Design, synthesis and biological evaluation of C(4) substituted monobactams as antibacterial agents against multidrug-resistant Gram-negative bacteria

Article abstract of DOI:10.1016/j.ejmech.2018.03.058

A series of novel pyridone conjugated monobactams with various substituents at the (4) position were synthesized and evaluated for their antibacterial activities against a panel of multidrug-resistant (MDR) Gram-negative bacteria in vitro. Compounds 46d, 54 and 75e displayed good to moderate activities against P. aeruginosa, among which the activity of 75e against P. aeruginosa was comparable to that of BAL30072 under iron limitation condition. Compounds 35, 46d, 54, 56a, 56c and 56d exhibited good to excellent antibacterial activities against E. coli and K. pneumoniae, which were comparable or superior to that of BAL30072. In vitro liver microsomal stability was further evaluated and the results manifested that Compounds 35, 46d and 54 were metabolically stable in human liver microsomes.

Full text of DOI:10.1016/j.ejmech.2018.03.058

Accepted Manuscript
Design, synthesis and biological evaluation of C(4) substituted monobactams as
antibacterial agents against multidrug-resistant Gram-negative bacteria
Qunhuan Kou, Ting Wang, Feng Zou, Shuhua Zhang, Qian Chen, Yushe Yang
PII:
S0223-5234(18)30301-5
10.1016/j.ejmech.2018.03.058
EJMECH 10323
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 February 2018
Revised Date: 15 March 2018
Accepted Date: 20 March 2018
Please cite this article as: Q. Kou, T. Wang, F. Zou, S. Zhang, Q. Chen, Y. Yang, Design, synthesis
and biological evaluation of C(4) substituted monobactams as antibacterial agents against multidrug-
resistant Gram-negative bacteria, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.03.058.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of C(4) Substituted
Monobactams as Antibacterial Agents Against Multidrug-Resistant
Gram-Negative Bacteria
Qunhuan Koua, b, Ting Wangc, Feng Zouc, Shuhua Zhangc, Qian Chena,b, Yushe Yanga,
b, *
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203, China.
bUniversity of Chinese Academy of Sciences, Beijing 100049, China.
cDepartment of Microbiology, Sichuan Primed Shines Bio-tech Co., Ltd, Chengdu, Sichuan
Province 610041, China.
*Corresponding author: Yushe Yang. Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:
ysyang@simm.ac.cn.
Abstract: A series of novel pyridone conjugated monobactams with various
substituents at the C(4) position were synthesized and evaluated for their antibacterial
activities against a panel of multidrug-resistant (MDR) Gram-negative bacteria in
vitro. Compounds 46d, 54 and 75e displayed good to moderate activities against P.
aeruginosa, among which the activity of 75e against P. aeruginosa was comparable to
that of BAL30072 under iron limitation condition. Compounds 35, 46d, 54, 56a, 56c
and 56d exhibited good to excellent antibacterial activities against E. coli and K.
pneumoniae, which were comparable or superior to that of BAL30072. In vitro liver
microsomal stability was further evaluated and the results manifested that Compounds
35, 46d and 54 were metabolically stable in human liver microsomes.
Keywords: monobactam; C(4) substituents; pyridone; MDR Gram-negative
bacteria; antibacterial activity.
1. Introduction
The continuing emergence of multidrug-resistant (MDR) Gram-negative bacteria
such as Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa
and Escherichia coli has become a significant and ever increasing threat to public
health [1]. Unfortunately few antibiotics are in the late-stage clinical development
pipeline to treat these intractable infections caused by such strains [2, 3]. Therefore,
there is an urgent need to discover and develop new antibiotics to fight against MDR
Gram-negative bacteria.
One of the largest challenges in the discovery of new Gram-negative
antibacterial agents is the reduction of outer membrane permeability [4, 5]. One useful
strategy to circumvent the penetration issue is to exploit the iron uptake pathway of
Gram-negative pathogens [6, 7]. Bacteria requires iron as an essential nutrient for
survival. In order to scavenge iron from the host, bacteria manufactures and secretes
iron chelators known as siderophores. The iron-complexed siderophores are
recognized by specific outer membrane receptors and translocated into the bacterial
cell [8]. Conjugation of siderophore to antibacterial agent enables active delivery of
the antibacterial moiety to the bacteria to circumvent the permeability-mediated
resistance, known as a “Trojan Horse” strategy [6, 9-12]. Natural siderophores usually
most often contain three bidentate ligands for stoichiometric binding of iron (III) [9].
To reduce structural complexity, a large number of simplified conjugates, which
utilize a single bidentate ligand such as a catechol, have been studied since the 1980s
[13, 14]. However, catechol moiety has been shown to undergo metabolism in vivo by
catechol O-methyltransferase (COMT) and loose its antibacterial activity [15, 16].
Nobuhiro Ohi et al. reported that the resistance of penicillin to metabolism by COMT
was increased by introduction of the chlorine atom into the catechol moiety [17]. One

