Synthesis, structure and antibacterial activity of novel 1-(5-substituted-3-substituted-4,5-dihydropyrazol-1-yl)ethanone oxime ester derivatives

Article abstract of DOI:10.1016/j.bmc.2008.01.035

A series of novel 1-(5-substituted-3-substituted-4,5-dihydropyrazol-1-yl)ethanone oxime ester derivatives are synthesized. The results show that compounds 14 and 26c can strongly inhibit Staphylococcus aureus DNA gyrase and Escherichia coli DNA gyrase (with IC₅₀ of 0.25 and 0.125 μg/mL against S. aureus DNA gyrase, 0.125 and 0.25 μg/mL against E. coli DNA gyrase). On the basis of the biological results, structure-activity relationships are also discussed.

Full text of DOI:10.1016/j.bmc.2008.01.035

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4075–4082
Synthesis, structure and antibacterial activity
of novel 1-(5-substituted-3-substituted-4,5-dihydropyrazol-
1-yl)ethanone oxime ester derivatives
a
b,
b
Xin-Hua Liu,a,* Pin Cui, Bao-An Song, Pinaki S. Bhadury,
*
Hai-Liang Zhua,c and Shi-Fan Wanga
aKey Laboratory of Anhui Educational Department, Anhui University of Technology, Maanshan 243002, PR China
bKey Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education,
Guizhou University, Guiyang 550025, PR China
cState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
Received 10 December 2007; accepted 11 January 2008
Available online 29 January 2008
Abstract—A series of novel 1-(5-substituted-3-substituted-4,5-dihydropyrazol-1-yl)ethanone oxime ester derivatives are synthesized.
The results show that compounds 14 and 26c can strongly inhibit Staphylococcus aureus DNA gyrase and Escherichia coli DNA
gyrase (with IC50 of 0.25 and 0.125 lg/mL against S. aureus DNA gyrase, 0.125 and 0.25 lg/mL against E. coli DNA gyrase).
On the basis of the biological results, structure–activity relationships are also discussed.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
of the known DNA gyrase inhibitors, it has become
imperative to identify new class of compounds.
DNA gyrase, a typical of type II topoisomerase, has been
known to cause DNA replication, transcription, and
recombination.1 DNA gyrase catalyzes the ATP-depen-
dent introduction of negative supercoils into bacterial
DNA as well as the decatenation and unknotting 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., ciprofloxa-
cin).3,4 Unfortunately, of late, multidrug-resistant
Gram-positive bacteria, such as methicillin-resistant
Staphylococcus aureus, and penicillin-resistant Strepto-
coccus pneumoniae have started posing serious issues in
medical science to deal with. To overcome the limitations
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 anti-
microbial agent after the discovery of the natural pyra-
zole C-glycoside, pyrazofurin which demonstrated a
broad spectrum of antimicrobial activity.9 The pyrazole
derivatives used as a potent and selective inhibitor
against DNA gyrase capable of causing bacterial cell
death have also been reported. 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 compounds 2a, 2b, and 3
(Fig. 1) as potent and selective inhibitors of DNA gyr-
ase. From the structures shown in Figure 1, it may be
noticed that all the selective inhibitors of DNA gyrase
contain the characteristic arylpyrazole template. In our
previous work, we reported that some 1-acetyl-5-(substi-
tuted-phenyl)-3-methy-4,5-dihydropyrazole derivatives
showed antibacterial activity against Bacillus subtilis
ATCC 6633 and Pseudomonas fluorescens ATCC
13525.13 Earlier, the derivatives of 3,5-diaryl-4,5-dihyd-
ropyrazole were also well known to possess bioactivities,
such as Christopher D. Cox14,15 disclosed 3,5-diaryl-4,
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 bro-
mide; MICs, minimum inhibitory concentrations; NMM, N-methyl-
morpholine; DMAP, N,N-dimethyl-4-aminopyridine; DMSO, dimethyl
sulfoxide; MH, Mueller–Hinton.
Keywords: Synthesis; 5-Arylpyrazole; Oxime ester; Antibacterial
activity.
