DNA gyrase inhibitory and antimicrobial activities of some diphenic acid monohydroxamides

Article abstract of DOI:10.1021/jm9701583

The synthesis and inhibitory activity against DNA gyrase of a series of diphenic acid Monchydroxamides 4a-f are described. A protocol of two biological assays showed conclusively that inhibition occurs specifically at the DNA-DNA gyrase complex and is not

Full text of DOI:10.1021/jm9701583

3292
J . Med. Chem. 1997, 40, 3292-3296
DNA Gyr a se In h ibitor y a n d An tim icr obia l Activities of Som e Dip h en ic Acid
Mon oh yd r oxa m id es
Kwasi A. Ohemeng,* Brent L. Podlogar, Van N. Nguyen, J effrey I. Bernstein, Heather M. Krause,
J amese J . Hilliard, and J ohn F. Barrett
Discovery Research, The R.W. J ohnson Pharmaceutical Research Institute, Raritan, New J ersey 08869
Received March 10, 1997X
The synthesis and inhibitory activity against DNA gyrase of a series of diphenic acid
monohydroxamides 4a -f are described. A protocol of two biological assays showed conclusively
that inhibition occurs specifically at the DNA-DNA gyrase complex and is not attributable to
nonspecific inhibition. In the enzyme assays, 4c was as potent as the prototypical quinolone,
nalidixic acid (1), with an IC50 value of 58.3 µg/mL compared to 52 µg/mL for 1. MIC activity
against bacterial strains showed a systematic drop for all compounds relative to 1. For
compounds 4c-e, the addition of PMBN produced dramatic increases in MIC activity indicating
that activity is likely to be related to membrane transport. Molecular modeling of 4a indicates
that the diphenic acid monohydroxamides can bind to the DNA-DNA gyrase complex in a
similar fashion as that hypothesized for the quinolone series according to the hypothesis
suggested by Shen et al. but may not self-associate by π-π stacking. In contrast to the
quinolone series, as the diphenic acid monohydroxamides are shown by molecular mechanics
minimizations to be nonplanar, they may present novel approaches for chemotherapeutic
intervention with a potential for decreased side effects.
In tr od u ction
templates upon which inhibitors of DNA gyrase could
be based.11
The quinolones have become one of the most clinically
useful classes of broad spectrum antibacterial drugs
available today.1,2 Nalidixic acid (1), the first prototypic
“quinolone”, exhibited activity against Gram-negative
bacteria but lacked substantial Gram-positive activity.3
Recently newer broad-spectrum agents, for example,
ofloxacin (2), have been discovered as a result of
extensive variations of the ring system and substituents
of 1.4 In general, the clinically useful members of this
class of antibacterials contain a â-keto acid moiety.
Because the two carbonyl groups of the â-keto acid are
part of a rigid aromatic ring system, they are confor-
mationally restricted to a coplanar orientation which
has been postulated to be optimally positioned to form
critical hydrogen bonds with the DNA-DNA gyrase
complex.5 The extended aromatic ring system, however,
may itself be related to the side effects on the central
nervous system (CNS) and the articular cartilage which
has been reported for members of the quinolone series.6-9
O
O
3a
3b
The results of our searches generated a list of ∼200
potential inhibitors. These were subsequently screened
for DNA gyrase and antibacterial activity. Among the
active compounds in this list were the diphenic acid
monohydroxamide series exemplified by 4a . This class
bears an obvious structural similarity to the diphenic
acid monoamides, which were reported as having DNA
gyrase and antibacterial activity by Kiely and co-
workers.12 We have pursued this series and report
herein the synthesis, molecular modeling studies, DNA
gyrase inhibitory activities, and antibacterial activities
of compound 4a and its analogs 4b-f (Chart 1).
O
O
O
O
F
Ch em istr y
OH
OH
The synthesis of compounds 4a -f is shown in Scheme
1. The hydroxylamine 7c was synthesized as published
earlier.13 The ketone 5f was synthesized from 4-fluo-
roacetophenone and phenylpiperizine (Scheme 2); 5a -e
were obtained from commercial sources. The ketones
were converted to the corresponding oximes with hy-
droxylamine hydrochloride and NaOH, followed by
reduction with sodium cyanoborohydride to give the
hydroxylamines 7a -f.14 The final products (4a -c,e)
were obtained by reaction of the respective hydroxyl-
amine with diphenic anhydride (9) in THF. Compounds
4d ,f were obtained by reacting the hydroxylamines with
the half-ester 8 to give derivative 10 which was selec-
tively hydrolyzed with LiOH to give the final products.
