Synthesis of new 4-thiazolidinone-, pyrazoline-, and isatin-based conjugates with promising antitumor activity

Article abstract of DOI:10.1021/jm300789g

The synthesis and antitumor activity screening of novel 3-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-4-oxo-4,5-dihydro-1, 3-thiazol-5-ylidene]-2,3-dihydro-1H-indol-2-ones 1-23 and 3-(3,5-diarylpyrazol- 1-yl)-2,3-dihydro-1H-indol-2-ones 24-39 are performed. I

Full text of DOI:10.1021/jm300789g

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Article
Synthesis of New 4-Thiazolidinone, Pyrazoline and Isatin
Based Conjugates with Promising Antitumor Activity
Dmytro Havrylyuk, Borys Zimenkovsky, Olexandr Vasylenko, Andrzej Gzella, and Roman Lesyk
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 20 Sep 2012
Downloaded from http://pubs.acs.org on September 20, 2012
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Synthesis of New 4ꢀThiazolidinone, Pyrazoline and
Isatin Based Conjugates with Promising Antitumor
Activity
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Dmytro Havrylyuk†, Borys Zimenkovsky†, Olexandr Vasylenko‡, Andrzej Gzella§║ and
Roman Lesyk*,†
† Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv
National Medical University, Pekarska 69, Lviv, 79010, Ukraine
‡ Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Science of Ukraine
Murmanska 1, Kyiv, 02094, Ukraine
§ Department of Organic Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6,
Poznan, 60780, Poland
║Faculty of Pharmacy, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus
Copernicus University in Torun’, M. Curie Skłodowskiej 9, Bydgoszcz, 85094, Poland
KEYWORDS 4ꢀthiazolidinones, pyrazolines, isatins, Xꢀray analisys, anticancer activity
ABSTRACT The synthesis and antitumor activity screening of novel 3ꢀ[2ꢀ(3,5ꢀdiarylꢀ4,5ꢀ
dihydropyrazolꢀ1ꢀyl)ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ1,3ꢀthiazolꢀ5ꢀylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones 1ꢀ23
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and 3ꢀ(3,5ꢀdiarylpyrazolꢀ1ꢀyl)ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones 24ꢀ39 are performed. In vitro
anticancer activity of the synthesized compounds was tested by the National Cancer Institute.
Most of them displayed anticancer activity on leukemia, melanoma, lung, colon, CNS, ovarian,
renal, prostate and breast cancers cell lines. The structureꢀactivity relationship is discussed. The
most effective anticancer compound 10 was found to be active with a mean GI50 and TGI values
of 0.071 ꢁM and 0.76 ꢁM, respectively. It demonstrated the highest antiproliferative influence on
the nonꢀsmall cell lung cancer cell line HOPꢀ92 (GI50 < 0.01 ꢁM), colon cancer line HCTꢀ116
(GI50 = 0.018 ꢁM), CNS cancer cell line SNBꢀ75 (GI50 = 0.0159 ꢁM), ovarian cancer cell line
NCI/ADRꢀRES (GI50 = 0.0169 ꢁM) and renal cancer cell line RXF 393 (GI50 = 0.0197 ꢁM).
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INTRODUCTION
The synthesis and biological activity evaluation of nonꢀcondensed systems containing
thiazolidine,1 pyrazoline2 and isatin3 fragments are based upon previous results of experimental
studies of these heterocycles derivatives as potential chemotherapeutic agents. Antitumor activity
evaluation is actual and promising for these compounds (Figure 1). Antitumor mechanisms of 4ꢀ
thiazolidinones can be associated with their affinity to JNKꢀstimulating phosphataseꢀ1 (JSPꢀ1),4
tumor necrosis factor TNFα,5 antiꢀapoptotic biocomplex BclꢀXLꢀBH3,6 integrin αvβ3 receptor,7
nonmembrane protein tyrosine phosphatase (SHPꢀ2),8 etc. Besides, 1Hꢀindoleꢀ2,3ꢀdione
derivatives possess potent anticancer activity as tyrosine kinase inhibitors,9 inhibitors of cyclinꢀ
dependent kinases (CDKs),10 inhibitors of plasma membrane electron transport11 and carbonic
anhydrase isozymes inhibitors (CAIs).12 On the other hand, various pyrazole and pyrazoline
derivatives have been identified as inhibitors of cyclinꢀdependent kinase,13 heat shock proteins,14
vascular endothelium growth factors15 and Pꢀglycoprotein.16
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Figure 1.
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A pharmacophore hybrid approach for exploring of novel and highly active compounds is an
effective and commonly used direction in the modern medicinal chemistry. Hybridization of two
different bioactive molecules with complementary pharmacophoric functions or with different
mechanisms of action often showed synergistic effects.17 This hypothesis was confirmed by the
results of our previous studies that allowed identification of the high antitumor activity of 4ꢀ
thiazolidinone derivatives coupled with other heterocyclic fragments in one molecule.18
In continuation of this theme, we performed design (Figure 2) and synthesis of new nonꢀ
condensed heterocyclic compounds containing 4ꢀthiazolidinone, 2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone and
pharmacologically attractive pyrazole or pyrazoline moieties. The evaluation of their antitumor
activity of was carried out in vitro and in vivo. Combination of pyrazoline, 4ꢀthiazolidinone and
2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone moieties in one molecule allowed to gain of one or two logs of
antitumor cytotoxicity at GI50 level (ꢀ5.5 to ꢀ7.5, activity range) in comparison with pyrazoleꢀ
indolineꢀ2ꢀone conjugates (ꢀ4.8 to ꢀ5.5) or pyrazolineꢀthiazolidinone analogous compounds (ꢀ4.1
to ꢀ5.6), reported previously.19
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Figure 2
RESULTS AND DISCUSSION
Chemistry
The general methods for synthesis of target pyrazolineꢀthiazolidinoneꢀisatin conjugates and
pyrazoleꢀindolꢀ2ꢀone analogs are depicted in Schemes 1 and 2.
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3ꢀ[2ꢀ(3,5ꢀDiarylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ1,3ꢀthiazolꢀ5ꢀylidene]ꢀ2,3ꢀ
dihydroꢀ1Hꢀindolꢀ2ꢀones 1ꢀ23 were synthesized via oneꢀpot methodology involving reaction of
3,5ꢀdiarylꢀ1ꢀthiocarbamoylꢀ2ꢀpyrazolines with chloroacetic acid and appropriate isatins in the
presence of fused sodium acetate in refluxing acetic acid (Scheme 1). Preparation of compound 3
as well as structures of the compounds 4 and 11 were described in our previous reports.20
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Scheme 1.
