New C-3′ hydroxamate-substituted and more lipophilic cyclic hydroxamate cephalosporin derivatives as a potential new generation of selective antimicrobial agents

Article abstract of DOI:10.1039/b612475e

Syntheses of a series of new C-3′ hydroxamate-substituted cephalosporin derivatives with potent antibacterial and media-dependent anti-TB activity are described. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b612475e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
New C-3ꢀ hydroxamate-substituted and more lipophilic cyclic hydroxamate
cephalosporin derivatives as a potential new generation of selective
antimicrobial agents
Marvin J. Miller,*a Gaiying Zhao,a Sergei Vakulenko,a Scott Franzblaub and Ute Mo¨llmannc
Received 29th August 2006, Accepted 22nd September 2006
First published as an Advance Article on the web 12th October 2006
DOI: 10.1039/b612475e
Syntheses of a series of new C-3ꢀ hydroxamate-substituted cephalosporin derivatives with potent
antibacterial and media-dependent anti-TB activity are described.
Introduction
Tuberculosis (TB) is one of the leading causes of death and suf-
fering worldwide. The increasing drug resistance, toxicity and side
effects of currently used anti-tuberculosis drugs also emphasizes
the urgent need for new, safer and more effective anti-tuberculosis
agents.1–5 Although mycobacteria produce b-lactamases and are
intrinsically resistant to b-lactam antibiotics, there are reports
that the addition of b-lactamase inhibitors to penems or cephems
greatly improved their in vitro activity against tuberculosis.6,7 Since
the loss of activity of b-lactam antibiotics could also be attributed
to the lack of cell envelope permeability and variations in certain
peptidoglycan biosynthetic enzymes,8,9 we initiated a study to
identify structures with increased lipophilicity and/or improved
anti-b-lactamase activity. Here, we report the syntheses and results
of some biological assays of new C-3ꢀ hydroxamate-substituted
Fig. 1 New C-3ꢀ hydroxamate-substituted cephalosporins (lipophilic
cyclic form and hydrolytically generated hydrophilic free carboxy-
late/hydroxamate form) and mycobactin T.
cephalosporins and their more lipophilic cyclic hydroxamate esters
shown in Fig. 1.
Compared to the common cephalosporins, these 2,3-cyclic
hydroxamate-fused cephalosporins were anticipated to be more
hydrophobic and possibly able to diffuse through the lipid-
rich cell wall of mycobacteria. As further shown in Fig. 1, the
cyclic hydroxamate component is an “active ester” that was
also anticipated to be able to hydrolytically generate the normal
cephalosporin carboxylate subsequent to cellular uptake. The
same intracellular hydrolytic process would also release a free
hydroxamic acid that could influence either growth promotion
or inhibition by affecting iron assimilation or metabolism since
hydroxamates are iron binding components of iron sequestering
agents (siderophores), including the mycobactins. Mycobactin T,
a siderophore selective for growth promotion and virulence of
Mycobacterium tuberculosis contains two hydroxamates and a
phenolic oxazoline for iron chelation. We have previously shown
that mycobactin analogs can have potent anti-tuberculosis activity
and that, among other factors, activity does require a lipophilic
component as does the natural mycobactin T with its long acyl
moiety on the e-amino group of the constituent linear N-hydroxy
lysine residue.10,11
With these considerations in mind, our first goal was to
synthesize representatives of our targeted structure (Fig. 1) and
determine if this new class of cephalosporins has antibiotic activity.
The retrosynthetic disconnections are shown in Scheme 1. A key
point was the introduction of a C-3ꢀ hydroxamate side chain by
coupling of a cephalosporin to an O-protected hydroxamic acid.
Scheme 1 Retrosynthetic analysis.
aDepartment of Chemistry and Biochemistry, University of Notre Dame,
Notre Dame, IN 46556, USA. E-mail: mmiller1@nd.edu; Fax: +1 574-631-
6652
Results and discussion
bCollege of Pharmacy Institute for Tuberculosis Research, M/C 964, Rm
412, University of Illinois at Chicago, 833 S. Wood St., Chicago, Illinois,
60612-7231, (USA)
The syntheses of the hydroxamic acids (2a–e, 6) used for the
alkylation of 3-chloromethyl-7-phenylacetylamino cephalospo-
ranic acid p-methoxybenzyl ester (GCLE) are shown in Scheme 2.
O-tert-Butyldimethylsilyl benzohydroxamic acid 2c was obtained
cLeibniz-Institute for Natural Product Research and Infection Biology-Hans
Knoell Institute, Beutenbergstrasse 11a, D-07745, Jena, Germany
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protected hydroxamic acids (2a–e) provided key intermediates 7a–
e in 21–30% yield with 20–40% of GCLE recovered. Though some
D-3 to D-2 double bond isomerization (7ꢀa–e) was observed during
these alkylation reactions, the desired products were isolated after
purification by careful chromatography. Following deprotection
with TFA in the presence of anisole17,18 or triethylsilane with
CH2Cl2 as the solvent, the desired products 8a–e were obtained
in 21–36% yield along with the corresponding ring-opened free
carboxylic acid hydroxamates 9a–e in 20–32% yield. Use of TFA
alone as reactant and solvent led to isolation of only the free acids
9a–e.
Scheme 2 Syntheses of hydroxamic acids 2a–e, 6. a) NH2OH hydrochlo-
ride (1.0 equiv.), 1,2-diaminoethane (1.0 equiv.), CH2Cl2, rt, 20 h; then
TBSCl (1.0 equiv.), rt, 4 d, 69%; b) benzoic acid (1.0 equiv.), Et3N
(1.0 equiv.), EDC (1.1 equiv.), CH2Cl2, 16 h, 69%; c) NH2OH hydrochloride
(1.0 equiv.), Et3N (2.0 equiv.), 3a–b, 3d–e (1.0 equiv.), CHCl3, rt, 1 h; d)
imidazole (1.3 equiv.), TBSCl (0.95 equiv.), DMF, rt, 16 h; e) octanoic acid
(1.0 equiv.), EtOCOCl (1.2 equiv.), NMM (1.3 equiv.), Et2O, 0 ◦C, 10 min;
then NH2OH (1.5 equiv.), ether–methanol, rt, 15 min, 91%. NMM =
N-methyl morpholine.
