Synthesis of the β-lactamase indicators Cefesone and Nitrocefin

Article abstract of DOI:10.1055/s-1998-2018

The synthesis of the β-lactamase indicators Cefesone [(6R,7R)-3-(2,4- dinitrostyryl)-7-(phenylacetamido)ceph-3-em-4-carboxylic acid] and Nitrocefin [(6R,7R)-3-(2,4-dinitrostyryl)-7-(2-thienylacetamido)ceph-3-em-4-carboxylic acid] from tert-butyl (1S,6R,7R)-3-bromomethyl-1-oxo-7-(phenylacetamido)ceph- 3-em-4-carboxylate is reported. Phosphonylation of the latter compound with triphenylphosphine gave the corresponding phosphonium derivative in 93% yield. Reduction followed by Wittig coupling with 2,4-dinitrobenzaldehyde gave a 1:12 mixture of E- and Z-isomers in 83% yield. The tert-butyl protecting group was removed with titanium tetrachloride to give Cefesone as the pure crystalline E-isomer in 63% yield. De-acylation was achieved by enzymatic hydrolysis in 77% yield. Finally the 2-thienylacetyl side chain was introduced to give crystalline Nitrocefin in 67% yield.

Full text of DOI:10.1055/s-1998-2018

February 1998
SYNTHESIS
145
b
Synthesis of the -Lactamase Indicators Cefesone and Nitrocefin
Nico C. M. E. Barendse, Pieter A. M. van der Klein, Jan Verweij, Henk A. Witkamp, Wim J. van Zoest, Erik de Vroom*
Gist-brocades B. V., P. O. Box 1, NL-2600 MA Delft, The Netherlands
E-mail: ERIK.DE VROOM@GIST-BROCADES.INFONET.COM
Received 28 July 1997
drolysis of cephalosporin esters is a procedure already
reported for benzyl,5 diphenylmethyl,6 and 4-
methoxybenzyl7 esters using aluminum trichloride.8The
use of tin tetrachloride8 and titanium tetrachloride5, 8 has
also been advocated. However, a high yielding Lewis acid
mediated method for deprotection of cephalosporin tert-
butyl esters has not been reported thus far. In our labora-
tories we developed a procedure to hydrolyze tert-butyl
esters of cephalosporins with titanium tetrachloride.9
Thus, upon treatment of 3 with titanium tetrachloride, the
b-lactamase indicator Cefesone (4) can be obtained after
workup and crystallization in 63% yield as the single E-
isomer with a purity of 99%. It should be noted that during
titanium tetrachloride mediated ester hydrolysis, almost
complete Z to E isomerization takes place.
Abstract: The synthesis of the b-lactamase indicators Cefesone
[(6R,7R)-3-(2,4-dinitrostyryl)-7-(phenylacetamido)ceph-3-em-4-carb-
oxylic acid] and Nitrocefin [(6R,7R)-3-(2,4-dinitrostyryl)-7-(2-
thienylacetamido)ceph-3-em-4-carboxylic acid] from tert-butyl
(1S,6R,7R)-3-bromomethyl-1-oxo-7-(phenylacetamido)ceph-3-em-4-
carboxylate is reported. Phosphonylation of the latter compound with
triphenylphosphine gave the corresponding phosphonium derivative
in 93% yield. Reduction followed by Wittig coupling with 2,4-dini-
trobenzaldehyde gave a 1:12 mixture of E- and Z-isomers in 83%
yield. The tert-butyl protecting group was removed with titanium
tetrachloride to give Cefesone as the pure crystalline E-isomer in
63% yield. De-acylation was achieved by enzymatic hydrolysis in
77% yield. Finally the 2-thienylacetyl side chain was introduced to
give crystalline Nitrocefin in 67% yield.
Key words: Cefesone, cephalosporin, Nitrocefin, b-lactamase indi-
cator, penicillin acylase
Cefesone (4) and Nitrocefin (6) are chromogenic cepha-
losporins useful in the determination of.b-lactamase activi-
ty in biological samples. Upon enzymatic hydrolysis of the
b-lactam amide linkage by ab-lactamase, a yellow colored
solution containing Cefesone or Nitrocefin turns red as a re-
sult of the formation of a highly conjugated compound.
Thus, determination of.b-lactamase activity, of utmost im-
portance in clinical microbiology, is easily achieved in a
visual manner. The application of Cefesone has been de-
scribed by Sutton et al.1Although a synthetic approach was
given by the authors, no experimental details were dis-
closed. To our knowledge, the synthesis of Nitrocefin has
only been performed by workers from the Glaxo Laborato-
ries.2 Following this approach, Nitrocefin can be obtained
starting from the diphenylmethyl ester of the 3¢-phosphoni-
um bromide of Cephalothin in 45% yield.