It is known that the reaction of secondary amines with isatins passes through the formation of
3ꢀdialkylaminoꢀ3ꢀhydroxyꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones and further indole cycle opening.21
However, according to other published data, the reaction of isatin derivatives with 5ꢀ(2ꢀ
hydroxyphenyl)ꢀ3ꢀphenylpyrazoline yielded
a
spirosystem with 2ꢀoxoindole and
benzoxazinopyrazoline fragments.22 Considering these ambiguity information reaction features
were studied between 3,5ꢀdiarylꢀ4,5ꢀdihydroꢀ1Hꢀpyrazoles and isatins and the structures of
obtained products were identified. Abovementioned reaction yielded the 3ꢀ(3,5ꢀdiarylpyrazolꢀ1ꢀ
yl)ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones 24ꢀ39 (Scheme 2), that is not consistent with previously
published data.21,22
Scheme 2
The first stage of the reaction potentially follows a classic mechanism of a nucleophilic attack
of the secondary amine in position C3 on isatin through the formation of 3ꢀ(3,5ꢀdiarylꢀ4,5ꢀ
dihydropyrazolꢀ1ꢀyl)ꢀ3ꢀhydroxyꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones21 following by intramolecular
redox reaction with dehydrogenation of pyrazoline cycle.23
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Scheme 3
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The analytical and spectral data (1H NMR, 13C NMR, LCMS) confirmed the structure and
purity of 1ꢀ39.
1H NMR spectra of 1ꢀ23 show characteristic patterns of an AMX system for CH2ꢀCH protons
of pyrazoline fragment. The chemical shifts of the protons HA, HM, and HX have been assigned to
about δ~3.41ꢀ3.66, δ~4.10ꢀ4.26, and δ~5.82ꢀ6.05 with corresponding coupling constants of JAM
= 17.5ꢀ18.7, JAX = 10.4ꢀ11.3, and JMX = 2.3ꢀ4.1 Hz, respectively. CHꢀGroup proton of 2,3ꢀ
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dihydroꢀ1Hꢀindolꢀ2ꢀone fragment in the H NMR spectra of 24ꢀ39 appears as singet at δ~6.78ꢀ
7.18 ppm and the CH proton of pyrazole cycle has a singlet at δ~5.79ꢀ6.21.
Structural features of synthesized 3ꢀ[2ꢀ(3,5ꢀdiarylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ
1,3ꢀthiazolꢀ5ꢀylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones and 3ꢀ(3,5ꢀdiarylpyrazolꢀ1ꢀyl)ꢀ2,3ꢀdihydroꢀ
1Hꢀindolꢀ2ꢀones were confirmed by Xꢀray crystallographic analysis of 23 (confirmed the
structure of 1ꢀmethylꢀ3ꢀ[4ꢀoxoꢀ2ꢀ(3ꢀphenylꢀ5ꢀthiophenꢀ2ꢀylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4Hꢀthiazolꢀ
5ꢀylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone) and 24 (confirmed the structure of {3ꢀ[5ꢀ(2ꢀ
hydroxyphenyl)ꢀ3ꢀphenylpyrazolꢀ1ꢀyl]ꢀ2ꢀoxoꢀ2,3ꢀdihydroindolꢀ1ꢀyl}ꢀacetic
disolvate).
acid
ethanol
Figure 3.
The geometry of 23 is shown at Figure 3 and its structure can be represented by the mesomeric
structures A and B in which the C2 atom of 1,3ꢀthiazolꢀ4ꢀone system is connected to the endoꢀ
(N3) or exocyclic (N6) nitrogen atom via a double bond (Figure 4).
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Figure 4.
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In the crystal lattice, the molecules take up a structure intermediate (form C) between
structures A and B (Figure 4). This conclusion was drawn on the basis of similar values of the
interatomic distances for C2–N3 [1. 1.300(3) Å] and C2–N6 [1.328(3) Å] indicating that these
two bonds have a partial doubleꢀbond character in the molecules. The distances are by 7 and 15σ,
respectively, longer than a regular double bond length C=N [1.279(1) Å].24
In the molecule 23 only the 4,5ꢀdihydropyrazole ring is slightly puckered (r.m.s. deviation:
0.0382 Å) and has an envelopeꢀoriented conformation [Cremer & Pople 25 puckering parameters:
Q(2) = 0.086(3) Å,
ϕ(2) = 318.2(18)°]. The four atoms N6, N7, C8 and C9 are coplanar
[maximum deviation from the leastꢀsquares plane of 0.0049(14) Å] while the fifth atom C10 is
out of this plane [0.141(4) Å].
The asymmetric unit of 24 contains one solute molecule (host) and two solvent (ethanol)
molecules (guests) (Figure 5).
Figure 5.
The architecture of the crystal structure of 24 is determined mainly by O–HꢂꢂꢂO hydrogen bonds
involving ethanol molecules, which act both as a donor and an acceptor.
Evaluation of anticancer activity in vitro and in vivo
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Synthesized
3ꢀ[2ꢀ(3,5ꢀdiarylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ1,3ꢀthiazolꢀ5ꢀ
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ylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones (1ꢀ20) and 3ꢀ(3,5ꢀdiarylpyrazolꢀ1ꢀyl)ꢀ2,3ꢀdihydroꢀ1Hꢀ
indolꢀ2ꢀones (24ꢀ39) were submitted and evaluated at the single concentration of 10 ꢁM towards
a panel of approximately sixty cancer cell lines. The human tumor cell lines were derived from
nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and
breast cancers. Primary anticancer assays were performed according to the US NCI protocol
(http://dtp.nci.nih.gov), as described elsewhere.26 The compounds were added at the single
concentration and the cell culture was incubated for 48 h. The end point determinations were
made with a protein binding dye, sulforhodamine B (SRB). The results for each compound are
reported as the growth percentage (GP%) of treated cells when compared to untreated control
cells (Table 1). A range of growth % shows the lowest and the highest growth % found among
different cancer cell lines.
