2-(Substituted phenyl)amino analogs of 1-methoxyspirobrassinol methyl ether: Synthesis and anticancer activity

DOI: 10.1016/j.bmc.2009.03.064

Source and publish data:

Bioorganic and Medicinal Chemistry p. 3698 - 3712 (2009)

Update date:2022-08-11

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Authors:

Kutschy, Peter

Salayova, Aneta

Curillova, Zuzana

Kozar, Tibor

Mezencev, Roman

Mojzis, Jan

Pilatova, Martina

Balentova, Eva

Pazdera, Pavel

Sabol, Marian

Zburova, Michaela

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Article abstract of DOI:10.1016/j.bmc.2009.03.064

New analogs of indole phytoalexin 1-methoxyspirobrassinol methyl ether have been designed by replacement of its 2-methoxy group with 2-(substituted phenyl)amino group. Synthesized by spirocyclization methodology, trans- and cis-diastereoisomers of target

Full text of DOI:10.1016/j.bmc.2009.03.064

Bioorganic & Medicinal Chemistry 17 (2009) 3698–3712

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

2-(Substituted phenyl)amino analogs of 1-methoxyspirobrassinol methyl

ether: Synthesis and anticancer activity

Peter Kutschy ^a,, Aneta Salayová , Zuzana Curillová , Tibor Kozár , Roman Mezencev , Ján Mojziš ,

Martina Pilátová ^d, Eva Balentová ^a, Pavel Pazdera ^e, Marián Sabol ^f, Michaela Zburová ^a

^aDepartment of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science, P.J. Šafárik University, Moyzesova 11, 040 01 Košice, Slovak Republic

^bDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Košice, Slovak Republic

^cGeorgia Institute of Technology, College of Science, School of Biology, 315 Ferst Drive, Atlanta, GA 30332, USA

^dDepartment of Pharmacology, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66 Košice, Slovak Republic

Centre for Syntheses at Sustainable Conditions and Their Management, Department of Chemistry, Masaryk University, Kotlárská 2, 611 37 Brno, Czech Republic

^fDepartment of Microbiology, Faculty of Medicine, P.J. Šafárik University, SNP 1, 040 66 Košice, Slovak Republic

a r t i c l e i n f o

a b s t r a c t

Article history:

New analogs of indole phytoalexin 1-methoxyspirobrassinol methyl ether have been designed by

replacement of its 2-methoxy group with 2-(substituted phenyl)amino group. Synthesized by spirocyc-

lization methodology, trans- and cis-diastereoisomers of target compounds were isolated and evaluated

as potential anticancer and antimicrobial agents. Their molecular geometries were reﬁned by ab initio

minimizations. Pharmacophore modeling and QSAR studies were performed in order to correlate their

molecular structure and biological activity.

Received 20 February 2009

Revised 25 March 2009

Accepted 25 March 2009

Available online 11 April 2009

Keywords:

Phytoalexins

Spiroindolines

Anticancer activity

QSAR

1. Introduction

While the anticancer properties of natural (S)-(ꢀ)-spirobrassinin

(5) have not yet been studied, the natural isomers of (R)-(+)-1-

Indole-derived sulfur-containing phytoalexins are structurally

unique natural products isolated from economically and dietary

important plants of the family Cruciferae (syn. Brassicaceae). Cru-

ciferous indole phytoalexins have been reviewed with regard to

their isolation, occurrence, synthesis, biosynthesis, biotransforma-

tion, and their biological activity and role in plant defenses.^1–5Be-

sides their antimicrobial properties, several indole phytoalexins

also exhibit anticancer activity.⁴Speciﬁcally, brassinin (1, Fig. 1),

cyclobrassinin (3), and ( )-spirobrassinin [( )-5] demonstrated

cancer chemopreventive activity in the 7,12-dimethyl-

benz[a]anthracene (DMBA)-induced precancerous lesions in

mouse mammary gland organ culture.⁶In addition, brassinin (1)

