Preparation of three impurities in cefoxitin

Article abstract of DOI:10.3184/174751918X15402956899595

Three known impurities of cefoxitin have been prepared and characterised. One is a double-bond isomer of cefoxitin, ?³-cefoxitin, which was easily prepared by base-catalysed isomerisation of cefoxitin. The second is a side-chain methoxylated derivative, methoxycefoxitin, which required a four-step synthesis from cephalothin. The last impurity is a lactone lacking the carbamyl grouping in cefoxitin, cefoxitin lactone, which was also prepared in four steps from cephalothin. The accessibility of these three impurities and methods for their analysis could be important for quality control in the manufacture of cefoxitin.

Full text of DOI:10.3184/174751918X15402956899595

JOURNAL OF CHEMICAL RESEARCH 2018
VOL. 42 NOVEMBER, 541–546
RESEARCH PAPER 541
Preparation of three impurities in cefoxitin
Jun Yu, Luo-bing Jiang, Wei-dong Feng, Chuan-meng Zhao and Fu-li Zhang*
Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, 285 Gebaini Road, Pudong, Shanghai 201203, P.R. China
3
Three known impurities of cefoxitin have been prepared and characterised. One is a double-bond isomer of cefoxitin, ∆ -cefoxitin, which
was easily prepared by base-catalysed isomerisation of cefoxitin. The second is a side-chain methoxylated derivative, methoxycefoxitin,
which required a four-step synthesis from cephalothin. The last impurity is a lactone lacking the carbamyl grouping in cefoxitin, cefoxitin
lactone, which was also prepared in four steps from cephalothin. The accessibility of these three impurities and methods for their analysis
could be important for quality control in the manufacture of cefoxitin.
3
Keywords: cefoxitin impurity, ∆ -cefoxitin, cefoxitin lactone, methoxycefoxitin
1
13
Cefoxitin (1, Fig. 1), a second-generation cephalosporin
antibiotic, shows excellent efficacy due to its long-acting and
broad-spectrum antibacterial properties as well as less potential
their characterisation by H NMR, C NMR and 2D-NMR
spectroscopy.
1,2
Results and discussion
Preparation and characterisation of impurity ∆ -cefoxitin 2
Rao and co-workers reported that treatment of natural
∆ -cephalosporin derivatives with triethylamine in methanol
nephrotoxicity and ototoxicity. The mechanism of action of
cefoxitin is similar to other second-generation cephalosporins.
However, the methoxy group of cefoxitin significantly reduces
3
8
3
3
the hydrolysis by β-lactamases in bacteria. Therefore, the drug-
resistance problem is much less serious than for other second-
generation cephalosporins.
converted them into an equilibrium mixture of the natural
3
∆ -cephalosporin
derivatives
and
the
corresponding
2
8
As treatment of bacterial infections requires a large dose
∆ -cephalosporin derivatives. Using this methodology, we
found that cefoxitin 1 was isomerised at room temperature to an
equilibrium mixture of 1 and ∆ -cefoxitin 2 (Scheme 1). High
performance liquid chromatography (HPLC) analysis was used to
establish the ratio of 1 and 2 as 8:1 (Fig. 2), and preparative HPLC
of the mixture permitted the isolation of a pure sample of 2. The
HRMS spectrum of impurity 2 showed a protonated molecular
peak at 450.0404 [M + Na] in positive-ion mode, indicating the
mass of this impurity to be 427.04, which is the same mass as 1. The
isolated impurity 2 and cefoxitin 1 were subjected to 1D NMR ( H,
C and DEPT) and 2D NMR (HSQC and HMBC) analysis. As
an isomer of 1, impurity 2 has many closely comparable chemical
shift values, but some are different (see Table 1 for H and
assignments of 1 and 2). As expected, the most dramatic changes
affect carbons 9, 10 and 11 and the attached protons. In the H NMR
spectrum of the isomer 2, the double-bond proton at H11 and the
vinylic proton at H9 were observed at δ 6.25 (1H) and 4.80 (1H)
ppm, respectively, whereas in cefoxitin 1 no proton was observed
at C9 and H11 showed two geminally coupled signals (J = 18 ppm)
(
adult daily i.v. dose is 3 g), impurities related to the active
4
3
pharmaceutical ingredient is a significant concern. The
structures of seven impurities for cefoxitin appeared in the
European Pharmacopoeia 9th edition (2016). Vundavilli and
5
co-workers also reported an impurity in cefoxitin resulting
6
from stress stability studies. Owing to the poor stability of
+
cephalosporins, the synthesis of impurities can be difficult.
The structures and simplified names of three of the impurities,
1
2
, 3 and 4, are shown in Fig 1, none of which are available
13
commercially and no preparation method has been reported.