The syntheses of compounds 11 and 12, with a more lipophilic
acyl group that was expected to more effectively permeate
the mycobacterial envelope, are outlined in Scheme 4. Direct
substitution reaction between 3-iodomethyl cephalosporin and
the corresponding hydroxamic acid 6 gave the desired product
10 in very low yield (8%). Pd(0)-catalyzed reaction only gave a
trace of the desired product. However, using the direct alkylation
route, enough material was generated to allow us to proceed with
syntheses of the target molecules needed for biological studies.
Following deprotection with TFA in the presence of anisole and
CH2Cl2 as solvent, the desired product 11 was obtained in 31%
yield, along with the corresponding ring opened free carboxylic
acid hydroxamate 12 in 24% yield.
by an EDC coupling reaction between benzoic acid and O-tert-
butyldimethylsilyl hydroxylamine. The latter was prepared by
treating hydroxylamine with O-tert-butyldimethylsilyl chloride in
1,2-diaminoethane and methylene chloride.12 4-Substituted benzo-
hydroxamates 4a–b, 4d–e, which were easily obtained by reaction
of hydroxylamine with the corresponding acyl chlorides 3a–b,
3d–e,13 were treated with O-tert-butyldimethylsilyl chloride and
imidazole in DMF to give 4-substituted O-tert-butyldimethylsilyl
benzohydroxamates 2a–b, 2d–e. Octanoic acid was activated with
ethyl chloroformate and N-methyl morpholine (NMM), followed
by coupling with freshly-generated free hydroxylamine to give
heptanohydroxamic acid (5) in 91% yield.14 Then compound 5 was
protected with TBSCl and imidazole (imid) to provide protected
hydroxamic acid 6 in 84% yield.
The syntheses of the target hydroxamate-containing
cephalosporins are outlined in Scheme 3. After several methods
were attempted, the approach that efficiently introduced the
C-3ꢀ hydroxamate side chain of cephalosporins was a palladium-
catalyzed nucleophilic reaction.15,16 A short survey of reaction
conditions indicated that use of 0.12 equivalent of Pd(0) catalyst
from 0 ◦C to rt for 3 h was most effective. Palladium-catalyzed
nucleophilic reaction between GCLE and variously substituted
Scheme 4 Synthesis of 11 and 12. a) GCLE (1.0 equiv.), NaI (1.25 equiv.),
acetone, rt, 1.5 h, 94%; b) 6 (0.74 equiv.), NaH (0.70 equiv.), DMF, rt, 16 h,
8%; c) TFA (14 equiv.), anisole (4 equiv.), CH2Cl2, 0 ◦C–rt, 1.5 h.
All of the synthesized cephalosporins were tested for their anti-
bacterial activities against various strains of Gram-positive and
Gram-negative bacteria using standard MIC determinations.19,20
The results showed that they are active only against Gram-positive
strains such as Micrococcus luteus ATCC 10240, Bacillus subtilis
ATCC 6633 and Staphylococcus aureus SG 511 (Table 1). The
assays also showed that the ring opened free carboxylic acid
hydroxamates 9a–e, 12 are superior in activity compared to
the corresponding cyclic hydroxamate esters 8a–e, 11. This is
consistent with the requirement of a free ionizable carboxyl
acid for activity of classical b-lactam antibiotics. For the cyclic
hydroxamate esters 8a–e, 11, the more lipophilic compound 11
with a long chain gave better activity against all the strains listed.
Generally, both electron-donating and electron-withdrawing
substituents on the phenyl ring retained or improved the
biological activities. For Micrococcus luteus ATCC 10240,
compound 9e with a p-trifluoromethyl substituent on the phenyl
ring gave the best activity with an MIC of 0.4 lM. Compound 9d
with a p-fluoro substituent on the phenyl ring and compound 12
with a long chain gave the best activity with an MIC of 0.2 lM
Scheme 3 Synthesis of 8a–e and 9a–e. a) GCLE (1.0 equiv.), Pd(OAc)2
(0.12 equiv.), PPh3 (0.60 equiv.), 2a–e (1.1 equiv.), NaH (1.0 equiv.), 0 ◦C–rt,
3 h; b) TFA (14 equiv.), anisole (or triethylsilane) (4.0 equiv.), CH2Cl2,
0
◦C–rt, 1.5 h.
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d
Table 1 Activity against representative bacteria (MIC in lM) and toxicity against VERO cells (IC50 in lM)
Species/Strains
Micrococcus luteus
ATCC 10240
Bacillus subtilis
ATCC 6633
Staphylococcus
aureus SG 511
M. tuberculosis H37Rv
M. tuberculosis H37Rv
Compound
MABAa/GASb
MABA/GASTc
VERO cells
8a
6.25
6.25
12.5
12.5
12.5
1.56
3.12
1.56
6.25
1.56
0.4
0.4
0.4
0.78
0.4
0.4
0.2
<0.1
<0.1
0.2
0.2
<0.1
0.2
1.56
1.56
1.56
1.56
1.56
0.4
0.78
0.78
0.78
0.2
31.16
12
>128
15.3
10.3
124.1
61.6
26.7
63.7
120.5
86.0
69.3
3.2
2.8
126
23.4
9.8
10.1
46.2
31.0
53.0
17.1
126.6
9.1
>128
NT
NT
8b
8c
8d
>128
>128
19.5
>128
>128
>128
>128
NTe
8e
11
9a
9b
9c
9d
9e
0.4
0.2
3.12
12
1.56
50
14.6
NT
GCLE
0.78
>128
13.9
a Microplate Alamar Blue assay.22 b GAS: glycerol-alanine-salts.23 c GAST: GAS without added iron and containing Tween 80.23 d Concentration resulting
in 50% inhibition of the growth of VERO cells.24 e NT = not tested.
against Staphylococcus aureus SG 511. Against Bacillus subtilis
ATCC 6633, compound 9a with a p-methoxy substituent, 9b with
a p-methyl substituent and compound 9e with a p-trifluoromethyl
on the phenyl ring showed excellent activity. Compounds 8c, 9c
were also tested for their activity as b-lactamase inhibitors by
methods described in the literature,21 but were found to be inactive.
Compounds 8a–e, 9a–e, 11, 12 were also tested against M.
tuberculosis H37Rv (Table 1). Consistent with our hypothesis,
several of the more lipophilic cephalosporin derivatives were found
to be more active against TB. Especially notable is the comparison
of the results of assays of the cyclic hydroxamates 8 relative to the
free carboxylate/hydroxamate series 9 in normal iron sufficient
media (GAS) and in iron deficient media (GAST). In some cases
(8c/9c and 8d/9d in the GAST medium), there was little difference
side chain was found to be more pronounced than that of
compound 8c without a substituent on the phenyl ring, but the
activity was not as good as that of compounds 8a or 8b with
electron-donating groups on the phenyl ring. The length and
type of the carbon chain might need to be optimized in further
SAR studies, especially, and not surprisingly, since use of aliphatic
groups also reduced selectivity relative to VERO cells.