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The synthesized pyrazolineꢀthiazolidinoneꢀisatin conjugates 1ꢀ4, 7ꢀ12, 14, 15, 17ꢀ20 displayed
significant activity in the in vitro screen on tested cell lines in 10ꢁM concentration (Table 1).
These compounds demonstrated a cytotoxic effect on the most of the tumor cell lines. One
derivative of this group 6 showed moderate activity and three compounds 5, 13, 16 were inactive.
The pyrazole derivatives with 2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone fragment (24ꢀ39) were far less active
than the nonꢀcondensed systems with three heterocycles (1ꢀ20), emphasizing the importance of
presence and positioning of central linker between two terminal heterocycles, well represented by
4ꢀthiazolidinone moiety, which is coplanar with the indolone part in the target compounds with
potent antitumor properties. Only four compounds from this group 32ꢀ34 and 38 demonstrated
moderate activity in 10ꢁM concentration (GP = 50.79ꢀ70.89%) with the selective influence on
several leukemia (CCRFꢀCEM and HLꢀ60) and ovarian cancer (OVCARꢀ4) cell lines.
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Finally, compounds 1ꢀ4, 6ꢀ15, 17ꢀ20, 32ꢀ34 and 38 were selected in advanced assay against a
panel of approximately sixty tumor cell lines at 10ꢀfold dilutions of five concentrations (100ꢀM,
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10ꢀM, 1ꢀM, 0.1ꢀM and 0.01ꢀM).26 Based on the cytotoxicity assays, three antitumor activity
doseꢀresponse parameters were calculated for experimental agents against each cell line: GI50 –
molar concentration of the compound that inhibits 50% net cell growth; TGI – molar
concentration of the compound leading to total inhibition; and LC50 – molar concentration of the
compound leading to 50% net cell death. Furthermore a mean graph midpoints (MG_MID) were
calculated for each of the parameters, giving an average activity parameter over all cell lines for
the tested compounds. For the calculation of the MG_MID, insensitive cell lines are included
with the highest concentration tested (Table 2). Some of the compounds were retested on the
basis of the results of the first screen: compounds 4, 7ꢀ12, 14, 15, 17, and 19 twice, compound 18
in triple, and all other compounds were tested once.
Table 2.
The above NCI screening data analysis indicated that most of members of the pyrazolineꢀ
thiazolidinoneꢀisatin series (1ꢀ4, 7ꢀ12, 14, 15, 17ꢀ20) possessed potent in vitro antiproliferative
activity, with mean GI50 values across the 60 cell lines ranging from 0.071 ꢁM (10) to 8.05 ꢁM
(18). Activities of 6 and 14 were insignificant. The pyrazoleꢀindole conjugates 32ꢀ34, 38 were
less active compare to their 4ꢀthiazolidinoneꢀcontaining analogues with mean GI50 values from
4.47 ꢁM to 18.06 ꢁM.
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The most active compound in the series of pyrazolineꢀthiazolidinoneꢀisatin conjugates is the 5ꢀ
bromoꢀ3ꢀ{2ꢀ[5ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀnaphthalenꢀ2ꢀylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl]ꢀ4ꢀoxoꢀ4,5ꢀ
dihydroꢀ1,3ꢀthiazolꢀ5ꢀylidene}ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone 10, both in terms of the mean GI50
(0.071 ꢁM) and TGI (0.76 ꢁM). Notably, this compound ranks as near 100ꢀfold more potent
(mean GI50) compared to the most potent member of the 2ꢀ[3,5ꢀdiarylꢀ4,5ꢀdihydropyrazolꢀ1ꢀ
yl]thiazolꢀ4(5H)ꢀones, reported previously.19 At the second assay (Table 3) the compound 10 was
found to be a highly active growth inhibitor of the nonꢀsmall cell lung cancer cell line HOPꢀ92
(GI50 < 0.01 ꢁM), colon cancer line HCTꢀ116 (GI50 = 0.018 ꢁM), CNS cancer cell line SNBꢀ75
(GI50 = 0.0159 ꢁM), ovarian cancer cell line OVCARꢀ3 (GI50 = 0.0216 ꢁM) and renal cancer cell
line RXF 393 (GI50 = 0.0197 ꢁM). Mean pGI50, pTGI and pLC50 of compound 10 in comparison
with standard anticancer agent are given at Figure 6.
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Figure 6
The SAR study revealed that: (1) potent anticancer activity of tested compounds depended on
the presence of a combination of three heterocycles in one molecule, thus the pyrazolineꢀ
thiazolidinoneꢀisatin conjugates were more active in comparison with pyrazolineꢀthiazolidinone19
or pyrazoleꢀindolineꢀ2ꢀone systems; (2) attachment of halogen (Br or Cl) to 5ꢀposition of isatin
scaffold (2, 4, 7, 8, 10, 14, 15, 19, 20, 32, 33, 38) allowed us to gain of one log unit of activity
(GI50 level), in comparison with 5ꢀunsubstituted isatin analogs; (3) introduction of a methyl
group or CH2COOH group at 1Nꢀposition of isatin fragment (5, 16, 24, 30, 39) led to the loss of
activity; (4) the nature of a substituent in the 5ꢀaryl fragment also had an influence on the
antitumor activity. Therefore, the introduction of electronꢀwithdrawing group – 4ꢀchlorine (11)
improved the antiproliferative activity in comparison with electronꢀdonor groups 2ꢀOH (1), 4ꢀ
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OMe (3) and NMe2 (18) groups. This would indicate that decreased of electron density on 5ꢀaryl
moiety is important for the inhibitory activity; and (5) substitution of the phenyl fragment in 3
position of pyrazoline cycle on naphthaleneꢀ2ꢀyl substituent (compounds 9, 10, 17, 35ꢀ37 and 39,
except 38) did not have significant influence on antitumor activity, but introduction of the 4ꢀ
methoxyphenyl group in the mentioned position (6, 13) limited this effect (Figure 7).
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Figure 7
Nonꢀtumored Animal Toxicity Assays for 4, 10, 13, 17, and 18 were performed according to
NCI protocol (http://dtp.nci.nih.gov/branches/btb/acute_tox.html). The results have shown the
maximum tolerated dose was more than 400 mg/kg. It can be used to calculate the amount of
material to be given to experimental mice during further antitumor testing in vivo.