Impurity 2 is a double-bond isomer of cefoxitin, impurity 3 is a
side-chain methoxylated derivative and impurity 4 is a lactone
derived by ring closure of the 10-hydroxymethyl derivative
formed by cleavage of the carbamate residue of cefoxitin.
According to the guidelines on impurities in drug substances
1
13
C
1
7
suggested by the International Conference on Harmonisation,
it is necessary to have samples of these impurities in pure
form for analytical method development. Herein, we report
methods for the preparation of impurities 2, 3 and 4 and
13
at δ 3.55 and 3.21 ppm due to a methylene group. In the C NMR
16
1
6
COOH
O
13
COOH
O
O
9
8
10
1
3
14
NH2
N
12
O
O
9
8
H
7
N
12
O
14 NH2
5
1
0
N
6
11
H
7
S
5
N
4
6
11
S
O
H
15
S
4
O
S
O
H
1
3
2
O
15
1
2
3
1
2
O
13
17
O
COOH
O
O
9
14
13
8
O
9
8
N
12
1
0
14 NH2
15
N
O
7
1
0
O
H
N
5
3
6HN
7
12
11
S
8
6
11
4
S
S
O
H
4
5
S
O
H
15
O
O
16
1
1
3
4
2
2
3
3
Fig. 1 Chemical structures of cefoxitin 1 and three impurities, ∆ -cefoxitin 2, (R,S)-5-methoxycefoxitin 3 and cefoxitin lactone 4.
*
Correspondent. E-mail: zhangfuli1@sinopharm.com

5
42 JOURNAL OF CHEMICAL RESEARCH 2018
9
spectrum of isomer 2, the C11 and C9 signals were observed at
using a reported method, it was found that a by-product 7
2
3
δ 120.5 and 54.9 ppm due, respectively, to sp and sp carbons
was easily generated (Scheme 2). As the amount of t-butyl
10
in contrast to those of cefoxitin 1, which appeared at δ 25.2 and
hypochlorite was increased, the yield of by-product 7 increased
simultaneously. The molecular weight of 7 was determined by
liquid chromatography–mass spectrometry (LC-MS), which
3
2
1
15.3 ppm, due, respectively, to sp and sp carbons. The above
data and the results of 1D NMR and 2D NMR experiments
confirmed the structure of impurity 2.
+
displayed a protonated molecular ion peak at m/z 474 [M + NH4]
Preparation and characterisation of impurity 3
in positive-ion mode, indicating the mass of this by-product to be
456, which is 30 amu more than 6. The extra 30 amu is equivalent
to a methoxy group. The exact structure of by-product 7 was
During optimisation of the synthesis of the 7-methoxylate-
dintermediate 6 from the known compound, cephalothin 5,
O
COOH
O
H
COOH
O
O
N
O
NH2
N
H
O
NH2
Triethylamine
MeOH, r.t.
H
H
N
N
S
S
S
S
O
H
O
O
O
2
1
Scheme 1
Fig. 2 A typical trace of a HPLC run (for eluting conditions, see Experimental), which shows the separation of cefoxitin 1 and isomeric impurity 2.
1
13
Table 1 H and C NMR data and the results of DEPT experiments for 1 and impurity 2
1
2
a
Position
1
13
1
13
H
J (Hz)
C
DEPT
CH
H
J (Hz)
dd, J = 1.2 Hz, J = 5.4 Hz, 1H
C
DEPT
CH
1
2
7.30
dd, J = 1.2 Hz, J = 5.4 Hz, 1H
124.5
7.29
126.5
128.3,
128.5
1
2
1
2
1
26.3,
126.5
–
135.9
–
172.6
95.1
159.8
115.3
132.5
25.2
6.97
dd, J = 3.6 Hz, J = 5.4 Hz, 1H
CH
6.97
dd, J = 3.6 Hz, J = 5.4 Hz, 1H
CH
1
2
1
2
3
4
5
6
7
8
9
7.02
m, 1H
–
–
CH2
–
–
–
–
–
CH2
7.01
–
3.91
–
–
–
4.80
–
6.25
m, 1H
–
–
–
CH2
–
–
–
CH
–
CH
–
3.86
–
–
–
–
–
137.5
38.0
174.7
97.4
162.6
54.9
124.3
120.5
dd, J = 15.0 Hz, J = 33.0 Hz, 2H
dd, J = 15.6 Hz, J = 44.4 Hz, 2H
1
2
1
2
–
–
–
–
–
–
–
–
–
d, J = 1.2 Hz, 1H
–
d, J = 1.2 Hz, 1H
10
–
3
3
.55
.21
d, J = 18.0 Hz, 1H
d, J = 17.4 Hz, 1H
11
1
2
5.05
4.85
4.77
–
3.49
–
s, 1H
d, J = 12.0 Hz, 1H
d, J = 12.0 Hz, 1H
–
s, 3H
–
63.4
63.8
63.8
158.4
52.1
167.4
CH
CH2
CH2
–
CH3
–
5.48
4.77
4.62
–
3.49
–
d, J = 1.2 Hz, 1H
d, J = 12.0 Hz, 1H
d, J = 12.6 Hz, 1H
–
s, 1H
–
61.5
67.9
67.9
160.0
54.3
174.0
–
CH2
CH2
–
CH3
–
1
3
1
3
1
4
1
5
16
a
s, singlet; d, doublet; dd, doublet of doublet; m, multiplet; for numbering, see chemical structure in Fig. 1.

