Synthesis and anti-MRSA activity of novel cephalosporin derivatives

Article abstract of DOI:10.1016/S0040-4020(00)00418-X

Cephalosporin derivatives containing a unique combination of lipophilic C-7 sidechains and polar C-3 thiopyridinium groups were synthesized and found to exhibit potent anti-MRSA activity in vitro and in vivo. The optimum C-7 sidechains utilized were 2,5-dichlorophenylthioacetamido and 2,6-dichloropyrid-4-ylthioacetamido. The C-3 thiopyridinium rings were substituted at nitrogen with amino acid and pyruvic acid groups that were designed to confer aqueous solubility as required for IV formulation. This paper describes the characteristics of these novel cephalosporins and highlights synthetic methods developed to allow their practical, large-scale syntheses. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00418-X

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 5687±5698
Synthesis and anti-MRSA Activity of Novel Cephalosporin
Derivatives
b
a
b
b
Stan V. D'Andrea,a, Daniel Bonner, Joanne J. Bronson, Junius Clark, Ken Denbleyker,
*
Joan Fung-Tomc,b Shelley E. Hoeft,a Thomas W. Hudyma,a John D. Matiskella,a
Raymond F. Miller,a Peter F. Misco,a Michael Pucci,b Roman Sterzycki,a Yuan Tsai,
²,b
Yasutsuga Ueda,a John A. Wichtowski,a Janak Singh,c,³ Thomas P. Kissick,c Jeffery T. North,c
Annie Pullockaran,c Michael Humora,d Brenda Boyhan,d Truc Vu,d Alan Fritz,d J. Heikes,d
Rita Fox,c Jollie D. Godfrey,c Robert Perrone,e Murray Kaplan,e,² David Kronenthalc
and Richard H. Muellerc
aAnti-infective Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
bMicrobiology, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
cChemical Process Research, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
dChemical Process Research, Bristol-Myers Squibb Pharmaceutical Research Institute, New Brunswick, NJ 08903, USA
ePharmaceutics, Chemical Process Research, Bristol-Myers Squibb Pharmaceutical Research Institute, Syracuse, NY 13057, USA
Received 7 December 1999; accepted 14 February 2000
AbstractÐCephalosporin derivatives containing a unique combination of lipophilic C-7 sidechains and polar C-3 thiopyridinium groups
were synthesized and found to exhibit potent anti-MRSA activity in vitro and in vivo. The optimum C-7 sidechains utilized were 2,5-
dichlorophenylthioacetamido and 2,6-dichloropyrid-4-ylthioacetamido. The C-3 thiopyridinium rings were substituted at nitrogen with
amino acid and pyruvic acid groups that were designed to confer aqueous solubility as required for IV formulation. This paper describes
the characteristics of these novel cephalosporins and highlights synthetic methods developed to allow their practical, large-scale syntheses.
q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
bacterial agents. Another effective strategy to identify
compounds with activity against resistant organisms is to
improve on a known class of antibacterial agents.9 Since
cephalosporins have an excellent safety pro®le and are
synthetically accessible from commercial sources, we
initiated a program to develop a cephalosporin with anti-
MRSA activity. The goals of the program were to identify a
cephalosporin with (1) in vitro and in vivo activity against
MRSA, (2) suitable pharmacokinetics in animals to predict
convenient dosing in humans, (3) suf®cient water solubility
to allow I.V. dosing, and (4) an excellent safety pro®le. The
2,5-dichlorophenylthioacetamido C-7 group was disclosed
in the late 1970s by researchers at Lilly as a lipophilic
sidechain that conferred excellent gram-positive activity
to the cephem class.10 For instance, their C-3 acetate
Methicillin-resistant Staphylococcus aureus (MRSA)1 is
resistant to b-lactam antibiotics due to the presence of an
additional penicillin-binding protein (PBP2a) for which
b-lactams have poor af®nity.2 Vancomycin3 and Synercid4
are the only marketed drugs that are effective against MRSA
infections. In addition to the complications caused by
limited treatment options, vancomycin intermediate-
resistant MRSA5 has recently been detected in hospitals in
Japan6 and the USA.7 Furthermore, the hospital setting is no
longer the exclusive domain of MRSA infections. Recently,
there have been alarming reports of its spread in nursing
homes, extended-care facilities, and a day care center.8
The need for new anti-MRSA agents is clear.
Many companies are looking for novel classes of anti-
Keywords: anti-MRSA activity; 2,5-dichlorophenylthioacetamido; 2,6-
dichloropyrid-4-ylthioacetamido; novel cephalosporins.
* Corresponding author. E-mail: stanley.dandrea@bms.com
²
Retired.
E-mail: janak.singh@bms.com
³
Figure 1.
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00418-X

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S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
Figure 2.
derivative (Fig. 1) had MICs of ,1 mg/mL against
Methicillin-susceptible Staphylococcus aureus (MSSA). In
our own hands, this Lilly compound had MICs of 2 mg/mL
against MRSA (strain A 2722311). Unfortunately, this
acetate derivative suffered from poor pharmacokinetics.
Our efforts at Bristol±Myers Squibb were aimed at ®nding
an improved cephalosporin containing a lipophilic C-7
sidechain. Some of our earlier work in this area has been
published.12
1. Cephems 1±4 have excellent activity against homoge-
neous and heterogeneous methicillin-resistant strains of
MRSA.14 In addition, these cephems have excellent in
vitro activity against a variety of gram-positive bacteria
including resistant strains (e.g., penicillin-resistant S. pneu-
moniae, methicillin-resistant S. epidermitis, and methicillin-
resistant S. haemolyticus). In terms of PBP2a inhibition,
these new cephalosporins have a much greater af®nity for
this critical PBP than traditional cephalosporins (methicillin
PBP2a IC50 ,100 mg/mL). Cephems 1±4 also retained
good binding af®nities (,0.6 mg/mL) to the other important
staphylococcal PBPs.
Results and Discussion
Through a series of optimizations at the C-7 and C-3
positions, we discovered cephems 1, 2, 3, and 4 as having
excellent properties for use as anti-MRSA agents (Fig. 2).
This paper describes the activity and synthesis of these four
compounds.
Table 2 summarizes the ef®cacy and solubility properties of
1, 2, 3, and 4. All were ef®cacious in a systemic murine
model of infection (MRSA, homogeneous) with PD50s
ranging from 8±9.6 mg/Kg.15 The aqueous solubility of
the pyruvate derivatives 3 and 4 is greatly improved over
that of cephems 1 and 2 especially at neutral pH.
All four cephems contain a lipophilic C-7 group (2,5-
dichlorophenylthioacetamido or 2,6-dichloropyrid-4-ylthio-
acetamido) and a thiopyridinium group at C-3. Compounds
1 and 2 are substituted with an amino acid sidechain while 3
and 4 contain a pyruvic acid moiety. Key parameters that
were optimized included MICs, IC50s vs. PBP2a, PD50s, and
solubility. The MICs13 and PBP2a IC50s are shown in Table
The synthetic approaches to cephems 1±4 are shown retro-
synthetically in Scheme 1. The thiopyridinium sidechain at
C-3 can be introduced by reacting allylic chloride B with
thiopyridone C. Alternatively, the thiopyridine nitrogen of
D can be quaternized with an electrophile such as bromo-
pyruvic acid E. Cephem D is synthesized by reacting allylic
chloride B with 4-thiopyridine.
Table 1. Minimum Inhibitory Concentrations (MICs) in mg/mL
(MRˆmethicillin-resistant; P-ˆpenicillinase negative; MˆMethicillin;
IMˆimipenem; NTˆnot tested; IC50ˆthe concentration at which 50% inhi-
bition has occurred)
The preparation of the C-7 sidechains is shown in Schemes
2 and 3. The earlier synthesis of 616 relied on the expensive
thiophenol 5. Alternatively, careful diazotization of aniline
7 followed by reaction with the copper salt of thioglycolic
acid (mercaptoacetic acid) produced 6 in 70% overall
yield.17 The initial route from 6 to 10 utilized a DCC
coupling of the acid 6 with 7-aminocephalosporonic acid
diphenylmethane ester 8 to provide DPM ester 9. While
this proved satisfactory for small scale reactions, the large
scale preparations of 10 were best accomplished by convert-
ing 6 to its acid chloride followed by coupling with amine 8
to give ester 9. Deprotection of 9 with tri¯uoroacetic acid
gave the chloroacid 10 (Scheme 2).
Organism
A No.
