Synthesis, structure and antibacterial activity of new 2-(1-(2-(substituted-phenyl)-5-methyloxazol-4-yl)-3-(2-substitued-phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-7-substitued-1,2,3,4-tetrahydroisoquinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2008.12.034

A series of new 2-(1-(2-(substituted-phenyl)-5-methyloxazol-4-yl)-3-(2-substitued-phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-7-substitued-1,2,3,4-tetrahydroisoquinoline derivatives were synthesized. The results showed that compounds 9q and 10q can strongly inhi

Full text of DOI:10.1016/j.bmc.2008.12.034

Bioorganic & Medicinal Chemistry 17 (2009) 1207–1213
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure and antibacterial activity of new 2-(1-(2-(substituted-
phenyl)-5-methyloxazol-4-yl)-3-(2-substitued-phenyl)-4,5-dihydro-1H-
pyrazol-5-yl)-7-substitued-1,2,3,4-tetrahydroisoquinoline derivatives
a
a
b,
Xin-Hua Liu a, , Jing Zhu , An-na Zhou , Bao-An Song , Hai-Liang Zhu a,c, Lin-Shan bai a,
*
*
Pinaki S. Bhadury b, Chun-Xiu Pan a
a School of Chemistry and Chemical Engineering, Anhui Key Laboratory of Coal Clean Conversion and Utilization, Anhui University of Technology, Maanshan 243002, PR China
b Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
c State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 2-(1-(2-(substituted-phenyl)-5-methyloxazol-4-yl)-3-(2-substitued-phenyl)-4,5-dihy-
dro-1H-pyrazol-5-yl)-7-substitued-1,2,3,4-tetrahydroisoquinoline derivatives were synthesized. The
results showed that compounds 9q and 10q can strongly inhibit Staphylococcus aureus DNA gyrase and
Received 18 October 2008
Revised 10 December 2008
Accepted 12 December 2008
Available online 24 December 2008
Bacillus subtilis DNA gyrase (with IC50s of 0.125 and 0.25
0.125
tionships were also discussed.
l
g/mL against S. aureus DNA gyrase, 0.25 and
l
g/mL against B. subtilis DNA gyrase). On the basis of the biological results, structure–activity rela-
Keywords:
Synthesis
Ó 2008 Elsevier Ltd. All rights reserved.
Methyloxazol pyrazole
Antibacterial activity
DNA gyrase inhibitor
1. Introduction
Many pyrazole derivatives are well acknowledged to possess a
wide range of antibacterial bioactivities.5–8 Much attention was
paid to pyrazole as a potential antimicrobial agent after the discov-
ery of the natural pyrazole C-glycoside pyrazofurin which demon-
strated a broad spectrum of antimicrobial activity.9 Some pyrazole
derivatives used as potent and selective inhibitors against DNA
gyrase are capable to cause bacterial cell death, for example, Hoff-
mann–La Roche’s group10,11 has developed a new lead DNA gyrase
inhibitor (compound 1, Fig. 1). Recently, Tanitame et al.12 have
found compound 2 (Fig. 1) as potent and selective inhibitors of
DNA gyrase. In our previous work, we13 found compound 3
(Fig. 1) 2,5,5-trimethyl-1,5,6,10b-tetrahydropyrazolo(5,1-a)iso-
quinoline to possess potent antibacterial activity capable of inhib-
iting Staphylococcus aureus DNA gyrase and Escherichia coli DNA
DNA gyrase, a typical of type II topoisomerase, has been known
to cause DNA replication, transcription and recombination.1 DNA
gyrase catalyzes the ATP-dependent introduction of negative
supercoils into bacterial DNA as well as the decatenation and unk-
notting of DNA.2 DNA gyrase is mainly inhibited by quinolones and
coumarins, some of which are widely used for the treatment of
bacterial infectious diseases (e.g., ciprofloxacin).3,4 Unfortunately,
of late, multidrug-resistant Gram-positive bacteria have started
posing serious issues in medical science to deal with. To overcome
the limitations of the known DNA gyrase inhibitors, it has become
imperative to identify new class of compounds.
gyrase (with IC50s of 0.25
lg/mL against S. aureus DNA gyrase,
Abbreviations: B. subtilis, Bacillus subtilis; E. coli, Escherichia coli; P. fluorescens,
Pseudomonas fluorescens; S. aureus, Staphylococcus aureus; MTT, 3-(4,5-dimethyl-2-
thiazyl)-2,5-diphenyl-2H-tetrazolium bromide; MICs, minimum inhibitory concen-
trations; NMM, N-methylmorpholine; DMSO, dimethyl sulfoxide; DMF, dimethyl
formamide, DMAP, N,N-dimethyl aminopyridine, MH, Mueller–Hinton; HEPES, N-2-
hydroxyethylpiperazine-N0-2-ethanesulfonic acid; PMSF, phenylmethyl-sulfonyl
fluoride.