exhibited dose-dependent inhibition of DMBA-induced and TPA-

promoted skin tumorigenesis in CD-1 mice.⁷Cytotoxic effects

against various solid tumor and leukemia cell lines in vitro were

reported for brassinin (1),⁸( )-spirobrassinin [( )-5],^8,9camalexin

(4),¹⁰1-methoxyspirobrassinol (6, mixture of diastereoisomers

that easily isomerize owing to their hemiaminal structure¹¹),⁹

1-methoxybrassinin (2),⁹and ( )-1-methoxyspirobrassinin [( )-7)].⁹

methoxyspirobrassinin (7) and (2R,3R)-(ꢀ)-1-methoxyspirobrassi-

nol methyl ether [(ꢀ)-8] were recently obtained in their individual

enantiomeric forms and (2R,3R)-(ꢀ)-1-methoxyspirobrassinol

methyl ether [(ꢀ)-8] was found to be a more potent inhibitor of

Jurkat cell proliferation than its (2S,3S)-(+)-enantiomer [(+)-8]

and racemic ( )-8. On the other hand, in the case of 1-methoxy-

spirobrassinin (7) there was no difference in potencies between

the enantiomers, and both displayed overall weak activity against

Jurkat cells.¹²Lately, the trans- and cis-diastereoisomers¹³of

2-piperidyl analogs were evaluated on the NCI₆₀panel of human

cancer cell lines. Introduction of a piperidyl moiety resulted in en-

hanced antiproliferative activity with the most potent anticancer

effect exhibited by trans-( )-9 against leukemic cell line CCRF-

CEM with IC₅₀= 0.0263

mol ꢁ L^{ꢀ1 14}

Even though the spiroindoline structures are present in many

biologically attractive natural products,¹⁵the anticancer and anti-

microbial properties of 1-methoxyspiroindoline phytoalexins and

their analogs are still almost unexplored. For this reason, we chose

1-methoxyspirobrassinol methyl ether (8), as a lead compound and

designed its 2-(substituted phenyl)amino analogs via the replace-

ment of 2-methoxy by 2-(substituted phenyl)amino group. The

introduction of 2-(substituted phenyl)amino substituent enables

* Corresponding author. Tel.: +421 552341256; fax: +421 55 62 22124.

E-mail address: peter.kutschy@upjs.sk (P. Kutschy).

doi:10.1016/j.bmc.2009.03.064

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

3699

Figure 1. Structures of selected indole phytoalexins.

the tuning of physico-chemical properties and hence the biological

properties as well. Furthermore, both secondary amino and aro-

matic moieties appear very frequently in drugs and other biologi-

cally active compounds. In addition, compared to the natural

lead 8 with no hydrogen bond donor, the secondary amino group

introduces a H-bond donor into designed analogs that might be

beneﬁcial for drug-target interactions. We determined cytostatic/

cytotoxic and antimicrobial activities of the synthesized com-

pounds, identiﬁed molecular fragments responsible for anticancer

activities on different cell lines, and predicted topoisomerase II as

their prospective drug target.

and 11b) and isosteric 2-phenylamino (12a and 12b) derivatives

that afforded isolable diastereoisomers. Since the activity of 2-phe-

nylamino derivative was notably higher, in the next study substi-

tuted phenylamino groups were introduced into position 2 by

reaction of methoxyiminium intermediate B with substituted

anilines as nucleophiles. Selection of substituents was inspired

by the Topliss operational scheme¹⁹enabling the choice of the

substituents with expected higher and lower activity compared

to the parent unsubstituted lead 12 based on Hammett electronic,

Taft steric and Hantzsch lipophilic substituent parameters. Thus a

suitable series of compounds for a SAR study was obtained. In all

cases both diastereoisomers were isolated (Scheme 1) and their

2. Results and discussion

2.1. Chemistry

Table 1

Chemical shifts of H-2 proton in cis- and trans-diastereoisomers

Compound

Isomer

¹H NMR (ppm)

The dioxane dibromide mediated spirocyclization of 1-meth-

oxybrassinin (2) in dioxane developed in our laboratory¹⁶opened

a new way for synthesis of 1-methoxyspiroindoline phytoalexins

and various related derivatives. Starting 1-methoxybrassinin (2)

can be prepared in small quantities from unstable 1-methoxyin-

dole-3-ylmethylamine (obtained by the reduction of 1-methoxyin-

dole-3-carboxaldehyde oxime) by the reaction with CS₂and CH₃I

in a mixture of pyridine and triethylamine as a solvent in 60–

64% yield.^17,18If we performed the reaction with a quantity of

starting oxime over 120 mg, the yields were not higher than 40%.

