Isothiocoumarin-3-carboxylic acid derivatives: Synthesis, anticancer and antitrypanosomal activity evaluation

Article abstract of DOI:10.1016/j.ejmech.2014.01.028

A series of new isothiocoumarin-3-carboxylic acids derivatives had been obtained based on the 5-arylidenerhodanines hydrolysis. Anticancer activity screening allowed identification of 7,8-dimethoxy-1-oxo-1H-isothiochromene-3- carboxylic acid (4-phenylthia

Full text of DOI:10.1016/j.ejmech.2014.01.028

Accepted Manuscript
Isothiocoumarin-3-carboxylic acid derivatives: synthesis, anticancer and
antitrypanosomal activity evaluation
Danylo Kaminskyy, Anna Kryshchyshyn, Ihor Nektegayev, Olexandr Vasylenko,
Philippe Grellier, Roman Lesyk
PII:
S0223-5234(14)00074-9
10.1016/j.ejmech.2014.01.028
EJMECH 6681
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 November 2013
Revised Date: 7 January 2014
Accepted Date: 14 January 2014
Please cite this article as: D. Kaminskyy, A. Kryshchyshyn, I. Nektegayev, O. Vasylenko, P. Grellier, R.
Lesyk, Isothiocoumarin-3-carboxylic acid derivatives: synthesis, anticancer and antitrypanosomal activity
evaluation, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.01.028.
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ACCEPTED MANUSCRIPT
Isothiocoumarin-3-carboxylic acid derivatives: synthesis, anticancer and antitrypanosomal
activity evaluation
Danylo Kaminskyy, Anna Kryshchyshyn, Ihor Nektegayev, Olexandr Vasylenko, Philippe
Grellier & Roman Lesyk
Ph
O
N
O
Me
H
O
N
N
O
S
H
S
OH
R1
S
O
S
Me
O
O
GI50, µM
1.80
S
RPMI-8226 (Leukemia)
R
NCI-H322M (NSC Lung Cancer) 1.28
O
SNB-75
MALME-3M (Melanoma)
TK-10 (Renal Cancer)
(CNS Cancer)
1.54
1.60
1.43
R
R
R

ACCEPTED MANUSCRIPT
Isothiocoumarin-3-carboxylic acid derivatives: synthesis, anticancer and antitrypanosomal
activity evaluation
Danylo Kaminskyya, Anna Kryshchyshyna, Ihor Nektegayeva, Olexandr Vasylenkob, Philippe
Grellierc & Roman Lesyka,*
a Department of Pharmaceutical, Organic and Bioorganic Chemistry
Danylo Halytsky Lviv National Medical University,
Pekarska 69, Lviv, 79010, Ukraine
b Institute of Bioorganic Chemistry and Petrochemistry,
National Academy of Science of Ukraine,
Murmanska 1, Kyiv, 02094, Ukraine
c National Museum of Natural History,
UMR 7245 CNRS-MNHN, team APE, CP 52,
57 Rue Cuvier, 75005 Paris, France
*corresponding author
dr_r_lesyk@org.lviv.net (R. Lesyk)
tel. +38 0322 75-59-66
fax. +38 0322 75-77-34
Keywords: synthesis, 5-ylidenerhodanines, isothiocoumarins, anticancer activity,
antitrypanosomal activity
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Abstract
A series of new isothiocoumarin-3-carboxylic acids derivatives had been obtained based on the
5-arylidenerhodanines hydrolysis. Anticancer activity screening allowed identification of 7,8-
dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (4-phenylthiazol-2-yl)-amide (30) with
the highest level of antimitotic activity (GI50 NCI-H322M/NSC Lung Cancer = 1.28 µM).
Evaluation of the antitrypanosomal activity against Trypanosoma brucei brucei showed that
investigated compounds did not exhibit significant antiparasitic effects. Additionally, the most
pharmacologically attractive compounds were non-toxic and well tolerated by the experimental
animals.
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1. Introduction
(Iso)coumarins and dihydroquinolines as representatives of biological active compounds
are promising scaffolds in the search of new drug-like small molecules [1-3] including those
showing antitumor [4] and antitrypanosomal activity [5]. In the contrast information on
isothiocoumarin (1-oxo-1Н-2-benzothiopyrane or 1-oxo-1H-isothiochromene) derivatives which
can be considered as bioisosters of the above mentioned compounds is rather scarce both
concerning the synthetic protocols and biological activities [6,7]. Although 1-oxo-1Н-2-
benzothiopyrane is simple hetero-bicyclic system, the methods of its synthesis are limited,
difficult in performance and often ineffective [8]. In the synthesis of mentioned heterocyclic
derivatives methyl-, 2-aroylmethyl- [9], carboxymethyl- and 2-cyanomethylbenzoates [10]; 2-
diphenyldithiochloroformates [11]; homophthalic acids [12]; S-(substituted-phenyl)-3-
oxobutanethioates or benzylthiolatе/benzenethiols [13] for example are used as starting reactants.
The efficient approach for isothiocoumarin derivatives obtaining including
isothiocoumarin-3-carboxylic acids is based on the 5-ylidene-4-thiazolidinones hydrolysis with
further intermediates (3-substituted-2-mercaptoacrylic acids) cyclization described in the early
50-ies [14-16] and improved in 2000 years [17-19]. Starting substances are easily accessible 5-
arylidene-2-thioxothiazolidine-4-ones (5-arylidenerhodanines) with carboxylic group in ortho
position of benzene ring. At the base hydrolysis of mentioned rhodanines two-step reaction
occurs in which intermediates of the hydrolysis undergo heterocyclization with the formation of
the 1-oxo-1H-2-benzothiopyran-3-carboxylic acids. This approach is efficient for not only
isothiocoumarins synthesis, but also for the obtaining of 3-mercaptocoumarins [20,21],
ketocinchonic acids, 2-indole-carboxylic acids etc. structure of which depends on the nature of
substituent in the ortho- position of aryl(heter)ylidene fragment [22,23] (Scheme 1).
Scheme 1
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The same protocol is described for the isocoumarin-3-carboxylic acids as well as
dihydroisoquinoline-carboxylic acids synthesis as intermediates in the reaction of hippuric acid
azlactones base hydrolysis yielding yohimbine alkaloids [3,24]. Moreover mentioned α-
mercapto-β-substituted acrylic acids, as well as 5-ylidenethiazolidinones used therein deserve
special attention as multifunctional reactants for various heterocyclic derivatives synthesis [25-
29].
The aim of our work was the search of new possible anticancer and anti-trypanosomal
agents among isothiocoumarin-3-carboxylic acids derivatives. The argument of the possibility of
simultaneous anticancer and antitrypanosomal activities identification is the data about
chemopreventive action of known antitrypanosomal drugs [30].
2. Results and discussion
2.1. Chemistry
Following our previous research in the thiazolidinone field [31-35] and proceeding from
the above mentioned approach a series of isothiocoumarin-3-carboxylic acid derivatives had
been obtained. The starting 5-arylidenerhodanines were prepared through condensation of 2-
formylbenzoic acid or opianic (6-formyl-2,3-dimethoxybenzoic) acid with rhodanine (acetic acid
medium in the presence of sodium acetate). Mentioned thiazolidinones derivatives are unstable
in strongly alkaline medium via hydrolysis to the 3-substituted-2-mercapto-acrylic acids. In case
of compounds with substituent in ortho position the two-step reaction occurs in which
intermediates of the hydrolysis undergo heterocyclization with the formation of the 1-oxo-1H-2-
benzothiopyran-3-carboxylic acids (Scheme 1). The heterocyclization reaction was performed in
the 30% sodium hydroxide water solution and followed by acidification with hydrochloric acid
the target 1 and 2 were obtained in high yield (Scheme 2). Aiming isothiocoumarin-3-carboxylic
acid functionalization the latter were converted with thionyl chloride to give corresponding acid
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chlorides [17]. For the target compounds (3-39) synthesis isothiocoumarin-3-carboxylic acids
chlorides were utilized in the acylation reactions of aliphatic alcohols, amines, and amino acids.