In Vivo Anticancer Activity: Hollow Fiber Assay. Compounds 4, 10, 13, 17, and 18 which
possessed a promising activity during in vitro anticancer drug screening were evaluated by NCI
using the hollow fiber assay. This assay provided quantitative indications of the compound’s
drug efficacy (http://dtp.nci.nih.gov/branches/btb/hfa.html).27 In a hollow fiber model,
polyvinylidene fluoride fibers containing various human cancer cell cultures were implanted
intraperitoneally (ip) and subcutaneously (sc) into athymic nude mice and compounds were
administrated by ip route. The effects of the compounds on reduction of the viable cancer cell
mass compared to control were determined. To simplify evaluation, the NCI protocol adopts a
point system that allows rapid viewing of the activity of a given compound. For this, a value of 2
is assigned for each compound dose that results in 50% or greater reduction in the viable cell
mass. Compounds with a combined ip + sc score ≥ 20, sc score ≥ 8, or net cell kill of one or
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more cell lines were considered significantly active. The tested compounds did not show
significant activity in the hollow fiber assay. Compound 4 resulted in the sc score of 2,
compound 11 exhibited the sc score 4 and compound 10 resulted in the ip score 6, with low
cytotoxic activity (no cell death).
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COMPARE analysis. The NCI’s COMPARE algorithm (http://dtp.nci.nih.gov/compare/)28
allows to assume biochemical mechanisms of action for the novel compounds on the basis of
their in vitro activity profiles when comparing with those of standard agents. We performed
COMPARE computations for synthesized highlyꢀactive compounds against the NCI “Standard
Agents” database at the GI50 and TGI levels (Supporting information).
COMPARE analysis hypothesis precludes, that the compound may have the same mechanism
of action as the agent with a known action mechanism. The majority of significant correlations of
tested compounds, in particular for 3ꢀ[2ꢀ(3,5ꢀdiarylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ
1,3ꢀthiazolꢀ5ꢀylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones, have been found with echinomycin and
bruceantin (inhibitors of protein or RNA synthesis). Interestingly, mean graph fingerprints of
compounds 1, 4, 9, 19 showed significant similarity at TGI level with tamoxifen (antagonist of
the estrogen receptor) in the COMPARE test.
Table 4.
CONCLUSIONS
In the present paper the novel 3ꢀ[2ꢀ(3,5ꢀdiarylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ1,3ꢀ
thiazolꢀ5ꢀylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones 1ꢀ23 and 3ꢀ(3,5ꢀdiarylpyrazolꢀ1ꢀyl)ꢀ2,3ꢀdihydroꢀ
1Hꢀindolꢀ2ꢀones 24ꢀ39 were described. The majority of these novel compounds were found to
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possess in vitro human tumor cell line activity substantially more potent than the recently
reported antitumor 4ꢀ(3,5ꢀdiarylꢀ4,5ꢀdihydroꢀ1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,3ꢀthiazolꢀ2(5H)ꢀones. The nonꢀ
thiazolidine derivatives 24ꢀ39 were far less active than conjugates with three heterocycles 1ꢀ20,
emphasizing the importance of presence and positioning of a central linker (4ꢀthiazolidinone
moiety) between two terminal heterocycles (indolone and pyrazoline). The most potent
compound 10 in the series of pyrazolineꢀthiazolidinoneꢀisatin conjugates exhibits the mean GI50
value of 0.071 ꢁM and mean TGI value of 0.76 ꢁM in the NCI 60ꢀcellꢀline screen with the low
toxicity and moderate activity level in vivo Hollow Fiber Assay.
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EXPERIMENTAL SECTION
Chemistry
The starting 3,5ꢀdiarylꢀ2ꢀpyrazolines29 and 3,5ꢀdiarylꢀ1ꢀthiocarbamoylꢀ2ꢀpyrazolines30 were
obtained according to the methods described previously.
Melting points were measured in open capillary tubes on a BŐCHI Bꢀ545 melting point
apparatus and are uncorrected. The elemental analyses (C, H, N) were performed using the
PerkinꢀElmer 2400 CHN analyzer. Analyses indicated by the symbols of the elements or
1
functions were within ±0.4% of the theoretical values. The HꢀNMR spectra were recorded on
Varian Gemini 400 MHz and 13C NMR spectra on Varian Mercuryꢀ400 100MHz in DMSOꢀd6 or
DMSOꢀd6+CCl4 mixture using tetramethylsilane (TMS) as an internal standard. Chemical shifts
are reported in ppm units with use of δ scale. Mass spectra were obtained using electrospray (ES)
ionization techniques on an Agilent 1100 Series LCMS. Analytical HPLC were performed on an
Agilent 1100 HPLC with DAD (Diode Array Detection). ESI refers to electrospray ionization,
HPLC refers to high pressure liquid chromatography, LCMS refers to liquid chromatography
coupled with a mass spectrometer, M in the context of mass spectrometry refers to the molecular
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peak, MS refers to mass spectrometer. Purity of all compounds was determined to be ≥95% by
HPLC (Agilent 1100 HPLC) with Diode Array Detection. The peak purity was checked with UV
spectra.
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General procedure for synthesis of 3ꢀ[2ꢀ(3,5ꢀdiarylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4ꢀoxoꢀ4,5ꢀ
dihydroꢀ1,3ꢀthiazolꢀ5ꢀylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones (1ꢀ23). A mixture of 3,5ꢀdiarylꢀ1ꢀ
thiocarbamoylꢀ2ꢀpyrazoline (10 mmol), chloroacetic acid (10 mmol), appropriate isatin (12
mmol) and anhydrous sodium acetate (10 mmol) was refluxed for 5 h in glacial acetic acid (10
mL). Precipitate obtained upon cooling was filtered off, washed with water and methanol and
recrystallized with DMF : ethanol mixtures (1:2 vol.).
