Synthesis of (5S)-tricyclic penems as novel and potent inhibitors of bacterial signal peptidases

Article abstract of DOI:10.1055/s-2003-40882

(5S)-Tricyclic penems were synthesized for the first time via intramolecular cyclization of penem epoxy amides catalyzed by a weak Lewis acid [Mg(ClO₄)₂]. Due to the high degree of ring strain in these molecules, mild reaction conditions were developed to successfully construct penem intermediates and the tricyclic penem final products. These tricyclic penems and other newly synthesized (5S)-penem esters and amides exhibited good-to-high potency when tested as inhibitors of bacterial signal peptidases.

Full text of DOI:10.1055/s-2003-40882

1732
PAPER
Synthesis of (5S)-Tricyclic Penems as Novel and Potent Inhibitors of Bacterial
Signal Peptidases
S
ynthesis of (5
S
)-T
.
ricyclic Pen
E
ems ric Hu,* Nick K. Kim, Leo Grinius, Charles M. Morris, Cynthia D. Wallace, Glen E. Mieling,
Thomas P. Demuth Jr.
Procter & Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, Mason, OH 45040, USA
E-mail: hu.xe@pg.com
Received 16 May 2003
known -lactam antibiotics such as (5R)-penems 1,6 ap-
peared in the literature as SPase inhibitors (Figure 1). In
this context, we wish to report our endeavors toward the
design and synthesis and biological evaluation of novel
Abstract: (5S)-Tricyclic penems were synthesized for the first time
via intramolecular cyclization of penem epoxy amides catalyzed by
a weak Lewis acid [Mg(ClO4)2]. Due to the high degree of ring
strain in these molecules, mild reaction conditions were developed
to successfully construct penem intermediates and the tricyclic pen- (5S)-tricyclic penems SPase inhibitors.
em final products. These tricyclic penems and other newly synthe-
sized (5S)-penem esters and amides exhibited good-to-high potency
when tested as inhibitors of bacterial signal peptidases.
R1
O
R2
R1
O
R2
R1
O
5
S
S
S
X
X
X
N
N
N
Key words: tricyclic penems -lactams, cyclization, epoxidation,
signal peptidase, inhibitors
O
O
O
1 (5S)-Penem
X = OH
2 (5S)-Penem
X = OR, NHR
3 (5S)-Tricyclic Penem
Figure 1 Structures of (5S)-Penems 1–3
Over recent years, drug researchers have increasingly
sought to find new antibiotics that inhibit novel targets or
possess novel mechanisms of antibacterial action.1 Bac-
terial signal peptidase (SPase) as a protease involves in
protein translocation through the cytoplasmic membrane
in the final step of the bacterial protein secretion path-
way.2 In this process, an amino-terminal signal peptide is
cleaved and the release of mature protein is facilitated into
the outer medium or periplasm.3,4 They are essential for
cell viability,5 and therefore represent a class of novel an-
tibacterial targets, providing an alternative approach to
conquer bacterial infections, including those caused by
drug resistant pathogens. Recently, (5S)-penems 2, which
are stereochemical isomers and structural analogs of the
Our design strategy for the synthesis of tricyclic penems
was based on the construction of an additional ring to an
existing penem bicyclic ring system. Due to the sensitivity
of the penem functionality toward nucleophiles, we
sought to develop mild reaction conditions, so as to mini-
mize the destruction of penem molecules during the syn-
thesis. As shown in Scheme 1, the (5S)-penem 5 was
readily derived from the corresponding (5R)-counterpart
47 via photochemical isomerization.3,8 The process typi-
cally yielded a mixture of nearly 3:2 ratio of 5S/5R-iso-
mers in 1.5 hours, which were readily separated by flash
chromatography.9 Extended reaction time did not consid-
TBDMSO
O
TBDMSO
6
5
S
S
hν / EtOAc
MnO2
OH
O
OH
O
N
N
or Dess-Martin
Periodinane
O
O
O
4
5
TBDMSO
O
O
O
TBDMSO
O
TBDMSO
O
O
O
S
CH2N2
Solvent
S
S
N
H
O
+
N
N
O
O
O
O
8
6
7 (α and β)
in CH2Cl2 , 7:8 ~ 4:1
in MeOH, 7:8 > 20:1
Scheme 1
Synthesis 2003, No. 11, Print: 05 08 2003.
Art Id.1437-210X,E;2003,0,11,1732,1738,ftx,en;C03303SS.pdf.
