) d: 7.32 (m, 5H, Ph), 5.71(s, 1H, NH), 5.13 (s, 2H,
CH2Ph), 4.45 (m, 1H, CH), 4.00 (dd, J = 11.2 and 3.0, 1H, CH'OH),
3.92 (dd, J = 11.2 and 3.0, 1H, CHOH), 3.78 (s, 3H, CH3). 13C NMR
(100 MHz, CDCl3) d: 171.8, 157.0, 136.7, 129.2, 128.9, 128.7, 67.8,
63.7, 56.7, 53.3. IR (cm-1, KBr): 3358 (O-H), 1778 (CO2Me), 1653
(CONR). GC/MS: m/z [M]+: 253. [a]D30: + 10.0 (c = 1.0, CHCl3)
Reaction between pyrazinecarbonyl chloride (3), and l-serine
methyl ester hydrochloride salt (1). Formation of methyl (S)-(+)-3-
hydroxy-2-[(pyrazinecarbonyl)amino]propionoate (7), and methyl 2-
[(pyrazinecarbonyl)-amino]acrylate (8)
Pyrazinecarbonyl chloride (3), was prepared from pyrazinecarboxylic
acid (1 g, 8.06 mmol), thionyl chloride (2.4 ml, 32.24 mmol) and
DMF (0.06 ml, 0.81 mmol) in dichloromethane (30 ml) at room
temperature.
To the stirred solution of l-serine methyl ester hydrochloride salt
(1)(860 mg, 7.25 mmol) and triethylamine (1.5 ml, 10.9 mmol) in
anhydrous THF (25 ml) at 0°C was added pyrazinecarbonyl chloride
(3) concentrate from above. The reaction mixture was stirred for
5 h at RT, quenched with water (20 ml), and extracted with ethyl
acetate. The combined organic phases were dried over anhydrous
sodium sulfate and concentrated under reduced pressure. The residue
was purified by column chromatography on silica gel, eluting with
hexane: ethyl acetate gradient (up to 20%) to give 7 and 8 in 35 and
30% yields, respectively.
Table 1 The in vitro activity of compounds against M.
tuberculosis H37Rv strain (ATCC 27294, susceptible both to
rifampin and isoniazid)
Compound
MIC (mg/ml)
2
Resistant
Resistant
Resistant
100
Resistant
50
4
5
6
7
8
Methyl (S)-(+)-3-hydroxy-2-[(pyrazinecarbonyl)amino]propionoate
(7): Yield: 35%, as a colourless oil. H NMR (500 MHz, CDCl3)
d: 9.38 (s; 1H; H3); 8.78 (d; J = 2; 1H; H5 or H6); 8.62 (d; J = 7.5;
9
Resistant
100
1
Pyrazinamide
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184 JOURNAL OF CHEMICAL RESEARCH 2007
1H; NH); 8.58 (d; J = 2; 1H; H5 or H6); 4.89 (m; 1H; CH); 4.15 (dd;
J = 11.5 and 3.5; 1H; CH'OH); 4.06 (dd; J = 11.5 and 3.5; 1H;
CHOH); 3.84 (s; 3H; CH3); 2.37 (s; 1H; OH). 13C NMR (100 MHz;
CDCl3) d: 171.2; 164.0; 148.2; 145.0; 144.6; 143.5; 63.8; 55.4; 53.6.
IR (cm-1; KBr): 3384 (O-H); 1743 (C(O)OCH3); 1674 (C(O)NR).
[a]D30: + 33.0 (c = 1.0, CHCl3). HRMS-ESI (m/z): [M + Na]+ calcd
for C9H11N3O4· Na+, 248.0647; found, 248.0636.
located in difference maps, and then treated as riding atoms. The H
atoms bonded to C atoms were placed geometrically, with Uiso(H) =
1.5Ueq(C) for methyl groups and Uiso(H) = 1.2Ueq(C) for others. The
H atoms bonded to N and O atoms were allowed to ride at the sites
located from difference maps with iso(H) = 1.2Ueq(N) and Uiso(H)
= 1.2Ueq(O) respectively. PLATON was used for the data analysis,
including conformational and H-bonding analysis, was performed
using PLATON.12 The program ORTEP-3 for Windows was used
to obtain the figures.23 Crystal data and structure refinement details
are listed in Table 2. “CCDC 633020 contains the supplementary
crystallographic data for this paper. These data can be obtained free
of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif”.
