Direct diazo-transfer reaction on β-lactam: Synthesis and preliminary biological activities of 6-triazolylpenicillanic acids

Article abstract of DOI:10.1016/j.bmc.2007.08.035

In this study we report the first example of a direct diazo-transfer reaction on readily available 6-aminopenicillanates to give 6-azidopenicillanates in high yield. Subsequent Cu(I)-catalyzed Huisgen cycloaddition between these 6-azidopenicillanates and assorted terminal alkynes facilely furnished 6-triazolylpenicillanic acids. Preliminary biological screening indicates that these triazolylpenicillanic acids possess low to moderate antibacterial activities.

Full text of DOI:10.1016/j.bmc.2007.08.035

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7288–7300
Direct diazo-transfer reaction on b-lactam: Synthesis
and preliminary biological activities of 6-triazolylpenicillanic acids
Po C. Chen, Rebekah E. Wharton, Pratiq A. Patel and Adegboyega K. Oyelere*
School of Chemistry and Biochemistry, Parker H. Petit Institute for Bioengineering and Biosciences,
Georgia Institute of Technology, Atlanta, GA 30332-0400, USA
Received 18 June 2007; revised 14 August 2007; accepted 20 August 2007
Available online 25 August 2007
Abstract—In this study we report the first example of a direct diazo-transfer reaction on readily available 6-aminopenicillanates to
give 6-azidopenicillanates in high yield. Subsequent Cu(I)-catalyzed Huisgen cycloaddition between these 6-azidopenicillanates and
assorted terminal alkynes facilely furnished 6-triazolylpenicillanic acids. Preliminary biological screening indicates that these tria-
zolylpenicillanic acids possess low to moderate antibacterial activities.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
the N-5 amide moiety in penicillins is highly reactive
and has been proposed to facilitate the hydrolysis of
the b-lactam ring in acidic media (Fig. 1)12 Incorpora-
tion of electron-withdrawing groups and steric bulk at
the N-acyl side chain has been shown to reduce the acid
sensitivity of penicillins.13,14
b-Lactam antibiotics such as penicillin and cephalospo-
rins are the most commonly prescribed antibacterial
agents. Their antibacterial efficacy derives from ability
to acylate a serine residue at the penicillin binding pro-
teins (PBPs) active site.1,2 Serine acylation inhibits the
enzymatic activities of PBPs, thus interfering with the
essential polymerization and cross-linking of the pepti-
doglycan components of the bacterial cell wall.1 How-
ever, the emergence of multi-drug resistant bacteria
strains such as Staphylococcus aureus is threatening the
clinical effectiveness of b-lactam antibiotics.3 The com-
mon resistance mechanism in Gram-negative bacteria
is the cellular expression of b-lactamases (or penicillin-
ase), which hydrolyze the b-lactam ring and thus
inactivate the antibiotics.4–7 Therapeutic agents targeting
b-lactamase resistance include clavulanate, sulbactam,
and tazobactam. These are generally administered in
combination with b-lactamase susceptible b-lactams.8,9
However, the emergence of multi-strains b-lactamase
has reduced the efficacy of some of these b-lactamase
inhibitors.10,11
Another shortcoming to the effectiveness of b-lactamase
antibiotics is the bacterial outer membrane, which is a
formidable barrier in Gram negative organisms. In addi-
tion, the bacterial membrane could acquire efflux pumps
that facilitate active elimination of drug from the bacte-
rial cytosol.15,16 All these changes could prevent the
intracellular accumulation of pharmacologically rele-
vant concentrations of the b-lactam antibiotics. One cle-
ver strategy to facilitate b-lactams’ diffusion across
membrane is to covalently link them to membrane-pene-
trating groups such as sideophores and peptides.1–3,17
However, the synthesis of such b-lactam conjugates of-
ten involves long synthetic schemes and complicated
compound purification protocols.2b–e
One of the most fruitful b-lactam SAR studies has been
the modification of the N-acyl groups. These studies
have led to the discovery of b-lactam analogs with supe-
rior antibacterial activity (Fig. 2).16,18–20 Much recent
studies have shown that attachment of N-acyl groups
incorporating the structural feature of peptidoglycan re-
sults in PBP specific b-lactams and cephalosporins.3,17,21
PBPs can also tolerate an assorted variety of non-amide
groups at the C-6 and C-7 positions of penicillins and
cephalosporins, respectively.20 Some of these non-amide
There are also other intracellular factors that attenuate
the antibacterial activities of b-lactams. For example,
Keywords: b-Lactam; Antibiotic; Diazo-transfer; Penicillin; Huisgen
cycloaddition; 6-Triazolylpenicillanic acids; Click chemistry; Terminal
alkynes; Azide.
*
Corresponding author. Tel.: +1 404 894 4047; fax: +1 404 894
2291; e-mail: aoyelere@gatech.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.035

P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
7289
R
O
H
N
R
N
S
-H+
N
O
S
S
R
O
N
N
HN
O
H+
O
O
CO2H
CO2H
CO2H
Penicillin
Penicillenic acid
R = C6H5CH2 - or -CH2OC6H5
H+
R
HO2C
S
NH
HS
HN
O
HO
N
N
O
CO2H
R
CO2H
Penillic acid
Penamaldic acid
Figure 1. Self-induced acid hydrolysis of penicillin.
O
O
H2N
NH
O
NH
O
NH
S
S
S
O
O
O
N
N
N
O
CO2H
CO2H
CO2H
Phenethicillin
Propicillin
Ampicillin
O
O
N
O
N
Cl
H
N
NH
O
O
NH
S
S
S
O
O
O
X
N
N
O
N
O
CO2H
CO2H
CO2H
Methicillin
Oxacillin
X=H, Cloxacillin
X=F, Floxacillin
Figure 2. The structure of some acid- and b-lactamase-resistant penicillins.
b-lactam analogs, in addition to possessing potent anti-
bacterial activity, have differential affinity for PBP iso-
forms (Fig. 3). For example, mecillinam (amdinocillin),
a b-lactam analog with an unusual 6-formylimido moi-
ety, derives its antibacterial activity from its specific
interaction with PBP-2.22 Very few b-lactams with tradi-
tional amide bond isosteres have been reported to date.
One such example is the sulfonamide derivatives. A sub-
set of these compounds have been synthesized and
tested. However these compounds are still prone to
chemical degradation and hydrolysis by b-lactamases.23
isostere.24,25 Unlike an amide bond, the triazole moiety
is not susceptible to hydrolysis.24 Replacement of the
reactive b-lactam acyl bond with a triazole ring may lead
to b-lactams that are more stable in acidic environment.
In addition, the triazole ring could in principle facilitate
facile conjugation of b-lactams to assorted cell perme-
able peptides and peptoids for promoted uptake of b-
lactams into bacterial cell. Here, we report our efforts
on the design, synthesis, and preliminary biological
activities of 6-triazolylpenicillanic acid.
We proposed that 1,4-disubstituted-1,2,3-triazoles could
serve as an isostere for the N-acyl group of penicillins
and cephalosporins. Due to the similarities between
some of its bond characteristics with an amide moiety,
the triazole group is commonly used as an amide bond
2. Results and discussion
The key intermediate in the synthesis of the proposed 6-
triazolylpenicillanic acids 1 is the 6-azidopenicillanates
2. It is anticipated that Cu(I)-catalyzed cycloaddition be-

