Synthesis and antibacterial evaluation of a novel series of 2-(1,2-dihydro-3-oxo-3H-pyrazol-2-yl)benzothiazoles

Article abstract of DOI:10.1002/cbdv.201000241

The 2-(1,2-dihydro-3-oxo-3H-pyrazol-2-yl)benzothiazole scaffold was selected as a central core structure for the discovery of novel antibacterial compounds. A systematic variation of the substituents on the oxo-pyrazole moiety, as well as on the benzo moiety, led to the creation of a small and focused library of benzothiazoles that was subjected to antibacterial screening. In a first round of screening, activity of the compounds against six representative microorganisms was established. For the most potent congeners, MIC values against S. aureus and P. aeruginosa were determined. The structure-activity relationship study clearly revealed that subtle structural variations influence the antibacterial activity to a large extent. The most potent congeners displayed MIC values of 3.30 μM.

Full text of DOI:10.1002/cbdv.201000241

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
253
Synthesis and Antibacterial Evaluation of a Novel Series of 2-(1,2-Dihydro-3-
oxo-3H-pyrazol-2-yl)benzothiazoles
by Alessandro Stellaa), Kenneth Segersb), Steven De Jonghea), Bart Vanderhoydoncka),
Jef Rozenskia), Jozef Anne´ b), and Piet Herdewijn*a)
a) Katholieke Universiteit Leuven, Rega Institute for Medical Research, Laboratory of Medicinal
Chemistry, Minderbroedersstraat 10, BE-3000 Leuven
(phone: þ32-16-337387; fax: þ32-16-337340; e-mail: piet.herdewijn@rega.kuleuven.be)
b) Katholieke Universiteit Leuven, Rega Institute for Medical Research, Laboratory of Bacteriology,
Minderbroedersstraat 10, BE-3000 Leuven
The 2-(1,2-dihydro-3-oxo-3H-pyrazol-2-yl)benzothiazole scaffold was selected as a central core
structure for the discovery of novel antibacterial compounds. A systematic variation of the substituents
on the oxo-pyrazole moiety, as well as on the benzo moiety, led to the creation of a small and focused
library of benzothiazoles that was subjected to antibacterial screening. In a first round of screening,
activity of the compounds against six representative microorganisms was established. For the most potent
congeners, MIC values against S. aureus and P. aeruginosa were determined. The structureꢀactivity
relationship study clearly revealed that subtle structural variations influence the antibacterial activity to a
large extent. The most potent congeners displayed MIC values of 3.30 mm.
Introduction. – Many of the currently marketed antibiotics (penicillins, cephalo-
sporins, aminoglycosides, tetracyclines, erythromycin and related macrolides, vanco-
mycin, and teicoplanin) have been discovered through the empirical (i.e., non-target,
but whole cell-based) antibacterial screening of natural compounds [1]. In 1995, the
determination of the complete DNA sequence of the bacterial genome of Haemophilus
influenza has revolutionized the field [2]. Since then, an explosion in microbial genome
sequencing yielded critical information about essential genes and encoded proteins.
Genomic sequences of many important bacterial pathogens are available, resulting in
almost 100 microbial genomes deposited in the Comprehensive Microbial Resource [3].
There have been many efforts, using a variety of genetic approaches, to define the
subset of essential bacterial genes that would be privileged targets for inhibitor design.
As a result, over the past twenty years, antibiotic drug discovery has been shifted from
natural-product screening to synthetic antibacterials, in which high-throughput screen-
ing (HTS) campaigns against novel targets were used to disclose new classes of drugs
with novel modes of action. The only known synthetic antibacterial compounds
currently licensed for clinical use are sulfamethoxazole and the fluoroquinolones. Both
compound classes have, however, been discovered before the era of genomics,
proteomics, HTS, and combinatorial chemistry. A more recent example of synthetic
antibacterials include the oxazolidinones (e.g., linezolide). It is clear that the screening
of synthetic libraries of compounds against isolated bacterial targets to generate novel
antibacterial leads is very inadequate. As a result, only two new classes of antibiotics
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
with novel mechanisms of action (i.e., daptomycin and linezolide) have been
introduced into the market during the last three decades. Owing to the development
of resistance that follows the introduction of a novel antibiotic on the market, there is a
constant need for new antibacterial drugs. It has been suggested that it is easier to find
the cellular target of an antibacterial compound than it is to engineer permeability into
an enzyme inhibitor [4]. Therefore, we focused on whole-cell assays for finding novel
lead compounds with antibacterial activity.
Benzothiazoles have been extensively studied in medicinal chemistry and have
been associated with a broad range of biological activities. Benzothiazole derivatives
are known to act as kinase inhibitors [4], as microsomal triglyceride transfer protein
inhibitors [5], as neuroprotective agents [6], and as antitumoral agents [7]. A number
of substituted benzothiazoles have been reported as having antibacterial activities. For
example, 2-(pyrazol-2-yl)benzothiazoles display strong antibacterial activities with
MIC values ranging from 2 to 4 mg/ml, depending on the bacterial strain [8]. Recently, a
6-chloro-2-(pyrazol-2-yl)benzothiazole was identified in a HTS program as capable of
curing C. elegans nematodes infected with Enterococcus faecalis [9].
In view of the interesting antibacterial activity associated with this particular
compound class, a systematic structureꢀactivity relationship (SAR) study was set up in
which substituents on the benzo part (substituent R) and the pyrazolone moiety
(substituents R1, R2, and R3) were varied in a systematic way. The compounds were
evaluated for their antibacterial activities against a panel of six microorganisms.
Results and Discussion. – Chemistry. The synthesis of compounds 3a–3u started
from commercially available 2-aminobenzothiazoles, bearing different substituents on
the benzo moiety (Scheme 1). The 2-NH2 group was converted to 2-Cl via diazotation
by treatment with NaNO2 in H2O under acidic conditions. Although, in the classical
Sandmeyer reaction, CuCl is used to introduce a Cl-atom, we observed the formation of
the 2-chlorobenzothiazole derivatives without adding CuCl. The subsequent nucleo-
philic substitution with NH2NH2 afforded 2-hydrazinobenzothiazole derivatives 2a–2g.
Finally, a cyclocondensation reaction with differently substituted b-keto esters, in the
presence of a catalytic amount of AcOH in EtOH, furnished the final compounds 3a–
3u (Table 1).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
255
Scheme 1
i) NaNO2, HCl, H2O, 08. ii) NH2NH2 ·H2O, EtOH, reflux. iii) b-Keto ester, Et3N, AcOH (cat.), EtOH
reflux.
Table 1. Synthesized 2-(Pyrazol-2-yl)benzothiazoles
Compound
R
R1
R2
R3
Compound
R
R1
R2
R3
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Me
6-MeO
6-F
4,6-F2
H
6-Me
5,6-Me2
6-MeO
6-F
Me
Et
i-Pr
Ph
H
H
H
H
Me
Et
i-Pr
Ph
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3s
3t
5-Cl
4-Cl
6-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
Me
Me
Me
Me
Et
Ph
Ph
Bn
H
H
H
H
H
H
H
H
H
H
H
3u
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
9g
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
i-Pr
Ph
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
i-Pr
H
H
H
Ph
Ph
Ph
Ph
H
H
Me
Et
Bn
Me
Et
Pr
Bn
4,6-F2
By selecting the appropriate keto ester, different alkyl groups (Me, Et, i-Pr, and Ph)
at C(4) and C(5) of the pyrazole ring could be introduced.