* Corresponding authors. Tel.: +86 555 23111551; fax: +86 555 2311552 (X.-H.
Liu).
E-mail addresses: xhliuhx@163.com (X.-H. Liu), songbaoan22@yahoo.com (B.-A.
Song).
0.125 g/mL against E. coli DNA gyrase). We also reported that
some 1-acetyl-5-(substituted-phenyl)-3-methy-4,5-dihydropyraz-
ole derivatives and some 1-(5-substituted-3-substituted-4,5-dihy-
l
dropyrazol-1-yl) ethanone oxime ester derivatives
4 (Fig. 1)
showed antibacterial bioactivities.13,14 All these selective inhibitors
of DNA gyrase shown in Figure 1 contain diarylpyrazole template.
Oxazoles are key building elements of natural products. Among
the numerous heterocyclic moieties of biological and pharmaco-
logical interest, the oxazole ring is endowed with various activi-
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.034

1208
X.-H. Liu et al. / Bioorg. Med. Chem. 17 (2009) 1207–1213
H
O
H
N
N
O
N
N
S
N
N
HN
O
Ph
O
3
1
2
O
S
O
N
H2N
N
CF3
S
O
COOMe
CF3
NOC
N
N
H3C
O
N
N
MeOOC
N
Me
Celecoxib
O
CH3
H3CO
4
5
Figure 1. Recently disclosed pyrazole and oxazole as antibacterial inhibitors.
ties.15,16 The substituted oxazole system occurs in various antivi-
rals17 and antibiotics.18 For example, sulfomycin I, a novel thiopep-
the reaction mixture was refluxed for 15 h in ethanol catalyzed
by NaCH3CO2 and pyridine.
tide antibiotic produced by
a
subspecies of Streptomyces
Microwave chemistry, a non conventional popular technique,
has been successfully employed in the preparation of oxazole.
Lee et al. developed a solvent free method in which (hydroxy-
(2,4-dinitrobenzenesulfonyloxy)iodo)benzene reacted with vari-
viridochromogenes, which exhibits strong inhibitory activity
against gram-positive bacteria (the oxazole structure 5 (Fig. 1)),
was isolated from the sulfomycin I. Furthermore, it was found that
the same heterocycle subunit is present in berninamycin A,19 thi-
oxamycin20 and A10255.21
ous ketones in a household microwave to form a-((2,4-dinitroben-
zene)sulfonyl)oxy ketones, which were then converted into
oxazoles through treatment with amides.29 Microwave irradiation
is also known to promote the rapid O,N-acylation–cyclodehydra-
tion cascade reaction of oximes and acid chlorides to give oxaz-
oles.30 Malamas et al.’s method31 for converting oxime to oxazole
under microwave irradiation at various temperatures in pyridine/
toluene (5.6:1, moL) actually turned out to be a failure in our
hands. Catalytic amount of NMM or DMAP in DMF was added in
an attempt to facilitate acyl transfer process which led to a much
faster conversion. The major advantage from using microwave irra-
diation was noted in terms of considerable increase in the product
yield. Control reactions with oximes 8 and acid chloride under a
range of thermal and optimized microwave conditions are shown
in Table 1. Physical appearance, melting points and yields of title
compounds 9–10 are shown in Table 2.
Motivated by the aforementioned findings, we anticipate that
the presence of oxazole and aryl-4,5-dihydropyrazole moiety in
quinoline part should play an important role in the antimicrobial
activities. However, to date, only a few reports have been dedicated
to the synthesis and antimicrobial activity evaluation of quinoline
containing oxazole and aryl-4,5-dihydropyrazole moiety. Herein,
in continuation to extend our research on antibacterial compounds
containing aryl-4,5-dihydropyrazole group,22,23 we designed a ser-
ies of new 2-(1-(2-(substituted-phenyl)-5-methyloxazol-4-yl)-3-
(2-substitued-phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-7-substitued-
1,2,3,4-tetrahydroisoquinoline derivatives. Fluorinated compounds
in general are of significant interest in modern medicinal chemis-
try. The trifluoromethyl-substituted compounds have been re-
ported to possess biological activities and are often employed as
fungicides,24 analgesic agents,25 antipyretic agents.26 So, in our
present design of new compounds with improved activity, the role
of incorporation of fluorinated moiety into the parent structure
was investigated. The results showed that some 2-(1-(2-(substi-
tuted-phenyl)-5-methyloxazol-4-yl)-3-(2-substitued-phenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-7-substitued-1,2,3,4-tetrahydroisoquin-
oline compounds in this series exhibited potent antibacterial
activity.