Conclusions
In summary, we have synthesized and studied several C-3ꢀ-
hydroxamate substituted cephalosporins, including cyclic forms
8a–e, 11 with enhanced lipophilicity and possible alternate modes
of activity. For various Gram-positive strains listed in Table 1,
the ring-opened free carboxylic acid hydroxamates 9a–e and 12
are superior in activity compared to the corresponding cyclic
hydroxamate esters 8a–e and 11. This is consistent with SAR of
classical cephalosporins that seem to require a free carboxylate
for activity and do not suffer from limited microbial permeability.
Of the cyclic hydroxamate esters 8a–e and 11, compound 11 was
more active against all of the non-mycobacterial strains listed
and these cyclic, more lipophilic compounds were generally more
active against TB. These studies have accomplished our first goal to
demonstrate that novel hydroxamate-containing cephalosporins
can have interesting and potentially useful antimicrobial activity.
These results provide new leads for the possible generation
of species-selective antimicrobial agents based on variation of
peripheral functionality and substituents. Additional synthetic
and antimicrobial studies are planned to further test this con-
cept and to test our hypothesis related to alternate modes of
action.
(
one dilution) of activity, but in others there were notable
differences in activity. While the free carboxylate/hydroxamates
would be able to bind iron, they are very polar and are not likely to
be able to permeate the lipophilic outer layers of the mycobacterial
cell. However, the cyclic hydrophobic forms 8 might effectively
penetrate and then either have intrinsic activity themselves or,
upon either enzymatic or general intracellular hydrolysis, convert
to forms of 9. Once generated in the targeted cells, the resulting
new cephalosporins might cause cellular disruption by classical
modes of action and/or by affecting critical iron assimilation and
metabolism processes, especially under iron restricted conditions.
The possible influence of iron regulation is suggested by the
notably enhanced activity of some forms of 8 in the GAST
(“iron deficient”) medium. Thus, while additional studies are
needed to determine details related to the possible reasons for
differences in activity, the results are consistent with our initial
hypotheses. Of additional interest was the observation that for
the cyclic hydroxamate-containing cephalosporin compounds 8a–
e, the presence of either an electron-donating or an electron-
withdrawing substituent on the phenyl ring (Rꢀ of the generalized
structure) seemed to notably increase anti-TB activity. This
suggests that possible additional substitution studies will further
help define important SAR relationships. The growth inhibitory
activity of compound 11 with the lipophilic acyclic seven-carbon
Experimental
General
All NMR spectra were recorded on a Varian instrument at
300 MHz (1H), 75 MHz (13C), or 500 MHz (1H), 125 MHz (13C)
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unless otherwise noted. Chemical shifts are indicated in d values
(ppm) from internal reference peaks of TMS, CDCl3, CD3OD or
DMSO-d6. Melting points were taken on a Thomas Hoover melt-
ing point apparatus and are uncorrected. TLC was performed with
aluminium backed Merck silica gel 60-F254 using 254 nm light,
aq. KMnO4, FeCl3, or ninhydrin for visualization. Commercial
reagents were used without purification. Flash chromatography
was performed on Sorbent Technologies silica gel 60 (32–63 lm).
All reagents were purchased from Aldrich, Acros, Advanced
ChemTech, and Fisher and used without purification unless
otherwise indicated. THF was distilled from a mixture of sodium
metal and benzophenone and CH3CN and TEA were distilled
from CaH2. DI water was further purified through a mixed
bed type II filter made by US Filter. All other solvents were
used without purification. 3-Chloromethyl-7-phenylacetylamino
cephalosporanic acid p-methoxybenzyl ester (GCLE) was gener-
ously provided by Otsuka Chemical Co., Ltd.
(s, 1H). 13C NMR (DMSO-d6, 75 MHz) d: 21.41, 127.34, 129.36,
130.44, 141.46, 164.69.
p-Fluorobenzoylhydroxamic acid (4d). 34% yield. Mp 161–
◦
1
162 C. H NMR (CD3OD, 300 MHz) d: 7.17 (t, J = 8.7 Hz,
2H), 7.79 (dd, J = 5.1 Hz, J = 8.7 Hz, 2H). 13C NMR (CD3OD,
75 MHz) d: 116.52, 116.81, 130.75, 130.87, 164.67, 168.00.
p-◦Trifluorobenzoylhydroxamic acid (4e). 37% yield. Mp 134–
136 C. 1H NMR (CD3OD, 300 MHz) d: 7.18 (t, J = 8.4 Hz, 2H),
7.81 (dd, J = 5.1 Hz, J = 8.4 Hz, 2H).
O-tert-Butyl-dimethylsilyl-p-methoxybenzohydroxamic acid (2a)
To a stirred solution of p-methoxybenzohydroxamic acid 4a
(1.06 g, 0.064 mol) in DMF (10 mL), imidazole (566 mg, 0.083 mol)
and tert-butyldimethylsilyl chloride (961 mg, 0.061 mol) were
added successively. The resulting mixture was allowed to stir
overnight at room temperature. Then 10 mL of water was added
followed by extraction with EtOAc (3 × 20 mL). The combined
organic layers were washed with brine (50 mL), dried over
anhydrous MgSO4, filtered and concentrated under reduced pres-
sure. The residue was purified by flash column chromatography
(hexanes–EtOAc: 5 : 1) to give 812 mg (86% based on recovered
starting material) of compound 2a as a white solid. 500 mg of
p-methoxybenzohydroxamic acid was recovered. Mp 132–134 ◦C.
1H NMR (CDCl3, 300 MHz) d: 0.24 (s, 6H), 1.00 (s, 9H), 3.83 (s,
3H), 6.90 (d, J = 9.0 Hz, 2H), 7.69 (d, J = 9.0 Hz, 2H). 13C NMR
(CD3OD, 75 MHz) d: −5.50, 18.29, 25.96, 55.37, 113.87, 128.41,
205.28.