2-Amino-5-chlorobenzothiazole is not commercially available. Therefore, 5-chloro-
2-(3-oxopyrazol-2-yl)benzothiazole derivatives (¼2-(5-chlorobenzothiazol-2-yl)-1,2-
dihydro-3H-pyrazol-3-ones) were prepared from sulfanyl-aniline 4 (Scheme 2). Con-
struction of the thiazole ring was achieved by heating compound 4 with CS2 under
alkaline conditions affording benzothiazole-2-thiol 5 [10]. Methylation of the SH group
using MeI yielded the 2-methylsulfanyl derivative. Direct substitution of the latter with
NH2NH2 did not work, and, therefore, the MeS group was first oxidized to the
corresponding sulfone with meta-chloroperbenzoic acid (m-CPBA) to furnish com-

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
pound 6. Finally, nucleophilic substitution with NH2NH2 in EtOH as solvent and
subsequent cyclocondensation with b-keto esters afforded the desired compounds 8a–
8g (Table 1).
Scheme 2
i) CS2, KOH, EtOH, reflux. ii) MeI, NaOH, H2O, r.t. iii) m-CPBA, CH2Cl2, 08. iv) NH2NH2 ·H2O, EtOH
reflux. v) b-Keto ester, Et3N, AcOH (cat.), EtOH reflux.
The structures of compounds 3a–3u and 8a–8g were consistent with the 1H-NMR
and high-resolution mass spectra. In addition, for some representative examples,
13C-NMR spectra have been recorded. Due to the existence of different tautomeric
structures of the pyrazolone moiety, some signals are broadened, and, hence, not all C-
atom signals are visible. This problem can be overcome by recording the spectra under
acidic conditions by the addition of one drop of concentrated HCl into the NMR tube
[11].
Alkylations of the pyrazolone ring with alkyl and benzyl halides have also been
performed, with DMF as solvent and Et3N as base (Scheme 3 and Table 1).
Scheme 3
In principle, N, O, and/or C-alkylation is possible. According to NMR spectra and
TLC, only one compound was isolated, indicating the selectivity of the alkylation
reaction. The regioselectivity of the alkylation reaction was established by NMR
spectroscopy. Based on the chemical-shift data of the H-atoms of the alkyl and benzyl
moiety (Table 2), C-alkylation could be ruled out. In fact, CH2 groups resonate
between 3.68 and 5.65 ppm (depending on the group; see Table 2), and these values are
clearly in accordance with a CꢀX bond, where X¼heteroatom.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
257
Table 2. 1H and 13C Chemical-Shift Values of Compounds 9a–9g. d in ppm.
Compound
R2
R4
d(H)
d(C)
9a
9b
9c
9d
9f
9g
H
H
H
H
Ph
Ph
Ph
3.68
4.46
5.61
3.77
4.48
5.65
37.4
44.3
55.6
37.9
50.7
52.5
Me
Ph
H
Et
Ph
However, these data are inconclusive to distinguish between O- and N-alkylation.
From the 13C-NMR data, the C-atoms directly linked to the heteroatom resonate
between 37 and 44 ppm for alkyl groups and at ca. 50 ppm for benzyl group (Table 2).
These values are fully in agreement with literature data of similar compounds [12], and
clearly support N-alkylation. This was further supported by a Heteronuclear Multiple
Bond Correlation (HMBC) experiment. Compound 9b was selected as a representa-
tive example: a long-distance correlation (3J) between the CH2 H-atoms signal of the
Et and the C(3) 13C signal of the pyrazole ring established the regioselectivity of the N-
alkylation The results are in complete agreement with N-alkylation of pyrazolone rings
as recently reported by Janin and co-workers [12].
Biological Evaluation and StructureꢀActivity Relationship (SAR) Study. In a first
round of screening, all compounds were assayed for antibacterial activity against three
representative Gram-positive bacteria (Staphylococcus aureus ATCC 65388, Staph-
ylococcus epidermidis RP62A ATCC 35984, and Bacillus subtilis ATCC 6633) and three
Gram-negative bacteria (Escherichia coli NCIB 8743, Pseudomonas aeruginosa PAO1,
and Salmonella typhimurium sp.) using a growth-inhibition plate assay. The zones of
bacterial growth were visually compared with the control plate containing DMSO
without compound. Results are compiled in Table 3.
From the 35 analogues tested, 13 compounds showed activity against at least one
bacterial strain, indicating that the 2-(3-oxopyrazol-2-yl)benzothiazole scaffold is
ideally suited as a basic heterocyclic structure for searching novel antibacterials. This
first broad screening indicated that N-alkylated derivatives, compounds 9a–9g, are
totally devoid of antibacterial activity, pointing to an essential role of the NH group for
biological activity. The substitution pattern of the benzo moiety influences the
antibacterial activity to a large extent. Compounds 3o (5,6-Me2), 3p (6-MeO), and 3r
(4,6-F2) all contain a 1,2-dihydro-5-methyl-3-oxo-4-phenyl-3H-pyrazol-2-yl moiety at
C(2) of the benzothiazole scaffold and completely lack bacterial activity. Closely
related analogues 3h (6-Cl), 3n (6-Me), 3q (6-F), 3s (5-Cl), and 3t (4-Cl) with an
identical pyrazole moiety, but having a different substitution pattern of the benzo
moiety, do show activities against at least one bacterial strain. A similar trend is

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 3. Antibacterial Screening. Compounds (10 mg/ml) were evaluated for their antibacterial activities
against six representative bacterial strains in a growth inhibition plate assay. Symbols used: þ þ: no
inhibition; þ : intermediate inhibition; –: total inhibition.
Compound
S. aureus
S. epidermidis
P. aeruginosa
E. coli
S. typhimurium
B. subtilis
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
þ þ
þ þ
þ þ
–
þ þ
þ þ
þ
þ þ
þ
þ þ
þ
þ
þ þ
þ
–
þ
þ
–
–
–
þ
þ
þ
þ
þ þ
þ þ
þ
þ
þ/–
þ
þ/–
þ
þ
þ
þ
þ
–
–
þ
þ
þ
–
–
–
–
þ
þ
þ
þ þ
þ þ
þ þ
þ þ
þ þ
–
þ þ
þ þ
–
þ þ
þ
þ
–
þ
þ
þ
þ
þ
þ
þ þ
þ þ
þ þ
–
þ þ
þ þ
þ þ
–
þ
þ
þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ
þ þ
þ þ
þ þ
þ þ
þ þ
–
þ þ
þ þ
–
þ þ
þ þ
–
þ þ
þ þ
þ þ
þ þ
þ þ
3s
3t
–
–
–
–
–
–
–
–
–
þ
þ þ
–
þ þ
–
3u
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
9d
9e
9f
9g
–
þ þ
þ þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
–
–
–
þ
þ
þ þ
þ þ
þ
–
þ
–
þ
þ
þ
þ
þ
þ
þ
–
–
–
þ
þ
þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
observed for benzothiazoles with a 5-methyl-3-oxo-3H-pyrazol-2-yl moiety at C(2).