3. In vitro antibacterial assay
The activities of synthesized compounds were tested against
Bacillus subtilis ATCC 6633, E. coli ATCC 35218, Pseudomonas fluores-
cens ATCC 13525 and S. aureus ATCC 6538 which may be causal
agents of some serious infections in humans using MH medium
(Mueller-Hinton medium: casein hydrolysate 17.5 g, soluble starch
1.5 g, beef extract 1000 mL). The MICs of the compounds against
four bacteria are presented in Table 3. Also included are the activ-
ities of reference compounds kanamycin, penicillin and novobio-
cin. The results revealed that some of the synthesized
compounds exhibited significant antibacterial activity, especially
against B. subtilis ATCC 6633 and S. aureus ATCC 6538.
2. Chemistry
The synthetic route to target compounds is shown in Scheme 1.
Dehydrogenation of ketone using mild oxidizing agent HIO3ꢀDMSO
at 100 °C, by following a reported method,27 proved to be an effi-
cient alternative for the synthesis of
a,b-unsaturated ketone 6.
The compounds 9e, 9s, 9r and 10s, 10r, 10f showed antibacte-
Compounds 7 were synthesized by cyclization of
a
,b-unsatu-
rial activities against B. subtilis with the MIC of 1.562
parable to that of positive control penicillin. Compounds 10e and
10q with MIC value of 0.78 g/mL exhibited promising antibacte-
rial activities against B. subtilis which were even better than that
of the commercial penicillin. Compound 9q with the MIC of
lg/mL, com-
rated ketone with hydrazine monohydrate in propionic acid.28
The Compounds 7 were converted into their corresponding oxi-
mes 8 by hydroxylamine. Spectroscopic studies of the oximes in
solution confirmed the presence of anti configuration. The prepara-
tion of oximes was the key step for the synthesis of the title com-
pounds. In order to optimize the reaction conditions for
preparation of oximes, the syntheses were carried out in presence
of various bases, for example: NaHCO3, KHCO3, NaCH3CO2, pyri-
dine. It was found that a yield up to 65% could be attained when
l
0.39 lg/mL exhibited potent antibacterial activity against B. subtilis
which was even better than those of the commercial penicillin,
kanamycin and novobiocin. The compounds 9g, 9h, 9r, 9s, 10h
and 10t showed antibacterial activities against S. aureus with the
MIC of 3.125 lg/mL, comparable to that of positive control novobi-

X.-H. Liu et al. / Bioorg. Med. Chem. 17 (2009) 1207–1213
1209
O
O
b
a
N
N
R2
R2
R1
R1
6
R1
R2
R1
N
N
R1
R2
N
N
c
d
O
N
N
N
N
N
O
NOH
R3
N
9-10
8
7
R2
9a-9t
R1: H
10a-10t
R1: 7-OMe
R2: 2-CF3, 2,4-2Cl, 2-F, -H, 4-furan
R3: 2,4-2F, 2-F, 2-CF3, 2-CH=CH2
R2: 2-CF3, 2,4-2Cl, 2-F, -H, 4-furan
R3: 2,4-2F, 2-F, 2-CF3, 2-CH=CH2
Scheme 1. Synthesis of new 2-(1-(2-(substituted-phenyl)-5-methyloxazol-4-yl)-3-(2-substitued-phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-1,2,3,4-tetrahydroisoquinoline
derivatives. Reagent and conditions: (a) HIO3, DMSO, 100 °C, 15 h; (b) N2H4ꢀH2O, 98% CH3CH2COOH, reflux, 4 h; (c) NH2OHꢀHCl, NaCH3CO2, pyridine, reflux, 15 h; (d)
substituted-benzoyl chloride, catalytic NMM, DMSO (dry 4 h), 100 °C, 10 min.