*
Corresponding authors. Tel.: +86 851 3620521; fax: +86 851
3622211; e-mail: songbaoan22@yahoo.com
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.035

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X.-H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 4075–4082
O
S
H
O
N
N
O
H2N
N
N
CF3
S
O
O
Celecoxib
1
CH3
H
H
N
N
MeOOC
N
N
H
N
HN
O
2a: R1 = 4- Bu
2b: R2 = 3,4-diCl
O
3
R
2
Figure 1. Recently disclosed pyrazole as antibacterial inhibitors.
5-dihydropyrazole as a potent, selective inhibitor of
kinesin spindle protein.
monohydrate in acetic acid.22 But, on the other hand,
when the cyclization with hydrazine monohydrate was
carried out under neutral condition, instead of the re-
ported 3-methyl-4,5-dihydro-1H-pyrazole,an unusual
product 2,5,5-trimethyl-1,5,6,10b-tetrahydropyrazolo[5,
1-a]isoquinoline (14) was formed which was character-
ized by single X-ray crystallographic data.
Recently, oxime ester derivatives have attracted consid-
erable attention in medicinal research due to their anti-
phytoviral, antitumor, and fungicidal bioactivities. A
large number of investigations on their synthesis and
biological activities have been reported during the last
20 years.16,17
The ketones 6, 7, 17, 18, and 24 were converted into
their corresponding oximes by hydroxylamine. Spectro-
scopic studies of the oximes and the oxime esters in
solution confirmed the presence of anti configuration.
The preparation of oximes was the key step for the
synthesis of the title compounds. In order to optimize
the reaction conditions for preparation of oximes, the
synthesis was carried out in presence of various bases
(Table 1), for example: NaHCO3, KHCO3, NaCH3-
CO2, NaOH, pyridine. It was found that a yield up
to 60% (62) could be attained when the reaction mix-
ture was refluxed for 10 h (15) in ethanol catalyzed
by NaCH3CO2.
Motivated by the afore-mentioned findings, we antici-
pate that the presence of aryl-4,5-dihydropyrazole group
in the oxime ester part should play an important role in
the antimicrobial activities. But few reports have been
dedicated to the synthesis and antimicrobial activities
evaluation of oxime ester containing aryl-4,5-dihydropy-
razole group. Herein, in continuation to extend our
research on antibacterial compounds containing aryl-
4,5-dihydropyrazole group,18,19 we designed a series of
new aryl-4,5-dihydropyrazole oxime ester derivatives.
In the molecule design against antimicrobial activities,
considering to detecting the role of the presence position
of aryl in the 4,5-dihydropyrazole, we designed and syn-
thesized 3-methyl (or aryl)-5-aryl (or methoxyl)-pyrazole
oxime ester and 3,5-diaryl-pyrazole oxime ester deriva-
tives. The results showed that some 3-aryl-5-methoxyl-
pyrazole oxime ester compounds in this series exhibited
potent antibacterial activity.
The esterification of compounds 10, 11, 19, 20, and 25
with acyl chloride produced 12, 13, 21, 22, and 26,
respectively, in moderate yield. In order to optimize
the reaction conditions, the esterification reaction was
carried out under various conditions. First, the effect
of temperature was studied and the best result could
be obtained between 0 and 20 ꢁC. While negligible
amount of product was obtained at temperature below
0 ꢁC, on increasing the reaction temperature above
20 ꢁC, a number of side products were obtained causing
significant reduction in the yield of title compounds. In
addition, role of various bases, such as pyridine, trieth-
ylamine, sodium carbonate, potassium carbonate,
NMM, and DMAP was also studied. The result demon-
strated that the presence of NMM could accelerate the
esterification reaction. Further, the effect of solvent,
reaction time, and molar ratio of the components on
the esterification reaction was examined. The best result
was obtained when oxime was reacted with 1.25 equiv of
acyl chloride and 1.5 equiv of NMM in chloroform at 0–
20 ꢁC for 10 h.
2. Chemistry
The synthetic routes to target compounds are shown in
Schemes 1–3. Claisen–Schmidt condensation20 of a ke-
tone with an aldehyde in the presence of NaOH or
H2SO4 gave the enones (4, 15, and 16) in generally good
yields (highest for acetone). Dehydrogenation of ketone
using mild oxidizing agent HIO3ÆDMSO, by following a
reported method,21 proved to be an efficient alternative
for the synthesis of a, b unsaturated ketone 23.