Compounds 3a (6-Cl), 3i (3-Me), 3j (6-MeO), and 3k (6-F) exhibit antibacterial
activities against at least one of the six microorganisms tested, whereas the closely
related analogues 3l (4,6-F2) and 3m (unsubstituted benzo moiety) are devoid of any
activity.
To obtain more detailed SAR information, the most potent compounds were
further evaluated for their antibacterial activities according to the standard NCCLS
protocol following the broth-microdilution method [13]. Gentamycin, a marketed
aminoglycoside antibiotic, was used as positive control. For two microorganisms (S.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
259
aureus and P. aeruginosa), minimal inhibitory concentrations (MICs) were determined
(Table 4). The MIC is defined as the lowest concentration of a compound (in mm) that
showed no detectable bacterial growth. From Table 4, it turns out that the two most
potent congeners are compounds 3h (MIC: 3.43 mm for S. aureus and P. aeruginosa) and
compound 3u (MIC: 3.30 mm for S. aureus and P. aeruginosa), with a Ph and Bn group
on the pyrazol-2-yl moiety, respectively. These compounds are only 20-fold less potent
than gentamycin. The presence of an aromatic moiety on the 3-oxo-pyrazol-2-yl
substructure is necessary for biological activity. This is evident from the comparison of
the MIC data of compounds 3f, 3g, and 3h. An Et (compound 3f; MIC: 63.98 mm for S.
aureus and P. aeruginosa) or an i-Pr substituent (compound 3g; MIC: 122.1 mm for P.
aeruginosa and 244.2 mm for S. aureus) are much less active than the corresponding Ph
derivative (compound 3h; MIC: 3.43 mm for P. aeruginosa and S. aureus).
Table 4. Antibacterial Activities of Active Compounds. Minimum inhibitory concentration (MIC) values
against P. aeruginosa PAO1 and S. aureus ATCC 65388 were determined using the broth microdilution
method. The antibiotic gentamycin was used as a positive control.
Compound
MIC [mm]
P. aeruginosa
S. aureus
3d
3f
3g
3h
14.31
63.98
122.1
3.43
14.31
63.98
244.2
3.43
3n
3q
3s
3t
14.58
14.42
13.75
110.0
3.30
29.16
14.42
6.88
27.5
3.30
3u
8b
8c
134.4
32
67.20
8
8d
8e
8f
28.68
67.20
15.97
0.16
28.68
134.4
31.94
0.16
Gentamycin
From the first screening campaign (Table 3), it was apparent that the 6-Cl and 6-Me
substituents are optimal for activity. Comparison of their MIC values reveals that a 6-Cl
(MIC of 3h is 3.43 mm for S. aureus and for P. aeruginosa) is favored over a 6-Me
derivative (MIC of 3n is 29.16 mm against S. aureus and 14.58 mm for P. aeruginosa). The
6-F congener (compound 3q) is equally active as the 6-Me derivative (compound 3n)
against P. aeruginosa (MIC: 14.42 mm) and twofold more potent against S. aureus
(14.42 mm).
As it was clear that Cl is the best substituent, a number of regioisomers (with Cl at
different positions of the phenyl moiety) have been prepared, and MIC values were
determined. The 4-Cl isomer (compound 3t) was the least active (MIC: 27.5 mm for S.
aureus and 110.0 mm for P. aeruginosa), whereas the 6-Cl regioisomer (compound 3h)
turned out to be the most potent. The 5-Cl derivative 3s displayed intermediate
antibacterial activity (MIC for S. aureus: 6.88 mm and for P. aeruginosa: 13.75 mm). As

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
this 5-Cl-benzothiazole displayed reasonable antibacterial activity, a series of analogs
has been synthesized (compounds 8a–8g), and MIC values for selected examples
(compounds 8b–8f) were determined. Compound 8f (the direct analogue of compound
3s, in which the Ph is replaced with an Et group) had a similar activity as compound 3s
against P. aeruginosa (MIC of 15.97 mm), whereas it was less active against S. aureus
(MIC of 31.94 mm versus 6.88 mm). An i-Pr analog, compound 8c, was as active as
compound 3s against S. aureus (MIC of 8 mm) and only threefold less active than
compound 3s against P. aeruginosa (MIC of 32 mm vs. 9.38 mg/ml). A Ph group on the 3-
oxo-pyrazole moiety (compound 8d) gave rise to MIC values of 28.68 mm against S.
aureus as well as against P. aeruginosa.
Conclusions. – A number of novel benzothiazoles, bearing a 3-oxo-pyrazol-2-yl
moiety at C(2), have been synthesized and screened for antibacterial activity in two
stages. In a first screening, antibacterial activities against six representative micro-
organisms (S. aureus, S. epidermidis, B. subtilis, E. coli, P. aeruginosa, and S.
typhimurium) at a concentration of 10 mg/ml were determined. In a follow-up
screening, the most potent congeners were selected for MIC determination against S.
aureus and P. aeruginosa. The SAR clearly demonstrated that the substitution pattern
on the benzothiazole core structure, as well as the substituents on the 3-oxo-pyrazol-2-
yl moiety, influence the antibacterial activity to a large extent. A Cl-atom at C(6) of the
benzo part and an aromatic moiety (Ph or Bn) on the pyrazole moiety are crucial for
biological activity. This strategy led to the discovery of potent antibacterials (MIC of
3.43 mm for S. aureus and P. aeruginosa), and, in this way, we have contributed to the
study of the SAR of potential novel antibiotics based on the benzothiazole scaffold.
Additional studies are ongoing in order to elucidate the molecular target and to
establish in vivo activity of these novel antibacterials.
Experimental Part
General. For all reactions, anal. grade solvents were used. All moisture-sensitive reactions were
carried out in oven-dried glassware (1358). TLC: Precoated aluminum sheets (Fluka silica gel (SiO2)/
TLC plates, 254 nm). Column chromatography (CC): ICN SiO2 63–200, 60 ꢂ. M.p.: Barnstead IA 9200;
uncorrected. 1H- and 13C-NMR spectra: Bruker Avance 300 (1H: 300 and 13C: 75 MHz), using Me4Si as
1
internal standard for H-NMR spectra and (D6)DMSO (39.5 ppm) or CDCl3 (77.2 ppm) for 13C-NMR
spectra; coupling constants in Hz. MS: Finnigan LCQ advantage Max (ion trap) mass spectrometer from
Thermo Finnigan, San Jose, CA, USA; exact mass determinations performed on a quadrupole time-of-
flight mass spectrometer (Q-tof-2, Micromass, Manchester, UK) equipped with a standard electrospray-
ionization (ESI) interface; samples infused in i-PrOH/H2O 1:1 at 3 ml/min.