Table 1
other compounds containing substituted phenyl moieties only
Yields of 9a under various reaction conditions
showed moderate antibacterial activity (9a–9d, 9i–9p, 10i–10p).
So the group of compounds having furan-phenyl moieties has a
much higher inhibitory potency than the rest.
Compd
Solvent
Conditions
Additive
Yield (%)
9a
9a
9a
9a
9a
Pyridine
1,2-Dichlorobenzene
DMF
DMF
DMF
lW, 180 °C, 10 min
lW, 160 °C, 10 min
lW, 100 °C, 10 min
lW, 120 °C, 20 min
lW, 100 °C, 10 min
—
—
—
—
36
32
40
Among the other compounds containing substituted phenyl
moieties (difference R2, Scheme 1), they could be divided into
two subunits: A-substituted phenyl ring and A, B-substituted phe-
nyl ring. A comparison of the substitution pattern against the B.
subtilis ATCC 6633 and S. aureus ATCC 65385 strains demonstrated
that 2,4-disubstituted-phenyl analogs afforded compounds with
much higher potency (9e, 9f, 9g, 9h, 10e, 10f, 10g and 10h) than
2 or 4-substituted-phenyl analogs (9i–9p and 10i–10p).
NMMa
DMAPb
NMMa
—: No product.
a
5 mol % of NMM was used.
7 mol % of DMAP was used.
b
The rationale behind selecting a large number of compounds
bearing fluorinated functionalities is to ascertain the role of fluo-
rine in imparting bioactivity. The influence of fluorinated substitu-
ent on bioactivity is clearly demonstrated by the observed higher
activities against B. subtilis ATCC 6633 and S. aureus ATCC 6538.
Similarly, it can be derived from the data presented in Table 3 that
the nature of R3 group in the title compounds significantly influ-
ences the antibacterial activity. With a fluorinated substituent
(2,4-2F) in the phenyl ring, the compounds exhibited enhanced
bioactivity against B. subtilis ATCC 6633 and S. aureus ATCC 6538
(9e, 9q, 10e, 10q).
To elucidate the mechanism by which the pyrazole derivatives
induce antibacterial activity, the inhibitory activities of selected
compounds (9e, 9l, 9f, 9q, 10e, 10j, 10q and 10s) were examined
against DNA gyrase isolated from B. subtilis and S. aureus. As shown
in Table 4, compounds 9q and 10q with potent antibacterial activ-
ities strongly inhibited S. aureus DNA gyrase and B. subtilis DNA
ocin, compounds 9e, 9q, 9t and 10e, 10f, 10g, 10r, 10s with the
MIC of 1.562 g/mL, exhibited antibacterial activities against S.
aureus surpassing that of the commercial novobiocin. The com-
pound 10d showed antibacterial activity against P. fluorescens with
MIC of 3.125 lg/mL, comparable to the positive control kanamycin
l
and even better than that of the commercial penicillin. The com-
pounds 9a, 9n, 9p, 10c and 10j showed moderate antibacterial
activities against P. fluorescens with MIC of 12.5
pound 9n showed antibacterial activity against E. coli with MIC
of 6.25 g/mL, comparable to the positive control penicillin. The
compounds 9m and 9p showed moderate antibacterial activities
against E. coli with MIC of 12.5 g/mL.
lg/mL. The com-
l
l
From the structure–activity relationships presented in Table 3,
it can be concluded that some 2-(1-(2-(substituted-phenyl)-5-
methyloxazol-4-yl)-3-(2-substitued-phenyl)-4,5-dihydro-1H-
pyrazol-5-yl)-7-substitued-1,2,3,4-tetrahydroisoquinoline deriva-
tives showed good activity against Gram positive strains (B. subtilis
ATCC 6633 and S. aureus ATCC 65385), but most of the derivatives
displayed poor activity against Gram negative strain (P. fluorescens
ATCC 13525 and E. coli ATCC 35218).