Methyl 2-[(pyrazinecarbonyl)amino]acrylate (8): Yield: 30%.
1
m.p. 95.5°C. H NMR (500 MHz, CDCl3) d: 10.2 (s; 1H; NH); 9.44
(s; 1H; H3); 8.80(d; J = 2; 1H; H5 or H6); 8.61 (d; J = 2; 1H; H5 or
H6); 6.85 (s; 1H; CCH'); 6.07 (s; 1H; CCH); 3.92 (s; 3H; CH3). 13C
NMR (100 MHz; CDCl3) d: 163.6; 161.0; 147.1; 143.9; 143.6; 142.2;
130.3; 109.4; 52.5. IR (cm-1; KBr): 1744 C(O)OCH3; 1636 (C(O)NR).
HRMS-ESI (m/z): [M + H]+ calcd for C9H9N3O3· H+, 208.0722; found,
208.0728.
Biological testing
1,3-Dicyclohexyl-1-(pyrazinecarbonyl)urea (9): To
a stirred
solution of pyrazinecarboxylic acid (500 mg, 4.1 mmol) in anhydrous
CH2Cl2 (25 ml) were added DCC (645 mg, 3.15 mmol) and HOBt,
1-hydroxybenzotriazole (13 mg, 0.1 mmol). After 2 h at room
temperature, the precipitate of 1,3-dicyclohexylurea was removed by
filtration, and the filtrate was poured into saturated aqueous NaHCO3
solution (20 ml) and extracted with EtOAc (3 ¥ 20 ml). The combined
organic layers were dried ( MgSO4), filtered and concentrated.
The residue was chromatographed (10%→50% EtOAc/hexanes)
providing compound 9 (707 mg, 68%).
The antimycobacterial activity to the compounds was assessed against
M. tuberculosis ATTC 27294 using the micro plate Alamar Blue assay
(MABA).17 This methodology is nontoxic, temperature–stable reagent
and show good correlation with proportional method and BACTEC
radiometric method.24-25 Sterile deionised water (200 μl) were added
to all outer-perimeter wells of sterile 96 well plates (Falcon, 3072:
Becton Dickinson, Lincoln Park, NJ, USA) to minimise evaporation
of the medium in the test wells during incubation. The 96 plates
received 100ml of the Middlebrook 7H9 broth (Difco laboratories,
Detroit, MI, USA) and a serial dilution of the compounds 9–16 was
made directly on the plate. The final drug concentrations tested were
between 0.01 and 2.0 ml/ml. Plates were covered and sealed with
parafilm and incubated at 37°C for 5 days. After this time, 25 ml of a
freshly prepared 1:1 mixture of Alamar Blue (Accumed International,
Westlake, Ohio, USA) reagent and 10% tween 80 was added to the
plate and incubated for 24 h. A blue colour in the well was interpreted
as no bacterial growth, and pink colour was scored as growth. The
MIC (Minimal Inhibition Concentration) is defined as the lowest drug
concentration, which prevents the colour change from blue to pink.
1
Yield: 68%. m.p. 164.5°C. H NMR (400 MHz, CDCl3) d: 8.99
(d; J = 1.6; 1H; H3); 8.65 (d; J = 2.4; 1H; H6); 8.51 (dd; J = 2.4 and
1.6; 1H; H5); 5.94 (s; 1H; NH); 4.22 (m; 1H; NCH); 3.56 (m; 1H;
NHCH); 1–2 (m; 20H; cyclohexyl). 13C NMR (100 MHz; CDCl3) d:
166.7; 154.2; 149.6; 146.6; 145.4; 143.2; 57.2; 50.6; 33.1; 31.4; 26.8;
26.0; 25.9; 25.3. IR (cm-1; KBr): 1698 (CONH) and 1677 (CON).
HRMS-ESI (m/z): [M + Na]+ calcd for C18H26N4O2· Na+, 353.1953;
found, 353.1967.
Crystallography
The crystals of 8 were grown from EtOH. The intensity data
were collected at 120K using synchrotron radiation on a Bruker
SMART APEX2 CCD diffractometer at Daresbury SRS station 9.8.
The process of data collection was accomplished by means of the
program Bruker APEX2.18 Cell refinement and data reduction were
accomplished by the program BrukerSAINT.19 The structures were
solved by direct methods in SHELXS-9720 within the OSCAIL
suite of programs21 and refined in SHELXL-9722. All H atoms were
We are indebted to Prof W. Clegg and the Synchrotron
component of the EPSRC X-ray service for data collection.