General Procedure for the Synthesis of 2-Hydrazinylbenzothiazole Analogs. To a soln. of 2-
aminobenzothiazole (3 mmol) in conc. HCl (7 ml) was added NaNO2 (30 mmol) at 08. The mixture was
stirred for 24 h at r.t. After complete consumption of the starting material (TLC monitoring), the soln.
was diluted with H2O/Et2O, and the org. phase was extracted twice with AcOEt, dried (MgSO4), filtered,
and evaporated under reduced pressure. This crude residue was used in the next step without any further
purification. To a soln. of 2-chlorobenzothiazole (0.5 mmol) in EtOH (5 ml) was added NH2NH2 ·H2O
(64% H2O soln., 2.5 mmol), and the mixture was heated to reflux for 3 h. The solvents were evaporated,
and the crude residue was purified by flash chromatography (FC) on SiO2 using heptane/AcOEt as a
mobile phase (in a ratio from 1:1 to 1:2). The following compounds were prepared according to this
procedure.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
261
6-Chloro-2-hydrazinylbenzothiazole (R¼6-Cl; 2a). Yield: 78%. 1H-NMR (300 MHz, (D6)DMSO):
9.19 (br. s, 1 H); 7.80 (d, J¼2.21, 1 H); 7.30 (d, J¼8.53, 1 H); 7.21 (dd, J¼8.53, 2.21, 1 H); 5.11 (br. s,
2 H). ESI-MS (pos.): 200.1 ([MþH]þ ).
4-Chloro-2-hydrazinylbenzothiazole (R¼4-Cl; 2b). Yield: 61%. 1H-NMR (300 MHz, (D6)DMSO):
8.69 (s, 1 H); 7.79 (d, J¼8.21, 1 H); 7.59 (br. t, 1 H); 7.22 (d, J¼8.12, 1 H); 5.16 (br. s, 2 H). ESI-MS
(pos.): 200.1 ([MþH]þ ).
2-Hydrazinyl-6-methoxybenzothiazole (R¼6-MeO; 2c). Yield: 77%. 1H-NMR (300 MHz,
(D6)DMSO): 8.77 (br. s, 1 H); 7.32 (s, 1 H); 7.25 (d, J¼8.6, 1 H); 6.80 (d, J¼8.6, 1 H); 4.94 (br. s,
2 H); 3.74 (s, 3 H). 13C-NMR (75 MHz, (D6)DMSO): 172.5; 154.0; 147.6; 131.6; 118.4; 113.0; 105.7; 55.7.
ESI-MS (pos.): 196.0 ([MþH]þ ).
2-Hydrazinyl-6-methylbenzothiazole (R¼Me; 2d). Yield: 56%. 1H-NMR (300 MHz, (D6)DMSO):
8.89 (br. s, 1 H); 7.47 (s, 1 H); 7.20 (d, J¼8.13, 1 H); 7.00 (d, J¼8.13, 1 H); 4.96 (br. s, 2 H); 2.31 (s, 3 H).
ESI-MS (pos.): 180.1 ([MþH]þ ).
1
6-Fluoro-2-hydrazinylbenzothiazole (R¼6-F; 2e). Yield: 71%. H-NMR (300 MHz, (D6)DMSO):
9.03 (br. s, 1 H); 7.60 (dd, J¼8.8, 2.7, 1 H); 7.29 (d, J¼8.8, 1 H); 7.02 (td, J¼8.8, 2.7, 1 H); 5.05 (br. s, 2 H).
ESI-MS (pos.): 184.0 ([MþH]þ ).
2-Hydrazinyl-5,6-dimethylbenzothiazole (R¼5,6-Me2; 2f). Yield: 51%. 1H-NMR (300 MHz,
(D6)DMSO): 8.85 (s, 1 H); 7.70 (s, 1 H); 7.63 (s, 1 H); 5.14 (br. s, 2 H). ESI-MS (pos.): 194.1 ([Mþ
H]þ ).
4,6-Difluoro-2-hydrazinylbenzothiazole (R¼4,6-F2 ; 2g). Yield: 59%. 1H-NMR (300 MHz,
(D6)DMSO): 9.27 (s, 1 H); 7.50 (dd, Jm(H,H)¼1.5, Jo(H,F)¼8.4, 1 H); 7.10 (dt, Jm(H,H)¼1.5,
Jo(H,F)¼8.4); 5.16 (s, 2 H). ESI-MS (pos.): 202.0 ([MþH]þ ).
General Procedure for the Formation of the Pyrazolone Moiety. To a soln. of a 2-hydrazinylbenzo-
thiazole (0.8 mmol) in EtOH (3 ml) was added an appropriate b-keto ester (0.8 mmol) and a cat. amount
of AcOH (0.08 mmol). The mixture was heated to reflux overnight. The soln. was then cooled to r.t., and
the precipitate was filtered off. The crude products were purified by FC on SiO2 using AcOEt as eluent.
The following compounds were synthesized according to this procedure.
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-3H-pyrazol-3-one (3a). Yield: 42%. 1H-NMR
(300 MHz, (D6)DMSO): 12.75 (br. s, 1 H); 8.18 (s, 1 H); 7.81 (d, J¼8.58, 1 H); 7.50 (d, J¼8.58, 1 H); 5.28
(s, 1 H); 2.23 (s, 3 H). 13C-NMR (75 MHz, (D6)DMSO): 162.0; 154.4; 153.5; 147.4; 133.6; 128.1; 126.9;
122.0; 121.9; 91.3; 12.6. HR-ESI-MS (pos.): 266.0159 ([MþH]þ , C11H9ClN3OSþ ; calc. 266.0155).
2-(6-Chlorobenzothiazol-2-yl)-5-ethyl-1,2-dihydro-3H-pyrazol-3-one (3b). Yield: 46%. 1H-NMR
(300 MHz, (D6)DMSO): 12.7 (br. s, 1 H); 8.19 (s, 1 H); 7.82 (d, J¼8.21, 1 H); 7.52 (d, J¼8.21, 1 H); 5.33
(s, 1 H); 2.56 (br. s, 2 H); 1.21 (br. s, 3 H). HR-ESI-MS (pos.): 280.0316 ([MþH]þ , C12H11ClN3OSþ
;
calc. 280.0311).
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-(1-methylethyl)-3H-pyrazol-3-one (3c). Yield: 46%.
1H-NMR (300 MHz, (D6)DMSO): 12.78 (br. s, 1 H); 8.19 (s, 1 H); 7.83 (d, J¼8.67, 1 H); 7.52 (d, J¼
8.67, 1 H); 5.33 (s, 1 H); 2.92 (sept., J¼6.87, 1 H); 1.25 (d, J¼6.87, 6 H). HR-ESI-MS (pos.): 294.0464
([MþH]þ , C13H13ClN3OSþ ; calc. 294.0468).
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-phenyl-3H-pyrazol-3-one (3d). Yield: 70%. 1H-NMR
(300 MHz, (D6)DMSO): 8.22 (d, J¼1.8, 1 H); 7.91 (m, 3 H); 7.52 (m, 4 H); 6.12 (s, 1 H). HR-ESI-MS
(pos.): 328.0314 ([MþH]þ , C16H11ClN3OSþ ; calc. 328.0311).
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-4,5-dimethyl-3H-pyrazol-3-one (3e). Yield: 35%.
1H-NMR (300 MHz, (D6)DMSO): 12.2 (br. s, 1 H); 8.19 (s, 1 H); 7.80 (d, J¼8.5, 2 H); 7.50 (d, J¼8.5,
2 H); 2.19 (s, 3 H); 1.75 (s, 3 H). HR-ESI-MS (pos.): 280.0313 ([MþH]þ , C12H11ClN3OSþ ; calc.
280.0311).