N
N
CH3
N
N
N
O
C2H5
CH3
CH3
1
2
It has therefore been our aim to identify novel
inhibitors of DNA gyrase that retain the hydrogen-
accepting capabilities of the quinolones but lack the
planar ring system.10 We have employed database-
searching techniques using fragments of known inhibi-
tors (3a ,b, together in a single search query) against
our proprietary compound library to identify alternative
X Abstract published in Advance ACS Abstracts, August 15, 1997.
S0022-2623(97)00158-1 CCC: $14.00 © 1997 American Chemical Society

Activities of Some Diphenic Acid Monohydroxamides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3293
Ch a r t 1
HO
O
O
CH3
R1
R2
N
OH
R3
R1
H
OCH3
R2
H
OCH3
R3
H
OCH3
4a
4b
HO
HO
O
CH3
O
CH3
H
N
O
O
N
N
OH
OH
O
S
4c
4d
HO
HO
O
CH3
O
CH3
O
O
N
N
OH
OH
N
N
4f
4e
Ta ble 1. Inhibition of DNA Gyrase Activity
Sch em e 1. Synthesis of Compounds 4a -f
CH3
CH3
CH3
supercoiling inhibition
cleavable complex
H
compd
SCIC50 (µg/mL)
CC50 (µg/mL)a
R1
R2
R1
R2
R1
R2
O
N
N
4a
4b
4c
4d
4e
4f
1
>500
>500
58.3
658
NT
NT
62.5
750
97.7
NT
41
OH
OH
R3
R3
R3
5a–f
6a–f
7a–f
95
>500
O
O
52
EtO
O
a
NT ) not tested if catalytic activity > 500 µg/mL.
O
Cl
O
introduced, inhibitory activity was observed. Three of
the compounds inhibited the DNA gyrase catalytic
activity.
8
9
The use of the DNA gyrase supercoiling inhibition
assay has been the classical approach in identifying and
quantitating the inhibition of DNA gyrase by quino-
lones.15 This assay can detect both GyrA and GyrB
subunit inhibitors, since the reaction exposes the ho-
loenzyme to the potential inhibitor. Unfortunately, in
catalytic assays, reaction conditions such as pH, ionic
strength, intercalation, chelation, and other nonspecific
effects can arise and be mistakenly read as specific
inhibition. Since diphenic acid monohydroxamides may
inhibit by nonspecific mechanisms, it cannot be con-
cluded based on the catalytic activity that they are
bonafide inhibitors of the enzyme. A more specific
variation of the supercoiling inhibition assay is the DNA
gyrase “cleavable complex” assay.16,17 A major differ-
ence in this assay is that the generation of the linearized
DNA fragment requires the catalytic activity of the
holoenzyme to be retained (i.e., free from nonspecific
inhibition) in order for the “cleavable complex” inter-
mediate of DNA gyrase-DNA-drug to be formed. Any
activity that prevents the binding or catalytic activity
of DNA gyrase in a non-mechanism-based manner
would preclude “cleavable complex” formation. Thus,
any gyrase inhibitor that specifically targets the GyrA
subunit in a mechanism-based manner, such as the
quinolones, would generate a “cleavable complex” and
EtO
HO
O
CH3
O
CH3
O
R1
R2
O
R1
R2
N
N
OH
OH
R3
R3
10a–f
4a–f
Sch em e 2. Synthesis of compound 5f
O
EtN3, ∆
N
N H + F
CH3
11
12
O
N
N
CH3
5f
Resu lts a n d Discu ssion
The inhibitory activities of the six diphenic acid
monohydroxamides against the Escherichia coli DNA
gyrase supercoiling activity and their ability to facilitate
the “cleavable complex” are shown in Table 1. Also
shown in Table 1 are the comparative DNA gyrase
inhibitory activities of nalidixic acid. The initial com-
pound 4a did not have appreciable activity against the
enzyme, but as larger hydrophobic substituents were

3294 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Ohemeng et al.