7290
P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
R1
O
n
H
O
S
R2
N
N
S
S
N
O
S
R1
N
OAc
O
N
O
N
O
COONa
CO2H
CO2H
n = 0, 2
R1, R2 =Alkyl group
R1 = Alkyl group
7-Alkylidenecephalosporins
Sulfamidopenicillins
Mecillinam
(Amdinocillin)
Figure 3. Structure of amide isosteric b-lactams.
tween 2 and appropriate alkynes (Sharpless Click Chem-
istry)26 followed by deprotection of the ester 6-azidope-
nicillanate protection group will furnish the desired
triazolylpenicillanic acid 1 (Scheme 1). The synthesis of
protected 6-azidopenicillanates similar to 2a–c has been
described in the literature.27–30 These compounds are
generally synthesized, in moderate yields, using multi-
step reactions. For example, Barrett and Sakadarat
described the synthesis of benzyl protected 6-azidopenic-
illanate, the final intermediate in their total synthesis of
6-aminopenicillanic acid (6-APA), in a six-step reaction
scheme with a total yield of about 15%.30 For the synthe-
sis of 6-triazolylpenicillanates, we desired a more general
and mild synthetic strategy to 6-azidopenicillanates.
fer reaction with freshly prepared triflyl azide under ba-
sic conditions in the classic CH2Cl2/MeOH/H2O solvent
mixture.31b,e These conditions, however, gave a complex
mixture from which we only isolated a minute amount
of the ring-opened methyl ester azide 4. This product
was presumably obtained from Et3N promoted methan-
olysis of compound 3a in addition to the desired
diazo-transfer reaction. The reaction was repeated in
anhydrous CH2Cl2 and Et3N. Gratifyingly, we obtained
the desired azide 5a in yields of 50–68% within 2–2.5 h
of reaction, though other uncharacterized degradation
products persisted. Because of the potential hazard of
handling triflyl azide in halogenated solvent, we investi-
gated the compatibility of this reaction with non-haloge-
nated solvents. The reaction worked equally well when
CH2Cl2 was replaced with toluene, yielding azide 5a in
70% yields. Similarly, azides 5b and 5c were obtained
from amines 2b and 2c in moderate to good yields
(Scheme 2). The b orientation of the azide group in
5a–c was authenticated by 1H NMR (J5,6 coupling
constant = 4 Hz),29 thereby confirming that the reaction
occurred with the characteristic retention of
configuration.31b,e
2.1. Synthesis of 6-azidopenicillanic acid by diazo-transfer
reaction
As a new general alternative, we envisioned that azid-
openicillanic acid could be obtained from commercially
available 6-aminopenicillanic acids through a diazo-
transfer reaction. The diazo-transfer reaction is a mild,
high yielding reaction that has been used to effect direct
conversion of assorted amines to azides.31 In addition, a
recent observation from our lab has extended the scope
of this versatile reaction to include azide functionaliza-
tion of amine-coated solid supports under heteroge-
neous reaction conditions.32 However, our initial
efforts at initiating diazo-transfer reaction on unpro-
tected 6-aminopenicillanic acid were unsuccessful. To
thoroughly probe the feasibility of this reaction on b-
lactam moiety, we decided to investigate various car-
boxyl protected 6-aminopenicillanates. The requisite
carboxyl protected aminopenicillanates 3a–c were syn-
thesized adapting literature protocols.21,33–37 Using the
PNB protected compound 3a, we initiated diazo-trans-
2.2. Cu(I)-catalyzed cycloaddition reaction between
6-azidopenicillanates and terminal alkynes
To identify optimum conditions for Cu(I)-catalyzed
cycloaddition reaction, we investigated the reaction of
azides 5b and 5c with 3-phenyl-1-propyne 6 and 2-ethy-
nyl-1,3-dimethoxybenzene 7. Our choice of terminal
alkynes 6 and 7 is partly informed by the possibility that
cycloaddition between these alkynes and azides 5 will,
respectively, furnish triazolyl isosteres of penicillin G
and methicillin, two historically useful penicillin deriva-
tives. Cu(I)-catalyzed reaction of azide 5b or 5c with
N
N
R1
H3N
N
N3
O
S
S
S
N
O
N
N
O
CO2
CO2H
CO2R2
6-Aminopenicillanic acid
2a-c
1
a, R2 = PNB
b,
c,
R2 = PMB
R2 = BzD
Scheme 1. Synthetic approach to 6-triazolylpenicillanic acids. Abbreviations: PNB, para-nitrobenzyl; PMB, para-methoxylbenzyl; BzD, benzhydryl.

P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
7291
N3
H3N
N3
O
S
p-TsO
S
S
O
b or c
a
HN
OMe
N
N
O
CO2R
CO2R
CO2R
4,
R = PNB
+
3a-c
5a-c
a, R = PNB
b,
a, R = PNB
b,
R = PMB
c, R = BzD
R = PMB
c, R = BzD
Complex
degradation
mixture
Scheme 2. Synthesis of protected 6-azidopenicillanic acid. Reagents and conditions: (a) TfN3, Et3N, CH2Cl2/MeOH/H2O, rt; (b) TfN3, Et3N,
DMAP, rt, 2–2.5 h; (c) TfN3, Et3N, toluene, rt, 1.5–2.5 h.
alkyne 6 resulted in triazoles 8a and 8b in excellent yield.
Subsequent TFA deprotection of the PMB and the BzD
group furnished the desired penicillanic acid 9a in excel-
lent yield. Similarly, the reaction of azide 5c with alkyne
7 followed by deprotection of the BzD group gave pen-
icillanic acid 9 in 72% overall yield (Scheme 3). Because
of the rapidity of deprotection of the BzD group and the
product quality, we focused much of our attention on
the reactivity of azide 5c.
tious Disease (NIAID) through the In vitro and Animal
Models for Emerging Infectious Diseases and Biode-
fense Screening Program. Compounds were screened
following the procedures recommended by the Clinical
Laboratory Standard Institute (CLSI). Compounds 9a,
12e, 12g, and 12l exhibited some antibacterial activity
against the Gram-positive bacteria strain Streptococcus
pneumoniae with MIC value of 4–8 lg/mL. Similarly,
compound 12l showed a moderate activity (MIC
ꢀ8 lg/mL) against Bacillus anthracis. Other compounds
listed in Table 1 displayed comparatively poor antibac-
terial activity (MIC >8 lg/mL).
We turned to investigate the reactivity of azide 5c with
assorted terminal alkynes in order to probe the scope
of this reaction. We selected a subset of terminal
alkynes whose closely related carboxylic acid analogs
have been shown to support the antimicrobial activities
of b-lactams.38–40 Alkynes 7, 10h, 10i, and 10k that we
could not obtain from commercial sources were
synthesized from the corresponding carboxylic acid,
through the intermediacy of aldehyde, using the
Bestmann-Ohira reagent.41–43 Hydroxyl alkynes 10f
and 10l were obtained by a direct Grignard reaction
of ethynylmagnesium bromide with the appropriate
aldehydes. Detailed protocols for the synthesis of these
alkynes are reported in Section 4.
3. Conclusion
We have reported a convenient route for the synthesis of
6-triazolylpenicillanic acid. Preliminary biological evalu-
ations of compounds described in this report demon-
strated that they possess low to moderate antibacterial
activity. The triazole ring lacks the H-bonding donating
N–H group which could act as an additional recognition
feature in the binding of some b-lactams to the PBPs.
This may partly explain the relatively low antimicrobial
activities of these 6-triazolyl-penicillanic acids. Cur-
rently, we are investigating the SAR of the lead com-
pounds in order to identify 6-triazolylpenicillanic acid
with improved antibacterial activity against drug resis-
tant bacterial strains.
Cu(I)-catalyzed reaction of alkynes 10a–l with azidope-
nicillanate 5b or 5c proceeded smoothly at ambient tem-
perature, leading to uneventful formation of triazole
11a–l in good to excellent yields. Subsequent carbonyl
group deprotection furnished the desired 6-triazolylpe-
nicillanic acid 12a–l (Scheme 4).
4. Experimental
2.3. Biological evaluation
6-Aminopenicillanoic acid (6-APA) was purchased from
Sigma Aldrich. Anhydrous solvents and other reagents
were purchased and used without further purification.
The synthesized 6-triazolylpenicillanic acids were
screened at the National Institute of Allergy and Infec-
N
N
N
N
R2
R2
N3
N
N
S
S
S
a
b
N
N
O
N
O
O
CO2R1
CO2H
CO2R1
9a, R2 = Bz
8a,
R1 = PMB, R2 = Bz
8b, R1 = BzD, R2 = Bz
8c,
5b, R1 = PMB
5c, R1 = BzD
9b,
R2 = 2,6-DMP
R1 = BzD, R2 = 2,6-DMP
Scheme 3. Cu(I)-catalyzed cycloaddition reaction between 6-azidopenicillanates and representative terminal alkynes. Reagents and conditions: (a)
CuI, Hunig’s base, alkynes 6 or 7, THF, rt; (b) TFA, anisole, CH2Cl2, À5 ꢁC.