Scheme 2
The increasing of the pharmacological activity caused by the introduction of the substituent
in the (iso)coumarin core C-3 position defined the modification direction [36]. The data
characterizing synthesized compounds are presented in the experimental part. Analytical and
spectral data (1H NMR, 13C NMR) confirmed the structure of the synthesized compounds. In the
1H NMR spectra the isothiocoumarin fragment is characterized by the singlet of =CH group in
the 4th position at 7.10-8.30 ppm, and the subspectrum of two triplets and two doublets at 7.00-
8.30 ppm (R = H) or by two doublets at 7.30-7.70 and 7.40-7.90 ppm (R = OMe).
2.2. Anticancer activity
Newly synthesized compounds were selected by the National Cancer Institute (NCI)
Developmental Therapeutic Program (www.dtp.nci.nih.gov) for the in vitro cell line screening to
investigate their anticancer activity. Anticancer assays were performed according to the US NCI
protocol, which was described elsewhere [37-39]. The compounds were first evaluated at one
dose primary assay towards three cell lines (panel consisting of three types of human cancers:
breast (MCF7), lung (NCI-H460) and CNS (SF-268) – concentration 10-4M) or approximately 60
cell lines (concentration 10-5M) for compounds 14 (743078), 30 (748183), 9 (748491) and 29
(748231) (here and hereafter in brackets: NCS – NCI code). In the screening protocol, each cell
line was inoculated and preincubated for 24–48 h on a microtiter plate. Test agents were then
added at a single concentration and the culture was incubated for further 48 h. End point
determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each
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test agent were reported as the percent growth (GP) of the treated cells when compared to the
untreated control cells. The screening results are shown in Table 1.
Table 1
For the compound 14 the average index of cell growth (GPmean) was 86.7%, however it
should be noted high selectivity to the NCI-Н572 (Non-Small Cell Lung Cancer) – GP = -
11.00%, UO-31 (renal cancer) – GP = -62.11%, and to the CCRF-CEM (Leukemia) – GP = -
27.96%. The most prominent results were observed for 30: GPmean = 23.84% and the most
sensitive cell lines were as follows: A549/ATCC (Non-Small Cell Lung Cancer) GP = -50.81%;
SF-539 (CNS Cancer) GP = -47.97%; SNB-75 (CNS Cancer) GP = -43.79%; OVCAR-3 (Ovarian
Cancer) GP = -67.02%; UO-31 (Renal Cancer) GP = -65.51%; CAKI-1 (Renal Cancer) GP = -
70.76%. Negative values of GP indicated the cytotoxicity of tested compounds. In contrast
structurally related 1-oxo-1H-isothiochromene-3-carboxylic acid (4-phenyl-thiazol-2-yl)-amide
(29) did not possess any anticancer effect (GPmean = 98.97%), as well as [(1-oxo-1H-
isothiochromene-3-carbonyl)-amino]-acetic acid (9) (GPmean = 100.94%).
For the other derivatives, the prescreening assay realized on three cell lines identified the
compounds 7, 19, 24, 31, 30 with the highest selectivity towards the NCI-H460 cell line. Data
analysis showed an increase of biological activity when there is: i) substitution of chlorine atom
in the amide fragment (25) with fluorine (24), ii) moving trifluoromethyl moiety in amide
fragments from position 2 (26) in position 3 (19), and iii) the absence of methoxy-groups in
isothiocoumarin fragment (comparison of 31 and 32).
The most active compounds in the one dose assay (7, 19, 24, 30 and 31) as well as 8, 15,
16, 23, 36, 37, were further selected for an advanced assay against a panel of approximately 60
tumor cell lines (cell lines were derived from nine different cancer types: leukemia, melanoma,
lung, colon, CNS, ovarian, renal, prostate and breast cancers) at concentrations 10-fold diluted
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(100µM, 10µM, 1µM, 0.1µM and 0.01µM). The percentage of growth was evaluated
spectrophotometrically versus controls not treated with test agents after 48-h exposure and using
the SRB protein assay to estimate cell viability or growth. Three antitumor activity dose-
response parameters were calculated for each cell line: GI50 – molar concentration of the
compound that inhibits 50% net cell growth; TGI - molar concentration of the compound leading
to the total inhibition; and LC50 - molar concentration of the compound leading to 50% net cell
death (presented in negative logarithm). 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 compound. For the MG_MID calculation, insensitive cell lines were included with the
highest concentration tested (Table 2).
Table 2
Obtained data indicate in general low to moderate anticancer activity, the highest activity
was observed for aromatic amides 19 and 24. Comparison of anticancer profiles of isopropyl
esters 7 and 8 showed a slight loss of activity in the absence of methoxy-groups. Secondary
amides were characterized by somewhat less antitumor activity in comparison with esters. The
introduction of morpholine fragment (36) leaded to the complete loss of activity (comparison of
36 and 37). Likewise esters compound 37 revealed slight selectivity of action to melanoma and
lung cancer cell lines. Worth mentioning was the antitumor effect of 31 from a series of
heterocyclic amides of isothicoumarin-3-carboxylic acids. Against the backdrop of slight total
activity 31 revealed the highest antimitotic effect among all tested compounds against leukemia
line SR (pGI50= 6.47) and selectivity towards CNS cancer cell lines. Moreover, 7,8-dimethoxy-
1-oxo-1H-isothiochromene-3-carboxylic acid (4-phenylthiazol-2-yl)-amide (30) showed the
highest anticancer activity among all tested compounds. These findings are also interesting in the
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context of a comparison with the related heterocyclic amides activities (28 and 29) that do not
possess anti-tumor activity.
Aiming to investigate the possible mechanisms of cell growth inhibition we carried out
the COMPARE-analysis, namely comparison of selectivity patterns (mean graph fingerprints
according DTP protocol) of tested compounds with standard anticancer agents, NCI active
synthetic compounds and natural extracts (http://dtp.nci.nih.gov/docs/compare/compare.html). It
is based on the comparison of the patterns of differential growth inhibition for cultured cell lines
and can potentially gain insight into the mechanism of the cytotoxic action. Unfortunately no
significant correlations were detected.
2.3. Antitrypanosomal activity
Series of isothiocoumarin-3-carboxylic acids derivatives (1, 5, 9, 17-19, 21-23, 25, 26,
29, 30, 32, 35) had been tested for their antitrypanosomal activity against Trypanosoma brucei
brucei (Tbb) and the results are shown in Table 3.
Table 3
Bloodstream forms of Tbb strain 90-13 were cultured in HMI9 medium supplemented
with 10% FCS at 37°C under an atmosphere of 5% CO2 [40]. In all experiments, log-phase cell
cultures were harvested by centrifugation at 3000 x g and immediately used. Drug assays were
based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable
cells [41]. Compounds were tested at concentrations of 10 and 1 µg/mL. Investigated compounds
did not exhibit significant antitrypanosomal effects except several substances, namely 1-oxo-1H-
isothiochromene-3-carboxylic acid naphthalen-1-ylamide (22) and 7,8-dimethoxy-1-oxo-1H-
isothiochromene-3-carboxylic acid (4-sulfamoyl-phenyl)-amide (17) that inhibited growth of
Tbb bloodstream forms by more than 50% at 10ꢀg/mL (table 3). In the spirit of such results little
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information is available regarding the antiprotozoal effects of not only isothiocoumarins but
coumarins and isocoumarins as well. 3-Aryl-4-hydroxycoumarins had been reported to show low
to moderate antitrypanosomal activity against Trypanosoma cruzi [42]; 4-oxo-1,4-
dihydroquinoline-3-carboxamides exhibited antitrypanosomal activity against Tbb and
Trypanosoma brucei rhodesience [5].