Compounds 6, 7, 17, 18, and 24 were synthesized by
cyclization of a, b-unsaturated ketone with hydrazine

X.-H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 4075–4082
4077
c
O
CHO
N
N
CH=CH
C CH3
a
HO
HO
14
b
H3C
4
N
d
OH
N
Ac
6:
7:
O
-OH
H3C
H3C
p-OH
N
N
e
O
O
f
N
N
C
F
Ac
NOH
F
H2
O C
H2
H3C
8:
o
F
F
o
O C
F
10:
H2
O C
H2
O C
9: p-
p
F
11:
H3C
N
O
N
C
F
NOCOR1
H3C
H2
O C
H2
O C
p
F
13a-13k:
o
F
12a-12e:
R1: CH2CH3, (CH2)2CH3, C6H4-p-CF3,C6H4-o-F, C6H4-p-F, C6H4-p-Cl, C6H4-o-Cl, 2,4-2Cl-C6H3, 3-pyridine, CH2=CH,
Ph-CH=CH
Scheme 1. Synthesis of novel isoquinoline and 1-(5-aryl-3-methyl-4,5-dihydropyrazol-1-yl)ethanone oxime ester derivatives. Reagents and
conditions: (a) CH3COCH3, NaOH, C2H5OH, 25 ꢁC, 15 h; (b) N2H4ÆH2O, 98% CH3COOH, reflux, 2 h; (c) N2H4ÆH2O, n-butanol, reflux, 10 h;
(d) p-F-Ph-CH2Cl, NaOH, CHCl3, reflux, 3 h; (e) NH2OHÆHCl, NaCH3CO2, pyridine, reflux, 8 h; (f) RCOCl, NMM, CHCl3.
O
Cl
Cl
O
O
g
CH3
R2
H
+
R2
Cl
Cl
N
15:
16:
o
p
-F
Ac
-CF3
NOH
H3C
N
h
N
N
i
Cl
R2
Cl
R2
Cl
Cl
17:
18:
o
-F
-CF3
19:
20:
o
-F
-CF3
p
p
NOCOR3
H3C
N
N
j
21:
o-F
Cl
22:
p-CF3
Cl
R2
R2: o-F, p-CF3
R3: CH2CH3, C6H4-p-CF3, C6H4-o-F, C6H4-p-F, C6H4-p-Cl, CH2=CH, Ph-CH=CH, C6H4-o-NO2, C6H4-o-Me, 2-furan
Scheme 2. Synthesis of novel 1-(3,5-diaryl-4,5-dihydropyrazol-1-yl)ethanone oxime ester derivatives. Reagents and conditions: (g) H2SO4, MeOH,
reflux, 10 h; (h) N2H4ÆH2O, 98% CH3COOH, reflux, 4 h; (i) NH2OHÆHCl, NaCH3CO2, pyridine, reflux, 8 h; (j) RCOCl, NMM, CHCl3.

4078
X.-H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 4075–4082
Ac
O
O
N
N
k
l
H3CO
OMe
OMe
23
24
NOCOR4
NOH
H3C
H3C
N
N
N
n
m
N
H3CO
H3CO
25
26
R4: CH2CH3, C6H4-p-CF3, C6H4-p-F, C6H4-p-Cl, Ph-CH=CH, C6H4-o-NO2, C6H4-o-Me, 2-furan
Scheme 3. Synthesis of novel 1-(5-methoxy-3-aryl-4,5-dihydropyrazol-1-yl)ethanone oxime ester derivatives and isoquinoline. Reagents and
conditions: (k) HIO3ÆDMSO, 60 ꢁC, 15 h; (l) N2H4ÆH2O, 98% CH3COOH, reflux, 2 h; (m) NH2OHÆHCl, NaCH3CO2, reflux, 10 h; (n) RCOCl,
NMM, CHCl3.
