Synergism of fused bicyclic 2-aminothiazolyl compounds with polymyxin B against: Klebsiella pneumoniae

Article abstract of DOI:10.1039/c7md00354d

A series of fused bicyclic 2-aminothiazolyl compounds were synthesized and evaluated for their synergistic effects with polymyxin B (PB) against Klebsiella pneumoniae (SIPI-KPN-1712). Some of the synthesized compounds exhibited synergistic activity. When 4 μg ml^-1 compound B1 was combined with PB, it showed potent antibacterial activity, achieving 64-fold reduction of the MIC of PB. Furthermore, compound B1 showed prominent synergistic efficacy in both concentration gradient and time-kill curves in vitro. In addition, B1 combined with PB also exhibited synergistic and partial synergistic effect against E. coli (ATCC25922 and its clinical isolates), Acinetobacter baumannii (ATCC19606 and its clinical isolates), and Pseudomonas aeruginosa (Pae-1399).

Full text of DOI:10.1039/c7md00354d

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Page 1 of 12
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Synergism of Fused Bicyclic 2-Aminothiazolyl compounds with
polymyxin B against Klebsiella pneumonia
Rong Wang,a Shuang Hou,a Xiaojing Dong,b Daijie Chen,b ∗ Lei Shao,c Liujia Qian, c
Zhong Lia, d and Xiaoyong Xua, d∗
a Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China
University of Science and Technology, Shanghai 200237, China;
b School of Pharmacy, Shanghai Jiaotong University
CShanghai Institute of Pharmaceutical Industry
dShanghai Collaborative Innovation Center for Biomanufacturing Technology,
Shanghai 200237, China
∗
Corresponding author. Tel: +86ꢀ21ꢀ64252945; fax: +86ꢀ21ꢀ64252603; eꢀmail:
xyxu@ecust.edu.cn
A series of fused bicyclic 2ꢀaminothiazolyl compounds were synthesized and
evaluated for their synergistic effects with polymyxin B (PB) against Klebsiella
pneumonia (SIPIꢀKPNꢀ1712). Some of the synthesized compounds exhibited
synergistical activity. When 4 ꢁg/ml compound B1 was combined with PB, it showed
potent antibacterial activity, achieving 64ꢀfold reduction for the MIC of PB.
Furthermore, compound B1 showed prominent synergistic efficacy in both
concentration gradient and time–kill curves in vitro. In addition, B1 combined with
PB also exhibited synergistic and partial synergistic against E. coli (ATCC25922 and
its clinical isolates), Acinetobacter baumanii (ATCC19606 and its clinical isolates),
and Pseudomonas aeruginosa (Paeꢀ1399).
Introduction
The misuse of antibiotics in clinical and nonꢀclinical field has led to the rapid
emergence of widespread multiꢀdrug resistant (MDR) Pathogenic bacteria, especially
ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella
pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa) which were
highlighted by the Infectious Diseases Society of America. 1ꢀ3 Klebsiella pneumonia,
as a representative of antibioticꢀresistant Gramꢀnegative bacteria, has caused many
kinds of infections which threaten the global public health, including pneumonia,
bloodstream infections, foodꢀborne disease, wound or surgical site infections and so
4, 5
on.
More troubling, high antimicrobial resistance rate of Klebsiella bacterias was
observed for βꢀlactamase inhibitors and many other antibiotics, which has caused