2-(6-Chlorobenzothiazol-2-yl)-4-ethyl-1,2-dihydro-5-methyl-3H-pyrazol-3-one (3f). Yield: 34%.
1H-NMR (300 MHz, (D6)DMSO): 12.20 (br. s, 1 H); 8.20 (s, 1 H); 7.80 (d, J¼8.67, 1 H); 7.50 (d, J¼
8.67, 1 H); 2.28 (s, 3 H); 2.23 (br. d, 2 H); 1.08 (t, J¼7.41, 3 H). HR-ESI-MS (pos.): 294.0464 ([MþH]þ
,
C13H13ClN3OSþ ; calc. 294.0468).
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-(1-methylethyl)-3H-pyrazol-3-one (3g).
1
Yield: 34%. H-NMR (300 MHz, (D6)DMSO): 12.10 (br. 1 H); 8.20 (d, J¼2.1, 1 H); 7.81 (d, J¼8.67,

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1 H); 7.51 (dd, J¼2.1, 8.67, 1 H); 2.75 (m, 1 H); 2.21 (s, 3 H); 1.22 (d, J¼7, 6 H). HR-ESI-MS (pos.):
308.0630 ([MþH]þ , C14H14ClN3OSþ ; calc. 308.0625).
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-phenyl-3H-pyrazol-3-one (3h). Yield: 51%.
1H-NMR (300 MHz, (D6)DMSO): 8.21 (s, 1 H); 7.84 (d, J¼7.77, 1 H); 7.63 (d, J¼7.83, 2 H); 7.52 (d, J¼
7.77, 1 H); 7.41 (t, J¼7.23, 2 H); 7.39 (t, J¼7.78, 1 H); 2.44 (s, 3 H). HR-ESI-MS (pos.): 342.0476 ([Mþ
H]þ , C17H13ClN3OSþ ; calc. 342.0468).
1,2-Dihydro-5-methyl-2-(6-methylbenzothiazol-2-yl)-3H-pyrazol-3-one (3i). Yield: 81%. 1H-NMR
(300 MHz, (D6)DMSO): 7.82 (s, 1 H); 7.72 (d, J¼8.16, 1 H); 7.30 (d, J¼8.16, 1 H); 5.27 (s, 1 H); 2.42 (s,
3 H); 2.23 (s, 3 H). HR-ESI-MS (pos.): 246.0707 ([MþH]þ , C12H12N3OSþ ; calc. 246.0701).
1,2-Dihydro-2-(6-methoxybenzothiazol-2-yl)-5-methyl-3H-pyrazol-3-one (3j). Yield: 41%. 1H-NMR
(300 MHz, (D6)DMSO): 12.73 (br. s, 1 H); 7.71 (d, J¼8.89, 1 H); 7.64 (s, 1 H); 7.09 (d, J¼8.89, 1 H); 5.26
(s, 1 H); 3.82 (s, 3 H); 2.22 (s, 3 H). HR-ESI-MS (pos.): 262.0658 ([MþH]þ , C12H12N3O2Sþ ; calc.
262.0650).
2-(6-Fluorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-3H-pyrazol-3-one (3k). Yield: 55%. 1H-NMR
(300 MHz, (D6)DMSO): 7.98 (dd, Jm(H,H)¼2.61, Jo(H,F)¼8.76, 1 H); 7.81 (dd, Jm(H,F)¼4.77,
Jo(H,H)¼8.88, 1 H); 7.34 (dt, Jm(H,H)¼2.7, Jo(H,H)¼9.09, 1 H); 5.27 (s, 1 H); 2.28 (s, 3 H). HR-ESI-
MS (pos.): 250.0457 ([MþH]þ , C11H9FN3OSþ ; calc. 250.0450).
2-(4,6-Difluorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-3H-pyrazol-3-one (3l). Yield: 81%.
1H-NMR (300 MHz, (D6)DMSO): 12.82 (br. s, 1 H); 7.85 (d, J¼6.99, 1 H); 7.44 (dt, J¼2.37, 8.76,
1 H); 5.30 (s, 1 H); 2.24 (s, 3 H). HR-ESI-MS (pos.): 268.0359 ([MþH]þ , C11H8F2N3OSþ ; calc.
268.0356).
2-(Benzothiazol-2-yl)-1,2-dihydro-5-methyl-3H-pyrazol-3-one (3m). Yield: 37%. 1H-NMR
(300 MHz, (D6)DMSO): 8.06 (d, J¼7.38, 1 H); 7.87 (d, J¼7.98, 1 H); 7.54 (t, J¼7.98, 1 H); 7.41 (t, J¼
7.38, 1 H); 5.31 (s, 1 H); 2.27 (s, 3 H). 13C-NMR (75 MHz, CDCl3): 161.9; 154.0; 152.9; 148.4; 131.8;
126.6; 124.2; 122.2; 120.8; 91.4; 12.5. ESI-MS (pos.): 232.0 ([MþH]þ ).
1,2-Dihydro-5-methyl-2-(6-methylbenzothiazol-2-yl)-4-phenyl-3H-pyrazol-3-one (3n). Yield: 48%.
1H-NMR (300 MHz, (D6)DMSO): 12.0 (br. s, 1 H); 7.86 (s, 1 H); 7.75 (d, J¼8.31, 1 H); 7.61 (d, J¼8.31,
1 H); 7.30 (m, 5 H); 2.43 (s, 3 H); 1.90 (s, 3 H). HR-ESI-MS (pos.): 322.1016 ([MþH]þ , C18H16N3OSþ
;
calc. 322.1014).
2-(5,6-Dimethylbenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-phenyl-3H-pyrazol-3-one (3o). Yield:
1
59%. H-NMR (300 MHz, (D6)DMSO): 7.78 (s, 1 H); 7.65 (m, 3 H); 7.39 (t, J¼7.50, 2 H); 7.23 (d, J¼
7.50, 1 H); 2.42 (s, 3 H); 2.35 (s, 3 H); 2.33 (s, 3 H). HR-ESI-MS (pos.): 335.1095 (Mþ, C19H17N3OSþ
;
calc. 335.1092).
1,2-Dihydro-2-(6-methoxybenzothiazol-2-yl)-5-methyl-4-phenyl-3H-pyrazol-3-one (3p). Yield: 62%.
1H-NMR (300 MHz, (D6)DMSO): 12.98 (br. s, 1 H); 7.65 (m, 5 H); 7.41 (br. d, 1 H); 7.26 (br. d, 1 H); 7.15
(s, 1 H); 3.84 (s, 3 H); 2.44 (s, 3 H). HR-ESI-MS (pos.): 338.0966 ([MþH]þ , C18H16N3O2Sþ ; calc.
338.0963).
2-(6-Fluorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-phenyl-3H-pyrazol-3-one (3q). Yield: 41%.
1H-NMR (300 MHz, (D6)DMSO): 8.02 (dd, Jo(H,H)¼8.76, Jm(H,H)¼2, 1 H); 7.86 (dd, Jo(H,H)¼8.76,
Jm(H,F)¼4.62, 1 H); 7.63 (d, Jo(H,F)¼7.8, 1 H); 7.42 (m, 4 H); 7.26 (br. t, 3 H); 2.42 (s, 3 H). HR-ESI-
MS (pos.): 326.0773 ([MþH]þ , C17H13FN3OSþ ; calc. 326.0763).
