Synthesis and Antibacterial Activity of Novel 4-Bromo-1H-Indazole Derivatives as FtsZ Inhibitors

Article abstract of DOI:10.1002/ardp.201400412

A series of novel 4-bromo-1H-indazole derivatives as filamentous temperature-sensitive protein Z (FtsZ) inhibitors were designed, synthesized, and assayed for their in vitro antibacterial activity against various phenotypes of Gram-positive and Gram-negative bacteria and their cell division inhibitory activity. The results indicated that this series showed better antibacterial activity against Staphylococcus epidermidis and penicillin-susceptible Streptococcus pyogenes than the other tested strains. Among them, compounds 12 and 18 exhibited 256-fold and 256-fold more potent activity than 3-methoxybenzamide (3-MBA) against penicillin-resistant Staphylococcus aureus, and compound 18 showed 64-fold better activity than 3-MBA but 4-fold weaker activity than ciprofloxacin in the inhibition of S. aureus ATCC29213. Particularly, compound 9 presented the best activity (4 μg/mL) against S. pyogenes PS, being 32-fold, 32-fold, and 2-fold more active than 3-MBA, curcumin, and ciprofloxacin, respectively, but it was four times less active than oxacillin sodium. In addition, some synthesized compounds displayed moderate inhibition of cell division against S. aureus ATCC25923, Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC27853, sharing a minimum cell division concentration of 128 μg/mL.

Full text of DOI:10.1002/ardp.201400412

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Full Paper
Synthesis and Antibacterial Activity of Novel 4-Bromo-1H-Indazole
Derivatives as FtsZ Inhibitors
Yi Wang1, Mi Yan1, Ruixin Ma2, and Shutao Ma1
1
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School
of Pharmaceutical Sciences, Shandong University, Jinan, P. R. China
Affiliated Hospital of Medical College, Qingdao University, Qingdao, P. R. China
2
A series of novel 4-bromo-1H-indazole derivatives as filamentous temperature-sensitive protein Z (FtsZ)
inhibitors were designed, synthesized, and assayed for their in vitro antibacterial activity against various
phenotypes of Gram-positive and Gram-negative bacteria and their cell division inhibitory activity. The
results indicated that this series showed better antibacterial activity against Staphylococcus epidermidis
and penicillin-susceptible Streptococcus pyogenes than the other tested strains. Among them,
compounds 12 and 18 exhibited 256-fold and 256-fold more potent activity than 3-methoxybenzamide
(3-MBA) against penicillin-resistant Staphylococcus aureus, and compound 18 showed 64-fold better
activity than 3-MBA but 4-fold weaker activity than ciprofloxacin in the inhibition of S. aureus
ATCC29213. Particularly, compound 9 presented the best activity (4 mg/mL) against S. pyogenes PS, being
32-fold, 32-fold, and 2-fold more active than 3-MBA, curcumin, and ciprofloxacin, respectively, but it was
four times less active than oxacillin sodium. In addition, some synthesized compounds displayed moderate
inhibition of cell division against S. aureus ATCC25923, Escherichia coli ATCC25922, and Pseudomonas
aeruginosa ATCC27853, sharing a minimum cell division concentration of 128 mg/mL.
Keywords: Antibacterial evaluation / FtsZ inhibitors / Indazole derivatives / Resistant bacteria / Synthesis
Received: November 6, 2014; Revised: February 4, 2015; Accepted: February 19, 2015
DOI 10.1002/ardp.201400412
Additional supporting information may be found in the online version of this article at the publisher's web-site.
:
been either weak or not active against the resistant bacteria.
For purpose of preventing serious medical problem, there is
Introduction
Nowadays, the extensive use and misuse of antibiotics have
resulted in the emergence and prevalence of bacterial
resistance, which has seriously threatened human health. In
particular, methicillin-resistant Staphylococcus aureus
(MRSA), vancomycin-resistant enterococci (VRE), and penicil-
lin-resistant Streptococcus pneumoniae contribute to the
enormous difficulties in fighting against bacterial infec-
tions [1–3]. Many initial clinical effective antibiotics have
an urgent need for development of new types of antibacterial
agents or the expansion of bioactivity of the previous drugs,
especially with novel mechanisms of action [4].
Bacterial cell division protein FtsZ (filamentous tempera-
ture-sensitive protein Z) is conserved in almost all bacterial
pathogens and in several genetic studies has been shown to
be essential for bacterial viability [5–8]. Cell division in
bacteria occurs at the site of formation of a cytokinetic
Z-ring polymeric structure, which comprises FtsZ subunits [9].
During bacterial cell division, FtsZ offers an essential skeleton
for the formation of Z-ring in the presence of guanosine
triphosphate (GTP) [10–12]. Thus, if FtsZ assembly is restrained
or removed, the bacterial cell division will eventually fail,
thereby resulting in bacterial apoptosis. The vital role of FtsZ
Correspondence: Dr. Shutao Ma, Department of Medicinal
Chemistry, School of Pharmaceutical Sciences, Shandong Uni-
versity, 44, West Culture Road, Jinan 250012, P. R. China.
E-mail: mashutao@sdu.edu.cn
in bacterial cell division renders FtsZ as
a promising
Fax: þ86-531-88382009
therapeutic target to develop antibacterial agents with
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selective toxicity to bacterial pathogens. At present, a certain
amount of FtsZ-targeting antibacterial agents have been
screened from natural sources such as plants, microbial
fermentations metabolites, or small molecules designed
and synthesized, such as curcumin, 3-MBA, sanguinarine,
and chelerythrine (Fig. 1). FtsZ inhibitors can show their
destructive effects on the Z-ring by either enhancing or
inhibiting FtsZ self-polymerization [13–20].