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precedent example is compound S-649266, a cephalosporin conjugated with catechol
carrying a chloride atom the position adjacent to the hydroxyl group, which has
progressed into phase III clinical trial [18, 19]. Hydroxypyridone moiety, “unnatural”
siderophore mimic, also appears to circumvent this undesired metabolic pathway and
can be recognized by siderophore receptors, which has been employed successfully as
siderophore mimic [20-22].
Monocyclic β-lactam antibiotics display excellent antimicrobial activities against
Gram-negative bacteria [23, 24]. They target the penicillin-binding proteins (PBPs),
which are located in the periplasm. Only the outer membrane has to be crossed by
monobactams to get to their targets. So monobactams are attractive drug moieties to
conjugate to siderophore mimics [6]. Structures of the representative monocyclic
β-lactams are shown in Figure 1. Aztreonam (1) and Carumonam (2) have been used
in the clinic for several decades. However, they were reported to be ineffective against
MDR Gram-negative bacteria in recent years [25, 26]. LYS-228 (3) with
oxazolidinonemethyl group at the C(4) position, has progressed into phaseⅡclinical
trial at Novartis for the treatment of Enterobacteriaceae infection [27]. MB-1 (4), 5
and BAL30072 (6) are novel monobactams that are conjugated to siderophore mimics.
The Pfizer group revealed that the monobactam MB-1 with pyridone as siderophore
mimic showed good in vitro antibacterial activity against clinically relevant
Gram-negative bacteria. However, it was reported to be ineffective in vivo [21, 28].
Recently, Marvin J. Miller group reported the synthesis of monobactam 5 with
bis-catechol siderophore mimic at the C(4) position, which had potent activity against
Acinetobacter baumannii [29]. BAL30072 with pyridone at oxime side chain,
discovered by Basilea Pharmaceutica, has ever been investigated in phase I clinical
studies. But it was dropped because of liver toxicity in healthy humans in
multiple-dose clinical studies [30]. BAL30072 showed good activity against P.
aeruginosa and Acinetobacter baumannii. However, it was reported to be ineffective
against K. pneumoniae [31, 32].
Figure 1. Structures of the representative monocyclic β-lactams
Our group previously reported that antibacterial activity improved greatly when a
isopropyl substituent was introduced to the oxime side chain on the basis of the
structure of BAL-30072 [33], possibly due to the interaction with a hydrophobic
pocket formed by Tyr503, Tyr532 and Phe533 [34]. It has been shown that
substitution at the C(4) position by alkyl groups led to compounds with improved
antibacterial activities against Gram-negative bacteria and better stability against

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β-lactamases [29, 35-37]. In this study, we designed and synthesized a series of C(4)
substituted monobactams and evaluated their antibacterial activities against a panel of
MDR Gram-negative bacteria.
Figure 2. Design of C(4) substituted monobactams
2. Results and discussion
2.1 chemistry
The synthetic route of target compounds 17a-b is described in Scheme 1. The
amino groups of compounds 7a-b were protected with (Boc)2O seperately, and then
converted 7a-b to ester 8a-b through methylation reaction. Oxidation of 8a-b using
SeO2 and t-BuOOH afforded 9a (dr 4:1) and 9b (dr 1:1) [38]. Compounds 9a-b were
converted to hydroxamic acids, followed by acetylation to afford intermediates 10a-b.
The Mitsunobu reaction was performed to transform 10a-b to the corresponding
ring-closed products 11a-b seperately. Removal of the acetyl group using Na2CO3
yielded intermediates 12a-b [39]. Sulfonation of 12a-b with an excess of SO3·DMF
complex gave tetra-n-butylammonium salt 13a-b, followed by deprotection of the
Boc group under acid condition to give the corresponding amines 14a-b. A
condensation reaction was carried out between acid 15 and amines 14a-b to afford
intermediates 16a-b [33]. Subsequent deprotection of the protecting groups provided
the target compounds 17a-b.

