Synthesis and antibacterial evaluation against resistant Gram-negative bacteria of monobactams bearing various substituents on oxime residue

Article abstract of DOI:10.1016/j.bioorg.2019.103487

Based on the structural characteristics of aztreonam (AZN) and its target PBP3, a series of new monobactam derivatives bearing various substituents on oxime residue were prepared and evaluated for their antibacterial activities against susceptible and res

Full text of DOI:10.1016/j.bioorg.2019.103487

Journal Pre-proofs
Synthesis and antibacterial evaluation against resistant Gram-negative bacte-
ria of monobactams bearing various substituents on oxime residue
Zhi–Wen Li, Xi Lu, Yan–Xiang Wang, Xin–Xin Hu, Hai–Gen Fu, Li–Mei
Gao, Xue–Fu You, Sheng Tang, Dan–Qing Song
PII:
S0045-2068(19)30881-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.103487
YBIOO 103487
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
30 May 2019
30 September 2019
27 November 2019
Please cite this article as: Z. Li, X. Lu, Y. Wang, X. Hu, H. Fu, L. Gao, X. You, S. Tang, D. Song, Synthesis and
antibacterial evaluation against resistant Gram-negative bacteria of monobactams bearing various substituents on
oxime residue, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103487
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Synthesis and antibacterial evaluation against resistant
Gram-negative bacteria of monobactams bearing various
substituents on oxime residue
Zhi–Wen Li†, Xi Lu†, Yan–Xiang Wang, Xin–Xin Hu, Hai–Gen Fu, Li–Mei Gao,
Xue–Fu You, Sheng Tang**, Dan–Qing Song*
Beijing Key Laboratory of Antimicrobial Agents, Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China
† These authors made equal contributions to the work.
** Corresponding authors.
*Corresponding authors.
E-mail address: tangsheng@imb.pumc.edu.cn (Sheng Tang); songdanqing@imb.
pumc.edu.cn (Danqing Song).
1

Abstract
Based on the structural characteristics of aztreonam (AZN) and its target PBP3,
a series of new monobactam derivatives bearing various substituents on oxime residue
were prepared and evaluated for their antibacterial activities against susceptible and
resistant Gram-negative bacteria. Among them, compounds 8p and 8r displayed
moderate potency with MIC values of 0.125−32 μg/mL against most tested
Gram-negative strains, comparable to AZN. Meanwhile, the combination of 8p and 8r
with avibactam as a β-lactamases inhibitor, in a ratio of 1:16, showed a promising
synergistic effect against both ESBLs- and NDM-1-producing K. pneumoniae, with
significantly reduced MIC values up to 8-fold and >256-fold respectively.
Furthermore, both of them demonstrated excellent safety profiles both in vitro and in
vivo. The results provided powerful information for further structure optimization of
monobactam antibiotics to fight β-lactamase-producing resistant Gram-negative
bacteria.
Keywords:
monobactam; structure–activity relationship; antibacterial; Gram-negative bacteria;
synergistic effect.
2

1. Introduction
Multidrug-resistant Gram-negative bacteria, especially Pseudomonas aeruginosa
(P. aeruginosa), Klebsiella pneumonia (K. pneumoniae), Escherichia coli (E. coli)
and Acinetobacter baumannii (A. baumannii), listed by the WHO as critical priority
concerns [1], have become one of the most complex and gravest global threats [2–4].
One prevailing drug-resistant mechanism is the presence of β-lactamases including
serine hydrolases (SBLs), extended spectrum β-lactamases (ESBLs) and New Delhi
metallo-β-lactamase-1 (NDM-1), which could hydrolyze most clinically used
β-lactam antibiotic and made more bacteria become resistant to even some of the
strongest antibiotics. Despite of the limited therapeutic options for treating infections
caused by Gram-negative bacteria, pharmaceutical research and development failed to
meet the clinical needs, due to the “auto-obsolescence” of antibacterials [5–7].
Currently, discovering new antibiotics from medicinal chemistry efforts to improve
the profiles of existing classes is still recognized as one of the most effective
strategies, and the resulted derivatives can enhance potency and counter bacterial
resistance mechanisms [8].
OH
O
O
O
OH
COOH
O
OH
O
O
NH
N
OH
O
O
N
N
H2N
O
O
H
N
H
N
N
H
N
N
N
N
N
N
N
H
N
O
N
H2N
H2N
N
H2N
N
N
ONa
N
S
SO3H
S
S
S
O
O
S
OSO3H
SO2Me
O
O
O
O
O
O
O
O
Avi
AZN 1
,
BAL30072
mono24c
Figure 1. Structures of AZN, BAL30072, Mono24c and Avi
Aztreonam (AZN, 1, Fig. 1), the only monobactam antibiotic in clinic [9,10], has
been successfully used for the treatment of Gram-negative bacteria infections for
several decades [11–15]. The combination of AZN and avibactam sodium (Avi, Fig.1)
as a β-lactamase inhibitor has just been completed the phase II clinical trial for
3

treatment of infections caused by resistant Gram-negative bacteria [16]. Owing to the
advantages of simple structure and strong efficacy against Gram-negative bacteria,
AZN becomes an ideal parent for conducting structure optimization, and then several
monobactam candidates, such as BAL30072 [17] and Mono 24c [18] (Fig. 1), are
active in different stages of clinical or preclinical investigation.
As monobactams act upon bacteria by targeting penicillin-binding proteins
(PBPs) located in periplasm [11,12], we first conducted the structural characteristics
analyses between this kind of analogues and target protein PBP3. The results revealed
there is a large space between oxime-linked group in monobactams and active site in
PBP3[19], which is tolerable and can bear various substituents with different
polarities or size [20a–c]. For AZN, as displayed in Fig. 2, salt bridge between
carboxyl and amino acid residues Arg489 was formed and gem-dimethyl group
interacted favorably with the hydrophobic wall (Tyr503-Tyr532-Phe533). On the
other hand, 4-dihydroxypyridone moiety on the oxime side chain of BAL30072
located near the hydrophobic wall and a favorable interaction was well formed. The
results suggested various substituents could be introduced on the oxime residue so as
to improve the effect against Gram-negative bacteria.
Based on the structural characteristics mentioned above, in the present study,
structural modification strategy was mainly focused on the nitrogen-based groups on
oxime side-chain as depicted in Fig. 2. Various substituents, such as hydrophobic
unsaturated aliphatic chains, phenyl groups and functional amino, carboxyl and
siderophores catecholates fragments, were introduced respectively, by which several
series of new monobactams were created. Herein, thirty-four new monobactam
derivatives were synthesized and evaluated for their antibacterial activities using
phenotypic screening assay. Additionally, the synergistic effects with β-lactamase
4

inhibitor, safety profiles of the representative compounds were also expounded.
A
B
substituted aliphatic chain, pyridine and phenyl groups, etc.
interact with hydrophobic pocket
X
=
C
X
O
O
N
O
H
H
N
OH
OH
R
N
R1
N
S
R
HN
H2N
N
O
NH2
OH
O
SO3H
O
8a t
-
12a f
-
12g h
-
22a c
-
Figure 2. Crystallographic reported in literature of the complex of monobactam derivatives with
PBP3 and structural modification strategy proposed. (A) Active site in AZN-acyl-PaePBP3
complex. AZN is shown in stick rendering with green carbons. (B) Active site of PaePBP3 bound
to BAL30072 colored in green. (C) Structural modification strategy proposed.
2. Results and discussion
2.1 Chemistry
O
O
R
O
OH
R
OH
N
N
N
N
c
b
N
O
N
O
OH
a
N
TrtHN
O
N
TrtHN
79-84%
67-75%
TrtHN
H2N
77%
S
O
S
O
S
O
S
3
5
4
2
H2N
R
d
N
O
SO3H
O
64-85%
N
6
H2N
O
N
O
H
R1
NH2
N
R1
S
HN
TrtHN
HN
e
O
N
R
NHBoc
N
54-64%
O
N
g
60%
R= -CH2NH2
S
h
8a-r
O
SO3H
O
O
(8o)
50-60%
N
NH
N
H
O
H
N
N
S
O
N
O
N
O
H2N
N
O
H2N
R1
R1
O
N
S
O
O
S
f
N
OH
O
R2
OH
S
O
O
OH
7
O
SO3H
NHBoc
NHBoc
12a-k
9
11
10
O
N
or
N
R2=
O
O
N
Scheme 1 (a) Ph3CCl, TEA, DMF, r.t.; (b) ArCH2Br, K2CO3, NaI, DMF, r.t.; (c) 2 N NaOH (aq.);
then 1N HCl, pH = 1−2; (d) DIC, HOBt, DMAP, DMF, r.t.; (e) TFA, SiEt3H, DCM, −10 ◦C; (f)
EDCI, NHS, anhydrous DMF, r.t. or CDI, anhydrous DMF, r.t.; (g) Anhydrous DMF, r.t.
The synthesis route of target analogues 8a−t was shown in Scheme 1, taking
commercially available (Z)-ethyl 2-(2-aminothiazol-4-yl)-2-(hydroxyimino)acetate 2
as the starting material. Firstly, the bare amino group in 2 was protected by
5