JOURNAL OF CHEMICAL RESEARCH 2018 543
Scheme 2
Scheme 3
1
13
characterised by H NMR and C NMR, confirming that the
introduced methoxy group was located at C5.
switch to tetrahydrofuran. Then chlorosulfonyl isocyanate
underwent a nucleophilic addition with a slurry of intermediate
10 in tetrahydrofuran at −40 °C to yield the corresponding
N-chlorosulfonyl carbamate, further hydrolysis of which gave
crude 3. Crude 3 was subjected to semi-preparative liquid
chromatography and the fractions collected were lyophilised to
remove the solvent to obtain 3 with a purity of 96%.
11–13
According to literature,
a possible mechanism for
the formation of by-product 7 was proposed (Scheme 2).
Intermediate 5 undergoes N-chlorination of the amide bond
and chlorination of the thienobenzyl side chain by t-butyl
hypochlorite to generate 8, which suffers elimination of HCl
−
from the chloroamide and nucleophilic substitution by MeO of
The high-resolution mass spectrum of 3 showed a molecular
+
the side-chain Cl group to give the acylimine intermediate 9.
The strongly electrophilic acylimine 9 readily adds methanol to
afford by-product 7. By-product 7 was a key intermediate in the
synthesis of impurity 3 described below.
ion peak at m/z 480.0511 [M + Na] in positive-ion mode,
indicating the mass of 3 to be 457.05, which is 30 amu more
than cefoxitin (1). The characterisation of impurity 3 was
1
13
achieved by 1D NMR ( H NMR, C NMR and DEPT) and 2D
9
1
13
Based on the reported synthesis route of cefoxitin (1),
NMR (HSQC and HMBC) analysis (see Table 2 for H and C
impurity 3 was prepared in four steps starting from cephalothin
assignments). Compared with cefoxitin (1), impurity 3 showed
(
5) (Scheme 3). Compound 5 was methoxylated with 2.5 equiv.
one more methoxy group (–OCH ). In the HMBC spectrum,
3
of t-butyl hypochlorite and 6 equiv. of sodium methoxide
at −75 °C to give 7. Intermediate 7 was treated with sodium
hydroxide in a mixture of methanol and water at −30 °C to
afford hydroxymethylated derivative 10. The intermediate 10 is
extremely unstable under acidic conditions, readily cyclising to
a lactone. We tried to apply the cefoxitin synthetic procedure
the C15 protons at δ 3.47 ppm are coupled with C5 (δ 79.3 and
78.7 ppm), since impurity 3 is a pair of diastereomers and H5
appears as two signals (δ 5.14 and 5.10 ppm). The protons at δ
3.52 and 3.43 ppm are coupled with C7 (δ 95.7 and 95.6 ppm)
and the C5 protons at δ 5.14 and 5.10 ppm are coupled with C1,
C2, C3, C4 and C6 and C15, respectively. The C13 protons at
9
1
of salt precipitation , but failed. After the base hydrolysis was
δ 5.05 ppm are coupled with C7, C8 and C11. In the H NMR
completed, the reaction mixture was adjusted to pH 6–7 by acetic
acid to prevent the formation of lactone. Removal of methanol
and water under reduced pressure was followed by a solvent
spectrum, the protons of the thiophene ring appeared at δ 7.45–
7.03 ppm (3H). In the DEPT spectrum of 3, the negative-ion
peaks of methylene carbons at δ 62.7 and 25.7 ppm correlated

5
44 JOURNAL OF CHEMICAL RESEARCH 2018
1
13