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of (5S)-Tricyclic Penems
1733
erably improve the isomerization ratio, but only resulted desired tricyclic penem 10 under basic conditions includ-
in a lower yield.10 Oxidation of the (5S)-penem alcohol to ing K2CO3 and NaH. Instead, only an unidentified mixture
aldehyde 6 by conventional conditions such as Swern and of degradation products was obtained. Alternatively, we
Jones oxidation methods were impractical, due to sensi- decided to focus on an approach involving amide forma-
tivity of the -lactam toward the reaction conditions. tion followed by ring construction. This structural elabo-
11
However, two mild methods using MnO2 and Dess– ration included the conversion of the epoxy ester to the
Martin Periodinane12 were applied to produce the alde- corresponding amide 11 in 75% yield through a one-pot
hyde in >98% yield. Subsequent epoxidation of the alde- three-step sequence: deallylation, mixed anhydride for-
hyde with diazomethane13 in CH2Cl2 cleanly provided a mation and subsequent amidation. Attempt to isolate the
mixture, identified as diastereomeric epoxide isomers 7 , acid intermediate was unsuccessful, due largely to its ap-
7 , and methyl ketone 8 in a ratio of 4:1 of 7:8. The latter parent instability.14 Finally, the lactam ring closure via
was likely derived from rearrangement of an initially with the epoxide ring-opening took place in refluxing
formed diazonium species. The ratio of the designed ep- THF in the presence of a weak Lewis acid, Mg(ClO4)215 to
oxides and the unwanted ketone was increased to >20:1, form tricyclic penem 10 in only 5% yield after purifica-
when the epoxidation took place in MeOH. The epoxides tion. It was noticed that the complete consumption of the
7 and 7 in ca.1:4 ratio were readily separated by flash starting epoxide under the reaction conditions (in reflux-
chromatography, but the assignment of the relative stere- ing THF for 3 h) gave only low yield of the desired prod-
ochemistry by 2D NMR analyses remained inconclusive. uct. The poor chemical yield in the cyclization step was
Therefore, the mixed epoxides (obtained in 95% opti- due partially to the formation of methyl ketone 13 (isolat-
mized yield) were utilized to carry out the synthesis of the ed and characterized), potentially from rearrangement of
tricyclic penems.
the initially formed tricyclic penem alcohol. The mecha-
nism of this rearrangement is not completely understood,
but we suspect that the free hydroxyl group generated in
the cyclization might be involved in the rearrangement
process. Therefore, the procedure was modified such that
the cyclized product was isolated and immediately acylat-
ed to give the more stable acetate 15a in 30% yield in a
shorter reaction time (in refluxing THF for 1.5 h) with re-
covery of 12a. Surprisingly, the removal of the silyl group
The robust preparation of the penem epoxy ester provided
a viable building block for adding another ring to the ex-
isting penem skeleton. Our initial attempt to form an addi-
tional ring on the -lactam scaffold was carried out by
benzylamine nucleophilic addition to the epoxide 7 in hot
i-PrOH to give the hydroxylamine intermediate 9 in 55%
yield. However, the final cyclization failed to produce the
R1O
R1O
OH
TBDMSO
OH
H
O
BnNH2
i-PrOH
N
S
S
S
Bn
N
N
N
N
O
O
Bn
O
O
O
O
O
O
9 R1 = TBDMS
10 R1 = TBDMS
7 (α and β)
1) Pd(0)(PPh3)4/PPh3
Na 2-Et-Hexanoate
2) IPCF/TEA
3) BnNH2
R1O
O
TBDMSO
O
O
O
Mg(ClO4)2, THF
S
S
1) NH4F•HF
2) Acylation
H
H
N
N
N
N
Bn
Bn
O
O
12a R1 = TBDMS
12b R1 = H
11
12c R1 = Ac
12d R1 = Allyl-OC(=O) (Alloc)
R1O
O
R1O
R1O
OAc
O
OH
S
S
S
AcCl/TEA
+
N
O
15a R1 = TBDMS
15b R1 = H
15c R1 = Ac
15d R1 = Alloc
N
N
N
O
N
O
or Alloc-Cl/TEA
Bn
Bn
O
O
HN
Bn
13 R1 = TBDMS
10 R1 = TBDMS
14c R1 = Ac
14d R1 = Alloc
Scheme 2
Synthesis 2003, No. 11, 1732–1738 © Thieme Stuttgart · New York

1734
X. E. Hu et al.
PAPER
in 15a in the final step failed to give the expected product was converted to the corresponding alcohol 16a and ace-
15b under the conditions of NH4F·HF, a very mild proce- tate 16b via a three-step sequence: acylation, desilylation
dure used successfully in carbapenem16 and penem17 des- and then acylation. The (5R)-penem ester was protected
ilylation. This problem was circumvented by utilizing the with 3,4-dihydro-2H-pyran (DHP) to give intermediate
acetylated precursor 12c and 12d, generated from desily- 17. The silyl protecting was removed and then acylated
lation of 11 followed by acylation, prior to the ring closure with Alloc-Cl. The deprotection of the THP group result-
in the identical overall process. Thus, the final (5S)-tricy- ed in the (5R)-penem alcohol 18. The similar procedure
clic penem diacetates 15c and 15d (ca.1:4 ratio of diaste- was used to prepare the corresponding alcohol 19a, ace-
reomers) were obtained using the aforementioned tate 19b and allyloxycarbonyl 19c starting from the (5S)-
cyclization procedure in slightly improved chemical yield penem alcohol 5. In all cases, satisfactory chemical yields
(38%).
were achieved in a range of 62–75%. The DHP protected
(5S)-penem ester 20 was converted to the amide 22 using
the palladium(0) catalysis conditions as previously de-
scribed followed by mixed anhydride coupling reaction.