3ꢀ{2ꢀ[5ꢀ(2ꢀHydroxyphenyl)ꢀ3ꢀphenylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl]ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ1,3ꢀ
1
thiazolꢀ5ꢀylidene}ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone (1). Yields 69%, mp 310ꢀ313 °C. H NMR
(400 MHz, DMSOꢀd6+CCl4): δ 11.15 (s, 1H), 9.92 (s, 1H), 8.92 (d, J = 7.6 Hz, 1H), 7.92 (d, J =
7.6 Hz, 2H), 7.54ꢀ7.59 (m, 3H), 7.35 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.7 Hz, 1H), 7.02ꢀ7.06 (m,
2H), 6.92 (d, J = 7.5 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 5.97 (dd, J =
13
11.2, 3.9 Hz, 1H), 4.13 (dd, J = 17.9, 11.2 Hz, 1H), 3.41 (dd, J = 17.9, 3.9 Hz, 1H). C NMR
(100 MHz, DMSOꢀd6): δ 179.5 (C=O), 173.1 (C=O), 169.5 (C=N, thiaz.), 163.0 (C=N, pyraz.),
154.9 (CꢀOH), 143.6, 137.7, 132.2, 132.1, 130.2, 129.5, 128.5, 127.9, 127.7, 126.3, 125.9, 122.3,
120.9, 119.5, 116.2, 110.6, 61.5 (CHCH2), 42.7 (CHCH2). LCMS (ESI+) m/z 467 (M+H)+.
Calcd. for C26H18N4O3S: C, 66.94; H, 3.89; N, 12.01; Found: C, 67.10; H, 3.73; N, 11.87%
3ꢀ{2ꢀ[3,5ꢀBisꢀ(4ꢀmethoxyphenyl)ꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl]ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ1,3ꢀthiazolꢀ
1
5ꢀylidene}ꢀ5ꢀbromoꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone (7). Yields 80%, mp 316ꢀ318 °C. H NMR
(400 MHz, DMSOꢀd6+CCl4): δ 11.02 (s, 1H), 9.08 (s, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.49 (d, J =
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7.5 Hz, 1H ), 7.24 (d, J = 8.7 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 6.89ꢀ6.96 (m, 3H), 5.90 (dd, J =
10.6, 3.8 Hz, 1H), 4.15 (dd, J = 18.6, 10.6 Hz, 1H), 3.83 (s, 3H), 3.71 (s, 3H), 3.52 (dd, J = 18.6,
3.8 Hz, 1H). 13C NMR (100 MHz, DMSOꢀd6): δ 179.4 (C=O), 173.2 (C=O), 169.1 (C=N, thiaz.),
163.1 (C=N, pyraz.), 162.7 (CꢀO), 159.5 (CꢀO), 142.1, 139.4, 132.5, 131.3, 130.0, 127.9, 127.8,
126.1, 125.1, 122.5, 122.1, 115.0, 114.8, 111.9, 63.8 (CHCH2), 56.1 (OCH3), 55.7 (OCH3), 43.7
(CHCH2). LCMS (ESI+) m/z 589/591 (M+H)+. Calcd. for C28H21BrN4O4S: C, 57.05; H, 3.59; N,
9.50; Found: C, 57.21; H, 3.67; N, 9.43 %
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5ꢀBromoꢀ3ꢀ{2ꢀ[5ꢀ(4ꢀmethoxyphenyl)ꢀ3ꢀnaphthalenꢀ2ꢀylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl]ꢀ4ꢀoxoꢀ
4,5ꢀdihydroꢀ1,3ꢀthiazolꢀ5ꢀylidene}ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone (10). Yields 67%, mp 283ꢀ286
°C. 1H NMR (400 MHz, DMSOꢀd6+CCl4): δ 11.10 (s, 1H), 9.12 (s, 1H), 8.40 (s, 1H), 7.99ꢀ8.08
(m, 4H), 7.61ꢀ7.64 (m, 2H), 7.49 (d, J = 7.5 Hz, 1H), 7.29 (d, J = 7.6 Hz, 2H), 6.94 (d, J = 7.6
Hz, 2H), 6.89 (d, J = 8.0 Hz, 1H), 5.98 (dd, J = 10.9, 2.3 Hz, 1H), 4.29 (dd, J = 17.9, 10.9 Hz,
1H), 3.76 (s, 3H), 3.66 (dd, J = 17.9, 2.3 Hz, 1H). 13C NMR (100 MHz, DMSOꢀd6): δ 179.5
(C=O), 173.6 (C=O), 169.5 (C=N, thiaz.), 162.9 (C=N, pyraz.), 159.5 (CꢀO), 143.7, 139.4, 134.4,
133.1, 132.2, 132.1, 129.7, 129.3, 129.1, 128.6, 128.4, 128.3, 127.9, 127.6, 127.4, 126.7, 123.7,
122.1, 120.9, 114.8, 110.6, 64.0 (CHCH2), 55.6 (OCH3), 43.8 (CHCH2). LCMS (ESI+) m/z
609/611 (M+H)+. Calcd. for C31H21BrN4O3S: C, 61.09; H, 3.47; N, 9.19; Found: C, 61.25; H,
3.62; N, 9.33 %
1ꢀMethylꢀ3ꢀ[4ꢀoxoꢀ2ꢀ(3ꢀphenylꢀ5ꢀthiophenꢀ2ꢀylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4Hꢀthiazolꢀ5ꢀ
1
ylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone (23). Yields 72%, mp 248ꢀ250 °C. H NMR (400 MHz,
DMSOꢀd6+CCl4): δ 8.96 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 7.4 Hz, 2H), 7.56ꢀ7.63 (m, 3H), 7.50
(d, J = 4.2 Hz, 1H), 7.42 (t, J = 7.1 Hz, 1H), 7.22 (d, J = 2.7 Hz, 1H), 7.11 (t, J = 7.7 Hz, 1H),
7.06 (d, J = 7.8 Hz, 1H), 7.02 (t, J = 4.2 Hz, 1H), 6.28 (dd, J = 10.6, 2.8 Hz, 1H), 4.18 (dd, J =
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18.3, 10.6 Hz, 1H), 3.72 (dd, J = 18.3, 2.8 Hz, 1H), 3.28 (s, 3H). C NMR (100 MHz, DMSOꢀ
d6): δ 179.2 (C=O), 173.5 (C=O), 167.9 (C=N, thiaz.), 162.8 (C=N, pyraz.), 144.2, 142.3, 137.8,
132.5, 132.2, 129.9, 129.6, 128.3, 128.1, 127.5, 126.7, 126.6, 125.7, 122.9, 120.1, 109.4, 59.9
(CHCH2), 43.6 (CHCH2), 26.9 (CH3). LCMS (ESI+) m/z 471 (M+H)+. Calcd. for C25H18N4O2S2:
C, 63.81 H, 3.86; N, 11.91; Found: C, 63.69; H, 3.95; N, 11.81 %
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Crystal data: C25H18N4O2S2, Mr = 470.55, triclinic, space group Pꢀ1, a = 8.8197(9), b =
10.7567(11), c = 12.0420(12) Å, α = 94.037(8),
β = 108.803(9), γ = 101.738(9)º, V =
1047.39(18) Å3, Z = 2, Dcalc = 1.492 g/cm3, ꢀ = 2.578 mmꢀ1, T = 130(2) K
Data collection: A brown plate crystal (DMF) of 0.30 x 0.20 x 0.03 mm was used to record
7343 (CuKα radiation, θmax = 73.5º) intensities on an Oxford Diffraction SuperNova fourꢀcircle
diffractometer equipped with Atlas CCD detector31 using mirrorꢀmonochromatized CuKα
radiation from highꢀflux microꢀfocus source (λ = 1.54178 Å). Accurate unit cell parameters were
determined by least squares techniques from the Ө values of 2906 reflections, Ө range 3.9ꢀ73.5°.