Table 2 H and C NMR data and the results of COSY, DEPT, HSQC and HMBC experiments for impurity 3
a
1
13
Position
H
J (Hz)
m, 1H
m, 1H
m, 1H
–
s, 1H
–
–
COSY
H2
H1, H3
C
DEPT
CH
CH
CH
–
CH
–
–
HMBC
HSQC
1
2
3
4
5
6
7
8
9
7.45
7.03
7.24
–
5.14, 5.10
–
–
–
C2, C3, C4
C1, C3, C4
C1, C2, C4, C5
–
C1, C2, C3, C4, C6, C15 79.3, 78.7
–
–
–
–
–
C9, C10, C12, C13
C9, C10, C12, C13
C7, C8, C11
C9, C10, C11, C14
C9, C10, C11, C14
–
C5
C7
–
1
26.6, 126.4
126.6, 126.4
126.3, 126.2
1
26.3, 126.2
H2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
127.3, 127.1
139.0
79.3, 78.7
172.0
95.7, 95.6
160.5
128.9, 128.5
123.5
25.7
25.7
64.2
62.7
62.7
158.0
56.5, 56.4
52.6, 52.5
163.6
127.3, 127.1
–
–
–
–
–
–
–
–
–
–
–
25.7
25.7
64.2
62.7
62.7
–
–
–
1
0
1
1
3.59–3.55
3.37–3.30
5.08
5.05
4.84
–
3.47
3.46, 3.43
–
m, 1H
m, 1H
CH2
CH2
CH
CH2
CH2
–
CH3
CH3
–
1
1
1
2
d, J = 1.2 Hz, 1H
dd, J = 2.4 Hz, J = 13.8 Hz,1H
1
3
1
2
1
3
d, J = 4.2 Hz, J = 13.2 Hz, 1H
1
2
1
4
–
1
5
s, 3H
s, 3H
–
56.5, 56.4
52.6. 52.5
–
1
6
17
a
s, singlet; d, doublet; dd, doublet of doublet; m, multiplet; for numbering, see chemical structure in Fig. 1.
Scheme 4
with C13 and C11, respectively. The above data and the 1D
NMR and 2D NMR experiments confirmed the structure of 3.
Since impurity 3 was a pair of diastereomers, HPLC analysis
showed two peaks.
spectrum of 4, the negative-ion peaks of methylene carbons at δ
71.9, 36.3 and 23.3 ppm were assigned, respectively, to C14, C5
and C11. In addition, the FTIR spectrum of impurity 4 exhibited
three strong characteristic absorption bands at ν = 1789, 1751 and
−1
1
666 cm indicating the presence of three carbonyl groups.
Preparation and characterisation of impurity 4
To determine ways of improving the synthetic pathway of an
active pharmaceutical ingredient, it is necessary to identify the
impurities formed. The preparation of three impurities of cefoxitin
described in this paper has never been reported. In addition, these
impurities were characterised by NMR and HRMS spectral data.
This work will be useful for quality control in the manufacture of
cefoxitin.
During our studies of the hydrolysis of 11 to 12 by a reported
method, we found that the hydroxymethyl acid 12 was easily
converted to lactone impurity 4 at pH 0–2. Therefore, we carried
out the hydrolysis of 12 in a mixture of methanol and 10% HCl at
room temperature and pH 0–2 for 5 h to transform 12 into impurity
9
4
1
completely (Scheme 4). To obtain high purity 4, the precursor of
2, the methoxylated intermediate 6 was purified by formation of
its cyclohexylamine salt 11, which was isolated as a pale yellow
solid. The crude 4 was purified by column chromatography to
yield a pale yellow solid (m.p. 161–162 °C). The HRMS spectrum
Experimental
Chemical, reagents and samples
+
Cefalothin (5) and cefoxitin (1) were procured from Hubei Jusheng
Technology Co., Ltd. Analytical reagents formic acid, acetic acid,
ammonium acetate and HPLC grade acetonitrile and ethanol were
procured from Shanghai Titan Technology Co., Ltd. Conventional solvents
and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.