Subsequently, the silyl and THP protecting groups of the
compound 22 were removed to give the diol 23a, which
was acylated to afford the amide 23b. Alternatively, the
We also prepared substituted (5R)-penem and (5S)-penem
esters and amides as shown in Scheme 3. Again, we uti-
lized conventional mild reaction conditions to embark on
the functional group transformation to prevent degrada-
tion of the penem structure. The (5R)-penem alcohol 4
R1O
R1O
5
1). NH4F.HF
S
S
OH
OTHP
O
N
O
N
2). Alloc-Cl/Et3N
3). p-TsOH.H2O
O
O
O
O
18 R1 = Alloc
17 R1 = TBDMS
DHP/p-TsOH
R1O
O
R1O
O
5
1). Ac2O/Et3N
S
S
OAc
OH
N
O
N
O
2). NH4F.HF
3). Alloc-Cl/Et3N
O
O
16a R1 = H
16b R1 = Alloc
4 R1 = TBDMS
hν
R1O
R1O
O
1). Ac2O/Et3N
S
S
OH
OAc
N
O
N
O
2). NH4F.HF
O
3). Ac2O/Et3N
or Alloc-Cl/Et3N
O
O
5 R1 = TBDMS
5a R1 = H
19a R1 = H
NH4F.HF
19b R1 = Ac
19c R1 = Alloc
DHP/p-TsOH
R1O
O
R1O
THPO
1). Pd(0)(PPh3)4/PPh3
Na 2-Et Hexanoate
S
S
OTHP
O
N
N
N
O
2). EtOCOCl/Et3N
then Pyrrolidine
O
O
22 R1 = TBDMS
20 R1 = TBDMS
1). p-TsOH.H2O
1). NH4F.HF
2). Ac2O/Et2N
or Alloc-Cl/Et3N
3). p-TsOH.H2O
2). NH4F.HF
3). Ac2O/Et3N
R1O
O
R1O
O
OR2
S
S
OH
O
N
N
N
O
O
21a R1 = Ac
21b R1 = Alloc
23a R1 = R2 = H
23b R1 = R2 = Ac
Scheme 3
Synthesis 2003, No. 11, 1732–1738 © Thieme Stuttgart · New York

PAPER
Synthesis of (5S)-Tricyclic Penems
1735
(5S)-penem diol 5a was obtained by deprotection of the moderate in vitro inhibitory activity against both E. coli
silyl ether 5, and the hydroxyl acylates 21a and 21b were and MRSA SPases. However, enhanced activity was seen
prepared from 20 by sequential desilylation, acylation and in mono acetates 19b and 21a against E. coli with only
then removal of the THP protecting group.18
weak activity against MRSA, and it was also true for the
diacetate 21b. One of the most intriguing finding in sub-
stitution variations was the incorporation of the Alloc
group at the R1 position in analog 19c and the activity was
Inhibition of SPase
A gene encoding SPase was isolated from methicillin-re- significantly increased in both E. coli (IC50 at 0.5 M)
sistant Staphylococcus aureus (MRSA, strain MI339) us- and MRSA (IC50 at 6 M) assays. Our structure-activity
ing reported techniques.19 The gene was cloned in a pET learnings were further expanded in the case of (5S)-penem
vector (Novagen) and overexpressed as a His-tagged pro- amides. The free hydroxyl amide 23a displayed about 25-
tein in Escherichia coli. The SPase was isolated using a fold greater potency than the acetyl amide 23b in E. coli
described protocol.20 Determination of IC50 values for the assay, but possessed similarly low activity in the MRSA
test compounds against MRSA SPase and E. coli21 was assay. When the amides were compared with the esters
performed by a fluorescence polarization assay using a having the same variations, unsubstituted diol 23a exhib-
fluorescent preprotein as the substrate and FPM-2 (Jolley ited 47-fold increase in its potency against E. coli vs. its
Inc.) as the reader. These two SPase enzymes were select- counterpart ester analog 5a, although the activity against
ed to evaluate new analogs as representatives of a broad MRSA SPace was worsened by about 4-fold. In contrast,
spectrum profile for their in vitro activity.