1
2
3
4
M
IM
S. aureus/Hetero MR A27218 0.5
S. aureus/150% calf serum A27218 0.5
1
1
0.5
1
1
1
1
1
2
2
2
2
2
2
32
8
64
128
128 32
64 NT
64 16
128 64
64
128 NT
1
NT
1
S. aureus/Hetero MR
S. aureus/Hetero MR
S. aureus/Homo MR
S. aureus/150 calf serum
S. aureus/Homo MR
S. aureus/Homo MR
S. aureus/Homo MR
S. aureus/MR, P-
A27217 0.5 0.5 0.5
A25795
A27223
A27223
A27621
A27295
A27226
A27225
1
2
4
1
2
1
1
2
2
4
2
4
2
2
1
2
16
2
4
1
8
4
2
PBP2a IC50 (mg/mL)
28 NT 10 4.5 100 NT
For the C-7 dichloropyrid-4-yl derivatives, the key acid 14
was prepared from 2,6-dichloropyridine as shown in
Scheme 3. While the general route from 11 to 14 was
previously reported,18 modi®cations were made to facilitate
work on a larger scale. Commercially available 2,6-dichlor-
opyridine (11) was oxidized with hydrogen peroxide to give
the N-oxide 12. A major improvement over the literature
conditions for the large scale conversion of 12 to 2,4,6-
trichloropyridine (13) was realized when LiCl was added
to the reaction mixture. This moderated an otherwise
Table 2. PD50s and solubility (NTˆnot tested)
Cmpd
PD50
(mg/Kg/total dose)
vs. MRSA (A27223)
Aqueous solubility
(mg/mL at pH 7, rt)
1
2
3
4
8.2
4.8
8.0
9.6
2±3
NT
23
40

S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
5689
Scheme 1.
Scheme 2. Synthesis of chloroester 9 and chloroacid 10. Reaction conditions and yields: (a) BrCH2CO2H, aq NaOH, 1008C; ,100%; (b) HCl, NaNO2;
(c) HSCH2CO2H, CuSO4; 55±70% from 7; (d) (COCl)2, NMM, DMF; 88%; (e) TFA, anisole, CH2Cl2, 08C, 94%.
Scheme 3. Synthesis of chloroester 15 and chloroacid 16. Reaction conditions and yields: (a) 30% H2O2, TFA, 1008C, 3 h, 84%; (b) LiCl, POCl3, 1158C, 6 h,
71%; (c) aq. NaOH, MeOH, 258C, 17 h, 70%; (d) EDC, NMM, CH2Cl2, 08C, 1 h, 93%; (e) TFA, anisole, 08C, 1 h, 98%.
overly vigorous reaction. Acid 14 was coupled ef®ciently on
a large scale to the cephem nucleus using EDC. The TFA
deprotection of DPM ester 15 gave an excellent yield of the
acid 16.
with Lawesson's reagent under anhydrous conditions
afforded 19 with minimal amounts (,3%) of the side
product 22. Puri®cation of 19 by bulb-to-bulb distillation
rather than chromatography effectively removed the
impurities 22 and 23. Reaction of N-Boc-ornithine 20 with
19 gave the expected thiopyridone 21 plus small amounts of
the dimer 24. Method development work revealed that
replacing NaOEt with pyridine minimized the formation
of colored impurities during the formation of 21.
Scheme 4 illustrates the synthesis of thiopyridone 21.
Improvements were made to the literature preparations of
1819 and 19.20 The expensive g-pyrone (18) was prepared by
decarboxylating chelidonic acid (17) in di-n-butylphthalate
(bp 3408C), a solvent with a considerably higher boiling
point than 18 (bp ,2108C). Isolation by distillation rather
than extraction led to an 81% yield of 18. Treatment of 18
With the requisite cephem and thiopyridone units in hand,
several approaches to cephems 1 and 2 were studied. The

5690
S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
Scheme 4. Synthesis of 4-mercaptopyridyl-N-Boc-l-ornithine 21. Reaction conditions and yields: (a) Cu-bronze, 2,20-dipyridyl, n-butylphthalate, ,2108C;
80±90% distilled; (b) Lawesson's reagent, toluene, 808C; 98% distilled; (c) N-Boc-l-ornithine 20, pyridine, aq EtOH, re¯ux; 91%.
Scheme 5. Synthesis of cephems 1 and 2. Reaction conditions: (a) 16125!29: DMF, 5 h, rt; (b) 10121!27: THF, 3 h, rt; (c) TFA, anisole, CH2Cl2;
(d) HCO2H, HCl; (e) NaOAc, MeOH.
coupling of thiopyridone 21 (acid form) with DPM ester 9
gave cephem 26 in a crude yield of 80%; however, the
presence of the corresponding delta-2 isomer (10%) made
puri®cation dif®cult. Based on this result, the analogous
approach to cephem 28 was not attempted. An improvement
in purity was realized when thiopyridone 25 (Na salt) was
reacted with the chloroacids 10 and 16 to give the thio-
pyridinium compounds 27 and 29 (97 and 71% yield,
respectively) (Scheme 5). Work to optimize the large
scale synthesis of 1 led to the coupling of thiopyridone 21
with chloroacid 10. This procedure minimized the formation
of the delta-2 isomer and other impurities and allowed the
isolation of cephem 27 in 97% yield. Cephems 27 and 29
typically were deprotected using TFA/anisole/CH2Cl2
conditions (70±90% yield). However, a mixture of formic
acid and hydrochloric acid was also effective for the large
scale deprotection of 27 (99.7% yield). The crude depro-
tected salts (TFA or HCl) of cephems 1 and 2 were puri®ed
further by conversion to the bis-zwitterionic cephems 1 and
2, respectively.
The pyruvate derivatives 3 and 4 were prepared by the
alternative quaternization route (Scheme 1: D1E!A).
Our initial route to compound 4 (and 3) involved the use
of the DPM esters, which underwent quaternization at the C-
3 thiopyridine group when treated with 5 mol equiv. of
bromopyruvic acid (Scheme 6). This procedure was compli-
cated by (1) incomplete quaternization, (2) secondary
reaction of the product with bromopyruvic acid,21 and (3)
the necessity of C-18 column chromatography to purify the
crude product.
It was subsequently found that the DPM esters 9 and 15
were not the preferred starting materials. In an improved

S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
5691
Scheme 6. Synthesis of 4 via thionation of chloroester 15. Reaction conditions and yields: (a) 2,6-lutidine, NaI, DMF, 258C, 1.5 h, 80%; (b) DMF, 258C, 18 h,
80%; (c) TFA, anisole, 0±258C, 1.5 h, 90%; aq NaOH, C-18 chromatography; 40%.
Scheme 7. Synthesis of 3 and 4 via chloro acids 10 and 16. Reaction conditions: (a) 2,6-lutidine, NaI, DMF, 258C, 40 min; (b) BSA, THF, 258C, 2 h.
sequence, chloroacids 10 and 16 were treated with
4-mercaptopyridine to give cephems 32 and 33, respec-
tively. Quaternization of 32 and 33 with bromopyruvic
acid was best accomplished using a novel in situ silylation
procedure (Scheme 7).22 This involved pretreatment of 32
and 33 with BSA in THF to afford a homogeneous solution.
The addition of 2.5 equiv. of bromopyruvic acid led to the
ef®cient quaternization of the pyridyl groups in just 2 h at rt.
The ®nal products 3 and 4 were isolated in excellent yield
and high purity while avoiding the pitfalls of the DPM ester
route (Scheme 6).
chemistry also proved critical in the synthesis of a variety
of related analogs. Our continuing studies on the optimiza-
tion of anti-MRSA cephalosporins will be reported in future
publications.
Experimental
Abbreviations: rtˆroom temperature, EDCˆN-(3-dimethy-
laminopropyl)-N0-ethylcarbodiimide hydrochloride, DCUˆ
dicyclohexylurea, DCCˆdicyclohexylcarbodiimide, BSAˆbis-
trimethylsilylacetamide, TFAˆtri¯uoroacetic acid, NMMˆN-
Methyl Morpholine.