Replacement of the basic solvent by methanol improved the over-

all yield to about 70%, shortened the reaction time from 24 h to

15 min and is suitable for a higher quantity of starting oxime. With

a sufﬁcient amount of 1-methoxybrassinin (2) available, syntheses

of new analogs of 1-methoxyspirobrassinol methyl ether can be

studied more conveniently since the pure 1-methoxybrasinin (2)

is a stable oily compound that can be stored for several months

without decomposition. Spirocylization of 1-methoxybrassinin

(2) with bromine in dry dichloromethane, which appeared to be

more suitable compared to dioxane,^13,14proceeds via sulfenyl bro-

mide A which after electrophilic cyclization produces 1-methoxy-

spiroindoleninium intermediate B. After trapping by nucleophiles

it afforded products 10a–21b (Scheme 1). In this way, the target

spiroindolines were obtained by efﬁcient electrophilic-nucleo-

philic difunctionalization of the indole moiety. First we studied

the synthesis of 2-methylamino derivative, isosteric to natural

product 8. In this case only the cis-isomer 10b was isolated as a

pure substance since the trans-diastereoisomer 10a owing to its

isomerization during the separation on alumina or silica gel was

obtained only as a mixture with cis-diastereoisomer in the ratio

4:1 in favor of trans-isomer. Next we prepared 2-phenoxy (11a

d(H-2)

d^a

12a

12b

13a

13b

13a

13b

14a

14b

15a

15b

16a

16b

17a

17b

18a

18b

18a

18b

19a

19b

20a

20b

21a

21b

10a^d

10b

11a

11b

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

5.38^b

5.19^b

5.31^b

5.13^b

5.22^c

4.95^c

5.31^b

5.12^b

5.15^c

4.89^c

5.27^c

4.98^c

5.19^c

4.92^c

5.41^b

5.24^b

5.16^c

4.89^c

5.36^b

5.20^b

5.35^c

5.04^c

5.30^c

5.01^c

4.44^c

4.05^c

5.78^c

5.49^c

4.94^c

4.62^c

0.19

0.18

0.27

0.19

0.26

0.29

0.27

0.17

0.27

0.16

0.31

0.29

0.39

0.29

0.32

d = d(H-2)_transꢀ d(H-2)_cis

CD₃COCD₃.

CDCl₃.

Measured in a mixture containing 25% of cis-diastereoisomer.

3700

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

Scheme 1.

Figure 2. NOESY interactions evidencing the diastereoisomeric structure.

ratio was determined by ¹H NMR spectra of crude reaction prod-

ucts obtained after dilution with dichloromethane, washing with

1 M HCl, brine, drying and evaporation of solvent. The ratio of dia-

stereoisomers was determined by integration of separated signals

corresponding to the H-2, H_a, H_b, OCH₃and SCH₃protons (for pro-

tons identiﬁcation see Fig. 2). The reaction of intermediate B with

used nucleophiles did not exhibit any diastereoselectivity indicat-

ing no difference in steric hindrance for approach of nucleophiles

under study from any side of the C@N double bond. Cooling of

the reaction mixture up to ꢀ20 °C did not change the diastereose-

lectivity. On the other hand, availability of both diastereoisomers

allowed sufﬁcient quantity for examination of biological activity.

Structure of individual diastereoisomers was unequivocally

proved by NMR studies, including 2D HSQC, HMBC, and NOESY

experiments. In the NOESY spectra of cis-diastereoisomers of

2-amino derivatives (Fig. 2) the expected interaction between H_b

and H-2 protons, whereas with trans-diastereoisomers the interac-

tion between H_band NH protons was observed (for full details see

experimental). 2-Phenoxy derivative 11b exhibited in its NOESY

spectrum the cross peak between H-2 and H_bprotons evidencing

its cis-diastereoisomeric structure (Fig. 2). The spectrum of com-

pound 11a did not show the interaction between H_band aromatic

protons of phenoxy group, which would conﬁrm its trans-conﬁgu-

ration. However, the interaction between H-2 and H_bwas also not

observed and thus the structure of trans-distereoisomer was

assigned to this product. Figure 3 illustrates the interatomic dis-

tances between the above mentioned hydrogens in energy-mini-

mized structures of 2-phenyamino derivatives 12a (trans-) and

12b (cis-).