tri-phenylmethyl (Trt) to achieve intermediate 3 with the yield of 77%. Then, the key
intermediate 5 was generated by a two-step sequence including an etherification with
corresponding halides and an ester hydrolysis in ideal yields. Then condensation of 5
and (2S,3S)-3-amino-2-methyl-4-oxoazetidine-1-sulfonic acid
6
generated
intermediate 7, which underwent an amino deprotection to achieve the desired
products 8a−t in fair yields.
Next, to increase the substituent diversity on the oxime side chain, another series
of target compounds 12a−k were obtained taking 8o with a terminal amino group as
the starting material in overall yields of 30−36%, as shown in Scheme 1. First, the
carboxylic acid group in amino acids 9a–k was activated by EDCI/NHS or CDI to
form intermediate 10a−k., which coupled with 8o in anhydrous DMF directly without
further purification to produce intermediate 11a-k. Removal of the BOC protective
group led to the final products 12a−k.
O
O
O
O
R
R
R
OtBu
OtBu
R
a
b
OtBu
O
OtBu
c
NH
O
O
NH
HN
NH2 HCl
O
N
~90%
O
O
O
Cl
NH2
13
14
15
16
O
O
O
TrtHN
N
H2N
N
d
O
TrtHN
N
e
40%
f
S
O
78%
S
80%
OH
S
O
O
O
17
18
19
H2N
S
TrtHN
R
H
H
N
N
H2N
R
R
OtBu
N
N
N
N
HN
S
O
O
O
N
S
O
O
S
O
OH
OH
OtBu
O
O
g
O
O
O
6
NH
h
NH
O
O
O
N
40-50%
74%
TrtHN
O
O
N
O
N
OH
N
S
O
O
OH
OH
O
O
S
20
22a-c
21
◦
Scheme 2 (a) Chloroacetyl chloride, TEA, DCM, 0◦C; (b) PhtNOH, K2CO3, DMF, 40 C; (c)
Hydrazine hydrate, in MeOH, r.t.; (d) Ph3CCl, TEA, DMF, r.t.; (e) 2 N NaOH; then 1 N HCl, pH
= 1-2; (f) MeOH, r.t; (g) DIC, HOBt, TEA, DMF, r.t.; (h) TFA, SiEt3H, DCM, −10 ◦C.
Scheme 2 illustrated the synthesis of 22a−c taking the commercially available
amino acids (13a–c) as the starting material. The treatment of 13a–c with chloroacetyl
6

chloride gave 14a–c, which were converted into 15a–c through nucleophilic
substitution with N-hydroxyphthalimide. After removal of the phthaloyl group, the
key intermediate alkoxyamine 16a-c were obtained, which were then condensed with
2-oxo-2-(2-(tritylamino)thiazol-4-yl) acetic acid 19 generated by a two-step procedure
from ethyl 2-oxo-2-(2-(amino)thiazol-4-yl) acetate 17, to give Z-configuration 20a–c
as stable isomers. On one hand, the strong steric hindrance of triphenylmethyl
aminothiazoly is not conductive to the formation of E-isomer. On the other hand, the
intra-molecular hydrogen bond between carboxyl and the oxygen of oxime makes
Z-isomer more stable [21a–c]. Then, the condensation of 6 and 20a–c rendered
intermediates 21a–c, which underwent a Boc removal to afford the target compounds
22a−c in moderate yields of 40–50%.
2.2. SAR analysis for the antibacterial activity
The structures and activities against Gram-negative bacteria of the total 32 target
compounds were listed in Table 1. Minimum inhibitory concentration (MIC) was
determined by the agar dilution assay described by the Clinical and Laboratory
Standards Institute with AZN as the reference drug. Bacterial strains including
drug-resistant E. coli, K. pneumoniae, A. baumannii, P. aeruginosa and
drug-susceptible Enterobacter cloacae (E. cloacae), Enterobacter aerogenes (E.
aerogenes) were from the ATCC collection and clinical isolates from Chinese
hospitals.
Firstly, a series of hydrophobic groups including substituted pyridine, phenyl,
alkene and alkyne were respectively incorporated on the oxime side chain of AZN,
and a set of monobactam analogues (8a−k, 8p−t) were prepared and measured for
their activities against drug-susceptible and resistant Gram-negative bacteria.
Compounds 8a−h with substituted pyridine moieties displayed decreased antibacterial
7

activities against the tested Gram-negative strains with MICs values ranging from 8 to
>32 μg/mL, compared with the lead AZN. Only compound 8k gave moderate activity
against tested E. coli and E. cloacae strains with MICs values in the range of 1 to 2
μg/mL.
At the meantime, compounds 8l−n bearing saturated heterocyclic substituents
instead also exhibited reduced antibacterial potencies against all tested strains,
compared with AZN. To our excitement, compounds 8p and 8r bearing a unsaturated
allyl and propargyl group respectively showed comparable potencies to AZN with
MIC values of 0.125−0.5 μg/mL against tested E. coli strain. More importantly, both
of them exhibited a higher potency against A. baumannii strain than AZN with the
MIC value of 8 μg/mL. Then the unsaturated allyl was retained, and methyl and
bromo atom were respectively introduced on the double bond, by which new
compounds 8q, 8s and 8t were then created. However, a significant decrease in
antibacterial potency was witnessed, compared with 8p, which might originate from
the steric hinderance of the extra substitutions on the alkene bond. Therefore, it was
suggested that the presence of free allyl and propargyl moieties might be beneficial
for antibacterial potency.
Table 1 Antibacterial activities of the target compounds against Gram-negative bacteria
O
R
R
N
R
H
O
O
O
N
O
O
N
H
N
H
N
(S)
H
N
(S)
N
N
(S)
(S)
N
N
(S)
O
(S)
H2N
H2N
O
N
H2N
O
N
O
S
N
S
S
S
OH
S
S
O
OH
O
O
OH
O
O
8a-t
O
22a-c
12a-k
Minimum Inhibitory Concentration (MIC*, μg/mL)
E. colia
K. pneumb
P. aeruf
E.cloac
ATCC
43560
E. aerogd
ATCC
13048
A. baume
ATCC
19606
ATC
C
Comp. R
ATCC
25922
CDC
ATCC
700603
ESBLs(+)
2146
NDM-1(+)
16-11
CRE
1001728
13-66
16-14
27853
BL(+)
ESBLsa(-) NDM-1(+)
8a
8b
32
64
64
>64
>64
>32
>32
>32
>32
>64
>64
32
>32
>32
>32
>32
>32
>32
>64
>64
Br
N
N
CF3
>32
>32
8

8c
16
16
>64
>32
>32
>64
8
32
>32
>32
>64
F
N
NH2
8d
8e
8f
8
>32
16
>32
>64
>64
>32
>32
>32
>32
>32
>32
>32
>64
>64
8
16
>32
>32
>32
>32
>32
>32
>32
>64
>64
N
O
16
>32
16
>32
32
N
N
64
>32
O
CF3
N
8g
8h
>32
16
64
16
>64
32
>32
>32
>32
>32
>64
>64
>32
16
>32
32
>32
>32
>32
>32
>64
>64
O
O
N
N
N
CF3
F
8i
64
8
32
8
>64
64
>128
>128
>64
>128 >64
>128 >64
64
4
128
16
128
64
>128
>128
>64
>64
>64
>64
8j
8k
CN
2
1
>64
>64
>64
1
32
64
O
8l
16
4
64
4
>64
>64
>64
>128
>128
>64
>128 >64
>128 >64
4
2
4
64
4
>128
>128
>64
>128
>128
>64
>64
>64
>64
N
NH
8m
8n
N
N
16
32
>64
>64
32
NH2
8o
8p
1
2
4
8
4
128
4
8
1
2
>128
8
>128
32
>64v
64
0.25
16
16
>128
0.125
0.25
8q
8r
8s
1
32
16
8
32
16
32
128
>128 32
>128 16
0.25
0.125
1
2
>128
8
64
>64
>64
>64
0.5
2
>128
>64
0.5
2
128
64
>64
>64
64
64
Br
8t
4
8
>64
>64
>64
>32
>64
4
8
>64
>32
>64
>32
>64
>64
O
O
OH
12a
>32
32
>32
>32
>32
>64
64
>32
>32
NH2
O
12b
12c
>32
>32
16
16
>64
>64
>32
>32
4
2
4
32
>32
>32
>64
>64
NH2
NH2
O
O
S
64
16
>32
O
12d
12e
>32
>32
32
>64
>64
>32
>32
>32
>32
>64
64
2
4
>32
>32
>32
>32
>64
>64
OH
NH2
O
OH
>64
16
32
N
O
N
12f
12g
12h
>64
>32
>32
>64
>64
>64
>64
>64
>64
>64
>32
>32
>64
>32
>32
>64
>64
>64
>64
8
>64
2
>64
>32
>32
>64
>32
>32
>64
>64
>64
NH2
NH
OH
O
OH
OH
OH
O
>32
32
O
12i
>32
64
>64
>32
>32
>64
>32
32
>32
>32
>64
NH2
NH
O
N
12j
>32
>32
64
64
>64
>64
>32
>32
>32
>32
>64
>64
8
32
32
>32
>32
>32
>32
>64
>64
O
N
12k
16
N
S
22a
>128
32
>64
>128
>128 32
>128
>128
>128
>128
>64
OH
N
H
O
9