7292
P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
N
N
N
N
N
N
R2
R2
N3
O
S
S
S
a
b
N
N
N
O
O
CO2X
OR1
O
OR1
R1 = PMB
O
12a: R2 = 4-Py, X = H
12b: R2 = 3-Py, X = H
5b,
5c, R1 = BzD
11a: R1 = BzD, R2 = 4-Py
11b: R1 = BzD, R2 = 3-Py
12c:
R
2 = 2-Py, X = H
12d: R2 = Tp, X = H
12e:
11c:
R1 = BzD, R2 = 2-Py
11d: R1 = BzD, R2 = Tp
11e:
R
2 = Ph, X = H
R1 = PMB, R2 = Ph
12f: R2 = BzOH, X = H
12g: R2 = 4-Bp, X = H
11f: R1 = BzD, R2 = BzOH
11g: R1 = BzD, R2 = 4-Bp
12h:
R2 = 3-Bp, X= H
12i: R2 = 2-Bp, X = H
12j:
11h:
R1 = BzD, R2 = 3-Bp
11i: R1 = BzD, R2 = 2-Bp
11j:
R2 = MetNap, X=H
R1 = BzD, R2 = MetNap
11k: R1 = BzD, R2 = 4-PyP
11l: R1 = BzD, R2 = 4-PyBzOH
12k: R2 = 4-PyP, X = H
12l: R2 = 4-PyBzOH, X = Na
Scheme 4. Scope of Cu(I)-catalyzed cycloaddition between 6-azidopenicillanate and terminal alkynes. Reagents and conditions: (a) CuI, Hunig’s
base, alkynes 10, THF, rt; (b) TFA, anisole CH2Cl2, À5 ꢁC.
Analtech silica gel plates (60 F254) were used for analyt-
ical TLC, and UV light was used to examine the spots.
Two hundred to four hundred mesh silica gel was used
in column chromatography. NMR spectra were re-
corded on a Varian-Gemini 400 magnetic resonance
4.1.2. 4-(4-Ethynylphenyl)pyridine (10k). Reaction of 4-
(4-formylphenyl)pyridine (0.3 g, 1.64 mmol) and Best-
mann-Ohira reagent (0.627 g, 3.27 mmol) within 24 h
as described for the synthesis of compound 7 followed
by flash chromatography (silica gel, 2:1 hexane/EtOAc)
1
1
spectrometer. H NMR spectra are recorded in parts
gave 261 mg (89%) of 10 k as a white solid. H NMR
per million (ppm) relative to the peak of CDCl3,
(7.24 ppm), acetone-d6 (2.09 ppm), or DMSO-d6
(2.49 ppm). 13C spectra were recorded relative to the
central peak of the CDCl3 triplet (77.0 ppm), acetone-
d6 (205.8 ppm), or the DMSO-d6 septet (39.7 ppm),
and were recorded with complete hetero-decoupling.
High resolution mass spectra were recorded at the Geor-
gia Institute of Technology mass spectrometry facility in
Atlanta. Triflyl azide was prepared as described before
and used without storage.31h,i The Bestmann-Ohira re-
agent was prepared as described by Ghosh et al.42 while
diphenyldiazomethane was prepared by using the proce-
dure described by Ko and Kim.37 6-Aminopenicillanates
3a–c were synthesized adapting literature proce-
dures.21,33–37
(CDCl3, 400 MHz) d 3.16 (1H, s), 7.47 (2H, d,
J = 6.4 Hz), 7.59 (4H, s), 8.66 (2H, d, J = 6.4 Hz).
HRMS (EI) calcd for [C13H9N] 179.0735, found
179.0746.
4.1.3. 2-Ethnylbiphenyl (10i). The required biphenyl-2-
carbaldehyde was synthesized from the corresponding
carboxylic acid through NaBH4/BF3:OEt2 reduction to
the primary alcohol.43 Subsequent PDC oxidation of
the primary alcohol furnished biphenyl-2-carbaldehyde.
Reaction of biphenyl-2-carbaldehyde (0.66 g, 3.63 mmol)
and Bestmann-Ohira reagent (1.72 g, 7.27 mmol) within
24 h followed by flash chromatography (silica gel, 5:1
hexane/EtOAc) yielded 523 mg (81%) of 10i as a colorless
oil. 1H NMR (CDCl3, 400 MHz) d 3.03 (1H, s), 7.28–7.32
(1H, m), 7.36–7.45 (5H, m), 7.57–7.63 (3H, m).
4.1. Representative procedure for the alkyne transforma-
tion reaction
4.1.4. 3-Ethynylbiphenyl (10h). The required biphenyl-3-
carbaldehyde was synthesized from the corresponding
carboxylic acid through NaBH4/BF3:OEt2 reduction to
the primary alcohol.43 Subsequent PDC oxidation of
the primary alcohol furnished biphenyl-3-carbaldehyde.
4.1.1. 2-Ethynyl-1,3-dimethoxybenzene (7). 2,6-Dimeth-
oxybenzaldehyde (0.45 g, 2.71 mmol) was first dissolved
in anhydrous MeOH (25 mL) and stirred under argon at
room temperature. Anhydrous K2CO3 (1.12 g,
8.12 mmol) and Bestmann-Ohira reagent (1.03 g,
5.42 mmol) were added to the reaction mixture and stir-
ring continued for 24 h at room temperature. Solvent
was evaporated off, and the remaining residue was dis-
solved in CH2Cl2 (25 mL) and washed with saturated
NH4Cl (3 · 20 mL). The organic layer was dried over
Na2SO4 and concentrated in vacuo. The crude product
was purified by flash chromatography (silica gel, 5:1
hexane/EtOAc) to give 220 mg (50%) of 7 as a white so-
Reaction
of
biphenyl-3-carbaldehyde
(0.87 g,
4.78 mmol) and Bestmann-Ohira reagent (1.72 g,
9.56 mmol) within 24 h followed by flash chromatogra-
phy (silica gel, 5:1 hexane/EtOAc) gave 850 mg (100%)
1
of 10h as a reddish oil. H NMR (CDCl3, 400 MHz) d
3.03 (1H, s), 7.33–7.47 (5H, m), 7.55–7.57 (3H, m),
7.72 (1H, s). HRMS(EI) calcd for [C14H10] 178.0782,
found 178.0797.
4.2. Representative procedure for the alkyne transforma-
tion via Grignard reaction
1
lid. H NMR (CDCl3, 400 MHz) d 3.55 (1H, s), 3.88
(6H, s), 6.53 (2H, d, J = 8.4 Hz), 7.24 (1H, t, 7.4 Hz);
13C NMR (CDCl3, 100 MHz) d 56.0, 76.2, 85.7, 100.0,
103.5, 130.3, 162.0.
4.2.1. 1-Phenylprop-2-yn-1-ol (10f). To a solution of
benzaldehyde (0.5 g, 4.71 mmol) in anhydrous THF

P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
7293
Table 1. Minimal inhibitory concentration (MIC) of 6-triazolylpenicillanic acids in lg/mL: each MIC value was obtained from an average of three
independent experiments
N
N
R2
N
S
N
O
CO2X
X = H, Na
Compound
R2
S. aureus ATCC
29213 (lg/mL)
Escherichia coli ATCC
25922 (lg/mL)
S. pneumoniae ATCC
49619 (lg/mL)
B. anthracis AMES
(lg/mL)
9a
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
4
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
>8
O
9b
>8
>8
>8
>8
8
O
12a
12b
12c
12d
12e
12f
12g
12h
12i
N
N
N
S
4
OH
>8
P8b
>8
>8
>8
>8
O
12j
12k
>8
N
(continued on next page)