O-tert-Butyldimethylsilyl benzohydroxamic acid (2c)
To a stirred solution of benzoic acid (610 mg, 5.0 mmol) in methy-
lene chloride (10 mL), a solution of O-tert-butyldimethylsilyl
hydroxylamine12 (735 mg, 5.0 mmol) in methylene chloride (20 mL)
and triethylamine (0.7 mL, 5.0 mmol) were added. Then a
suspension of EDC·HCl (1054 mg, 5.5 mmol) in methylene
chloride (25 mL) was added. The reaction mixture was allowed to
stir overnight at room temperature. Then the mixture was diluted
with methylene chloride (60 mL), washed with saturated NaHCO3
solution (60 mL), water (60 mL) and brine (60 mL). The organic
layer was dried over anhydrous Na2SO4, filtered and concentrated
under reduced pressure at room temperature. The residue was
purified by flash column chromatography (hexanes–EtOAc: 5 : 1)
to give (872 mg, 3.4◦7 mmol) (69.5%) of compound 2c as a white
O-tert-Butyl-dimethylsilyl-p-methylbenzohydroxamic acid (2b).
82% yield (based on recovered starting material). Mp 128–130 ◦C.
1H NMR (CDCl3, 300 MHz) d: 0.25 (s, 6H), 1.01 (s, 9H), 2.39 (s,
3H), 7.31 (m, 4H). 13C NMR (CD3OD, 75 MHz) d: −5.42, 17.79,
20.94, 25.75, 126.86, 128.87, 129.84, 140.94, 164.19.
1
solid. Mp 138–140 C. H NMR (CDCl3, 300 MHz) d: 0.26 (s,
3H), 0.38 (s, 3H), 1.02 (s, 9H), 7.46 (m, 5H).
O-tert-Butyl-dimethylsilyl-p-fluorobenzoylhydroxamic acid (2d).
61% yield (based on recovered starting material). Mp 152–153 ◦C.
1H NMR (CD3OD, 300 MHz) d: 0.06 (s, 6H), 0.89 (s, 9H), 7.17
(t, J = 8.7 Hz, 2H), 7.79 (dd, J = 5.1 Hz, J = 9.0 Hz, 2H). 13C
NMR (CD3OD, 75 MHz) d: −5.28, 26.48, 116.49, 116.78, 130.73,
130.85, 164.64, 167.96.
Representative procedure for the syntheses of hydroxamic acids
starting from the corresponding acyl chloride
p-Methoxybenzohydroxamic acid (4a). To a suspension of
hydroxylamine hydrochloride (815 mg, 0.012 mol) in chloroform
(20 mL), was added triethylamine (3.3 mL, 0.024 mol) and p-
methoxybenzoyl chloride (2.0 g, 0.012 mol) in chloroform (10 mL)
dropwise. The resulting mixture was stirred for 1 h. Then the
solvent was removed under vacuum. The residue was dissolved
in water and cooled in an ice bath. Then 1 N HCl was added.
The resulting precipitate was filtered, washed with cold water and
dissolved in dilute NaOH. Small pieces of dry ice were added to
the alkaline solution and a precipitate was obtained, which was
recrystallized from acetone–ether to give (996 mg, 5.96 mmol)
(51%) of compound 4a as colorless crystals. Mp 159–159.5 ◦C. 1H
NMR (CD3OD, 300 MHz) d: 3.85 (s, 3H), 6.98 (dd, J = 2.1 Hz,
J = 9.0 Hz, 2H), 7.83 (dd, J = 2.1 Hz, J = 9.0 Hz, 2H), 8.27 (br,
1H), 10.65 (br, 1H). 13C NMR (CD3OD, 75 MHz) d: 55.78, 114.08,
125.38, 129.10, 161.95.
O-tert-Butyl-dimethylsilyl-p-trifluoromethylbenzoylhydroxamic
acid (2e). ◦53% yield (based on recovered starting material). Mp
129.5–131 C. MS (FAB) (M + H)+ m/z 320. 1H NMR (CD3OD,
300 MHz) d: 0.02 (s, 6H), 1.02 (s, 9H), 7.78 (d, J = 8.1 Hz, 2H),
7.90 (d, J = 8.1 Hz, 2H).
Heptanohydroxamic acid (5)
To a solution◦of octanoic acid (2.88 g, 0.02 mol) in diethyl ether
(60 mL) at 0 C, ethyl chloroformate (2.60 g, 0.024 mol) and N-
methyl morpholine (2.63 g, 0.026 mol) were added and the mixture
was stirred for 10 min. The solid was filtered off and the filtrate was
added to freshly-prepared hydroxylamine (0.03 mol) in methanol
(8 mL). The resulting mixture was stirred for 15 min at room
temperature. Then the solvent was removed and the residue was
purified by iron-free silica gel column chromatography (hexanes–
EtOAc: 1 : 2) to give 2.92 g (91.7%) of desired compound 5 as a
white solid. Mp 76–78 ◦C. 1H NMR (CDCl3, 300 MHz) d: 0.89 (t,
p-Methylbenzohydroxamic acid (4b). 44% yield. Mp 148–
◦
1
149.5 C. H NMR (DMSO-d6, 300 MHz) d: 2.35 (s, 3H), 7.26
(d, J = 8.1 Hz, 2H), 7.67 (d, J = 8.1 Hz, 2H), 8.98 (s, 1H), 11.19
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J = 7.2 Hz, 3H), 1.30 (m, 8H), 1.63 (m, 2H), 2.16 (t, J = 7.5 Hz,
2H), 4.90 (br, 1H), 7.50 (br, 1H). 13C NMR (CDCl3, 75 MHz) d:
14.06, 22.60, 25.44, 28.95, 29.11, 31.67, 33.03, 172.02.
127.70, 127.74, 128.62, 128.99, 129.14, 129.44, 130.83, 130.93,
133.76, 141.59, 159.95, 161.47, 164.66, 171.18.
3-[(tert-Butyldimethylsilyloxy)benzamido]methyl-7-phenylaceta-
mido-3-cephem-4-carboxylic acid p-methoxybenzyl ester (7c).