Table 1. Yields of 10 under various reaction conditions
bered ring [C(3)–C(4)–C(12)–C(11)–C(7)–N(2)] and the
benzene ring is 7.44 (48)ꢁ. The angle between the five-
membered ring [N(1)–N(2)–C(7)–C(8)–C(9)] and the
Entry Solvent
Base
Time (h) Temperature Yield
(%)
aromatic ring is 77.40 (28)ꢁ.
l
n-Butanol NaHCO3
n-Butanol KHCO3
C2H5OH NaOH
10
10
10
15
20
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
—
2
3
4
5
6
7
10
—
6
3. In vitro antibacterial assay
CH3OH NaOH
n-Butanol NaOH
The activities of synthesized compounds were tested
against B. subtilis ATCC 6633, Escherichia coli ATCC
35218, P. fluorescens ATCC 13525, and S. aureus ATCC
6538 using MH medium. The MICs of the compounds
against four bacteria are presented in Table 2. Also in-
cluded was the activities of reference compounds kana-
mycin, penicillin, and novobiocin. The compounds
22k, 22n, 26c, and 26k showed antimicrobial activities
against B. subtilis with the MIC of 1.562 lg/mL, compa-
rable to that of positive control penicillin. Compounds
14 and 26n with the MIC of 0.78 and 1.25 lg/mL,
respectively, exhibited promising antimicrobial activities
against B. subtilis which were even better than that of
the commercial fungicide penicillin. The compounds
14, 21j, and 22n showed antimicrobial activities against
S. aureus with the MIC of 3.125 lg/mL, comparable to
that of positive control novobiocin, compounds 21n
and 26c with the MIC of 1.562 lg/mL, exhibited antimi-
crobial activities against S. aureus surpassing that of the
commercial fungicide novobiocin. The compounds 14,
21k, 22k, 22n, 26c, 26k, and 26n showed antimicrobial
activities against P. fluorescens with MIC values in the
range 1.562–3.125 lg/mL, comparable to the positive
control kanamycin and penicillin. The compounds 14,
21n, 26c, and 26n showed promising antimicrobial activ-
ities comparable to the positive control kanamycin and
novobiocin and even better than that of the commercial
fungicide penicillin against E. coli with MIC values
ranging from 1.562–3.125 lg/mL.
C2H5OH NaCH3CO2 15
C2H5OH Pyridine 10
62
32
2.1. Crystal structure analysis
The skeleton of the new unusual compound 14 contains
a benzene ring, a six-membered non-aromatic heterocy-
clic ring, a five-membered ring with two nitrogens, and
three methyl groups directly attached to the ring car-
bons (Fig. 2). The bond length of C(9)–N(1) (1.320
˚
(12) A) is slightly shorter than that of typical C@N
(1.34 A), which is indicative of significant double bond
˚
˚
character; C(11)–N(2) (1.426 (11) A) is in good agree-
˚
ment with normal single C–N (1.47 A) bond, N(1)–
˚
N(2) (1.379 (10) A) is comparable with the general
N–N single bond length. The bond angles of C(11)–
N(2)–C(7) and C(9)–N(1)–N(2) are 112.9 (6)ꢁ and
108.9 (7)ꢁ, respectively. The angle between the six-mem-
From the structure–activity relationships presented in
Table 2, it can be concluded that all 5-phenyl-3-
methyl-4,5-dihydropyrazole derivatives displayed poor
activity against four strains, but only some 3-phenyl-5-
phenyl-4,5-dihydropyrazole derivatives showed good
activity against bacterial strains, specially against S. aur-
eus ATCC 6538 and Pseudomonas eruginosa ATCC
13525. The most active agent against the bacterial
Figure 2. The molecular structure of compound 14.