In summary, cephalosporins 1±4 with potent anti-MRSA
activity and promising physical characteristics have been
identi®ed. In 1±4, the cephalosporin core is appended
with a novel combination of a lipophilic C-7 substituent
and a polar thiopyridinium group at C-3. The original routes
to cephems 1±4 were modi®ed substantially to allow the
practical, large scale synthesis of these derivatives. This
[6R-[3(Sp),6a,7b]]-1-(4-Amino-4-carboxybutyl)-4-[[[2-
carboxy-7-[[[(2,5-dichlorophenyl)thio]-acetyl]amino]-8-
oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]thio]-
pyridinium, hydroxide, inner salt (1). Powdered chloride
salt 27 (122.44 g, 141.9 mmol) was added in portions to

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S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
98% formic acid (236 mL) with rapid stirring so that the
solid dissolved. The solution was stirred for 4 h. Conc. HCl
(14.07 mL, 170.3 mmol) was added and the solution was
stirred for 20 min. The mixture was added rapidly over
2 min to EtOAc (2600 mL) in a Morton ¯ask with mechani-
cal stirring. The residue from the reaction ¯ask was washed
into the slurry with HCO2H (2£5 mL) and EtOAc
(2£10 mL). The mixture was stirred for 15 min, ®ltered,
and the ®lter cake was transferred to a Morton ¯ask with
1000 mL EtOAc and stirred for 1 h to remove most of the
entrained HCO2H. The slurry was ®ltered and the ®lter cake
was washed thoroughly with EtOAc (2£100 mL). The solid
was dried under vacuum overnight to give 117.11 g (99.7%)
of the chloride salt of 1 as a golden yellow powder, mp
,1008C (eff.). [a]Dˆ128.0 (cˆ1, CH3CN/H2O 2:1,
pHˆ1.8). Anal. Calcd for C26H26N4O6S3Cl2´2 HCl´1.0
H2O´0.5 EtOAc´0.24 HCO2H: C, 42.20; H, 4.32; N, 6.97;
S, 11.97; Cl, 17.65; H2O, 2.24. Found: C, 41.99; H, 4.41; N,
6.73; S, 11.53; Cl, 17.46; H2O, 2.26 (KF).
and TFA. The residue was stirred with diethyl ether
(100 mL) for 15 min, and the resulting solid was collected
by ®ltration. This solid was stirred with acetone (25 mL) for
20 min, and then the solid was collected by ®ltration to give
386 mg (68%) of 2 as the monochloride, mono TFA salt. 1H
NMR (DMSO-d6, 300 MHz) d 1.72 (br s, 2H), 1.98 (br s,
2H), 3.42 (d, Jˆ17 Hz, 1H), 3.58±3.74 (m, 3H), 3.61 (d,
Jˆ17 Hz, 1H), 3.99 (d, Jˆ15 Hz, 1H), 4.07 (d, Jˆ15 Hz,
1H), 4.37±4.58 (m, 4H), 5.02 (d, Jˆ5 Hz, 1H), 5.57 (dd,
Jˆ5.8 Hz, 1H), 7.51 (s, 2H), 8.10 (d, Jˆ6 Hz, 2H), 8.73 (d,
Jˆ6 Hz, 2H), 9.42 (d, Jˆ8 Hz, 1H); MS (ESI) M1Hˆ658.
(6R-trans)-4-[[[2-Carboxy-7-[[[(2,5-dichlorophenyl)thio]-
acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-
3-yl]methyl]thio]-1-(2-carboxy-2-oxoethyl)-pyridinium,
hydroxide, inner salt (3). (Method A) The sodium salt form
of 3 was prepared from (6R)-trans-3-[(4-pyridylthio-
methyl]-7-[(2,5-dichlorophenyl)-thioacetamido]-8-oxo-5-
thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, diphenyl-
methyl ester23 using the same procedure described for
1
The chloride salt of 1 (115.12 g, 143.25 mmol) was
powdered with a Te¯on spatula and added slowly over
20 min to mechanically stirred 50% aqueous EtOH (6 L)
at 258C (pHˆ3.0). After 15 min a mixture of a solution of
1N NaOH (23.6 mL) and NaOAc (40.21 g, 295.7 mmol) in
50% aqueous EtOH (492 mL) was added via an addition
funnel as a rapid stream of droplets over 20 min. The
mixture (pH 5.2) was stirred for 1.5 h and the solid was
®ltered through a medium porosity sintered glass funnel.
The product was washed by stirring it in the funnel with
50% aqueous EtOH (2 L). The material was transferred to a
Buchner funnel containing a Whatman 1 (18.5 cm) ®lter
paper for a faster ®ltration. The washing was repeated two
more times with EtOH/H2O (1:1, 1 L), each time transfer-
ring the product to a beaker for slurrying and then ®ltering
through a fresh ®lter paper in the Buchner funnel. After
further washing with EtOH (3£800 mL) and EtOAc
(3£800 mL) the product was dried for 17 h under vacuum
to give 78.4 g (77.4%) of the double zwitterion 1, mp
compound 4 (method A): H NMR (DMSO-d6, 300 MHz)
d 3.35 (d, Jˆ17 Hz, 1H), 3.51 (d, Jˆ17 Hz, 1H), 3.91 (s,
2H), 4.30 (d, Jˆ14 Hz, 1H), 4.74 (d, Jˆ14 Hz, 1H), 4.97 (d,
Jˆ5 Hz, 1H), 5.46 (dd, Jˆ5.8 Hz, 1H), 5.75 (s, 2H), 7.24
(dd, Jˆ9.2 Hz, 1H), 7.45±7.50 (m, 2H), 8.38 (d, Jˆ7 Hz,
2H), 8.48 (d, Jˆ7 Hz, 2H), 9.20 (d, Jˆ8 Hz, 1H); MS (ESI)
M1ˆ627. Anal. Calcd for C24H18N3O7S3Cl2Na´3 H2O: C,
40.91; H 3.43; N, 5.96; S, 13.65; Na, 3.26. Found: C, 40.62;
H, 3.26; N, 5.82; S, 13.78; Na, 3.30.
(Method B) Bistrimethysilylacetamide (BSA)(3.4 g, 14.9
mmol) was added to a stirred suspension of mercaptopyridyl
acid 32 (2.03 g, 3.5 mmol) in anhydrous CH3CN (11.0 mL)
at 258C under an argon atmosphere. A clear orange solution
was obtained after 5 min. Anhydrous bromopyruvic acid
(1.46 g, 8.8 mmol) was added and the reaction mixture
was stirred for 3 h. The solution was diluted with CH3CN
(11.0 mL) and H2O (30 mL) was added dropwise with good
stirring to obtain a precipitate. After 1.5 h the solid was
®ltered, washed with water/CH3CN (2:1, 30 mL), and
CH3CN and dried with suction under nitrogen overnight to
give 2.23 g of 3. A suspension of 2.1 g of this solid in
CH3CN (20.0 mL) was sonicated for 10 min, ®ltered
and dried under vacuum to give 1.85 g (yield ,84%) of
zwitterion 3.
1
,1658C (dec.). H NMR (DMSO-d6-TFA, 270 MHz) d
1.8±2.1 (m, 4H), 3.7 (ABq, Jˆ18.2 Hz, 2H), 4.0 (s, 2H),
4.4±4.7 (m, 4H), 5.2 (d, Jˆ4.7 Hz, 1H), 5.8 (dd, Jˆ4.7 and
8.0 Hz, 1H), 7.3 (dd, Jˆ2.3 and 8.2 Hz, 1H), 7.5 (d,
Jˆ9.4 Hz, 1H), 8.1 (d, Jˆ7.0 Hz, 2H), 8.4 (br s, 3H), 8.8
(d, Jˆ7.0 Hz, 2H), 9.4 (d, Jˆ8.2 Hz, 1H); 13C NMR
(DMSO-d6-TFA, 68 MHz) d, 26.9, 27.7, 34.1, 38.3, 51.9,
58.0, 60.0, 123.4, 124.1, 126.8, 127.5, 129.5, 131.1, 133.1,
138.2, 143.1, 158.5, 159.0, 159.6, 160.1, 162.6, 163.4,
164.7, 168.9, 171.1; IR (KBr) 3500, 1763, 1630 cm21; MS
(ESI) M1Hˆ657, M2Hˆ655; [a]Dˆ135.7 (cˆ1, 0.1N
(6R-trans)-4-[[[2-Carboxy-7-[[[(2,6-dichloro-4-pyridinyl)-
thio]acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-
2-en-3-yl]methyl]thio]-1-(2-carboxy-2-oxoethyl)-pyri-
dinium, hydroxide, inner salt (4). (Method A) A stirred
solution of DPM ester 15 (10 g, 15.75 mmol) in 100 mL
of DMF was cooled to 08C (ice bath) under a N2 atmosphere.
To this solution was added NaI (2.36 g, 15.75 mmol)
followed by 4-mercaptopyridine (1.92 g, 17.3 mmol) and
2,6-lutidine (2.02 g, 18.9 mmol). The ice bath was removed
and the reaction was allowed to stir at rt for 1.5 h. Water was
added dropwise to the reaction mixture to precipitate a
yellow solid which was collected by ®ltration, washed
with water, and dried on the ®lter while under suction.
This gave 12 g of solid which was stirred with 200 mL of
ether for 1 h. The solid was again collected by ®ltration and
dried on the ®lter to give 10 g of 30 as a yellow solid.
Bromopyruvic acid (12.5 g, 74.9 mmol) was added all at
HCl/CH3CN
2:3,
pHˆ3.2).
Anal.