Inspection of ¹H NMR spectra of 2-arylamino derivatives 12a–

21b revealed the signiﬁcant difference in chemical shifts between

H-2 protons of trans- and cis-diastereoisomers. In all cases the

d(H-2)_transappeared at lower ﬁeld compared to d(H-2)_cis

(Table 1). The difference in chemical shifts

d = d(H-2)_trans

ꢀ

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

3701

diastereoisomers were spotted 10–60 min before developing the

plate. In order to study this isomerization quantitatively the

4-methylphenyl (13a, 13b), 3,4-dichlorophenyl (18a, 18b) and

4-nitrophenyl (20a, 20b) derivatives were selected and their sepa-

rated pure cis- and trans-isomers dissolved in deuteriochloroform

in the presence of 10 mol % of para-toluenesulfonic acid (PTS).

The isomerization was monitored by ¹H NMR spectroscopy at

certain periods of time. It was found that both diastereoisomers

isomerize and after sufﬁcient time an equilibrium mixture is

formed with the cis-diastereoisomer prevailing. Both diastereoiso-

mers of 4-methylphenyl derivative (13a and 13b) achieved the

equilibrium after 24 h with the ratio cis:trans = 88:12. In the case

of cis-3,4-dichlorophenyl derivative 18b the equilibrium was

reached after 3 days (cis:trans = 68:32), whereas in the equilibrium

mixture obtained from the corresponding trans-isomer 18a, the

ratio of cis:trans was 73:27 after three days. With 4-nitrophenyl

derivatives 20a and 20b the isomerization was slowed down by

the electron accepting effect of the nitro group. Equilibrium was

not achieved even after three weeks and vast decomposition did

not allow completion of the experiment. Isomerization can be of

advantage if cis-diastereoisomer is required. It can be obtained

by isomerization of a crude reaction mixture preceding the chro-

matographic separation of isomers. Thus if the spirocyclization of

1-methoxybrassinin (2) was performed with 2 equiv of 4-methy-

laniline and the reaction time was prolonged from 5 min to

60 min, the excess of HBr liberated during the reaction caused

isomerization, and the cis-distereoisomer 13b was obtained in

65% isolated yield, compared to 42% afforded by standard proce-

dure (see experimental). Analogously, if crude product of a mixture

of diastereoisomers 13a and 13b was dissolved in dichloromethane

and 10 molar % of triﬂuoroacetic acid was added, cis-diastereoiso-

mer 13b was isolated in 67% yield after 24 h. It is supposed that

in acidic medium an amino group can be cleaved off with the for-

mation of 1-methoxyiminium intermediate B that subsequently

reacts with amine from both sides of C@N double bond with the

formation of both isomers (Scheme 2). When equilibrium is

achieved, the thermodynamically more stable diastereoisomer

prevails.

Figure 3. Molecular geometries of 12a (trans; grey carbons) and 12b (cis; green

carbons) resulting from DFT B3LYP 6-31^**minimizations. The green-boxed part

(upper right corner) illustrates the four most stable conformations of 12b (C1 being

the lowest-energy form), demonstrating the inﬂuence of the thiazoline ring on the

molecular shape (showed as semitransparent surfaces). The molecular shape of

conformations is inﬂuenced also by ﬂipping of the NOCH₃group (compare C1 vs C2,

C3 vs C4). The conformations and shapes of 12a and the protonized form of both

molecules resembles the shapes of 12b thus they were not incorporated into the

ﬁgure. The transition state structures are in the yellow-framed lower part of the

ﬁgure with superimposition of the energetically close IM forms with their

values (in kJ/mol). Selected distances between hydrogen atoms for the most stable

conformations of 12a and 12b are also indicated.