7294
P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
Table 1 (continued)
Compound
R2
S. aureus ATCC
29213 (lg/mL)
Escherichia coli ATCC
25922 (lg/mL)
S. pneumoniae ATCC
49619 (lg/mL)
B. anthracis AMES
(lg/mL)
OH
12la
>8
>8
P8b
P8b
N
H
N
S
1
>2
NDc
60.125
O
O
N
CO2H
Penicillin G
O
O
N
N
NDc
1
NH
H
O
2
4
N
S
O
O
N
CO2H
Piperacillin
a X = Na.
b Denotes that one of the 3 replicates had MIC value of 8 lg/mL.
c No data.
(2 mL) was added ethynylmagnesium bromide (14.0 mL,
0.5 M in THF) at room temperature under argon. The
reaction mixture was stirred for 1 h and quenched with
distilled water. The reaction mixture was partitioned be-
tween CH2Cl2 (10 mL) and water (15 mL), and the two
layers separated. The organic layer was washed in suc-
cession with distilled water (2 · 10 mL) and saturated
brine (2 · 10 mL), and dried over Na2SO4. Solvent was
evaporated off to give 0.626 g (100%) of 10f as a brown-
ish oil. 1H NMR (CDCl3, 400 MHz) d 2.66 (1H, d,
J = 2.4 Hz), 5.45 (1H, d, J = 4.0 Hz), 7.30–7.40 (3H,
m), 7.54 (2H, d, J = 7.2 Hz); HRMS (FAB, thioglycerol)
calcd for [C9H8O] 132.0575, found 132.0566.
9.5 mmol) was suspended in EtOAc (40 mL) and washed
with saturated NaHCO3 (2 · 30 mL). The organic layer
was dried over Na2SO4 and concentrated in vacuo to
give pure 4-nitrobenzyl 6-aminopenicillanate. To a solu-
tion of 4-nitrobenzyl 6-aminopenicillanate in anhydrous
CH2Cl2 (10 mL) were added triflyl azide solution
(25 mmol) in CH2Cl2 (25 mL), and Et3N (2.1 mL,
15.0 mmol). The reaction mixture was stirred at room
temperature for 2 h. Solvent was evaporated off and
the residue was directly purified by flash chromatogra-
phy (silica, gradient 4:1; 3:1 hexane/EtOAc) to give
1
2.35 g (68%) of 5a as a colorless gel. H NMR (CDCl3,
400 MHz) d 1.38 (3H, s), 1.61 (3H, s), 4.49 (1H, s), 4.92
(1H, d, J = 4.0 Hz), 5.23 (2H, q, J = 24.0, 12.0 Hz), 5.43
(1H, d, J = 4.0 Hz), 7.50 (2H, d, J = 8.8 Hz), 8.17 (2H,
d, J = 8.8 Hz); 13C NMR (CDCl3, 100 MHz) d 26.4,
31.5, 64.3, 65.7, 66.5, 67.6, 70.0, 123.7, 128.0, 141.5,
147.9, 167.0, 169.8.
4.2.2. 1-4-(pyrdin-4-yl)prop-2-yn-1-ol (10l). To a solution
of benzaldehyde (0.5 g, 2.73 mmol) in anhydrous THF
(2 mL) was added ethynylmagnesium bromide (8.2 mL,
0.5 M in THF) at room temperature under argon. The
reaction mixture was stirred for 1 h and quenched with
distilled water. The reaction mixture was partitioned be-
tween CH2Cl2 (10 mL) and water (15 mL), and the two
layers separated. The organic layer was washed in suc-
cession with distilled water (2 · 10 mL) and saturated
brine (2 · 10 mL), and dried over Na2SO4. Solvent was
evaporated off to give 0.491 (86%) of 10l as a brownish
solid. 1H NMR (CD3OD, 400 MHz) d 3.05 (1H, s), 5.46
(1H, s), 7.66–7.78 (6H, m), 8.56 (2H, d, J = 9.2 Hz);
HRMS (FAB, thioglycerol) calcd for [C14H11NO+H]+
210.0918, found 210.0909.
4.3.2. Method B: benzhydryl 6-azidopenicillanate (5c).
Benzhydryl 6-aminopenicillanate salt 3c (3.5 g,
6.32 mmol) was suspended in EtOAc (60 mL) and
washed with saturated NaHCO3 (3 · 40 mL). The or-
ganic layer was dried over Na2SO4 and concentrated
in vacuo to give 2.4 g of pure benzhydryl 6-aminopenic-
illanate. To a solution of benzhydryl 6-aminopenicilla-
nate in anhydrous CH2Cl2 (15 mL) were added triflyl
azide solution (14 mmol) in CH2Cl2 (35 mL), DMAP
(1.15 g, 9.24 mmol), and Et3N (0.1 mL). The reaction
mixture was stirred at room temperature for 1.5 h and
solvent was evaporated off. The residue was directly
purified by flash chromatography (silica, gradient 1:0;
5:1 hexane/EtOAc) to give 1.38 g (54%) of 5c as a color-
less gel. 1H NMR (CDCl3, 400 MHz) d 1.26 (3H, s), 1.64
4.3. Representative procedures for diazo-transfer reaction
4.3.1. Method A: p-nitrobenzyl 6-azidopenicillanate (5a).
4-Nitrobenzyl 6-aminopenicillanate salt 3a (5.0 g,

P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
7295
(3H, s), 4.57 (1H, s), 4.89 (1H, d, J = 4.0 Hz), 5.47 (1H,
d, J = 4.0 Hz), 6.93 (1H, s), 7.28–7.35 (10H, m); 13C
NMR (CDCl3, 100 MHz) d 26.2, 31.9, 64.7, 66.6, 67.8,
70.2, 78.4, 126.8, 127.5, 128.1, 128.4, 128.5, 138.8,
166.5, 169.8; HRMS (FAB, mnba) calcd for
[C21H20N4O3S+H]+ 409.1334, found 409.1357.
400 MHz) d 1.35 (3H, s), 1.57 (3H, s), 3.79 (3H, s),
4.08 (2H, s), 4.47 (1H, s), 5.12 (2H, app. q, J = 24.0,
12.0 Hz), 5.69 (1H, d, J = 4.0 Hz), 6.27 (1H, d,
J = 4.4 Hz), 6.88 (2H, d, J = 8.8 Hz), 7.20–7.30 (7H,
m), 7.42 (1H, s); 13C NMR (CDCl3, 100 MHz) d 26.9,
30.9, 32.0, 55.2, 65.3, 66.2, 67.3, 67.5, 70.5, 114.0,
122.4, 126.5, 128.5, 128.6, 130.6, 138.5, 147.2, 160.0,
167.1, 168.1; HRMS (FAB, thioglycerol) calcd for
[C25H26N4O4S+H]+ 479.1753, found 479.1756.
4.3.3. Method C: p-nitrobenzyl 6-azidopenicillanate (5a).
4-Nitrobenzyl 6-aminopenicillanate salt 3a (0.75 g,
1.4 mmol) was suspended in EtOAc (30 mL) and washed
with saturated NaHCO3 (2 · 20 mL). The organic layer
was dried over Na2SO4 and concentrated in vacuo to
give pure 4-nitrobenzyl 6-aminopenicillanate. To a solu-
tion of pure 4-nitrobenzyl 6-aminopenicillanate in anhy-
drous toluene(5 mL) were added triflyl azide solution
(2.97 mmol) in toluene (25 mL), and Et3N (0.3 mL,
2.1 mmol). The reaction mixture was stirred at room
temperature for 2 h. The reaction was diluted with
EtOAc (10 mL) and washed with 1 N HCl (20 mL), dis-
tilled water (2 · 20 mL), and saturated brine (20 mL).
The organic layer was dried over Na2SO4 and concen-
trated in vacuo to give 361 mg (70%) of 5a as a yellowish
4.4.2. Benzhydryl benzyl-6-triazolylpenicillanate (8b).
Reaction of benzhydryl 6-azidopenicillanate 5c
(0.270 g, 0.661 mmol) and 3-phenyl-1-propyne
6
(0.17 mL, 1.39 mmol) within 2.5 h followed by flash
chromatography (silica, gradient 3:1; 2:1; 3:2 hexane/
EtOAc) gave 246 mg (71%) of 8b as a white solid. H
1
NMR (CDCl3, 400 MHz) d 1.61 (3H, s), 1.57 (3H, s),
4.09 (2H, s), 4.59 (1H, s), 5.73 (1H, d, J = 4.0 Hz),
6.29 (1H, d, J = 4.4 Hz), 6.94 (1H, s), 7.21–7.35 (15H,
m), 7.43 (1H, s); 13C NMR (CDCl3, 100 MHz) d 26.7,
31.2, 32.0, 65.4, 66.3, 67.4, 70.6, 78.6, 122.4,
126.5,126.9, 127.6, 128.3, 128.5, 128.6, 128.7, 138.5,
138.7, 138.8, 147.3, 166.3, 168.1; HRMS (FAB, mnba)
calcd for [C30H28N4O3S+H]+ 525.1956, found 525.1960.
1
solid. H NMR (CDCl3, 400 MHz) d 1.42 (3H, s), 1.66
(3H, s), 4.52 (1H, s), 4.92 (1H, d, J = 4.0 Hz), 5.26
(2H, q, J = 24.0, 12.0 Hz), 5.46 (1H, d, J = 4.0 Hz),
7.53 (2H, d, J = 8.8 Hz), 8.24 (2H, d, J = 8.8 Hz).
4.4.3. Benzhydryl 2,6-dimethoxyphenyl-6-triazolylpenic-
illanate (8c). Reaction of benzhydryl 6-azidopenicilla-
nate 5c (0.227 g, 0.555 mmol) and 2-ethynyl-1,3-
dimethoxybenzene 7 (0.06 g, 0.37 mmol) within 4 h fol-
lowed by flash chromatography (silica gel, 1:1 hexane/
4.3.4. p-Methoxybenzyl 6-azidopenicillanate (5b). 6-Azid-
openicillanate 5b was prepared from p-methoxybenzyl 6-
aminopenicillanate 3b (4.0 g, 7.9 mmol) and triflyl azide
(25 mmol) in CH2Cl2 (total volume = 25 mL), and Et3N
(1.44 mL, 10.3 mmol) using Method A. The reaction
mixture was stirred at room temperature for 2 h. Purifi-
cation of the crude product by flash chromatography
(silica, gradient 4:1; 3:1 hexane/EtOAc) afforded 1.40 g
(49%) of 5b as a colorless gel. 1H NMR (CDCl3,
100 MHz) d 1.36 (3H, s), 1.61 (3H, s), 3.79 (3H, s),
4.45 (1H, s), 4.88 (1H, d, J = 4.0 Hz), 5.10 (2H, app.
q, J = 24.0, 12.0 Hz), 5.44 (1H, d, J = 4.0 Hz), 6.87
(2H, d, J = 8.8 Hz), 7.27 (2H, d, J = 8.8 Hz); 13C
NMR (CDCl3, 400 MHz) d 26.6, 31.7, 55.3, 64.6, 66.5,
67.4, 67.7, 70.2, 113.9, 126.5, 130.4, 159.7, 167.2,
1
EtOAc) gave 120 mg (57%) of 8c as a white solid. H
NMR (CDCl3, 400 MHz) d 1.26 (3H, s), 1.69 (3H, s),
3.78 (6H, s), 4.64 (1H, s), 5.80 (1H, d, J = 4.8 Hz),
6.42 (1H, d, J = 4.4 Hz), 6.62 (2H, d, J = 8.4 Hz), 6.96
(1H, s), 7.26–7.35 (11H, m), 7.95 (1H, s); 13C NMR
(CDCl3, 100 MHz) d 26.7, 31.4, 56.0, 65.2, 66.4,
67.7, 70.6, 78.6, 104.1, 125.2, 126.9, 127.6,
128.3, 128.5, 128.6, 128.7, 129.9 138.9, 139.9, 158.3,
166.4, 168.6; HRMS (FAB, thioglycerol) calcd for
[C31H30N4O5S+H]+ 571.2015, found 571.2049.
4.4.4. Benzhydryl 4-pyridyl-6-triazolylpenicillanate (11a).
4-Ethynylpyridyl hydrochloride (0.06 g, 0.414 mmol)
was suspended in EtOAc (30 mL) and washed with sat-
urated NaHCO3 (1 · 30 mL). The organic layer was
dried over Na2SO4 and concentrated in vacuo to give
4-ethynylpyridine 10a as a white brown solid. The reac-
tion of benzhydryl 6-azidopenicillanate 5c (0.15 g,
0.367 mmol) and 10a (0.076 g, 0.734 mmol) within 3 h
followed by flash chromatography (silica gel, gradient
1:1; 1:4 hexane/EtOAc) gave 166 mg (74%) of 11a as a
169,7;
HRMS
(FAB,
mnba)
calcd
for
[C16H18N4O4S+H]+ 363.1127, found 363.1151.
4.4. Representative procedure for Cu(I)-catalyzed cyclo-
addition reaction
4.4.1. p-Methoxybenzyl benzyl-6-triazolylpenicillanate
(8a). p-Methoxybenzyl 6-azidopenicillanate 5b (0.27 g,
0.635 mmol) and 3-phenyl-1-propyne
6
(0.17 mL,
1
1.39 mmol) were dissolved in anhydrous THF (8 mL)
and stirred under argon at room temperature. Copper
(I) iodide (0.011 g, 0.06 mmol) and Hunig’s base
(0.1 mL) were then added to the reaction mixture, and
stirring was continued for 2 h. The reaction mixture
was diluted with CH2Cl2 (40 mL) and washed with 1:4
NH4OH/saturated NH4Cl (3 · 30 mL) and again with
saturated NH4Cl (30 mL). The organic layer was dried
over Na2SO4 and concentrated in vacuo. The crude
product was purified by flash chromatography (silica,
gradient 3:1; 2:1; 3:2 hexane/EtOAc) to give 246 mg
(71%) of 8a as a white solid. 1H NMR (CDCl3,
white solid. H NMR (CDCl3, 400 MHz) d 1.26 (3H,
s), 1.69 (3H, s), 4.65 (1H, s), 5.80 (1H, d, J = 4.0 Hz),
6.39 (1H, d, J = 4.0 Hz), 6.96 (1H, s), 7.32–7.36 (10H,
m), 7.77 (2H, d, J = 5.20 Hz), 8.19 (1H, s), 9.03(2H, d,
J = 5.20 Hz); 13C NMR (CDCl3, 100 MHz) d 26.8,
31.1, 65.7, 66.2, 67.2, 70.8, 78.8, 120.1, 122.1, 126.7,
127.5, 128.2, 128.4, 128.5, 138.6, 138.6, 144.5, 149.2,
165.9, 167.5; HRMS (FAB, thioglycerol) calcd for
[C28H25N5O3S+H]+ 512.1756, found 512.1763.
4.4.5. Benzhydryl 3-pyridyl-6-triazolylpenicillanate (11b).
The reaction of benzhydryl 6-azidopenicillanate 5c