Ch a r t 2
this feature, the interatomic distance between the two
hydrogen bond acceptors is slightly increased compared
to a â-keto acid. The R and S stereoisomers were
evaluated in both conformations, but the stereogenic
center appears sufficiently removed from the pharma-
cophore center and renders only a minimal effect on the
conformational energies and interatomic distances.
Both conformations are capable of satisfying the dis-
tance requirements as defined by the nalidixic acid and
ofloxacin series and can form multiple hydrogen bond
interactions with a strand of “relaxed” DNA.
h
P
1
The antibacterial activities of the compounds are
shown in Table 2. Antibacterial activity was only
observed with the enzyme-active diphenic acid mono-
hydroxamides against Gram-positive organisms. Gram-
negative antibacterial activity was not observed with
any of the compounds and was presumed to be related
to their poor penetration into the bacterial cell. There-
fore, we studied antibacterial activity in a modified MIC
assay by using polymyxin B nonapeptide (PMBN),
which semipermeabilizes Gram-negative cells by form-
ing selective membrane channels.22 In the presence of
PMBN, compounds exhibited Gram-negative antibacte-
rial activity with MICs as low as 2 µg/mL (Table 2).
We have identified the diphenic acid monohydroxa-
mides as a class of antibacterials which exhibit bonafide
DNA gyrase inhibition. The potencies of this series are
comparable to the first-generation quinolone antibac-
terials, and the compounds lack a planar structure. This
lack of planarity may come at the expense of decreased
π-π stacking, which has been postulated to be a driving
force of the observed quinolone drug cooperativity in the
drug-DNA-DNA gyrase complex.5 Design modifica-
tions aimed at increasing the potencies of this series
are currently underway.
would be reported as a bonafide active inhibitor. How-
ever, nonspecific inhibition that would normally be
detected as a “false positive” in the DNA gyrase super-
coiling inhibition assay would not appear as active in
the DNA “cleavable complex” assay.
As seen in Table 1, all of the diphenic acid monohy-
droxamides that inhibited the DNA gyrase catalytic
activity also facilitated the cleavable complex. We
therefore concluded that the activity of all diphenic acid
monohydroxamides with supercoiling inhibitory activi-
ties is in fact against the GyrA subunit of DNA gyrase.
The potencies of the most active diphenic acid mono-
hydroxamides at the enzyme level were in the range of
the first-generation 4-quinolone, nalidixic acid (IC50
)
52 µg/mL; 4c, IC50 ) 58.3 µg/mL). To characterize the
structural features of this series as related to the
quinolone series, a molecular modeling study of the
initial lead structure 4a was conducted. The carbonyl
and carboxylic acid interatomic distance, which is a
prominent feature in the nalidixic acid and ofloxacin
series and constitutes an obvious pharmacophore ele-
ment that should be reproduced in novel DNA gyrase
inhibitors, was used as the basis for the initial sub-
structure searches.18 The optimal distance between the
ring carbonyl oxygen and a carboxylate oxygen of
nalidixic acid was determined from the X-ray structure
to be ∼3.9 Å.19 Additionally, in accord with the coop-
erative drug-DNA binding model proposed by Shen et
al.,5 mimics of quinolone inhibitors must also possess
the ability to self-assemble via intermolecular π-π
interactions and form a tight stacking arrangement
within the active site. In the present series, the ortho-
substituted acid prohibits the biphenyl rings from
adopting a coplanar geometry suitable for π-π stacking
analogous to that postulated in the quinolone binding
model.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined on a
Meltemp II melting point apparatus and are uncorrected.
Thin-layer chromatography (TLC) was performed on silica gel
plates with a fluorescent indicator and visualized with light
at 254 nm. 1H NMR spectra were recorded on GE-300
spectrometers; signals are reported in ppm from tetrameth-
ylsilane. MS data were collected on a double-focusing mag-
netic sector Finnigan MAT 8230 mass spectrometer. Com-
bustion analyses were obtained with a Perkin-Elmer Model
2400 elemental analyzer and are reported within 0.4% of the
theoretical values unless otherwise indicated.