the diacetate amide 23b was less active than the ester
compound 18b. Again, the Alloc substitution displayed
the tendency of enhancing the in vitro activity in the case
of epoxides, in which the Alloc analog 12d was over 60-
fold more active than the acetate 12c against E. coli SPase
and over 4-fold more active against MRSA. The same
trend was sustained in the case of tricyclic penems, but
with more pronounced potency. The Alloc substituted tri-
The newly synthesized (5R)-, (5S)-penems and tricyclic
penems were tested against the isolated E. coli and MRSA
enzymes and exhibited interesting biological activity as
shown in Table 1. The stereochemical effect at the C-5
position of the penems was evident in examples of (5R)-
penem 16a and (5S)-penem 19a, the latter showed at least
4-fold more potent against MRSA. This effect was more
pronounced when the counterparts were compared be-
tween the Alloc derivatives 18 and 19c; greater than 33-
fold potency discrepancy observed in favor of the (5S)-
penem isomer. When both R1 and R2 substituents were
kept as free hydroxyl groups, compound 5a showed only
cyclic penem 15d exhibited IC50 at 0.2 M and 5
M
against E. coli and MRSA SPases, respectively, the best
compound evaluated in our isolated enzyme assays,
whereas the acetate 15c showed similar activity to that of
the diacetate 21b. Overall, the Alloc group presented a
significant influential enhancement in the SPase inhibi-
Table 1 Biological and Stability Data of New (5S)-Penems and (5S)-Tricyclic Penemsa
Product
Config. at C5
R1
R2
SPase IC50 ( M)
E. coli
MIC MRSAb
( g/mL)
Stability
T1/2 (h)
MRSA
16a
19b
5a
R
S
S
S
S
S
R
S
S
S
S
S
S
H
Ac
Ac
H
n.d.
12
47
3
>200
50
n.d.
>250
90
4.6
H
1.6
H
50
n.d.
n.d.
<1
21a
21b
19c
18
Ac
H
50
90
Ac
Ac
H
6
100
6
>256
120
Alloc
Alloc
H
0.5
n.d.
1
n.d.
n.d.
21
H
>200
>200
>100
>100
25
n.d.
23a
23b
12c
12d
15c
15d
H
>128
>128
n.d.
Ac
Ac
–
25
38
0.6
12
0.2
n.d.
n.d.
n.d.
0.5
Ac
Alloc
Ac
–
n.d.
–
>100
5
>128
>128
Alloc
–
n.d.
a Ac = Acetyl, Alloc = Allylxoycarbonyl, n.d. = Not determined.
b MRSA = strain MI339.
Synthesis 2003, No. 11, 1732–1738 © Thieme Stuttgart · New York

1736
X. E. Hu et al.
PAPER
tory assays, which was especially pronounced in the (5S)- ing with Phe84. These additional binding features may
tricyclic penem.
contribute to the observed high in vitro potency of the
(5S)-tricyclic penem in bacterial SPase.
When the newly synthesized (5S)-penems and tricyclic
penems were tested for MIC measurement in whole cell In conclusion, (5S)-tricyclic penems containing a highly
assay, only the free diol 5a, the mono acetate 21a and Al- strained, fused 4-, 5- and 6-membered ring system were
loc ester 19c exhibited weak antibacterial activity against synthesized for the first time by intramolecular cycliza-
MRSA. We subsequently determined solution stability of tion of penem epoxy-amides. The accomplishment of the
these analogs at 37 °C in 50 mM pH 7.4 potassium phos- synthesis was based on careful control of reaction condi-
phate buffer. The half-life for disappearance of the test tions to preserve the sensitive 5S-penem functionality.
compounds was measured by reverse phase HPLC. As The tricyclic penems were good inhibitors of bacterial
shown in Table 1, most of the compounds displayed short SPases from E. coli and MRSA. Other (5S)-penems were
half-life property, which may explain their poor antibac- also prepared and exhibited activity against the SPases.
terial activity.
Molecular modeling of the (5S)-tricyclic penems in E. coli
crystallographic structure was used to rationalize the high
potency in SPase assay. However, the disappearance of
antibacterial activity for MIC determination may be relat-
ed to instability of this class of molecules.
Figure 2 below shows the published X-ray crystallograph-
ic structure of a (5S)-penem inhibitor covalently bound in
the active site of E. coli SPase,22 where it was observed
that the dihydrothiazole ring formed -stacking with
Tyr143 residue. We also examined our novel (5S)-tricy-
clic penem 15d in the same crystal structure of E. coli
SPase using a molecular modeling approach as shown in
Figure 3. Interestingly, a similar binding pattern of the tri-
cyclic penem with the SPase to that of the penem was pre-
dicted, in which the dihydrothiazolopyridone ring also
exhibited -stacking with Tyr143. In addition, the N-ben-
zyl group was positioned along the peptide cleft, and the
All reagents were commercial grade and were used as received
without further purification, unless otherwise specified. Commer-
cially available anhydrous solvents were used for reactions con-
ducted under inert atmosphere. Reagent grade solvents were used in
all other cases, unless otherwise specified. Flash chromatography
was performed on E. Merck silica gel 60 (230–400 mesh). TLC was
performed on E. Merck 60 F-254 precoated silica plates (250 mm
layer thickness). 1H NMR spectra were recorded at 300 MHz.