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P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
(0.15 g, 0.441 mmol) and 3-ethynylpyridine 10b (0.068 g,
0.661 mmol) within 3 h followed by flash chromatogra-
phy (silica gel, gradient 1:1; 1:3 hexane/EtOAc) gave
170 mg (76%) of 11b as a white solid. 1H NMR (CDCl3,
400 MHz) d 1.26 (3H, s), 1.70 (3H, s), 4.66 (1H, s), 5.80
(1H, d, J = 4.0 Hz), 6.40 (1H, d, J = 4.0 Hz), 6.96 (1H,
s), 7.32–7.36 (10H, m), 8.13 (1H, s), 8.26 (1H, d,
J = 8.24 Hz), 8.58 (1H, br s), 9.03(1H, br s); 13C NMR
(CDCl3, 100 MHz) d 26.9, 31.1, 65.7, 66.3, 67.3,
70.7, 78.7, 120.8, 126.7, 127.5, 128,2, 128,4, 128,5,
133.8, 138.5, 138.6, 138.6, 144.0, 146.1, 148.3, 166.0,
167.6; HRMS (FAB, thioglycerol) calcd for
[C28H25N5O3S+H]+ 512.1756, found 512.1773.
4.4.9. Benzhydryl a-hydroxylbenzyl-6-triazolylpenicilla-
nate (11f). Reaction of benzhydryl 6-azidopenicillanate
5c (0.2 g, 0.49 mmol) and 1-phenylprop-2-yn-1-ol 10f
(0.13 g, 0.985 mmol) within 2 h followed by flash chro-
matography (silica gel, 2:1 hexane/EtOAc) gave
191 mg (72%) of 11f as a white solid. 1H NMR (CDCl3,
400 MHz) d 1.23 (3H, d, J = 12 Hz), 1.59 (3H, d,
J = 8.0 Hz), 4.61 (1H, s), 5.71 (1H, d, J = 4.4 Hz), 5.99
(1H, d, J = 5.2 Hz), 6.23 (1H, dd, J = 4.0, 1.6 Hz), 6.95
(1H, s), 7.24–7.42 (15H, m), 7.57 (1H, s). 13C NMR
(CDCl3, 100 MHz) d 26.6, 31.2, 31.3, 65.3, 65.3, 66.3,
66.4, 67.3, 67.4, 68.7, 68.9, 70.4, 70.5, 78.5, 122.0,
122.1, 126.1, 126.2, 126.3, 126.4, 126.6, 126.7, 127.1,
127.3, 127.6, 127.7, 128.0, 128.2, 128.3, 128.4, 138.5,
138.6, 141.5, 150.9, 166.0, 167.4, 167.5; HRMS (FAB,
thioglycerol) calcd for [C30H28N4O4S+H]+ 541.1909,
found 541.1936.
4.4.6. Benzhydryl 2-pyridyl-6-triazolylpenicillanate (11c).
The reaction of benzhyldryl 6-azidopenicillanate 5c
(0.15 g, 0.367 mmol) and 2-ethynylpyridine 10c
(0.06 mL, 0.55 mmol) within 6 h followed by flash
chromatography (silica gel, gradient 4:1; 2:1; 1:2; hex-
ane/EtOAc) gave 145 mg (64%) of 11c as a white so-
lid. 1H NMR (CDCl3, 400 MHz) d 1.25 (3H, s),
1.71 (3H, s), 4.64 (1H, s), 5.78 (1H, d, J = 4.0 Hz),
6.39 (1H, d, J = 4.0 Hz), 6.96 (1H, s), 7.32–7.36
(10H, m), 7.78 (1H, m), 8.18 (1H, d, J = 7.69 Hz),
8.45 (1H, s), 8.58 (1H, d, J = 4.39 Hz); 13C NMR
(CDCl3, 100 MHz) d 26.9, 31.3, 65.7, 66.5, 67.4,
70.8, 78.7, 120.5, 123.0, 126.8, 127.5, 128.2, 128.4,
128.5, 128.6, 138.6, 138.7, 166.1, 167.8; HRMS
(FAB, thioglycerol) calcd for [C28H25N5O3S+H]+
512.1756, found 512.1765.
4.4.10. Benzhydryl 4-biphenyl-6-triazolylpenicillanate
(11g). Reaction of benzhydryl 6-azidopenicillanate 5c
(0.15 g, 0.367 mmol) and 4-ethynylbiphenyl 10g
(0.098 g, 0.55 mmol) within 5 h followed by flash chro-
matography (silica gel, gradient 1:0; 5:1; 3:1 hexane/
1
EtOAc) gave 158 mg (73%) of 11g as a white solid. H
NMR (DMSO-d6, 400 MHz) d 1.26 (3H, s), 1.69 (3H,
s), 4.92 (1H, s), 5.88 (1H, d, J = 2.0 Hz), 6.81 (1H, d,
J = 2.0 Hz), 6.96 (1H, s), 7.32 (2H, d, J = 8.0 Hz), 7.38
(5H, t, J = 7.2 Hz), 7.47 (5H, t, J = 6.8 Hz), 7.52 (2H,
d, J = 8.0 Hz), 7.77 (2H, d, J = 8.0 Hz), 7.90 (2H, d,
J = 8.0 Hz), 8.66 (1H, s).
4.4.7. Benzhydryl 2-thiopyl-6-triazolylpenicillanate (11d).
Reaction of benzhydryl 6-azidopenicillanate 5c (0.15 g,
0.367 mmol) and 3-ethynylthiopene 10d (0.06 g,
0.55 mmol) within 5 h followed by flash chromatogra-
phy (silica gel, gradient 1:0; 5:1; 3:1 hexane/EtOAc) gave
150 mg (79%) of 11d as a white solid. 1H NMR (CDCl3,
400 MHz) d 1.25 (3H, s), 1.69 (3H, s), 4.64 (1H, s), 5.78
(1H, d, J = 4.4 Hz), 6.36 (1H, d, J = 4.0 Hz), 6.96 (1H,
s), 7.30–7.38 (11H, m), 7.44 (1H, dd, J = 4.0, 1.2 Hz),
7.70 (1H, dd, J = 2.0, 1.2 Hz), 7.90 (1H, s); 13C NMR
(CDCl3, 100 MHz) d 26.7, 30.9, 66.1, 67.3, 70.8, 78.7,
120.1, 121.6, 125.7, 126.4, 126.9, 127.6, 128.3, 128.6,
128.7, 131.1, 138.7, 138.8, 143.8, 166.3, 168.2; HRMS
(FAB, thioglycerol) calcd for [C27H24N4O3S2+H]+
517.1368, found 517.1374.
4.4.11. Benzhydryl 3-biphenyl-6-triazolylpenicillanate
(11h). Reaction of benzhydryl 6-azidopenicillanate 5c
(0.15 g, 0.367 mmol) and 3-ethynylbiphenyl 10h
(0.098 g, 0.55 mmol) within 5 h followed by flash chro-
matography (silica gel, gradient 1:0; 5:1; 3:1 hexane/
EtOAc) gave 215 mg (100%) of 11h as a white solid.
1H NMR (CDCl3, 400 MHz) d 1.14 (3H, s), 1.58 (3H,
s), 4.54 (1H, s), 5.68 (1H, d, J = 4.4 Hz), 6.28 (1H, d,
J = 4.0 Hz), 6.86 (1H, s), 7.12 (1H, s), 7.19–7.24 (10H,
m), 7.31–7.39 (3H, m), 7.45 (1H, d, J = 8.0 Hz), 7.52
(2H, d, J = 8.0 Hz), 7.69 (2H, d, J = 8.0 Hz), 7.95 (1H,
s); 13C NMR (CDCl3, 100 MHz) d 26.7, 30.9, 65.5,
66.2, 67.3, 70.7, 78.7, 120.5, 124.6, 124.7, 126.9, 127.2,
127.5, 127.6, 128.3, 128.5, 128.6, 128.7, 129.3, 130.4,
138.7, 138.8, 140.6, 141.9, 147.5, 166.3, 168.2; HRMS
(FAB, thioglycerol) calcd for [C35H30N4O3S+H]+
587.2116, found 587.2143.
4.4.8. p-Methoxybenzyl phenyl-6-triazolylpenicillanate
(11e). Reaction of p-methoxybenzyl-6-azidopenicilla-
nate 5b (0.15 g, 0.414 mmol) and phenylacetylene 10e
(0.14 mL, 1.24 mmol) within 2 h followed by flash chro-
matography (silica gel, gradient 3:1; 3:2 hexane/EtOAc)
4.4.12. Benzhydryl 2-biphenyl-6-triazolylpenicillanate
(11i). Reaction of benzhydryl 6-azidopenicillanate 5c
(0.15 g, 0.367 mmol) and 2-ethynylbiphenyl 10i
(0.098 g, 0.55 mmol) within 5 h followed by flash chro-
matography (silica gel, gradient 1:0; 5:1; 3:1 hexane/
EtOAc) gave 150 mg (70%) of 11i as a white solid.
1H NMR (CDCl3, 400 MHz) d 1.20 (3H, s), 1.47
(3H, s), 4.47 (1H, s), 5.63 (1H, d, J = 4.0 Hz), 6.26
(1H, d, J = 4.4 Hz), 6.80 (1H, s), 6.93 (1H, s), 7.22–
7.45 (18H, m), 8.15 (1H, d, J = 8.0 Hz); 13C NMR
(CDCl3, 100 MHz) d 26.7, 31.3, 65.4, 66.3, 67.4,
70.4, 78.6, 123.0, 126.9, 127.2, 127.5, 127.8, 128.1,
128.2, 128.5, 128.6, 128.6, 128.7, 128.8, 129.2, 130.3,
138.8, 140.2, 141.5, 166.3, 167.0; HRMS (FAB, thio-
1
gave 165 mg (86%) of 11e as a white foam. H NMR
(CDCl3, 400 MHz) d 1.38 (3H, s), 1.66 (3H, s), 3.80
(3H, s), 4.54 (1H, s), 5.14 (2H, app. q, J = 24.0,
12.0 Hz), 5.75 (1H, d, J = 4.0 Hz), 6.37 (1H, d,
J = 4.0 Hz), 6.89 (2H, d, J = 8.8 Hz), 7.29–7.35 (3H,
m), 7.39–7.42 (2H, m), 7.82 (2H, m), 7.99 (1H, s); 13C
NMR (CDCl3, 100 MHz) d 27.0, 30.8, 55.2, 65.4, 66.1,
67.2, 67.6, 70.7, 114.0, 120.3, 125.8, 126.5, 128.4,
128.8, 129.9, 130.6, 147.5, 160.0, 167.1, 168.2; HRMS
(FAB, thioglycerol) calcd for [C24H24N4O4S+H]+
465.1596, found 465.1571.