Calcd
for
C26H26N4O6S3Cl2´2.73 H2O: C, 44.18; H, 4.49; N, 7.93; S,
13.61; Cl, 10.03; H2O, 6.96. Found: C, 44.04; H, 4.33; N,
7.69; S, 13.50; Cl, 10.00; H2O, 6.93 (KF).
[6R-[3(Sp),6a,7b]]-1-(4-Amino-4-carboxybutyl)-4-[[[2-
carboxy-7-[[[(2,6-dichloro-4-pyridinyl)thio]-acetyl]-
amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]-
methyl]thio]pyridinium, hydroxide, inner salt (2). The
sodium salt of 29 (576 mg, 0.704 mmol) was stirred with
CH2Cl2 (24 mL) at 08C while anisole (2.4 mL) and TFA
(8.7 mL) were added. This solution was stirred for 1 h at
08C followed by concentration in vacuo to remove CH2Cl2

S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
5693
once to a DMF solution (120 mL) of cephem 30 (9.0 g,
12.8 mmol). This mixture was stirred at rt for 18 h at
which time the DMF was removed in vacuo (,1 torr,
#408C) to give a dark brown residue. The residue was
stirred with 200 mL of water for 30 min and the resulting
tan solid was collected by ®ltration and dried (with P2O5 in a
vacuum dessicator). The dry solid was slurried in EtOAc
and stirred for 1 h. The solid was collected by ®ltration to
give 9 g (81%) of 31 as a tan solid. DPM ester 31 (10 g,
11.4 mmol) was slurried with CH2Cl2 (50 mL) and anisole
(15 mL), and then cooled to 08C. TFA (50 mL) was added
and the mixture was stirred at rt for 1.5 h. The reaction was
concentrated in vacuo to remove most of the CH2Cl2 and
TFA. The residue was stirred with 100 mL of ether and the
resulting solid was collected by ®ltration. This solid was
slurried in 100 mL of ethyl acetate for 0.5 h, collected by
®ltration, and dried in vacuo to give 7.4 g (89%) of 4 as the
TFA salt. The TFA salt of 4 was dissolved in ,200 mL of
water adjusted to pH 8 with NaOH. Puri®cation over a C18
column (,125 g) was accomplished by eluting with water
followed by increasing amounts of acetonitrile up to 30% to
give 3.25 g of 4 as the monosodium salt. 1H NMR (DMSO-
d6, 300 MHz) d 3.39 (d, Jˆ17 Hz, 1H), 3.53 (d, Jˆ17 Hz,
1H), 3.96 (d, Jˆ16 Hz, 1H), 4.03 (d, Jˆ16 Hz, 1H), 4.31 (d,
Jˆ14 Hz, 1H), 4.76 (d, Jˆ14 Hz, 1H), 4.99 (d, Jˆ5 Hz,
1H), 5.46 (dd, Jˆ5.8 Hz, 1H), 5.76 (s, 2H), 7.54 (s, 2H),
8.39 (d, Jˆ7 Hz, 2H), 8.49 (d, Jˆ7 Hz, 2H), 9.25 (d,
Jˆ8 Hz, 1H); MS (ESI) M1ˆ628.
(75 mL) was added dropwise to the slurry. The salt initially
dissolved, then a gummy precipitate formed which solidi-
®ed upon stirring after 15 min. The solid was ®ltered and the
®lter cake was washed twice by slurrying with 5% H2O/
acetone in the ®ltration funnel. The product was dried
under vacuum to give 2.21 g (57% overall yield from
zwitterion) of the meglumine salt of 4, mp 1308C (dec.).
1H NMR (THF-d8-TFA, 270 MHz) d 2.7 (m, 1H), 2.8 (t,
Jˆ5.3 Hz, 3H), 2.9 (m, 1H), 3.3±3.4 (m, 2H), 3.6±4.0 (m,
8H), 4.1±4.2 (m, 4H), 4.6 (m, 2H), 4.8 (s, 1H), 5.3 (d,
Jˆ4.7 Hz, 1H), 5.8 (dd, Jˆ4.7 and 8.2 Hz, 1H), 6.2 (s,
1H), 7.5 (s, 2H), 8.0 (s, 2H), 8.1 and 8.2 (pair of doublets
due to TFA, Jˆ6.5 Hz each, 2H), 8.6 (br s, 2H), 8.8 (m, 2H),
9.3 (d, Jˆ8.2 Hz, 1H); [a]Dˆ1.5 (cˆ1.5, H2O, pHˆ6.1). IR
(KBr) 3368, 1750, 1661, 1630 cm21
.
MS (ESI)
M1H2O1Hˆ647, M1H2O2Hˆ645. Anal. Calcd for
C23H18N4O7S3Cl2´C7H17NO5´1.0 H2O: C, 42.75; H 4.43;
N, 8.31; S, 11.41; Cl, 8.41; H2O 2.14. Found: C, 42.60; H,
4.26; N, 8.11; S, 11.50; Cl, 8.40; H2O, 1.83 (KF).
[(2,5-Dichlorophenyl)thio]acetic acid (6). (Method A)
Conc. HCl (76.5 mL, 925.9 mmol) was added to 2,5-
dichloroaniline
7
[50.0 g, 308.6 mmol, (CAUTION,
lachrymator)] in a 2 L beaker. The large lumps that
developed were broken up with a stirring rod. The mixture
of white crystals was stirred for 30 min to ensure that all of
the aniline was converted to the HCl salt. Water (400 mL)
was added to form a suspension, which was stirred mag-
netically. Ice (,450 mL) was added to bring the tempera-
ture to 108C. A solution of NaNO2 (22.36 g, 324.0 mmol) in
H2O (80 mL) was added dropwise with rapid stirring over
5 min. Small portions of ice were added to keep the
temperature at 108C throughout the diazotization reaction.
The resulting solution contained a small amount of insoluble
material. After stirring at 108C for 1 h the excess nitrite was
destroyed by addition of a solution of sulfamic acid (2.10 g,
21.6 mmol) in H2O (10 mL) dropwise (carefully so that the
resulting foam didn't spill out of the beaker). The mixture
was ®ltered and the ®lter cake was washed with a small
amount of H2O. Mercaptoacetic acid (26.5 mL, 432.0
mmol) was added to a solution of CuSO4´5H2O (77.05 g,
308.6 mmol) in water (300 mL) stirred mechanically under
N2 in a 2 L round-bottomed ¯ask. After stirring for 30 min a
dark precipitate formed. The solution of the diazonium salt
was added in small portions through a dropping funnel to the
insoluble copper complex with rapid stirring. The tempera-
ture of the mixture was kept at 208C with a water bath. The
mixture was stirred for 30 min, conc. HCl (80 mL) was
added, and the mixture was stirred at 40±508C for 1 h.
The warm reaction mixture was ®ltered to collect the
solid, which was washed twice with 1% HCl and water.
The ®lter cake was washed into a 4 L beaker with 1.5 L
H2O and dissolved by the addition of 38 g Na2CO3. After
heating to boiling, 30 g Darco was added and the mixture
was boiled for 15 min. The mixture was ®ltered hot through
Celite and washed with water. Conc. HCl (30 mL) was
added to the warm ®ltrate dropwise until the pH reached 1
(foaming!). The precipitated product was collected by ®ltra-
tion and thoroughly washed with water three times. Each
time the ®lter cake was sucked dry on the ®lter funnel. The
lightly colored solid was dried under vacuum over P2O5
overnight to give 6: 43.81 g (60%). Hexane (300 mL) was
added to a solution of crude material in hot EtOAc
(Method B) Bistrimethysilylacetamide (BSA)(7.99 mL,
32.4 mmol) was added to a suspension of the mercapto-
pyridyl acid 33 (5.0 g, 8.59 mmol) stirred in THF
(28.6 mL) at 258C under an argon atmosphere. A clear solu-
tion was obtained after 30 min. Anhydrous bromopyruvic
acid (3.59 g, 21.5 mmol) was added and the reaction
mixture was stirred for 2 h. The solution was chilled in an
ice-bath and H2O (120 mL) was added dropwise with good
stirring to obtain a precipitate. After 15 min the solid was
®ltered, washed twice with water, and dried under vacuum
overnight to give 5.04 g (89%) of the zwitterion 4. 1H NMR
(DMSO-d6, 300 MHz) d 3.39 (d, Jˆ17 Hz, 1H), 3.53 (d,
Jˆ17 Hz, 1H), 3.96 (d, Jˆ16 Hz, 1H), 4.03 (d, Jˆ16 Hz,
1H), 4.31 (d, Jˆ14 Hz, 1H), 4.76 (d, Jˆ14 Hz, 1H), 4.99 (d,
Jˆ5 Hz, 1H), 5.46 (dd, Jˆ5.8 Hz, 1H), 5.76 (s, 2H), 7.54 (s,
2H), 8.39 (d, Jˆ7 Hz, 2H), 8.49 (d, Jˆ7 Hz, 2H), 9.25 (d,
Jˆ8 Hz, 1H); 13C NMR (DMSO-d6, 68 MHz) d 27.1, 33.2,
33.7, 57.4, 59.3, 66.2, 119.2, 122.2, 122.4, 127.7, 143.6,
144.3, 149.1, 155.3, 161.9, 162.4, 163.1, 163.9, 167.8,
194.3; IR (KBr) 3265, 1770, 1629 cm21; MS (ESI)
M1ˆ628; Anal. Calcd for C23H18N4O7S3Cl2´0.72 H2O: C,
41.82; H 3.27; N, 8.48; S, 14.56; Cl, 10.73; H2O 4.69.