Gen er a l P r oced u r e for th e P r ep a r a tion of Oxim es 6a -
f. The following procedure for the preparation of N-h yd r oxy-
N-[2-(3,4,5-tr im eth oxyp h en yl)-2-eth yl]im in e (6b) is rep-
resentative. To a solution of 3′,4′,5′-trimethoxyacetophenone
(44 g, 0.21 mol) and hydroxylamine hydrochloride (50 g, 0.72
mol) in 90% EtOH (700 mL) was added powdered NaOH (75
g, 1.88 mol) in small portions. The mixture was allowed to
stir at 25 °C for 30 min and then refluxed for another 30 min.
The reaction mixture was then cooled to 25 °C and poured into
a mixture of concentrated HCl (80 mL) and water (320 mL).
The resulting precipitate was filtered, washed with water, and
dried to give 46 g (98% yield) of product. The oximes were
taken directly to the next step without purification and
characterization.
Molecular mechanics and molecular orbital optimiza-
tions of 4a confirm the existence of two prominent local
minima (conformers 1 and 2, shown in Chart 2). The
preferred conformer (1) is lower in energy by ∼10 kcal/
mol and is dominated by an internal hydrogen bond
between the N-hydroxyl hydrogen and carboxylate
group. Additionally, the phenyl ring bearing the car-
boxylic acid and the terminal phenyl ring form a
hydrophobically collapsed π-π interaction.20 Effec-
tively, the carboxylic acid and carbonyl groups, compa-
rable to those of the quinolone series, are in a transoid
configuration and would not be capable of binding in a
similar fashion as a â-keto acid to a common DNA
strand. However, the N-hydroxyl group may act as a
hydrogen bond acceptor in place of the carbonyl oxy-
gen.21 While the higher energy conformer 2 possesses
Gen er a l P r oced u r e for th e P r ep a r a tion of Hyd r oxyl-
a m in es 7a -f. The following procedure for the preparation
of N-h ydr oxy-N-[2-(3,4,5-tr im eth oxyph en yl)-2-eth yl]am in e
(7b) is representative. To a solution of N-hydroxy-N-[2-(3,4,5-
trimethoxyphenyl)-2-ethyl]imine (6b) (46 g, 0.20 mol) in MeOH
(500 mL) were added NaBH3CN (15 g, 0.24 mol) and a trace
of methyl orange; 12 N HCl was added dropwise until the color
remained pink. After 1 h at 25 °C, TLC showed starting
materials, and more NaBH3CN (9.23 g, 146.9 mmol) was

Activities of Some Diphenic Acid Monohydroxamides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3295
Ta ble 2. MIC (µg/mL) of Compounds 4a -f and Nalidixic Acid on Gram-Positive and Gram-Negative ((PMBN) Bacteria
E.coliss
E. coli KL-16
S. epidermidis
S. aureus
compd
PMBN (0 µg/mL)
PMBN (10 µg/mL)
PMBN (0 µg/mL)
PMBN (10 µg/mL)
strain OC2603
strain OC6538
4a
4b
4c
4d
4e
4f
1
1000
>1000
>1000
>1000
>1000
>1000
15.6
>1000
>1000
>1000
>1000
>1000
>1000
8
>1000
>1000
31.2
125
31.25
>1000
1000
1000
15.6
62.5
31.25
500
1000
1000
15.6
62.5
31.25
500
32.5
2.0
500
31.2
>1000
1
250
500
PMBN
500
>500
added. Additional 12 N HCl was added dropwise to retain an
acidic mixture. The reaction mixture was allowed to stir at
25 °C for 3.5 h. The reaction mixture was reduced in vacuo,
and 6 N NaOH was added until the pH was ∼9. The product
was extracted with CH2Cl2 (3 × 250 mL), dried over anhydrous
MgSO4, filtered, and concentrated. The product was purified
by column chromatography on silica gel with 1:4 EtOAc:hexane
chloride (1.94 g, 6.73 mmol) was allowed to stir at 25 °C for 2
h. The solvent was removed in vacuo, and water was added.
