Syntheses and antibacterial activity of N -acylated ciprofloxacin derivatives based on the trimethyl lock

Article abstract of DOI:10.1021/acsmedchemlett.5b00146

Several N-acyl ciprofloxacin quinone derivatives based on a trimethyl lock structure were synthesized, and their in vitro antibacterial activity against a panel of clinically relevant bacteria was evaluated. A few new analogues displayed enhanced activity

Full text of DOI:10.1021/acsmedchemlett.5b00146

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Letter
Syntheses and Antibacterial Activity of N-acylated
Ciprofloxacin Derivatives Based on the Trimethyl Lock
Marvin J. Miller, Cheng Ji, and Patricia A. Miller
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 11 May 2015
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Syntheses and Antibacterial Activity of N-acylated Ciproflox-
acin Derivatives Based on the Trimethyl Lock
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Cheng Ji, Patricia. A. Miller, and Marvin J. Miller*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
ABSTRACT: Several N-acyl ciprofloxacin quinone derivatives based on a trimethyl lock structure were synthesized and their in
vitro antibacterial activity against a panel of clinically relevant bacteria was evaluated. A few new analogs displayed enhanced ac-
tivity against Gram-positive species compared to the parent drug. Additionally, studies of 8-Cip, which was the most potent com-
pound tested, indicate that it may act through a dual-action mechanism.
Since their discovery in 1970s, fluoroquinolone antibiotics
have played an important role in treatment of bacterial infec-
tions and have saved countless millions of lives. Targeting
bacterial DNA gyrase and topoisomerase, fluoroquinolones
exhibit their significant bactericidal activity by forming ter-
nary complexes of enzyme-DNA-drug, and consequently
blocking bacterial replication.1,2 Their notable antimicrobial
activity, excellent pharmacokinetic properties and few side
effects have led to the widespread use of fluoroquinolones
and, unfortunately, thus also the development of bacterial re-
sistance.3 Therefore, although many advances in the search for
new fluoroquinolones have been already made, there is a sense
of urgency for new and more effective derivatives.
The syntheses of trimethyl locks with different substituents
on the quinone are shown in Scheme 2. Both forms 5H and
8H were derived from lactone 4 by oxidative ring opening of
the lactone using bromine8 and N-bromosuccinimide,12 respec-
tively. The acids were conveniently converted to their methyl
esters 5Me and 8Me via thionyl chloride-mediated or acid
catalyzed esterification in methanol. Nucleophilic substitution
of 5Me with NaN3 in aqueous methanol provided azide 6 in
excellent yield. A triphenylphosphine-mediated reduction of
the azide afforded vinylogous amide 7Me in 66% yield, which
was subsequently hydrolyzed to furnish acid 7H. Water-
soluble carbodiimide-mediated coupling of ciprofloxacin to
quinone trimethyl locks 5H, 7H and 8H provided N-acyl
ciprofloxacin derivatives 5-Cip, 7-Cip and 8-Cip with differ-
ent substituents (Br-, NH2-, H-) on the quinone moieties in 44-
71% yield (Scheme 3).
Recently, in our development of linkers to incorporate a
drug release function in iron transport-mediated drug delivery,
a series of carboxylic acids with a quinone “trimethyl lock”
structure and their methyl esters were obtained as synthetic
intermediates.4,5 The chemical nature of the trimethyl lock is
an o-hydroxylcinnamic acid derivative.6 Reduction of the qui-
none trimethyl lock system generalized by structure 1 would
lead to the corresponding hydroquinones 2 in which severe
steric repulsion between three methyl groups promotes rapid
lactonization with concomitant release of an alcohol or amine
(Scheme 1).7,8 Several quinone trimethyl lock compounds have
been investigated for reductase-activated prodrugs such as
those based on mustard9 and oxindoles10 for anticancer thera-
py. In ciprofloxacin, one of the best known fluoroquinolones,
it has been demonstrated that chemical modification of the
secondary amine in the piperazinyl ring by acylation does not
always alter the effect of these molecules towards their bacte-
rial target.11 With the aim of probing the effects of the quinone
trimethyl lock on antibacterial activity of fluoroquinonlones,
we herein describe the syntheses of several ciprofloxacin de-
rivatives with quinone trimethyl lock precursors as N-acyl
substituents and studies of their in vitro antibacterial activity.
Scheme 2. Synthesis of quinone trimethyl locks 5H, 7H, 8H
and their methyl estersa
O
O
O
O
OR
O
O
O
OR
Br
N3
a
c
d
H2N
O
O
O
O
6
5H R = H
5Me R= Me
7Me R=Me
7H R=H
O
e
b
O
OH
O
OR
4
f
O
8H R=H
8Me R=Me
g
aReagents and conditions: (a) Br2, AcOH, rt, 80% (b) SOCl2,
MeOH, reflux, 77%; (c) NaN3, MeOH-H2O, rt, 91%; (d) 1. PPh3,
CH2Cl2, rt; 2. AcOH, THF-H2O, reflux, 66% for 2 steps; (e) Li-
OH, MeOH-H2O, rt, 91%; (f) NBS, CH3CN-H2O-acetone, rt,
79%; (g) H2SO4 (cat.), MeOH, reflux, 95%.