of 4 exhibited a molecular ion peak at m/z 389.0240 [M + Na] in
1
positive-ion mode, indicating the mass of 4 to be 366. H NMR and
13
C NMR analyses revealed that the impurity 4 had one amide N–H
proton (s, 9.54 ppm, 1H) and three well-resolved carbonyl carbon
signals were observed at δ 170.1, 166.5 and 159.6 ppm. The proton
signals of the thiophene ring appeared at δ 7.38 (1H) and 6.97–6.94
High-performance liquid chromatography
(
2H) ppm, and the carbon signals at δ 125.6, 126.9 and 127.1 ppm,
All reactions were monitored by HPLC on a Dionex Ultimate 3000 HPLC
instrument using Shiseido ACR C18 (5 μm, 4.6 mm × 250 mm). The
which are characteristic of thiophene carbon signals. In the DEPT

JOURNAL OF CHEMICAL RESEARCH 2018 545
−
1
mobile phase consisting of A (100 mmol L NH OAc, pH 5.9, adjusted
reaction mixture was cooled to −70 ± 5 °C in a liquid nitrogen bath, and
4
−1
by acetic acid) and B (acetonitrile:ethanol = 15:7) was used in the gradient
then sodium methoxide solution (5.4 mol L ; 84.1 mL, 454.1 mmol) was
added into the solution at −70 ± 5 °C. Next, t-butyl hypochlorite (20.5 g,
188.8 mmol) was slowly added dropwise to control the temperature at −70
± 5 °C. After the reaction was completed, the reaction mixture was treated
with sodium metabisulfite (28.7 g, 107.8 mmol) and stirred for 10 min at
−70 °C. The pH value of the solution was adjusted to 6–7 at −60 °C with
acetic acid and then further adjusted to pH 1–2 with 10% hydrochloric
acid at 0–5 °C. The aqueous layer was separated and was extracted again
with dichloromethane (100 mL). The combined organic phase was washed
once with water (100 mL) and the organic phase was treated with saturated
sodium bicarbonate solution to adjust the aqueous phase to pH 7–8. The
organic phase was discarded. The obtained aqueous phase was adjusted
carefully to pH 1–2 with 10% hydrochloric acid and extracted with ethyl
acetate (200 mL). The organic phase was concentrated to generate 7 a light
brown oil, which was not further purified for the next step; yield 24.6 g;
−1
mode at a flow rate of 1.0 mL min . The column was thermostated at a
temperature of 40 °C and UV detection at 254 nm was used. The initial
gradient was 10% of mobile phase B and at 10 min it was set to 15%. The
ratio was set to 20% at 15 min, and then 35% at 30 min, followed by the
ratio of mobile phase B set to 10% at 40 min, which was continued for
50 min. HPLC purity was reported in area %.
Preparative liquid chromatography
Preparative HPLC separation and fraction collections were carried out
using a Waters 2767-2454 preparative liquid chromatograph equipped
with a 2996 PDA detector. The sample solutions (acetonitrile:water =
−1
1
:1), about 40 mg mL concentration, were prepared in diluent. The
column, a Waters Xbridge C18 with 150 mm × 19 mm, 5 μm particle size
was used for chromatographic separation. A 0.1% aqueous solution of
trifluoroacetic acid and acetonitrile were used as solvent A and solvent
B, respectively, as the mobile phase. A flow rate of 15 mL min was
employed throughout the process and the wavelength of detection in UV
was 254 nm. The linear gradient programmes were set as time in minutes
1
H NMR (600 MHz, CDCl ): δ 7.63 (d, J = 21.6 Hz, 1H), 7.27–7.25 (m,
−1
3
1
H), 7.11 (dd, J = 3 Hz, J = 17.4 Hz, 1H), 6.95–6.92 (m, 1H), 5.06–5.01
1
2
(
7
m, 3H), 4.95 (d, J = 7.8 Hz, 1H), 4.88 (d, J = 14.4 Hz, 1H), 3.52–3.35 (m,
H), 3.29–3.19 (m, 1H), 2.02 (d, 3H, J = 4.2 Hz, 3H); C NMR (150 MHz,
13
(
Tmin)/solvent A:solvent B T0.01/90:10, T /40:60, T /05:95 and T /05:95 for
10 11 15
CDCl ): δ 170.0, 169.8, 162.1, 159.7, 130.1, 129.2, 126.8, 126.3, 126.0,
3
the isolation of impurities 2 and 3. The crude sample was dissolved in 50%
125.9, 125.5, 125.4, 124.6, 124.5, 93.9, 78.7, 78.4, 63.4, 63.3, 61.8, 56.7, 56.4,
−1
acetonitrile aqueous solution (concentration of 40 mg mL ). The solution
was loaded into the preparative liquid chromatograph using the conditions
mentioned in the above analytical liquid chromatography. Fractions
were collected through repeated injections. The collected fractions were
lyophilised to afford the pure products. The fractions containing impurity
were collected at retention time 9.4 min and those containing impurity
were collected at retention time 8.7 min. The respective fractions were
combined and the solvent removed using a lyophiliser (Labconco®
5
2.9, 52.7, 25.8, 19.7; MS (ESI) m/z: 474.14 [M + H O].