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Scheme 1. Synthetic route of the title compounds 17a-b. Reagents and conditions: (a) (Boc)2O,
NaOH, 1,4-dioxane/H2O = 1/1, room temp, overnight; (b) MeI, K2CO3, acetone, room temp, 10 h;
(c) SeO2, t-BuOOH, dichloroethane, 70°C, 24 h; (d) H2NOH·HCl, KOH, MeOH, 0°C, 45 min; (e)
Ac2O, MeOH, 0°C, 1 h; (f) for 10a, PPh3, DIAD, THF, 0°C, 1 h; (g) for 10b, PPh3, DEAD, THF,
0°C, 1 h; (h) Na2CO3, MeOH, 0°C, 1 h; (i) SO3·DMF, Bu4NHSO4, DMF, room temp, 1 h; (j) TFA,
Et3SiH, DCM, 0°C then room temp, 2 h; (k) 15, HATU, NaHCO3, DMSO, room temp, 3 h; (l)
TFA, Et3SiH, DCM, -15°C then room temp, 15 h.
The target compounds 27a-e were afforded according to the procedure illustrated
in Scheme 2. The aldol reaction was carried out between 1,3-difluoroacetone or
various cyclic ketones and 18 to give 19a-e seperately [40]. Then hydrogenolytic
removal of the Bn group yielded intermediates acids 20a-e, which were coupled with
BnONH2 to give amides 21a−e. The Mitsunobu reaction was carried out to transform
21a-b to the corresponding ring-closed products 22a-b seperately. The hydroxyl
groups in 21c-e were reacted with SO3·Py complex, followed by intramolecular
nucleophilic substitution reaction to afford 22c-e. Deprotection of Bn group afforded
hydroxamic acids 23a-e. Intermediates 23a-e were sulfonated with SO3·DMF
complex to give tetra-n-butylammonium salt 24a-e, which were then converted to the
target monobactams 27a-e by the same manner as described for 17a-b.
Scheme 2. Synthetic route of the title compounds 27a-e. Reagents and conditions: (a) for a,
1,3-difluoroacetone, for b-e, Cyclic ketones, n-BuLi, i-Pr2NH, THF, -78°C then -20°C, 2 h; (b) 10%
Pd/C, H2, EtOH, room temp, 7 h; (c) BnONH2·HCl, EDCI·HCl, HOBT, NEt3, DCM, room temp,
overnight; (d) for 21a-b, PPh3, DEAD, THF, 0°C, 1 h; (e) for 21c-e, 1), SO3·Py, Py, 60°C, 2.5 h;
2), K2CO3, EA/H2O (3/1), 80°C, 5 h; (f) 10% Pd/C, H2, MeOH, room temp, 2 h; (g) SO3·Py,
Bu4NHSO4, Py, 60°C, 3 h; (h) TFA, Et3SiH, DCM, 0°C then room temp, 2 h; (i) 15, HATU,
NaHCO3, DMSO, room temp, 3 h; (j) TFA, Et3SiH, DCM, -15°C then room temp, 15 h.

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The synthetic route of target compound 35 is described in Scheme 3. 29 was
synthesized by addition of Allylmagnesium bromide to N-(Boc)serine methyl ester.
Ring-closure metathesis reaction of 29 was performed using 2nd Grubbs catalyst to
afford 30 [41]. Reduction of the double bond in the five-membered ring of 30 with
Wilkinson’s catalyst gave saturated product 31. Intermediate 32 was obtained by
primary alcohol oxidation. Then 32 was converted to the target monobactam 35 by the
same manner as described for 17a-b.
Scheme 3. Synthetic route of the title compounds 35. Reagents and conditions: (a)
Allylmagnesium bromide, THF, -78°C then room temp, 2h; (b) 2nd Grubbs catalyst, DCM, 40°
C, overnight; (c) Wilkinson’s catalyst, H2, EtOH, 60°C, 7 h; (d) TEMPO, NaClO2, NaClO,
CH3CN/H2O, 35°C, 24 h; (e) BnONH2·HCl, EDCI·HCl, HOBT, NEt3, DCM, room temp,
overnight; (f) SO3·Py, Py, 60°C, 2.5 h; (g) K2CO3, EA/H2O (3/1), 80°C, 5 h; (h) 10% Pd/C, H2,
MeOH, room temp, 2 h; (i) SO3·Py, Bu4NHSO4, Py, 60°C, 3 h; (j) TFA, Et3SiH, DCM, 0°C then
room temp, 2 h; (k) 15, HATU, NaHCO3, DMSO, room temp, 3 h; (l) TFA, Et3SiH, DCM, -15°C
then room temp, 15 h.
Key intermediate 43 was afforded according the procedure illustrated in Scheme
4. 37 was obtained from L-Ascorbic acid (36) upon treatment with
2,2-dimethoxypropane. Oxidation of 37 with hydrogen peroxide yielded the threonic
acid derivative, which was then transformed to the methyl ester 38 [42]. 38 was
treated with Tf2O under basic condition, followed by nucleophilic substitution with
NaN3 to give intermediate 39. Protecting group in 39 was quickly removed to prevent
the production of lactone, and then selective protection of primary hydroxyl with
Ph3CCl afforded intermediate 40. Compound 40 was subjected to hydrolysis under
alkaline condition, followed by condensation with BnONH2 to give 42. The key
intermediate 43 was obtained through Mitsunobu reaction. The stereochemistry of 43
was confirmed by X-ray crystallography.