Received 10 January 2007; accepted 30 March 2007
Paper 07/4408
doi: 10.3184/030823407X200001
Table 2 Crystal data and structure refinement for 8
References
Empirical formula
Formula weight
Temperature, K
Wavelength, Å
Crystal system, space group
Unit cell dimensions
a, Å
C9H9N3O3
207.19
1
http: //www.who.int/tb/en/
2
Y. Zhang, M.M. Wade and A. Scorpio. J. Antimicrob. Chemoth., 2003,
52, 790.
120(2)
0.69050
Monoclinic, C2/c
3
4
5
6
S.S. Brown and R.J. Wade. J. Am. Chem. Soc., 1962, 84, 3280.
E. Baer, J. Maurukas and D. Clarke, Canad. J. Chem., 1956, 34, 1182.
A.J. Kolar and R.K. Olsen, Synthesis, 1977, 457.
T.C. Smale and S. Bailey, GBP 1354571/1940530, 1974; Chem. Abs.,
1974, 81, 63353.
41.838(7)
b, Å
3.7273(6)
c, Å
24.707(4)
7
8
K. Goodall and A.F. Parsons, Tetrahedron Lett., 1995, 36, 3259.
P.M.T. Ferriera, H.L.S. Maia and L.S. Monteiro, Eur. J. Org. Chem., 2003,
2635.
b, °
101.835(2)
3771.0(11)
16
Volume, Å3
Z
9
S. Meunier, P. Cristau and F. Taran, Synthesis, 2005, 2415.
Density (calculated), Mg/m3
Absorption coefficient, mm-1
F(000)
1.460
10 C. Najera, Synlet, 2002, 1388.
11 U. Schmidt, A. Lieberknecht and J. Wild, Synthesis, 1988, 159.
12 A.L. Spek, J. Appl. Crystallogr., 2003, 36, 7.
13 S.A. Hall and P.E. Spoerri, J. Am. Chem. Soc., 1940, 62, 664.
14 M. Slebioda, Tetrahedron, 1995, 51, 7829.
15 D.F. DeTar and R. Silverstein, J. Am. Chem. Soc., 1966, 88, 1013.
16 A. Ljundqvist and K. Folkers, Acta Scand. B., 1988, 42, 408.
17 S.G. Franzblau, R.S. Witzig, J.C. McLaughlin, P. Torres, G. Madico,
A. Hernandez, M.T. Degnan, M.B. Cook, V.K. Quenzer, R.M. Ferguson
and R.H. Gilman, J. Clin Microbiol, 1998, 36, 362.
0.113
1728
Crystal size, mm
Theta range for data collection
Index ranges, °
0.10 × 0.05 × 0.05
2.90 to 29.55
–58< = h < = 58
–5< = k < = 5
–35< = l < = 28
14573
Reflections collected
Independent reflections
Reflections observed (>2 sigma)
Data Completeness
5391 [R(int) = 0.0567]
3975
18 APEX2. Bruker AXS Inc., Madison, USA, 2004.
19 SAINT. Bruker AXS Inc., Madison, USA, 2004.
0.934
None
20 G.M. Sheldrick, SHELXS-97. University of Göttingen, Germany, 1997.
21 P. McArdle, OSCAIL for Windows, National University of Ireland,
Galway, Ireland, 2002.
22 G.M. Sheldrick, SHELXL-97. University of Göttingen, Germany, 1997.
23 L.J. Farrugia, J. Appl. Crystallogr., 1997, 30, 565.
24 J.D. Vanitha and C.N. Paramasivan, Mycobacteriology, 2004, 49, 179.
25 R.S. Reis, I. Neves Jr., S.L.S. Lourenço, L.S. Fonseca and M.C.S. Lourenço,
J. Clin. Microbiol., 2004, 42, 2247.
Absorption correction
Refinement method
Full-matrix least-squares
on F2
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2 sigma(I)]
R indices (all data)
5391/0/292
1.029
R1 = 0.0546 wR2 = 0.1452
R1 = 0.0745 wR2 = 0.1596
0.309 and –0.265
Largest diff. peak and hole, e. Å3
26 A.M. Mattocks, W.H. Hartung. J. Biol. Chem., 1946, 165, 501.
PAPER: 07/4408