Among all the synthetic compounds, two classifications can be
made, one containing furan-phenyl moieties which include 9q–9t
and 10q–10t, and the other containing substituted phenyl moieties
(see Scheme 1). From Table 3 we can see that compounds 9q–9t
gyrase (with IC50s of 0.125 and 0.25
gyrase, 0.25 and 0.125 g/ml against B. subtilis DNA gyrase). Com-
pound 9f showed moderate inhibition against the S. aureus DNA
gyrase (IC50 = 1.0 g/mL). Compound 9l showed poor inhibition
against the S. aureus DNA gyrase and B. subtilis DNA gyrase (IC50
>125 g/mL).There was a good correlation between the MICs and
lg/ml against S. aureus DNA
l
l
l
the IC50s of 9q and 10q (Tables 3 and 4), indicating that inhibition
of the DNA gyrase by the pyrazole-oxazole derivatives caused inhi-
bition of bacterial cell growth. Bacterial topoisomerase inhibitors
sometimes have poor selectivity against human topoisomerase,
for example, the compound 9f showed the same activities against
(with MICs of 0.39, 1.562, 1.562 and 3.125
ATCC 6633, with MICs of 1.562, 3.125, 3.125 and 1.562
against S. aureus ATCC 65385) and 10q–10t (with MICs of 0.78,
1.562, 1.562 and 3.125 g/mL against B. subtilis ATCC 6633, with
MICs of 0.78, 1.562, 1.562 and 3.125 g/mL against S. aureus ATCC
l
g/mL against B. subtilis
l
g/mL
l
S. aureus and B. subtilis with the MIC of 3.125
different inhibition against the S. aureus DNA gyrase and B. subtilis
DNA gyrase (IC50 = 5.5 g/mL, 1.0 g/mL respectively).
lg/mL, but it showed
l
65385) showed obvious antibacterial activity against the B. subtilis
ATCC 6633 and S. aureus ATCC 65385 strains, while most of the
l
l

1210
X.-H. Liu et al. / Bioorg. Med. Chem. 17 (2009) 1207–1213
Table 2
Structure, appearance, melting points and yields of title compounds 9–10
Compound
R1
R2
R3
Appearance
Mp (°C)
Yielda (%)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-CF3
2-CF3
2-CF3
2-CF3
2,4-2Cl
2,4-2Cl
2,4-2Cl
2,4-2Cl
2-F
2-F
2-F
2-F
H
2,4-2F
2-F
2-CF3
2-CH=CH2
2,4-2F
2-F
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
Colorless crystals
62–63
59–60
59–60
61–63
66–67
65–66
66–67
65–66
60–61
57–58
61–62
62–63
60–61
59–60
61–61
62–63
68–69
66–67
68–69
67–68
65–66
64–65
65–66
66–67
70–71
68–69
69–70
70–71
64–65
63–64
64–65
65–66
63–64
61–62
63–64
64–65
71–72
69–70
68–69
99–70
40
38
39
34
35
38
33
36
41
42
36
33
45
35
33
31
42
38
36
38
32
31
32
35
34
31
40
30
41
43
44
39
38
42
42
39
34
36
37
33
2-CF3
2-CH@CH2
2,4-2F
2-F
2-CF3
2-CH@CH2
2,4-2F
2-F
H
H
H
2-CF3
2-CH@CH2
2,4-2F
2-F
H
H
H
H
4-Furan
4-Furan
4-Furan
4-Furan
2-CF3
2-CF3
2-CF3
2-CF3
2,4-2Cl
2,4-2Cl
2,4-2Cl
2,4-2Cl
2-F
2-F
2-F
2-F
H
H
H
H
9r
9s
9t
2-CF3
2-CH@CH2
2,4-2F
2-F
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
10t
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
2-CF3
2-CH@CH2
2,4-2F
2-F
2-CF3
2-CH@CH2
2,4-2F
2-F
2-CF3
2-CH@CH2
2,4-2F
2-F
2-CF3
2-CH@CH2
2,4-2F
2-F
2-CF3
2-CH@CH2
4-Furan
4-Furan
4-Furan
4-Furan
a
Yields are based on oxime.
chemically pure. All solvents were dried, deoxygenated, and redis-
tilled before use. Compound 6 was prepared according to a previ-
ously published report.27 Compounds 7 and 8 were prepared
according to literature method as described.5
4. Conclusion
A series of new 2-(1-(2-(substituted-phenyl)-5-methyloxazol-
4-yl)-3-(2-substitued-phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-7-
substitued-1,2,3,4-tetrahydroisoquinoline derivatives
9 and 10
were synthesized. The compounds were evaluated and assayed
for their antibacterial (B. subtilis ATCC 6633, E. coli ATCC 35218,
P. fluorescens ATCC 13525 and S. aureus ATCC 6538) activities by
MTT method. The results show that compounds 9p and 10p pos-
sess potent antibacterial activity and can strongly inhibit S. aureus
DNA gyrase and B. subtilis DNA gyrase, with IC50s of 0.125 and
5.2. Syntheses
5.2.1. General synthetic procedure for 1-(5-(substituted-3,4-
dihydroisoquinolin-2(1H)-yl)-3-(2-(trifluoromethyl)phenyl)-
4,5-dihydropyrazol-1-yl)propan-1-one oxime (8)
The appropriate 1-(5-(substituted-3,4-dihydroisoquinolin-
2(1H)-yl)-3-(2-(trifluoromethyl)phenyl)-4,5-dihydropyrazol-1-yl)
0.25 lg/ml against S. aureus DNA gyrase, 0.25 and 0.125 lg/ml
against B. subtilis DNA gyrase.