1
29.3% yield. MS (FAB) (M + H)+ m/z 702. H NMR (CDCl3,
O-tert-Butyl-dimethylsilyl heptanohydroxamic acid (6)
300 MHz) d: −0.07 (s, 3H), 0.01 (s, 3H), 0.83 (s, 9H), 3.28 (d, J =
18 Hz, 1H), 3.64 (d, J = 18 Hz, 1H), 3.66 (dd, J = 16 Hz, 2H),
3.83 (s, 3H), 4.93 (dd, J = 15 Hz, 2H), 4.93 (d, J = 5 Hz, 1H),
5.13 (dd, J = 16 Hz, 2H), 5.85 (dd, J = 5 Hz, J = 9 Hz, 1H), 6.08
(d, J = 9 Hz, 1H), 7.30 (m, 14H); 13C NMR (CDCl3, 75 MHz)
d: −4.99, 18.01, 25.83, 26.31, 43.57, 55.49, 57.64, 59.31, 68.28,
92.03, 114.16, 125.41, 126.87, 127.69, 128.01, 128.57, 128.66,
129.44, 129.67, 131.04, 131.29, 133.78, 134.08, 160.15, 161.64,
164.79, 171.24.
To a stirred solution of 5 (500 mg, 3.2 mmol) in DMF (7 mL),
imidazole (279 mg, 4.1 mmol) and O-tert-butyldimethylsilyl
chloride (468 mg, 3.1 mmol) were added successively. The resulting
mixture was allowed to stir overnight at room temperature. Then
water (10 mL) was added followed by extraction with EtOAc (3 ×
20 mL). The combined organic layers were washed with brine
(50 mL), dried over anhydrous MgSO4, filtered and concentrated
under reduced pressure. The residue was recrystallized from
hexanes–EtOAc twice to give 520 mg (84%) of compound 6 as
white solid. Mp 58.5–61 ◦C. 1H NMR (CDCl3, 300 MHz) d: 0.18
(s, 6H), 0.95 (m, 12H), 1.27 (m, 8H), 1.63 (m, 2H), 2.09 (m, 2H),
7.65 (br, 1H).
3-[tert-Butyldimethylsilyloxy-(4-fluorobenzoyl)amino]methyl-7-
phenylacetamido-3-cephem-4-carboxylic acid p-methoxybenzyl es-
1
ter (7d). 27% yield. MS (FAB) (M + H)+ m/z 720. H NMR
(CDCl3, 300 MHz) d: −0.12 (s, 3H), −0.02 (s, 3H), 0.82 (s, 9H),
3.30 (d, J = 18.6 Hz, 1H), 3.61 (d, J = 18.6 Hz, 1H), 3.64 (dd,
J = 15.9 Hz, 2H), 3.82 (s, 3H), 4.93 (dd, J = 15.0 Hz, 2H), 4.93
(d, J = 4.8 Hz, 1H), 5.14 (dd, J = 12.0 Hz, 2H), 5.83 (dd, J =
4.8 Hz, J = 9.0 Hz, 1H), 6.08 (d, J = 9.3 Hz, 1H), 7.30 (m, 13H);
13C NMR (CDCl3, 300 MHz) d: −5.13, 17.80, 25.62, 26.21, 43.30,
55.29, 57.60, 59.17, 68.13, 113.97, 115.27, 115.57, 125.30, 126.66,
127.26, 127.70, 129.15, 129.43, 129.90, 130.86, 131.02, 131.14,
133.74, 159.98, 161.44, 162.52, 164.64, 165.86, 171.18.
3-[tert-Butyldimethylsilyloxy-(4-methoxybenzoyl)amino]methyl-7-
phenylacetamido-3-cephem-4-carboxylic acid p-methoxybenzyl
ester (7a)
To a suspension of GCLE (1.302 g, 2.675 mmol) in THF (20 mL)
was added a solution of Pd(OAc)2 (72.1 mg, 0.321 mmol) and
PPh3 (420.9 mg, 1.605 mmol) in THF (30 mL) under N2 over
15 min. Then a solution of 2a (790.0 mg, 2.809 mmol) and sodium
hydride (60% oil suspen◦sion) (107.1 mg, 2.675 mmol) in THF
(20 mL) was added at 0 C. The resulting mixture was stirred at
room temperature for 3 h under N2. Then 70 mL of water was
added and the resulting mixture was extracted with EtOAc (3 ×
150 mL). The organic layers were combined and washed with
saturated NaCl solution (400 mL), dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (hexanes–
EtOAc: 3 : 1) to give 453 mg (30.2%) of compound 7a as a light
yellow oil. 1H NMR (CDCl3, 300 MHz) d: −0.101 (s, 3H), −0.03
(s, 3H), 0.81 (s, 9H), 3.22 (d, J = 18.3 Hz, 1H), 3.58 (d, J =
16.0 Hz, 1H), 3.59 (d, J = 18.3 Hz, 1H), 3.65 (d, J = 16.0 Hz,
1H), 3.79 (s, 3H), 3.83 (s, 3H), 4.81 (d, J = 16.2 Hz, 1H), 4.89
(d, J = 4.8 Hz, 1H), 4.94 (d, J = 16.2 Hz, 1H), 5.10 (dd, J =
11.7 Hz, 2H), 5.80 (dd, J = 4.8 Hz, J = 9.0 Hz, 1H), 6.16 (d, J =
9.0 Hz, 1H), 7.31 (m, 13H); 13C NMR (CDCl3, 75 MHz) d: −5.18,
−5.15, 17.84, 25.71, 26.27, 43.18, 55.29, 55.42, 57.64, 59.16, 68.04,
113.57, 113.95, 125.08, 125.90, 126.76, 127.53, 127.77, 129.01,
129.38, 130.72, 130.84, 133.99, 159.93, 161.47, 161.84, 164.72,
171.33.
3-[tert-Butyldimethylsilyloxy-(4-trifluoromethylbenzoyl)amino]-
methyl-7-phenylacetamido-3-cephem-4-carboxylic acid p-methoxy-
1
benzyl ester (7e). 21% yield. MS (FAB) (M + H)+ m/z 770. H
NMR (CDCl3, 300 MHz) d: −0.13 (s, 3H), −0.02 (s, 3H), 0.80 (s,
9H), 3.32 (d, J = 18.0 Hz, 1H), 3.63 (d, J = 17.0 Hz, 1H), 3.67 (dd,
J = 16.0 Hz, 2H), 3.84 (s, 3H), 4.95 (dd, J = 15.0 Hz, 2H), 4.95 (d,
J = 4.8 Hz, 1H), 5.16 (dd, J = 12.0 Hz, 2H), 5.85 (dd, J = 4.8 Hz,
J = 9.0 Hz, 1H), 6.07 (d, J = 9.0 Hz, 1H), 7.30 (m, 13H); 13C NMR
(CDCl3, 75 MHz) d: −5.07, 17.78, 25.53, 26.15, 43.32, 55.29, 57.61,
59.21, 68.17, 94.78, 113.98, 125.24, 126.63, 126.90, 127.74, 129.00,
129.17, 129.43, 130.85, 133.71, 160.00, 161.44, 164.65, 171.17.