2.4. Acute toxicity in vivo
The most active of the synthesized compounds were evaluated for their approximate LD50
in mice [43,44]. The stock solutions of the compounds used in this study were prepared
immediately before usage and increasing amounts of substances (100-1000 mg/kg) were injected
intraperitoneally. The LD50 were calculated according to Litchfield and Wilcoxon [44]. The
results indicated that tested compounds were non-toxic and well tolerated by the experimental
animals as demonstrated by their LD50 (870±112.0 mg/kg (17), 750±31.0 mg/kg (19), 410±48.5
mg/kg (22), 515±61.4 mg/kg (30)).
3. Conclusions
In the presented paper the series of isothiocoumarin-3-carboxylic acids derivatives had
been obtained based on the 5-arylidenerhodanines hydrolysis. Anticancer activity screening
allowed establishing the influence of carboxylic group modification and led to the 7,8-
dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic
acid
(4-phenylthiazol-2-yl)-amide
identification with the highest level of antimitotic activity. Antitrypanosomal activity evaluation
showed that investigated compounds did not exhibit significant antiparasitic effects. Worth
mentioning is that the pharmacologically attractive tested compounds were non-toxic and well
tolerated by mice.
4. Experimental
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4.1. Materials and methods
Melting points were measured in open capillary tubes on a BŰCHI B-545 melting point
apparatus and are uncorrected. The elemental analyses (C, H, N) were performed using the
Perkin-Elmer 2400 CHN analyzer. Analysis results indicated by the symbols of the elements or
1
functions were within ±0.4% of the theoretical values. The H-NMR spectra were recorded on
13
Varian Gemini 400 MHz spectrometer and C NMR spectra on Varian Mercury-400 100MHz
spectrometer in DMSO-d6 using tetramethylsilane (TMS) as an internal standard. Chemical
shifts are reported in ppm units with the use of δ scale. Mass spectra were obtained using
electrospray (ES) ionization techniques on an Agilent 1100 series LCMS instrument.
4.2. Chemistry
General procedure for synthesis of 1-oxo-1H-isothiochromene-3-carboxylic acids (1, 2)
Appropriate 5-arylidenerhodanine (20 mmol) was added to 30% NaOH aqueous solution (13
mL) and the mixture was boiled for 0.5 h. After cooling the reaction mixture was poured into
conc. HCl (40 mL) and ice (100 g). The precipitate was filtered off and purified by
recrystallization (BuOH).
1-Oxo-1H-isothiochromene-3-carboxylic acid (1). Yield 78%, mp 258-260оС, lit 260-262оС [6].
1H NMR (400 MHz, DMSO-d6): δ 7.77 (t, 1H, J = 8.4 Hz, 6-H), 7.90 (t, 1Н, J = 8.0 Hz, 7-H),
13
7.99 (d, 1Н, J = 8.0 Hz, 5-Н); 8.20 (d, 1Н, J = 8.8 Hz, 8-Н), 8.21 (s, 1Н, 4-Н). C NMR (100
MHz, DMSO-d6): δ 186.1, 163.7, 136.4, 134.6, 132.7, 131.4, 130.2, 129.0, 128.3, 125.7. EI-MS
(m/z): 206 (М+). Calcd. for C10H6O3S: C, 58.24; H, 2.93; Found: C, 58.40; H, 3.05%
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (2). Yield 80%, mp 252-254оС, lit
1
257-258оС [14]. H NMR (400 MHz, DMSO-d6): δ 3.82 (s, 3H, OCH3), 3.97 (s, 3Н, OCH3),
13
7.38 (d, 1Н, J = 8.7 Hz, 6-Н), 7.48 (d, 1Н, J = 8.7 Hz, 5-Н), 7.89 (s, 1Н, 4-Н). C NMR (100
MHz, DMSO-d6): δ 183.7, 163.7, 155.3, 147.7, 130.6, 129.6, 128.5, 126.8, 123.3, 118.7, 60.9,
56.5. EI-MS (m/z): 266 (M+). Calcd. for C12H10O5S: C, 54.13; H, 3.79; Found: C, 54.30; H,
3.85%.
General procedure for synthesis of 1-oxo-1H-isothiochromene-3-carboxylic acid esters (3-8)
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The mixture of appropriate acid (1 or 2) (13 mmol) and thionyl chloride (30 mL) was boiled for
0.5 h. The mixture was cooled, and hexane (60 mL) was added, the precipitated acid chloride
was filtered off and washed with hexane. The mixture of obtained acid chloride (8 mmol) and
appropriate alcohol (20 mL) was boiled for 1 h. The obtained precipitate was filtered off and
purified by recrystallization [17].
1
1-Oxo-1H-isothiochromene-3-carboxylic acid methyl ester (3). Yield 81%, mp 133-135оС. H
NMR (400 MHz, DMSO-d6): δ 3.93 (s, 3Н, CH3), 7.80 (t, 1H, J = 8.2 Hz, 6-H), 7.92 (t, 1H, J =
8.2 Hz, 7-H), 8.03 (d, 1Н, J = 7.6 Hz, 5-Н), 8.21 (d, 1Н, J = 8.0 Hz, 8-Н), 8.29 (s, 1Н, 4-Н). 13C
NMR (100 MHz, DMSO-d6): δ 185.7, 162.7, 136.1, 135.3, 133.4, 132.3, 129.3, 128.8, 128.0,
125.6, 53.8. Calcd. for C11H8O3S: C, 59.99; H, 3.66; Found: C, 60.10; H, 3.80%.
7,8-Dimethoxy-1-Oxo-1H-isothiochromene-3-carboxylic acid methyl ester (4). Yield 86%, mp
150-153оС. 1H NMR (400 MHz, DMSO-d6): δ 3.77 (s, 3Н, COOCH3), 3.89 (s, 3Н, ОCH3), 3.96
(s, 3Н, ОCH3), 7.62 (d, 1H, J = 8.7 Hz, 6-Н), 7.76 (d, 1Н, J = 8.8 Hz, 5-Н), 8.10 (s, 1Н, 4-Н).
13C NMR (100 MHz, DMSO-d6): δ 184.0, 162.9, 155.7, 148.0, 130.6, 129.8, 128.3, 126.7, 122.9,
118.5, 60.8, 56.6, 53.1. Calcd. for C13H12O5S: C, 55.71; H, 4.32; Found: C, 55.83; H, 4.49%.
1
1-Oxo-1H-isothiochromene-3-carboxylic acid ethyl ester (5). Yield 78%, mp 120-122оС. H
NMR (400 MHz, DMSO-d6): δ 3.40 (t, 3Н, J = 7.2 Hz, OCH2CH3), 4.39 (q, 2Н, J = 7.2 Hz,
OCH2CH3), 7.80 (t, 1H, J = 8.4 Hz, 6-H), 7.92 (t, 1Н, J = 7.6 Hz, 7-H), 8.03 (d, 1Н, J = 8.0 Hz,
5-Н), 8.21 (d, 1Н, J = 7.6 Hz, 8-Н), 8.26 (s, 1Н, 4-Н). 13C NMR (100 MHz, DMSO-d6): δ 185.6,
161.9, 136.1, 134.8, 133.0, 131.7, 129.0, 128.9, 128.7, 125.7, 62.4, 14.4. Calcd. for C12H10O3S:
C, 61.52; H, 4.30; Found: C, 61.65; H, 4.50%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid ethyl ester (6). Yield 86%, mp 144-
1
146оС. H NMR (400 MHz, DMSO-d6): δ 1.33 (t, 3H J = 7.1 Hz, OCH2CH3), 3.76 (s, 3Н,
ОCH3), 3.96 (s, 3Н, ОCH3), 4.34 (q, 2H, J = 6.8 Hz, OCH2CH3), 7.68 (d, 1H, J = 8.5 Hz, 6-Н),
7.84 (d, 1Н, J = 8.6 Hz, 5-Н), 8.13 (s, 1Н, 4-Н). 13C NMR (100 MHz, DMSO-d6): δ 183.6,
162.6, 156.1, 147.9, 130.7, 130.6, 129.7, 125.3, 123.5, 120.0, 62.8, 61.4, 57.2, 14.7. Calcd. for
C14H14O5S: C, 57.13; H, 4.79; Found: C, 57.30; H, 43.85%.