d(H-2)_cisfalls in the range of 0.16–0.39 ppm. The higher shielding

of the H-2 proton in cis-diastereoisomers is probably caused by

anisotropic shielding imposed by the C@N double bond of the

thiazoline moiety. This relation is also valid for 2-methylamino-

(10a, 10b), 2-phenoxy- (11a, 11b) as well as natural trans-[( )-

8a], and unnatural cis-[( )-8b] diastereoisomer of 1-methoxyspiro-

brassinol methyl ether. The chemical shift difference was observed

in deuterated chloroform or acetone as demonstrated for 4-meth-

ylphenyl (13a, 13b) and 3,4-dichlorophenyl (18a, 18b) derivatives.

Consequently the chemical shifts of H-2 protons can be used for

assignment of diastereoisomeric structure of 1-methoxyspiro-

brassinol methyl ether analogs without the study of Nuclear

Overhauser Effects (NOE).

High-level quantum chemical studies (DFT B3LYP with 6-31^**

basis set) on all diasteroisomers of 2-amino analogs proved higher

stability for the cis-isomers than for the trans-isomers. Both ﬁve-

membered heterocycles in the molecules under study exhibited

distinct nonplanar conformation, as illustrated in Figure 3 for the

model case of 12a and 12b. The nonplanar shape is caused by

sp³characters of the indoline C-2 and C-3 carbons and methoxy

substituted nitrogen. The pyramidal inversion of sp³nitrogen in

the indoline moiety (‘up’ and ‘down’ oscillation of the unshared

Whereas under neutral or basic conditions the synthesized

diastereoisomers are stable, during the separation by column chro-

matography the partial isomerisation of trans- to cis-isomers was

observed and conﬁrmed also on TLC plates when samples of pure

Scheme 2.

3702

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

pair and hence the methoxy group as well from one side of the ind-

oline ring to the other) introduces conformational ﬂexibility into

these molecules inﬂuencing the orientation of the arylamino

group. Although the overall molecular shape looks similar for C1,

C2, C3 and C4 conformations, the shape difference is more evident

when comparing the difference in upper (C1, C2) and lower (C3, C4)

conformations in Figure 3. The inﬂuence of the conformational

change of the thiazoline ring exhibits signiﬁcant effect on the

molecular shape. The left-turned thiazoline in the C1 and C2 con-

formations turns right in the C3 and C4 conformations. These forms

also remain present in the protonized species of the molecules (see

Scheme 2 for protonization). The most stable conformation for the

cis-isomer 12b is the left-turned C1 conformation, whereas the

most stable conformation of the trans-isomer 12a (shown in the

upper-left corner of Figure 3) has a right-oriented thiazoline ring.

The indoline part of the structures becomes fully planar in the

transition state of trans–cis interconversion. The corresponding

molecular structures for the transition state models, resulting from

the transition-state guess option of the ^JAGUARprogram²⁰with the

LST (linear synchronous transit) protocol are in the yellow framed

part of Figure 3 together with their relative energies and reaction

intermediate (IM-12abH⁺).

the neutral and down by 1% for the protonized forms. Solvation

calculated in this way has only minor effect on cis:trans equilibria

for the molecules under study.

2.2. Biological evaluation

2.2.1. Antiproliferative/cytotoxic activity

IC₅₀values of newly synthesized compounds and conventional

anticancer agents doxorubicin, etoposide and cisplatin on the pa-

nel of 6 human cancer cell lines are presented in Table 2. Average

potencies of these compounds across all cell lines (expressed as

averaged—log IC₅₀), similarity of their ﬁngerprints (growth inhibi-

tory proﬁles) to that of doxorubicin, etoposide and cisplatin, and

p-values for statistical signiﬁcance of the similarity are presented

in Table 3.