The pH of the aqueous solution was adjusted to 7 with 6 N
HCl, and then the mixture was extracted with EtOAc. The
product was extracted with EtOAc, dried over anhydrous
MgSO4, filtered, and concentrated. The product was purified
by column chromatography on silica gel (230-400 mesh) eluted
with 1-2% MeOH/CH2Cl2 to give 1.8 g (49% yield): mp 133.5-
135.1 °C; 1H NMR (DMSO-d6) δ 7.83 (bs, 2 H), 7.45-6.78 (m,
15 H), 4.03 (bs, 1 H), 3.33 (s, 3 H), 3.25 (bs, 8 H), 1.16 (q, 2 H),
0.87 (t, 3 H); MS m/z 550 (M + 1). Anal. (C34H35N3O4) C, H,
N.
1
to give 45 g (97% yield) of product: mp 84-85 °C; H NMR
(CDCl3) δ 6.54 (s, 2 H), 4.00 (q, 1 H), 3.85 (s, 9 H), 3.84 (d, 3
H), 1.35 (d, 1 H); MS m/z 228 (M + 1). Anal. (C11H17NO4) C,
H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Hy-
d r oxa m ic Acid s (4a -c,e). The following procedure for the
preparation of N-h yd r oxy-N-[2-(3,4,5-tr im eth oxyp h en yl)-
eth yl]-2-(2-ca r boxyp h en yl)ben za m id e (4b) is representa-
tive. A mixture of N-hydroxy-N-[2-(3,4,5-trimethoxyphenyl)-
2-ethyl]amine (7b) (2.23 g, 9.81 mmol) in THF (100 mL) and
diphenic anhydride (2.23 g, 9.95 mmol) was refluxed for 5 h.
The solvent was removed in vacuo, and the residue was put
on a silica gel column and eluted with 5% MeOH/CH2Cl2 to
give 3 g (68% yield) of product: mp 55-57 °C; 1H NMR (CDCl3)
δ 7.95 (m, 2 H), 7.51-7.13 (m, 6 H), 6.44 (d, 2 H), 3.92 (q, 1
H), 3.83 (d, 3 H), 3.78 (s, 9 H); MS m/z 228 (M + 1). Anal.
(C25H25NO7) C, H, N.
4′-(1-P h en ylp ip er a zin e)a cetop h en on e (5f). To a solu-
tion of 1-phenylpiperazine (10.0 g, 62 mmol) in DMSO (150
mL) were added 4′-fluoroacetophenone (8.5 g, 62 mmol) and
Et3N (10 mL). The solution was refluxed for 23 h and then
poured into ice water (500 mL). The product was collected by
vacuum filtration to give 16.7 g (97% yield) of product. The
ketone was recrystallized from MeOH: mp 179.9-181.5 °C;
1H NMR (CDCl3) δ 7.17 (d, 2 H), 7.33-6.91 (m, 7 H), 3.44 (d,
8 H), 2.54 (s, 3 H); MS m/z 281 (M + 1). Anal. (C18H20NO2)
C, H, N.
N-[1-[4-(4-P h en ylp ip er a zin -4-yl)p h en yl]et h yl]-N-h y-
d r oxyla m in e (7f). To a solution of 4′-(1-phenylpiperazine)-
acetophenone (5f) (16 g, 57 mmol) and hydroxylamine hydro-
chloride (13.8 g, 0.20 mol) was added powdered NaOH (20.2
g, 0.50 mol) in 90% EtOH (200 mL) in small portions. The
mixture was allowed to stir at 25 °C for 30 min and then
refluxed for another 30 min. The reaction mixture was then
cooled to 25 °C and poured into a mixture of concentrated HCl
(80 mL) and water (320 mL). The resulting precipitate was
filtered, washed with water, and dried. The oxime was
dissolved in MeOH (500 mL), and to this solution were added
NaBH3CN (15 g, 0.24 mol) and a trace of methyl orange; 12 N
HCl was added dropwise to retain an acidic media. The
reaction mixture was allowed to stir at 25 °C for 3.5 h. The
reaction mixture was reduced in vacuo, and 6 N NaOH was
added until the pH was ∼9. The product was extracted with
CH2Cl2 (3 × 250 mL), dried over anhydrous MgSO4, filtered,
and concentrated. The product was purified by column chro-
matography on silica gel with 1:4 EtOAc:hexane to give
hydroxylamine 7b: 9.8 g (58% yield overall); mp 149.5-151.7
°C; 1H NMR (CDCl3) δ 7.32-6.89 (m, 9 H), 4.05 (q, 1 H), 3.33
(d, 8 H), 1.38 (d, 3 H); MS m/z 298 (M + 1). Anal. (C18H23N3O)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Hy-
d r oxa m ic Acid s 4d ,f. The following procedure for the