22b
>128
32
32
>128
>128
8
>128
>128
>128
128
>64
OH
OH
N
H
O
22c
>128
>64
4
64
16
>128
4
>128 >64
64
>128
0.5
>128
>128
>32
>128
8
>64
64
N
H
O
AZN
-
≤0.25
2
≤0.25
a
b
c
*MIC (μg/mL) was determined by agar dilution; E. coli; K. pneumoniae; E. cloacae 435
60; E. aerogenes 13048; A. baumannii; P. aeruginosa.
d
e
f
In addition, it is worth to mention that compound 8o with propylamine was
effective against both NDM-1-producing E. coli strain and ESBLs-producing K.
pneumonia with MIC values of 2−8 μg/mL. Thus, the substituents with siderophores
catecholate fragments [22] which might easily penetrate the cell membranes, free
amine groups and carboxyl groups were introduced through flexible and long linkers
in compound 8o respectively. Compounds 12a−k were synthesized and screened
accordingly as described in Table 1. However, most of them exhibited obviously
decreased activities against all Gram-negative strains. Only compounds 12b and 12d
showed moderate potencies against some drug-susceptible strains, such as E. cloacae
and E. aerogenes with MICs of 2−4 μg/mL. It seemed the flexible substituents would
be detrimental to maintain antibacterial potency.
Finally, to simulate the salt bridge and Pi-Alkyl interactions between PBP3 and
AZN, three analogues 22a−c bearing different amino acid fragments on oxime side
chain were prepared. However, compounds 22a−c showed minimal activity against
the tested strains as depicted in Table 1. Based on these results, compounds 8p and 8r
were selected as the representative compounds for further investigations.
2.3. Safety evaluations of compounds 8p and 8r in vitro and in vivo
To evaluate the safety profiles of the representative compounds, the cytotoxic
effects of the compounds 8p and 8r on H460 cells were carried out using MTT assay.
As displayed in Fig. 3, both of them showed none cytotoxicity up to the concentration
of 30 μg/mL to give the CC50 values of over 30 μg/mL, suggesting high safety
10

profiles in vitro.
CC50 > 31.12 ± 1.1 μg/mL
CC50 > 30.96 ± 1.4 μg/mL
Figure 3. Cytotoxic effects of compounds 8p and 8r on H460 cells. Following pre-treatment with
compounds 8p and 8r at the indicated concentrations for 24 h, the viability of cells was
determined by MTT assay. Control cells were treated with 0.4 % (v/v) DMSO.
Acute toxicity of compounds 8p and 8r in mice was evaluated as well.
Compounds 8p and 8r were given intravenously in a single-dosing experiment at 100,
200, 300 or 400 mg/kg, respectively. The mice were closely monitored for 7 days. No
mouse died in the 7-day observation duration, indicating that the LD50 of 8p and 8r
via intravenous route was more than 400 mg/kg, suggesting that both of them had a
wide therapeutic window and deserved further investigations.
Table 2. Acute toxicity of compounds 8p and 8r in mice
Compd
8p
LD50 (mg/kg)
> 400
8r
> 400
2.4. Molecular docking
In order to further understand their modes of interactions with P. aeruginosa
PBP3, molecular docking between P. aeruginosa PBP3 and compounds 8p and 8r
were performed with Discovery Studio 4.5 docking program. As depicted in Fig. 4,
Pi-alkyl interactions between allyl of compound 8r and hydrophobic wall composed
of Tyr-Tyr-Phe were well formed. These interactions might be helpful for inducing-fit
conformational changes (Figure 4 IIA/IIB) and contribute to the formation of different
hydrogen bonds between 8r and other residues (SER294, LYS484, SER485, etc.) in
11

the active site of PBP3. And compound 8p interacted in the hydrophobic pocket in a
similar way to 8r (Fig. 4, IA/IB). In addition to hydrophobic interaction of Pi-alkyl
with PBP3, several other factors could contribute to the antibacterial effect, such as
permeability, binding to PBP3, efflux pump. These data may be helpful to include
PBP3 binding data for those specific compounds 8p and 8r.
IA
IB
IIB
IIA
Figure 4. Interactions of compounds 8p, 8r in the active site of the P. aeruginosa PBP3 (Docking
with Discovery Studio). (I A/B) compound 8p; (II A/B) compound 8r; Compounds are shown as
stick models with green carbon atoms, blue nitrogen atoms, red oxygen atoms, and yellow sulfur
atoms. Hydrogen bonds are depicted as red dashes in three-dimensional view and green dashes in
two-dimensional view; Salt bridge interactions are depicted as brown dashes; Pi-Sulfur
interactions are depicted as yellow dashes; Pi-Alkyl interactions are depicted as purple dashes.
2. 5. Synergic Studies
The combination of antibiotics and β-lactamase inhibitor has been widely used in
clinic studies owing to their exciting synergy effect [23]. To find the strategy against
resistant bacteria, synergistic studies of compounds 8p and 8r with β-lactamase
inhibitor Avi were conducted on ESBLs- or NDM-1-producing K. pneumoniae
strains, using microdilution checkerboard technique [24−26]. As shown in Table 3,
taking AZN as the positive reference, significant synergies were observed in
12

combinations of 8p or 8r and Avi respectively, based on the fractional inhibitory
concentration index (FICI) was ≤ 0.5. The combination of 8p/8r with avi in a ratio of
1:16, showed a promising synergistic effect against two drug-resistant K. pneumoniae
strains, with significantly reduced MIC values up to 8-fold and >256-fold. It also
confirmed that β-lactam ring hydrolysis by β-lactamase might be the main resistant
mechanism of its kind [27], other than the outer membrane permeability barrier and
efflux pump mechanism.
Table 3. Antibacterial activities of compounds 8p and 8r alone and in combination with Avi
against K. pneumoniae strains (MICa)
alone
combination
AZN
(+Avi)
0.5c
Strain
8p
8r
>128 32
Avi AZN 8p (+Avi)b 8r (+Avi)b
700603 ESBLs (+)
4
4
0.5
0.5
0.5
0.5
2146 NDM-1(+)
>128 >128 32
64
0.5d
a, MIC (μg/mL) was determined by agar dilution; FICI<0.5; b, combination ratio in 1:16
(samples/Avi, m/m); c, combination ratio in 1:8; d, combination ratio in 1:2
3. Conclusion
In summary, thirty-four new monobactam derivatives were prepared and
evaluated for their antibacterial activities against Gram-negative bacteria, taking AZN
as the lead. Among them, compounds 8p and 8r showed satisfactory potencies with
MIC values of 0.125−32 μg/mL against most tested Gram-negative strains. Both of
them displayed high safety profiles both in vitro and in vivo. Synergistic effect of
8p/8r combined with Avi significantly enhanced their antibacterial activity against
ESBLs and NDM-1-producing K. pneumoniae, with significantly reduced MIC value
up to 8-fold and >256-fold in a ratio of 1:16. It further confirmed that main resistant
mechanism of its kind might originate from β-lactam ring hydrolysis by β-lactamase.
Further structure modifications and optimizations of this kind for overcoming
β-lactamase-producing resistant Gram-negative bacteria are actively underway in our
laboratory.
13

4. Experimental section
4.1 Chemistry
Melting points (mp) were measured with an MPA 100 OptiMelt automated
1
melting point system (Stanford Research Systems, California, USA). H NMR and
13C NMR spectra were performed on a Varian Inova spectrometer (San Francisco,
CA, USA) or a Bruker Avance III spectrometer (Bruker Co., Zurich, Switzerland) in
DMSO-d6 with Me4Si as the internal standard. ESI high-resolution mass spectrometry
(HRMS) was performed on an AutospecUltima-TOF mass spectrometer (Micromass
UK Ltd, Manchester, UK). Flash chromatography was performed on a CombiFlash Rf
200 system (Teledyne, Nebraska, USA), with a particle size of 38 µm. Lyophilization
was performed on CHRIST ALPHA 2-4 LD plus freeze drier (Marin Christ, Osterode,
Germany).
4.2 General procedures for synthesis of compounds 8a–r
4.2.1 2S,3S-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[(6-bromopyridin-2-yl)methoxy]imino]
acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8a). Et3SiH (0.06 g, 0.53 mmol)
was added to a suspension of 7a (0.2 g, 0.26 mmol) in DCM (5 mL) under Nitrogen at
–15 oC. The reaction mixture was allowed to stir at –15 oC for 20 min, then TFA (1.8
g, 15.75 mmol) was added. After stirring at –15 oC for 5 h, the solvent was evaporated
in vacuo at r.t.. EA and petroleum ether (1:1, 10 mL) was added to the residue and
stirred at r.t. for 1 h. The mixture was filtered to give the title product 8a (65.0 mg,
48%) as off-white solid, mp: 180.8–182.1 oC. 1H NMR (500 MHz) δ 9.65 (s, 1H), 7.81
(t, J = 7.8 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 6.97 (d, J = 3.4
Hz, 1H), 5.28 (s, 2H), 4.51 (dd, J = 7.7, 2.3 Hz, 1H), 3.80–3.62 (m, 1H), 1.54–1.31 (d,
13
3H). C NMR (126 MHz, DMSO-d6) δ 170.3, 162.3, 161.5, 159.7, 141.2, 141.0,
14