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serious sequel that a variety of traditional antibiotics are not able to achieve a
therapeutic effect.6ꢀ10
This trend has driven the use of polymyxins as the ultimate weapon due to their
effective antibacterial activity against MDR Gramꢀnegative bacteria, such as
Acinetobacter baumannii, E. coli, and Klebsiella pneumonia.11ꢀ13 Polymyxins are
decapeptide antibiotics and mainly include five different types, polymyxin AꢀE.
Among these polymyxins, polymyxin B (PB) is widely used. The only difference
between polymyxin B and polymyxin E is that DꢀLeu (Dꢀleucine) replaced the
position of a DꢀPhe (Dꢀphenylalanine) in polymyxin B.14 PB can effectively treat
some diseases caused by microorganisms, such as skin diseases in combination with
Bꢀbacitracinꢀneomycin and it can also suppress the growth of Enterobacteriaceae
15
when it is used in selective decontamination regimes. However, the use of PB in
clinical field is restricted on account of its nephrotoxicity and neurotoxicity.
According to the recent clinical studies, above 60% of patients have been found with
polymyxins induced nephrotoxicity after administration intravenously. Therefore, the
toxicity of polymyxin precludes the use of appropriate dosing for treatment of MDR
Gramꢀnegative infections, and even worse is that inferior dosing may promote the
emergence of polymyxin resistance.16, 17 Thus, it is necessary to find new strategies to
reduce the dose of polymyxin in order to make these antibiotics effective.
Combination therapies have been proposed as good options. Nowadays, the hot
directions of combination therapies include combining two ‘old’ antibiotics,
traditional antibiotic with natural products or peptide antibiotics.18, 19 For instance,
combining chloramphenicol with PB to synergistically kill NDMꢀproducing MDR
Klebsiella pneumonia and combining curcumin with PB against MDR bacteria
associated with traumatic wound infections.15, 20, 21Another way to prolong the life
span of ineffective antibiotics is to employ adjuvants, mainly include βꢀlactamase
inhibitors, pump inhibitors, outer membrane permeabilizers.22ꢀ24
Thiazole scaffold are widely used in medicine, pesticides, fine chemicals and
other fields due to the unique nitrogen, sulfur heterocyclic structure and wide
bioactivities. In medicinal chemistry, many thiazole derivatives have shown effective
activities in antiꢀinflammation25, anticancer26, antituberculous27, antiꢀHIV28 and
antiꢀbacterial infection29.
Enlightened by all of the descriptions above, we tried to screen thiazole compounds
synthesized in our group30 and test their synergistic activities with PB against
Klebsiella pneumonia (SIPIꢀKPNꢀ1712). Luckily, we found that compound 1108
exhibited effective synergism with PB，achieving 16ꢀfold reduction for the MIC of PB
against SIPIꢀKPNꢀ1712. Then, we designed and synthesized three series of fused
bicyclic 2ꢀaminothiazolyl compound derivatives (A, B, C) on the basis of the structure
of compound 1108 and studied their synergistic effects with PB against

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SIPIꢀKPNꢀ1712 in vitro by evaluating the Minimum Inhibitory Concentration (MIC,
the lowest concentration of a drug preventing visible growth of a bacterium) and
Fractional Inhibitory Concentration Indice (FICI, the value to predict the synergy).
Figure 1. Design strategy of the title thiazole compounds A, B, C
Results and discussion
Chemistry
The intermediate thioureas 5 were synthesized referring to the method by Rodl et
al31. As described in Scheme 1, the benzoyl isothiocyanate 2 was prepared by the
reaction of benzoyl chloride and KSCN in acetone at room temperature under inert
atmosphere conditions, and then reacted with aniline derivatives 3 in ethyl acetate
under reflux condition to get compounds 4. Finally, following debenzoylation of 4,
intermediate thioureas 5 were obtained in 80ꢀ90% yield.
The synthesis of the target compounds A were outlined in Scheme 2a, according
to the method by Liu et al30. The mixture of thioureas 5 and α, βꢀepoxycyclohexanone
6 in alcohol were heated under microwave irradiation to cyclocondensate to get title
compounds A in 70ꢀ90% yield. While, target compounds B were gained in moderate
yields (60ꢀ75%) by the mixture of thioureas 5 and α, βꢀepoxycyclohexanone 6 in H2O
under the condition of reflux which is shown in Scheme 2b. Then, target compounds
C was synthesized by route as described by Yao et al32 shown in Scheme 2c and this
method afford compounds C in70ꢀ90% yields. Compounds C could be obtained by a
copperꢀcatalyzed tandem reaction of 2ꢀiodoaniline derivatives 7 with isothiocyanates
8 using CuSO4 as catalyst and Bu3N as a base under ligandꢀ and solventꢀfree
conditions in air.