Found: C, 41.55; H, 3.09; N, 8.43; S, 14.47; Cl, 11.11;
H2O, 4.71 (KF). Cephem 4 could be further puri®ed as the
meglumine (MGA) salt as described below. Powdered
zwitterion 4 (3.0 g, 4.54 mmol) was added in portions to a
solution of methyl-d-glucamine (0.887 g, 4.54 mmol) in
20 mL H2O. The solid dissolved after stirring for 15 min
(pHˆ6.8). Acetone (80 mL) was added dropwise over
15 min to form a precipitate, which was stirred for
15 min. The solid was ®ltered, washed twice with 10%
H2O/acetone, and dried under vacuum to give 2.93 g
(73%) of the meglumine salt of 4. The salt was powdered
and added to water (15 mL) with stirring at 258C. Acetone

5694
S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
(100 mL); crystals formed immediately. The mixture was
cooled to room temperature and let stand for 2 h. The crys-
tals were ®ltered, washed with hexane, and dried under
vacuum over P2O5 at 508C overnight to give 32.48 g (®rst
crop corrected yield 44%) of 6, mp 128±1298C. The mother
liquor was evaporated to ,10% volume. After standing for
4 h, the crystals were ®ltered, washed with hexane, and
dried under vacuum overnight to give a second crop of 6:
0.09. Found: C, 54.53; H, 3.69; N, 4.20; S, 9.87; Cl, 16.75;
H2O, 0.10 (KF).
(6R)-trans-3-Chloromethyl-7-amino-8-oxo-5-thia-1-aza-
bicyclo[4.2.0]oct-2-ene-2-carboxylate, diphenylmethyl ester,
HCl salt (8). (Method B) The mentioned compound (248 g,
0.55 mol) was treated with NaHCO3 (56 g, 0.66 mol) in
water (1.6 L) at 08C. The mixture was stirred at 08C for
0.5 h and then CH2Cl2 (1.5 L) was added. The biphasic
mixture was ®ltered through Celite and the Celite pad was
washed with CH2Cl2 (2 L total). The layers were separated
and the organic solution was dried over anhydrous MgSO4,
®ltered, and concentrated to a volume of ca. 2 L. The free
amine solution was then added to a mixture of 2,5-dichloro-
thiophenylacetic acid (130 g, 0.55 mol) and dicyclohexyl-
carbodiimide (144 g, 0.70 mol) in THF (1 L) at rt. The
reaction mixture was stirred for 2.5 h and then was ®ltered
through Celite, washing the Celite pad with several portions
of acetone. The ®ltrate was concentrated in vacuo to give a
solid mass. The solid was slurried in Et2O and then collected
by ®ltration, washing the solid with several portions of
Et2O. The solid was dried under high vacuum over P2O5
1
7.19 g (,10%), total yield for two crops ,54%. H NMR
(DMSO-d6, 270 MHz) d 4.0 (s, 2H), 7.24±7.27 (d,
Jˆ8.8 Hz, 1H), 7.37 (s, 1H), 7.47±7.50 (d, Jˆ8.8 Hz,
1H), 13.0 (br s, 1H). 13C NMR (DMSO-d6, 68 MHz) d
33.6, 126.0, 128.9, 130.7, 132.4, 137.8, 169.9. IR (KBr)
3500, 1707 cm21. MS (ESI) M2Hˆ235. Anal. Calcd for
C8H6Cl2SO2´0.081 H2O: C, 40.25; H, 2.60; Cl, 29.72; S,
13.44; H2O, 0.61. Found: C, 40.53; H, 2.49; Cl, 29.51; S,
13.18; H2O, 0.61 (KF).
(Method B) A mixture of 2,5-dichlorothiophenol (10.3 g,
57.5 mmol) and bromoacetic acid (8.03 g, 57.8 mmol) in
water (225 mL) was treated with 10N NaOH (13 mL,
130 mmol) and the mixture was heated at 1008C for 1 h.
The reaction mixture was then cooled to 08C and acidi®ed
to pH 1 with 6N HCl. The product precipitated and was
collected by ®ltration to give 13.0 g (95% yield) of 2,5-
dichlorophenylthioacetic acid as white crystals, mp 1188C.
1H NMR (300 MHz, CDCl3) d 3.74 (s, 2H), 7.15 (dd,
Jˆ2.9 Hz, 1H), 7.32 (d, Jˆ9 Hz, 1H), 7.36 (d, Jˆ2 Hz,
1H). Anal. Calcd for C8H6O2SCl2: C, 40.53; H, 2.55.
Found: C, 40.46; H, 2.64.
1
to give 268 g (77%) of 9, mp 1208C. H NMR (300 MHz,
CDCl3) d 3.43 (d, Jˆ18 Hz, 1H), 3.59 (d, Jˆ18 Hz, 1H),
3.69 (d, Jˆ17 Hz, 1H), 3.79 (d, Jˆ17 Hz, 1H), 4.36 (d,
Jˆ12 Hz, 1H), 4.41 (d, Jˆ12 Hz, 1H), 4.98 (d, Jˆ5 Hz,
1H), 5.81 (dd, Jˆ5.9 Hz, 1H), 6.98 (s, 1H), 7.14±7.44 (m,
14H). Anal. Calcd for C29H23N2O4S2Cl3: C, 54.94; H, 3.66;
N, 4.42. Found: C, 55.18; H, 3.84; N, 4.62.
(6R,7R)-7-[(2,5-Dichlorophenyl)thiomethylcarbonyl-
amino]-3-chloromethyl-8-oxo-5-thia-1-azabicyclo[4.2.0]-
oct-2-ene-2-carboxylic acid (10). To a slurry of benzhydryl
ester 9 (93.0 g, 146.7 mmol) in CH2Cl2 (465 mL) chilled in
an ice-bath was added anisole (18.6 mL) and then TFA
(139.5 mL, added over 5 min). After 40 min the ice-bath
was removed and the solution was diluted with toluene
(930 mL). The mixture was evaporated on a rotary evapora-
tor at a bath temp. of 408C to ,500 mL volume. During the
evaporation a solid formed. CH2Cl2 (300 mL) was added
and the resulting solution was evaporated to ,500 mL
volume. This last process was repeated two more times to
remove any residual TFA. The mixture was stirred at rt for
1 h. The solid was ®ltered and the ®lter cake was washed
with toluene (4£270 mL); each time the solid was slurried
in the funnel and sucked dry by compacting. Finally, the
product was dried under vacuum overnight to give 75.28 g
of microscopic glassy beads. The material was dissolved in
THF (120 mL) and hexane (10 mL) was added. A massive
precipitate formed immediately. The mixture was stirred as
more hexane (400 mL) was added slowly. After stirring for
1 h the solid was ®ltered, washed with hexane, and dried
under vacuum overnight to give 70.08 g (93.8%) of 10, mp
,136±1408C dec. 1H NMR (THF-d8, 270 MHz) d 3.4±3.7
(ABq, Jˆ18.1 Hz, 2H), 3.7 (s, 2H), 4.5±4.6 (AB q,
Jˆ11.7 Hz, 2H), 5.0 (d, Jˆ4.7 Hz, 1H), 5.7±5.8 (dd,
Jˆ4.7 and 8.5 Hz, 1H), 7.0±7.1 (dd, Jˆ2.4 and 8.5 Hz,
1H), 7.3 (d, Jˆ8.2 Hz, 1H), 7.5 (d, Jˆ2.3 Hz, 1H), 8.4
(d, Jˆ8.5 Hz, 1H); 13C NMR (THF-d8, 68 MHz) d, 27.4,
35.9, 44.4, 58.9, 60.7, 125.7, 127.2, 128.3, 131.2, 134.0,
140.0, 163.1, 165.2, 168.6; IR (KBr) 3500, 3270, 1775,
1653 cm21, [a]Dˆ61.3 (cˆ1, THF); Anal. Calcd for
C16H13N2O4S2Cl3´0.13 H2O´O.85 THF: C, 44.05; H, 3.73;
(6-R-trans)-3-(chloromethyl)-7-[[[(2,5-dichlorophenyl)thio]-
acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-
2-carboxylic acid, diphenylmethyl ester (9). (Method A)
Oxalyl chloride (7.06 mL, 80.9 mmol) and DMF (0.10 mL.