The phenylacetyl side chain in 4 can be replaced by a 2-
thienylacetyl side chain to produce Nitrocefin (6). Various
methods exist for removal of the phenylacetyl function.
Chemical deacylation3,10 using silyl carboxyl protection,
PCl5 mediated imide chloride formation, and subsequent
alcoholysis, is a commonly accepted method in many syn-
thetic schemes giving satisfactory yields in most exam-
ples. However, this procedure employs toxic chemicals
and solvents at sub-zero temperatures. Fortunately, an at-
tractive alternative may be found in enzymatic hydrolysis
using penicillin acylase (EC 3.5.1.11) under mild condi-
tions. Thus, the phenylacetyl side chain is removed using
immobilized penicillin-G acylase in water at neutral pH.11
The nucleus 5 is obtained by precipitation at pH 3 in 77%
yield.
In the final step, the 2-thienylacetyl side chain is coupled
in a two-step process. Compound 5 is silylated using 1,3-
bis(trimethylsilyl)urea and in the next step 2-thiophene-
acetyl chloride is added. After workup and crystallization
from CH2Cl2, Nitrocefin is obtained as the single E-iso-
mer in 67% yield with a purity of 95%.
In this paper we present a straightforward synthesis of
Cefesone and Nitrocefin starting from readily available
tert-butyl (1S,6R,7R)-3-bromomethyl-1-oxo-7-(phenyl-
acetamido)ceph-3-em-4-carboxylate3 (3-BMC™).
When a suspension of 3-BMC™ in MeOAc/MeOH is
treated with 1.5 equivalents of triphenylphosphine, the
corresponding 3¢-phosphonium bromide 2 is obtained as
white crystals in a yield of 93%. The 7-(2-bromo-2-
phenylacetamido) derivative of 3-BMC™, present in
small quantities in commercial samples, is also converted
into the desired product. The latter phenomenon does not
occur in the absence of MeOH. The debromination reac-
tion is in accordance with the observation of alcohol-pro-
moted dehalogenation of a-carbonyl compounds with
triphenylphosphine.4
The above-mentioned procedure enabled us to repro-
ducibly prepare the chromogenic cephalosporin Cefe-
sone in 49% yield from 3-BMC™ on a multigram scale.
In two additional steps, Nitrocefin can be prepared in
52% yield.
All reagents were of commercial quality. TiCl4 was purchased fromAl-
drich. 1,3-Bis(trimethylsilyl)urea, 2-thiopheneacetyl chloride and PPh3
were purchased from Acros. PCl3 was purchased from Merck. 3-
BMC™ was from Gist-brocades (industrial quality). 2,4-Dinitrobenz-
aldehyde, required for the introductionof the 2,4-dinitrophenyl moiety,
was synthesized in 48% yield from 2,4-dinitrotoluene (Acros) as fol-
lows: according to the procedure developed by Kalir for the synthesis
of 2-nitrobenzaldehyde,12 N-(2,4-dinitrobenzyl)pyridinium bromide
was obtained from 2,4-dinitrotoluene after bromination with NBS in
the presence of benzoyl peroxide followed by reaction with pyridine.
Reaction with N,N-dimethyl-4-nitrosoaniline yielded N-(4-dimethyl-
aminophenyl)-a-2,4-dinitrophenyl nitrone as reported by Behr and co-
workers.13 The nitrone was converted into the desired aldehyde us-
ing the Kröhnke method.14 Immobilized penicillin-G acylase15
In the next step, 2 is reduced with 1.5 equivalents of phos-
phorus trichloride and subsequently coupled with 2,4-
dinitrobenzaldehyde. After purification, the 3¢-alkenyl-
cephalosporin 3 is obtained in 83% yield and consists of a
1:12 mixture of E- and Z-isomers.
Deprotection of the tert-butyl ester is achieved using rela-
tively mild acidic conditions. Lewis acid promoted hy-

146
Short Papers
SYNTHESIS
(150 units/gram; 1 unit corresponds to the amount of enzyme that hy-
drolyzes per minute 1 mmol of penicillin-G potassium salt at a con-
centration of 100 g L-1 in 0.05 M potassium phosphate buffer at pH
8.0 and 28 °C) was repeatedly washed with water before use and
weighed without drying. Mass spectra were obtained with an AMD
402 mass spectrometer.1H spectra were recorded on a Bruker AM
360 MHz instrument. Purities were determined with 1H spectroscopy
using an internal reference.