P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
7297
glycerol) calcd for [C35H30N4O3S+H]+ 587.2116, found
587.2143.
CH2Cl2 (1 mL) at À5 ꢁC. Anhydrous anisole (0.2 mL,
1.83 mmol) and trifluoroacetic acid (0.5 mL, 6.49 mmol)
were added, and the reaction mixture stirred for 2 h. The
reaction mixture was diluted with cold Et2O (10 mL),
and the solvent was evaporated off in an ice bath. The
residue was concentrated in vacuo on an ice bath for
15 min and redissolved in THF (5 mL) and 1/2 saturated
NaHCO3 (15 mL) at 0 ꢁC. The resulting mixture was
stirred at 0 ꢁC for 15 min and partitioned between
deionized water (5 mL) and EtOAc (20 mL). The two
layers were separated and the aqueous layer was ex-
tracted with EtOAc (2 · 20 mL). The aqueous layer
was acidified to pH 3 in an ice bath with 1 N HCl and
extracted with EtOAc (3 · 20 mL). The combined or-
ganic layer was dried over Na2SO4 and concentrated
in vacuo to give 89 mg (100%) of 9a as a white solid.
1H NMR (acetone-d6, 400 MHz) d 1.55 (3H, s), 1.70
(3H, s), 4.07 (2H, s), 4.55 (1H, s), 5.85 (1H, d,
J = 4.4 Hz), 6.58 (1H, d, J = 4.4 Hz), 7.26–7.28 (5H,
m), 7.79 (1H, s); 13C NMR (acetone-d6, 100 MHz) d
26.8, 31.0, 31.8, 65.1, 67.0, 67.8, 70.7, 123.2, 126.7,
128.8, 128.9, 129.0, 139.9, 168.4, 168.5; HRMS (FAB,
mnba) calcd for [C17H18N4O3S+H]+ 359.1177, found
359.1177.
4.4.13. Benzhydryl 6-methoxynapthalyl-6-triazolylpenic-
illanate (11j). Reaction of benzhydryl 6-azidopenicilla-
nate 5c (0.15 g, 0.367 mmol) and 2-ethynyl-6-
methoxylnaphthalene (0.067 g, 0.37 mmol) within 5 h
followed by flash chromatography (silica gel, gradient
1:0; 5:1; 3:1 hexane/EtOAc) gave 175 mg (81%) of 11j
1
as a white solid. H NMR (CDCl3, 400 MHz) d 1.27
(3H, s), 1.72 (3H, s), 3.91 (3H, s), 4.67 (1H, s), 5.80
(1H, d, J = 4.4 Hz), 6.41 (1H, d, J = 4.4 Hz), 6.97 (1H,
s), 7.13–7.16 (2H, m), 7.31–7.35 (10H, m), 7.77 (2H,
dd, J = 6.4, 2.8 Hz), 7.88 (1H, dd, J = 6.4, 1.6 Hz),
8.08 (1H, s), 8.26 (1H, s); 13C NMR (CDCl3,
100 MHz) d 26.7, 31.0, 55.3, 65.5, 66.2, 67.3, 70.8,
78.7, 119.3, 120.2, 124.3, 124.6, 125.1, 126.9, 127.4,
127.6, 128.3, 128.6, 128.7, 128.9, 129.7, 134.4, 138.7,
138.8, 147.7, 158.0, 166.3, 168.2; HRMS (FAB, mnba)
calcd for [C34H30N4O4S+H]+ 591.2066, found 591.2061.
4.4.14. Benzhydryl 4-pyridylphenyl-6-triazolylpenicilla-
nate (11k). Reaction of benzhydryl 6-azidopenicillanate
5c (0.15 g, 0.367 mmol) and 4-(4-ethynylphenyl)pyridine
10k (0.098 g, 0.55 mmol) within 5 h followed by flash
chromatography (silica gel, gradient 1:1; 1:2; 1:4
hexane/EtOAc) gave 202 mg (94%) of 11 k as a white so-
4.5.2. 2,6-Dimethoxyphenyl-6-triazolylpenicillanic acid
(9b). The reaction of a CH2Cl2 solution of 8c with ani-
sole and TFA within 2 h, as described for the synthesis
of 9a from 8a, afforded 85 mg (100%) of 9b as a white
1
lid. H NMR (CDCl3, 400 MHz) d 1.27 (3H, s), 1.71
(3H, s), 4.66 (1H, s), 5.80 (1H, d, J = 4.0 Hz), 6.40
(1H, d, J = 4.0 Hz), 6.97 (1H, s), 7.31–7.35 (12H, m),
7.61 (2H, br s), 7.72 (2H, d, J = 8.0 Hz), 7.97 (2H,
d, J = 8.0 Hz), 8.09 (1H, s); 13C NMR (CDCl3,
100 MHz) d 26.7, 30.9, 65.6, 66.2, 67.3, 70.7, 78.7,
120.8, 126.5, 126.9, 127.5, 127.6, 128.3, 128.6,
128.7, 131.2, 137.3, 138.7, 138.8, 146.6, 148.8,
148.9166.3, 168.1; HRMS (FAB, thioglycerol) calcd
for [C34H29N5O3S+H]+ 588.2069, found 588.2100.
1
solid. H NMR (acetone-d6, 400 MHz) d 1.59 (3H, s),
1.77 (3H, s), 3.77 (6H, s), 4.62 (1H, s), 5.93 (1H, d,
J = 4.4 Hz), 6.73 (1H, d, J = 4.4 Hz), 6.74 (2H, d,
J = 8.4 Hz), 7.35 (1H, t, J = 8.4 Hz), 8.15 (1H, s); 13C
NMR (acetone-d6, 400 MHz) d 26.9, 31.2, 55.9, 65.2,
67.2, 68.0, 70.7, 104.6, 126.2, 131.0, 158.7, 168.6; HRMS
(FAB, mnba) calcd for [C18H20N4O5S+H]+ 405.1232,
found 405.1214.
4.4.15.
Benzhydryl
4-pyridylbenzylhydroxyl-6-tria-
4.5.3. 4-Pyridyl-6-triazolylpenicillanic acid (12a). The
reaction of a CH2Cl2 solution of 11a with anisole and
TFA within 0.5 h, as described for the synthesis of 9a
from 8a, afforded 80 mg (100%) of 12a as a white solid.
1H NMR (acetone-d6, 400 MHz) d 1.56 (3H, s), 1.73
(3H, s), 4.60 (1H, s), 5.95 (1H, d, J = 4.0 Hz), 6.78
(1H, d, J = 4.0 Hz), 8.57 (2H, br s), 9.01 (2H, br s),
9.07 (1H, s); 13C NMR (acetone-d6, 100 MHz) d 27.1,
31.6, 65.7, 67.7, 68.0, 71.4, 122.8, 126.8, 142.8, 143.6,
146.6, 167.5, 170.7; HRMS (FAB, mnba) calcd for
C15H15N5O3S+H]+ 346.0973, found 346.0972.
zolylpenicillanate (11l). Reaction of benzhydryl 6-azid-
openicillanate 5c (0.15 g, 0.367 mmol) and 1-4-(pyrdin-
4-yl)prop-2-yn-1-ol 10l (0.115 g, 0.55 mmol) within 2 h
followed by flash chromatography (silica gel, gradient
1:2; 1:3 hexane/EtOAc) gave 190 mg (84%) of 11l as a
1
white solid. H NMR (CDCl3, 400 MHz) d 1.22 (3H,
d, J = 12 Hz), 1.60 (3H, d, J = 5.6 Hz), 4.58 (1H, s),
5.72 (1H, d, J = 4.4 Hz), 6.08 (1H, s), 6.28 (1H, d,
J = 4.4 Hz), 6.92 (1H, s), 7.24–7.32 (10H, m), 7.44
(2H, dd, J = 6.0, 1.6 Hz) 7.52–7.58 (4H, m), 7.67 (1H,
s), 8.53 (2H, dd, J = 6.4, 1.6 Hz). 13C NMR (CDCl3,
100 MHz) d 26.7, 30.6, 31.1, 31.2, 64.3, 65.4, 66.2,
66.3, 67.3, 68.4, 68.5, 70.5, 78.6, 121.4, 122.0, 126.6,
126.9, 127.1, 127.4, 128.1, 128.3, 128.4, 128.5, 137.1,
137.2, 138.5, 142.9, 147.8, 149.6, 150.8, 150.9, 166.0,
167.5, 167.6; HRMS (FAB, thioglycerol) calcd for
[C35H31N5O4S+H]+ 618.2175, found 618.2180.
4.5.4. 3-Pyridyl-6-triazolylpenicillanic acid (12b). The
reaction of a CH2Cl2 solution of 11b with anisole and
TFA within 0.5 h, as described for the synthesis of 9a
from 8a, afforded 80 mg (100%) of 12b as a reddish
white solid. 1H NMR (acetone-d6, 400 MHz) d 1.57
(3H, s), 1.74 (3H, s), 4.60 (1H, s), 5.95 (1H, d,
J = 4.0 Hz), 6.76 (1H, d, J = 4.0 Hz), 8.15 (1H, br s),
8.84 (1H, s), 8.92 (2H, m), 9.41 (1H, br s); HRMS
(FAB, mnba) calcd for [C15H15N5O3S+H]+ 346.0973,
found 346.0972.
4.5. Representative procedure for deprotection of car-
boxyl protecting group
4.5.1. Benzyl-6-triazolylpenicillanic acid (9a). p-Meth-
oxybenzyl benzyl-6-triazolylpenicillanate 8a (0.13 g,
0.26 mmol) was dissolved and stirred in anhydrous
4.5.5. 2-Pyridyl-6-triazolylpenicillanic acid (12c). The
reaction of a CH2Cl2 solution of 11c with anisole and