1.35 mmol) were added to a stirred suspension of acid 6
(16.08 g, 64.7 mmol) in 50 mL CH2Cl2 at 2258C. Vigorous
gas evolution occurred. The solution was stirred for 2 h,
evaporated to dryness, and the residue was re-evaporated
from toluene (10 mL£3). The product acid chloride was
dissolved in 50 mL CH2Cl2 and the solution was chilled to
2308C. In a separate ¯ask, N-methylmorpholine (13.7 mL,
124.7 mmol) was added to a suspension of 8 (25.8 g,
57.3 mmol) in 300 mL CH2Cl2. The resulting solution was
added dropwise over 20 min to the above acid chloride solu-
tion, keeping the temperature ,2208C. The bath tempera-
ture was kept at 235 to 2408C to allow a fast rate of
addition. After stirring at 2258C for 20 min, the bath was
removed, and the reaction was quenched with water and
stirred for 5 min. The mixture was washed with 400 mL
water (pH 1), 200 mL water, 400 mL 5% NaHCO3, and
200 mL water. All aqueous layers were backwashed with
CH2Cl2 (20 mL£2). The combined organic layers were
dried (MgSO4), stirred with Darco (30 g) for 15 min, and
®ltered through a pad of Celite. The ®ltrate was evaporated
to ,100 mL, and t-butylmethyl ether (300 mL) was added.
The mixture was stirred at 258C for 1 h and overnight at 58C.
The crystals were ®ltered and the ®lter cake was stirred with
t-butylmethyl ether on the funnel and ®ltered. This washing
process was repeated one more time and then the product
was dried overnight under vacuum to give 31.96 g (88%) of
9, mp 157±1588C. Anal. Calcd for C29H23N2O4S2Cl3´0.03
H2O: C, 54.89; H, 3.66; N, 4.42; S, 10.11; Cl, 16.76; H2O,

S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
5695
N, 5.14; S, 11.80; Cl, 19.62; H2O 0.41. Found: C, 43.85; H,
3.81; N, 5.27; S, 12.07; Cl, 20.01; H2O 0.44 (KF).24
(100 mL) was added to a solution of NaOH (16.06 g,
401.56 mmol) in H2O (30 mL) at 258C and the solution
was sparged with argon for 15 min. After cooling to 08C,
the reaction vessel was charged with mercaptoacetic acid
(17.99 g, 195.34 mmol) and the colorless solution was
sparged with argon for an additional 15 min. In a separate
¯ask a solution of 2,4,6-trichloropyridine (13) (35.64 g,
195.3 mmol) in MeOH (65 mL) was sparged with argon
for 15 min at 258C and then added in one portion to the
above solution. The transfer was completed with MeOH
(25 mL), the resulting heterogeneous mixture was further
diluted with MeOH (250 mL) to obtain a clear solution.
The solution was stirred at 08C for 1.5 h and then warmed
to rt and stirred overnight. After a total reaction time of
16.5 h the milky mixture was diluted with MeOH and
H2O to solubilize the insoluble material. The solution was
concentrated to remove most of the MeOH and then parti-
tioned between H2O (600 mL) and CH2Cl2 (250 mL). The
aqueous layer was separated and washed with CH2Cl2
(100 mL). The volatile organics were removed from the
milky aqueous phase in vacuum and the resulting clear solu-
tion was stirred and heated to 808C. The pH of the hot
solution was lowered from 9.90 to 3.45 with conc. H3PO4
and then the mixture was seeded with crystals of acid 14 and
stirred for 1 h. Since there was no evidence of crystalliza-
tion, the pH of the solution was lowered further to 3.15 with
H3PO4. After 1 h a signi®cant amount of a white crystalline
material had formed. The pH was lowered further stepwise
in a fashion for slow crystallization at 808C; pHs: 3.00
(15 min), 2.85 (15 min) and 2.35 (2.5 h). The heating was
stopped and the mixture was stirred overnight at 258C. The
solid was ®ltered, washed with H2O (2£250 mL), air-dried
(45 min), and then placed under vacuum overnight to
give 14 as a white crystalline material (39.07 g, 84%).
Recrystallization of 25.12 g of this material from EtOH
(50 mL)/heptane (450 mL) furnished 20.46 g (68% overall
yield) of 14 as ¯uffy white crystals, mp 1498C. Anal. Calcd
for C7H5NO2SCl2: C, 35.31; H, 2.12; N, 5.88; S, 13.47; Cl,
29.78. Found: C, 35.19; H, 1.86; N, 5.79; S, 13.35; Cl,
30.09.
2,6-Dichloropyridine, 1-oxide (12). An aqueous solution
of H2O2 (30%, 62.3 mL, 550 mmol) was added dropwise
over 5 min to a solution of 2,6-dichloropyridine (11)
(44.4 g, 300 mmol) in TFA (350 mL). The solution was
heated to re¯ux with a heating mantle behind a safety shield;
vigorous evolution of gas was observed. After 1 h, HPLC
showed starting material and product in a ratio of 8.5:91.5.
This ratio was unchanged after 2 h. After 3 h, the reaction
mixture was cooled to 258C and evaporated to 2/3 volume
behind a safety shield. The residue was added with stirring
to a mixture of 700 mL H2O and 100 mL ice. After stirring
for 10 min, the precipitated solid was ®ltered off and washed
with 50 mL of H2O. The ®lter ¯ask was changed and the
solid on the ®lter was washed further with H2O (4£50 mL).
The solid was dried under vacuum overnight to give 6.48 g
of 2,6-dichloropyridine (15% recovered, contained ,10%
product 12). The pH of the aqueous ®ltrate was carefully
adjusted to pH 9 (pH meter) with solid K2CO3 (foaming!)
and then extracted with CH2Cl2 (4£200 mL). The organic
extract was dried (MgSO4) and evaporated to ,50 mL.
Crystals started forming in the ¯ask. Heptane (200 mL)
was added slowly with rapid stirring. The mixture was
evaporated to remove ,50 mL of the solvent. The precipi-
tated solid was ®ltered, washed with heptane, and dried
under vacuum to give 41.63 g (84% yield, 99% based on
the recovered starting material) of 12, mp 137±1388C. Anal.
Calcd for C5H3NOCl2´0.04 H2O´0.02 CH2Cl2: C, 36.23; H,
1.89; N, 8.42; Cl, 43.46; H2O, 0.43. Found: C, 36.60; H,
1.81; N, 8.33; Cl, 43.82; H2O, 0.49 (KF).
2,4,6-Trichloropyridine (13). A 5 L four-necked Morton
¯ask was equipped with a mechanical stirrer, a liquid
addition funnel, a thermocouple, and a nitrogen inlet. The
¯ask was ¯ushed with N2 gas and charged with ®nely
powdered LiCl (87 g, 2.05 mol) and then POCl3 (520 mL)
via the addition funnel. The mixture was heated to 758C and
the liquid addition funnel was replaced with a solid addition
funnel. N-Oxide 12 (350 g, 2.13 mol) was introduced
portionwise via the solid addition funnel. An exotherm
was observed in a few min. The temperature was maintained
at 85±908C by controlling the rate of addition of 12 and by
external cooling when necessary; the addition was complete
in 2 h. After an additional 3 h at 858C the reaction was
complete by HPLC. The mixture was allowed to slowly
cool to 608C and then poured in portions into a mixture of
1 kg ice and 500 mL H2O in a beaker cooled in an ice-bath.
The temperature of the mixture was ,308C during the later
part of the quenching process. After stirring for 0.5 h 1 L of
hexane was added. Aqueous NaOH (1N, 900 mL) was
added with cooling at a rate such that the temperature
remained 30±408C. The organic layer was separated and
the aqueous layer was extracted with n-heptane (2£1 L).
The combined organic extract was dried over ®nely
powdered anhydrous Na2SO4 and ®ltered. The product partly
crystallized on concentration of the ®ltrate. After cooling at
258C for 4 h the liquid portion was decanted and then the
solid was ®ltered with suction and dried under vacuum for a
few minutes to give 231.8 g (60%) of product 13.
(6R-trans)-3-(Chloromethyl)-7-[[[(2,6-dichloro-4-pyri-
dinyl)thio]acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]-
oct-2-ene-2-carboxylic acid, diphenylmethyl ester (15).
NMM (1.93 mL, 17.54 mmol) was added to a suspension
of acid 14 (4.16 g, 17.5 mmol) and 8 (7.91 g, 17.54 mmol)
in CH2Cl2 (88 mL) stirred at ice-bath temperature to obtain
a clear solution.25 EDC26 (4.03 g, 21.0 mmol) was added as
a solid and the resulting solution was stirred in the ice-bath.