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Scheme 4. Synthetic route of the key intermediate 43. Reagents and conditions: (a)
2,2-dimethoxypropane, HCl·MeOH, acetone, room temp, 4 h; (b) H2O2, K2CO3, H2O, 0°C then
room temp, 16 h; (c) MeI, DMF, room temp, overnight; (d) Tf2O, Py, DCM, -15°C, 1 h; (e)
NaN3, Acetone /Water=3/1, room temp, overnight; (f) HCl·MeOH, MeOH, room temp, 2 min; (g)
Ph3CCl, Py, DMAP, DCM, room temp, overnight; (h) NaOH, CH3CN/H2O = 1/1, room temp, 0.5
h; (i) BnONH2·HCl, EDCI·HCl, HOBT, NEt3, DCM, room temp, overnight; (j) PPh3, DEAD,
THF, 0°C, 1 h.
The preparation route for compounds 46a-b is outlined in Scheme 5.
Hydrogenolytic removal of the Bn group and reduction of the azide group in 43
afforded N-hydroxy-2-azetidinone, followed by reduction of the N-O bond using
TiCl3 to give intermediate 44 [43]. A condensation reaction was carried out between
acid 15 and 44 to afford intermediate 45. The sulfonation reaction of the lactam
nitrogen in 45 was performed, and then deprotection of the protecting group afforded
target compound 46a. The CPh3 group in intermediate 44 was removed under acidic
condition to yield 47, followed by condensation reaction with acid 15 to give key
intermediate 48. Compound 49 was obtained through sulfonation reaction of both the
hydroxyl and lactam nitrogen, followed by deprotection to afford target compound
46b.
Scheme 5. Synthetic route of the title compounds 46a-b. Reagents and conditions: (a) 10% Pd/C,
H2, THF, room temp, 7 h; (b) TiCl3, NaOH, MeOH/H2O, room temp, 0.5 h; (c) 15, HATU,
NaHCO3, DMSO, room temp, 3 h; (d) SO3·DMF, DMF, room temp, 1 h; (e) TFA, Et3SiH, DCM,
-15°C then room temp, 15 h.
Target compounds 46c-d, 54 and 56a-b were prepared from intermediate 48 as
shown in Scheme 6. Acetylation of hydroxyl in 48 yielded 50. Then 50 was converted
to the target compound 46c by the same manner as described for 46a. 48 was treated
with trichloroacetyl isocyanate, followed by sulfonation reaction and deprotection of
the trichloroacetyl group to afford 4-carbamoyloxymethyl derivative 51. Target
compound 46d was obtained by removal of the protection group of 51. Mesylation of
48 gave the 4-mesyloxymethyl intermediate 52, which was then transformed into the
4-chloromethyl derivative 53. 53 was converted to the target compound 54 by the
same manner as described for 46a. The Mitsunobu reaction was performed between
48 and 4-methylthiazole-2-thiol or 2-mercaptobenzoxazole to afford 55a-b smoothly.
Then 55a-b were converted to the target compounds 56a-b by the same manner as
described for 46a.
Target compounds 46e-g could not be obtained from intermediates 48 or 52.
Methylation of 48 failed with iodomethane or Meerwein salt. Treatment of 52 with
nucleophiles under basic condition could not afford substituted products. Instead

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intramolecular reaction of 52 was carried out. So we had to prepare 46e-g from 57, as
shown in Scheme 7. Methylation of 57 afforded intermediate 58a using Meerwein
salt. Trifluoromethanesulfonylation of hydroxyl group in compound 57 gave
intermediate 59, followed by nucleophilic substitution with tritylpiperazine or
N1-tritylethane-1,2-diamine separately to afford 58b and 60. Then the ring closing
reaction of 60 was carried out using triphosgene to obtain 58c. Intermediates 58a-c
were then converted to the target monobactams 46e-g by the same manner as
described for 46a.
Scheme 6. Synthetic route of the title compounds 46c-d, 54, 56a-b. Reagents and conditions: (a)
Ac2O, Py, DCM, room temp, 1 h; (b) SO3·DMF, DMF, room temp, 1 h; (c) TFA, Et3SiH, DCM,
-15°C then room temp, 15 h; (d) Trichloroacetyl isocyanate, DCM, room temp, 2 h; (e) NaHCO3,
MeOH, room temp, 1 h; (f) MsCl, NEt3, DMAP, DCM, room temp, room temp, 24 h; (g) LiCl,
acetone, 50°C, 36 h; (h) ArSH, PPh3, DEAD, DCM, 0°C then room temp, overnight.