X.-H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 4075–4082
4079
Table 2. Minimum inhibitory concentrations (MIC-lg/mL) of the title compounds negative control DMSO, no activity
Compound
Gram-positive
Staphylococcus aureus
Gram-negative
Pseudomonas fluorescens
Bacillus subtilis
Escherichia coli
12a
12b
CH2CH3
50
50
50
50
50
50
50
50
(CH2)2CH3
C6H4-p-CF3
C6H4-o-F
12c
12d
50
50
50
50
50
50
50
50
12e
13a
C6H4-p-F
CH2CH3
>50
50
>50
50
>50
50
>50
50
13b
13c
(CH2)2CH3
C6H4-p-CF3
C6H4-o-F
50
25
>50
25
50
25
>50
50
13d
13e
25
50
50
50
25
50
50
50
C6H4-p-F
13f
13g
C6H4-p-Cl
C6H4-o-Cl
2,4-2Cl–C6H3
3-Pyridine
CH2@CH
Ph-CH@CH
Isoquinoline
CH2CH3
>50
50
>50
50
>50
50
>50
12.5
12.5
6.25
50
13h
13i
12.5
6.25
12.5
12.5
1.25
50
12.5
6.25
25
50
12.5
50
13j
13K
14
12.5
3.125
50
12.5
3.125
50
12.5
1.562
50
21a
21c
C6H4-p-CF3
C6H4-o-F
C6H4-p-F
12.5
12.5
25.0
12.5
3.125
6.25
25
12.5
25.0
25.0
50
50
12.5
12.5
12.5
50
21d
21e
6.25
25.0
50
21f
21j
C6H4-p-Cl
CH2@CH
Ph-CH@CH
C6H4-o-NO2
C6H4-o-Me
2-Furan
3.125
6.25
50
12.5
3.125
50
25.0
50
21k
21l
50
50
21m
21n
50
3.125
50
50
50
1.562
50
50
6.25
50
3.125
50
50
22a
22c
CH2CH3
C6H4-p-CF3
C6H4-o-F
C6H4-p-F
25.0
12.5
25.0
12.5
3.125
1.562
50
50
22d
22e
50
12.5
25.0
50
50
50
25.0
50
22f
22j
C6H4-p-Cl
CH2@CH
Ph-CH@CH
C6H4-o-NO2
C6H4-o-Me
2-Furan
50
6.25
12.5
50
12.5
3.125
50
12.5
6.25
50
22k
22l
22m
22n
50
50
50
50
1.562
12.5
1.562
3.125
3.125
1.562
6.25
12.5
0.78
1.562
0.39
0.78
3.125
12.5
1.562
6.25
6.25
6.25
6.25
12.5
6.25
1.562
1.562
3.125
1.562
12.5
1.562
12.5
6.25
3.125
12.5
25.0
1.562
6.25
3.125
1.562
12.5
25.0
3.125
6.25
25.0
12.5
12.5
12.5
3.125
6.25
3.125
3.125
26a
26c
CH2CH3
C6H4-p-CF3
C6H4-p-F
26e
26f
C6H4-p-Cl
Ph-CH@CH
C6H4-o-NO2
C6H4-o-Me
2-Furan
26k
26l
26m
26n
Penicillin
Kanamycin
Novobiocin
strains was 5-methoxy-3-phenyl-4,5-dihydropyrazol,
such as 26c (with MICs of 1.562, 1.562, 1.562, and
3.125 lg/mL against B. subtilis ATCC 6633, S. aureus
ATCC 65385, P. fluorescens ATCC 13525, and E. coli
ATCC 35218) and 26n (with MICs of 0.78, 1.562, and
3.125 lg/mL against B. subtilis ATCC 6633, P. fluores-
cens ATCC 13525, and E. coli ATCC 35218). Further,
the presence of furan group in the oxime ester part
played an important role in the antimicrobial activities
(such as 21n, 22n, and 26n), however, introduction of al-
kyl group in the oxime ester depressed the antimicrobial
activities. Compound 14 (a saturated analogue of iso-
quinoline) has potentially high antimicrobial activity.
To elucidate the mechanism by which the pyrazole
derivatives induce antibacterial activity, the inhibitory
activity of selected compounds (12a, 13i, 14, 21n, 21j,
22n, 26c, and 26n) was examined against DNA gyrase
isolated from S. aureus and E. coli. As shown in Table
3, compounds 14 and 26c with potent antibacterial
activities strongly inhibited S. aureus DNA gyrase and
E. coli DNA gyrase (with IC50s of 0.25 and 0.125 lg/
mL against S. aureus DNA gyrase, 0.125 and 0.25 lg/
mL against E. coli DNA gyrase). Compounds 21j and
22n showed moderate inhibition against the S. aureus
DNA gyrase (IC50 = 0.5 lg/mL). There was a good cor-
relation between the MICs and the IC50s of 14 and 26c

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X.-H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 4075–4082
Table 3. Inhibitory effects of the selected title compounds against
DNA gyrase
hydrazine hydrate (2.2 mmol) in n-butanol (30 mL).