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Scheme 1. Synthesis of intermediates. (i) KSCN, acetone, Ar, r.t., 1 h; (ii) EA, reflux,
2h; (iii) NaOH, EtOH, reflux, 2h.
Scheme 2. Synthesis of compounds A, B, C. (i) R2OH, MW, 15ꢀ30 min. (ii) H2O,
reflux, 12 h. (iii) CuSO4, Et3N, 80℃,5 h.
Synergistic activities with polymyxin B (PB) against Klebsiella pneumonia and
the SAR study
Antibacterial synergists combined with PB can be used for many Gramꢀnegative
bacterias. For example, curcumin combined with PB produced the antimicrobial
synergy against MDR bacteria associated with traumatic wound infections, including
E. coli, Acinetobacter baumanii, and Pseudomonas aeruginosa.15 However, there was
no reported synergistic compound combined with PB against Klebsiella pneumonia
available at present. So we started our study further based on compound 1108.
To study the structure–activity relationships (SAR) preliminarily, 63 compounds
of structure A, eight compounds B and three compounds of structure C with different
substituents at R1, and R2, were synthesized, and the synergistic effects between these
compounds and PB against SIPIꢀKPNꢀ1712 were studied by the determination of
MICs and the calculation of FICIs, which is shown in Table 1. Firstly, every
synthesized compound individual does not possess any antibacterial effects against
the test isolated strain (MICs >128ꢁg mlꢀ1), and the MIC of PB is 32ꢁg mlꢀ1 before

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combination. When synthesized compounds combined with PB, the results showed
that most of these compounds exhibited synergism or partial synergism with PB
against SIPIꢀKPNꢀ1712, the MICs of PB ranged from 0.5 to 32 ꢁg mlꢀ1. When we
tried different groups at R1 position, it was found that aromatic heterocyclic
(A32ꢀA38，such as pyridine, thiazole, pyrimidine, quinoline and naphthalene),
heterocyclic (A39) and benzylated (A40) compounds exhibited lower activity than the
corresponding phenylated counterparts (A1-A31). Then, we focused on the effect of
various substituents for benzene ring. As shown in Table 1, the activity data revealed
that when the substituents were electronꢀwithdrawing groups (A1ꢀA17), compounds
exhibited better activity than the compounds bearing electronꢀdonating groups
(A18ꢀA31). Further investigations were performed for compounds A1ꢀA17 and we
found that halogen groups at paraꢀsubstituted showed a clear preference for the
potency among monoꢀsubstituted compounds. A1 and A3, pꢀCl and pꢀCF3 separately,
reduced the MIC of PB to 1 ꢁg mlꢀ1 and 8 ꢁg mlꢀ1 respectively. Besides, when
bisꢀsubstituted compounds contained at least one Cl group, the synergistic
antibacterial activity were better than that of the others, such as A13, A15, A16.
Compound A13 exhibited the same synergism as compound A1. Then, we tried to
introduced different substitution for R2 group including methyl, ethyl, linear alkanes,
branched alkanes, cycloalkane and 1Hꢀimidazoleꢀ1ꢀcarbonyl, but these substituents
had no obvious influence on synergistic effect. And then, when the R2 group was
replaced with H to generate structure B, it was inspiring that compound B1 (the
substitution of pꢀCl of benzene ring) can cause a 64ꢀfold reduction for the MIC of PB,
reducing to 0.5 ꢁg mlꢀ1. However, changing tetrahydrobenzothiazole to benzothiazole
(structure C) did not show any improvement for the synergism. Overall, the
relationship between the structure and the synergistic activity exhibited that
4ꢀClꢀphenyl at R1, no substitution or alkyl substitution at R2 for structure A and B was
benefit for the synergistic effect.
Table 1
The MICs and FICIs of PB in combination with compounds (4ꢁg mlꢀ1) against
SIPIꢀKPNꢀ1712.
SIPIꢀKPNꢀ1712
compounds
R1
R2
MIC
FICI
0.09
0.06
Compound
1108
4ꢀBrꢀphenyl
4ꢀClꢀphenyl
Me
Me
2
1
A1