propan-1-one
(1 mol equiv), hydroxylamine hydrochloride
(3.0 mol equiv), pyridine (2.0 mol equiv) and sodium acetate
(3.0 mol equiv) in absolute ethanol (20 L per mol of ethanone)
were heated under reflux for 15 h. The mixture was allowed to cool
to room temperature and water (30 L per mol ethanone oxime)
was added. The aqueous layer was extracted with dichlorometh-
ane, washed with water and dried. The solvent was removed in va-
cuo and the crude oxime mixture was recrystallized from ethanol
to give the compounds 8. The structures were confirmed by 1H
NMR and 13C NMR spectral data.
5. Experimental
5.1. Analysis and instruments
Melting points were measured and not corrected. The 1H NMR
spectra were recorded on a Varian INOVA500 (500 MHz) pulse Fou-
rier-transform NMR spectrometer in CDCl3. Elemental analysis was
performed by a Vario-III CHN analyzer and were within 0.4% of the
theoretical values. ESI mass spectra were obtained on a Mariner
System 5303 mass spectrometer. Analytical TLC was performed
on Silica Gel GF254. Column chromatographic purification was car-
ried out using silica gel. All reagents were of analytical grade or
5.2.1.1. 8a:1-(5-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-(2-(trifluoro-
methyl)phenyl)-4,5-dihydropyrazol-1-yl)propan-1-oneoxime. 1H
NMR (CDCl3, 500 MHz): d 1.18 (t, 3H, Me, J = 7.2 Hz), 2.07 (q,

X.-H. Liu et al. / Bioorg. Med. Chem. 17 (2009) 1207–1213
1211
Table 3
Minimum inhibitory concentrations (MIC-
l
g/mL) of the title compounds Negative control DMSO, no activity
Gram positive
Compd
Gram negative
Pseudomonas fluorescens
Bacillus subtilis
Staphylococs aureus
Escherichia coli
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
12.5
12.5
6.25
12.5
1.562
3.125
3.125
6.25
25.0
12.5
6.25
50
6.25
6.25
6.25
12.5
1.562
3.125
3.125
3.125
6.25
12.5
6.25
50
12.5
50
50
25
50
50
50
>50
>50
50
>50
>50
25
>50
>50
50
>50
>50
>50
50
25
50
>50
50
9m
9n
9o
9p
9q
9r
9s
9t
12.5
25.0
12.5
12.5
0.39
1.562
1.562
3.125
3.125
12.5
3.125
6.25
0.78
1.562
3.125
3.125
3.125
12.5
25.0
12.5
6.25
25.0
12.5
50
12.5
6.25
25
12.5
1.562
3.125
3.125
1.562
12.5
50
3.125
6.25
1.562
1.562
1.562
3.125
6.25
50
25.0
50
6.25
25.0
25.0
50
0.78
1.562
1.562
3.125
50
12.5
50
12.5
25.0
25.0
50
50
25.0
50
12.5
3.125
50
>50
>50
>50
50
12.5
25.0
50
25.0
>50
>50
>50
50
12.5
6.25
50
12.5
>50
>50
50
>50
25.0
50
25.0
50
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
10t
50
>50
>50
50
50
50
50
50
50
>50
50
>50
25.0
25.0
50
0.78
1.562
1.562
3.125
>50
25.0
25.0
>50
Penicillin
Kanamyci
Novobiocin
1.562
0.39
0.78
1.562
1.562
3.125
6.25
3.125
1.562
6.25
3.125
3.125
Table 4
5.2.1.2. 8b: 1-(5-(7-Methoxy-3,4-dihydroisoquinolin-2(1H)-yl)-
3-(2-(trifluoromethyl)phenyl)-4,5-dihydropyrazol-1-yl)propan-
1-one oxime. 1H NMR (CDCl3, 500 MHz): d 1.21 (t, 3H, Me,
J = 7.2 Hz), 2.01 (q, 2H, CH2), 2.52 (dd, J = 18.0 and 3.0 Hz, 1H, pyr-
azole, 4-Ha), 2.64 (t, 2H, isoquinoline-H), 2.79 (t, 2H, isoquinoline-
H), 2.81 (dd, J = 18.0 and 11.0 Hz, pyrazole, 1H, 4-Hb), 3.68 (t, 2H,
isoquinoline-H), 3.79 (S, 3H, OMe), 4.55 (dd, J = 11.0 and 3.0 Hz,
1H, pyrazole, 5-H), 6.46–7.88 (m, 7H, ArH), 10.08 (S, 1H, –NOH);
13C NMR (CDCl3, 125 MHz): d 5.9, 19.0, 27.7, 42.9, 48.0, 55.5,
57.7, 65.8, 119.0, 126.3, 126.7, 126.9, 128.1, 128.4, 128.6, 129.9,
130.6, 131.2, 133.4, 135.6, 138.2, 154.1, 168.7.