7298
P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
TFA within 0.5 h, as described for the synthesis of 9a
from 8a, afforded 80 mg (100%) of 12c as a white solid.
1H NMR (acetone-d6, 400 MHz) d 1.57 (3H, s), 1.76
(3H, s), 4.63 (1H, s), 5.95 (1H, d, J = 4.0 Hz), 6.79
(1H, d, J = 4.0 Hz), 7.79 (1H, m), 8.39 (1H, m), 8.48
(1H, m), 8.90 (1H, d, J = 5.1 Hz), 8.95 (1H, s); 13C
NMR (acetone-d6, 100 MHz) d 27.1, 31.5, 65.7, 67.6,
68.0, 71.2, 122.8, 125.3, 125.7, 143.2, 145.9, 146.7,
160.2, 167.8, 168.6; HRMS (FAB, mnba) calcd for
[C15H15N5O3S+H]+ 346.0973, found 346.0974.
(3H, s), 4.61 (1H, s), 5.93 (1H, d, J = 4.0 Hz), 6.71
(1H, d, J = 4.0 Hz), 7.38 (1H, m), d 7.48 (2H, m), 7.54
(1H, t, J = 8.0 Hz), 7.64 (1H, m), 7.72 (2H, m), 7.96
(1H, dt, J = 8.0, 1.5 Hz), 8.23 (1H, t, 1.5 Hz), 8.58
(1H, s); 13C NMR (acetone-d6, 100 MHz) d 27.2, 31.2,
65.5, 67.3, 68.0, 71.3, 122.0, 124.6, 125.1, 127.2, 127.5,
128.1, 129.4, 130.0, 131.9, 141.1, 142.2, 147.2,
168.4, 168.9; HRMS (ESI, mnba) calcd for
[C22H20N4O3S+H]+ 421.1329, found 421.1312.
4.5.11. 2-Biphenyl-6-triazolylpenicillanic acid (12i). The
reaction of a CH2Cl2 solution of 11i with anisole and
TFA within 1 h, as described for the synthesis of 9a
from 8a, afforded 65 mg (100%) of 12i as a white solid.
H NMR (acetone-d6, 400 MHz) d 1.53 (3H, s), 1.59
(3H, s), 4.41 (1H, s), 5.78 (1H, d, J = 4.0 Hz), 6.55
(1H, d, J = 4.0 Hz), 7.00 (1H, s), 7.22–7.25 (2H, m),
7.32–7.52 (6H, m), 8.07 (1H, dd, J = 5.8, 1.5 Hz);
HRMS (ESI, mnba) calcd for [C22H20N4O3S + H}+
421.1329, found 421.1359.
4.5.6. 2-Thiopyl-6-triazolylpenicillanic acid (12d). The
reaction of a CH2Cl2 solution of 11d with anisole and
TFA within 1 h, as described for the synthesis of 9a
from 8a, afforded 81 mg (100%) of 12d as a yellowish so-
lid. 1H NMR (acetone-d6, 400 MHz) d 1.57 (3H, s), 1.74
(3H, s), 4.59 (1H, s), 5.90 (1H, d, J = 4.0 Hz), 6.65 (1H,
d, J = 4.0 Hz), 7.54 (1H, m), 7.57 (1H, dd, J = 3.0,
1.5 Hz), 7.87 (1H, dd, J = 3.0, 1.5 Hz), 8.34 (1H, s);
13C NMR (acetone-d6, 100 MHz) d 27.1, 31.3, 65.4,
67.3, 68.0, 71.2, 121.4, 121.6, 126.3, 127.1, 132.4,
143.7, 168.3, 168.9; HRMS (FAB, mnba) calcd for
[C14H14N4O3S2+H]+ 351.0585, found 351.0577.
4.5.12. 6-Methoxynaphthalyl-6-triazolylpenicillanic acid
(12j). The reaction of a CH2Cl2 solution of 11j with ani-
sole and TFA within 1 h, as described for the synthesis
of 9a from 8a, afforded 56mg (89%) of 12j as a white
4.5.7. Phenyl-6-triazolylpenicillanic acid (12e). The reac-
tion of a CH2Cl2 solution of 11e with anisole and TFA
within 2 h, as described for the synthesis of 9a from 8a,
afforded 68.5 mg (93%) of 12e as a white solid. 1H NMR
(acetone-d6, 400 MHz) d 1.47 (3H, s), 1.67 (3H, s), 4.54
(1H, s), 5.80 (1H, d, J = 4.0 Hz), 6.76 (1H, d,
J = 4.4 Hz), 7.34 (1H, m), 7.44 (2H, m), 7.89 (2H, m),
8.60 (1H, s); 13C NMR (acetone-d6, 100 MHz) d 27.2,
31.2, 65.4, 67.3, 68.0, 71.2, 121.6, 126.1, 126.1, 128.6,
129.4, 131.3, 147.3, 168.4; HRMS (FAB, mnba) calcd
for [C16H16N4O3S+H]+ 345.1021, found 345.1015.
1
foam. H NMR (DMSO-d6, 400 MHz) d 1.58 (3H, s),
1.78 (3H, s), 3.91 (3H, s), 4.17 (1H, s), 5.80 (1H, d,
J = 4.0 Hz), 6.62 (1H, d, J = 4.0 Hz), 7.16 (1H, dd,
J = 6.5, 2.2 Hz), 7.30 (1H, m), 7.85 (2H, dd, J = 3.0,
5.0 Hz), 7.98 (1H, m), 8.39 (1H, m), 8.55 (1H, s); 13C
NMR (acetone-d6, 100 MHz) d 26.7, 30.7, 55.1, 65.0,
67.0, 67.6, 70.8, 106.1, 119.5, 121.3, 124.4, 124.5,
126.2, 127.7, 129.3, 129.9, 134.9, 147.4, 158.4, 168.3,
168.4; HRMS (FAB, thioglycerol) calcd for
[C21H20N4O4S+H]+ 425.1283, found 425.1264.
4.5.8. a-Hydroxylbenzyyl-6-triazolylpenicillanic acid
(12f). The reaction of a CH2Cl2 solution of 11f with
thioanisole and TFA within 0.5 h, as described for the
synthesis of 9a from 8a, afforded 76 mg (100%) of 12f
as a pale yellowish solid. 1H NMR (acetone-d6,
400 MHz) d 1.53 (3H, s), 1.67 (3H, s), 3.37 (1H, s),
4.55 (1H, s), 5.84 (1H, br s), 5.97 (1H, s), 6.55 (1H, br
s), 7.22–7.43 (4H, m), 7.80 (1H, s); HRMS (FAB, thio-
glycerol) calcd for [C17H18N4O4S+H]+ 375.1127, found
375.1192.
4.5.13. 4-Pyridylphenyl-6-triazolylpenicillanic acid (12k).
The reaction of a CH2Cl2 solution of 11k with anisole
and TFA within 1 h, as described for the synthesis of
9a from 8a, afforded 37 mg (65%) of 12k as a white
1
foam. H NMR (acetone-d6, 400 MHz) d 1.58 (3H, s),
1.77 (3H, s), 4.61 (1H, s), 5.94 (1H, d, J = 4.0 Hz),
6.74 (1H, d, J = 4.0 Hz), 8.17 (4H, m), 8.47 (2H, br s),
8.64 (1H, s), 9.04 (2H, br s); HRMS (FAB, mnba) calcd
for [C21H19N5O3S+H]+ 422.1287, found 422.1301.
4.5.14.
4-Pyridylbenzylhydroxyl-6-triazolylpenicillanic
4.5.9. 4-Biphenyl-6-triazolylpenicillanic acid (12g). The
reaction of a CH2Cl2 solution of 11g with anisole and
TFA within 2 h, as described for the synthesis of 9a
from 8a, afforded 65 mg (100%) of 12g as a yellowish
acid salt (12l). Benzhydryl 4-pyridylphenyl-6-triazolylpe-
nicillanate 11l (0.09 g, 0.14 mmol) was dissolved and
stirred in anhydrous CH2Cl2 (1 mL) at À5 ꢁC. Anhy-
drous thioanisole (0.2 mL, 1.83 mmol) and TFA
(0.5 mL, 6.49 mmol) were added, and the reaction mix-
ture stirred for 0.5 h. The reaction mixture was diluted
with cold Et2O (10 mL), and the solvent was evaporated
off in an ice bath. The residue was concentrated in vacuo
on an ice bath for 15 min and redissolved in aqueous
NaHCO3 (0.018 g, 0.21 mmol) at 0 ꢁC. The resulting
mixture was stirred at 0 ꢁC for 15 min and partitioned
between deionized water (5 mL) and EtOAc (20 mL).
The two layers were separated and the aqueous layer
was extracted with EtOAc (2 · 20 mL). The aqueous
layer was frozen in acetone dry ice bath and concen-
trated in vacuo to give 35 mg (52%) of 12l as a white
1
solid. H NMR (DMSO-d6, 400 MHz) d 1.48 (3H, s),
1.65 (3H, s), 4.38 (1H, s), 5.79 (1H, d, J = 4.0 Hz),
6.72 (1H, d, J = 4.0 Hz), 7.35–7.40 (1H, m), 7.47 (2H,
t, J = 8.0 Hz), 7.74 (4H, dd, J = 11.2, 8.0 Hz), 7.99
(2H, d, J = 8.0 Hz), 8.63 (1H, s); HRMS (FAB, mnba)
calcd for [C22H20N4O3S+H]+ 421.1334, found 421.1333.
4.5.10. 3-Biphenyl-6-triazolylpenicillanic acid (12h). The
reaction of a CH2Cl2 solution of 11h with anisole and
TFA within 2 h, as described for the synthesis of 9a
from 8a, afforded 54 mg (83%) of 12h as a white solid.
1H NMR (acetone-d6, 400 MHz) d 1.59 (3H, s), 1.77