N-[6-(4-Methoxybenzoyl)-1,8-dioxo-2,2a,4,5,6,8-hexahydro-1H-
7-oxa-3-thia-6,8b-diazacyclobuta[a]naphthalen-2-yl]-2-
phenylacetamide (8a)
To a mixture of compound 7a (60 mg, 0.082 mmol) and tri-
ethylsilane (0.01 mL) in methylene chloride (0.5 mL), was added
TFA (0.1 mL) at 0 ◦C. The resulting mixture was stirred at room
temperature for 1.5 h. Then the TFA was removed under reduced
pressure and the residue was taken up in EtOAc–H2O (4 mL of a
1 : 1 mixture). The pH value of the aqueous layer was adjusted to
7.5 using saturated NaHCO3. The aqueous layer was separated,
washed with EtOAc (3 × 1 mL) (The aqueous layer was used
for the synthesis of compound 9a). The combined organic layers
were washed with brine, dried over anhydrous MgSO4, filtered and
concentrated to give a yellow oil. The residue was purified by flash
column chromatography on silica gel (hexanes–EtOAc: 2 : 1) to
give 14 mg (35.6%) of compound 8a as a light yellow solid. MS
(FAB) (M + H)+ m/z 480. 1H NMR (CDCl3, 300 MHz) d: 3.31 (d,
J = 18.6 Hz, 1H), 3.64 (dd, J = 16.2 Hz, 2H), 3.69 (d, J = 18.6 Hz,
1H), 3.84 (s, 3H), 4.65 (dd, J = 18.0 Hz, 2H), 4.98 (d, J = 5.0 Hz,
3-[tert-Butyldimethylsilyloxy-(4-methylbenzoyl)amino]methyl-7-
phenylacetamido-3-cephem-4-carboxylic acid p-methoxybenzyl
ester (7b)
29.1% yield. 1H NMR (CDCl3, 300 MHz) d: −0.08 (s, 3H), −0.02
(s, 3H), 0.81 (s, 9H), 2.38 (s, 3H), 3.59 (dd, J = 18 Hz, 2H), 3.62
(dd, J = 16 Hz, 2H), 3.79 (s, 3H), 4.89 (dd, J = 16 Hz, 2H), 4.90
(d, J = 4.8 Hz, 1H), 5.10 (dd, J = 12 Hz, 2H), 5.80 (dd, J =
4.8 Hz, J = 9.0 Hz, 1H), 6.10 (d, J = 9.0 Hz, 1H), 7.31 (m, 13H);
13C NMR (CDCl3, 75 MHz) d: −5.91, −5.11, 17.87, 21.55, 25.68,
26.16, 43.29, 55.29, 57.51, 59.13, 68.05, 113.97, 125.13, 126.73,
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1H), 5.84 (dd, J = 5.0 Hz, J = 0.9 Hz, 1H), 6.22 (d, J = 9.0 Hz, 1H),
7.32 (m, 9H). 13C NMR (CDCl3, 150 MHz) d: 25.43, 43.26, 46.42,
55.48, 57.26, 59.66, 113.77, 121.03, 122.55, 127.81, 129.23, 129.47,
132.02, 133.60, 134.27, 157.75, 163.23, 163.68, 170.20, 171.27.
1H), 7.34 (m, 9H). 13C NMR (CD3OD, 75 MHz) d: 25.91, 41.75,
50.24, 54.47, 57.54, 59.23, 112.86, 125.39, 126.59, 128.17, 128.81,
130.53, 131.43, 135.04, 161.92, 164.74, 170.40, 173.21.
3-[Hydroxy-(4-methylbenzoyl)-amino]methyl-7-phenylacetamido-
N-[6-(4-Methylbenzoyl)-1,8-dioxo-2,2a,4,5,6,8-hexahydro-1H-
7-oxa-3-thia-6,8b-diazacyclobuta[a]naphthalen-2-yl]-2-phenylace-
tamide (8b). 23% yield. MS (FAB) (M + H)+ m/z 464. 1H NMR
(CDCl3, 300 MHz) d: 2.39 (s, 3H), 3.31 (d, J = 19.2 Hz, 1H),
3.64 (dd, J = 16.0 Hz, 2H), 3.68 (d, J = 19.2 Hz, 1H), 4.67
(dd, J = 18.0 Hz, 2H), 4.98 (d, J = 4.8 Hz, 1H), 5.84 (dd, J =
3-cephem-4-carboxylic acid (9b). 29% yield. MS (FAB) (M +
1
H)+ m/z 482, 504 (M + Na). H NMR (CD3OD, 300 MHz) d:
2.37 (s, 3H), 3.45 (d, J = 18.0 Hz, 1H), 3.59 (dd, J = 15.0 Hz,
2H), 3.67 (d, J = 18.0 Hz, 1H), 4.89 (overlap with the solvent
peak, 2H), 5.06 (d, J = 4.8 Hz, 1H), 5.70 (d, J = 4.8 Hz, 1H),
7.42 (m, 9H). 13C NMR (CD3OD, 75 MHz) d: 21.61, 27.44, 43.29,
51.70, 59.08, 60.77, 128.14, 129.71, 129.78, 129.84, 130.36, 132.29,
136.58, 142.62, 165.25, 166.25, 172.39, 174.75.
4.8 Hz, J = 8.4 Hz, 1H), 6.20 (d, J = 8.4 Hz, 1H), 7.41 (m, 9H). 13
C
NMR (CDCl3, 75 MHz) d: 21.67, 25.42, 43.27, 46.32, 57.29, 59.65,
121.05, 127.69, 127.83, 129.15, 129.24, 129.49, 129.65, 133.61,
134.18, 143.56, 157.68, 163.74, 170.53, 171.29.