Sanguinarine with a benzo[c]phenanthridine structure is
an antibacterial plant alkaloid, which has been identified as
a small molecule altering the Z-ring formation [14]. Cheler-
ythrine, an analog of sanguinarine, has also similar effects on
S. aureus and Bacillus subtilis to sanguinarine. In addition,
the presence of hydrophobic functionality at the 1-position
of the benzo[c]phenanthridines such as that of compound 1
(Figure 1), has been shown to significantly enhance
antibacterial activity relative to either sanguinarine or
chelerythrine [21, 22]. More importantly, 5-substituted
1-phenylnaphthalene has been found to be the pharmaco-
phore of these benzo[c]phenanthridines [23]. For example,
several 5-substituted 1-phenylnaphthalene derivatives have
exerted noteworthy antibacterial activity against methicillin-
resistant S. aureus with minimum inhibitory concentration
(MIC) values from 2.0 to 4.0 mg/mL. Moreover, FtsZ polymeri-
zation test results have suggested that the antibacterial
activity of the phenylnaphthalenes is associated with their
stimulatory impact on the dynamics of FtsZ polymerization.
Indazole molecule has a wide range of biological proper-
ties, such as tuberculostatic activity, analgesic and anti-
inflammatory [24], hepatoprotective [25], antibacterial [26],
antiangiogenic [27], and cytostatic [28]. Therefore, we
designed and synthesized a novel series of 4-bromo-1H-
indazole derivatives (Fig. 1) by replacing the naphthalene and
phenyl group of 5-substituted 1-phenylnaphthalene with the
indazole and bromine atom, respectively, on the basis of the
principle of bioisosterism. Our objective was to explore FtsZ-
targeting antibacterial agents with potent antibacterial
activity against a wide range of bacteria.
Results and discussion
Chemistry
In the present work, the methods used for preparation of the
4-bromo-1H-indazole derivatives 9–16 and 17–19 are outlined
in Scheme 1. Commercially available starting material 2 was
converted to 1-bromo-2-methyl-3-nitrobenzene
3 in the
presence of ammonium sulfide in ethanol by reflux followed
by Sandmeyer reaction. 3 was reduced with ferrum and
ammonium chloride in refluxing ethanol and water to give
Figure 1. Structures of curcumin, 3-MBA, sanguinarine, chelerythrine, and 1-phenyl-5-methyl-2,3,7,8-tetramethoxybenzo[c]-
phenanthridine 1, and the design of target compounds.
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Scheme 1. Reagents and conditions: (a) (NH4)2S, EtOH, reflux, 79%; (b) HBr/H2O, NaNO2/H2O, 0°C, 84%; (c) CuBr/HBr, 25–35°C; (d) Fe,
NH4Cl, N2, EtOH, reflux; (e) AcOK, (AcO)2, toluene, 0°C, 35°C; (f) iso-amyl nitrite, 80°C; (g) 6 N HCl, MeOH; (h) Br(CH2)nBr, K2CO3, DMF,
29–42%; (i) BrCH2CO2Et, K2CO3, DMF, rt., 71.5%; (j) amines, K2CO3, DMF, 80°C, or TEA, CH3CN, rt.; (k) NH2R2, MeOH, reflux, 35–51%.
3-bromo-2-methylaniline 4 in 81% yield. 4 reacted with acetic
anhydride in the presence of potassium acetate and then
condensed with iso-amyl nitrite to produce the N-acetyl-4-
bromo-1H-indazole 5. 5 was hydrolized in mixture solution of
6 N hydrochloric acid in methanol to yield 4-bromo-1H-
indazole 6 as the key intermediate. 6 reacted with alkyl
bromides or bromo esters in the presence of potassium
carbonate to give intermediates 7–8 in 29–42% yields and 17
in 71.5% yield. 7 or 8 was heated with morpholine at 80°C in
the presence of potassium carbonate and catalytic sodium
iodide to afford compounds 9 and 13. At room temperataure,
7 or 8 was converted to compounds 10–12 and 14–16 by
reacting with amines in acetonitrile. 17 was subjected to
subsequent nucleophilic substitution with hydroxyamine or
hydrazine hydrate to provide compounds 18–19. The struc-
tures of the newly synthesized compounds were characterized
by MS, 1H NMR, and all the spectral data were in agreement
with the proposed structures.
S. aureus ATCC31007, S. epidermidis, S. pyogenes PS,
S. pyogenes PR, Escherichia coli ATCC25922, B. subtilis
ATCC9372, and Pseudomonas aeruginosa ATCC27853. S.
aureus ATCC25923, B. subtilis ATCC9372, E. coli ATCC25922,
and P. aeruginosa ATCC27853 were used to determine the cell
division inhibitory activity of the synthesized compounds. The
results in the unit of mg/mL are shown in Tables 1 and 2.