J2 = 17.7 Hz, CH2P), 5.41 (dd, 1H, J1 = J2 = 14.0 Hz, CH2P), 6.02 (dd,
1H, J6,7 = 4.3 Hz, J7,NH = 10.1 Hz, H7), 6.75 (d, 1H, J = 10.1 Hz, NH),
7.2–7.7 (m, 20H, ArH).
tert-Butyl (6R,7R)-3-(2,4-Dinitrostyryl)-7-(phenylacetamido)-
ceph-3-em-4-carboxylate, Z-Isomer (3):
A stirred solution of 2 (39.72 g, purity 93.9%, 50.0 mmol) in CH2Cl2
(125 mL) was cooled under N2 to –20°C. PCl3 (6.55 mL, 75.1 mmol)
was added at such a rate that the temperature never exceeded –10°C.
After stirring for 1 h at –10°C, the solution was cooled to –55 °C and
slowly added to ice water (250 mL) under vigorous stirring while
maintaining the pH of the aqueous phase at 4.0 with 4 M NaOH. The
organic phase was separated and extracted with ice water (250 mL).
The combined aqueous phases were back-extracted with CH2Cl2
(75 mL). The combined organic phases were concentrated at 20°C
under reduced pressure to a volume of 250 mL. To this solution was
added a solution of 2,4-dinitrobenzaldehyde (29.41 g, purity 95%,
142.5 mmol) in CH2Cl2 (1 L) and 1 M aq NaHCO3 (1240 mL). After
vigorous stirring for 2 h, the phases were separated and the organic
phase was extracted with brine (500 mL), dried (MgSO4), and con-
centrated under reduced pressure to a volume of 100 mL. The solution
of crude 3 thus obtained was purified by short-column chromatogra-
phy on silica gel (565 g) using CH2Cl2 with a 0 to 3% gradient of ac-
etone as elution solvent. The appropriate fractions, as judged by TLC
(CH2Cl2/acetone 10:1, v/v), were pooled and concentrated under re-
duced pressure to give 25.43 g (purity 7% E-isomer, 85% Z-isomer,
41.3 mmol; 83%) of 3 as an orange foam.
tert-Butyl (1S,6R,7R)-1-Oxo-7-(phenylacetamido)-3-(triphenyl-
phosphoniomethyl)ceph-3-em-4-carboxylate Bromide (2):
A suspension of 1 {52.14 g, purity 87.3%, 94.2 mmol, contaminated
with 6.3% of the 7-[2-bromo-2-(phenylacetamido)] derivative,
5.8 mmol} and PPh3 (39.74 g, purity 99%, 150 mmol) in MeOAc
(50 mL) and MeOH (10 mL) was boiled under reflux for 1 h. MeOAc
(875 mL) was added in 1,5 h and reflux was continued for 2 h. The
mixture was cooled to 0 °C and stirred for 1.5 h. The white crystals
were collected by filtration, washed with MeOAc (250 mL) and dried
at 40 °C under reduced pressure for 12 h to give 74.12 g (purity
93.9%, 93.3 mmol; 93%) of 2.
IR (KBr): n = 3380 (NH), 1790 (CO, b-lactam), 1705 (CO, ester),
1680 (CO, amide), 1030 cm-1 (SO).
FABMS: m/z = 665.2 (M+-Br).
1H NMR (CDCl3; 360 MHz): d = 1.23 (s, 9H, tert-butyl), 3.20 (d, 1H,
J = 19.1 Hz, H2), 3.57 (s, 2H, ArCH2), 4.87 (dd, 1H, J1 = 5.5 Hz, J2 =
19.1 Hz, H2), 5.03 (d, 1H, J = 4.3 Hz, H6), 5.16 (dd, 1H, J1 = 14.0 Hz,

February 1998
SYNTHESIS
147
(6R,7R)-3-(2,4-Dinitrostyryl)-7-(2-thienylacetamido)ceph-3-em-
4-carboxylic Acid, E-Isomer (6):
IR (KBr): n = 3290 (NH), 1785 (CO, b-lactam), 1715 (CO, ester),
1665 (CO, amide), 1525 cm–1 (NO2).
MS (DCI): m/z = 584.2 (MNH4+).