3-[Hydroxy(benzoyl)amino]methyl-7-phenylacetamido-3-cephem-
4-carboxylic acid (9c). 31% yield. MS (FAB) (M + H)+ m/z
468. 1H NMR (DMSO-d6, 300 MHz) d: 3.34 (d, J = 18 Hz, 1H),
3.67 (dd, J = 16.2 Hz, 2H), 3.77 (d, J = 18 Hz, 1H), 4.75 (dd,
J = 15 Hz, 2H), 5.08 (d, J = 5 Hz, 1H), 5.69 (dd, J = 5 Hz, J =
8 Hz, 1H), 7.43 (m, 10H), 9.13 (d, J = 8 Hz, 1H), 10.05 (s, 1H).
13C NMR (CD3OD, 75 MHz) d: 27.29, 43.57, 49.68, 55.52, 57.61,
59.44, 68.39, 95.05, 114.21, 119.60, 124.95, 125.21, 126.91, 128.02,
128.19, 128.54, 129.43, 129.46, 129.70, 130.67, 131.06, 131.59,
133.11, 133.85, 136.41, 151.41, 160.19, 161.81, 164.89, 171.31.
N-[6-Benzoyl-1,8-dioxo-2,2a,4,5,6,8-hexahydro-1H-7-oxa-3-thia-
6,8b-diazacyclobuta[a]naphthalen-2-yl]-2-phenylacetamide
(8c).
◦
24% yield. MS (FAB) (M + H)+ m/z 450 decompose at 122 C.
1H NMR (CDCl3, 300 MHz) d: 3.34 (d, J = 18.6 Hz, 1H), 3.67
(dd, J = 16.2 Hz, 2H), 3.75 (d, J = 18.6 Hz, 1H), 4.72 (dd, J =
18 Hz, 2H), 5.01 (d, J = 4.8 Hz, 1H), 5.88 (dd, J = 4.8 Hz, J =
9 Hz, 1H), 6.10 (d, J = 9 Hz, 1H), 7.45 (m, 10H). 13C NMR
(CDCl3, 75 MHz) d: 25.66, 43.55, 46.38, 57.53, 59.90, 121.33,
128.10, 128.69, 129.52, 129.71, 129.73, 130.86, 132.93, 133.78,
134.23, 157.70, 163.95, 170.56, 171.44.
3-[Hydroxy-(4-fluorobenzoyl)-amino]methyl-7-phenylacetamido-
3-cephem-4-carboxylic acid (9d). 31% yield. MS (FAB) (M +
H)+ m/z 486. 1H NMR (CD3OD, 300 MHz) d: 3.48 (d, J =
18.0 Hz, 1H), 3.60 (dd, J = 14.1 Hz, 2H), 3.68 (d, J = 18.0 Hz,
1H), 4.92 (s, 2H), 5.07 (d, J = 4.8 Hz, 1H), 5.71 (d, J = 4.8 Hz,
1H), 7.13–7.78 (m, 9H). 13C NMR (CD3OD, 75 MHz) d: 27.50,
43.28, 51.48, 59.08, 60.79, 115.89, 116.18, 127.96, 128.13, 129.70,
130.34, 131.44, 132.42, 132.54, 136.57, 163.96, 166.25, 167.27,
171.16, 174.76.
N-[6-(4-Fluorobenzoyl)-1,8-dioxo-2,2a,4,5,6,8-hexahydro-1H-7-
oxa-3-thia-6,8b-diazacyclobuta[a]naphthalen-2-yl]-2-phenylaceta-
1
mide (8d). 21% yield. MS (FAB) (M + H)+ m/z 486. H NMR
(CDCl3, 300 MHz) d: 3.37 (d, J = 19.2 Hz, 1H), 3.68 (dd, J =
16.2 Hz, 2H), 3.75 (d, J = 19.2 Hz, 1H), 4.73 (dd, J = 18.0 Hz,
2H), 5.03 (d, J = 5.1 Hz, 1H), 5.90 (dd, J = 4.8 Hz, J = 8.7 Hz,
1H), 6.10 (d, J = 8.7 Hz, 1H), 7.12–7.88 (m, 9H). 13C NMR
(CDCl3, 75 MHz) d: 25.44, 43.28, 46.07, 57.29, 59.68, 115.57,
115.86, 121.01, 127.86, 129.27, 129.49, 132.22, 132.35, 133.54,
134.16, 163.60, 163.74, 166.97, 169.15, 171.27.
3-[Hydroxy-(4-trifluoromethylbenzoyl)-amino]methyl-7-phenyl-
acetamido-3-cephem-4-carboxylic acid (9e). 31% yield. MS
(FAB) (M + H)+ m/z 536. 1H NMR (CD3OD, 300 MHz) d: 3.52
(d, J = 18.0 Hz, 1H), 3.63 (dd, J = 15.0 Hz, 2H), 3.74 (d, J =
18.0 Hz, 1H), 4.96 (s, 2H), 5.11 (d, J = 4.8 Hz, 1H), 5.75 (d, J =
4.8 Hz, 1H), 7.26–7.87 (m, 9H). 13C NMR (CD3OD, 75 MHz) d:
26.01, 41.75, 49.72, 57.55, 59.27, 122.13, 124.56, 124.61, 125.80,
126.60, 126.77, 128.17, 128.71, 128.81, 131.54, 131.97, 135.04,
137.87, 163.67, 164.69, 169.37, 173.23.
N -[6-(4-Trifluoromethylbenzoyl)-1,8-dioxo-2,2a,4,5,6,8-hexa-
hydro-1H-7-oxa-3-thia-6,8b-diazacyclobuta[a]naphthalen-2-yl]-2-
phenylacetamide (8e). 21% yield. MS (FAB) (M + H)+ m/z 518.
1H NMR (CDCl3, 300 MHz) d: 3.39 (d, J = 19.2 Hz, 1H), 3.69
(dd, J = 16.0 Hz, 2H), 3.77 (d, J = 19.0 Hz, 1H), 4.78 (dd, J =
18.0 Hz, 2H), 5.04 (d, J = 5.1 Hz, 1H), 5.91 (dd, J = 5.1 Hz,
J = 8.7 Hz, 1H), 6.19 (d, J = 8.4 Hz, 1H), 7.29–7.89 (m, 9H). 13
C
NMR (CDCl3, 75 MHz) d: 25.63, 29.91, 43.45, 46.03, 57.52, 59.92,
121.21, 125.63, 125.68, 128.06, 129.37, 129.46, 129.67, 129.99,
133.71, 134.18, 157.22, 163.97, 168.75, 171.54.