The synthesized compounds universally exhibited better
antibacterial activity against S. epidermidis and S. pyogenes PS
than the other tested strains. Among them, many compounds
showed superior or comparable activity against S. epidermidis
to ciprofloxacin. Especially, compound 9 bearing the morpho-
line group in this series displayed the best antibacterial activity
with an MIC value of 4 mg/mL against S. pyogenes PS, showing
over 32-fold, 32-fold, and 2-fold better activity than 3-MBA,
curcumin, and ciprofloxacin, respectively, but it was four times
less active than oxacillin sodium. In the inhibition of S. aureus
PR, the most active compounds 12 and 18 with the benzyl-
aminoethyl and hydroxycarbamoylmethyl groups at 1-position
of the indazole, respectively, exhibited the same MIC value of
16 mg/mL, being 256-fold, 8-fold, 8-fold, and 8-fold better than
3-MBA, curcumin, oxacillin sodium, and ciprofloxacin, respec-
tively. Furthermore, compound 18 also showed the strongest
activity with an MIC value of 32 mg/mL against S. aureus
ATCC29213 in all of the tested compounds, being 64-fold,
4-fold, and 4-fold more potent than 3-MBA, curcumin, and
oxacillin sodium, but 4-fold weaker than ciprofloxacin. Besides,
all of the compounds showed same activity as oxacillin
Antibacterial evaluation
The 4-bromo-1H-indazole derivatives (6, 9–16, and 17–19)
were determined for their in vitro antibacterial activity and
cell division inhibitory activity, respectively. The in vitro
antibacterial activity was tested using tube dilution method
recommended by NCCLS [29] and the cell division inhibitory
activity was measured by assessing cell morphology using
phase-contrast light microscopy [30]. The tested strains
included S. aureus ATCC25923, S. aureus ATCC29213 (MRSA),
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Table 2. The cell division inhibitory activity of the 4-bromo-1H-indazole derivatives (mg/mL).
S. aureus
B. subtilis ATCC
E. coli
P. aeruginosa
Compounds
ATCC25923a)
9372b)
ATCC25922c)
ATCC 27853d)
6
9
10
11
12
13
14
15
16
17
18
19
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
WT
256
128
>128
128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
ND
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
512
128
>128
>128
128
>128
>128
128
128
128
ND
3-MBA
Curcumin
Oxacillin sodium
Ciprofloxacin
>128
>128
>128
16
>128
>128
256
>128
>128
ND
>128
>128
WT, no effect on morphology at 2048–4096 mg/mL; ND, not determined.
a) S. aureus ATCC25923: penicillin-susceptible strain.
b) B. subtilis ATCC9372: penicillin-susceptible strain.
c) E. coli ATCC25922: penicillin-susceptible strain.
d) P. aeruginosa ATCC27853: penicillin-susceptible strain.
sodium, but weaker activity than curcumin and ciprofloxacin in
the activity against B. subtilis ATCC9372, and most of all the
compounds displayed comparable potency to oxacillin sodium
and much better activity than curcumin, but weaker activity
than ciprofloxacin against E. coli ATCC25922, P. aeruginosa
ATCC27853.
coli ATCC25922. Among them, compound 12 showed improved
on-targetactivityagainstS.aureusATCC25923incomparisonto
3-MBA. In the inhibition of B. subtilis ATCC9372, few
compounds displayed improved on-target activity. In contrast,
compounds 6, 10, 11, 14, and 17–19 showed slightly improved
on-target activity against E. coli ATCC25922 compared with
curcumin. In all synthesized compounds, only compound 11
against P. aeruginosa ATCC27853 shared a minimum cell
division concentration of 128 mg/mL. Therefore, the above
results indicated that they were promising FtsZ inhibitors with
on-target activity. However, the cell inhibitory activity of some
compounds was not accordant to their in vitro antibacterial
activity, which implied that they could have other mechanism
against the bacteria.
Moreover, although precursor 6 exerted moderate or weak
activity against all the tested bacterial strains, many deriva-
tives of 6 displayed greatly improved antibacterial activity, in
which compounds 9 exhibited 16-fold improved activity
against S. pyogenes PS while compound 12 showed 8-fold
improved activity against S. aureus ATCC29213 in comparison
with 6. The subseries of 13–16 with a linkage of the three
carbon atoms connected between the indazole and alkyl-
amino group displayed a similar trend in antibacterial activity
to the subseries of 9–12 with a linkage of the two carbon
atoms connected between them. All the above results
indicated that the alkylamino side chains at the 1-position
of the indazole were beneficial to the antibacterial activity,
and the length of the linkage between the indazole and
alkylamino group had almost no effect on the antibacterial
activity. In another subseries of 18–19, compound 18 bearing
the amide side chain at the 1-position of the indazole showed
significantly increased activity 4-fold and 8-fold better than 6
against S. aureus ATCC29213 and S. aureus PR.
Conclusion
In conclusion, a series of 4-bromo-1H-indazole derivatives
were designed, synthesized, and evaluated for in vitro
antibacterial activity and cell inhibitory activity against
various Gram-positive and Gram-negative bacteria. Com-
pounds bearing the alkylamino side chains 9–16 generally
displayed better in vitro activity aganist S. epidermidis and S.
pyogenes PR than the other tested strains. In particular,
compound 9 showed the best activity with an MIC value of
4 mg/mL against S. pyogenes PS in the tested compounds,
displaying 32-fold, 32-fold, and 2-fold more potent activity
than 3-MBA, curcumin, and ciprofloxacin, respectively, but it
AsseenfromTable2,somesynthesizedcompoundsdisplayed
moderate inhibition of cell division against E. coli ATCC25922
sharing a minimum cell division concentration of 128mg/mL,
whichbasicallyfittedtheinvitroantibacterialactivity againstE.