P. C. Chen et al. / Bioorg. Med. Chem. 15 (2007) 7288–7300
7299
1
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solid. H NMR (DMSO-d6, 400 MHz) d 1.46 (3H, s),
1.61 (3H, m), 4.51 (1H, s), 5.75 (1H, d, J = 4.4 Hz),
5.94 (1H, s), 6.67 (1H, d, J = 4.0 Hz), 7.57 (2H, d,
J = 8.0 Hz), 7.86 (4H, m), 8.75 (2H, m); HRMS (FAB,
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474.1234.
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Acknowledgments
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We thank Professor James Power and Josh Canzoneri
for a helpful discussion on the manuscript. This work
was financially supported by Georgia Institute of Tech-
nology. Compound screening was supported, in part, by
the National Institute of Allergy and Infectious Dis-
eases, under contract number N01-AI-30061. C.P.C. is
a recipient of a GAANN predoctoral fellowship from
the Gatech Center for Drug Design, Development and
Delivery. P.A.P. is a Gatech PURA fellow.
24. (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew.
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ChemBioChem 2005, 6, 1167–1169; (e) Whiting, M.;
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Supplementary data
Proton and carbon NMR spectra for all compounds de-
scribed in Section 4. This material is available free of
charge via the Internet at http://pubs.acs.org. Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2007.08.035.
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