The reaction mixture was refluxed for 8 h and washed
with 5% HCl solution (10 mL). The solvent was re-
moved in vacuo, added dropwise acetone/petroleum
(10 mL, volume ratio 2:1). The resultant solution was
kept to evaporate slowly in air. When the solvent vol-
ume was reduced to almost half of the original, large
brown slab crystals of the title compound were depos-
ited and collected by filtration (yield 56%). Mp 155–
156 ꢁC. Elemental analysis: Anal. Calcd for C14H18N2:
C, 78.46; H, 8.47; N, 13.07. Found: C, 78.40; H, 8.33;
N, 12.97.
a
Compound
IC50 (lg/mL)
S. aureus DNA gyrase
E. coli DNA gyrase
12a
13i
>125
3.5
>125
4.0
14
21n
0.25
0.125
0.5
0.125
1.0
100
21j
22n
0.5
8.0
26c
26n
0.125
4.0
0.28
0.25
0.25
0.31
Novobiocin
a DNA gyrase supercoiling activity.
5.2.2. General synthetic procedure process for 1-
(5-substituted-3-substituted-4,5-dihydro-pyrazol-1-yl)eth-
anone oxime (10, 11, 19, 20, and 25). The appropriate
1-(5-substituted-3-methyl-4,5-dihydropyrazol-1-yl)etha-
none (1 mol equiv), hydroxylamine hydrochloride
(4.0 mol equiv), and sodium acetate (2.5 mol equiv) in
absolute ethanol (30 L per mol of ethanone) were heated
under reflux for 10 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
dichloromethane, washed with water and dried. The sol-
vent was removed in vacuo and the crude oxime mixture
was recrystallized from ethanol to give the compound
(10, 11, 19, 20, and 25). The structure was confirmed
by 1H NMR, 13C NMR, IR, and elemental analysis
(see the Supporting information).
(Tables 2 and 3), indicating that inhibition of the DNA
gyrase by the pyrazole derivatives caused inhibition of
bacterial cell growth.
4. Conclusion
A series of novel 1-(5-substituted-3-substituted-4,5-dihy-
dropyrazol-1-yl)ethanone oxime ester derivatives and an
unexpected compound 2,5,5-trimethyl-1,5,6,10b-tetra-
hydro-pyrazolo[5,1-a]isoquinoline (14) were synthesized.
The compounds are 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 14 and 26c possess potent antibacterial
activity and can strongly inhibit S. aureus DNA gyrase
and E. coli DNA gyrase (with IC50s of 0.25 and
0.125 lg/mL against S. aureus DNA gyrase, 0.125 and
0.25 lg/mL against E. coli DNA gyrase).
5.2.3. General synthetic procedure for 1-(5-substituted-3-
substituted-4,5-dihydropyrazol-1-yl)ethanone oxime ester
(12, 13, 21, 22, and 26). To a solution of 1-(5-substi-
tuted-3-methyl-4,5-dihydropyrazol-1-yl)ethanone oxime
(2 mmol) and NMM (0.010 mmol) in chloroform
(30 mL) at 10 ꢁC was added dropwise acid chloride
(3.0 mmol) for 30 min. The reaction mixture was stirred
at room temperature for 8 h and washed with 20 mL
H2O, 10 mL 5% NaHCO3, respectively, then dried on
anhydrous MgSO4. The solvent was removed in vacuo
and the crude residue was purified by chromatography
on SiO2 (acetone/petroleum, 5:1, v/v) to give title com-
pound as a colorless solid. The spectral data can be
found in the Supporting information.