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A2
A3
A4
A5
A6
A7
A8
A9
4ꢀIꢀphenyl
4ꢀCF3ꢀphenyl
4ꢀNO2ꢀphenyl
4ꢀCNꢀphenyl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
16
8
16
16
32
8
16
32
16
32
16
1
0.53
0.28
0.53
0.53
1.03
0.28
0.53
1.03
0.53
1.03
0.53
0.06
1.03
0.16
0.28
0.53
1.03
0.53
1.03
1.03
0.53
0.53
0.53
0.53
1.03
0.53
1.03
1.03
1.03
0.53
0.16
0.53
0.53
1.03
0.53
1.03
1.03
0.53
1.03
0.53
1.03
0.53
0.09
0.53
4ꢀCH3CONHꢀphenyl
3ꢀClꢀphenyl
3ꢀBrꢀphenyl
3ꢀFꢀphenyl
3ꢀCF3ꢀphenyl
2ꢀClꢀphenyl
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
A33
A34
A35
A36
A37
A38
A39
A40
A41
A42
A43
A44
A45
2ꢀCF3ꢀphenyl
2,6ꢀ(Cl)2ꢀphenyl
2,6ꢀ(F)2ꢀphenyl
2ꢀClꢀ4ꢀ CF3ꢀphenyl
2,4ꢀ(Cl)2ꢀphenyl
3ꢀCF3ꢀ4ꢀCNꢀphenyl
phenyl
4ꢀCH3ꢀphenyl
4ꢀCH2CH3ꢀphenyl
4ꢀOHꢀphenyl
32
4
8
16
32
16
32
32
16
16
16
16
32
16
32
32
32
16
4
16
16
32
16
32
32
16
32
16
32
16
2
4ꢀOCH3ꢀphenyl
4ꢀC(CH3)3ꢀphenyl
4ꢀN(CH3)2ꢀphenyl
1ꢀCH3ꢀ2ꢀphenoxybenzyl
1ꢀFꢀ4ꢀphenoxybenzyl
4ꢀCH3COꢀphenyl
2ꢀCH3ꢀphenyl
3ꢀCH3ꢀphenyl
2,3ꢀ(CH3)2ꢀphenyl
3,4,5ꢀ(OCH3)3ꢀphenyl
4ꢀ(2ꢀchloropyridyl)
2ꢀ(5ꢀchloropyridyl)
3ꢀchloropyridyl
1ꢀthiazolyl
1ꢀpyrimidinyl
2ꢀquinolyl
1ꢀnaphthyl
1ꢀmorpholyl
1ꢀbenzyl
1ꢀpyrimidinyl
2ꢀquinolyl
Et
Et
Et
Et
2ꢀ(5ꢀchloropyridyl)
4ꢀBrꢀphenyl
4ꢀFꢀphenyl
16

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A46
A47
A48
A49
A50
A51
A52
A53
A54
A55
A56
A57
A58
4ꢀClꢀphenyl
4ꢀCH3ꢀphenyl
1ꢀCH3ꢀ2ꢀphenoxybenzyl
4ꢀFꢀphenyl
Et
Et
Et
1
4
0.06
0.16
1.03
0.53
0.53
0.53
1.03
0.06
0.53
1.03
0.16
0.28
32
16
16
16
32
1
16
32
4
CH2CH2CH3
CH2CH2CH3
CH2(CH2)2CH3
CH2(CH2)2CH3
4ꢀCH3ꢀphenyl
4ꢀClꢀphenyl
4ꢀCH3ꢀphenyl
4ꢀClꢀphenyl
4ꢀCH3ꢀphenyl
4ꢀCH3ꢀphenyl
4ꢀClꢀphenyl
CH(CH3)2
CH(CH3)2
CH2CH2OCH3
CH(CH2CH2CH2CH3)2
4ꢀClꢀphenyl
4ꢀClꢀphenyl
cyclopentyl
1Hꢀimidazoleꢀ1ꢀ
carbonyl
1Hꢀimidazoleꢀ1ꢀ
carbonyl
1Hꢀimidazoleꢀ1ꢀ
carbonyl
1Hꢀimidazoleꢀ1ꢀ
carbonyl
1Hꢀimidazoleꢀ1ꢀ
carbonyl
1Hꢀimidazoleꢀ1ꢀ
8
32
32
16
16
16
32
1.03
1.03
0.53
0.53
0.53
1.03
A59
A60
A61
A62
A63
4ꢀFꢀphenyl
4ꢀCF3ꢀphenyl
2ꢀ(5ꢀchloropyridyl)
phenyl
4ꢀCH3ꢀphenyl
carbonyl
B1
B2
B3
B4
B5
B6
B7
B8
C1
C2
C3
PB
4ꢀClꢀphenyl
4ꢀFꢀphenyl
4ꢀCF3ꢀphenyl
phenyl
/
/
/
/
/
/
/
/
H
0.5
32
16
32
32
16
16
16
32
32
16
32
0.05
1.03
0.53
1.03
1.03
0.53
0.53
0.53
1.03
1.03
0.53
1.03
4ꢀCH3ꢀphenyl
4ꢀCH3COꢀphenyl
2ꢀ(5ꢀchloropyridyl)
1ꢀnaphthyl
4ꢀCl
4ꢀCl
H
5ꢀCl
4ꢀ CF3
In order to further study the synergism of compound B1 with PB, we combined
PB with different concentrations of B1 to investigate the changes of the MICs. As
seen in Table 2, the MIC of PB alone is 32 ꢁg mlꢀ1, and the MIC sharply decreased
with the participation of B1. When combined 4 ꢁg mlꢀ1 of B1, the MIC of PB
achieved 0.5 ꢁg mlꢀ1, which means 64 folds reduction. When the dosage of B1
continuously increased, the MIC slightly decreased. The MICs of PB in combination