1
1-Oxo-1H-isothiochromene-3-carboxylic acid isopropyl ester (7). Yield 77%, mp 90-92оС. H
NMR (400 MHz, DMSO-d6): δ 1.32 (d, 6H, J = 6.2 Hz, CH(CH3)2), 5.20 (m, 1H, CH), 7.79 (t,
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1H, J = 8.2 Hz, 6-H), 7.91 (t, 1Н, J = 7.6 Hz, 7-H), 8.05 (d, 1Н, J = 8,0 Hz, 5-Н), 8.24 (d, 1Н, J
= 7.6 Hz, 8-Н), 8.27 (s, 1Н, 4-Н). 13C NMR (100 MHz, DMSO-d6): δ 185.3, 162.9, 136.1, 134.8,
133.1, 132.4, 129.2, 128.6, 128.0, 125.2, 70.6, 22.1. Calcd. for C13H12O3S: C, 62.88; H, 4.87;
Found: C, 62.95; H, 4.55%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid isopropyl ester (8). Yield 80%, mp
126-128оС. 1H NMR (400 MHz, DMSO-d6): δ 1.32 (d, 6H, J = 6.4 Hz, CH(CH3)2), 3.75 (s, 3Н,
ОCH3), 3.95 (s, 3Н, ОCH3), 5.13 (m, 1H, CH), 7.69 (d, 1H, J = 8.7 Hz, 6-Н), 7.86 (d, 1Н, J =
13
8.7 Hz, 5-Н), 8.12 (s, 1Н, 4-Н). C NMR (100 MHz, DMSO-d6): δ 183.7, 162.1, 156.0, 147.9,
130.7, 130.6, 129.6, 125.7, 123.5, 120.0, 70.7, 61.4, 57.2, 22.2. Calcd. forC15H16O5S: C, 58.43;
H, 5.23; Found: C, 58.30; H, 5.05%.
General procedure for synthesis of 3-(1-oxo-1H-isothiochromene-3-carbonyl-amino)-carboxylic
acids (9-11)
The mixture of the acid chloride (5 mmol) and appropriate amino acid (10 mmol) in acetic acid
medium (8 mL) was boiled for 1 h. After cooling the resulting precipitate was filtered, washed
with water and recrystallized [25].
[(1-Oxo-1H-isothiochromene-3-carbonyl)-amino]-acetic acid (9). Yield 77%, mp 122-124оС. 1H
NMR (400 MHz, DMSO-d6): δ 3.95 (d, 2H, J = 5.6 Hz, CH2), 7.08 (t, 1H, J = 7.6 Hz, 6-H), 7.89
(d, 1H, J = 7.6 Hz, 5-H), 7.97 (t, 1H, J = 7.6 Hz, 7-H), 8.17 (s, 1H, 4-H), 8.20 (d, 1H, J = 8.0 Hz,
13
8-H), 9.24 (br.s, 1H, NH), 12.75 (br.s, 1H, COOH). C NMR (100 MHz, DMSO-d6): δ 187.0,
171.4, 163.3, 136.9, 135.6, 133.8, 132.8, 131.8, 128.8, 125.9, 124.6, 42.0. Calcd. for
C12H9NO4S: C, 54.75; H, 3.45; N,5.32; Found: C, 54.90; H, 3.65; N, 5.48%.
[(7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carbonyl)-amino]-acetic acid (10). Yield 69%,
mp 221-223оС. 1H NMR (400 MHz, DMSO-d6): δ 3.77 (s, 3H, ОCH3), 3.96 (s, 3Н, ОCH3), 3.89
(d, 2Н, J = 5.6 Hz, СН2), 7.57 (d, 1Н, J = 8.2 Hz, Н6), 7.61 (d, 1Н, J = 8.2 Hz, Н5), 7.96 (s, 1Н,
Н4), 8.90 (t, 1Н, J = 5.6 Hz, NH). 13C NMR (100 MHz, DMSO-d6): δ 184.1, 172.3, 164.1, 156.1,
147.9, 131.5, 129.9, 128.5, 127.1, 123.8,119.0, 61.0, 56.8, 42.2. Calcd. for C14H13NO6S: C,
52.01; H, 4.05; N, 4.33; Found: C, 51.90; H, 3.95; N, 4.48%.
3-[(7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carbonyl)-amino]-propionic acid (11). Yield
1
56%, mp 214-216оС. H NMR (400 MHz, DMSO-d6): δ 3.77 (s, 3H, ОCH3), 3.95 (s, 3Н,
12

ACCEPTED MANUSCRIPT
ОCH3), 3.45 (m, 4Н, СH2СН2), 7.53 (d, 1Н, J = 8.8 Hz, 6-H), 7.59 (d, 1Н, J = 8.8 Hz, 5-H), 7.89
(s, 1Н, 4-Н), 8.62 (t, 1Н, J = 4.2 Hz, NH), 12.26 (br.s, 1Н, СООН). 13C NMR (100 MHz,
DMSO-d6): δ 183.5, 171.9, 163.3, 155.8, 148.0, 130.7, 129.8, 128.6, 126.9, 123.6, 118.9, 60.6,
60.0, 56.6, 42.5. Calcd. for C15H15NO6S: C, 53.41; H, 4.48; N, 4.15; Found: C, 53.52; H, 4.60;
N, 4.03%.
General procedure for synthesis of 1-oxo-1H-isothiochromene-3-carboxylic acid amides (12-39)
To the solution of appropriate amine (20 mmol) in a/h dioxane (30 mL) the solution of
appropriate isothiocoumarin-3-carboxylic acid chloride (10 mmol) in a/h dioxane (20 mL) was
added. The mixture was refluxed for 1 h. Resulting precipitate was filtered, washed with water
and recrystallized from appropriate solvent. When the precipitate wasn’t formed the reaction
mixture was poured into the water [25].
1
1-Oxo-1H-isothiochromene-3-carboxylic acid phenylamide (12). Yield 81%, mp 193-195оС. H
NMR (400 MHz, DMSO-d6): δ 7.13 (t, 1H, J = 7.6 Hz, 6-H), 7.35 (t, 2H, J = 7.6 Hz, arom.),
7.72 (d, 2Н, J = 8.0 Hz, arom.), 7.77 (t, 1H, J= 8.0 Hz, 7-H), 7.88-7.96 (m, 2H, arom.), 8.23 (d,
13
1Н, J = 8.4 Hz, 8-H), 8.28 (s, 1Н, 4-Н), 10.51 (s, 1H, NH). C NMR (100 MHz, DMSO-d6): δ
186.4, 161.3, 138.6, 136.7, 134.8, 134.7, 132.3, 130.9, 128.8, 125.6, 124.4, 120.8. Calcd. for
C16H11NO2S: C, 68.31; H, 3.94; N, 4.98; Found: C, 68.50; H, 4.12; N, 5.05%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid phenylamide (13). Yield 92%, mp
208-210оС. 1HNMR (400 MHz, DMSO-d6): δ 3.77 (s, 3Н, ОCH3), 3.96 (s, 3Н, ОCH3), 7.11-7.15
(m, 2H, arom.), 7.38-7.41 (m, 2H, arom.), 7.70-7.73 (m, 3Н, arom.), 8.19 (s, 1Н, 4-Н), 10.50 (s,
13
1H, NH). C NMR (100 MHz, DMSO-d6): δ 184.3, 161.6, 155.1, 147.6, 138.6, 130.9, 130.8,
129.4, 129.1, 125.0, 124.6, 122.9, 120.9, 119.7, 61.1, 56.8. Calcd. for C18H15NO4S: C, 63.33; H,
4.43; N, 4.10; Found: C, 63.50; H, 4.55; N, 4.30%.