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Scheme 7. Synthetic route of the title compounds 46e-g. Reagents and conditions: (a)
Trimethyloxonium Tetrafluoroborate, 2,6-di-tert-butyl-4-methylpyridine, DCM, room temp, 4 h;
(b) Tf2O, Py, DCM, -15°C, 0.5 h; (c) Tritylpiperazine, K2CO3, THF, room temp, 1 h; (d)
N1-tritylethane-1,2-diamine, K2CO3, THF, room temp, 1 h; (e) Triphosgene, Triethylamine, DCM,
0°C, 1 h; (f) 10% Pd/C, H2, THF, room temp, 7 h; (g) TiCl3, NaOH, MeOH/H2O, room temp, 0.5 h;
(h) 15, HATU, NaHCO3, DMSO, room temp, 3 h; (i) SO3·DMF, DMF, room temp, 1 h; (j) TFA,
Et3SiH, DCM, -15°C then room temp, 15 h.
Target compounds 56c-e could not be obtained through Mitsunobu reaction from
intermediate 48 or nucleophilic substitution reaction from 52. 56c-e had to be
synthesized from 57, as shown in Scheme 8. 57 was treated with TsCl to give 63.
Then intermediate 64 was obtained by deprotection of Bn group, reduction of the
azide group to amino group and protection of the amino group with TrocCl in one pot
under hydrogen atmosphere. 69a-c were obtained from 64 by two routes. For 69a-b,
64 was transformed into 4-iodomethyl derivative 65, which was treated with
5-Mercapto-1-methyltetrazole or 2-mercapto-5-methyl-1,3,4-thiadiazole separately to
give 66a-b, followed by reduction of N-O bond to afford 69a-b. For 69c, the N-O
bond in 64 was cleaved firstly, followed by transformation into 68 and substitution
with thiotriazinone to afford 69c. 70a-c were obtained through sulfonation reaction of
the lactam nitrogen in 69a-c, and then deprotection of Troc with Zn powder afforded
amines 71a-c. Intermediates 71a-c were converted to the target compounds 56c-e by
the same manner as described for 17a-b.

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Scheme 8. Synthetic route of the title compounds 56c-e. Reagents and conditions: (a) TsCl, NEt3,
DMAP, DCM, room temp, overnight; (b) 10% Pd/C, H2, TrocCl, THF, room temp, 7 h; (c) NaI,
Acetone, 50°C, 36h; (d) 5-mercapto-1-methyltetrazole, 2-mercapto-5-methyl-1,3,4-thiadiazole or
Thiotriazinone, NaH, DMF, 0°C then room temp, 30 h; (e) TiCl3, NaOH, MeOH/H2O, room temp,
0.5 h; (f) SO3·DMF, DMF, room temp, 1 h; (g) Zn, KH2PO4, THF/H2O (5:1) room temp, 1 h; (h)
15, HATU, NaHCO3, DMSO, room temp, 4 h; (i) TFA, Et3SiH, DCM, -15°C then room temp, 15
h.
The preparation of target compounds 75a-d is outlined in Scheme 9.
Condensation reaction was performed between 48 and 73a-d (details for the synthesis
of acid 73a-d could be found in Supporting Information) to afford intermediates
74a-d. Intermediates 74a-d were then converted to the target monobactams 75a-d by
the same manner as described for 46a.
OCHPh2
OCHPh2
OCHPh2
OCHPh2
O
OH
N
O
O
O
N
O
O
N
OH
O
O
O
O
O
R
R
O
O
N
N
H
OH
N
a
H
+
H
O
b, c
N
HO
R
N
N
N
N
N
Ph3CHN
73a, 74a R=
75a R=
Ph3CHN
73a-d
H2N
NH
NH
N
S
S
SO3H
S
O
O
74a-d
O
75a-d
48
OCHPh2
OCHPh2
OCHPh2
OCHPh2
O
Cl
Cl
Cl
73d, 74d R=
73c, 74c R=
H
N
73b, 74b R=
OCHPh2
OCHPh2
OCHPh2
OCHPh2
H
N
N
O
N
O
N
O
O
OH
OH
OH
Cl
OH
75c R=
H
75d R=
OH
OH
75b R=
OH
OH
H
N
N
N
N
N
H
O
OH
O
Scheme 9. Synthetic route of the title compounds 75a-d. Reagents and conditions: (a) DCC,
DMAP, DCM, room temp, 24 h; (b) SO3·DMF, DMF, room temp, 1 h; (c) TFA, Et3SiH, DCM,
-15°C then room temp, 15 h.
The synthetic route of target compound 75e is described in Scheme 10. 68 was
transformed into 76, followed by click reaction with 77 to afford 78. Removal of the