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with 8 ꢁg mlꢀ1 or 16 ꢁg mLꢀ1 of compound B1 remained the same value at 0.25 ꢁg
mlꢀ1, which is equal to 128 folds reduction.
As shown in Figure 2, 4 ꢁg mlꢀ1 of PB alone exhibited a strong bactericidal effect
within 4 h, showing a decrease of ≥ 4 log cfu mLꢀ1. Despite the great initial effect,
significant bacterial regrowth was observed. Within 8 h, a regrowth of ≥1 log cfu
mLꢀ1 was observed and the regrowth approached higher amount than the original at 24
h. Meanwhile, 4 ꢁg mlꢀ1 of B1 alone had no appreciable antibacterial activity against
SIPIꢀKPNꢀ1712, the growth curve was similar to that of the noꢀdrug control. When 4
ꢁg mlꢀ1 of B1 combined with 0.5 ꢁg mlꢀ1 and 1 ꢁg mlꢀ1 PB respectively, it showed the
similar trend as that of PB alone. However, when the amount of PB added up to 2 ꢁg
mlꢀ1, the antibacterial activity markedly enhanced, and complete cell death was
observed at 24 h. Besides, 4 ꢁg mlꢀ1 of B1 combined with 4 ꢁg mlꢀ1 of PB exhibited
the completely inhibited of bacteria growth within 2 h and no regrowth was observed
at 24 h. The results suggested that this combination of both 4 ꢁg mlꢀ1 of B1 and 2.0 ꢁg
mlꢀ1 or 4.0 ꢁg mlꢀ1 PB produced synergistic bactericidal effects.
Table 2
The MICs of PB in combination with different concentrations of compound B1
MIC of PB
Compound B1
concentration
Fold reduction
（MIC ꢁg/ml）
0ꢁg/ml
1ꢁg/ml
2ꢁg/ml
4ꢁg/ml
8ꢁg/ml
16ꢁg/ml
32
8
2
0.5
0.25
0.25
1
4
16
64
128
128
Figure 2．Time–kill curves of PB and B1 alone and in combination in different
concentrations versus SIPIꢀKPNꢀ1712
Due to the obvious synergistic efficacy of B1 combined with PB against Klebsiella
pneumonia (SIPIꢀKPNꢀ1712), we also tested its synergistic antibacterial activity