5. Experimental
5.1. Analysis and instruments
Melting points were measured and not corrected. The
1H NMR spectra were recorded on a Varian INOVA300
(300 MHz) pulse Fourier-transform NMR spectrometer
in CDCl3. Elemental analysis was performed by a Vario-
III CHN analyzer and was within 0.4% of the theoret-
ical values. ESI mass spectra were obtained on a Mari-
ner System 5303 mass spectrometer. Analytical TLC
was performed on silica gel GF254. Column chromato-
graphic purification was carried out using silica gel. All
reagents were of analytical grade or chemically pure. All
solvents were dried, deoxygenated, and redistilled before
use. Compounds 4, 6, 7, 8, 9, 15, 16, 17, 18 and 24
were prepared according to literature method as de-
scribed.13,18 Compound 23 was prepared according to
a previously published report.21
Compound 12a: Colorless crystals, yield, 51%; mp 170–
171 ꢁC,1H NMR (CDCl3, 300 MHz): d 1.16 (t, 3H, Me,
J = 7.2 Hz), 2.22 (s, 3H, Me), 2.35 (q like, 2H, COCH2),
2.40 (s, 3H, Me), 3.02 (dd, 1H, J1 = 18.3, J2 = 3.1 Hz, 4-
Ha), 3.44 (dd, 1H, J1 = 18.3, J2 = 11.1 Hz, 4-Hb), 5.33 (s,
2H, CH2–O), 5.59 (dd, 1H, J1 = 11.1, J2 = 3.1 Hz, 5-H),
6.81–7.32 (m, 8H, ArH); 13C NMR (CDCl3, 125 MHz):
d 9.7, 16.4, 21.5, 26.1, 43.4 (CH2-4), 53.8 (CH-5), 73.5,
114.9, 115.8, 117.8, 122.3, 128.8, 129.5, 130.9, 138.0,
157.2, 158.4 (C-3), 163.3, 169.6, 178.5 (C@N–OCO);
ESI-MS: 397.0 (C22H24FN3O3, [M+H]+). Anal. Calcd
for C22H24FN3O3: C, 66.48; H, 6.09; N, 10.57. Found:
C, 66.23; H, 6.21; N, 10.39.
5.2. Syntheses
5.3. Crystal structure determination
5.2.1. Synthesis of 2,5,5-trimethyl-1,5,6,10b-tetrahydro-
pyrazolo[5,1-a]isoquinoline (14). To a solution of the 4-
(2-hydroxyphenyl) but-3-en-2-one (2 mmol) was added
A sample of size 0.25 · 0.21 · 0.16 mm3 was selected for
the crystallographic study. The diffraction measurement

X.-H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 4075–4082
4081
was performed at room temperature (293 K) using
graphite monochromated MoKa radiation (k =
Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223
336033; e-mail: deposit@ccdc.cam.ac.uk). Supplemen-
tary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.01.035.
˚
0.71073 A) and an Enraf-Nonius CAD-4 four-circle dif-
fractometer. Accurate cell parameters and orientation
matrix were obtained by least-squares refinement of
the setting angles of 1542 reflections at the h range of
3.00 < h < 24.99. The systematic absences and intensity
symmetries indicated the Monoclinic Cc space group.
The corrections for LP factors were applied. The struc-
ture was solved by direct methods and refined by full-
matrix least-squares techniques on F2 with anisotropic
thermal parameters for all non-hydrogen atoms. The
calculations were performed with SHELXL-97 pro-
gram.23 The molecular structure of the compound 14
is shown in Figure 2. Crystallographic data (excluding
structure factors) for the structure have been deposited
with the Cambridge Crystallographic Data Center as
supplementary publication No. CCDC-668105.
References and notes
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37 ꢁC for 24 h. After the MICs were visually determined
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(phosphate buffered saline 0.01 mol/L, pH 7.4: Na2H-
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0.2 g, distilled water 1000 mL) containing 2 mg of
MTT/mL was added to each well. Incubation was con-
tinued at room temperature for 4–5 h. The content of
each well was removed, and 100 lL of isopropanol con-
taining 5% 1 mol/L HCl was added to extract the dye.
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550 nm. The MICs were observed.
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Acknowledgment
The work was financed by
a
Grant (Project
070416274X) from Anhui Natural Science Foundation,
Anhui Province, China.
Supplementary data
CCDC-668105 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained
free of charge via the URL http://www.ccdc.cam.ac.uk/
conts/retrieving.html (or from the CCDC, 12 Union

4082
X.-H. Liu et al. / Bioorg. Med. Chem. 16 (2008) 4075–4082
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