2
Sodium hydroxide solution (6.3 g NaOH dissolved in 40 mL water)
was added at −30 °C to a mixture of water (50 mL), methanol (100 mL)
and crude 7 (24.0 g) cooled to −30 °C, and the solution was stirred for
3
6
3
h. After completion of the hydrolysis reaction, the pH was adjusted to
–7 using acetic acid at −30 °C. The reaction temperature was raised to
0 °C. The solvent was distilled off under reduced pressure, followed
2
3
by a solvent switch to tetrahydrofuran. Anhydrous magnesium sulfate
was used to dry the tetrahydrofuran solution of compound 10. The
solvent was concentrated under reduced pressure to obtain a solid
FreeZone® Plus™ 12 L) to obtain impurities 2 and 3.
NMR, LC-MS, MS, IR and melting points apparatus
(30.0 g), which was directly used in the next step without purification.
NMR spectra were obtained on a Bruker Avance III spectrometer
H NMR at 600 Hz, C NMR at 150 Hz). Chemical shifts (δ) are given
in ppm and coupling constants (J) are given in Hertz (Hz). The solvents
used were CDCl , CD OD or DMSO-d . The mass spectra and high-
1
13
Crude compound 10 (30.0 g) in tetrahydrofuran was cooled to −40 °C
(
under liquid nitrogen, and a solution of chlorosulfonyl isocyanate (22.3 g,
57.6 mmol) was slowly added at −40 °C. The reaction was monitored by
1
3
3
6
HPLC. When the reaction was completed, the reaction mixture was added
into ice water and stirred for 3 h. Tetrahydrofuran was removed under
reduced pressure. The product was extracted with ethyl acetate and the
aqueous phase discarded. The organic layer was washed with saturated
sodium chloride solution. The organic phase was treated with sodium
bicarbonate solution to adjust the aqueous phase to pH 7–8. The obtained
aqueous phase was adjusted carefully to pH 1–2 with 10% hydrochloric
acid and extracted into ethyl acetate. Ethyl acetate was distilled off under
reduced pressure and crude 3 [yield 11.1 g (32%)] was obtained. The crude
3 (1.0 g) was further purified by semi-preparative liquid chromatography
to obtain 3 as a pale yellow solid; purity 96%; yield 400 mg; m.p.
resolution mass spectra were recorded on an Aglient 6210B series single
quadrupole LC-MS and a Q-Tof micro YA019 instrument. Melting points
were measured on a WRS-1B apparatus. FTIR spectra were recorded in
the solid state using attenuated total reflectance method on IRT racer-100
(Shimadzu Corporation, Kyoto, Japan).
Synthesis of (2R,6R,7S)-3-((carbamoyloxy)methyl)-7-methoxy-8-oxo-
7-(2-(thiophen-2-yl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-3-ene-2-
carboxylic acid (2); general procedure
Triethylamine (4.7 g, 46.5 mmol) was added to a solution of cefoxitin
1
(10.0 g, 23.4 mmol) in methanol (100 mL) and stirred for 24 h at room
1
temperature. HPLC showed about 13% of 1 had been converted to the
double-bond isomer 2. The solvent of the reaction mixture was removed
under reduced pressure at 25 °C. The residue was dissolved in ethyl acetate
53–55 °C ; H NMR (600 MHz, CD OD): δ 7.46–7.44 (m, 1H), 7.24–7.22
3
(m, 1H), 7.04–7.02 (m, 1H), 5.14 (s, 0.5H), 5.10 (s, 0.5H), 5.08 (d, J = 1.2
Hz, 1H), 5.05 (dd, J = 2.4 Hz, J = 13.8 Hz, 1H), 4.84 (dd, J = 4.2 Hz,
1
2
1
(
150 mL) and washed with 5% aqueous hydrochloric acid (100 mL) to
remove triethylamine. The ethyl acetate layer was washed with water
80 mL) and concentrated under reduced pressure to remove organic
J = 13.2 Hz, 1H), 3.59–3.55 (m, 1H), 3.52 (s, 1.5H), 3.47 (d, J = 4.2 Hz,
2
13
3H), 3.43 (s, 1.5H), 3.37–3.30 (m, 1H); C NMR (150 MHz, CD OD): δ
3
(
172.0, 163.6, 160.5, 158.0, 139.0, 128.9, 128.5, 127.3, 127.1, 126.6, 126.4,
solvent. The crude oil was further purified by semi-preparative liquid
126.3, 126.2, 123.5, 95.7, 95.6, 79.3, 78.7, 64.2, 62.7, 56.5, 56.4, 52.6, 52.5,
+
chromatography to obtain 2 a pale yellow solid; purity 98.8%; yield 800
25.7. HRMS m/z calcd for C H N NaO S [M + Na] : 480.0506; found:
17
19
3
8 2
1
mg (8%); m.p. 125–130 °C; H NMR (600 MHz, CD OD): δ 7.29 (dd,
480.0511.