Inhibitory effects of the selected title compounds against DNA gyrase
a
Compounds
IC50
(lg/mL)
S. aureus DNA gyrase
B. subtilis DNA gyrase
9e
9f
9l
9q
10e
10j
10q
10s
0.5
5.5
>125
0.125
0.5
0.25
1.0
>125
0.25
0.25
>125
0.125
0.25
0.5
4.0
0.25
0.25
0.25
Novobiocin
a
5.2.2. General synthetic procedure for 2-(1-(2-(substituted-
phenyl)-5-methyloxazol-4-yl)-3-(2-(substituted-phenyl)-4,5-
dihydro-1H-pyrazol-5-yl)-substituted-1,2,3,4-tetrahydro-
isoquinoline (9, 10)
To a solution of oxime 8 (2 mmol) and N-methylmorpholine
(0.010 mmol) in DMF (30 mL) at 20 °C was added dropwise acid
chloride (3.0 mmol) for 30 min. The reaction mixture was heated
in the microwave for 10 min at 100 °C and poured into water
(50 mL), then the solution was maintained at 0–5 °C for 10 h. The
product was collected by filtration, and the crude residue was puri-
fied by chromatography on SiO2 (acetone/petroleum, v/v = 4:1) to
DNA gyrase supercoiling activity.
2H, CH2), 2.50 (dd, J = 18.0 and 3.0 Hz, 1H, pyrazole, 4-Ha), 2.68
(t, 2H, isoquinoline-H), 2.73 (t, 2H, isoquinoline-H), 2.85 (dd,
J = 18.0 and 11.0 Hz, pyrazole, 1H, 4-Hb), 3.77 (t, 2H, isoquino-
line-H), 4.52 (dd, J = 11.0 and 3.0 Hz, 1H, pyrazole, 5-H), 6.80-
7.94 (m, 8H, ArH), 10.01 (S, 1H, –NOH); 13C NMR (CDCl3,
125 MHz): d 5.7, 19.1, 27.9, 42.5, 48.2, 57.0, 65.2, 119.3, 126.1,
126.6, 126.7, 128.1, 128.4, 128.8, 129.6, 130.4, 131.3, 133.1,
135.2, 138.2, 154.3, 169.3.

1212
X.-H. Liu et al. / Bioorg. Med. Chem. 17 (2009) 1207–1213
give 9 and 10 as colorless solids. Their spectra are provided in the
Supplementary data.
5.3.2.2. B. subtilis DNA gyrase supercoiling. The B. subtilis DNA
gyrase were purified by the methods of Elisha, O.35: Cells were sus-
pended in an equal volume of 25 mM HEPES-KOH (pH 8.0)–
100 mM KCl and stored frozen at ꢁ70 °C. The frozen cell suspen-
sion was thawed and diluted with an equal volume of 25 mM
HEPES-KOH (pH 8.0)–0.4 M sucrose–20 mM magnesium acetate–
1 mM dithiothreitol–5 mM PMSF. All operations were performed
at 0–4 °C. Lysozyme was added to a final concentration, and the
mixture was incubated for 2.5 h. One-third volume of 2 M KCl–
1.5% Brij was added, and the incubation was continued for 15
min. The lysate was then centrifuged for 90 min in tirotor. The
supernatant was adjusted to a KCl concentration of 0.2 M by dilu-
tion with 25 mM HEPES–KOH (pH 8.0)–1 mM dithiothreitol–1 mM
EDTA–0.5 mM pmsf–10% ethylene glycol and applied to a column.