Scheme 1. Reaction of a quinone trimethyl lock to form
lactone via reduction
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Table 1. Diameter of growth inhibition zones (mm) in the agar diffusion antibacterial susceptibility assay
3
4
5
6
7
8
9
5-Cip
35
5Me
0
7-Cip
36
7Me
0
8-Cip
39
8Me
0
Cip
1
B.subtilis ATCC 6633
S. aureus SG511
20/24Pb
21a
0a
23a
18/21Pa
15b
19/24Pb
23c
18a
2
25
0
31
0
33
0
3
M. luteus ATCC10240
M. vaccae IMET10670
A. baumannii ATCC17961
P. aeruginosa K799/wt
P. aeruginosa K799/61
E. coli X580
19
0
17V
0
28
0
4
37
0
24/30P
24/30P
22/32P
31/35P
31/37P
16/23P
23
0
32/42P
26/32P
41
0
5
18/23P
29/35P
29/38P
33/40P
17/21P
24
0
0
0
10
11
12
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6
0
0
0
7
0
0
35/42P
32/40P
21/29P
27
0
8
0
0
0
9
E. coli DC0
0
0
0
10
E. coli DC2
0
0
0
22a
P, unclear inhibition zone/many colonies in the inhibition zone; V, very unclear inhibition zone. Exactly 50 µL of a 0.1 mM solution of
each compound dissolved in 1:9 DMSO/MeOH was added to 9 mm wells in agar media (Standard I Nutrient Agar, Serva or Mueller Hin-
ton II Agar, Becton, Dickinson and Company). Inhibition zones were read after incubation at 37 ºC for 24 h. M. vaccae was incubated
37ºC for 44 h; a,b,cCiprofloxacin was tested at (a) 5 µg/mL, (b) 1.66 µg/mL, (c) 0.33 µg/mL in H2O.
Table 2. MIC values of the N-acyl ciprofloxacin derivatives against selected bacterial strains
5-Cip
<0.05
0.1
7-Cip
<0.05
<0.05
12.5
2
8-Cip
<0.05
<0.05
0.2
9
10
8Me
50
Cip
0.03
0.47
8c
B.subtilis ATCC 6633
S. aureus SG511
<0.05
0.4
<0.05
0.4
>100
50
M. luteus ATCC10240
M. vaccae IMET10670
A. baumannii ATCC17961
P. aeruginosa K799/wt
P. aeruginosa K799/61
E. coli DC0
1.56
2
>100
2
12.5
2
0.5
80
0.47
0.24
0.47
0.24
0.47
0.24
0.78
5
0.6
0.39
1.25
2.5
>100
>100
>100
>100
>100
25
>100
>100
>100
>100
>100
2.5
>100
>100
>100
100
2.5
5
12.5
8.33
25
12.5
6.25
E. coli DC2
12.5
aMIC values (µM) were determined using the broth microdilution method in Mueller-Hinton broth No.2 (MHII) with visual end point
analysis according to the CLSI guidelines.14; bEach compound was tested in triplicate; cData from ref 13.
Scheme 3. Synthesis of N-acyl ciprofloxacin derivatives
All ciprofloxacin derivatives were subjected to further as-
says to determine their minimum inhibitory concentrations
(MIC) (Table 2). Two additional N-acyl derivatives 9 and 10
that have similar trimethyl substituted side chain structures but
without the quinone fragment were also synthesized and in-
cluded in the assay. In general, the trimethyl lock ciprofloxa-
cin quinone derivatives possessed equal or enhanced in vitro
activity against Gram-positive species compared to the parent
drug. Among the three derivatives, compound 8-Cip was the
most active with MIC values less than 0.05 µM and 0.2 µM
against S. aureus and M. luteus, respectively, against which
N-Acyl ciprofloxacin quinone derivatives 5-Cip, 7-Cip and
8-Cip were initially screened for antibiotic activity against
representative Gram-positive and Gram-negative bacteria as
well as Mycobacterium vaccae using agar diffusion assays
(Table 1). All three ciprofloxacin derivatives displayed strong
antibacterial activities when tested at 0.1 mM, as indicated by
the large zones of inhibition they induced. The trimethyl lock
cores, as represented by methyl esters 5Me, 7Me and 8Me,
were generally inactive. Moreover, all three ciprofloxacin
derivatives showed moderate to good activity against Myco-
bacterium vaccae, a non-pathogenic model organism for My-
cobacterium tuberculosis that causes tuberculosis, suggesting
their potential in anti-tuberculosis agent development.
ciprofloxacin is less active (MIC in 0.47 µM and 8 µM, re-
spectively). On the other hand, 9 and 10 were less active than
the trimethyl lock ciprofloxacin derivatives, indicating that the
quinone structure plays a key role for the activity enhancement
observed. In fact, trimethyl lock compound 8Me has very
weak antibacterial activity (MIC=50-80 µM) against the
Gram-positive species tested except S. aureus, suggesting that
the trimethyl lock ciprofloxacin derivatives may be dual-
action antibiotics.