A suspension of 5 (392 mg, purity 82% E-isomer, 0.82 mmol) in
CH2Cl2 (100 mL) was evaporated under reduced pressure to a volume
of 25 mL. 1,3-Bis(trimethylsilyl)urea (216 mg, 1.06 mmol) and
ammonium bromide (1 mg) were added and the mixture was boiled
under reflux for 1.5 h. The red solution was cooled to –12°C and
2-thiopheneacetyl chloride (0.14 mL, purity 99%, 1.13 mmol) was
added. After stirring for 1.5 h at –10 °C, CH2Cl2 (1.5 L) and H2O
(125 mL) were added and the pH of the aqueous phase was adjusted
to 3.0 with 0.1 M NaOH. The organic phase was extracted with H2O
(3 ´ 500 mL), dried (Na2SO4), and concentrated under reduced pres-
sure to a volume of 25 mL. The yellow crystals formed were collected
by filtration, washed with CH2Cl2 (25 mL), and dried in a desiccator
over P2O5. Yield: 299 mg (purity 95% E-isomer, 0.55 mmol; 67%).
1H NMR (CDCl3; 360 MHz): d = 1.53 (s, 9H, tert-butyl), 2.75 (d, 1H,
J = 18.2 Hz, H2), 3.25 (d, 1H, J = 18.2 Hz, H2), 3.63 (ABq, 2H, J =
15.9 Hz, ArCH2), 4.91 (d, 1H, J = 4.9 Hz, H6), 5.84 (dd, 1H, J6.7 = 4.9
Hz, J7,NH = 9.1 Hz, H7), 6.02 (d, 1H, J = 9.1 Hz, NH), 6.84 (ABq, 2H,
J = 11.9 Hz, CH=CH), 7.3 (m, 5H, ArH), 7.57 (d, 1H, J = 8.6 Hz,
ArH), 8.88 (d, 1H, J = 2.3 Hz, ArH).
(6R,7R)-3-(2,4-Dinitrostyryl)-7-(phenylacetamido)ceph-3-em-4-
carboxylic Acid, E-Isomer (4):
A stirred solution of 3 (67.41 g, purity 8% E-isomer, 81% Z-isomer,
105.9 mmol) in CH2Cl2 (1685 mL) was cooled to –25 °C. TiCl4
(53 mL, 482 mmol) was added in 10 min and the temperature was
brought to 0°C. After 1.75 h, chilled 2 M HCl (1685 mL) was added
at such a rate that the temperature remained under 10 °C. The organic
phase was separated and extracted with 2 M HCI (2 ´ 1685 mL), H2O
(1685 mL) and brine (1685 mL). The organic phase was concentrated
under reduced pressure to give an orange foam. Crude 4 thus obtained
was crystallized by dissolving in acetone (1350 mL) at 65 °C and add-
ing H2O (675 mL). Crystallization was allowed to proceed for 16 h at
0°C and the crystals were collected by filtration. Recrystallization of
the product was performed by dissolving the material in acetone/
HOAc (2:1) at 53 °C, removing the solvent (1700 mL) by evaporation
under reduced pressure, and stirring for 16 h at 20 °C. Crystals were
collected by filtration, washed with HOAc (300 mL) and Et2O
(250 mL), and dried under vacuum at 45 °C to give 34.33 g (purity
99% E-isomer, 66.6 mmol; 63%) of 4 as yellow crystals.
IR (KBr): n = 3295 (NH), 1780 (CO, b-lactam), 1720 (CO, amide),
1630 (CO, carboxylic acid), 1525 cm–1 (NO2).
MS (DCI): m/z = 534.1 (MNH4+).
1H NMR (CDCl3/DMSO-d6; 360 MHz): d = 3.57/3.70 (ABq, 2H, J =
l7.4 Hz, H2), 3.78 (ABq, 2H, J = 15.9 Hz, thiophene-CH2), 5.00 (d,
1H, J = 4.9 Hz, H6), 5.77 (dd, 1H, J6,7 = 4.9 Hz, J7,NH = 8.4 Hz, H7),
6.89 (m, 2H, thiophene-H), 7.15 (m, 2H, CH=C + thiophene-H), 7.67
(d, 1H, J = 16.1 Hz, C=CH), 7.76 (d, 1H, J = 8.8 Hz, ArH), 8.27 (dd,
1H, J1 = 2.0 Hz, J2 = 8.8 Hz, ArH), 8.45 (d, 1H, J = 8.4 Hz, NH), 8.67
(d, 1H, J = 2.0 Hz, ArH).
The authors gratefully acknowledge M. M. Pootfor recording the 1H
NMR spectra and G. A. de Vogelfor recording the mass spectra.
IR (KBr): n = 3300 (NH), 1780 (CO, b-lactam), 1715 (CO, amide),
1625 (CO, carboxylic acid), 1525 cm–1 (NO2).
MS (DCI): m/z = 528.0 (MNH4+).