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was four times less active than oxacillin sodium. In the
inhibition of S. aureus PR, the most active compounds were 12
and 18 with an MIC value of 16 mg/mL, being 256-fold, 8-fold,
8-fold, and 8-fold better than 3-MBA, curcumin, oxacillin
sodium, and ciprofloxacin, respectively. Compound 18
showed 256-fold better activity than 3-MBA but 4-fold
weaker activity than ciprofloxacin against S. aureus
ATCC29213. In cell division inhibitory activity, some synthe-
sized compounds displayed moderate inhibition of cell
division against S. aureus ATCC25923, E. coli ATCC25922,
and P. aeruginosa ATCC27853 sharing a minimum cell division
concentration of 128 mg/mL.
and NH4Cl (0.24 g, 4.40 mmol) at room temperature. The
reaction was refluxed till complete conversion took place,
which was monitored by TLC. It took about 2 h. The reaction
mixture was then cooled to room temperature and filtered to
remove solid. The combined solutions were evaporated under
reduced pressure and the mixture was diluted with H2O and
extracted with ethyl acetate. The organic layer was washed
consecutively with water and saturated brine, and dried over
anhydrous Na2SO4. The dried organic layer was filtered and
evaporated under pressure to dryness and residue was
purified on a column of silica gel eluting with 10% ethyl
acetate: petroleum ether. 3-Bromo-2-methylaniline 4 was
obtained as a light yellow liquid. Yield: 81%.
N-Acetyl-4-bromo-1H-indazole (5)
Experimental
To 4 (1.43 g, 7.69 mmol) was added toluene (23 mL) and
potassium acetate (0.91 g, 9.23 mmol). This mixture was
cooled to 0°C with stirring, and then acetic anhydride
(2.35 g, 23.06 mmol) was added dropwise over 2 min. The
reaction mixture was allowed to gradually warm up to room
temperature and stirred for 1 h (monitored by TLC). The
solution was heated to 80°C and then isoamyl nitrite (2.2 g,
18.46 mmol) was added, which was stirred for 7 h at 80°C until
the reaction was completed (TLC, petroleum ether/ethyl
acetate 2:1). Cooled to room temperature, the reaction
mixture was washed with 5% NaHCO3 to pH 7 and then
washed with water again. The organic layer was dried over
anhydrous Na2SO4 and then evaporated in vacuo to give
N-acetyl-4-bromo-1H-indazole 5 as orange crystal. Yield:
89.4%.
Chemistry
Thin-layer chromatography (TLC) was done using 0.25 mm pre-
coated silica gel plates (Qingdong Yumingyuan silica gel
reagent factory, Shandong, China, Yuyuan). The 1H NMR
spectra wererecordedonBruker Avance DRX400 spectrometer
(Bruker, Switzerlands) at ambient temperature using TMS as
internal reference standard (chemical shift in d ppm); coupling
constants(J)weregiveninHertz.Massspectrawererecordedon
API 4000 instrument (Applied Biosystems, CT). All melting
points were determined by open capillary methods and are
uncorrected. Column chromatography was performed on a
neutral silica column (2.5 ꢀ 45 cm) using appropriate eluent.
1-Bromo-2-methyl-3-nitrobenzene (3)
To a solution of 2,6-dinitrotoluene 2 (9 g, 49.45 mmol) in
ethanol at 79°C was slowly added (NH4)2S (60 mL). The
reaction was refluxed till complete conversion took place,
which was monitored by TLC. It took about 2 h. The reaction
mixture was then cooled to room temperature and filtered to
remove solid. The combined solutions were evaporated in
vacuum and residue thus obtained was recrystallized from
ethanol and water to yield 2-methyl-3-nitroaniline as a yellow
crystalline solid. Yield: 79.0%, mp 87–91°C.
4-Bromo-1H-indazole (6)
To 5 (1.6 g, 6.69 mmol) was added methanol (25.9 mL) and
then 6 N HCl (15.6 mL). The resulting solution was stirred at
room temperature until the reaction was completed after
7 h (monitored by TLC, petroleum ether/ethyl acetate 2:1).
After evaporation of methanol in vacuo, the residue was
extracted with ethyl acetate (50 mL ꢀ 3). The combined
organic layers were dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude was purified by flash
chromatography (petroleum ether/ethyl acetate, 5:1) to
yield 4-bromo-1H-indazole (0.8 g) as milky white solid, mp
To
a
suspension of 2-methyl-3-nitroaniline (1.5 g,
9.87 mmol) in water (4 mL) was added concentrated HBr
(10 mL) at room temperature. The contents were cooled to
0–5°C and a solution of sodium nitrite (0.72 g, 10.35 mmol) in
water (3 mL) was added dropwise at 0–5°C over a period of
3–5 min. After stirring at 0–5°C for another 20–30 min, the
reaction mixture was then added to a cuprous bromide (1.4 g,
9.87 mmol) in concentrated HBr (14 mL) with stirring. The
resulting reaction mixture was stirred at 30–35°C for another
30 min. Finally, precipitated solid was filtered, washed with
water, saturated NaHCO3, water and dried under vacuum to
yield 1.79 g of 1-bromo-2-methyl-3-nitrobenzene 3 as milky
white solid. Yield: 84.0%, mp 38–40°C.
164–166°C, yield 67.1%. 1H NMR (400 MHz, CDCl3):
d
–
–
(ppm) ¼ 10.25 (s, 1H, NH), 8.11 (s, 1H, CH N), 7.45 (d,
J ¼ 8.0 Hz, 1H, Ar–H), 7.34 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.26 (t,
J ¼ 8.0 Hz, 1H, Ar–H). ESI–MS m/z calcd for C7H5BrN2, 195.96;
found: [MþHþ] 197.2.