After 1 h, TLC analysis27 showed no 8 remaining. The reac-
tion mixture was diluted with CH2Cl2 (10 mL) and washed
successively with very dilute aqueous HCl (0.04N, 50 mL),
brine, 5% NaHCO3, and brine. The solution was dried
(MgSO4) and evaporated to ,50 mL. Crystals formed
during evaporation of the solvent. t-Butylmethyl ether
(140 mL) was added slowly, and after stirring for 30 min,
the crystals were ®ltered, washed with t-butylmethyl ether,
and dried under vacuum to give 10.37 g (93%) of chloro-
1
ester 15 as microscopic needles, mp 178±1808C (dec.). H
NMR (CDCl3, 270 MHz) d 3.4±3.8 (m, 4H), 4.4 (d,
Jˆ3 Hz, 1H), 4.9 (d, Jˆ4.7 Hz, 1H), 5.8 (m, 1H), 6.9 (s,
1H), 7.3±7.4 (m, 11H); 13C NMR (CDCl3, 68 MHz) d, 27.3,
34.4, 43.0, 57.5, 59.3, 80.0, 119.1, 125.6, 127.0, 127.1,
[(2,6-Dichloropyrid-4-yl)thio]acetic acid (14). MeOH

5696
S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
127.8, 128.2, 128.4, 128.5, 128.6, 138.8, 138.9, 150.9,
151.8, 160.3, 163.8, 166.5; IR (KBr) 3500, 3271, 1777,
1723, 1653 cm21; MS (ESI) M1Hˆ635; [a]Dˆ24.1
(cˆ0.5, THF). Anal. Calcd for C28H22N3O4S2Cl3: C,
52.96; H, 3.49; N, 6.62; S, 10.10; Cl, 16.75. Found: C,
52.95; H, 3.26; N, 6.60; S, 10.11; Cl, 16.45.
under vacuum for 0.5 h to give 45.46 g (89%) of 4-pyranone
18.
4-Thiopyranone (19). A literature procedure20 was modi-
Ê
®ed as follows. Molecular sieves (2.0 g, 4 A, spherical)
were added to a solution of pyranone 18 (24.25 g,
250 mmol) in dry toluene (400 mL) and the mixture was
deoxygenated twice by evacuation and release of vacuum
with argon. After the mixture was stirred overnight,
Lawesson's reagent (57.26 g, 142 mmol) was added and
the mixture was heated to 808C in an oil-bath. The mixture
stayed heterogeneous due to the crystallization of the trimer
byproduct 23. After 1 h the mixture was cooled and ®ltered
through 200 mL of silica gel in a sintered glass funnel. The
cake was washed with 200 mL toluene and 300 mL 20%
EtOAc in toluene until the red-orange color was removed.
The ®ltrate was evaporated to give 62 g of a solid. The
product was distilled in a Kugelrohr assembly with the
receiver bulb cooled in a dry ice-acetone bath. Distillation
at 45±808C (oven temperature) under vacuum (1 mm) gave
28 g (quantitative, uncorrected yield) of 4-thiopyranone 19.
The product was redistilled as above to furnish 26.57 g
(95%) of 4-thiopyranone 19.
(6R-trans)-3-(Chloromethyl)-7-[[[(2,6-dichloro-4-pyri-
dinyl)thio]acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]-
oct-2-ene-2-carboxylic acid (16). TFA (25.0 mL) was
added dropwise to a stirred suspension of ester 15
(16.70 g, 26.3 mmol) and anisole (8.56 mL, 78.9 mmol) in
CH2Cl2 (83.0 mL) chilled in an ice-bath. After 40 min, TLC
(silica gel, EtOAc/hexane 3:7) showed no ester 15. After a
total of 1 h, toluene (217 mL) was added to the reaction
mixture and the mixture was evaporated to ,1/2 volume
(a precipitate formed during the evaporation). Toluene
(100 mL) was added and the mixture was evaporated to
,200 mL and then stirred in an ice-bath for 30 min. The
product was ®ltered, washed twice with cold toluene
(40 mL), and dried under vacuum over NaOH pellets
overnight to give 12.41 g (98%) of chloroacid 16,28 mp
1
173±1758C (dec). H NMR (CDCl3, DMSO-d6, 270 MHz)
d
3.5±3.7 (ABq, Jˆ17.8 Hz, 2H), 3.8±4.0 (ABq,
Jˆ15.6 Hz, 2H), 4.4±4.6 (ABq, Jˆ11.1 Hz, 2H), 5.0 (d,
Jˆ4.7 Hz, 1H), 5.2 (br s, 2H), 5.70±5.75 (dd, Jˆ4.7 Hz,
1H), 7.3 (s, 2H), 9.3 (d, Jˆ8.2 Hz, 1H); 13C NMR
(CDCl3, DMSO-d6, 68 MHz) d 26.8, 34.0, 43.5, 57.8,
59.8, 119.1, 124.9, 126.7, 150.0, 154.3, 162.8, 164.2,
168.1; IR (KBr) 3263, 1776, 1714, 1651 cm21; MS (ESI)
M1Hˆ464, M2Hˆ462; [a]Dˆ80.5 (cˆ0.8, THF). Anal.
Calcd for C15H12N4O4S2Cl3´0.065 H2O´0.085 TFA: C 37.99;
H, 2.57; N, 8.76; S, 13.37; Cl, 22.17; F, 1.01; H2O, 0.24.
Found: C, 38.31; H, 2.33; N, 8.61; S, 13.18; 22.23; F, 1.28;
H2O, 0.25 (KF).29
(S)-a-[[(1,1-Dimethylethoxy)carbonyl]amino]-4-thioxo-
1(4H)-pyridinepentanoic acid (21). A mixture of 4-thio-
pyrone 19 (7.43 g, 66.27 mmol)], l-a-N-Boc-ornithine 20
(15.39 g, 66.27 mmol), absolute ethanol (70 mL), distilled
water (70 mL), and pyridine (5.4 mL, 66.27 mmol) was
re¯uxed under an argon atmosphere. Within 5 min. all the
material was in solution. After 6.3 h the heating bath was
removed and stirring was continued at 258C for 30 min.
Methanesulfonic acid (4.4 mL, 66.27 mmol) was added
and the mixture was stirred for 10 min. The volatiles were
removed under vacuum and the resulting orange solution
was treated with EtOAc (200 mL) and H2O (200 mL). The
aqueous phase was saturated with NaCl, stirred vigorously
for 10 min, and then the phases were separated. The aqueous
fraction was re-saturated with NaCl and extracted with
EtOAc (4£200 mL). The combined organic fractions were
dried (MgSO4) and stirred with activated charcoal (0.226 g)
for 20 min. The mixture was ®ltered through a pad of Celite
and the ®ltrate was evaporated. The residue was dried under
high vacuum to a constant weight to give 21 as a yellow
amorphous solid (16.84 g, 78%). mp 908C (eff., contraction
at 608C). 1H NMR (DMSO-d6, 270 MHz) d 1.4 (s, 9H), 1.5±
1.8 (m, 4H), 3.9 (m, 3H), 7.1 (m, 1H), 7.2 (d, Jˆ7.0 Hz, 2H),
7.6 (d, Jˆ7.0 Hz, 2H); 13C NMR (DMSO-d6, 68 MHz) d
27.1, 27.4, 28.2, 53.0, 56.0, 78.1, 130.2, 136.2, 155.6, 173.8,
188.9; IR (KBr) 3490, 1703, 1624 cm21; MS (ESI)
M1Hˆ327; [a]Dˆ11.4 (cˆ1.0, CH3CN). Anal. Calcd
for C15H22N2O4S´0.16 H2O´0.31 EtOAc: C, 54.69; H, 7.01;
N, 7.86; S, 8.99; H2O, 0.8. Found: C, 54.73; H, 7.02; N,
7.78; S, 8.86; H2O, 0.8 (KF).
4-Pyranone (18). A literature procedure19 was modi®ed as
follows. A 1 litre, three-necked ¯ask was equipped with a
mechanical stirrer, thermometer, and a Claison adapter
connected to a condenser on top. The ¯ask was charged
with anhydrous chelidonic acid (17)(100 g, 530 mmol),
dibutylphthalate (300 mL), Cu-bronze (10.85 g), and 2,20-
dipyridyl (3.8 g, 24.4 mmol). The mixture was stirred
slowly under argon and heated with a heating mantle to
,2108C under an argon atmosphere. The ¯ow of argon
gas was stopped and the progress of the reaction was moni-
tored by the evolution of CO2. After ,1/2 h the rate of
evolution of CO2 decreased considerably. The mixture
was stirred vigorously for about 30 s, cooled, and then
®ltered through a glass wool plug for a rapid ®ltration.