Alloc group was extended into the S3 pocket by -stack- Chemical shifts for 1H NMR spectra are expressed in parts per mil-
Figure 2 Crystal structure of a (5S)-penem in E. coli SPase
Figure 3 Molecular modeling of (5S)-tricyclic penem 15d in E. coli SPase
Synthesis 2003, No. 11, 1732–1738 © Thieme Stuttgart · New York

PAPER
Synthesis of (5S)-Tricyclic Penems
1737
lion downfield from tetramethylsilane. Splitting patterns are desig-
nated as s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; m,
multiplet; br, broad. Coupling constants are given in Hertz (Hz).
The 13C NMR spectra were recorded at 75 MHz. Chemical shifts for
13C NMR spectra are expressed in parts per million downfield from
CHCl3. Mass spectra were obtained by electrospray mass spectrom-
etry utilizing an electrospray ionization (ESI) interface in flow in-
jection analysis (FIA) mode equipped with an ESI interface.
Melting points were taken on a capillary melting point apparatus.
High resolution mass spectra were obtained by fast atom bombard-
ment (FAB) with a p-nitrobenzyl alcohol matrix. IR spectra were re-
corded using attenuated total reflectance spectroscopy with a
germanium internal reflection element. Optical rotations were mea-
sured using a 10 cm cell at 20 °C.
0.422 mmol) was dissolved in anhyd CH2Cl2 (10 mL) and AcCl
(0.045 mL, 0.633 mmol) was added followed by Et3N (0.088 mL,
0.633 mmol) at r.t. The resulting solution was stirred for 30 min and
directly purified by flash chromatography using EtOAc–hexanes
(1:4 1:2); Rf 0.15 (EtOAc–hexanes, 1:2) to give 12c.
1H NMR (300 MHz, CDCl3): = 7.25 (m, 5 H), 6.95 (br, 1 H), 5.60
(d, J = 4.2 Hz, 1 H), 5.28 (m, 1 H), 4.73 (dd, J = 2.4, 4.2 Hz, 1 H),
4.52 (m, 2 H), 3.95 (dd, J = 3.6, 9.9 Hz, 1 H), 3.13 (dd, J = 4.5, 5.4
Hz, 1 H), 2.90 (dd, J = 2.1, 5.4 Hz, 1 H), 2.03 (s, 3 H), 1.53 (d,
J = 6.0 Hz, 3 H).
13C NMR (75 MHz, CDCl3): = 168.5, 163.3, 158.5, 139.8, 133.6,
130.8, 129.9, 128.8, 127.7, 70.6, 64.3, 62.9, 58.8, 40.0, 39.2, 22.8,
19.3.
HRMS: m/z calcd for C19H21N2O5S, 389.1171 (M + H+);. found,
6-[1-tert-Butyldimethylsilyl)ethyl]-3-formyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]hept-2-ene-2-carboxylic Acid Allyl Ester (6)
A suspension of penem alcohol 5 (1.96 g, 4.91 mmol) and MnO2
(8.54 g, excess) in CH2Cl2 (6 mL) was stirred at r.t. for 1.5 h. The
solid was removed by filtration and the filtrate was evaporated un-
der reduced pressure to give a light yellow solid (1.95 g, ca. 100%).
The crude product was pure by 1H NMR analysis and was used for
the next step without purification; mp 83–85 °C.
1H NMR (300 MHz): = 10.42 (s, 1 H), 5.98 (m, 1 H), 5.71 (d,
J = 4.8 Hz, 1 H), 5.47 (dd, J = 1.5, 17.1 Hz, 1 H), 5.38 (dd, J = 1.5,
10.8 Hz, 1 H), 4.81, (m, 2 H), 4.41 (m, 1 H), 3.94 (dd, J = 5.1, 12.3
Hz, 1 H), 1.42 (d, J = 6.6 Hz, 3 H), 0.85 (s, 9 H), 0.70 (s, 6 H).
389.1205.
Acetic Acid 1-(6-Acetoxy-4-benzyl-2,3-dioxo-1,3,4,5,6,7 -
hexahydro-2H-7-thia-2 ,4-diazacyclobuta[ ]inden-1-yl)ethyl
Ester (15c)
A solution of the epoxypenem amide 12c (62 mg, 0.160 mmol) and
Mg(ClO4)2 in anhyd THF (5 mL) was refluxed for 2 h. The solvent
was evaporated and the residue was washed with brine. The organic
layer was dried (MgSO4) and evaporated. The residue was quickly
loaded to a silica gel column and flash chromatographed eluting
with EtOAc–hexanes (1:4 1:1) to yield 14c as a pale oil (33 mg,
53%); Rf 0.15 (EtOAc–hexanes, 1:1). The purified alcohol 14c (20
mg, 0.052 mmol) was immediately dissolved in CH2Cl2 (2 mL) and
AcCl (6 L, 0.078 mmol) was added followed by Et3N (10 L,
0.078 mmol) at r.t. The resulting solution was stirred for 10 min and
then directly purified by flash chromatography using EtOAc–hex-
ESI-MS: m/z
=
398 [M
+
H+], 198 [M
+
H+
–
(O=C=CHCH(Me)OTBDMS)].