2-(4,6-Difluorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-phenyl-3H-pyrazol-3-one (3r). Yield:
71%. 1H-NMR (300 MHz, (D6)DMSO): 7.80 (d, J¼7.95, 1 H); 7.66 (br. d, 2 H); 7.35 (m, 3 H); 7.10
(br. t, 1 H); 5.76 (s, 1 H); 2.39 (s, 3 H). HR-ESI-MS (pos.): 344.0676 ([MþH]þ , C17H12F2N3OSþ ; calc.
344.0669).
2-(5-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-phenyl-3H-pyrazol-3-one (3s). Yield: 62%.
1H-NMR (300 MHz, (D6)DMSO): 13.2 (br. s, 1 H); 8.14 (d, J¼8.40, 1 H); 7.88 (s, 1 H); 7.64 (d, J¼7.26,
2 H); 7.44 (m, 3 H); 7.28 (d, J¼8.40, 1 H); 2.45 (s, 3 H). HR-ESI-MS (pos.): 342.0453 ([MþH]þ
,
C17H13ClN3OSþ ; calc. 342.0468).
2-(4-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-phenyl-3H-pyrazol-3-one (3t). Yield: 37%.
1H-NMR (300 MHz, (D6)DMSO): 12.89 (br. s, 1 H); 8.06 (d, J¼2.14, 1 H); 7.61 (m, 3 H); 7.40 (m, 4 H);
2.48 (s, 3 H). HR-ESI-MS (pos.): 342.0472 ([MþH]þ , C17H13ClN3OSþ ; calc. 342.0468).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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4-Benzyl-2-(6-chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-3H-pyrazol-3-one (3u). Yield: 59%.
1H-NMR (300 MHz, (D6)DMSO): 12.21 (br., 1 H); 8.20 (s, 1 H); 7.80 (d, J¼8.52, 1 H); 7.49 (d, J¼8.52,
1 H); 7.19 (m, 5 H); 3.59 (s, 2 H); 2.20 (s, 3 H). HR-ESI-MS (pos.): 356.0638 ([MþH]þ , C18H15ClN3OSþ ;
calc. 356.0624).
5-Chlorobenzothiazole-2-thiol (5). To a soln. of 5-chloro-2-sulfanylaniline (4, 500 mg, 3.13 mmol) in
EtOH/H2O 15 :3 were added CS2 (3.13 mmol, 189 ml) and KOH (3.13 mmol, 176 mg). The mixture was
refluxed overnight. Active carbon (500 mg) was added, and the soln. was stirred at r.t. for 1 h. The solid
was filtered off over Celite, and the solvent was evaporated under reduced pressure to yield 5 (473 mg,
1
75%). H-NMR (300 MHz, CDCl3): 13.85 (s, 1 H); 7.72 (d, J¼8.8, 1 H); 7.34 (d, J¼8.8, 1 H); 7.29 (s,
1 H). ESI-MS (pos.): 202.0 ([MþH]þ ).
5-Chloro-2-(methylsulfanyl)benzothiazole. To a soln. of 5 (700 mg, 3.47 mmol) in 1m NaOH (20 ml)
was added MeI (3.47 mmol, 216 ml). The resulting soln. was stirred at r.t. for 2 h. The precipitate was
filtered off and dried to yield the title compound (718 mg, 96%), which was used as such in the next step
without further purification. 1H-NMR (300 MHz, CDCl3): 7.86 (s, 1 H); 7.66 (d, J¼8.5, 1 H); 7.28 (d, J¼
8.5, 1 H); 2.80 (s, 3 H). ESI-MS (pos.): 216.0 ([MþH]þ ).
5-Chloro-2-(methylsulfonyl)benzothiazole (6). To a soln. of 5-chloro-2-(methylsulfanyl)benzothia-
zole (500 mg, 2.30 mmol) in CH2Cl2 (11 ml) was added m-CPBA (4.60 mmol, 794 mg) at 08. The soln. was
stirred at r.t. for 1 h. The mixture was diluted with an aq. NaHCO3 soln. The org. layer was separated and
washed twice with brine. The org. phase was dried (MgSO4), filtered, and evaporated under reduced
pressure. The solid was purified by FC (SiO2; heptane/AcOEt 1:1) to afford pure 6 (545 mg, 95%).
1H-NMR (300 MHz, CDCl3): 8.17 (s, 1 H); 8.04 (d, J¼8.14, 1 H); 7.41 (d, J¼8.14, 1 H); 3.40 (s, 3 H).
ESI-MS (pos.): 248.0 ([MþH]þ ).
5-Chloro-2-hydrazinylbenzothiazole (7). To a soln. of 6 (300 mg, 1.21 mmol) in EtOH (8 ml) was
added NH2NH2 ·H2O (64% H2O soln., 6.07 mmol, 250 ml). The mixture was heated to reflux for 3 h, until
complete consumption of the starting material (TLC monitoring). The solvent was evaporated in vacuo.
The crude product was purified by FC (SiO2; heptane/AcOEt 1:1) to yield pure 7 (178.9 mg, 74%).
1H-NMR (300 MHz, (D6)DMSO): 9.25 (br. s, 1 H); 7.70 (d, J¼8.19, 1 H); 7.33 (s, 1 H); 7.00 (d, J¼8.19,
1 H); 5.12 (s, 2 H). ESI-MS (pos.): 200.1 ([MþH]þ ).
General Procedure for the Synthesis of 2-(5-Chlorobenzothiazol-2-yl)-3H-pyrazol-3-ones 8a–8g.
These compounds have been synthesized according to the general procedure as described for the
synthesis of compounds 3a–3u.
2-(5-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-3H-pyrazol-3-one (8a). Yield: 37%. 1H-NMR
(300 MHz, (D6)DMSO): 12.3 (br. s, 1 H); 8.09 (d, J¼8.07, 1 H); 7.83 (s, 1 H); 7.40 (d, J¼8.07, 1 H); 5.29
(s, 1 H); 2.23 (s, 3 H). HR-ESI-MS (pos.): 266.0161 ([MþH]þ , C11H9ClN3OSþ ; calc. 266.0155).
2-(5-Chlorobenzothiazol-2-yl)-5-ethyl-1,2-dihydro-3H-pyrazol-3-one (8b). Yield: 67%. 1H-NMR
(500 MHz, (D6)DMSO): 12.86 (br., 1 H); 8.10 (d, J¼8.4, 1 H); 7.84 (s, 1 H); 7.42 (d, J¼8.4, 1 H); 5.33 (s,
1 H); 2.59 (t, J¼7.53, 2 H); 1.24 (t, J¼7.53, 3 H). 13C-NMR (75 MHz, (D6)DMSO): 162.1; 160.2; 154.6;
149.6; 131.2; 130.7; 124.2; 123.7; 120.2; 90.0; 20.1; 12.2. HR-ESI-MS (pos.): 280.0304 ([MþH]þ
,
C12H11ClN3OSþ ; calc. 280.0311).
2-(5-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-(1-methylethyl)-3H-pyrazol-3-one (8c). Yield: 30%.