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Troc group and reduction of N-O bond gave 79, which was reprotected with FmocCl
and oxidated with mCPBA to afford 81. Deprotection of Fmoc group in 81 afforded
82. 82 was then converted to the target compound 75e by the same manner as
described for 46a.
Scheme 10. Synthetic route of the title compounds 75e. Reagents and conditions: (a) NaN3, DMF,
room temp, 72 h; (b) CuSO4, Sodium Lascorbate, DMSO/H2O/i-PrOH=1/1/1, room temp, 24 h; (c)
Zn, KH2PO4, THF/H2O (5:1), room temp, 1 h; (d) FmocCl, NEt3, DCM, 0°C then room temp, 1 h;
(e) mCPBA, DCM, room temp, 1 h; (f) Piperidine, DCM, room temp, 1 h; (g) 15, HATU,
NaHCO3, DMSO, room temp, 4 h; (h) SO3·DMF, DMF, room temp, 1 h; (i) TFA, Et3SiH, -15°C
then room temp, 15 h.
2.2. In vitro antibacterial activity
The MIC values were determined using the broth microdilution protocol
according to the methods of the Clinical and Laboratory Standards Institute (CLSI).
Normal MIC values were determined in the Mueller−Hinton broth (Oxoid), while
MIC values under iron limitation were determined in the Mueller−Hinton broth
(Oxoid) supplemented with 16 mg/liter 2,2’-bipyridyl (BPL) to complex iron and
induce iron uptake systems [31]. The iron-limited growth condition was believed to
mimic the in vivo situation, in which iron was bound by a variety of proteins and has
previously been used to evaluate the activities of siderophore antibiotics [31].
Aztreonam, meropenem (provided by Sichuan Primed Shines Bio-Tech Group
Co.,Ltd.), and BAL30072 (synthesized according to published procedures [44]) were
served as the positive control drugs. The tested bacterial strains were all clinical
isolates. These strains included extended spectrum β-lactamases (ESBLs) producing E.
coli, Klebsiella pneumoniae carbapenemase (KPC-2) producing MDR K. pneumoniae,
and imipenemase (IMP-4) producing MDR P. aeruginosa, with five strains in each
family, respectively. All the target compounds were phenotypically screened against
these strains, and their activities are presented as minimum inhibitory concentrations
(MICs). The results are shown in Tables 1−3 as ranges of MIC values, and individual
MIC Data of tested compounds can be found in Supporting Information.
Ethenyl, ethynyl and gem-difluorine methylene were firstly introduced to the C(4)
position to replace gem-dimethyl of BAL30072, affording analogues 17a-b, 27a

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(Table 1). For the purpose of rapid biological evaluation of target compounds, their
racemates were synthesized in the beginning. Unfortunately, these slight
modifications led to a complete loss of antibacterial activity, no matter in normal or
iron limitation growth medium. It seems that unsaturated substituents and
gem-difluorine methylene were not tolerated at the C(4) position when
dihydroxypyridone moiety was at the oxime side chain. Constraining the
gem-dimethyl substituent via ring formation provided a series of spirocyclic scaffold
compounds. Spirocyclic scaffolds were considered to be privileged structures because
of their interesting conformational features and pharmacological properties [45].
27b-e displayed moderate activities against most tested strains. Activities of 27b-e
against Enterobacteriaceae strains (concluding E. coli and K. pneumoniae) under iron
limitation condition increased 2–16-fold relative to those of 27b-e at normal condition.
Iron uptake system was proved to be induced under iron limitation condition [31].
Compared with 27b, azaspiro[3.4]octane analogue 27c showed more potent
antibacterial activity. However, further increasing the size of the spirocyclic resulted
in significantly reduced activity (27d vs 27c). 27e bearing O atom in the spirocyclic
got the activity recovered against Enterobacteriaceae strains, but still had no activity
against P. aeruginosa. 27c was the most potent analogue among the spirocyclic
compounds and its stereoisomer 35 with preferred S configuration at C3 position of
monobactams was synthesized [36]. Activity of 35 improved significantly as
compared to that of 27c. 35 displayed moderate activity against P. aeruginosa and
excellent activity against Enterobacteriaceae strains.
Table 1. In vitro antibacterial Activity of Monobactams 17, 27 and 35 (MICa, ꢀg/mL)
Eco
iron
Kpn
iron
Pae
iron
Comp
d
normal
foldb
1-32
normal
fold
normal
fold
1-2
limitation
2-≥64
≥64
limitation
≥64
limitation
≥64
≥64
≥64
>64
>64
>64
≥64
17a
17b
27a
27b
27c
>64
32-≥64
≥64
>64
>64
32->64
8->64
2-8
4->64
4-16
8
>64
1-4
0.5-2
2-8
2-4
2-8
1-4
1-8
2
2-8
16-32
16-32
≥64
16-32
8-16
1-2
1-2
0.5-4
16->64
1-16
0.25-1
4->64
1-8
1
2-8
8-16
2-8
1-4
2-16
2-32
4-16
2-4
≥64
27d
27e
0.06-2
0.06-0.5
0.25->64
>64
≥64
0.5-1
0.5-1
0.5-32
0.06-0.5
0.25-0.5
1-2
8-16
2-8
8-16
1-2
1-2
35
BALc
AZTd
1->64
>64
1-4
32-64