3
1
1
H, J = 1.2 Hz, J = 5.4 Hz), 7.01 (m, 1H), 6.97 (dd, H, J = 3.6 Hz, J =
1
2
1
2
Synthesis of N-((5aR,6S)-6-methoxy-1,7-dioxo-1,4,5a,6-tetrahydro-
5
.4 Hz), 6.25 (s, 1H), 5.50 (s, 1H), 4.80 (s, 1H), 4.77 (d, J = 12.6 Hz, 1H),
3
H,7H-azeto[2,1-b]furo[3,4-d][1,3]thiazin-6-yl)-2-(thiophen-2-yl)
13
4.62 (d, J = 12.6 Hz, 1H), 3.87 (q, J = 15.6 Hz, 2H), 3.50 (s, 3H); C NMR
acetamide (4); general procedure
(
150 MHz, CD OD): δ 174.7, 174.0, 162.6, 160.0, 137.5, 128.5, 128.3,
3
Methylene chloride (100 mL), methanol (10 mL) and cephalothin 5
(10.0 g, 25.5 mmol) were added to a 250 mL three-necked flask. The
1
26.5, 124.3, 120.5, 97.5, 67.9, 61.5, 54.9, 54.3, 38.0. HRMS m/z calcd for
+
C H N O S [M + Na] : 450.0400; found: 450.0404.
1
6
17
3
7 2
reaction mixture was cooled to −70 ± 5 °C in a liquid nitrogen bath, and
then sodium methoxide solution (5.4 mol L−1; 23.6 mL, 127.5 mmol)
was added into the mixture at −80 °C. Next, t-butyl hypochlorite (3.3 g,
30.6 mmol) was slowly added dropwise to control the temperature at
−80 °C. After the reaction was completed, the reaction mixture was
treated with sodium metabisulfite (7.3 g, 38.4 mmol) and stirred for
Synthesis of (6R,7S)-3-((carbamoyloxy)methyl)-7-methoxy-7-((R)-2-
methoxy -2-(thiophen-2-yl)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]
oct-2-ene-2-carboxylic acid (3); general procedure
Methylene chloride (150 mL), methanol (15 mL) and cephalothin 5
(30 g, 75.7 mmol) were added to a 500 mL three-necked flask. The

5
46 JOURNAL OF CHEMICAL RESEARCH 2018
1
0 min at −70 °C. The pH value of the solution was adjusted to 6–7
at −60 °C with acetic acid and then further adjusted to pH 1–2 with
0% hydrochloric acid at 0–5 °C. The aqueous layer was separated and
Acknowledgement
The authors are grateful to the teachers of the China State
Institute of Pharmaceutical Industry for supporting the research.
1
was extracted again with dichloromethane (50 mL). The combined
organic phase was washed once with water (100 mL) and the organic
phase was treated with saturated sodium bicarbonate solution to
adjust the aqueous phase to pH 7–8. The organic phase was discarded.
The obtained aqueous phase was adjusted carefully to pH 1–2 with
Received 20 August 2018; accepted 12 September 2018
Paper 1805585
1
0% hydrochloric acid and extracted with ethyl acetate (100 mL).
https://doi.org/10.3184/174751918X15402956899595
Published online: 8 November 2018
The organic phase was washed with water (50 mL) and dried over
anhydrous sodium sulfate. A solution of cyclohexylamine (3 g,
3
0.2 mmol) in acetone (5 mL) was added to the ethyl acetate extract
References
at 25 °C. Intermediate 11 was precipitated from the reaction solution.
The isolated solid, a cyclohexylamine salt, was collected by filtration
1
J. Kosmidis, J.M. Hamilton, J.N. Gilchrist, D.W. Kerry and W. Brumfitt,
Br. Med. J., 1973, 4, 653.
and dried to yield 11 as a pale yellow solid; yield 10.3 g (78.1%); m.p.
1
1
39–142 °C; H NMR (600 MHz, DMSO-d ): δ 9.39 (s, 1H), 8.22 (br,
6
2
3
H. Fang, C. Edlund, K. Hultenby and M. Hedberg, Anaerobe, 2002, 8, 55.
J. Soki, S.M. Gonzalez, E. Urban, E. Nagy and J.A. Ayala, J. Antimicrob.
Chemother., 2011, 66, 2492.
s, 3H), 7.37 (dd, J = 1.2 Hz, J = 4.8 Hz, 1H), 6.96–6.94 (m, 2H), 4.97
1
2
(s, 1H), 4.86 (d, J = 12.0 Hz, 1H), 4.68 (d, J = 12.0 Hz, 1H), 3.80 (q,
J = 9.0 Hz, 2H), 3.44 (d, J = 18.0 Hz, 1H), 3.34 (s, 1H), 3.07 (d, J = 18.0
Hz, 1H), 2.94–2.90 (m, 1H), 1.99 (s, 3H), 1.91–1.89 (m, 3H), 1.71–1.68
4
Food and Drug Administration, Cefoxitin for injection and dextrose
injection.
https://www.accessdata.fda.gov/drugsatfda_docs/
(
m, 2H), 1.57 (d, J = 13.6 Hz, 1H), 1.30–1.19 (m, 4H), 1.11–1.06 (m,
13
label/2013/065214s013lbl.pdf [accessed 3 November 2018].