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versus other Gramꢀnegative bacterias including E. coli, Acinetobacter baumanii, and
Pseudomonas aeruginosa (containing their clinically isolated strains). The MICs and
FICIs of B1 and PB alone and in combination against these Gramꢀnegative bacterias
mentioned were shown in Table 3. Synergistic or partial synergistic effects were
observed in most of the tested strains except PaeꢀATCC27853 and Paeꢀ1292. As we
could see from the Table 3, B1 alone had no antibacterial activity against these strains
which was consistent with previous results. And B1 showed partial synergistic effects
in combination with PB against Acinetobacter baumanii (ATCC19606) and its four
clinical isolates, the FICIs were ranging from 0.28 to 0.53. Besides, B1 could
markedly decrease the MICs of PB against the other two Klebsiella pneumonia
clinical isolates, KPNꢀ2677 and KPNꢀ2967, showing the potent synergistic effects and
achieving 32 and 8 folds reduction, respectively. When 4ꢁg/ml B1 combined with PB,
it enhanced the activity of PB against E. coli (ATCC25922) and its two clinical
isolates, the MICs were decreased from 0.5 to 0.25ꢀ0.125 ꢁg/ml. While，B1 showed
partial synergistic effect combined with PB against clinical isolate Paeꢀ1399, but no
synergism with PB against Pseudomonas aeruginosa（ATCC27853）and its clinical
isolate Paeꢀ1292. These results demonstrated that B1 possessed broadly synergism
with PB against Gramꢀnegative bacterias. It suggested that the fused bicyclic
2ꢀaminothiazolyl structure was valuable to be modified further.
Table 3
Synergy between B1 and PB against Gramꢀnegative bacterias
MICs of B1 MICs of MICs of 4ꢁg/ml B1 in
strainsa
FICIb
alone
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
PB alone combination with PB
AbaꢀATCC19606
Abaꢀ1
1
4
0.5
2
0.53
0.53
0.28
0.53
0.53
0.06
0.16
0.53
0.28
0.28
1.03
0.53
1.03
Abaꢀ2
Abaꢀ3
Abaꢀ4
4
1
1
1
0.5
0.5
0.5
0.5
0.25
0.125
0.125
1
KPNꢀ2677
KPNꢀ2967
EcoꢀATCC25922
Ecoꢀ2944
Ecoꢀ2945
PaeꢀATCC27853
Paeꢀ1399
16
4
0.5
0.5
0.5
1
2
1
Paeꢀ1292
2
2
aAba, Acinetobacter baumanii; KPN, Klebsiella pneumonia; Eco, E. coli; Pae Pseudomonas
aeruginosa;
bFICI≤0.5=synergy, 0.5ꢀ0.75=partial synergy, 0.76ꢀ1= an additive effect.

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Conclusions
In conclusion, a series of fused bicyclic 2ꢀaminothiazolyl compounds were
synthesized and their synergistic effects with PB against Gramꢀnegative bacterias
were evaluated. For Klebsiella pneumonia (SIPIꢀKPNꢀ1712), significant synergistic
and partial synergistic interactions were observed by the determination of MICs and
the calculation of FICIs. A potent synergist B1 was found, which can reduce the MIC
of PB at 4ꢁg mlꢀ1 from 32ꢁg mlꢀ1 to 0.5ꢁg mlꢀ1, 64 folds reduction. And timeꢀkill
curves also suggested that the combination have significant bactericidal effect and can
prevent the regrowth of SIPIꢀKPNꢀ1712. B1 also could markedly decrease the MICs
of PB against the other two Klebsiella pneumonia clinical isolates, KPNꢀ2677 and
KPNꢀ2967. In addition, B1 combined with PB also showed synergistic and partial
synergistic against E. coli (ATCC25922 and its clinical isolates), Acinetobacter
baumanii (ATCC19606 and its clinical isolates), Paeꢀ1399 except Pseudomonas
aeruginosa（ATCC27853）and Paeꢀ1292. Such combination may be useful in the
treatment of infections caused by Gramꢀnegative bacterias. And the dosage reduction
of PB through efficacious synergist can provide a new strategy to decline its
nephrotoxicity and neurotoxicity. For this reason, further study on this structure is
valuable to find the optimized compounds with higher activity in vivo and its
synergistic mechanism.
Acknowledgments
This work was financial supported by Shanghai Foundation of Science and
Technology (15431902100) and part supported by National Natural Science
Foundation of China (21272071, 683788), the Fundamental Research Funds for
the Central Universities (222201718004).
Conflict of Interest
The authors declare no competing interests.
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Graphical Abstract
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1108
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