ACCEPTED MANUSCRIPT
MERe
0.125-8
≥64
32-64
a Eco, E. coli, 5 strains; Kpn, MDR K. pneumoniae, 5 strains; Pae, MDR P. aeruginosa, 5 strains.
b fold value= MIC(normal)/MIC(iron limitation)
.
c BAL = BAL30072; d AZT = Aztronam; e MER = Meropenem.
To further explore the SAR of this series of compounds, several polar
substituents were introduced to the C(4) position of monobactams to increase activity
against MDR Gram-negative bacteria [46]. As shown in Table 2, the 4-
hydroxymethyl compound 46a showed significant loss of activity. Sulfonation of the
hydroxymethyl group of 46a to 4-sulfonyloxymethyl compound 46b resulted in a
boost in potency against K. pneumoniae. The activity of 46b against K. pneumoniae
under iron limitation condition improved 4–16-fold compared with that of 46b under
normal condition. The hydroxyl derivatives 46c, 46d, 46e and 54 displayed good to
excellent antibacterial activities. Especially, 46d and 54 displayed 2–4-fold reduction
in activity against P. aeruginosa than BAL30072, and showed improved potency
against Enterobacteriaceae strains compared with BAL30072. Incorporation of
piperazine and imidazolidone to the C(4) position (46f-g) resulted in a loss of activity
against P. aeruginosa. Their activities against Enterobacteriaceae strains were
slightly reduced or maintained.
Table 2. In vitro antibacterial Activity of Monobactams 46, 54and 56 (MICa, ꢀg/mL)
Eco
iron
Kpn
iron
Pae
iron
Com
pd
normal
foldb
normal
fold
1-8
normal
fold
limitation
32->64
2-4
limitation
2-16
limitation
16->64
32
≥64
2-8
1-2
1-4
1-4
1-4
1-4
1-4
1-4
1-4
1-8
2-16
2-4
1-4
2-4
8-16
0.5-2
2-8
≥64
32->64
≥64
1-4
1-2
1-4
1-2
1-2
1-2
1
46a
46b
46c
46d
46e
54
0.125-0.5 4-16
0.5-1
0.25-1
0.5-1
0.5-1
1-2
0.25-1
0.125-0.25
0.125-0.5
0.25-0.5
0.5-1
0.5-1
0.25-8
0.125-4
0.25-8
0.5->64
0.125-4
0.5-2
2-16
2-4
2-8
1-4
1-8
2-4
1-2
2-8
1-8
2-8
1-8
16->64
4-8
1-32
0.5-16
0.5-32
2->64
0.25-16
1-2
4-8
8-16
8
8-16
4-8
32->64
32-64
≥64
32->64
32-64
≥64
46f
46g
56a
56d
56c
56e
56b
0.5-2
0.5-2
0.5-4
0.125-1
2-4
0.25-2
0.25-2
0.125-1
0.06-0.5
1-4
1
1
1-2
0.25-0.5
0.25-1
0.25-2
0.5-2
32->64
32->64
≥64
16->64
16-64
32->64
≥64
1-4
1-2
1
1-2
2-4
1-4
0.5-2
2-4
≥64
1

ACCEPTED MANUSCRIPT
BA
Lc
0.5-1
0.25-0.5
2
1->64
0.25->64
2-4
2-8
1-4
1-2
a Eco, E. coli, 5 strains; Kpn, MDR K. pneumoniae, 5 strains; Pae, MDR P. aeruginosa, 5 strains.
b fold value= MIC(normal)/MIC(iron limitation)
.
c BAL = BAL30072.
As observed for previously reported cephalosporin analogues, aromatic thioether
group was typically used as the C3 side chain of cephalosporin and usually endowed
compounds with good activity against P. aeruginosa [47]. In light of this
understanding, we try to incorporate aromatic thioether groups to C(4) position of
monobactam to increase antibacterial activity against P. aeruginosa. Unfortunately,
56a-e had no activity against P. aeruginosa at all. However, they exhibited excellent
activity against Enterobacteriaceae strains. Five-membered ring thioether substituents
at the C(4) position were more potent than six-membered ring and benzoxazole ring
thioether attachment. (56a, 56d and 56c vs 56e and 56b). The MIC values of 56a, 56d
and 56c ranged from 0.125 to 4 ꢀg/mL against E. coli and from 1 to 2 ꢀg/mL against
K. pneumonia. Activities of 56a, 56d and 56c against most test strains of K.
pneumoniae improved 32-fold as compared to that of BAL30072
.
Table 3. In vitro antibacterial activity of monobactams 75 (MICa, ꢀg/mL)
Eco
Kpn
Pae
Com
pd
normal
iron
foldb
normal
iron
fold
normal
iron
fold
limitation
8->64
2-64
limitation
4
limitation
32->64
16->64
32->64
2-8
16->64
8->64
16-32
4-8
1-2
1-8
1-8
2-4
2-16
2
16-32
>64
>64
8-32
2
4-8
8-16
4
≥64
32->64
≥64
1-2
1-2
1-2
1-2
1-8
1-2
75a
75b
75c
75d
75e
BA
Lc
4-8
4-16
16->64
2-4
1-4
2-16
8
4-16
4-16
2-8
16
1-8
0.25
1-4
0.5-1
0.25-0.5
1->64
0.25->64
2-4
1-4
a Eco, E. coli, 5 strains; Kpn, MDR K. pneumoniae, 5 strains; Pae, MDR P. aeruginosa, 5 strains.
b fold value= MIC(normal)/MIC(iron limitation)
.
c BAL = BAL30072.
Natural siderophores most often contained three bidentate ligands. They
possessed high affinity for iron and could scavenge iron at low concentration of iron