General methods for 1-N-bromoalkyl-4-bromo-1H-
indazoles (7 and 8)
In a round-bottomed flask with mechanical stirrer was added
6 (0.23 g, 1.17 mmol) and DMF and then K2CO3. The mixture
was stirred for 10 min and then 1,2-dibromoethane or
1,3-dibromopropane (5.85 mmol) was added dropwise. The
reaction mixture was stirred for 12 h at room temperature
and diluted with water. The mixture solution was extracted
3-Bromo-2-methylaniline (4)
To a solution of 3 (1.79 g, 8.29 mmol) in a mixture of ethanol
(21 mL) and water (7 mL) was added Fe (2.32 g, 41.45 mmol)
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with ethyl acetate (25mLꢀ 3) and washed with brine. The
organic was combined, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by flash
chromatography (petroleum ether/ethyl acetate, 30:1) to yield
compounds 7 and 8 in yields of 51.6% and 40.1%, respectively.
1-N-(2-Benzylaminoethyl)-4-bromo-1H-indazole (12)
Light yellow solid, mp 104–107°C, yield 54.5%, Rf ¼ 0.48
(petroleum ether/ethyl acetate, 1:2). 1H NMR (400 MHz,
–
DMSO-D ): d (ppm) ¼ 8.03 (s, 1H, CH N), 7.74 (d, J ¼ 8.0 Hz,
–
6
1H, Ar–H), 7.37–7.31 (m, 2H, Ar–H), 7.29–7.19 (m, 5H, Ar–H),
4.50 (t, J ¼ 6.4 Hz, 2H, CH2), 3.66 (s, 2H, CH2), 2.94 (t, J ¼ 6.4 Hz,
2H, CH2), 2.32 (s, 1H, NH). ESI–MS m/z calcd for C16H16BrN3,
329.05; found: [MþHþ] 330.4.
General methods for 1-N-aminoalkyl-4-bromo-1H-
indazoles (9–16)
A solution of 7 or 8 (0.63 mmol) in DMF was heated with
morpholine (1.2 mmol) at 80°C in the presence of K2CO3
(0.98 mmol) and NaI (0.06 mmol). The mixture solution was
extracted with ethyl acetate (15 mL ꢀ 3) and washed with
brine. The organic was combined, dried over anhydrous
Na2SO4, and concentrated in vacuo and the crude was purified
by flash chromatography (petroleum ether/ethyl acetate, 1:1)
to afford compounds 9 and 13. To a solution of 7 or 8
(0.63 mmol) in CH3CN was added triethylamine (0.17 mL). The
solution was stirred for 5–10 min and added amines
(1.2 mmol) gradually. The resulting mixture was stirred for
24 h at room temperature and then evaporated under
reduced pressure. The residue was dissolved in ethyl acetate
and washed with water and brine. The organic layer was dried
over anhydrous Na2SO4, filtered, and evaporated under
reduced pressure to dryness. The crude was purified by flash
chromatography (petroleum ether/ethyl acetate, 1:1) to yield
compounds 10–12, 14–16 in yields ranging from 22.3% to
61.5%.
1-N-[3-(4-Morpholinyl)propyl]-4-bromo-1H-indazole (13)
Yellow liquid, yield 57%, Rf ¼ 0.33 (petroleum ether/ethyl
acetate, 2:1). 1H NMR (400 MHz, MeOD): d (ppm) ¼ 7.99 (s, 1H,
–
CH N), 7.60 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.25–7.31 (m, 2H, Ar–H),
–
4.47 (t, J ¼ 6.4 Hz, 2H, CH2), 3.62 (t, J ¼ 6.4 Hz, 4H, 2CH2), 2.33
(brs, 4H, 2CH2), 2.25 (t, J ¼ 7.2 Hz, 2H, CH2), 2.12–2.06 (m, 2H,
CH2). ESI–MS m/z calcd for C14H18BrN3O, 323.06; found:
[MþHþ] 324.4.
1-N-(3-Isopropylaminopropyl)-4-bromo-1H-indazole (14)
White solid, mp 201–203°C, yield 35.7%, Rf ¼ 0.66 (dichloro-
methane/methanol, 8:1). 1H NMR (400 MHz, DMSO-D6): d
–
(ppm) ¼ 8.90 (s, 1H, NH), 8.08 (s, 1H, CH N), 7.83 (d, J ¼ 8.0 Hz,
–
1H, Ar–H), 7.41–7.34 (m, 2H, Ar–H), 4.58 (t, J ¼ 6.8 Hz, 2H, CH2),
3.23 (q, J ¼ 6.4 Hz, 1H, CH), 2.90 (t, J ¼ 7.2 Hz, 2H, CH2), 2.24–
2.17 (m, 2H, CH2), 1.20 (d, J ¼ 6.4 Hz, 6H, 2CH3). ESI–MS m/z
calcd for C13H18BrN3, 295.07; found: [MþHþ] 296.4.