1H NMR (CDCl3/DMSO-d6, 1:2; 360 MHz): d = 3.50/3.58 (ABq, 2H,
J = 14.1 Hz, ArCH2), 3.62/3.77 (ABq, 2H, J = 17.5 Hz, H2), 5.05 (d,
1H, J = 4.9 Hz, H6), 5.72 (dd, 1H, J6,7 = 4.9 Hz, J7,NH = 8.3 Hz, H7),
7.3 (m, 6H, ArH + CH=C), 7.63 (d, 1H, J = 16.1 Hz, C=CH), 7.82 (d,
1H, J = 8.8 Hz, ArH), 8.33 (dd, 1H, J1 = 2.1 Hz, J2 = 8.8 Hz, ArH),
8.66 (d, 1H, J = 2.1 Hz, ArH), 8.97 (d, 1H, J = 8.3 Hz, NH).
(1) Sutton, L. D.; Biedenbach, D. J.; Yen, A.; Jones, R. N. Diagn.
Microbiol. Infect. Dis. 1995, 21, 1.
(2) O’Callaghan, C. H.; Clark, J. C.; Kennedy, J.; Kirby, S. M.;
Long, A. G.; Moris, A.; Shingler, A. H.; Weir, N. G. Ger. Patent
2249165, 1973; Chem. Abstr. 1973, 79, 18736,
(3) Verweij, J.; de Vroom, E. Recl Trav. Chim. Pays-Bas 1993, 112,
66.
(4) Trippett, S. J. Chem. Soc. 1962, 2337.
(6R,7R)-3-(2,4-Dinitrostyryl)-7-aminoceph-3-em-4-carboxylic
Acid, E-Isomer (5):
(5) Tsuji, T.; Kataoka, T.; Yoshioka, M.; Sendo, Y.; Nishitani, Y.;
Hirai, S.; Maeda, T.; Nagata, W. Tetrahedron Lett. 1979, 2793.
(6) Yoshioka, M. Pure Appl. Chem. 1987, 59, 1041.
(7) Nagata, W. Pure Appl. Chem. 1989, 61, 325.
(8) Hamashima, Y. In Recent Advances in the Chemistry ofb-Lac-
tam Antibiotics; Bentley, P. H.; Southgate, R., Eds.; Special
Publication No. 70; Royal Society of Chemistry: London, 1989;
pp 77–90.
(9) De Vroom, E.; van der Does, T. Intern. Patent Application WO
97/17352, 1997.
(10) Weissenburger, H. W. O.; van der Hoeven, M. G. Recl Trav.
Chim. Pays-Bas 1970, 89, 1081.
Compound 4 (521 mg, purity 98% E-isomer, 1.00 mmol) was dis-
solved in water (20 mL) at pH 7.5 by the addition of 0.1 M NaOH.
The temperature of the solution was brought to 32 °C and immobi-
lized penicillin-G acylase (1 g) was added. The pH was kept at 7.5 by
the continuous addition of 0.1 M NaOH using an automatic titration
apparatus. After 3 h, when NaOH consumption had stopped, the im-
mobilized enzyme was removed by filtration and the pH of the red so-
lution was brought to 3.0. After stirring for 1 h, orange crystals were
collected by filtration, washed with H2O (25 mL), and dried under
vacuum at 40 °C. Yield: 359 mg (purity 75% E-isomer, 9% Z-isomer,
0.77 mmol; 77%).
(11) Van der Meij, M.; de Vroom, E. BioMed. Chem. Lett. 1994, 4,
345.
(12) Kalir, A. Org. Synth. Coll. Vol. 1973, 5, 825.
(13) Behr, L. C.; Alley, E. G.; Levand, O. J. Org. Chem. 1962, 27,
65.
IR (KBr): n = 1800 (CO, b-lactam), 1610 (CO, carboxylic acid),
1535 cm–1 (NO2).
MS (DCI): m/z = 410. 1 (MNH4+).
1H NMR (DMSO-d6; 360 MHz): d = 3.67/3.74 (ABq, 2H, J = 17.5
Hz, H2), 4.87 (d, 1H, J = 5.1 Hz, H6/H7), 5.10 (d, 1H, J = 5.1 Hz, H7/
H6), 7.18 (d, 1H, J = 16.1 Hz, CH=C), 7.51 (d, 1H, J = 16.1 Hz,
C=CH), 7.95 (d, 1H, J = 8.7 Hz, ArH), 8.47 (dd, 1H, J1 = 2.1 Hz, J2
= 8.7 Hz, ArH), 8.70 (d, 1H, J = 2.1 Hz, ArH).
(14) Kröhnke, F.; Schmeiss, H. Ber. Dtsch. Chem Ges. 1939, 72,
440.
(15) Immobilized penicillin-G acylase is available from Gist-bro-
cades under the name Separase-G(r).