ACCEPTED MANUSCRIPT
[9]. In order to mimic the natural siderophore and increase the ability of iron chelation,
we designed and synthesized bis-siderophore mimic compounds 75a-e, which might
increase antibacterial activity, broaden the antibacterial spectrum and overcome
adaptation-based resistance [28]. Bacteria excreted a wide variety of siderophores
with different structural types, and catechol as a common structural type of
siderophore was introduced initially to the C(4) position [6]. As shown in Table 3,
antibacterial activity of 75a-b reduced noticeably. Then pyridone was introduced to
the C(4) position. The dihydroxypyridone group in 75d was more preferable than
hydroxypyridone group in 75c. Then we conserved dihydroxypyridone as siderophore
mimic and change the linker to triazole to afford 75e, which exhibited good to
moderate antibacterial activity against tested strains. The antibacterial activity of 75e
under iron limitation condition improved remarkably than that of 75e at normal
condition. Especially, the activity of 75e against P. aeruginosa under iron limitation
condition was equivalent to that of BAL30072 with MIC ranging from 1 to 4 ꢀg/mL.
To further investigate the pharmacokinetic profile of these compounds, we
evaluated the metabolic stability of compounds 35, 46d, 54, 75e and BAL30072 in
human and mouse liver microsomes (Table 4). Like BAL30072, compounds 35, 46d
and 54 exhibited a long half-life, low liver microsomal clearance and high metabolic
bioavailability in human liver microsomes which manifested that these three
compounds were metabolically stable. Compounds 75e showed relatively unfavorable
liver microsomal stability both in human and mouse liver microsomes.
Table 4. Half-lives and intrinsic clearances of selected compounds in liver
microsomesa
c
compounds
species
t1/2b (min)
Clint
MFd (%)
(ml/min/g protein)
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
271
375
176
616
189
72.3
80.1
77.4
285
199
7.76
5.59
11.9
3.41
11.1
29.0
26.2
27.1
7.38
10.5
71.6
88.8
62.2
92.9
63.9
60.5
42.8
62.1
72.7
80.8
35
46d
54
75e
BAL30072
a 0.33 mg/mL microsomal protein, NADP+-regenerating system, [inhibitor], 0.1 ꢀM, incubation at
37°C, samples taken at 0, 7, 17, 30, and 60 min, determination of parent compound by MS.
b t1/2: elimination half-life.
c Clint: intrinsic clearance.
d MF: metabolic bioavailability.
3. Conclusion
In conclusion, twenty-six C(4) substituted monobactams were synthesized and
evaluated for their antibacterial activities against a panel of MDR Gram negative
strains. C(4) substituents exerted significant influences on the antibacterial activities.
Antibacterial activities of most compounds under iron limitation condition improved
significantly compared with those at normal condition. Compounds with
azaspiro[3.4]octane, carbamoyloxymethyl, chloridemethyl and five-membered ring
thioether as C(4) substituents (35, 46d, 54, 56a, 56c and 56d) exhibited good to
excellent antibacterial activities against E. coli, which were comparable or slightly
superior relative to that of BAL30072. Especially, activities of these six compounds
against K. pneumonia improved remarkably than that of BAL30072. 46d, 54 and
bis-siderophore mimic 75e showed good to moderate activities against MDR P.

ACCEPTED MANUSCRIPT
aeruginosa under normal condition. Under iron limitation condition, the activity of
75e against P. aeruginosa was equivalent to that of BAL30072. Liver microsomal
stability assay manifested that compounds 35, 46d and 54 were metabolically stable
in human liver microsomes, while compound 75e showed relatively poor metabolic
stability. The synthetic strategy toward target compounds will facilitate the synthesis
of various C(4) substituted monobactams. These results will provide deeper insight
into further optimization of monobactam derivatives for the treatment of infections
caused by MDR Gram-negative bacteria.
Acknowledgment
This work was supported by grants from the National Natural Science Foundation of
China (no. 81573284) and the National Science and Technology Major Project of
China (no. 2014ZX09507009-013).
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novel pyridone conjugated monobactams with various substituents at the C(4)
position.
35, 46d, 54, and 56a showed potent activities against Enterobacteriaceae strains.
75e displayed good to moderate activities against multidrug-resistant P. aeruginosa.
35, 46d and 54 were metabolically stable in human liver microsomes.