Insoluble material was washed separately with EtOAc and
®ltered, and the ®ltrate was evaporated to remove EtOAc.
This mixture of product 18 and dibutyl phthalate was further
dried under pump vacuum for 1 h and combined with the
®rst bulk ®ltrate. The product, a solid, was distilled under
vacuum without the use of a condenser, leaving behind the
high boiling solvent dibutyl phthalate. The receiver ¯ask
was cooled in an ice-bath. During distillation a heat-gun
was used to melt any solid in the receiver adapter. The
product 18 distilled at 60±658C/0.6 mm, bath temp.
,110±1308C. The solid was melted and stirred with
50 mL heptane. After stirring in an ice-bath for 0.5 h the
solid product was ®ltered, washed with heptane, and dried
[6R-[3(Sp),6a,7b]]-(4-[[[2-Carboxy-7[[[(2,5-dichloro-
phenyl)thio]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-
2-ene-3-yl]methyl]thio]-1[[[4-carboxy-4(1,1-dimethyl-
ethoxy)-carbonyl]amino]butyl]pyridinium, chloride (27).
To a solution of 10 (80.93 g, 153 mmol) in THF (400 mL)
under an argon atmosphere was added 21 (54.89 g,
153 mmol), and the mixture was stirred at 258C. The
reaction was monitored by HPLC; after 2.8 h the solution

S. V. D'Andrea et al. / Tetrahedron 56 (2000) 5687±5698
5697
was added over 2 min to t-butylmethyl ether (1700 mL)
stirred mechanically in a Morton ¯ask. The mixture was
stirred for 1 h, ®ltered, and then the ®lter cake was washed
thoroughly by slurrying with t-butylmethyl ether
(3£200 mL) in the ®ltration funnel. The product was dried
under vacuum overnight to give 123.68 g (96.8%) of chlo-
16.49; Cl, 12.16; H2O, 1.18 (KF). This material was used in
the next reaction without further puri®cation. A 3 g sample
of crude product was crystallized from DMF±CH3CN to
give 2.71 g (90.3%) of 32, mp .1458C (dec.).
(6R-trans)-7-[[[(2,6-Dichloro-4-pyridinyl)thio]acetyl]-
amino]-3-[(4-pyridinyl-thio)methyl]-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (33). NaI
(0.937 g, 6.26 mmol) was added portionwise to a rapidly
stirred solution of 4-mercaptopyridine (0.768 g, 6.56
mmol) in DMF (15.6 mL) at 258C. Chloroacid 16 (3.0 g,
6.26 mmol) was added to the reaction mixture and after
2 min 2,6-lutidine (0.87 mL, 7.51 mmol) was added. The
product 33 precipitated from the reaction mixture. After
15 min, isopropyl alcohol (31 mL) was added dropwise
and the mixture was stirred for 15 min. The solid was
®ltered, washed with isopropyl alcohol (25 mL), water
(25£2 mL),30 acetone (25 mL), and dried under vacuum to
give 3.15 g of crude 33. This material was powdered with a
stirring rod and added to DMF (19 mL) with rapid stirring.
The solid initially dissolved and then precipitated. After
stirring the suspension for 10 min, THF (19 mL) was
added and the mixture was stirred further for 15 min. The
solid was ®ltered, washed with THF (15 mL£2), and dried
under vacuum overnight to give 2.99 g (82%) of the
1
ride salt 27 as a yellow powder, mp. 155±1608C (eff.). H
NMR (DMSO-d6, 270 MHz) d 1.4 (s, 9H), 1.7±1.9 (m, 5H),
3.3±3.9 (m, 4H), 4.0 (s, 2H), 4.5 (m, 4H), 5.2 (d, Jˆ5.3 Hz,
1H), 5.6±5.7 (dd, Jˆ4.7 and 8.2 Hz, 1H), 7.1 (d, Jˆ8.2 Hz,
1H), 7.2 (d, Jˆ2.3 Hz, 1H), 7.4 (d, Jˆ2.3 Hz, 1H), 7.5 (s,
1H), 8.0 (d, Jˆ7 Hz, 2H), 8.8 (d, Jˆ6.5 Hz, 2H), 9.4 (d,
Jˆ8.0, 1H); IR (KBr) 3500, 1778, 1695, 1630 cm21; MS
(ESI) M2Clˆ757, M2HCl2Hˆ755; [a]Dˆ25.9 [cˆ1,
THF]. Anal. Calcd for C32H35N4O8S3Cl3´0.65 H2O´0.2
THF´0.2 (CH3)3COCH3: C, 47.10; H 4.85; N, 6.69; S,
11.48; Cl, 12.69; H2O, 1.40. Found: C, 46.70; H 4.91; N,
6.64; S, 11.03; Cl, 12.81; H2O, 1.36 (KF).
[6R-[3(Sp),6a,7b]]-(4-[[[2-Carboxy-7[[[(2,6-dichloro-4-
pyridinyl)thio]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]-
oct-2-ene-3-yl]methyl]thio]-1[[[4-carboxy-4(1,1-dimethyl-
ethoxy)-carbonyl]amino]butyl]pyridinium, chloride (29).
Chloroacid 16 (517 mg, 1.1 mmol) was dissolved in DMF
(4 mL) and then thiopyridone 25 (348 mg, 1.0 mmol) was
added. This mixture was stirred for 5 h at rt. The DMF
solution was added dropwise to 150 mL of vigorously stir-
red diethyl ether. The precipitated solid was collected by
®ltration. This solid was stirred with 25 mL of acetone for
15 min and then collected by ®ltration to give 584 mg (71%)
of 29 as its sodium salt. This material was used without
further puri®cation for the preparation of cephem 2.
1
mercaptopyridyl acid 33 mp 160±1618C (effer.). H NMR
(DMSO-d6, 270 MHz) d 3.5±3.8 (ABq, Jˆ18.2 Hz, 2H),
4.0 (s, 1H), 4.2±4.3 (ABq, Jˆ12.9 Hz, 2H), 5.1 (d,
Jˆ4.7 Hz, 1H), 5.6±5.7 (dd, Jˆ4.7 Hz, 1H), 7.3 (m, 2H),
7.5 (s, 2H), 8.4 (d, Jˆ5.8 Hz, 2H), 9.3 (d, Jˆ8.3 Hz, 1H);
13C NMR (DMSO-d6, 68 MHz) d 27.2, 32.8, 33.2, 57.5,
59.3, 119.2, 121.3, 125.8, 126.2, 147.7, 149.1, 149.2,
155.3, 163.0, 164.1, 167.7. IR (KBr) 3272, 1779,
1672 cm21; [a]Dˆ34.3 (cˆ0.5, pyridine); MS (ESI)
M1Hˆ543, M2Hˆ541. Anal. Calcd for C20H16N4
O4S3Cl2´0.09 H20´0.5 DMF: C, 44.40; H, 3.41; N, 10.84;
Cl, 12.19; H2O, 0.28. Found: C, 44.06, H, 3.59, N, 11.12;
Cl, 12.22; H2O, 0.27 (KF).
(6R-trans)-7-[[[2,5-Dichlorophenyl)thio]acetyl]amino]-
3-[(pyridinylthio)methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]-
oct-2-ene-2-carboxylic acid (32). NaI (4.62 g, 30.8 mmol)
was added portionwise over 2 min to an orange solution of
4-mercaptopyridine (3.78 g, 34 mmol) in 100 mL of anhy-
drous DMF under an argon atmosphere. The mixture was
stirred vigorously with an overhead stirrer for 8 min to
dissolve most of the salt. Chloroacid 10 (14.4 g,
30.1 mmol) was added and, after stirring for 5 min
(exotherm 228 to 29.48C), 2,6-lutidine (3.82 g, 35.6 mmol)
was added. After 40 min the dark red-brown solution was
diluted with 60 mL CH3CN and 40 mL of water and stirred
for 5 min. The solution was further diluted with 140 mL of
CH3CN and 60 mL water to precipitate the product. The
suspension was stirred for 35 min at 258C. The mixture
was ®ltered and the solid was washed with 100 mL of
CH3CN, 100 mL of 1:1 CH3CN/H2O, and CH3CN
(2£50 mL). The isolated solid was dried under vacuum
over P2O5 for 60 h to give 14.3 g (83.0%) of 32 as a yellow
Acknowledgements
We would like to acknowledge the analytical chemistry
group at Bristol±Myers Squibb for the spectroscopic
analysis of all compounds.
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1
solid. H NMR (DMSO-d6, 270 MHz) d 3.5±3.6 (ABq,
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byproducts during HPLC analysis.
29. Due to the instability of 16 in DMSO-d6, a freshly prepared
solution in CDCl3/DMSO-d6 was used for the NMR analyses.
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