The data were corrected for Lorentzꢀpolarization as well as for absorption effects.31 The 4052
total unique reflections (Rint = 0.0258) were used for structure determination.
Structure solution and refinement: The structure was solved by the direct methods using the
program SHELXSꢀ97, and refinement was done against F2 for all data using SHELXLꢀ97.32 The
positions of the H atoms were positioned geometrically, and were refined with a riding model
and Uiso(H) values constrained to be 1.2 (1.5 for methyl group) times Ueq of the appropriate
carrier atom. The methylꢀH atoms were refined as rigid group which was allowed to rotate. The
final refinement converged with R = 0.0578 (for 3532 data with F2 > 4σ(F2)), wR = 0.1656 (on F2
for all data), and S = 1.062 (on F2 for all data). The largest difference peak an hole was 0.869 and
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ꢀ0.417 eÅꢀ3. Crystals 23 were twinned, hence lower accuracy of the final results. The molecular
illustration was drawn using ORTEPꢀ3 for Windows.33
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The supplementary crystallographic data are deposited at the Cambridge Crystallographic Data
Centre (CCDC), 12 Union ROAD, Cambridge CB2 1EZ (UK), Tel.: (+44) 1223/336ꢀ408, Fax:
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(+44)
1223/336ꢀ033,
Eꢀmail:
deposit@ccdc.cam.ac.uk,
World
Wide
Web:
http://www.ccdc.cam.ac.uk (deposition No. CCDC 859413).
General procedure for synthesis of 3ꢀ[3,5ꢀdiarylpyrazolꢀ1ꢀyl]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀ
ones (24ꢀ39). A mixture of 3,5ꢀdiarylpyrazoline (5 mmol) and appropriate isatin (5 mmol) was
refluxed for 1 h in ethanol (15 mL). Obtained precipitate was filtered off, washed with ethanol
and recrystallized with DMF : ethanol mixture (1:2 vol.).
{3ꢀ[5ꢀ(2ꢀHydroxyphenyl)ꢀ3ꢀphenylpyrazolꢀ1ꢀyl]ꢀ2ꢀoxoꢀ2,3ꢀdihydroindolꢀ1ꢀyl}ꢀacetic acid
(24). Yields 78%, mp 132ꢀ134 °C. 1H NMR (400 MHz, DMSOꢀd6+CCl4): δ 13.14 (s, 1H), 10.25
(s, 1H), 7.70 (d, J = 7.4 Hz, 1H), 7.27ꢀ7.42 (m, 8H), 7.09 (t, J = 8.7 Hz, 2H), 6.98ꢀ7.07 (m, 2H),
13
6.84 (s, 1H), 5.79 (s, 1H), 4.52 (d, J = 18.2 Hz, 1H), 4.47 (d, J = 18.2 Hz, 1H). C NMR (100
MHz, DMSOꢀd6): δ 172.0 (C=O), 169.5 (COOH), 155.4 (CꢀO), 151.5 (C=N), 143.8, 133.4,
123.3, 131.3, 130.0, 129.0, 128.2, 125.8, 125.7, 125.3, 123.1, 119.9, 117.1, 116.7, 109.8, 104.5,
60.7 (CH), 56.6 (CH2). LCMS (ESI+) m/z 426 (M+H)+. Calcd. for C25H19N3O4: C, 70.58; H,
4.50; N, 9.88; Found: C, 70.75; H, 4.69; N, 9.72 %
Crystal data: C25H19N3O4,2(C2H6O), Mr = 517.57, triclinic, space group Pꢀ1, a = 11.5227(7), b
= 11.8942(7), c = 12.2739(8) Å,
α
= 113.837(6),
β = 115.349(6), γ = 93.313(5)º, V =
1335.22(14) Å3, Z = 2, Dcalc = 1.287 g/cm3,
ꢀ
= 0.091 mmꢀ1, T = 130(2) K
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Data collection: A colourless block crystal (ethanol) of 0.40 x 0.40 x 0.20 mm was used to
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record 16524 (MoKα
radiation, θmax = 29.1º) intensities on an XcaliburꢀAtlas diffractometer.34
The data were corrected for Lorentzꢀpolarization as well as for absorption effects.34 Accurate
unitꢀcell parameters were determined by a leastꢀsquares fit of 9535 reflections of highest
intensity, chosen from the whole experiment. The 6388 total unique reflections (Rint = 0.0188)
were used for structure determination.