6-[1-tert-Butyldimethylsilyloxy)ethyl]-3-oxiranyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]hept-2-ene-2 carboxylic Acid Allyl Ester (7)
To a solution of the penem aldehyde 6 (1.95 g, 4.91 mmol) in
MeOH (100 mL) was added CH2N2 in Et2O dropwise at –30 °C. Af-
ter the addition, the temperature was raised to r.t. in 30 min and the
volatiles were evaporated under reduced pressure to afford a light
yellow oil (2.018 g, ca. 100%). The crude product was pure by 1H
NMR analysis and was used for the next step without purification.
1H NMR (300 MHz, CDCl3): = 5.96 (m, 1 H), 5.62 (m, 1 H), 5.41
(dd, J = 17.1 Hz, 1 H), 5.22 (dd, J = 10.8 Hz, 1 H), 4.86, (m, 3 H),
4.61 (dd, J = 3.3, 5.7 Hz, 1 H), 4.40 (m, 1 H), 3.88 (dd, J = 3.3, 7.2
Hz, 1 H), 3.14 (dd, J = 6.0, 7.2 Hz, 1 H), 2.93 (dd, J = 3.3, 6.0 Hz,
1 H), 1.42 (d, J = 6.6 Hz, 3 H), 0.85 (s, 9 H), 0.70 (s, 6 H).
anes (1:4
1:2) to yield 15c as a pale oil (16 mg, 72%); Rf 0.70
(EtOAc–hexanes, 1:2); [ ]D20 –125.3 (c = 0.32, CHCl3).
1H NMR (300 MHz, CDCl3): = 7.16–7.40 (m, 5 H), 5.56 (d,
J = 4.5 Hz, 1 H), 5.53 (t, J = 2.1 Hz, 1 H), 5.31 (m 1 H), 4.76 (d,
J = 15.3 Hz, 1 H), 4.61 (d, J = 15.3 Hz, 1 H), 4.42 (d, J = 2.7 Hz, 1
H), 4.11 (m, 1 H), 2.16 (s, 3 H), 2.04 (s, 3 H), 1.53 (d, J = 6.0 Hz, 3
H).
HRMS: m/z calcd for C21H23N2O6S, 431.1377 (M + H+); found,
431.1385.
6-[1-tert-Butyldimethylsilyloxy)ethyl]-2-(pyrrolidine-1-carbon-
yl)-3-(tetrahydropyran-2-yloxymethyl)-4-thia-1-azabicyclo-
[3.2.0]hept-2-en-7-one (22)
ESI-MS: m/z
=
412 [M
+
H+], 212 [M
+
H+
–
To a solution of (5S)-penem ester 20 (0.500 g, 1.04 mmol) in anhyd
THF (10 mL) were added Ph3P (82 mg, 0.312 mmol, 30%),
(PPh3)4Pd (0.120 g, 0.104 mmol, 10%) and sodium 2-ethylhex-
anoate (0.173 g, 1.04 mmol) at 0 °C. The resulting solution was
stirred for 15 min at that temperature and TLC analysis showed no
starting penem ester at this point. The mixture was evaporated under
reduced pressure to about 2 mL of the volume and partitioned be-
tween phosphate buffer (pH 4, 50 mL) and EtOAc (50 mL). The or-
ganic layer was separated, dried (MgSO4) and evaporated under
reduced pressure to give a yellow oil. The residue was dissolved in
CH2Cl2 (20 mL) and to it was added EtOCOCl (0.11 mL, 1.14
mmol) followed by Et3N (0.16 mL, 1.14 mmol). The resulting solu-
tion was stirred for 0.5 h at 0 °C. To the reaction mixture was then
added pyrrolidine (96 mL, 1.14 mmol). The stirring was continued
for another 20 min at 5–20 °C. The reaction mixture was directly
loaded onto a column of silica gel and eluted with EtOAc–hexane
(O=C=CHCH(Me)OTBDMS)].
Acetic acid 1-(2-Benzylcarbamoyl-3-oxiranyl-7-oxo-4-thia-1-
azabicyclo[3.2.0]hept-2-en-6-yl)ethyl Ester (12c)
To a solution of the epoxy penem ester 7 (0.268 g, 0.790 mmol), so-
dium 3-ethylhexanoate (0.092 g, 0.790 mmol) and Ph3P (0.062 g,
0.237 mmol) in anhyd THF (8 mL) was added (Ph3P)4Pd at 0 °C.