1H-NMR (300 MHz, (D6)DMSO): 8.09 (d, J¼8.49, 1 H); 7.85 (s, 1 H); 7.42 (d, J¼8.49, 1 H); 5.31 (s,
1 H); 2.93 (sept., J¼6.9, 1 H); 1.26 (d, J¼6.9, 6 H). HR-ESI-MS (pos.): 294.0470 ([MþH]þ
,
C13H13ClN3OSþ ; calc. 294.0468).
2-(5-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-phenyl-3H-pyrazol-3-one (8d). Yield: 46%. 1H-NMR
(300 MHz, (D6)DMSO): 7.91 (br. d, 1 H); 7.67 (br. d, 2 H); 7.51 (s, 1 H); 7.40 (m, 3 H); 7.32 (br. s, 1 H);
5.89 (s, 1 H). HR-ESI-MS (pos.): 328.0316 ([MþH]þ , C16H11ClN3OSþ ; calc. 328.0311).
2-(5-Chlorobenzothiazol-2-yl)-1,2-dihydro-4,5-dimethyl-3H-pyrazol-3-one (8e). Yield: 70%.
1H-NMR (300 MHz, (D6)DMSO): 12.19 (br. s, 1 H); 8.10 (d, J¼8.16, 1 H); 7.83 (s, 1 H); 7.41 (d, J¼
8.16, 1 H); 2.20 (s, 3 H); 1.76 (s, 3 H). HR-ESI-MS (pos.): 280.0314 ([MþH]þ , C12H11ClN3OSþ ; calc.
280.0311).
2-(5-Chlorobenzothiazol-2-yl)-4-ethyl-1,2-dihydro-5-methyl-3H-pyrazol-3-one (8f). Yield: 47%.
1H-NMR (300 MHz, (D6)DMSO): 12.21 (br. s, 1 H); 8.10 (d, J¼8.43, 1 H); 7.83 (s, 1 H); 7.41 (d, J¼

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
8.43, 1 H); 2.21 (br. s, 5 H); 1.08 (t, J¼7.11, 3 H). HR-ESI-MS (pos.): 294.0460 ([MþH]þ
,
C13H13ClN3OSþ ; calc. 294.0468).
2-(5-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-(1-methylethyl)-3H-pyrazol-3-one (8g).
1
Yield: 31%. H-NMR (300 MHz, (D6)DMSO): 12.91 (br. s, 1 H); 8.07 (d, 1 H); 7.84 (s, 1 H); 7.41 (d,
1 H); 5.28 (s, 1 H); 2.79 (m, 1 H); 1.27 (d, J¼7.2, 6 H). HR-ESI-MS (pos.): 308.0628 ([MþH]þ
,
C14H15ClN3OSþ ; calc. 308.0624).
General Procedure for N-Alkylation of the Pyrazolone Ring. To a soln. of a 2-(5-chlorobenzothiazol-
2-yl)-1,2-dihydro-3H-pyrazol-3-one (0.5 mmol) in DMF (3 ml) were added an appropriate alkyl halide
(0.5 mmol) and Et3N (0.5 mmol). The mixture was stirred at r.t. for 3 h. The soln. was then diluted with
brine and AcOEt. The org. phase was separated and washed twice with brine. The crude product was
purified by FC on SiO2 using the mobile phase as mentioned below for each derivative. The following
compounds were obtained according to this procedure.
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-1,5-dimethyl-3H-pyrazol-3-one (9a) was prepared using
MeI as alkylating agent in a yield of 46%. The compound was purified by FC (SiO2; heptane/AcOEt
3 :1). 1H-NMR (300 MHz, (D6)DMSO): 8.19 (s, 1 H); 7.86 (d, J¼8.67, 1 H); 7.52 (dd, J¼8.67, 1.83, 1 H);
5.57 (s, 1 H); 3.68 (s, 3 H); 2.31 (s, 3 H). 13C-NMR (75 MHz, (D6)DMSO): 164.5; 161.9; 151.2; 147.0;
133.2; 129.4; 126.5; 120.0; 120.5; 98.0; 37.4; 13.0. HR-ESI-MS (pos.): 280.0319 ([MþH]þ
,
C12H11ClN3OSþ ; calc. 280.0311).
2-(6-Chlorobenzothiazol-2-yl)-1-ethyl-1,2-dihydro-5-methyl-3H-pyrazol-3-one (9b) was prepared
using EtI as alkylating agent in a yield of 49%. The compound was purified by FC (SiO2; heptane/
1
AcOEt 3 :1). H-NMR (500 MHz, CDCl3): 7.81 (d, J¼2.0, 1 H); 7.77 (d, J¼8.6, 1 H); 7.41 (dd, J¼2.0,
8.6, 1 H); 5.51 (s, 1 H); 4.46 (q, J¼7.1, 2 H); 2.3 (s, 3 H); 0.99 (t, J¼7.1, 3 H ) . 13C-NMR (75 MHz,
CDCl3): 166.0; 160.8; 154.7; 147.3; 133.5; 129.7; 126.8; 122.3; 120.8; 99.5; 44.3; 13.2; 10.2. HR-ESI-MS
(pos.): 294.0474 ([MþH]þ , C13H13ClN3OSþ ; calc. 294.0468).
1-Benzyl-2-(6-chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-3H-pyrazol-3-one (9c) was prepared
using BnBr as alkylating agent in a yield of 39%. The compound was purified by FC (SiO2; heptane/
AcOEt 2 :1). 1H-NMR (300 MHz, (D6)DMSO): 8.18 (d, J¼2.01, 1 H); 7.92 (d, J¼8.64, 1 H); 7.52 (dd,
J¼8.64, 2.01, 1 H); 7.24 (m, 3 H); 6.99 (m, 2 H); 5.61 (s, 3 H); 2.49 (s, 3 H). 13C-NMR (75 MHz,
(D6)DMSO): 164.8; 161.9; 154.5; 147.1; 134.0; 133.1; 128.6; 128.4; 128.2; 127.7; 126.7; 122.4; 121.6; 97.8;
55.6; 13.2. HR-ESI-MS (pos.): 356.0641 ([MþH]þ , C18H15ClN3OSþ ; calc. 356.0624).
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-1,5-dimethyl-4-phenyl-3H-pyrazol-3-one (9d) was pre-
pared using MeI as alkylating agent in a yield of 58%. The compound was purified by FC (SiO2; heptane/
1
AcOEt 3 :1). H-NMR (300 MHz, (D6)DMSO): 7.52 (s, 2 H); 7.47 (s, 1 H); 7.44 (m, 3 H); 7.35 (d, J¼
8.23, 1 H). 13C-NMR (75 MHz, CDCl3): 157.3; 154.8; 147.1; 133.2; 129.4; 129.3; 128.5 (2C); 128.3 (2C);
127.2; 126.5; 124.8; 122.1; 120.6; 100.9; 37.9; 12.0. HR-ESI-MS (pos.): 356.0628 ([MþH]þ
,
C18H15ClN3OSþ ; calc. 356.0624).