1-Oxo-1H-isothiochromene-3-carboxylic acid (4-hydroxyphenyl)-amide (14). Yield 70%, mp
>240оС. 1H NMR (400 MHz, DMSO-d6): δ 6.74 (d, 2Н, J = 9.0 Hz, arom.), 7.45 (d, 2Н, J = 8.9
Hz, arom.), 7.65 (m, 1H, arom.), 7.78-7.85 (br.s, 2Н, arom.), 8.13 (s, 1Н, 4-Н), 8.21 (d, 1Н, J =
13
8.0 Hz, 8-Н), 8.80 (br.s, 1Н, ОН), 9.96 (s, 1H, NH). C NMR (100 MHz, DMSO-d6): δ 187.1,
161.4, 155.2, 137.0, 135.6, 135.0, 132.8, 131.7, 130.2, 128.8, 125.9, 124.5, 123.2, 115.9. Calcd.
for C16H11NO3S: C, 64.63; H, 3.73; N, 4.71; Found: C, 64.50; H, 3.85; N, 4.95%.
13

ACCEPTED MANUSCRIPT
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (4-hydroxyphenyl)-amide (15).
1
Yield 80%, mp 215-216оС. H NMR (400 MHz, DMSO-d6): δ 3.76 (s, 3Н, ОCH3), 3.95 (s, 3Н,
ОCH3), 6.76 (d, 2H, J = 8.7 Hz, arom.), 7.47 (d, 2H, J = 8.5 Hz, arom.), 7.67 (d, 1Н, J = 8.6 Hz,
13
6-H), 7.72 (d, 1Н, J = 8.6 Hz, 5-H), 8.12 (s, 1Н, 4-Н), 10.28 (s, 1H, NH). C NMR (100 MHz,
DMSO-d6): δ 184.7, 161.4, 155.3, 154.8, 147.9, 131.6, 131.2, 130.4, 129.6, 124.7, 123.2, 123.0,
120.1, 115.8, 61.4, 57.1. Calcd. for C18H15NO5S: C, 60.49; H, 4.23; N, 3.92; Found: C, 60.30; H,
4.35; N, 3.75%.
1-Oxo-1H-isothiochromene-3-carboxylic acid (4-sulfamoylphenyl)-amide (16). Yield 94%, mp
>240оС. 1H NMR (400 MHz, DMSO-d6): δ 7.20 (s, 2Н, NH2), 7.76-7.81 (m, 1H, arom.), 7.82 (d,
2H, J = 9.2 Hz, arom.), 7.90 (d, 2Н, J = 9.2 Hz, arom.), 7.90-7.95 (m, 2Н, arom.), 8.23 (d, 1Н, J
= 8.0 Hz, 8-Н), 8.31 (s, 1Н, 4-Н), 10.82 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.6,
162.0, 141.5, 139.8, 136.4, 135.4, 133.8, 132.7, 131.8, 128.6, 127.0, 125.7, 125.5, 120.6. Calcd.
for C16H12N2O4S2: C, 53.32; H, 3.36; N, 7.77; Found: C, 53.55; H, 3.25; N, 7.90%
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (4-sulfamoylphenyl)-amide (17).
1
Yield 90%, mp >240оС. H NMR (400 MHz, DMSO-d6): δ 3.79 (s, 3Н, ОCH3), 3.97 (s, 3Н,
ОCH3), 7.17 (s, 2Н, NH2), 7.64 (br.s, 2Н, 5-Н, 6-H), 7.80 (d, 2H, J = 8.8 Hz, arom.), 7.87 (d, 2Н,
13
J = 8.8 Hz, arom.), 8.15 (s, 1Н, 4-Н), 10.63 (s, 1H, NH). C NMR (100 MHz, DMSO-d6): δ
184.5, 162.3, 155.7, 147.9, 141.9, 139.9, 130.9, 130.7, 129.9, 127.3, 126.0, 123.3, 120.8, 120.1,
61.5, 57.1. Calcd. for C18H16N2O6S2: C, 51.42; H, 3.84; N, 6.66; Found: C, 51.55; H, 3.95; N,
6.55%.
1-Oxo-1H-isothiochromene-3-carboxylic acid (3-trifluoromethylphenyl)-amide (18). Yield 60%,
mp 195-197оС. 1H NMR (400 MHz, DMSO-d6): δ 7.36 (d, 1H, J = 8.6 Hz, arom.), 7.50 (t, 1H, J
= 8.2 Hz, 6-H), 7.70 (m, 1H, 7-H), 7.84 (m, 2Н, arom.), 8.11 (m, 2Н, arom.), 8.20 (s, 1Н, 4-Н),
8.24 (d, 1Н, J = 8.0 Hz, 8-Н), 10.52 (s, 1Н, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.5,
162.0, 139.3, 136.4, 135.4, 133.7, 132.7, 131.8, 130.5, 129.8 (q, J = 272.0 Hz), 128.6, 127.3 (q, J
= 32.0 Hz), 125.7, 125.4, 124.4, 121.1 (q, J = 3.4 Hz), 117.0 (q, J = 3.0 Hz). Calcd. for
C17H10F3NO2S: C, 58.45; H, 2.89; N, 4.01; Found: C, 58.60; H, 3.05; N, 4.20%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (3-trifluoromethylphenyl)-amide
(19). Yield 70%, mp 210-212оС. 1H NMR (400 MHz, DMSO-d6): δ 3.76 (s, 3Н, ОCH3), 3.96 (s,
3Н, ОCH3), 7.49 (d, 1H, J = 7.2 Hz, arom.), 7.63 (t, 1H, J = 7.9 Hz, arom.), 7.69 (d, 1H, J = 8.8
14

ACCEPTED MANUSCRIPT
Hz, 6-H), 7.73 (d, 1H, J = 8.8 Hz, 5-H), 8.00 (d, 2Н, J = 7.4 Hz, arom.), 8.14 (s, 1H, arom.), 8.21
13
(s, 1Н, 4-Н), 10.77 (s, 1H, NH). C NMR (100 MHz, DMSO-d6): δ 184.4, 162.3, 155.7, 148.0,
139.8, 131.0, 130.8, 130.7, 130.1 (q, J = 32.3 Hz), 129.9, 125.9, 125.1 (q, J = 273.0 Hz), 124.6,
123.3, 121.2 (q, J = 4.7 Hz), 120.1, 117.2 (q, J = 3.7 Hz), 61.44, 57.13. Calcd. for
C19H14F3NO4S: C, 55.74; H, 3.45; N, 3.42; Found: C, 55.90; H, 3.65; N, 3.12%.
4-[(1-Oxo-1H-isothiochromene-3-carbonyl)-amino]-benzoic acid ethyl ester (20). Yield 70%,
1
mp 194-196оС. H NMR (400 MHz, DMSO-d6): δ 1.33 (t, 3H, J = 7.0 Hz, OCH2CH3), 4.29 (q,
2Н, J = 7.0 Hz, OCH2CH3), 7.80-7.90 (m, 1H, 6-Н), 7.94-8.03 (m, 2Н, arom.), 8.23 (d, 1Н, J =
7.8 Hz, 8-Н), 8.34 (s, 1Н, 4-Н), 7.89 (d, 2H, J = 9.0 Hz, arom.), 8.00 (d, 2H, J = 9.0 Hz, arom.),
10.94 (s, 1Н, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.9, 165.9, 162.3, 143.2, 136.7, 136.0,
134.1, 133.0, 132.1, 130.8, 128.9, 125.9, 125.8, 120.5, 61.3, 14.9. Calcd. for C19H15NO4S: C,
54.58; H, 4.28; N, 3.96; Found: C, 54.70; H, 4.12; N, 4.15%.