Cefoxitin sodium. In European pharmacopoeia (Ph. Eur.) 9th edn. Council
of Europe, Strasbourg, 2016, pp. 1987–1989.
1
1
2
H); C NMR (150 MHz, DMSO-d ): 170.9, 164.1, 159.6, 137.3, 135.4,
6
5
6
27.1, 126.9, 125.2, 111.9, 94.9, 64.6, 62.7, 60.2, 52.7, 49.6, 36.4, 30.9,
5.6, 25.1, 24.3, 21.1, 14.6. HRMS m/z calcd for C H N NaO S [M +
17
18
2
7 2
+
Na] : 449.0448; found: 450.0451.
V.J. Kumar, P.B.K. Gupta, R.B.S Mukkanti, K.S.R.P. Kumar, G.K.A.S.S.
Narayan, Y.S. Somannavar, G.S.S. Kumar and H.K. Sharma, Anal.
Methods, 2011, 3, 181.
Sodium hydroxide solution (1.1 g NaOH dissolved in 20 mL water)
was added at −30 °C to a mixture of water (20 mL), methanol (20 mL)
and crude 11 (5.0 g) cooled to −30 °C, and the solution was stirred for 2
h. After completion of the hydrolysis reaction, the pH was adjusted to
7
ICHharmonisedtripartiteguideline:impuritiesinnewdrugsubstancesQ3A
(
R2). In Proceedings of the International Conference on Harmonization
6
0
–7 using acetic acid at −30 °C. The reaction temperature was raised to
–5 °C. The reaction solution was further adjusted to pH 1–2 with 10%
of Technical Requirements for Registration of Pharmaceuticals for
Human Use, Geneva, Switzerland, 25 October 2006. https://www.ich.org/
fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3A_R2/
Step4/Q3A_R2__Guideline.pdf [accessed 3 November 2018].
K.V.V.P. Rao, R. Dandala, V.K. Handa, A.G. Kamat, I.V.S. Rao, A. Rani and
A. Naidu, J. Heterocycl. Chem., 2007, 44, 1513.
hydrochloric acid and stirred at 5–10 °C for 3 h. The solvent methanol
was removed under reduced pressure and crude 4 extracted with
ethyl acetate (80 mL). Ethyl acetate was concentrated and the residue
was purified by silica gel column chromatography (ethyl acetate:n-
hexane = 1:1) to yield 4 as a pale yellow solid; yield 2.6 g (73.6%); m.p.
8
9
−
1
1
61–162 °C; IR (ν cm ): 1789 (C=O), 1751 (C=O), 1666.5 (C=O);
P.B. Deshpande and B.P. Khadangale, WO Patent: 2004083217, issued 30
September 2004.
1
H NMR (600 MHz, DMSO-d ): δ 9.54 (s, 1H), 7.38 (dd, J = 1.2 Hz, J
6
1
2
=
5.4 Hz, 1H), 6.97–6.94 (m, 2H), 5.17 (s, 1H), 5.02 (s, 2H), 3.82 (q, J =
10
M.J. Mintz and C. Walling, Org. Synth., 1969, 49, 9.
1
3
1
5.6 Hz, 2H), 3.76 (d, J = 18.6 Hz, 1H), 3.65 (d, J = 18.6 Hz, 1H), 3.41 (s,
13
11 G.A. Koppel and R.A. Koehler, J. Am. Chem. Soc., 1973, 95, 2403.
12 R.A. Firestone and B.G. Christensen, J. Org. Chem., 1973, 38, 1436.
13 J.E. Baldwin, R.M. Adlington and N. Moss, Tetrahedron, 1989, 45, 2841.
H); C NMR (150 MHz, DMSO-d ): 170.1, 166.5, 159.6, 142.9, 137.0,
6
27.1, 127.0, 125.6, 123.0, 96.3, 71.9, 63.1, 53.0, 36.3, 23.3. HRMS m/z
+
calcd for C H N NaO S [M + Na] 389.0236; found: 389.0240.
15
14
2
5 2