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Structure solution and refinement: The structure was solved by the direct methods using
SHELXSꢀ97, and refinement was done against F2 for all data using SHELXLꢀ97.32 The positions
of the H atoms bonded to O13, O32 and O33 atoms were obtained from difference Fourier map
and were refined freely. The remaining H atoms were placed in geometrically calculated
positions and were refined using a riding model, with C–H = 0.96 Å (CH3), 0.97 Å (CH2), 0.98 Å
(Csp3H), 0.93 Å (CarH) and Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C) for methyl H atoms. The methyl
groups were refined as a rigid groups, which were allowed to rotate. The alternative positions of
C / O atoms of disordered solvent molecule, designed as A and B, were constrained to have the
same Uij components. The sum of siteꢀoccupancy factors for the disordered molecule was
constrained to unity. The final refinement converged with R = 0.0370 (for 5016 data with F2 >
4σ
(F2)), wR = 0.1034 (on F2 for all data), and S = 1.081 (on F2 for all data). The largest
difference peak an hole was 0.293 and ꢀ0.237 eÅꢀ3. The molecular illustration was drawn using
ORTEPꢀ3 for Windows.33
The supplementary crystallographic data are deposited at the Cambridge Crystallographic Data
Centre (CCDC), 12 Union ROAD, Cambridge CB2 1EZ (UK), Tel.: (+44) 1223/336ꢀ408, Fax:
(+44)
1223/336ꢀ033,
Eꢀmail:
deposit@ccdc.cam.ac.uk,
World
Wide
Web:
http://www.ccdc.cam.ac.uk (deposition No. CCDC 859414).
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3ꢀ[3,5ꢀBisꢀ(4ꢀmethoxyphenyl)ꢀpyrazolꢀ1ꢀyl]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone (25). Yields 83%,
mp 216ꢀ219 °C. 1H NMR (400 MHz, DMSOꢀd6+CCl4): δ 10.68 (s, 1H), 7.58ꢀ7.65 (m, 4H), 7.28
(t, J = 7.4 Hz, 1H), 7.10ꢀ7.12 (m, 3H), 6.92ꢀ6.98 (m, 4H), 6.81 (s, 1H), 5.95 (s, 1H), 3.82 (s, 3H),
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56
57
58
59
60
13
3.76 (s, 3H). C NMR (100 MHz, DMSOꢀd6): δ 174.0 (C=O), 160.2 (CꢀO), 159.5 (CꢀO), 151.1
(C=N), 147.0, 143.1, 130.8, 129.9, 127.2, 127.1, 125.9, 124.7, 122.4, 122.3, 114.9, 114.5, 110.6,
60.6 (CꢀN), 55.8 (OCH3), 55.6 (OCH3). LCMS (ESI+) m/z 412 (M+H)+. Calcd. for C25H21N3O3:
C, 72.98; H, 5.14; N, 10.21; Found: C, 73.06; H, 5.01; N, 10.32 %
3ꢀ[5ꢀ(4ꢀMethoxyphenyl)ꢀ3ꢀnaphthalenꢀ2ꢀylꢀpyrazolꢀ1ꢀyl]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone
1
(35). Yields 80%, mp 208ꢀ211 °C. H NMR (400 MHz, DMSOꢀd6+CCl4): δ 10.72 (s, 1H), 8.27
(s, 1H), 7.91ꢀ7.96 (m, 4H), 7.62 (br.s, 1H), 7.47ꢀ7.52 (m, 2H), 7.31 (t, J = 7.6 Hz, 1H), 7.13ꢀ7.16
(m, 3H), 7.05 (s, 1H), 6.94ꢀ6.99 (m, 2H), 6.03 (s, 1H), 3.84 (s, 3H). 13C NMR (100 MHz,
DMSOꢀd6): δ 173.9 (C=O), 160.4 (CꢀO), 151.2 (C=N), 143.1, 133.6, 133.1, 130.8, 130.7, 129.9,
128.6, 128.5, 128.1, 127.1, 126.8, 126.5, 124.8, 124.2, 124.1, 122.5, 122.3, 114.9, 110.6, 55.8
(OCH3). LCMS (ESI+) m/z 432 (M+H)+. Calcd. for C28H21N3O2: C, 77.94; H, 4.91; N, 9.74;
Found: C, 77.81; H, 4.86; N, 9.52 %
Pharmacology
A primary anticancer assay was performed at approximately sixty human tumor cell lines panel
derived from nine neoplastic diseases, in accordance with the protocol of the Drug Evaluation
Branch, National Cancer Institute, Bethesda.26 Tested compounds were added to the culture at a
single concentration (10ꢀ5 M) and the cultures were incubated for 48 h. End point determinations
were made with a protein binding dye, sulforhodamine B (SRB). The results for each tested
compound were reported as the growth percentage of the treated cells when compared to the
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untreated control cells. The percentage growth was evaluated spectrophotometrically versus
controls not treated with test agents. The cytotoxic and/or growth inhibitory effects of the most
active selected compounds were tested in vitro against the full panel of about 60 human tumor
cell lines at 10ꢀfold dilutions of five concentrations ranging from 10ꢀ4 to 10ꢀ8 M. A 48ꢀh
continuous drug exposure protocol was followed and an SRB protein assay was used to estimate
cell viability or growth.
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Using the seven absorbance measurements [time zero, (Tz), control growth in the absence of
drug, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the
percentage growth was calculated at each of the drug concentrations levels. Percentage growth
inhibition was calculated as:
[(Ti ꢁ Tz) / (C ꢁ Tz)] x 100 for concentrations for which Ti ≥ Tz,
[(Ti ꢁ Tz) / Tz] x 100 for concentrations for which Ti < Tz.
Three dose response parameters (GI50, TGI, LC50) were calculated for each compound. Growth
inhibition of 50% (GI50) was calculated from [(Ti ꢁ Tz)/(C ꢁ Tz)] x 100 = 50, which was the drug
concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB
staining) as compared to the net protein increase seen in the control cells. The drug concentration
resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration
of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as
compared to that at the beginning) indicating a net loss of cells following treatment was
calculated from [(Ti ꢁ Tz)/ Tz] x 100 = ꢀ50. Values were calculated for each of these three
parameters if the level of activity is reached; however, if the effect was not reached or was
exceeded, the value for that parameter was expressed as more or less one than the maximum or
minimum concentration tested. The lowest values are obtained with the most sensitive cell lines.
The compounds having GI50 values ≤100 ꢃM were declared to be active.