4-[(7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carbonyl)-amino]-benzoic acid ethyl ester (21).
1
Yield 85%, mp 219-221оС. H NMR (400 MHz, DMSO-d6): δ 1.33 (t, 3H, J = 6.8 Hz,
OCH2CH3), 3.76 (s, 3Н, ОCH3), 3.96 (s, 3Н, ОCH3), 4.30 (q, 2H, J = 6.8 Hz, OCH2CH3), 7.69-
7.74 (m, 2H, 5-Н, 6-H), 7.86 (d, 2Н, J = 8.5 Hz, arom.), 7.98 (d, 2H, J = 8.6 Hz, arom.), 8.22 (s,
1Н, 4-Н), 10.74 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 184.4, 165.9, 162.3, 155.7,
147.9, 143.4, 131.0, 130.9, 130.8, 129.9, 126.0, 125.7, 123.3, 120.4, 120.1, 61.4, 61.2, 57.1,
14.9. Calcd. for C21H19NO6S: C, 61.01; H, 4.63; N, 3.39; Found: C, 60.85; H, 4.55; N, 3.24%.
1-Oxo-1H-isothiochromene-3-carboxylic acid naphthalen-1-ylamide (22). Yield 87%, mp 199-
1
201оС. H NMR (400 MHz, DMSO-d6): δ 7.54-7.64 (m, 4H, arom.), 7.81-7.86 (m, 1H, arom.),
7.90 (d, 1H, J = 8.2 Hz, arom.), 7.98-8.07 (m, 4H, arom.), 8.24 (d, 1H, J = 8.3 Hz, 8-H), 8.53 (s,
1Н, 4-Н), 10.85 (s, 1Н, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.5, 162.2, 136.8, 134.7,
134.4, 134.1, 133.1, 132.4, 131.0, 129.3, 128.8, 128.4, 126.9, 126.3, 126.2, 125.7, 124.8, 124.0,
123.5. Calcd. for C20H13NO2S: C, 72.49; H, 4.23; N, 3.95; Found: C, 72.60; H, 4.38; N, 3.75%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid naphthalen-1-ylamide (23). Yield
1
81%, mp >240оС. H NMR (400 MHz, DMSO-d6): δ 3.81 (s, 3Н, ОCH3), 3.98 (s, 3Н, ОCH3),
7.50-7.62 (m, 4H, arom.), 7.72 (br.s, 2Н, arom.), 7.88 (d, 2Н, J = 7.9 Hz, arom.), 7.98-8.05 (m,
2H, arom.), 8.33 (s, 1Н, 4-Н), 10.53 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 184.6,
162.9, 155.5, 147.9, 134.4, 133.5, 131.2, 130.9, 129.8, 129.6, 128.8, 127.4, 127.0, 126.9, 126.3,
15

ACCEPTED MANUSCRIPT
125.6, 124.5, 123.9, 123.4, 120.1, 61.5, 57.1. Calcd. for C22H17NO4S: C, 67.50; H, 4.38; N, 3.58;
Found: C, 67.72; H, 4.25; N, 3.40%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (4-fluorophenyl)-amide (24). Yield
1
77%, mp >240оС. H NMR (400 MHz, DMSO-d6): δ 3.79 (s, 3Н, ОCH3), 3.97 (s, 3Н, ОCH3),
7.11 (t, J = 8.8 Hz, 2H, arom.), 7.63 (br.s, 2Н, 5-Н, 6-H), 7.70-7.74 (m, 2Н, arom.), 8.10 (s, 1Н,
4-Н), 10.40 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): 184.0, 161.2, 159.2 (d, J = 221.0 Hz),
155.0, 135.0, 131.3, 131.0, 129.0, 124.6, 122.5 (d, J = 7.5 Hz), 119.1, 115.3 (d, J = 22.1 Hz),
60.9, 56.6. Calcd. for C18H14FNO4S: C, 60.16; H, 3.93; N, 3.90; Found: C, 60.30; H, 4.10; N,
4.05%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (4-chlorophenyl)-amide (25). Yield
1
62%, mp 231-233оС. H NMR (400 MHz, DMSO-d6): δ 3.79 (s, 3Н, ОCH3), 3.97 (s, 3Н,
ОCH3), 7.34 (d, 2H, J = 9.2 Hz, arom.), 7.63 (s, 2Н, 5-Н, 6-H), 7.74 (d, 2Н, J = 9.2 Hz, arom.),
13
8.10 (s, 1Н, 4-Н), 10.45 (s, 1H, NH). C NMR (100 MHz, DMSO-d6): δ 185.2, 161.8, 155.3,
154.9, 147.8, 131.8, 131.4, 130.5, 129.7, 125.0, 123.2, 123.0, 119.7, 115.8, 61.0, 57.2. Calcd. for
C18H14ClNO4S: C, 57.53; H, 3.75; N, 3.73; Found: C, 57.60; H, 3.90; N, 3.95%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (2-trifluoromethylphenyl)-amide
(26). Yield 65%, mp 199-201оС. 1H NMR (400 MHz, DMSO-d6): δ 3.77 (s, 3Н, ОCH3), 3.96 (s,
3Н, ОCH3), 7.50-7.62 (m, 2H, arom.), 7.64-7.85 (m, 4Н, arom.), 8.15 (s, 1Н, 4-Н), 10.40 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6): δ 184.0, 162.7, 155.3, 147.6, 135.1, 133.7, 131.6, 130.64,
129.9, 129.5, 128.4, 127.0 (q, J = 30 Hz), 127.0 (q, J = 3.0 Hz), 126.7, 125.4, 123.0, 119.8, 61.1,
56.8. Calcd. for C19H14F3NO4S: C, 55.74; H, 3.45; N, 3.42; Found: C, 55.85; H, 3.60; N, 3.20%.
1-Oxo-1H-isothiochromene-3-carboxylic acid thiazol-2-ylamide (27). Yield 70%, mp >240оС.
1H NMR (400 MHz, DMSO-d6): δ 7.25 (br.s, 1H, thiaz.), 7.56 (d, 1H, J = 3.6 Hz, thiaz.), 7.80
(m, 1H, arom.), 7.96 (m, 2Н, 5-H, 6-H), 8.20 (d, 1Н, J = 7.8 Hz, 8-Н), 8.46 (s, 1Н, 4-H), 13.27
(s, 1Н, NH). 13C NMR (100 MHz, DMSO-d6): δ 187.3, 137.0, 135.5, 133.1, 131.9, 129.0, 126.4,
125.9, 113.8. Calcd. for C13H8N2O2S2: C, 54.15; H, 2.80; N, 9.72; Found: C, 54.30; H, 3.00; N,
9.90%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid thiazol-2-ylamide (28). Yield 80%,
mp >240оС. 1H NMR (400 MHz, DMSO-d6): δ 3.79 (s, 3H, ОCH3), 3.96 (s, 3Н, ОCH3), 7.09 (d,
16

ACCEPTED MANUSCRIPT
1H, J= 3.4 Hz, thiaz.), 7.45 (d, 1Н, J = 3.5 Hz, thiaz.), 7.58 (s, 2Н, 5-Н, 6-H), 8.32 (s, 1Н, 4-Н).
13C NMR (100 MHz, DMSO-d6): δ 184.2, 155.2, 147.7, 131.0, 129.5, 126.4, 123.3, 119.0, 113.3,
60.9, 56.6. Calcd. for C15H12N2O4S2: C, 51.71; H, 3.47; N, 8.04; Found: C, 51.80; H, 3.55; N,
7.95%.