3-[tert-Butyldimethylsilyloxyl(octanoyl)amino]methyl-7-
phenylacetamido-3-
cephem-4-carboxylic acid p-methoxybenzyl ester (10)
3-[Hydroxy-(4-methoxybenzoyl)-amino]methyl-7-
phenylacetamido-3-cephem-4-carboxylic acid (9a)
To a solution of GCLE (900 mg, 1.80 mmol) in acetone (18 mL)
was added NaI (346.5 mg, 2.25 mmol). The mixture was stirred
at room temperature for 1.5 h. Then 18 mL of water was added
and the resulting mixture was extracted with methylene chloride
(2 × 18 mL). The combined organic layers were washed with 5%
aqueous Na2S2O3 (12 mL), dried over anhydrous Na2SO4, filtered
and concentrated under reduced pressure to give 1.0 g (94%) of the
3-iodomethyl cephalosporin intermediate as a yellow solid. Then
to a solution of O-tert-butyl-dimethylsilyl heptanohydroxamic
acid (300 mg, 1.156 mmol) in DMF (3 mL) cooled in an ice bath,
sodium hydride (61% oil dispersion) (44 mg, 1.098 mmol) was
The aqueous layer from initial part of the workup during the
synthesis of compound 8a was adjusted to pH 5.5 using diluted
HCl and extracted with EtOAc (3 × 2 mL). The combined organic
layers were dried over anhydrous MgSO4, filtered and concentrated
to give 8 mg (20.3%) of compound 9a as a light yellow solid. MS
(FAB) (M + H)+ m/z 498, 520 (its sodium salt). 1H NMR (CD3OD,
300 MHz) d: 3.46 (d, J = 18.0 Hz, 1H), 3.59 (dd, J = 14.4 Hz,
2H), 3.67 (d, J = 18.0 Hz, 1H), 3.83 (s, 3H), 4.88 (overlap with the
solvent peak, 2H), 5.06 (d, J = 4.8 Hz, 1H), 5.70 (d, J = 4.8 Hz,
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Org. Biomol. Chem., 2006, 4, 4178–4185 | 4183
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added. After stirring for 30 min, a solution of the 3-iodomethyl
cephalosporin intermediate (900 mg, 1.556 mmol) in DMF (3 mL)
was then added and the mixture was stirred at rt overnight. Then
6 mL of water was added. The resulting mixture was extracted with
EtOAc (3 × 6 mL). The organic layers were combined and washed
with brine (20 mL), dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. The residue was purified
by flash column chromatography on silica gel (hexanes–EtOAc:
5 : 1) to give 70 mg (8%) of compound 10 as a yellow solid.
Antibacterial activity
Antibacterial activity of the compounds was studied by deter-
mination of minimal inhibitory concentrations (MIC) according
to the NCCLS guidelines19,20 using the broth micro dilution
method.
Anti-tuberculosis and cytotoxicity assays
1
MS (FAB) (M + H)+ m/z 724. H NMR (CDCl3, 300 MHz)
Activity against replicating Mycobacterium tuberculosis H37Rv
(American Type Culture Collection, Rockville, MD) was deter-
mined using a fluorescence readout in the Microplate Alamar
Blue Assay (MABA)22,24 following incubation for one week with
test compounds in glycerol-alanine-salts medium (GAS) as well
as in medium without added iron but with Tween 80 (GAST).23
The MIC was defined as the minimum concentration inhibiting
fluorescence by 90% relative to bacteria-only controls. Toxicity to
African green monkey kidney cells (VERO) was determined using
a colorimetric assay as previously described.24
d: 0.07 (s, 3H), 0.19 (s, 3H), 0.94 (m, 12H), 1.30 (m, 8H), 1.63
(m, 2H), 2.38 (m, 2H), 3.31 (dd, J = 18.0 Hz, 2H), 3.67 (dd,
J = 16.2 Hz, 2H), 3.84 (s, 3H), 4.66 (d, J = 16.5 Hz, 1H),
4.90 (d, J = 4.8 Hz, 1H), 5.00 (d, J = 16.5 Hz, 1H), 5.83 (dd,
J = 4.8 Hz, J = 9.3 Hz, 1H), 6.11 (d, J = 9.3 Hz, 1H), 6.90–
7.42 (m, 9H); 13C NMR (CDCl3, 75 MHz) d: −4.78, −4.64,
14.09, 17.80, 22.63, 24.36, 25.67, 25.75, 29.06, 29.39, 29.74, 31.70,
32.59, 43.39, 55.30, 59.14, 68.04, 113.98, 124.82, 126.79, 127.83,
127.97, 129.25, 129.48, 130.87, 133.62, 159.95, 161.57, 164.55,
171.13.
Acknowledgements
N-(6-Octanoyl-1,8-dioxo-2,2a,4,5,6,8-hexahydro-1H-7-oxa-3-
thia-6,8b-diazacyclobuta[a]naphthalene-2-yl)-2-phenylacetamide
(11)
We gratefully acknowledge the NIH (R01 AI054193) for support
of this research and the LizzadroMagnetic Resonance Magnetic
Resonance Research Center at Notre Dame for NMR facilities,
as well as Dr B. Boggess and N. Sevova for mass spectrometry fa-
cilities and G. Moraski for sample and database organization. We
acknowledge Otsuka Chemical Co. Ltd. for GCLE. S. Franzblau
acknowledges the skilful technical assistance of Baojie Wan and
Yuehong Wang. U. Mo¨llmann acknowledges the skilful technical
assistance of Irmgard Heinemann.
To a mixture of compound 10 (50 mg, 0.069 mmol) and anisole
(29.8 mg, 0.276 mmol) in methylene chloride (0.5 mL), was added
TFA (110 mg, 0.967 mmol) at 0 ◦C. The resulting mixture was
stirred at room temperature for 0.5 h. Then TFA was removed
under reduced pressure and the residue was purified by flash
column chromatography on silica gel (hexanes–EtOAc: 2 : 1) to
give 10 mg (30.8%) of compound 11 as a light yellow solid. MS
1
(FAB) (M + H)+ m/z 472. H NMR (CDCl3, 300 MHz) d: 0.87
References
(t, J = 6.6 Hz, 3H), 1.28 (m, 8H), 1.61 (t, J = 6.9 Hz, 3H), 2.51
(m, 2H), 3.28 (d, J = 18.9 Hz, 1H), 3.66 (dd, J = 16.0 Hz, 2H),
3.65 (d, J = 18.6 Hz, 1H), 4.59 (dd, J = 18.0 Hz, 2H), 4.98 (d,
J = 5.0 Hz, 1H), 5.88 (dd, J = 4.8 Hz, J = 8.7 Hz, 1H), 6.10 (d,
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ration of 11 was eluted with 5% MeOH in CH2Cl2 to give 8 mg
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