After stirring for 6 min, isopropyl chloroformate (IPCF, 0.95 mL,
0.948 mmol) was added followed by Et3N at 0 °C. The stirring was
continued for another 7 min at the same temperature and then
BnNH2 (0.121 mL, 1.185 mmol) was added. The resulting mixture
was maintained at 0 °C for additional 5 min and then evaporated un-
der reduced pressure. The residue was purified by flash chromatog-
raphy using EtOAc–hexanes (1:4
1:2) to give the penem amide
11 as a foaming yellow solid (0.205 g, 67%). A mixture of this pen-
em amide 11 (0.200 g, 0.435 mmol) and NH4F·HF (0.248 g, 4.35
mmol) in anhyd DMF (20 mL) was heated at 60 °C for 1.5 h. After
cooling, the mixture was partitioned between brine and EtOAc. The
organic layer was separated and washed with brine (5 ×), dried
(MgSO4) and evaporated. The residue was purified by flash chro-
matography using EtOAc–hexanes (1:4 1:2) to give a yellow sol-
id (0.146 g, 97%). The obtained hydroxyl penem 12b (0.146 g,
(1:4
1:3) to afford a thick yellow oil (0.306 g, 59%); Rf 0.22
(EtOAc–hexanes, 1:2).
1H NMR (300 MHz, CDCl3): = 5.68 (d, J = 3.9 Hz, 1 H), 4.85 (t,
J = 15.0 Hz, 1 H), 4.74–4.66 (m, 1 H), 4.61 (dd, J = 8.4, 15.0 Hz, 1
H), 4.39 (dq, J = 6.3, 9.9 Hz, 1 H), 3.91–3.45, (m, 7 H), 1.98–1.48
Synthesis 2003, No. 11, 1732–1738 © Thieme Stuttgart · New York

1738
X. E. Hu et al.
PAPER
(m, 10 H), 1.40 (d, J = 6.3 Hz, 3 H), 0.87 (s, 9 H), 0.13 (s, 3 H), 0.11
(s, 3 H).
References
(1) (a) Trias, J.; Gordon, E. M. Curr. Opin. Biotechnol. 1997, 8,
757. (b) Brickner, S. J. Chem. Ind. (London) 1997, 131.
(2) Dalbey, R. E.; Lively, M. G.; Bron, S.; van Dijl, J. M.
Protein Sci. 1997, 6, 1120.
HRMS: m/z calcd for C24H40N2O5SSi, 497.2505 (M + H+); found,
497.2438.
6-[1-tert-Butyldimethylsilyloxy)ethyl]-3-hydroxymethyl-2-
(pyrrolidine-1-carbonyl)-4-thia-1-azabicyclo[3.2.0]hept-2-en-7-
one
A solution of penem THP ether 22 (0.300 g, 0.605 mmol) and p-
TsOH·H2O (12 mg, 0.061 mmol) in MeOH (10 mL) was stirred at
r.t. for 1.5 h. The solvent was evaporated to 1/3 of its volume under
reduced pressure. The mixture was partitioned between CH2Cl2 (5
mL) and sat NaHCO3 (5 mL). The organic layer was separated and
the aqueous layer was extracted with CH2Cl2 (5 mL). The combined
extracts were dried (MgSO4) and evaporated. The residue was puri-
(3) (a) Dulbey, R. E.; Wickner, W. J. Biol. Chem. 1985, 260,
15925. (b) Dalbey, R. E. Features 1995, 61, 586.
(c) Stephens, C.; Shapiro, L. Chem. Biol. 1997, 4, 637.
(4) (a) Allsop, A. E.; Brooks, G.; Bruton, G.; Coulton, S.;
Edwards, P. D.; Hatton, I. K.; Kaura, A. C.; McLean, S. D.;
Pearson, N. D.; Smale, T. C.; Southgate, R. Bioorg. Med.
Chem. Lett. 1995, 5, 444. (b) Allsop, A. E.; Brooks, G.;
Edwards, P. D.; Kaura, A. C.; Southgate, R. , J. Antibiotics
1996, 49, 921. (c) Black, M. T.; Bruton, G. Curr. Pharm.
Des. 1998, 4, 133.
fied by chromatography using EtOAc–hexane (1:4
1:1) to give
(5) Date, T. J. Bacteriol. 1983, 154, 76.
0.215 g (86%) of a yellow solid; Rf 0.33 (EtOAc–hexane, 1:1).
(6) (a) Ernst, I.; Gosteli, J.; Greengrass, C. W.; Holick, W.;
Jackman, D. E.; Pfaendler, H. R.; Woodward, R. B. J. Am.
Chem. Soc. 1978, 100, 8214. (b) McCombie, S. W.;
Ganguly, A. K. Med. Res. Rev. 1988, 8, 393.
(7) Georg, G. I. The Organic Chemistry of -Lactams; VCH:
Weinheim, 1993.
1H NMR (300 MHz, CDCl3): = 5.57 (d, J = 4.2 Hz, 1 H), 5.34 (dd,
J = 3.9, 9.6 Hz, 1 H), 4.40 (dd, J = 3.9, 14.7 Hz, 1 H), 4.32 (dq,
J = 6.3, 10.5 Hz, 1 H), 4.12 (dd, J = 9.6, 14.7 Hz, 1 H), 3.70 (dd,
J = 4.2, 9.9 Hz, 1 H), 3.57–3.34 (m, 4 H), 1.92–1.62 (m, 4 H), 1.28
(d, J = 6.3 Hz, 3 H), 0.75 (s, 9 H), 0.01 (s, 3 H), 0.00 (s, 3 H).