1-Oxo-1H-isothiochromene-3-carboxylic acid (4-phenylthiazol-2-yl)-amide (29). Yield 75%, mp
>240оС. 1H NMR (400 MHz, DMSO-d6): δ 7.37 (m, 1H, arom.), 7.47 (t, 2H, J = 7.0 Hz, arom.),
7.73 (br.s, 1H, arom.), 7.83-7.86 (m, 1H, arom.), 7.93-8.05 (m, 4H, arom.), 8.23 (d, 1Н, J = 7.7
Hz, 8-Н), 8.65 (s, 1Н, 4-Н), 13.23 (s, 1Н, NH). 13C NMR (100 MHz, DMSO-d6): δ 186.8, 136.7,
135.8, 133.3, 132.4, 129.5, 129.0, 128.7, 127.3, 126.5, 126.00, 109.7. Calcd. for C19H12N2O2S2:
C, 62.62; H, 3.32; N, 7.69; Found: C, 62.50; H, 3.10; N, 7.50%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (4-phenylthiazol-2-yl)-amide (30).
1
Yield 75%, mp >240оС. H NMR (400 MHz, DMSO-d6): δ 3.78 (s, 3H, ОCH3), 3.96 (s, 3Н,
ОCH3), 7.35 (t, 1H, J = 7.0 Hz, arom.), 7.45 (t, 2H, J = 7.3 Hz, arom.), 7.64 (s, 1H, arom.), 7.68
(t, 2H, J = 8.7 Hz, arom.), 7.93 (d, 2H, J = 7.4 Hz, arom.), 8.47 (s, 1Н, 4-Н), 12.86 (s, 1Н, NH).
13C NMR (100 MHz, DMSO-d6): δ 184.3, 161.9, 158.7, 155.9, 149.9, 148.0, 134.8, 130.9, 130.2,
129.4, 128.6, 127.6, 126.5, 123.3, 120.0, 109.5, 61.4, 57.1. Calcd. for C21H16N2O4S2: C, 59.42;
H, 3.80; N, 6.60; Found: C, 59.50; H, 3.90; N, 6.50%.
1-Oxo-1H-isothiochromene-3-carboxylic acid (5-methylisoxazol-3-yl)-amide (31). Yield 87%,
1
mp 232-234оС. H NMR (400 MHz, DMSO-d6): δ 2.43 (s, 3H, Me), 6.71 (s, 1H, =CH isoxaz.),
7.84 (t, 1H, J = 7.8 Hz, 6-Н), 7.93 (t, 1H, J = 7.8 Hz, 7-Н), 8.02 (d, 1Н, J = 8.0 Hz, 5-Н), 8.23
(d, 1Н, J = 8.0 Hz, 8-Н), 8.50 (s, 1Н, 4-Н), 11.77 (s, 1Н, NH). 13C NMR (100 MHz, DMSO-d6):
δ 186.5, 170.3, 161.6, 158.3, 136.3, 135.4, 132.9, 132.5, 132.0, 128.7, 126.6, 125.6, 97.1, 12.5.
Calcd. for C14H10N2O3S: C, 58.73; H, 3.52; N, 9.78; Found: C, 58.90; H, 3.65; N, 9.95%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (5-methylisoxazol-3-yl)-amide (32).
1
Yield 87%, mp 234-236оС. H NMR (400 MHz, DMSO-d6): δ 2.43 (s, 3H, Me), 3.78 (s, 3H,
ОCH3), 3.96 (s, 3Н, ОCH3), 6.68 (s, 1H, =CH isoxaz.), 7.55 (d, 1H, J = 8.8 Hz, 6-Н), 7.60 (d,
13
1Н, J = 8.8 Hz, 5-Н), 8.31 (s, 1Н, 4-Н), 11.45 (s, 1Н, NH). C NMR (100 MHz, DMSO-d6): δ
184.4, 170.5, 161.9, 158.7, 155.8, 147.9, 130.9, 130.1, 129.4, 127.2, 123.3, 120.1, 97.4, 61.5,
57.2, 12.8. Calcd. for C16H14N2O5S: C, 55.48; H, 4.07; N, 8.09; Found: C, 55.55; H, 4.19; N,
8.21%.
17

ACCEPTED MANUSCRIPT
1-Oxo-1H-isothiochromene-3-carboxylic acid (1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-amide (33). Yield 93%, mp 179-181оС. 1H NMR (400 MHz, DMSO-d6): δ 2.20 (s,
3Н, Me), 3.15 (s, 3Н, Me), 7.28 (t, 1H, J = 7.6 Hz, 6-H), 7.40 (d, 2H, J = 8.2 Hz, arom.), 7.46 (t,
2H, J = 8.2 Hz, arom.), 7.70 (t, 1H, J = 7.8 Hz, 7-H), 7.80-7.87 (m, 2Н, arom.), 8.22 (d, 1Н, J =
13
8.4 Hz, 8-Н), 8.33 (s, 1Н, 4-Н), 9.90 (s, 1H, NH). C NMR (100 MHz, DMSO-d6): δ 186.6,
161.8, 161.8, 152.7, 136.7, 135.3, 134.7, 133.7, 132.3, 131.0, 129.2, 128.8, 126.5, 125.6, 125.0,
123.9, 107.3, 36.3, 11.7. Calcd. for C21H17N3O2S: C, 64.44; H, 4.38; N, 10.73; Found: C, 64.55;
H, 4.45; N, 10.90%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (1,5-dimethyl-3-oxo-2-phenyl-2,3-
1
dihydro-1H-pyrazol-4-yl)-amide (34). Yield 93%, mp 220-221оС. HNMR (400 MHz, DMSO-
d6): δ 2.17 (s, 3Н, Me), 3.34 (s, 3Н, Me), 3.76 (s, 3H, ОCH3), 3.95 (s, 3Н, ОCH3), 7.34 (t, 1H, J
= 8.6 Hz, arom.), 7.37 (d, 2H, J = 8.2 Hz, arom.), 7.52 (t, 2H, J = 8.2 Hz, arom.), 7.63 (d, 1H, J =
8.8 Hz, 6-Н), 7.72 (d, 2Н, J = 8.8 Hz, 5-Н), 8.20 (s, 1Н, 4-Н), 9.85 (s, 1H, NH). 13C NMR (100
MHz, DMSO-d6): δ 184.2, 161.9, 155.0, 152.8, 147.7, 135.3, 131.0, 130.4, 129.2, 129.0, 126.4,
125.1, 123.8, 123.2, 119.1, 107.5, 60.9, 56.7, 36.4, 11.7. Calcd. for C23H21N3O5S: C, 61.19; H,
4.69; N, 9.31; Found: C, 61.30; H, 4.80; N, 9.20%.
1
3-(Morpholine-4-carbonyl)-isothiochromen-1-one (35). Yield 70%, mp 119-121оС. H NMR
(400 MHz, DMSO-d6): δ 3.60-3.70 (m, 8Н, 4 x СН2), 7.46 (br.s, 1H, 6-H), 7.66 (t, 1H, J = 8.2
Hz, 7-H), 7.78-7.87 (m, 2Н, 4-H, 5-H), 8.20 (d, 1Н, J = 8.2 Hz, 8-Н). 13C NMR (100 MHz,
DMSO-d6): δ 185.9, 164.0, 137.3, 135.5, 133.1, 132.4, 131.1, 128.0, 125.7, 122.6, 66.7, 48.1,
43.0. 13C NMR (100 MHz, DMSO-d6): δ 185.9, 164.0, 137.3, 135.5, 133.1, 132.4, 131.1, 128.0,
125.7, 122.6, 66.7, 48.1, 43.1. Calcd. for C14H13NO3S: C, 61.07; H, 4.76; N, 5.09; Found: C,
61.15; H, 4.60; N, 4.90%.
7,8-Dimethoxy-3-(morpholine-4-carbonyl)-isothiochromen-1-one (36). Yield 80%, mp 188-
190оС. 1HNMR (400 MHz, DMSO-d6): δ 3.60-3.70 (m, 8Н, 4 x СН2), 3.79 (s, 3H, ОCH3), 3.98
13
(s, 3Н, ОCH3), 7.30 (s, 1Н, 4-Н), 7.50 (s, 2Н, 5-Н, 6-H). C NMR (100 MHz, DMSO-d6): δ
183.4, 164.2, 154.8, 147.7, 131.5, 129.8, 129.0, 123.0, 122.6, 120.2, 66.7, 61.4, 57.1, 46.9, 43.9.