1-N-(3-n-Butylaminopropyl)-4-bromo-1H-indazole (15)
White solid, mp 153–157°C, yield 22.3%, Rf ¼ 0.58 (dichloro-
methane/methanol, 8:1). 1H NMR (400 MHz, DMSO-D6): d
1-N-[2-(4-Morpholinyl)ethyl]-4-bromo-1H-indazole (9)
Pale yellow crystalline solid, mp 80–82°C, yield 61.5%,
Rf ¼ 0.48 (petroleum ether/ethyl acetate, 1:3). 1H NMR
–
(ppm) 8.99 (s, 1H, NH), 8.08 (s, 1H, CH N), 7.82 (d, J ¼ 8.0 Hz,
–
–
(400 MHz, CDCl ): d (ppm) ¼ 8.01 (s, 1H, CH N), 7.38 (d,
1H, Ar–H), 7.40–7.34 (m, 2H, Ar–H), 4.57 (t, J ¼ 6.8 Hz, 2H, CH2),
2.90–2.80 (m, 4H, 2CH2), 2.21 (t, J ¼ 7.6 Hz, 2H, CH2), 1.58–1.54
(m, 2H, CH2), 1.33–1.27 (m, 2H, CH2), 0.91 (t, J ¼ 7.2 Hz, 3H,
CH3). ESI–MS m/z calcd for C14H20BrN3, 309.08; found: [MþHþ]
310.5.
–
3
J ¼ 8.0 Hz, 1H, Ar–H), 7.30 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.23 (t,
J ¼ 8.0 Hz, 1H, Ar–H), 4.50 (t, J ¼ 6.8 Hz, 2H, CH2), 3.65 (t,
J ¼ 4.8 Hz, 4H, 2CH2), 2.87 (t, J ¼ 6.8 Hz, 2H, CH2), 2.49 (t,
J ¼ 4.8 Hz, 4H, 2CH2). ESI–MS m/z calcd for C13H16BrN3O,
309.05; found: [MþHþ] 310.4.
1-N-(3-Benzylaminopropyl)-4-bromo-1H-indazole (16)
Colourless liquid, yield 31.8%, Rf ¼ 0.45 (dichloromethane/
methanol, 8:1). 1H NMR (400 MHz, MeOD): d (ppm) ¼ 7.85 (s,
1-N-(2-Isopropylaminoethyl)-4-bromo-1H-indazole (10)
White solid, mp 155–159°C, yield 46.1%, Rf ¼ 0.51 (dichloro-
methane/methanol, 8:1). 1H NMR (400 MHz, CDCl3): d (ppm)
–
1H, CH N), 7.45 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.21–7.10 (m, 7H, Ar–
–
–
¼ 8.01 (s, 1H, CH N), 7.56 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.28 (d,
H), 4.35 (t, J ¼ 6.8 Hz, 2H, CH2), 3.55 (s, 2H, CH2), 2.42 (t,
J ¼ 7.2 Hz, 2H, CH2), 2.02–1.95 (m, 2H, CH2). ESI–MS m/z calcd
for C17H18BrN3, 343.07; found: [MþHþ] 344.4.
–
J ¼ 8.0 Hz, 1H, Ar–H), 7.22 (t, J ¼ 8.0 Hz, 1H, Ar–H), 4.94 (t,
J ¼ 6.8 Hz, 2H, CH2), 3.52 (t, J ¼ 6.8 Hz, 2H, CH2), 3.29 (q,
J ¼ 6.4 Hz, 1H, CH), 1.45 (d, J ¼ 6.4 Hz, 6H, 2CH3). ESI–MS m/z
calcd for C12H16BrN3, 281.05; found: [MþHþ] 282.4.
1-N-Ethyl ethanoate-4-bromo-1H-indazole (17)
To a solution of 4-bromo-1H-indazole 6 (0.4 g, 2.03 mmol) in
DMF was added K2CO3 (0.42 g, 3.05 mmol). The mixture was
stirred for 5–10 min, and then ethyl bromoacetate (0.51 g,
3.05 mmol) was added. The resulting mixture was stirred
16 hours at room temperature and then diluted with water.
The mixture solution was extracted with ethyl acetate
(30 mL ꢀ 3) and washed with brine. The organic was com-
bined, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was purified by flash chromatography
(petroleum ether/ethyl acetate, 30:1) to yield compound 17.
1-N-(2-n-Butylaminoethyl)-4-bromo-1H-indazole (11)
White solid, mp 178–180°C, yield 34.3%, Rf ¼ 0.48 (dichloro-
methane/methanol, 8:1). 1H NMR (400 MHz, DMSO-D6): d
–
(ppm) ¼ 8.10 (s, 1H, CH N), 7.82 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.41–
–
7.34 (m, 2H, Ar–H), 4.71 (t, J ¼ 6.4 Hz, 2H, CH2), 3.26 (t,
J ¼ 6.4 Hz, 2H, CH2), 2.76 (t, J ¼ 7.6 Hz, 2H, CH2), 1.52–1.44 (m,
2H, CH2), 1.31–1.23 (m, 3H, CH2, and 1H of NH), 0.85 (t,
J ¼ 7.2 Hz, 3H, CH3). ESI–MS m/z calcd for C13H18BrN3, 295.07;
found: [MþHþ] 296.4.
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4-Bromo-1H-Indazole Derivatives as FtsZ Inhibitors
Archiv der Pharmazie
Light yellow solid, yield 71.5%, mp 58–60°C, Rf ¼ 0.65
for 24 h. The last tube with no growth of microorganism was
recorded to represent the MIC value expressed in mg/mL.
(petroleum ether/ethyl acetate, 1:2). 1H NMR (400 MHz,
–
DMSO-D ): d (ppm) ¼ 8.09 (s, 1H, CH N), 7.71 (d, J ¼ 8.0 Hz,
–
6
1H, Ar–H), 7.41 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.34 (d, J ¼ 8.0 Hz, 1H,
Ar–H), 5.43 (s, 2H, CH2), 4.15 (q, J ¼ 7.2 Hz, 2H, CH2), 1.19 (t,
J ¼ 7.2 Hz, 3H, CH3). ESI–MS m/z calcd for C11H11BrN2O2,
282.00; found: [MþHþ] 283.3.