(8) Tanaka, R.; Iwata, H.; Ishiguro, M. J. Antibiotics 1990, 43,
1608.
13C NMR (75 MHz, CDCl3): = 173.7, 159.3, 151.9, 123.3, 67,
66.3, 65.2, 57.4, 47.1, 26.3, 25.6, 23.4, 22.2, 17.6, –3.6, –4.6;
HRMS: m/z calcd for C19H32N2O4SSi, 413.1930 (M + H+); found,
(9) The characterization of (5S)-Penems was based on MS and
NMR analyses. In the 1H NMR spectrum, both 5S- and 5R-
isomers show similar peak patterns, but different chemical
shifts. The most diagnostic peaks are CH-5, CH-6 and CH3-
8. In the 5S-isomer, a large coupling constant J = >4 Hz (vs
J = <2 Hz for 5R-isomer) for CH-5 indicated a cis
relationship with CH-6.
(10) A thiozone by-product was isolated and characterized.
Similar observation was descried by authors in Ref.5
(11) For review, see: (a) Fatiadi, A. J. Synthesis 1976, 65.
(b) Fatiadi, A. J. Synthesis 1976, 133. (c) See also for an
experimental procedure: Gilow, H. M.; Jones, G. II. Org.
Synth., Coll. Vol. 7; Wiley: New York, 1990, 102.
(12) (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
(b) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277.
413.1927.
6-(1-Hydroxylethyl)-3-hydroxymethyl-2-(pyrrolidine-1-carbo-
nyl)-4-thia-1-azabicyclo[3.2.0]hept-2-en-7-one (23a)
A mixture of the above THP-deprotected penem silyl ether (70 mg,
0.170 mmol) and NH4F·HF (97 mg, 1.70 mmol) in anhyd DMF (10
mL) was heated at 60 °C (oil bath temperature) for 2.5 h and cooled
to r.t. The solid was removed by filtration and the filtrate was evap-
orated in high vacuum at 40 °C. The residue was triturated with hex-
ane (10 mL) and the solvent layer was decanted. The solid was
purified by chromatography using EtOAc–hexane (1:1
1:0) to
yield a white solid (46 mg, 91%); Rf 0.15 (EtOAc); mp 104–106 °C;
[ ]D20 –209.2 (c = 0.60, CHCl3).
FT-IR (NaCl): 3364, 2971, 2928, 1773, 1603 cm–1.
(13) For a review, see: Gutsche, D. Org. React. 1954, 8, 364.
(14) Although the initially formed penem sodium salt was readily
isolated and found relatively stable under ambiguous
conditions, the acid was obtained in <5% yield after flash
chromatography separation.
(15) Personal communication with Professor Henry Rapoport at
University of California, Berkeley.
(16) Seki, M.; Kondo, K.; Kuroda, T.; Yamanaka, T.; Iwasaki, T.
Synlett 1995, 609.
1H NMR (300 MHz, CDCl3): = 5.73 (d, J = 4.2 Hz, 1 H), 5.53 (dd,
J = 3.9, 9.3 Hz, 1 H), 4.54 (d, J = 14.1 Hz, 1 H), 4.42 (dq, J = 6.3,
10.2 Hz, 1 H), 4.22 (dd, J = 9.3, 14.1 Hz, 1 H), 3.83 (dd, J = 3.9,
10.2 Hz, 1 H), 3.70–3.48 (m, 4 H), 2.78–2.67 (br, 1 H), 2.06–1.76
(m, 4 H), 1.43 (d, J = 6.0 Hz, 3 H).
13C NMR (75 MHz, CDCl3): = 174.0, 159.8, 151.6, 123.8, 66.4,
66.2, 65.0, 57.7, 48.0, 47.6 (CH2N), 26.7 (CH2), 23.4 (CH2), 22.5
(CH3-9).
(17) Unpublished results..
ESI-MS: m/z
(O=C=CHCH(Me)OH)].
=
299 (M
+
H+), 213 [M
+
H+
–
(18) All compounds showed satisfactory analytical data.
(19) Cregg, K. M.; Wilding, E. I.; Black, M. T. J. Bacteriology
1996, 178, 5712.
(20) Tschantz, W. R.; Paetzel, M.; Cao, G.; Suciu, D.; Inouye,
M.; Dalbey, R. E. Biochemistry 1995, 34, 3935.
(21) The E. coli SPase was kindly provided by Dr. R. E. Dalbey
(Ohio State University).
Anal. Calcd for C13H18N2O4S·0.25H2O: C, 51.56; H, 6.16; N, 9.25.
Found: C, 51.60; H, 5.81; N, 9.17.
Acknowledgments
(22) Paetzel, M.; Dalbey, R. E.; Strynadka, N. C. J. Nature
(London) 1998, 396, 186.
The authors are grateful to Professor Henry Rapoport for helpful
discussions and the method suggested for cyclization of epoxy
amides.
Synthesis 2003, No. 11, 1732–1738 © Thieme Stuttgart · New York