Calcd. for C16H17NO5S: C, 57.30; H, 5.11; N, 4.18; Found: C, 57.45; H, 5.20; N, 4.25%.
18

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3-(Azepane-1-carbonyl)-7,8-dimethoxy-isothiochromen-1-one (37). Yield 60%, mp 101-103оС.
1H NMR (400 MHz, DMSO-d6): δ 1.55 (br.s, 4H, 2 x CH2), 1.69 (br.s, 4Н, 2 x СН2), 3.53 (br.s,
4H, 2 x CH2), 3.74 (s, 3H, ОCH3), 3.92 (s, 3Н, ОCH3), 7.40 (s, 1Н, 4-Н), 7.64 (d, 1H, J = 8.8
Hz, 6-H), 7.68 (d, 1Н, J = 8.7 Hz, 5-Н). 13C NMR (100 MHz, DMSO-d6): δ 182.9, 164.7, 154.2,
147.3, 131.5, 131.0, 128.5, 122.0, 121.1, 120.0, 61.1, 56.8, 49.6, 46.1, 29.2, 27.5, 26.9, 26.1.
Calcd. for C18H21NO4S: C, 62.23; H, 6.09; N, 4.03; Found: C, 62.30; H, 6.20; N, 4.15%.
1-Oxo-1H-isothiochromene-3-carboxylic acid (2-hydroxyethyl)-amide (38). Yield 62%, mp 178-
1
180оС. H NMR (400 MHz, DMSO-d6): δ 3.34 (q, 2H, СН2), 3.55 (q, 2Н, СН2), 4.81 (br.s, 1Н,
ОН), 7.80 (t, 1H, J = 7.8 Hz, 6-H), 7.88 (d, 1Н, J = 7.3 Hz, 5-Н), 7.97 (t, 1H, J = 7.8 Hz, 7-H),
8.16 (s, 1Н, 4-Н), 8.20 (d, 1Н, J = 8.2 Hz, 8-Н), 8.85 (br.s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6): δ 185.5, 162.0, 136.3, 134.6, 133.0, 131.8, 129.2, 129.0, 128.6, 125.7, 60.0, 42.9.
Calcd. for C12H11NO3S: C, 57.82; H, 4.45; N, 5.62; Found: C, 57.95; H, 4.60; N, 5.70%.
7,8-Dimethoxy-1-oxo-1H-isothiochromene-3-carboxylic acid (2-hydroxyethyl)-amide (39). Yield
1
60%, mp 182-184оС. H NMR (400 MHz, DMSO-d6): δ 3.30 (q, 2H, CH2), 3.50 (q, 2Н, СН2),
3.75 (s, 3Н, ОCH3), 3.94 (s, 3Н, ОCH3), 4.76 (t, 1Н, J = 4.2 Hz, ОН), 7.59 (d, 1H, J = 8.7 Hz, 6-
Н), 7.68 (d, 2Н, J = 8.7 Hz, 5-H), 7.98 (s, 1Н, 4-Н), 8.65 (t, 1H, J = 4.2 Hz, NH). 13C NMR (100
MHz, DMSO-d6): δ 184.3, 162.5, 154.7, 147.6, 131.3, 128.7, 123.6, 123.1, 119.0, 60.8, 60.0,
56.5, 42.7. Calcd. for C14H15NO5S: C, 54.36; H, 4.89; N, 4.53; Found: C, 54.50; H, 5.00; N,
4.60%.
4.3. Primary anticancer assay
Primary anticancer assay was performed on a panel of approximately sixty human tumor
cell lines derived from nine neoplastic diseases, in accordance with the protocol of the Drug
Evaluation Branch, National Cancer Institute, Bethesda [38-40]. Tested compounds were added
to the culture at a single concentration (10-5 M) and the cultures were incubated for 48 h. End
point determinations were made with a protein binding dye, sulforhodamine B (SRB). Results
for each tested compound were reported as the percent of growth of the treated cells when
compared to the untreated control cells. The percentage growth was evaluated
spectrophotometrically versus controls not treated with test agents. The cytotoxic and/or growth
19

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inhibitory effects of the most active selected compounds were tested in vitro against the full
panel of human tumor cell lines at concentrations ranging from 10-4 to 10-8 M. 48-h Continuous
drug exposure protocol was followed and an SRB protein assay was used to estimate cell
viability or growth.
Using absorbance measurements [time zero (Tz), control growth in the absence of drug
(C), and test growth in the presence of drug (Ti)], the percentage growth was calculated for each
drug concentration. 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.
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 is 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 parameters if
the level of activity was reached; however, if the effect was not reached or was excessive, the
value for that parameter was expressed as more or less than the maximum or minimum
concentration tested. The lowest values were obtained with the most sensitive cell lines.
4.4. Antitrypanosomal activity assay
Bloodstream forms of Trypanosoma brucei brucei strain 90-13 were cultured in HMI9
medium supplemented with 10% FCS at 37°C under an atmosphere of 5% CO2 [41]. In all
experiments, log-phase parasite cultures were harvested by centrifugation at 3,000 x g and
immediately used. Drug assays were based on the conversion of a redox-sensitive dye (resazurin)
20

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to a fluorescent product by viable cells [42]. Drug stock solutions were prepared in pure DMSO.
T. brucei bloodstream forms (105 cells/mL) were cultured in 96-well plates either in the absence
or in the presence of different concentrations of inhibitors in a final volume of 200 µL. After a
72-h incubation, resazurin solution was added in each well at the final concentration of 45 µM
and fluorescence was measured at 530 nm and 590 nm absorbance after a further 4-h incubation.
The percentage of inhibition of parasite growth rate was calculated by comparing the
fluorescence of parasites maintained in the presence of drug to that in the absence of drug.
DMSO was used as control.
4.4. Acute toxicity in vivo
The experiments were conducted on white male mice weighing 23-25 g. Compounds
were dissolved in saline solution (0.9% NaCl) with l-2 drops of Polysorbate 80 (Tween-80®).
After dissolution they were administered to mice via intraperitoneal route. The LD50 was
evaluated for 4 or 5 different doses each on 6 animals and calculated by the Litchfield-Wilcoxon
method [43].
Acknowledgements
We are grateful to Dr. V.L. Narayanan from Drug Synthesis and Chemistry Branch,
National Cancer Institute, Bethesda, MD, USA, for in vitro evaluation of anticancer activity.
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Scheme and table captions:
Scheme 1. Utilization of 5-ylidene-4-azolidinones in isothiocoumarins and related cores
synthesis.
Scheme 2. Synthesis of target isothiocoumarin-3-carboxylic acids derivatives. Reagents,
conditions: (i) 1. 30% NaOH (water solution), reflux 0.5h, 2. HCl conc.; (ii) 1. SOCl2, reflux
0.5h, 2. alcohol, reflux 1.0 h (3-8) or amino acid, AcOH, reflux 1.0 h (9-11) or amine, dioxane,
reflux 1.0 h (12-39).
Table 1. Anticancer activity (one dose primary assay) of synthesized compounds.
Table 2. Cytotoxic activity of the compounds (10-4–10-8 M) towards 60 cancer cell lines.
Table 3. Antitrypanosomal activity screening on the Tbb bloodstream forms at 10 and 1 µg/mL.
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Scheme 1.
COOH
S
R
A = COOH
O
O
O
A
OH
SH
O
NH
S
R
SH
A
O
R
X
A = OH
A = Ph
X= S, NH
R
COOH
O
H
N
HOOC
R
SH
O
S
S
COOH
A = NH2
N
N
H
N
H
H
O
O
COOH
O
X
O
N
COOH
R
R
Ph
X = O, NH
28