128.4, 128.2, 127.7, 127.3, 121.4, 112.1, 76.5, 61.0, 57.6, 18.7. HRMS: calcd for
C15H16BrN6O6S2 [M+H]+ 518.9751, found 518.9760.
4.2.2 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[[6-(trifluoromethyl)pyridin-3-yl]me-
thoxy]imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8b). Compound 8b
was prepared from 7b (0.30 g, 0.40 mmol) by the procedure as described for 8a as
off-white solid (99.0 mg, 49%), mp: 158.5–160.0 oC. 1H NMR (500 MHz) δ 9.61 (d, J
= 6.7 Hz, 1H), 8.80 (s, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 6.96 (s,
1H), 5.39 (s, 2H), 4.50 (d, J = 7.1 Hz, 1H), 3.71 (d, J = 4.1 Hz, 1H), 1.42 (d, J = 5.8
13
Hz, 3H). C NMR (151 MHz) δ 170.8, 170.1, 162.1, 161.3, 149.6, 146.3(t), 137.7,
137.6, 123.1, 121.2, 121.0, 111.9, 73.3, 60.8, 57.4, 40.4, 40.3, 40.1, 40.0, 39.9, 39.7,
39.6, 18.4. HRMS: calcd for C16H16F3N6O6S2 [M+H]+ 509.0519, found 509.0528.
4.2.3 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[(6-fluoropyridin-2-yl)methoxy]imino]
acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8c). Compound 8c was prepared
from 7c (0.20 g, 0.29 mmol) by the procedure as described for 8a as off-white solid
(70.6 mg, 54%), mp: 169.5–171.4 oC. 1H NMR (500 MHz) δ 9.65 (d, J = 7.7 Hz, 1H),
8.04 (dd, J = 15.9, 8.0 Hz, 1H), 7.39 (d, J = 7.3 Hz, 1H), 7.12 (d, J = 6.8 Hz, 1H),
6.97 (s, 1H), 5.24 (s, 2H), 4.51 (dd, J = 7.7, 2.3 Hz, 1H), 3.74–3.71 (m, 1H), 1.43 (d, J
= 6.1 Hz, 3H). 13C NMR (126 MHz) δ 170.3, 163.9, 162.3, 162.1, 161.4, 157.0, 143.6,
119.7, 112.1, 109.2, 108.9, 76.4, 61.0, 57.6, 18.7. HRMS: calcd for C15H16N6O6FS2
[M+H]+ 459.0551, found 459.0557.
4.2.4 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[(6-carbamoylpyridin-2-yl)methoxy]
imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8d). Compound 8d was
prepared from 7d (0.30 g, 5.0 mmol) by the same procedure as described for 8a as
off-white solid (0.22 g, 54%). mp:169.3–170.1 oC. 1H NMR (500 MHz) δ 9.65 (d, J =
7.5 Hz, 1H), 8.07 (s, 1H), 8.02 (t, J = 7.7 Hz, 1H), 7.96 (d, J = 7.5 Hz, 1H), 7.67 (s,
15

1H), 7.62 (d, J = 7.7 Hz, 1H), 6.95 (s, 1H), 5.37 (s, 2H), 4.52 (dd, J = 7.7, 2.4 Hz, 1H),
13
3.77–3.71 (m, 1H), 1.42 (d, J = 8.8 Hz, 3H). C NMR (151 MHz) δ 170.6, 169.9,
166.3, 162.2, 159.0, 157.1, 150.1, 138.8, 129.5, 124.4, 121.2, 111.5, 76.7, 60.9, 57.4,
18.5. HRMS: calcd for C16H18N7O7S2 [M+H]+ 484.0704, found 484.07092
4.2.5
(2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[(6-methylpyridin-2-yl)methoxy]
imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8e). Compound 8e was
prepared from 7e (0.25 g, 0.35 mmol) by the procedure as described for 8a as
off-white solid (0.11 g, 70%). mp: 152.0–154.0 oC. 1H NMR (500 MHz) δ 9.57 (d, J =
7.7 Hz, 1H), 8.34 (t, J = 7.8 Hz, 1H), 7.74 (d, J = 7.8 Hz, 3H), 6.92 (s, 1H), 5.42 (s,
2H), 4.49 (dd, J = 7.6, 2.2 Hz, 1H), 3.72 (dd, J = 6.1, 2.3 Hz, 1H), 2.69 (s, 3H), 1.43
(d, J = 6.1 Hz, 3H). 13C NMR (126 MHz) δ 169.8, 162.3, 162.0, 158.9, 155.4, 153.3,
150.8, 144.8, 126.3, 122.6, 112.3, 73.1, 61.0, 57.6, 21.1, 18.7. HRMS: calcd for
C16H19N6O6S2 [M+H]+ 455.0802, found 455.0808.
4.2.6
(2S,3S)-2-Methyl-3-{(Z)-2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]
methoxy]imino]-2-[2-(tritylamino)thiazol-4-yl]acetamido}-4-oxoazetidine-1-sulfonic
acid (8f). Compound 8f was prepared from 7f (0.30 g, 0.37 mmol) by the procedure
as described for 8a as off-white solid (0.11 g, 54%), mp: 156.7–158.9 oC. 1H NMR
(500 MHz) δ 9.41 (d, J = 7.8 Hz, 1H), 8.82 (d, J = 6.7 Hz, 1H), 7.69 (d, J = 6.8 Hz,
1H), 6.84 (s, 1H), 5.43 (s, 2H), 5.22 (d, J = 8.5 Hz, 2H), 4.44 (dd, J = 7.8, 2.5 Hz, 1H),
13
3.66 (dd, J = 6.2, 2.6 Hz, 1H), 2.29 (s, 3H), 1.40 (d, J = 6.1 Hz, 3H). C NMR (151
MHz) δ 169.3, 167.1, 162.2, 162.0, 158.7, 150.8, 142.6, 125.7, 124.7, 116.9, 112.0,
110.1, 71.0, 66.4, 60.8, 57.4, 18.4, 10.5. HRMS: calcd for C18H20F3N6O7S2 [M+H]+
553.0782, found 553.0789
4.2.7
(2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[[4-(3-methoxypropoxy)-3-methyl
pyridin-2-yl]methoxy]imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8g).
16

Compound 8g was prepared from 7g (0.30 g, 0.38 mmol) according to the procedure
as described for 8a as off-white solid (0.11 g, 52%), mp: 142.8–144.4 oC. 1H NMR
(500 MHz) δ 9.38 (d, J = 7.8 Hz, 1H), 8.73 (d, J = 6.8 Hz, 1H), 7.62 (d, J = 6.9 Hz,
1H), 6.83 (s, 1H), 5.41 (s, 2H), 4.51–4.35 (m, 3H), 3.67 (dd, J = 6.1, 2.5 Hz, 1H), 3.52
(t, J = 6.1 Hz, 2H), 3.26 (s, 3H), 2.27 (s, 3H), 2.12–2.02 (m, 2H), 1.41 (d, J = 6.1 Hz,
3H). 13C NMR (126 MHz) δ 169.3, 162.1, 158.8, 151.8, 149.0, 141.8, 141.3, 125.8,
117.3, 114.9, 111.9, 109.5, 70.4, 68.5, 60.7, 58.5, 57.4, 28.8, 18.5, 10.6. HRMS: calcd
for C20H27N6O8S2 [M+H]+ 543.1326, found 543.1334
4.2.8
(2S,3S)-3-{(Z)-2-[[[6-(1H-Pyrazol-1-yl)pyridin-2-yl]methoxy]imino]-2-(2-
acid (8h).
aminothiazol-4-yl)acetamido}-2-methyl-4-oxoazetidine-1-sulfonic
Compound 8h was prepared from 7h (0.30 g, 0.40 mmol) by the procedure as
described for 8a as off-white solid (0.10 g, 50%), mp: 156.7–158.9 oC. 1H NMR (500
MHz) δ 9.55 (d, J = 7.8 Hz, 1H), 8.63 (d, J = 2.4 Hz, 1H), 8.51 (d, J = 1.4 Hz, 1H),
8.02 (dd, J = 8.4, 2.0 Hz, 1H), 7.94 (s, 1H), 7.84 (s, 1H), 6.93 (s, 1H), 6.59–6.58 (m,
1H), 5.29 (s, 2H), 4.48 (dd, J = 7.8, 2.5 Hz, 1H), 3.76–3.59 (m, 1H), 1.40 (d, J = 6.1
13
Hz, 3H). C NMR (151 MHz) δ 170.7, 170.0, 162.8, 162.2, 151.0, 148.2, 142.8,
139.7, 131.4, 127.6, 119.8, 112.1, 111.4, 108.8, 73.6, 60.8, 57.4, 18.4. HRMS: calcd
for C18H19N8O6S2 [M+H]+ 507.0864, found 507.0875
4.2.9
(2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[[4-(trifluoromethyl)benzyl]oxy]
imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8i). Compound 8i was
prepared from 7i (0.24 g, 0.32 mmol) according to the procedure as described for 8a
as white solid (0.12 g, 68%), mp: 190.0–191.3 oC. 1H NMR (500 MHz) δ 9.43 (d, J =
7.9 Hz, 1H), 7.73 (d, J = 8.1 Hz, 2H), 7.58 (d, J = 8.1 Hz, 2H), 7.25 (s, 2H), 6.76 (s,
1H), 5.24 (s, 2H), 4.47 (dd, J = 7.9, 2.5 Hz, 1H), 3.72 (dd, J = 6.1, 2.6 Hz, 1H), 1.39
(d, J = 6.1 Hz, 3H). 13C NMR (151 MHz) δ 169.0, 162.9, 162.6, 150.8, 143.3, 142.9,
17