Cell division inhibitory assay
Cell division inhibitory activity of the tested compounds was
performed as described previously [30]. Overnight cultures
were grown in starvation medium supplemented with 1%
hydrolyzed casein and then diluted in starvation medium
supplemented with 3% hydrolyzed casein (B. subtilis) or in
Mueller Hinton medium (S. aureus, E. coli, and P. aeruginosa)
and grown at 37°C. The culture was diluted to A600 of ꢂ0.06,
and 10mL of aliquots were added to transparent 96-well
microtiter plates containing dilutions of the 4-bromo-1H-
indazoles and ciprofloxacin as controls in 100 mL volumes of
medium. After incubation for approximately 5 h (4–5 gener-
ations) at 37°C, 20 mL of culture samples were transferred to
poly-L-lysine-coated slides for microscopy. Cell morphology was
assessed by phase-contrast light microscopy. Lowest concentra-
tion at which filamentation of B. subtilis, E. coli, and P.
aeruginosaorballooningofS.aureuswasrecordedrepresented
the cell division inhibitory activity indicating on-target activity.
1-N-Hydroxycarbamoylmethyl-4-bromo-1H-indazole (18)
A solution of hydroxylamine was obtained by dissolving
H2NOH ꢁ HCl (0.37 g, 5.3 mmol) in 3 mL of methanol and
stirring it with a solution of KOH (0.3 g, 5.3 mmol) in 2 mL of
methanol for 20 min. After the KCl precipitated out was
filtered, the hydroxylamine solution was added dropwise to
the ice cooled solution of 17 (0.3 g, 1.06 mmol). The mixture
was refluxed until the reaction was completed. The solid that
precipitated out was filtered and was recrystallized from
ethanol to furnish compound 18. White crystalline solid, yield
37%, mp 188–201°C, Rf ¼ 0.51 (dichloromethane/methanol,
8:1). 1H NMR (400 MHz, DMSO-D6): d (ppm) ¼ 10.94 (s, 1H,
–
CONH), 9.08 (s, 1H, OH), 8.05 (s, 1H, CH N), 7.68 (d, J ¼ 8.0 Hz,
–
1H, Ar–H), 7.40–7.32 (m, 2H, Ar–H), 5.03 (s, 2H, CH2). ESI–MS
m/z calcd for C9H8BrN3O2, 268.98; found: [MþHþ] 270.4.
This research was supported financially by the National
Natural Science Foundation of China (20872081 and
21072114), the Natural Science Foundation of Shandong
(ZR2010HM092), and China–Australia Centre for Health
Sciences Research (CACHSR, 2014GJ06).
1-N-aminocarbamoylmethyl-4-bromo-1H-indazole (19)
17 (0.31 g, 1.10 mmol) and hydrazine hydrate (0.071 g,
1.21mmol) in methanol was refluxed for about 2 h. The
reaction mixture was then cooled to room temperature and
filtered. The residue was recrystallized from ethanol to
furnish compound 19. White crystalline solid, yield 51%,
mp 210–212°C, Rf ¼ 0.65 (dichloromethane/methanol, 8:1). 1H
NMR (400 MHz, DMSO-D6): d (ppm) ¼ 9.45 (s, 1H, CONH), 8.04
The authors have declared no conflicts of interest.
References
–
(s, 1H, CH N), 7.67 (d, J ¼ 8.0 Hz, 1H, ArH), 7.39–7.31 (m, 2H,
–
CH2), 5.07 (s, 2H, CH2), 4.32 (d, J ¼ 2.8 Hz, 2H, NH2). ESI–MS m/z
calcd for C9H9BrN4O, 268.00; found: [MþHþ] 269.4.
[1] K. J. Hardy, P. M. Hawkey, F. Gao, B. A. Oppenheim, Br. J.
Anaesth. 2004, 92, 121–130.
[2] R. H. Deurenberg, E. E. Stobberingh, Curr. Mol. Med.
2009, 9, 100–115.
[3] I. M. Gould, M. Z. David, S. Esposito, J. Garau, G. Lina,
T. Mazzei, G. Peters, Int. J. Antimicrob. Agents 2012, 39,
96–104.
[4] A. Khalafi-Nezhad, M. N. Soltani Rad, H. Mohabatkar,
Z. Asrari, B. Hemmateenejad, Bioorg. Med. Chem. 2005,
13, 1931–1938.
Antibacterial evaluation
In vitro antibacterial assay
The MIC values were determined using tube dilution method
recommended by NCCLS [29]. Bacterial strains were main-
tained on Mueller Hinton Agar (MHA) medium for 24 h at
37°C. The bacteria were prepared by suspension in 10 mL of
sterile water for colonies from culture on MHA medium.
Mueller Hinton Broth (MHB) was used for bacteria in this test.
The cell density of each inoculum was adjusted in sterile water
[5] S. G. Addinall, E. Bi, J. Lutkenhaus, J. Bacteriol. 1996, 178,
3877–3884.
of
a
0.5 McFarland standard. In this method, various
[6] M. G. Pinho, J. Errington, Mol. Microbiol. 2003, 50,
871–881.
[7] J. Lutkenhaus, S. G. Addinall, Annu. Rev. Biochem. 1997,
66, 93–116.
[8] J. Lowe, F. van den Ent, L. A. Amos, Annu. Rev. Biophys.
Biomol. Struct. 2004, 33, 177–198.