The column was washed with starting buffer and eluted succes-
sively with buffer containing 20 mM ATP–25 mM magnesium ace-
tate–0.2 M KCI, buffer (2 M KCI), and 5 M urea in buffer (0.2 M KCl).
Protein-containing fractions were dialyzed against buffer (0.05 M
KCI). Supercoiling and decatenation were carried out by the
Blanche, F.33 method.
5.2.2.1.
2-(1-(2-(2,4-Difluorophenyl)-5-methyloxazol-4-yl)-3-
(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-1,2,3,
4-tetrahydroisoquinoline (9a). 1H NMR (CDCl3, 500 MHz): d 2.38
(s, 3H, Me), 2.55 (dd, J = 18.0 and 3.0 Hz, 1H, pyrazole, 4-Ha), 2.62
(t, 2H, isoquinoline-H), 2.73 (t, 2H, isoquinoline-H), 2.87 (dd,
J = 18.0 and 11.0 Hz, pyrazole, 1H, 4-Hb), 3.67 (t, 2H, isoquino-
line-H), 4.48 (dd, J = 11.0 and 3.0 Hz, 1H, pyrazole, 5-H), 6.80–
7.91 (m, 11H, ArH); 13C NMR (CDCl3, 125 MHz): d 5.8, 28.2, 37.6,
47.9, 55.9, 69.2, 106.3, 111.0, 119.2, 119.8, 125.4, 125.5, 125.9,
126.0, 127.9, 128.1, 128.4, 128.8, 129.7, 130.6, 131.0, 132.9,
133.6, 134.1, 138.0, 139.2, 152.3, 161.2, 164.8; ESI-MS: 538.4
(C29H23F5N4O, [M+H]+); Anal. Calcd for C29H23F5N4O: C, 64.68; H,
4.30; N, 10.40. Found: C, 65.03; H, 4.21; N, 10.08.
5.2.2.2. 2-(1-(2-(2,4-Difluorophenyl)-5-methyloxazol-4-yl)-3-
(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-7-meth-
oxy-1,2,3,4-tetrahydroisoquinoline
(10a). 1H
NMR
(CDCl3,
500 MHz): d 2.34 (s, 3H, Me), 2.57 (dd, J = 18.0 and 3.0 Hz, 1H, pyr-
azole, 4-Ha), 2.68–2.73 (m, 4H, isoquinoline-H), 2.89 (dd, J = 18.0
and 11.0 Hz, pyrazole, 1H, 4-Hb), 3.63 (t, 2H, isoquinoline-H),
3.78 (s, 3H, OMe), 4.51 (dd, J = 11.0 and 3.0 Hz, 1H, pyrazole, 5-
H), 6.58–7.87 (m, 10H, ArH); 13C NMR (CDCl3, 125 MHz): d 5.2,
28.0, 38.1, 47.9, 56.2, 56.4, 68.8, 105.4, 112.3, 112.7, 113.0, 119.0,
120.2, 125.2, 125.8, 126.0, 126.2, 128.9, 129.1, 130.0, 131.5,
131.7, 132.2, 135.4, 138.8, 152.5, 158.7, 161.0, 162.0, 164.1; ESI-
MS: 567.7 (C30H25F5N4O2, [M+H]+); Anal. Calcd for C30H25F5N4O2:
C, 63.38; H, 4.43; N, 9.85. Found: C, 63.01; H, 4.67; N, 10.00.
Acknowledgment
The authors wish to thank Universities natural science key re-
search projects of Anhui Province (2009KJ0101AZC) and the open-
ing foundation of the Key Laboratory of Green Pesticide and
Agricultural Bioengineering, Ministry of Education, Guizhou Uni-
versity, grant No.2008GDGP0105. The work was supported by the
Young College Teachers Research Projects of Anhui Province
(2008JQ1030) and we also thank the Young College Teachers Re-
search Projects of Anhui Technology University (QZ200809).
5.3. Bioassay conditions
Supplementary data
5.3.1. In vitro antibacterial activity
The antibacterial activities of the synthesized compounds were
tested against B. subtilis, E. coli, P. fluorescens and S. aureus using
MH medium (casein hydrolysate 17.5 g, soluble starch 1.5 g, beef
extract 1000 mL). The MICs of the test compounds were deter-
mined by a colorimetric method using the dye MTT.32 A stock
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.12.034.
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