128.7, 128.3(2), 125.8, 125.8, 125.8, 125.8, 125.6(2), 110.6, 74.9, 60.8, 57.3, 18.44.
HRMS: calcd for C17H17F3N5O6S2 [M+H]+ 508.0567, found: 508.0577.
4.2.10
(2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[(4-fluorobenzyl)oxy]imino]
acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8j). Compound 8j was prepared
from 7j (0.25 g, 0.36 mmol) by the procedure as described for 8a as off-white solid
o
1
(0.1 g, 65%). mp: 198.5–200.3 C. H NMR (500 MHz) δ 9.38 (d, J = 7.9 Hz, 1H),
7.46–7.37 (m, 2H), 7.31 (s, 2H), 7.24–7.15 (m, 2H), 6.75 (s, 1H), 5.12 (s, 2H), 4.44
(dd, J = 7.9, 2.6 Hz, 1H), 3.67 (qd, J = 6.1, 2.6 Hz, 1H), 1.36 (d, J = 6.2 Hz, 3H). 13C
NMR (151 MHz) δ 169.1, 163.0, 162.9, 162.5, 161.3, 150.2, 134.3, 130.2, 130.1,
115.6, 115.4, 110.3, 75.2, 60.8, 57.3, 18.4. HRMS: calcd for C16H17FN5O6S2 [M+H]+
458.0599, found: 458.0615.
4.2.11 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[(4-cyanobenzyl)oxy]imino]acetami
do}-2-methyl-4-oxoazetidine-1-sulfonic acid (8k). Compound 8k was prepared from
7k (0.40 g, 0.56 mmol) by the procedure as described for 8a as off-white solid (0.15
g, 56%), mp: 176.5–178.3 oC. 1H NMR (500 MHz) δ 9.54 (d, J = 7.8 Hz, 1H), 7.85 (d,
J = 8.2 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 6.86 (s, 1H), 5.29 (s, 2H), 4.48 (dd, J = 7.8,
13
2.5 Hz, 1H), 3.72–3.68 (m, 1H), 1.40 (d, J = 6.0 Hz, 3H). C NMR (151 MHz) δ
169.8, 169.7, 162.2, 161.9, 144.5, 143.9, 132.8, 128.3, 119.3, 111.3, 110.8, 75.2, 60.8,
57.4, 18.5. HRMS: calcd for C17H17N6O6S2 [M+H]+ 465.0646, found 465.0655.
4.2.12 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[(2-morpholinoethoxy)imino]acetam-
ido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8l). Compound 8l was prepared from 7l
(0.30 g, 0.43 mmol) by the procedure as described for 8a as white solid (0.10 g, 52%),
1
mp: 147.0–148.1 oC. H NMR (600 MHz) δ 9.16 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H),
4.56 (dd, J = 16.3, 4.5 Hz, 2H), 4.42 (dd, J = 8.0, 2.6 Hz, 1H), 3.82 (s, 4H), 3.75–3.72
13
(m, 2H), 3.62–3.52 (m, 2H), 3.33 (s, 4H), 1.45–1.36 (d, J = 1.41Hz, 3H). C NMR
18

(151 MHz) δ 167.4, 163.2, 162.4, 129.5, 128.8, 117.1, 69.6, 63.8(2), 61.1, 57.3, 55.4,
52.2(2), 18.5. HRMS: calcd for C15H23N6O7S2 [M+H]+ 463.1064, found 463.1073.
4.2.14 (2S,3S)-3-{(Z)-2-(2-aminothiazol-4-yl)-2-[[2-(piperazin-1-yl)ethoxy]imino]ace
tamido}-2-methyl-4-oxoazetidine-1-sulfonic acid, trifluoroacetic acid salt (8m).
Compound 8m was prepared from 7m (0.30 g, 0.37 mmol) by the procedure as
o
1
described for 8a as white solid (0.14 g, 65%), mp: 192.7–194.0 C. H NMR (600
MHz) δ 9.37 (d, J = 7.4 Hz, 1H), 8.93 (s, 2H), 6.89 (s, 1H), 4.48–4.37 (m, 3H), 3.80
(dd, J = 6.1, 2.4 Hz, 1H), 3.40 (s, 10H), 1.43 (t, J = 7.0 Hz, 3H). 13C NMR (151 MHz)
δ 169.3, 162.8, 159.1, 158.9, 115.4, 111.3, 70.0, 61.0, 57.3(2), 55.5, 49.4(2), 41.1,
18.5. HRMS: calcd for C15H24N7O6S2 [M–CF3COOH+H]+ 462.1224, found 462.1236。
4.2.15 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[2-(piperidin-1-yl)ethoxy]imino]ace
tamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8n). Compound 8n was prepared
from 7n (0.30 g, 0.43 mmol) by the procedure as described for 8a as off-white solid
(0.11 g, 56%), mp: 150.3–152.1 oC . 1H NMR (600 MHz) δ 9.16 (d, J = 8.0 Hz, 1H),
7.57 (s, 1H), 4.53 (t, J = 4.6 Hz, 2H), 4.42 (dd, J = 7.9, 2.4 Hz, 1H), 3.72 (dd, J = 6.1,
13
2.4 Hz, 1H), 3.49 (s, 4H), 2.97 (s, 2H), 1.67 (s, 3H), 1.41 (d, J = 6.2 Hz, 3H). C
NMR (151 MHz) δ 167.3, 163.3, 162.4, 129.5, 117.0, 115. 6, 69.6, 61.1, 57.3, 55.1,
53.2, 23.0, 21.5, 18.5. HRMS: calcd for C16H25N6O6S2 [M+H]+ 461.1272, found:
461.1278.
4.2.16
(2S,3S)-3-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-ammonioethoxy)imino]acetamido}
-2-methyl-4-oxoazetidine-1-sulfonate (8o). Et3SiH (0.10 g, 0.82 mmol) was added to a
suspension of 7o (0.30 g, 0.41 mmol) in DCM (10 mL) under nitrogen at –15 oC. The
reaction mixture was allowed to stir at -15 oC for 20 min, then TFA (5.0 mL) was
added. After stirring at –15 oC for 5 h, the mixture was evaporated in vacuo at r.t.. The
19

mixture of EA and petroleum ether (1:1, 10 mL) was added to the residue, stirred for
1 h. The mixture was filtered to give crude product. The crude solid was dissolved the
in water, and purified by HP20 resin column. The column was rinsed with water and
finally 20% MeOH in water to elute the target product. MeOH was evaporated under
vacuum at 36 oC. The residual water solution was lyophilized to give the title product
1
8o (0.1 g, 64%) as white solid, mp: 230.1 oC (dec.) H NMR (600 MHz) δ 9.21 (s,
1H), 7.75 (s, 3H), 7.23 (s, 2H), 6.84 (s, 1H), 4.48 (s, 1H), 4.23 (d, J = 4.5 Hz, 2H),
3.76–3.63 (m, 1H), 3.16–3.01 (m, 2H), 1.43 (d, J = 5.7 Hz, 3H). 13C NMR (151 MHz)
δ 169.1, 162.7, 162.7, 151.2, 142.7, 110.8, 70.8, 60.8, 57.7, 38.8, 18.5. HRMS: calcd
for C11H17N6O6S2 [M+H]+ 393.0646, found 393.0651.
4.2.17 (2S,3S)-3-{(Z)-2-[(Allyloxy)imino]-2-(2-aminothiazol-4-yl)acetamido}-2-methy
-l-4-oxoazetidine-1-sulfonic acid (8p). Compound 8p was prepared from 7p (0.30 g,
0.47 mmol) by the procedure as described for 8o as white solid (0.12 g, 65%), mp:
o
1
189.2–190.1 C. H NMR (600 MHz) δ 9.47 (d, J = 7.6 Hz, 1H), 6.88 (s, 1H), 6.02–
5.95 (m, 1H), 5.36–5.23 (m, 2H), 4.65 (d, J = 5.0 Hz, 2H), 4.43 (d, J = 7.7 Hz, 1H),
13
3.70–3.60 (m, 1H), 1.38 (d, J = 6.1 Hz, 3H). C NMR (151 MHz) δ170.2, 162.0,
161.2, 146.6, 134.1, 118.2, 111.1 75.7, 60.8, 57.4, 18.5. HRMS: calcd for
C12H16N5O6S2 [M+H]+ 390.0537, found: 390.0541.
4.2.18
(2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[(2-methylallyl)oxy]imino]aceta
mido}-2-methyl-4-oxoazetidine-1-sulfonic acid (8q). Compound 8q was prepared
from 7q (0.30 g, 5 mmol) by the procedure as described for 8a as off-white solid
1
(0.10 g, 56%), mp: 181.4–183.2 oC. H NMR (600 MHz) δ 9.45 (d, J = 7.7 Hz, 1H),
6.86 (s, 1H), 4.94 (d, J = 40.8 Hz, 2H), 4.56 (s, 2H), 4.46 (dd, J = 7.8, 2.4 Hz, 1H),
13
3.76–3.59 (m, 1H), 1.71 (s, 3H), 1.41 (d, J = 6.1 Hz, 3H). C NMR (151 MHz) δ
20