preparation of 1-[4-[(N-h yd r oxy-2′-ca r boxy-2-bip h en ylca r -
boxa m id o)eth ylid en e]p h en yl]-4-p h en ylp ip er a zin e (4f) is
representative. To a solution of ethyl 2-[2-[N-hydroxy-N-[1-
[4-(4-phenylpiperazin-1-yl)phenyl]ethyl]formamido]phenyl]-
benzoate (1.6 g, 2.91 mmol) in 2-propanol (70 mL) and H2O
(5.7 mL) was added LiOH (1.13 g, 47.18 mmol). The mixture
was allowed to reflux for 12 h. The solution was then made
acidic by adding 12 N HCl. The product was extracted with
CH2Cl2 (3 × 250 mL), dried over MgSO4, and concentrated in
vacuo. Purification by column chromatography on silica gel
(230-400 mesh) eluted with 1-2% MeOH/CH2Cl2 gave 1.2 g
(79% yield) of product: mp 177.8-178.4 °C; 1H NMR (DMSO-
d6) δ 7.83-6.74 (m, 17 H), 5.28 (bs, 1 H), 3.33 (s, 3 H), 3.24 (d,
8 H); MS m/z 550 (M + 1). Anal. (C34H35N3O4) C, H, N.
Molecu la r Mod elin g. All molecular modeling was per-
formed with the use of the software package SYBYL23 running
on a Silicon Graphics Indigo 2 workstation. Geometry opti-
mizations were first performed with the molecular mechanics
SYBYL/Tripos force field24 using Gasteiger-Huckel charges.
The resulting structures were then geometry optimized using
the AM1 Hamiltonian25 in the molecular orbital package
SPARTAN.26 Database searching was accomplished using
ISIS 2.0118 with both 3a ,b present in the same search query.
DNA Gyr a se Clea va ble Com p lex Assa y.17 Supercoiled
BR322 DNA (0.4 µg) was added to a reaction mixture composed
of 0.2 mM ATP, 5 mM spermidine, 5 nM DTT, 0.2 mM
Na2EDTA, 1.0% glycerol, 10 mM MgCl2, 100 µg/mL E. coli
tRNA (Sigma type XXI), 50 µg/mL bovine serum albumin
(ultrapure-BRL), and 35 mM Tris-HCl, pH 7.5, in 30 µL
reaction mixtures. To this reaction was added drug from
DMSO-solubilized stocks (such that the final concentration of
DMSO e 3.5%) followed by 3 units of gyrase holoenzyme. The
DNA gyrase subunits A and B were purified to homogeneity
from E. coli-overproducing strains (provided by M. Gellert,
NIH) and purified by the procedure of Mizuuchi et al.27 The
individual subunits were combined in 1:1 molar ratios and
reconstituted as the holoenzyme for use in the supercoiling
and cleavable complex assay. The reaction mixture was
incubated for 45 min at 25 °C. The reaction was stopped by
the addition of SDS (to 0.2%) followed by the addition of
proteinase K (to 90 µg/mL final concentration) to digest the
enzyme-DNA complex and incubation at 37 °C for 30 min.
This denaturation of the gyrase-DNA complex, followed by
proteolytic digestion of the protein portion of the complex,
releases the linear DNA fragments from the complex which
can then be separated from open circular and supercoiled DNA
by agarose gel electrophoresis. One microliter tracking dye
(50% glycerol, 0.125% bromophenol blue) was added to the
reaction mixture, and the mixture was loaded onto a 0.7%
agarose gel for electrophoretic separation. The reaction
Gen er a l P r oced u r e for th e P r ep a r a tion of th e Hy-
d r oxa m ic Acid s 10d ,f. The following procedure for the
preparation of eth yl 2-[2-[N-h yd r oxy-N-[1-[4-(4-p h en ylp ip -
er a zin -1-yl)p h en yl]et h yl]for m a m id o]p h en yl]b en zoa t e
(10f) is representative. N-[1-[4-(4-Phenylpiperazin-4-yl)phen-
yl]ethyl]-N-hydroxylamine (7f) (2.0 g, 6.72 mmol) in THF (50
mL) and Et3N (6 mL) and ethyl-2-(2-carbonylphenyl)benzoyl

3296 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Ohemeng et al.