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O
O
OH
Me
R
H
H
Ar
N
N
N
N
S
Ar
N
S
Me
N
NH
N
H
Ar'
O
Br
N
Het
O
N
Inhibitor of antiapoptic proteins
interaction BclꢀXL and BH3
H
N
N
O
Me
H
N
Inhibitors of cyclin
dependent kinases
H
Tyrosine kinase
inhibitor
O
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Pꢀglycoprotein
inhibitors
Inhibitors of cyclin
dependent kinases
NH
Me
Ar
O
S
Cl
S
N
N
N
R
Cl
N
N
N
O
HO
Inhibitor of nonmembranic
protein tyrosine phosphatase
O
O
O2N
N
N
N
H
Me
N
H
Me
R
S
O
Inhibitor of plasma membrane
electron transport
N
S
Vascular endothelium
growth factor inhibitors
N
O
S
O
NH
H
N
N
O
RHN
O
Cl
S
N
H
S
S
Me
S
N
NH
OH
N
N
N
N
N
N+
O
N
H
NH
H
HO
O
N
O
CF3
F
Inhibitors of heat shock
protein HSP90
Carbonic anhydrases
isozymes inhibitors
TNFaꢀTNFRcꢀ1
inhibitors
Inhibitor of necroptosis
Figure 1. Structures of anticancer diazole, isatin and 4ꢀthiazolidinone derivatives.
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O
H
4
5
N
6
7
S
S
8
9
R2
Ar2
10
11
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O
R2
O
N
Ar1
N
O
Ar2
N
N
N
R1
H
N
S
N
R1
O
Ar1
R2
Ar1
Ar2
N
N
N
R1
O
Figure 2. Design of the new nonꢀcondensed heterocyclic compounds with 4ꢀthiazolidinone, 2,3ꢀ
dihydroꢀ1Hꢀindolꢀ2ꢀone and pyrazole or pyrazoline moieties.
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Figure 3. A view of 23. Displacement ellipsoids are drawn at the 30% probability level and H
atoms are depicted as spheres of arbitrary radii.
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_
S
S
O
O
N
N
+
7
8
9
N
N
S
S
N
N
N
N
Me
O
Me
O
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B
A
S
δ−
N
O
δ+
N
S
N
N
Me
O
C
Figure 4. Two mesomeric structures (A and B) of 23 and the molecular structure (form C) found
in the crystal.
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Figure 5. A view of 24. Displacement ellipsoids are drawn at the 30% probability level and H
atoms are depicted as spheres of arbitrary radii.
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Compound10
Tamoxifen
Echinomycin
Bruceantin
pGI50
pTGI
pLC50
Figure 6. Anticancer activity of the most active compound 10 in comparison with standart
agents.
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Attachment of Br or Cl allowed
to gain of 1 log of activity
R2
6
7
8
9
R2
O
S
O
Significant increase
of activity
Significant decrease
of activity
N
N
R1
N
Ar1
Ar3
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N
N
S
Ar2
R1
O
N
N
N
N
Ar1
Ar2
Ar1
N
O
Ar2
Introduction of methyl group or CH2COOH group
led to the loss of activity
Introduction of chlorine in 4 position of aryl cycle
led to improveing antiproliferative activity
Figure 7. SARꢀanalysis of the nonꢀcondensed heterocyclic compounds with 4ꢀthiazolidinone,
2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀone and pyrazole or pyrazoline moieties.
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O
Comp
1
Ar1
Ar2
Ph
Ph
Ph
Ph
Ph
R1
H
H
H
R2
H
Br
H
Br
H
H
Br
Cl
H
Br
H
Me
H
Br
Cl
H
2ꢀOHꢀC6H4
2ꢀOHꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
Ar1
Ar2
2
3
4
5
6
7
8
9
10
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22
23
H
a
10
11
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CH2COOH
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
naphthalenꢀ2ꢀyl
naphthalenꢀ2ꢀyl
Ph
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
Me
Ar2
Ar1
N
N
S
H2N
Ph
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
naphthalenꢀ2ꢀyl
Ph
b
O
H
H
O
N
4ꢀNMe2ꢀC6H4
4ꢀNMe2ꢀC6H4
4ꢀNMe2ꢀC6H4
thiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
thiophenꢀ2ꢀyl
Ar2
N
R1
Ph
Ph
Ph
Ph
Br
Cl
H
Br
H
N
N
S
Ar1
1ꢀ23
Ph
R2
Scheme 1. Synthesis of 3ꢀ[2ꢀ(3,5ꢀdiarylꢀ4,5ꢀdihydropyrazolꢀ1ꢀyl)ꢀ4ꢀoxoꢀ4,5ꢀdihydroꢀ1,3ꢀthiazolꢀ
5ꢀylidene]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones. Reagents, conditions and yields: (a) thiosemicarbazide
(1.2 equiv), КОН (2.5 equiv), EtOH, reflux 8h, (Ref. [30]); (b) appropriate isatin (1.2 equiv),
ClCH2COOH (1.0 eq.), AcONa (2.0 equiv), AcOH, reflux 5h, 62ꢀ85%.
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Ar1
Ar1
O
O
d
c
R1
Ar2
Ar2
HN
N
Ar1
Ar2
N
N
N
9
10
11
12
13
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R2
Comp
24
25
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39
Ar1
Ar2
Ph
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
Ph
R1
R2
H
H
Br
Cl
H
Br
H
2ꢀOHꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
CH2COOH
H
H
H
H
Ph
Ph
H
CH2COOH
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
Ph
naphthalenꢀ2ꢀyl
naphthalenꢀ2ꢀyl
naphthalenꢀ2ꢀyl
naphthalenꢀ2ꢀyl
naphthalenꢀ2ꢀyl
H
H
H
H
H
H
H
H
H
Br
Cl
H
H
Cl
H
4ꢀClꢀC6H4
4ꢀNMe2ꢀC6H4
4ꢀOMeꢀC6H4
4ꢀOMeꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
4ꢀClꢀC6H4
Br
H
CH2COOH
Scheme 2. Synthesis of 3ꢀ[3,5ꢀdiarylpyrazolꢀ1ꢀyl]ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones. Reagents,
conditions and yields: (c) hydrazine hydrate (1.0 equiv), EtOH, reflux 0.5h, (Ref. [29]); (d)
appropriate isatin (1.0 equiv), EtOH, reflux 1h, 68ꢀ83%.
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Ar2
N
4
5
6
7
8
9
Ar2
N
O
R2
Ar2
O
N
R1
Ar1
N
Ar1
N
N
HO
O
O
ꢀH2O
NH
10
11
12
13
14
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22
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Ar1
R2
N
N
R2
R1
R1
Scheme 3. Proposed mechanism of 3ꢀ(3,5ꢀdiarylpyrazolꢀ1ꢀyl)ꢀ2,3ꢀdihydroꢀ1Hꢀindolꢀ2ꢀones
formation.
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