170.0, 162.2, 161.6, 141.7, 112.9, 110.8, 100.0, 78.3, 60.8, 57.4, 19.7, 18.5. HRMS:
calcd for C13H18N5O6S2 [M+H]+ 404.0693, found 404.0698.
4.2.19 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[(prop-2-yn-1-yloxy)imino]acetamid
o}-2-methyl-4-oxoazetidine-1-sulfonic acid (8r). Compound 8r was prepared from 7r
(0.30 g, 0.47 mmol) by the procedure as described for 8o as light-yellow solid (0.10 g,
56%), mp: 180 oC (dec.). 1H NMR (500 MHz) δ 9.32 (d, J = 7.7 Hz, 1H), 7.22 (s, 2H),
6.72 (s, 1H), 4.66 (s, 2H), 4.39 (d, J = 7.8 Hz, 1H), 3.70–3.64 (m, 1H), 3.47 (s, 1H),
13
1.38 (d, J = 6.1 Hz, 3H). C NMR (151 MHz) δ 169.1, 162.4, 162.3, 150.1, 150.1,
110.9, 80.4, 78.3, 62.3, 60.9, 57.3, 18.5. HRMS: calcd for C12H14N5O6S2 [M+H]+
388.0380, found 388.0384.
4.2.20 (2S,3S)-3-((Z)-2-(2-aminothiazol-4-yl)-2-(((2-bromoallyl)oxy)imino)
acetamido)-2-methyl-4-oxoazetidine-1-sulfonic acid (8s). Compound 8s was prepared
from 7s (0.30 g, 5.0 mmol) by the same procedure as described for 8a as off-white
solid (0.12 g, 60%). mp:166.3–167.1 oC. 1H NMR (600 MHz) δ 9.47 (d, J = 7.4 Hz,
1H), 6.86 (s, 1H), 6.04 (s, 1H), 5.69 (s, 1H), 4.74 (s, 2H), 4.45 (d, J = 5.4 Hz, 1H),
13
3.71 (d, J = 3.6 Hz, 1H), 1.41 (d, J = 5.8 Hz, 3H). C NMR (151 MHz) δ 169.58,
162.18, 161.88, 128.26, 118.95, 111.39, 77.58, 60.81, 57.37, 18.49. HRMS: calcd for
C12H13N5O6S2Br [M-H]- 465.9493, found 465.9489
4.2.21 (Z)-3-(2-(2-aminothiazol-4-yl)-2-(((3-methylbut-2-en-1-yl)oxy)imino)
acetamido)-2-methyl-4-oxoazetidine-1-sulfonic acid (8t). Compound 8t was prepared
from 7t (0.30 g, 5.0 mmol) by the same procedure as described for 8a as off-white
solid (0.10 g, 53%). mp:153.3–54.1 oC. 1H NMR (600 MHz) δ 9.41 (d, J = 7.7 Hz,
1H), 6.87 (s, 1H), 5.41 – 5.39 (m, 1H), 4.64 (d, J = 6.8 Hz, 2H), 4.44 (dd, J = 7.8, 2.6
Hz, 1H), 3.69 – 3.66 (m, 1H), 1.74 (s, 3H), 1.68 (s, 3H), 1.41 (d, J = 6.1 Hz, 3H). 13C
NMR (151 MHz) δ 170.07, 162.09, 138.17, 128.24, 127.99, 127.10, 120.09, 110.56,
21

71.90, 60.81, 57.39, 25.95, 18.58, 18.44. HRMS: calcd for C14H18N5O6S2 [M-H]-
416.0702, found 416.0712
4.3 General procedures for synthesis of compound 12a–k
4.3.1
(S)-3-Amino-4-{[2-[[(Z)-[1-(2-aminothiazol-4-yl)-2-[[(2S,3S)-2-methyl-4-
oxo-1-sulfoazetidin-3-yl]amino]-2-oxoethylidene]amino]oxy]ethyl]amino}-4-oxobuta
noic acid (12a). EDCI (86.0 mg, 0.45 mmol) and NHS (48.0 mg, 0.42 mmol) was
added to a solution of Boc-L-aspartic acid 4-tert-butyl ester (0.10 g, 0.35 mmol) in
anhydrous DMF (1.0 mL). The reaction mixture was allowed to stir at r.t. until the
reaction fininsed. 8o (0.15 g, 0.30 mmol) in dry acetonitrile (3.0 mL) and absolute
DIEA (76.6 mg, 0.59 mmol) was added to above reaction mixture dropwise. After
stirring at r.t. overnight, the mixture was diluted with water (5.0 mL) and extracted
with EA (10.0 mL×3). The organic layers were combined and washed with brine (20
mL×3), concentrated under vacuum to give crude product 11a.
Et3SiH (0.07 g, 0.60 mmol) was added to the suspension of 11a (0.20 g, 0.30
mmol) in DCM (10 mL) under nitrogen at –15 oC. The reaction mixture was allowed
o
to stir at -15 C for 20 min, then TFA (5.0 mL) was added thereto. After stirring at –
o
15 C for 5 h, the mixture was evaporated in vacuo at r.t.. EA and petroleum ether
(1:1, 10 mL) was added to the residue and stirred in for 1 h, then filtered to give the
title product 12a (0.10 g, 65%) as off-white solid. mp: 172.5–173.2 ◦C. 1H NMR (500
MHz) δ 9.34 (d, J = 7.7 Hz, 1H), 8.52 (t, J = 5.4 Hz, 1H), 8.19 (s, 4H), 6.89 (s, 1H),
4.46 (dd, J = 7.7, 2.4 Hz, 1H), 4.14 (s, 2H), 4.10–4.01 (m, 1H), 3.78–3.69 (m, 1H),
3.48–3.39 (m, 2H), 2.86–2.74 (m, 2H), 1.43 (d, J = 6.1 Hz, 3H). 13C NMR (151 MHz)
δ 171.3, 169.8, 168.3, 162.5, 158.9, 117.0, 115.0, 110.8, 72.9, 60.9, 57.5, 49.5, 39.0,
35.8, 18.5. HRMS: calcd for C15H22N7O9S2 [M+H]+ 508.0915, found 508.0923.
22

4.3.2
(2S,3S)-3-{(Z)-2-[[2-[(S)-2-Aminopent-4-ynamido]ethoxy]imino]-2-(2-amino
thiazol-4-yl)acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (12b). Compound
12b was prepared from 8o (0.15 g, 0.30 mmol) and boc-L-methionine (0.10 g, 0.47
mmol) by the procedure as described for 12a as off-white solid (0.10 g, 70%), mp:
1
161.5–163.3 oC. H NMR (500 MHz) δ 9.38 (d, J = 7.7 Hz, 1H), 8.54 (t, J = 5.6 Hz,
1H), 8.28 (d, J = 4.0 Hz, 4H), 6.93 (d, J = 9.0 Hz, 1H), 4.47 (dd, J = 7.7, 2.5 Hz, 1H),
4.24–4.09 (m, 2H), 3.98–3.90 (m, 1H), 3.76–3.71 (m, 1H), 3.56–3.37 (m, 2H), 3.11 (t,
J = 2.6 Hz, 1H), 2.80–2.67 (m, 2H), 1.43 (d, J = 6.1 Hz, 3H). 13C NMR (151 MHz) δ
169.9, 167.7, 162.5, 159.0, 116.8, 114.9, 111.0, 78.1, 75.9, 73.1, 60.9, 57.5, 51.3, 38.9,
21.6, 18.5. HRMS: calcd for C16H22N7O7S2 [M+H]+ 488.1017, found 488.1026.
4.3.3 (2S,3S)-3-[(S,Z)-5-Amino-12-(2-aminothiazol-4-yl)-6-oxo-10-oxa-2-thia-7,11-
diazatridec-11-enamido]-2-methyl-4-oxoazetidine-1-sulfonic acid (12c). Compound
12c was prepared from 8o (0.15 g, 0.30 mmol) and boc-L-methionine (87.3 mg, 0.35
mmol) by the procedure as described for 12a as off-white solid (0.10 g, 65%), mp:
164.5–165.9 oC. 1H NMR (500 MHz) δ 9.46 (d, J = 7.5 Hz, 1H), 8.60 (s, 1H), 8.21 (d,
J = 3.8 Hz, 4H), 6.96 (s, 1H), 4.47 (dd, J = 7.7, 2.4 Hz, 1H), 4.20 (t, J = 5.2 Hz, 2H),
3.95–3.86 (m, 1H), 3.76 (dd, J = 6.1, 2.5 Hz, 1H), 3.46 (dd, J = 9.8, 4.8 Hz, 2H), 2.04
(s, 3H), 1.98 (dd, J = 16.0, 6.3 Hz, 2H), 1.43 (d, J = 6.1 Hz, 3H). 13C NMR (151 MHz)
δ 173.2, 170.2, 168.9, 162.4, 161.0, 144.6, 111.2, 73.5, 60.9, 57.5, 52.2, 39.0, 31.4,
28.7, 18.5, 14.9. HRMS: calcd for C16H26N7O7S3 [M+H]+ 524.1050, found 524.1060.
4.3.4 (S)-2-Amino-5-{[2-[[(Z)-[1-(2-aminothiazol-4-yl)-2-[[(2S,3S)-2-methyl-4-oxo-
1-sulfoazetidin-3-yl]amino]-2-oxoethylidene]amino]oxy]ethyl]amino}-5-oxopentanoi
c acid (12d). Compound 12d was prepared from 8o (0.15 g, 0.30 mmol) and
boc-L-glutamic acid 1-tert-butyl ester (0.10 g, 0.33 mmol) by the procedure as
1
described for 12a as off-white solid (89.2 mg, 57%), mp: 166.5–167.9 oC. H NMR
23