The trimethyl lock ciprofloxacin derivatives displayed de-
creased activity (2-50 fold) against mycobacteria and Gram-
negative bacteria compared to the parent drug, presumably
because of the loss of zwitterionic structure of ciprofloxacin,
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which favors membrane penetration.15 However, 9 and 10
drug. The superior activity of 8-Cip suggests that it may act
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7
8
through a dual-action mechanism by serving as a prodrug and
as a covalent thiol-containing enzyme inhibitor. This dual-
action mechanism also has great potential for use in enzyme-
activated prodrug strategies for site-selective drug delivery.
Future work will focus on syntheses of other drug derivatives
based on 8H and examine the release of drug in appropriate
enzyme assays.
were virtually inactive (MIC>100 µM) against Gram-negative
bacteria, suggesting that the quinone fragment partially com-
pensates for the loss of activity caused by the non-zwitterionic
structure.
Based on results from antibacterial assays and the chemical
nature of the trimethyl lock system, we have proposed modes
of action of the trimethyl lock ciprofloxacin derivatives. While
all three derivatives could serve as prodrugs which are activat-
ed by potential reductases as depicted in Scheme 1, compound
8-Cip may have an additional dual-action mechanism by serv-
ing as a prodrug and as a covalent enzyme inhibitor, which is
consistent with its superior antibacterial activity. Conjugate
addition of sulfur nucleophiles to quinones is well known and
has been exploited in design of certain enzyme inhibitors.16
Similarly, the activation of compound 8-Cip could be initiated
by the reductive alkylation with certain thiol-containing en-
zymes (Scheme 4). The resulting thiol-quinone adduct 11
would be expected to aromatize to the corresponding hydro-
quinone 12. The rapid lactonization of 12 induced by the tri-
methyl lock could release ciprofloxacin and meanwhile result
in a covalent inactivated enzyme 13. In contrast, such a path-
way with an aromatization step could not be established for
compound 5-Cip and 7-Cip.
Scheme 5
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Scheme 4. Proposed modes of action of compound 8-Cip
Top: model reaction for enzyme activation of trimethyl lock de-
rivatives; Bottom: HPLC studies of the activation of 8-PP: panel
A, 8-PP standard; panel B, 8-PP after treatment with methyl mer-
captoacetate in CH3CN/PBS solution at 37 °C for 3 h; panel C,
phenylpiperazine (PP) standard; panel D, 14 standard.
To determine whether 8-Cip could be activated in the pres-
ence of thiol-containing enzymes as proposed, model reactions
were examined for the reaction of thiols with N-acyl phenylpi-
perazine 8-PP, a more soluble alternative to 8-Cip, and the
reaction was monitored by HPLC (Scheme 5). The thiol me-
thyl mercaptoacetate was chosen for the model studies princi-
pally because its pKa (7.9) was similar to that of the thiol in
cysteine (pKa = 8), which is ubiquitous in biological systems.
We were pleased to find that in the presence of the thiol, 8-PP
(retention time = 11.1 min) was largely converted to lactone
14 (retention time = 9.2 min) with concomitant release of the
phenylpiperazine (retention time = 8.5 min) in buffered aque-
ous acetonitrile after 3 h. In sharp contrast, no reaction was
observed for compound 7-PP, a representative system lacking
the aromatization pathway, under the same conditions that led
to rapid lactonization for 8-PP. These data provide evidence
for 8-Cip being a dual-action prodrug that might utilize a
unique thiol-activated pathway.
ASSOCIATED CONTENT
Experimental procedures, copies of spectral data, and characteri-
zation data. This material is available free of charge via the Inter-
net at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
mmiller1@nd.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported by grant 2R01AI054193 from the
National Institutes of Health (NIH). C. J. also acknowledges a
fellowship from the Notre Dame CBBI training grant (NIH T32
GM075762). We gratefully acknowledge the use of the NMR
facilities provided by the Lizzadro Magnetic Resonance Research
Center at the University of Notre Dame (UND) under the direc-
tion of Dr. Jaroslav Zajicek and the mass spectrometry services
provided by the UND Mass Spectrometry & Proteomics Facility
In summary, we have described the syntheses of a series of
ciprofloxacin derivatives with a quinone trimethyl lock as the
N-acyl substituent and their antibacterial activity. Among the
derivatives, compound 8-Cip displayed enhanced activity
against several Gram-positive species compared to the parent
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(Mrs. N. Sevova, Dr. W. Boggess, and Dr. M. V. Joyce; supported
by the National Science Foundation under CHE-0741793).
1
2
3
ABBREVIATIONS
DNA, deoxyribonucleic acid; Cip, ciprofloxacin; MIC, minimum
inhibitory concentration required to inhibit the growth of 90% of
organisms; HPLC, high-performance liquid chromatography; PP,
phenylpiperazine.
4
5
6
7
8
9
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