[9] R. L. Lock, E. J. Harry, Nat. Rev. Drug Discov. 2008, 7,
324–338.
concentrations of the 4-bromo-1H-indazole derivatives were
prepared from 128 to 0.25 mg/mL in sterile tubes No. 1–10. A
hundred microliters of sterile MHB was poured in each sterile
tube followed by adding 200 mL of the test compound in tube
1. Twofold serial dilutions were carried out from tube 1 to
tube 10 and excess broth (100 mL) was discarded from the last
tube no. 10. To each tube, 100 mL of standard inoculum
(1.5 ꢀ 108 cfu/mL) was added. 3-MBA, curcumin, oxacillin
sodium, and ciprofloxacin were used as controls. Turbidity
was observed after incubating the inoculated tubes at 37°C
[10] S. Ma, Chem. Med. Chem. 2012, 7, 1161–1172.
[11] D. W. Adams, J. Errington, Nat. Rev. Microbiol. 2009, 7,
642–653.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
273

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 266–274
Y. Wang et al.
Archiv der Pharmazie
[12] M. Osawa, D. E. Anderson, H. P. Erickson, Science 2008,
320, 792–794.
[13] J. M. Andreu, C. Schaffner-Barbero, S. Huecas, D. Alonso,
M. L. Lopez-Rodriguez, L. B. Ruiz-Avila, R. Nunez-
Ramirez, O. Llorca, A. J. Martin-Galiano, J. Biol. Chem.
2010, 285, 14239–14246.
[21] A. Parhi, C. Kelley, M. Kaul, D. S. Pilch, E. J. LaVoie,
Bioorg. Med. Chem. Lett. 2012, 22, 7080–7083.
[22] Y. Zhang, D. Giurleo, A. Parhi, M. Kaul, D. S. Pilch,
E. J. LaVoie, Bioorg. Med. Chem. Lett. 2013, 23,
2001–2006.
[23] C. Kelley, S. Lu, A. Parhi, M. Kaul, D. S. Pilch, E. J. Lavoie,
Eur. J. Med. Chem. 2013, 60, 395–409.
[24] C. Cheptea, V. Sunel, J. Desbrieres, M. Popa, J. Hetero-
cycl. Chem. 2013, 50, 366–372.
[25] S. Berhe, A. Slupe, C. Luster, H. A. Charlier, Jr.,
D. L. Warner, L. H. Zalkow, E. M. Burgess,
N. M. Enwerem, O. Bakare, Bioorg. Med. Chem. 2010,
18, 134–141.
[26] J. Rodriguez, A. Gerpe, G. Aguirre, U. Kemmerling,
O. E. Piro, V. J. Aran, J. D. Maya, C. Olea-Azar,
M. Gonzalez, H. Cerecetto, Eur. J. Med. Chem. 2009,
44, 1545–1553.
[14] T. K. Beuria, M. K. Santra, D. Panda, Biochemistry 2005,
44, 16584–16593.
[15] P. N. Domadia, A. Bhunia, J. Sivaraman, S. Swarup,
D. Dasgupta, Biochemistry 2008, 47, 3225–3234.
[16] D. J. Haydon, J. M. Bennett, D. Brown, I. Collins,
G. Galbraith, P. Lancett, R. Macdonald, N. R. Stokes,
P. K. Chauhan, J. K. Sutariya, N. Nayal, A. Srivastava,
J. Beanland, R. Hall, V. Henstock, C. Noula, C. Rockley,
L. Czaplewski, J. Med. Chem. 2010, 53, 3927–3936.
[17] D. J. Haydon, N. R. Stokes, R. Ure, G. Galbraith,
J. M. Bennett, D. R. Brown, P. J. Baker, V. V. Barynin,
D. W. Rice, S. E. Sedelnikova, J. R. Heal, J. M. Sheridan,
S. T. Aiwale, P. K. Chauhan, A. Srivastava, A. Taneja,
I. Collins, J. Errington, L. G. Czaplewski, Science 2008,
321, 1673–1675.
[18] K. Kumar, D. Awasthi, S. Y. Lee, I. Zanardi, B. Ruzsicska,
S. Knudson, P. J. Tonge, R. A. Slayden, I. Ojima, J. Med.
Chem. 2011, 54, 374–381.
[19] T. Lappchen, A. F. Hartog, V. A. Pinas, G. J. Koomen,
T. den Blaauwen, Biochemistry 2005, 44, 7879–7884.
[20] S. Urgaonkar, H. S. La Pierre, I. Meir, H. Lund,
D. RayChaudhuri, J. T. Shaw, Org. Lett. 2005, 7,
5609–5612.
[27] L. J. Huang, M. L. Shih, H. S. Chen, S. L. Pan, C. M. Teng,
F. Y. Lee, S. C. Kuo, Bioorg. Med. Chem. 2006, 14,
528–536.
[28] T. Yakaiah, B. P. Lingaiah, B. Narsaiah, B. Shireesha,
B. Ashok Kumar, S. Gururaj, T. Parthasarathy, B. Sridhar,
Bioorg. Med. Chem. Lett. 2007, 17, 3445–3453.
[29] J. M. Andrews, J. Antimicro. Chemother. 2001, 48, 5–16.
[30] N. R. Stokes, J. Sievers, S. Barker, J. M. Bennett,
D. R. Brown, I. Collins, V. M. Errington, D. Foulger,
M. Hall, R. Halsey, H. Johnson, V. Rose, H. B. Thomaides,
D. J. Haydon, L. G. Czaplewski, J. Errington, J. Biol. Chem.
2005, 280, 39709–39715.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
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