(500 MHz) δ 9.20 (d, J = 8.2 Hz, 1H), 8.20 (d, J = 3.6 Hz, 4H), 7.90 (t, J = 5.7 Hz,
1H), 6.83 (s, 1H), 4.52 (dd, J = 8.2, 2.5 Hz, 1H), 4.07 (t, J = 5.5 Hz, 2H), 3.94 (d, J =
5.4 Hz, 1H), 3.79–3.67 (m, 1H), 3.40–3.23 (m, 2H), 2.42–2.22 (m, 2H), 2.13–2.03 (m,
1H), 1.96 (dd, J = 10.8, 5.6 Hz, 1H), 1.43 (d, J = 6.1 Hz, 3H). 13C NMR (151 MHz) δ
171.7, 171.3, 169.3, 162.9, 162.5, 158.9, 158.6, 110.3, 73.2, 60.7, 57.8 52.2, 38.7,
26.3, 18.4. HRMS: calcd for C16H24N7O9S2 [M+H]+ 522.1071, found 522.1078.
4.3.5 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[2-(5-hydroxypicolinamido)ethoxy]
imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (12e). To the solution of
5-hydroxypicolinic acid (0.10 g, 0.72 mmol) in DMF (1.0 mL) was added CDI (0.12 g,
0.75 mmol) and stirred at r.t. for 1 h. The above solution (0.75 mL) was added
dropwise into the solution of 8o (0.10 g, 0.20 mmol) in acetonitrile (3.0 mL) and
stirred at r.t. overnight. Removal of solvent was carried out to give a brown residue,
which was subjected to flash column chromatography (eluent: from 0–15% of
methanol in DCM) to afford 12e (75.0 mg, 73%), mp: 193.1–194.0 oC. 1H NMR (500
MHz) δ 10.60 (s, 1H), 9.28 (d, J = 8.0 Hz, 1H), 8.61 (t, J = 6.0 Hz, 1H), 8.16 (d, J =
2.7 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.31–7.24 (m, 3H), 6.74 (s, 1H), 4.44 (dd, J =
8.0, 2.6 Hz, 1H), 4.15 (t, J = 6.0 Hz, 2H), 3.79–3.74 (m, 1H), 3.62–3.48 (m, 2H), 1.36
(d, J = 6.1 Hz, 3H). 13C NMR (151 MHz) δ 169.0, 164.6, 163.0, 162.6, 156.7, 150.3,
143.2, 141.6, 137.2, 123.8, 123.1, 109.9, 73.0, 60.8, 57.2, 39.0, 18.4. HRMS: calcd
for C17H20N7O8S2 [M+H]+ 514.0809, found 514.0815.
4.3.6 (2S,3S)-3-{(Z)-2-[[2-[(S)-2-Amino-3-(1H-imidazol-4-yl)propanamido]ethoxy]
imino]-2-(2-aminothiazol-4-yl)acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid
(12f). Compound 12f was prepared from 8o (0.10 g, 0.20 mmol) and boc-L-histidine
(0.24 mmol) by the procedure as described for 12e as off-white solid (74.1 mg, 70%),
1
mp: 149.9–152.8 oC. H NMR (500 MHz) δ 9.28 (d, J = 7.9 Hz, 1H), 9.01 (s, 1H),
24

8.29 (s, 3H), 8.19 (t, J = 5.6 Hz, 1H), 7.49 (s, 1H), 6.86 (s, 1H), 4.53 (dd, J = 7.9, 2.5
Hz, 1H), 4.21 (t, J = 6.6 Hz, 1H), 4.12–4.04 (m, 2H), 3.86–3.81 (m, 1H), 3.41 (dd, J =
10.3, 5.4 Hz, 2H), 3.33–3.05 (m, 3H), 1.45 (d, J = 6.1 Hz, 3H). 13C NMR (151 MHz)
δ 169.2, 167.8, 163.9, 162.7, 135.1, 127.0, 119.8, 118.8, 115.8, 110.7, 72.7, 60.8,
57.9, 51.8, 39.0, 26.8, 18.3. HRMS: calcd for C17H24N9O7S2 [M+H]+ 530.1235, found
530.1245.
4.3.7
(2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[2-[(E)-3-(3,4-dihydroxyphenyl)
acrylamido]ethoxy]imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (12g).
Compound 12g was prepared from 8o (0.15 g, 0.30 mmol) and caffeic acid (57.0 mg,
0.32 mmol) by the procedure as described for 12e as off-white solid (0.10 g, 60%),
1
mp: 223.7 oC (dec.). H NMR (500 MHz) δ 9.37–9.16 (m, 2H), 7.68 (s, 1H), 7.46–
7.30 (m, 3H), 7.26 (s, 2H), 7.02 (d, J = 13.7 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.76 (d,
J = 1.9 Hz, 1H), 6.31 (d, J = 15.9 Hz, 1H), 4.47 (dd, J = 9.0, 3.5 Hz, 1H), 4.10 (t, J =
13
6.1 Hz, 2H), 3.76–3.70 (m, 1H), 3.38–3.31 (m, 2H), 1.42 (d, J = 6.1 Hz, 3H). C
NMR (151 MHz) δ 169.0, 162.9, 162.5, 162.1, 156.2, 152.2, 150.3, 143.1, 139.6,
126.3, 123.8, 121.5, 117.2, 116.2, 110.1, 72.8, 60.8, 57.4, 46.1, 18.5. HRMS: calcd for
C20H21N6O9S2 [M-H]- 553.0805, found 553.0819.
4.3.8 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[2-[2-(3,4-dihydroxyphenyl)acetami
do]ethoxy]imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic
acid
(12h).
Compound 12h was prepared from 8o (0.10 g, 0.20 mmol) and 3,4-dihydroxy
phenylacetic acid (37.0 mg, 0.22 mmol) by the procedure as described for 12e as
off-white solid (73.8 mg, 68%), mp: 194.5–195.4 oC. 1H NMR (500 MHz) δ 9.26 (d, J
= 7.6 Hz, 1H), 7.96 (s, 1H), 7.41 (s, 2H), 6.77 (s, 1H), 6.67 (d, J = 1.6 Hz, 1H), 6.63
(d, J = 7.9 Hz, 1H), 6.49 (d, J = 7.8 Hz, 1H), 4.46 (dd, J = 7.7, 2.4 Hz, 1H), 4.03 (s,
2H), 3.74–3.71 (m, 1H), 3.30 (d, J = 5.5 Hz, 2H), 3.23 (s, 2H), 1.42 (d, J = 6.0 Hz,
25

13
3H). C NMR (151 MHz) δ 173.2, 171.6, 169.1, 162.9, 162.6, 145.3, 144.2, 127.5,
120.3, 116.9, 115.8, 110.2, 73.0, 60.8, 57.4, 49.1, 42.5, 38.7, 18.5. HRMS: calcd for
C19H23N6O9S2 [M+H]+ 543.0962, found 543.0963.
4.3.9
(2S,3S)-3-{(Z)-2-[[2-[(R)-2-Amino-3-(1H-indol-3-yl)propanamido]ethoxy]
imino]-2-(2-aminothiazol-4-yl)acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid
(12i). Compound 12i was prepared from 8o (0.15 g, 0.30 mmol) and N-(tert-butoxy
carbonyl)-D-tryptophan (0.10 g, 0.33 mmol) by the procedure as described for 12a as
off-white solid (92 mg, 53%), mp: 156.9–158.5 oC. 1H NMR (500 MHz) δ 10.98 (d, J
= 1.8 Hz, 1H), 9.31 (d, J = 7.8 Hz, 1H), 8.49 (t, J = 5.6 Hz, 1H), 8.08 (d, J = 4.3 Hz,
4H), 7.62 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 2.3 Hz, 1H), 7.09
(t, J = 7.2 Hz, 1H), 7.01 (t, J = 7.2 Hz, 1H), 6.89 (s, 1H), 4.47 (dd, J = 7.8, 2.5 Hz,
1H), 4.13–3.92 (m, 3H), 3.78–3.74 (m, 1H), 3.38 (dd, J = 11.2, 5.6 Hz, 2H), 3.23–
13
3.12 (m, 2H), 1.42 (t, J = 8.8 Hz, 3H). C NMR (151 MHz) δ 169.7, 169.2, 162.6,
159.0, 158.8, 136.7, 127.6, 125.4, 121.6, 119.0, 118.9, 117.0, 115.0, 111.9, 110.9,
107.4, 72.8, 60.8, 57.5, 53.4, 38.8, 27.7, 18.5. HRMS: calcd for C22H27N8O7S2
[M+H]+ 579.1439, found 579.1439.
4.3.10 (2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[2-[(E)-3-(pyridin-4-yl)acrylamido]
ethoxy]imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (12j). Compound
12j was prepared from 8o (0.15 g, 0.30 mmol) and 4-pyridineacrylic acid (49.2 mg,
0.33 mmol) according to the procedure as described for 12e as off-white solid (72.2
mg, 46%), mp: 175.4–176.5 oC. 1H NMR (600 MHz) δ 9.24 (d, J = 8.1 Hz, 1H), 8.61
(d, J = 4.5 Hz, 2H), 8.16 (s, 1H), 7.70 (d, J = 4.2 Hz, 2H), 7.43 (d, J = 15.8 Hz, 1H),
7.25 (s, 2H), 6.96 (d, J = 15.8 Hz, 1H), 6.77 (s, 1H), 4.51 (d, J = 6.0 Hz, 1H), 4.11 (s,
2H), 3.75 (m, 1H), 3.55–3.40 (m, 3H), 1.40 (d, J = 5.9 Hz, 3H). 13C NMR (151 MHz)
δ 169.0, 164.9, 163.0, 162.8, 150.5, 149.7(2), 142.9, 136.7, 127.7, 122.8(2), 110.2,
26