2-(6-Chlorobenzothiazol-2-yl)-1-ethyl-1,2-dihydro-5-methyl-4-phenyl-3H-pyrazol-3-one (9e) was
prepared using EtI as alkylating agent in a yield of 64%. The compound was purified by FC (SiO2;
1
heptane/AcOEt 2 :1). H-NMR (300 MHz, (D6)DMSO): 8.23 (s, 1 H); 7.89 (d, J¼8.58, 1 H); 7.50 (m,
7 H); 4.56 (q, J¼6.42, 2 H); 2.46 (s, 3 H); 0.96 (t, J¼6.42, 3 H). HR-ESI-MS (pos.): 370.0785 ([MþH]þ
,
C19H17ClN3OSþ ; calc. 370.0781).
2-(6-Chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-phenyl-1-propyl-3H-pyrazol-3-one (9f) was
prepared using PrI as alkylating agent in a yield of 69%. The compound was purified by FC (SiO2;
heptane/AcOEt 2 :1). 1H-NMR (300 MHz, CDCl3): 7.85 (d, J¼1.92, 1 H); 7.83 (d, J¼8.64, 1 H); 7.55 (d,
J¼7.53, 2 H); 7.46 (d, J¼7.53, 2 H); 7.42 (br. t, 1 H); 7.35 (d, J¼6.8, 1 H); 4.48 (t, J¼7.65, 2 H); 2.46 (s,
3 H); 1.50 (m, J¼7.65, 2 H); 0.91 (t, J¼7.65, 3 H). 13C-NMR (75 MHz, CDCl3): 164.2; 155.6; 154.6; 147.1;
133.3; 129.6; 129.4; 128.5 (2C); 128.2 (2C); 127.0; 126.5; 122.1; 120.6; 110.5; 50.7; 19.3; 12.0; 10.8. HR-
ESI-MS (pos.): 384.0931 ([MþH]þ , C20H19ClN3OSþ ; calc. 384.0937).
1-Benzyl-2-(6-chlorobenzothiazol-2-yl)-1,2-dihydro-5-methyl-4-phenyl-3H-pyrazol-3-one (9g) was
prepared using BnBr as alkylating agent in a yield of 71%. The compound was purified by FC (SiO2;
heptane/AcOEt 2 :1). 1H-NMR (300 MHz, (D6)DMSO): 7.85 (s, 1 H); 7.84 (d, J¼2, 1 H); 7.51 (m, 5 H);
7.26 (br. t, 1 H); 7.20 (m, 3 H); 7.05 (m, 2 H); 5.65 (s, 2 H); 2.55 (s, 3 H). 13C-NMR (75 MHz, CDCl3):

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
265
164.0; 155.6; 154.6; 147.0; 133.3; 129.5; 129.4; 128.5; 128.2; 127.5; 127.2; 126.6; 122.1; 120.7; 12.0; 52.5;
12.5. HR-ESI-MS (pos.): 432.0862 ([MþH]þ , C24H19ClN3OSþ ; calc. 432.0937).
Antibacterial Evaluation. All compounds were assayed for antibacterial activity against three
representative Gram-pos. bacteria (Staphylococcus aureus ATCC 65388, Staphylococcus epidermidis
RP62A ATCC 35984, and Bacillus subtilis ATCC 6633) and three Gram-neg. bacteria (Escherichia coli
NCIB 8743, Pseudomonas aeruginosa PAO1, and S. typhimurium sp. using a growth-inhibition plate
assay.
Bacterial cell cultures were grown overnight in Luria Bertani (LB) broth at 378. The next morning,
50 ml of these overnight cultures was used to inoculate 5 ml of LB medium. Bacterial cultures were grown
at 378 until OD600 ¼0.1 (5·107 cells/ml) and diluted 1/10 (OD600 ¼0.01, 5·106 cells/ml).
Ten ml of the diluted bacterial cultures was spotted on agar plates containing 10 mg/ml compound
(0.5% final DMSO concentration) or DMSO alone. After overnight incubation of the plates at 378, the
zones of bacterial growth were visually compared with the control plate containing DMSO without
compound.
Further antibacterial activity testing was carried out on active compounds according to the standard
NCCLS protocol following the broth-microdilution method [13]. Gentamycin was used as pos. control
and was purchased from Sigma. Overnight cultures of S. aureus and P. aeruginosa were diluted in LB
medium to OD600 ¼0.01 and 10-ml fractions were inoculated into 90 ml of the broth containing serial
twofold dilutions of compounds. Compounds were tested at a final concentration ranging from 0 to 75 mg/
ml and a final DMSO concentration of 5%. Culture plates were incubated at 378 for 24 h and growth was
monitored visually and spectrometrically in comparison to neg. and pos. controls. The MIC value was
determined and defined as the lowest concentration of compound that showed no detectable bacterial
growth.
REFERENCES
[1] C. Walsh, Nat. Rev. Microbiol. 2003, 1, 65.
[2] J. D. Peterson, L. A. Umayam, T. Dickinson, E. K. Hickey, O. White, Nucleic Acids Res. 2001, 29,
123.
[3] D. J. Payne, M. N. Gwynn, D. J. Holmes, D. L. Pampliano, Nat. Rev. Drug Discovery 2007, 6, 29.
[4] Y. Yin, L. Lin, C. Ruiz, M. D. Cameron, J. Pocas, W. Grant, T. Schrçter, W. Chen, D. Duckett, S.
Schꢁrer, P. LoGrasso, Y. Feng, Bioorg. Med. Chem. Lett. 2009, 19, 6686.
[5] C. B. Vu, J. C. Milne, D. P. Carney, J. Song, W. Choy, P. D. Lambert, D. J. Gagne, M. Hirsch, A. Cote,
M. Davis, E. Lainez, N. Meade, K. Normington, M. R. Jirousek, R. B. Perni, Bioorg. Med. Chem.
Lett. 2009, 19, 1416.
[6] M. Anzini, A. Chelini, A. Mancini, A. Cappelli, M. Frosini, L. Ricci, M. Valoti, J. Magistretti, L.
Castelli, A. Giordani, F. Makovec, S. Vomero, J. Med. Chem. 2010, 53, 734.
[7] E. Y. Song, N. Kaur, M.-Y. Park, Y. Jin, K. Lee, G. Kim, K. Y. Lee, J. S. Yang, J. H. Shin, K.-Y. Nam,
K. T. No, G. Han, Eur. J. Med. Chem. 2008, 43, 1519.
[8] R. Aggarwal, V. Kumar, P. Tyagi, S. P. Singh, Bioorg. Med. Chem. 2006, 14, 1785.
[9] T. I. Moy, A. L. Conery, J. Larkins-Ford, G. Wu, R. Mazitschek, G. Casadei, K. Lewis, A. E.
Carpenter, F. M. Ausubel, ACS Chem. Biol. 2009, 4, 527.
[10] W.-B. Chen, G.-Y. Jin, Heteroatom Chem. 2001, 12, 151.
[11] N. P. Peet, S. Sunder, R. J. Barbuch, M. R. Whalon, J. C. Huffman, J. Heterocycl. Chem. 1988, 25,
543.
[12] S. Guillou, F. J. Bonhomme, Y. L. Janin, Synthesis 2008, 3504.
[13] European Committee on Antimicrobial Susceptibility Testing (2003) Determination of minimum
inhibitory concentrations (MICs) of antibacterial agents by broth microdilution, EUCAST
Discussion Document E.Def 2003, 5.1, Clin. Microbiol Infect. 2003, 9 (issue 7 insert), pp. 1–10.
Received August 13, 2010