(7) Akahane, K.; Sekiguchi, M. A.; Une, T.; Osada, Y. Structure-
Epileptogenicity Relationship of Quinolones with Special Refer-
ence to Their Interaction with Gamma-aminobutyric Acid Re-
ceptor Sites. Antimicrob. Agents Chemother. 1989, 33, 1704-
1708.
mixture was electrophoresed in a horizontal submarine gel
through a 0.7% Tris-acetate-EDTA agarose gel to separate
different DNA topoisomers and linear DNA fragments from
the denatured gyrase-DNA-compound complex. Linearized
DNA was detected as a single band between open circular DNA
and supercoiled DNA after separation of the products by
electrophoresis. The gel was stained with ethidium bromide,
visualized by UV fluorescence at 300 nm, and documented by
Polaroid film 665 photography. Percent supercoiling was
determined by the densitometric tracing (area determination
using Collage Image Analysis software, FOTODYNE, New
Berlin, WI) of the linearized DNA bands. The cleavable
complex CC50 was determined by measuring the amount of
drug required to induce 50% cleavage of the maximal amount
of linear DNA from supercoiled DNA. Under these conditions,
the linear range of quantitation of DNA is up to 1.2 µg (data
not shown).
DNA Gyr a se Su p er coilin g In h ibition Assa y.16 A Portion
of 0.25-0.40 µg of pBR322 DNA (previously relaxed with
topoisomerase I) was added to a reaction mixture composed
of 1.4 mM ATP, 1.8 mM spermidine, 5 mM DTT, 0.14 mM
Na2EDTA, 6.5% glycerol, 24 mM KCl, 4 mM MgCl2, 0.36 µg/
mL bovine serum albumin (molecular biology grade), and 35
mM Tris-HCl, pH 7.5. To this reaction was added drug from
DMSO-solubilized stocks (such that the final concentration of
DMSO e 3.5%), followed by 1 unit of gyrase holoenzyme
(reconstituted GyrA and GyrB subunits).27 Each reaction
mixture was incubated for 30 min at 37 °C, and reactions were
stopped by the addition of SDS (to 0.5%), Na2EDTA (to 6 mM),
and 5.35% glycerol containing 0.013% bromophenol blue (as
tracking dye). The total reaction mix was loaded onto either
a 1% TAE or TBE agarose gel and was electrophoresed in a
horizontal submarine apparatus to separate different DNA
topoisomers. Gels were stained with EtBr and visualized by
Polaroid film 667 photography of fluoresced gels. The percent
supercoiling was determined by the densitometric tracing
(Collage, Image Dynamics Corp.) of supercoiling versus relaxed
DNA, normalized against no-drug control lanes.
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Gyrase Inhibitory and Antibacterial Activity of Some Flavones.
I. Bioorg. Med. Chem. Lett. 1993, 3, 225-230.
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of the test compounds was determined in either Mueller
Hinton broth for Gram-negatives or Luria broth for Gram-
positives in a microtiter well dilution series; 2-fold serial
dilutions (range 0.015-1000 µg/mL) of compound in broth were
inoculated with adjusted suspensions of test organisms to
approximately 5 × 105 CFU/mL. Microtiter plates were
incubated overnight at 35 °C. MIC is defined as the well
concentration with no visible growth. PMBN was used in
conjunction with test compounds being screened at doses (at
levels of PMBN exhibiting no antibacterial activity in itself)
lower than the MIC and compared to PMBN-free controls.
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some that had been reported by Kiely,12 and found that the
diphenic acid monoamides had lower potencies in our enzyme
assay than the diphenic acid monohydroxamides. For example,
the diphenic acid monoamide 13 possessed an IC50 of 96 µg/mL
in our enzyme assay.
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