73.0, 60.7, 57.5, 38.8, 18.4. HRMS: calcd for C19H22N7O7S2 [M+H]+ 524.1017, found
524.1025.
4.3.11
(2S,3S)-3-{(Z)-2-(2-Aminothiazol-4-yl)-2-[[2-(pyrimidine-4-carboxamido)
ethoxy]imino]acetamido}-2-methyl-4-oxoazetidine-1-sulfonic acid (12k). Compound
12k was prepared from 8o (0.15 g, 0.30 mmol) and 4-pyrimidinecarboxylic acid (39.7
mg, 0.32 mmol) according to the procedure as described for 12e as off-white solid
1
(80.8 mg, 54%), mp: 185 oC (dec.). H NMR (500 MHz) δ 9.37 (s, 1H), 9.27 (d, J =
8.1 Hz, 1H), 9.10 (t, J = 5.9 Hz, 1H), 9.06 (d, J = 5.0 Hz, 1H), 8.03 (d, J = 5.0 Hz,
1H), 7.26 (s, 2H), 6.73 (s, 1H), 4.47 (dd, J = 8.1, 2.5 Hz, 1H), 4.23–4.15 (m, 2H),
3.82–3.77 (m, 1H), 3.68–3.49 (m, 2H), 1.33 (d, J = 6.1 Hz, 3H). 13C NMR (151 MHz)
δ 169.0, 163.3, 162.9, 162.7, 160.1, 158.5, 156.7, 150.4, 143.1, 118.9, 110.0, 72.7,
60.7, 57.3, 39.3, 18.4. HRMS: calcd for C16H19N8O7S2 [M+H]+ 499.0813, found
499.0818.
4.4 General procedures for synthesis of compounds 22a–c
4.4.1 (2S,3S)-3-{(S,Z)-5-(tert-Butoxycarbonyl)-7-oxo-11-[2-(tritylamino)thiazol-4-yl]-
9-oxa-2-thia-6,10-diazadodec-10-enamido}-2-methyl-4-oxoazetidine-1-sulfonic acid
(21a). Chloroacetyl chloride (0.29 g, 2.53 mmol) was added to a solution of
(S)-1-(tert-butoxy)-4-(methylthio)-1-oxobutan-2-aminium chloride 13 (0.47 g, 1.94
o
mmol) and DIEA (0.63 g, 4.86 mmol) in DCM (10 mL) dropwise at 0 C. After
stirring for 5 min, the reaction mixture was allowed to warm to r.t.. Extracted with EA
(10 mL×3) and combined organic layers was dried over anhydrous sodium sulfate,
filtrated and concentrated under vacuum to give a brown oil residue 14 (0.51 g, 93%).
K2CO3 (0.32 g, 2.33 mmol) was added to a solution of PhtNOH (0.35 g, 2.14
mmol) in DMF (5.0 mL) . The reaction mixture was allowed to stir at r.t. for 30 min,
then there was plenty of solid precipitated out of solution. Compound 14 in DMF (1.0
27

o
mL) was added to above reaction mixture. After stirring at 40 C overnight, the
reaction mixture was allowed to cool to r.t.. Water (10.0 mL) was added dropwise into
the mixture, white solid precipitated out, filtered and collected the cake to give the
product 15 (0.83 g, 95%).
Hydrazine hydrate (1 mL) was added to the solution of compound 15 (0.83 g) in
DCM (5.0 mL). The mixture was stirred at r.t. for 6 h, the filtered. The filtration was
washed with water (10 mL×3) and the organics were combined and evaporated to
give the oil 16 (0.57 g).
To a solution of key intermediate 19 (0.56 g, 1.37 mmol) in MeOH (5.0 mL) was
added the above hydroxylamine 16 (0.6 g, 2.16 mmol), then stirred at r.t. for 1 h.
Concentrated the reaction mixture to afford the light-yellow residue. The residue was
purified by flash column chromatography (eluent: from 0–12% of MeOH in DCM) to
afford the intermediate 20 as white solid (0.47 g, 51%).
To a solution of 20 (0.30 g, 0.44 mmol) in anhydrous DMF (6 mL) was added
DIC (95.0 mg, 0.76 mmol) and HOBT (0.1 g, 0.76 mmol) successively. After stirred
at r.t. for 1 h, a solution of 6 (0.11 g, 0.58 mmol) in dry DMF (2.0 mL) with absolute
triethylamine (0.14 g) was added thereto, then stirred at r.t. overnight. The mixture
was diluted with water (20 mL) and extracted with EA (50 mL×3). The organic layers
were combined and washed with brine (50 mL×3), concentrated under vacuum to give
a brown oily residue. The residue was purified via flash column chromatography
(eluent: from 0–10% of methanol in DCM) to afford 21a (0.23 g, 78%) as foamy
1
solid. H NMR (400 MHz) δ 9.53 (d, J = 7.7 Hz, 1H), 8.88 (s, 1H), 7.77 (d, J = 8.0
Hz, 1H), 7.36–7.22 (m, 15H), 6.83 (s, 1H), 4.55 (q, J = 15.9 Hz, 2H), 4.41 (dd, J =
7.7, 2.4 Hz, 1H), 4.36–4.31 (m, 1H), 3.67–3.62 (m, 1H), 2.47–2.34 (m, 2H), 1.96 (s,
3H), 1.94–1.84 (m, 2H), 1.50–1.30 (m, 12H).
28

4.4.2
(S)-2-{2-[[(Z)-[1-(2-Aminothiazol-4-yl)-2-[[(2S,3S)-2-methyl-4-oxo-1-sulfo
azetidin-3-yl]amino]-2-oxoethylidene]amino]oxy]acetamido}-4-(methylthio)butanoic
acid (22a). Compound 22a was prepared from 21a (0.20 g, 0.23 mmol) according to
the procedure as described for 12f as off-white solid (87.5 mg, 68%), mp: 156.9–
1
158.1 oC. H NMR (400 MHz) δ 9.61 (d, J = 7.3 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H),
6.89 (s, 1H), 4.77–4.32 (m, 4H), 3.79–3.67 (m, 1H), 2.48–2.34 (m, 2H), 2.00–1.92 (m,
13
5H), 1.44 (d, J = 4.7 Hz, 3H). C NMR (151 MHz) δ 173.3, 169.6, 169.1, 162.0,
146.4, 128.8, 111.8, 73.4, 60.8, 57.5, 51.1, 31.1, 30.1, 18.5, 15.0. HRMS: calcd for
C16H23N6O9S3 [M+H]+ 539.0683, found 539.0689.
4.4.3
azetidin-3-yl]amino]-2-oxoethylidene]amino]oxy]acetamido}-2-phenylacetic
(22b). Compound 22b was prepared according to the procedure as described for 22a
(S)-2-{2-[[(Z)-[1-(2-Aminothiazol-4-yl)-2-[[(2S,3S)-2-methyl-4-oxo-1-sulfo
acid
o
1
as off-white solid (0.10 g, 32% overall yield), mp: 178.8–180.7 C. H NMR (400
MHz) δ 9.58 (d, J = 7.5 Hz, 1H), 8.36 (d, J = 7.6 Hz, 1H), 7.46–7.28 (m, 7H), 6.90 (s,
1H), 5.45 (d, J = 7.4 Hz, 1H), 4.66 (q, J = 15.6 Hz, 2H), 4.48 (d, J = 5.8 Hz, 1H), 3.73
13
(d, J = 5.7 Hz, 1H), 1.42 (d, J = 5.7 Hz, 3H). C NMR (151 MHz) δ 171.9, 171.9,
170.8, 169.5, 168.5, 162.1, 137.5, 137.4, 129.0, 129.0, 128.4, 128.0, 111.6, 73.2, 60.9,
57.5, 56.1, 18.5. HRMS: calcd for C19H21N6O9S2 [M+H]+ 541.0805, found 541.0812.
4.4.4
(R)-2-{2-[[(Z)-[1-(2-Aminothiazol-4-yl)-2-[[(2S,3S)-2-methyl-4-oxo-1-sulfo
azetidin-3-yl]amino]-2-oxoethylidene]amino]oxy]acetamido}-3-phenylpropanoic acid
(22c). Compound 22c was prepared according to the procedure as described for 22a
1
as off-white solid (90.5 mg, 34% overall yield), mp: 163.1–164.4 oC. H NMR (400
MHz) δ 9.71 (d, J = 7.8 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.37–7.08 (m, 7H), 6.97 (s,
1H), 4.67–4.39 (m, 4H), 3.77–3.72 (m, 1H), 3.14–3.07 (m, 1H), 2.91 (dd, J = 13.7,
13
9.9 Hz, 1H), 1.44 (d, J = 6.1 Hz, 2H). C NMR (151 MHz) δ 173.0, 170.8, 169.7,
29