Among newly synthesized 2-amino analogs of 1-methoxyspiro-

brassinol methyl ether [(ꢀ)-8], eight compounds have their aver-

age potency higher by at least 50% than compound trans-( )-8a,

natural diastereoisomer and the more potent of two diastereoiso-

meric 1-methoxyspirobrassinol methyl ethers cis-( )-8b and

trans-( )-8a. All remaining 2-amino analogs display equal or

slightly higher average potency in comparison with compounds

cis-( )-8b and trans-( )-8a, while the other two 2-phenoxy com-

pounds, 11a and 11b, were found inactive on our panel. For this

reason, synthesis of 2-amino analogs of 1-methoxyspirobrassinol

methyl ether seems to be beneﬁcial for anticancer drug design.

In addition, comparison of potencies of newly synthesized com-

pounds and conventional anticancer agents demonstrates that

The cis:trans (12b:12a) equilibrium ratio based on all mini-

mized structures resulted 91% in favor of the cis-diastereoisomer.

The equilibrium of the protonized species was even more shifted

towards the cis-isomer, approaching 98%. When we reoptimized

the molecular geometries including the PBF (Poisson–Boltzmann)

solvation model the preference of cis-isomer went up by 2% for

Table 2

Antiproliferative activities of 1-methoxyspirobrassinol methyl ether and its analogs

SCH₃

OCH₃

trans-

cis-

Compound

Isomer

Cell line, IC₅₀

MDA-MB-231

mol ꢁ L^ꢀ1

)

Jurkat

MCF-7

HeLa

CCRF-CEM

A-549

10a

10b

11a

11b

12a

12b

13a

13b

14a

14b

15a

15b

16a

16b

17a

17b

18a

18b

19a

19b

20a

20b

OCH₃

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

trans-

cis-

—

30.2

57.4

NT^b

100

17.8

33.3

6.6

47.1

10.0

69.4

34.8

43.0

29.4

100

30.7

21.0

10.0

33.4

29.3

100

10.0

28.4

41.8

0.078

100

48.9

53.2

100

NHCH₃

OC₆H₅

NH–C₆H₅

100

58.0

100

28.5

100

22.6

100

34.1

100

88.0

100

66.8

100

0.5

100

62.0

100

78.7

100

64.0

100

20.7

100

20.7

35.5

100

31.1

33.6

100

31.9

25.8

36.8

100

23.8

54.9

100

81.5

100

76.8

22.3

44.8

21.8

15.4

81.5

55.7

100

50.0

34.9

81.9

0.2

100

31.8

100

64.7

45.5

100

15.5

10.0

75.8

36.0

100

37.4

22.6

100

28.7

54.8

54.5

100

54.0

74.3

0.09

4.4

100

42.6

100

73.9

82.0

100

22.3

85.9

21.1

24.7

35.1

100

30.2

100

46.6

1.0

NH-4-CH₃-C₆H₄

NH-3,4-di-CH₃–C₆H₃

NH-4-CH₃O–C₆H₄

NH-2-Cl–C₆H₄

NH-4-Cl–C₆H₄

NH-3,4-di-Cl–C₆H₃

NH-4-Br–C₆H₄

NH-4-NO₂-C₆H₄

NH-4-CF₃–C₆H₄

NH-4-CF₃-C₆H₄

21a

21b

Doxorubicin

Cisplatin

VP-16 (Etoposide)

0.2

14.7

21.2

1.9

12.2

14.3

11.4

10.9

7.7

3.9

1.2

1.1

Synthesized according the published procedure.¹²

The trans-diastereoisomer was not tested (NT) since it was obtained only in a mixture containing 25% of cis-distereoisomer 10b.

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

3703

Table 3

Similarity of growth inhibitory proﬁles of tested compounds on the panel of 6 cell lines, to the proﬁles of conventional anticancer agents doxorubicin, etoposide and cisplatin

(ꢀlog IC₅₀) values were calculated for any given compound across all cell lines; r—correlation coefﬁcient of a proﬁle to a selected reference proﬁle; p-value—statistical signiﬁcance

of correlation coefﬁcient tested with two-tailed t-test (NS: p > 0.05)

Compound

ꢀlog IC₅₀

mol ꢁ L^ꢀ1

)

Similarity to doxorubicin

Similarity to etoposide

Similarity to cisplatin

p-value

Doxorubicin

Etoposide

Cisplatin

18a

18b

21b

17a

21a

12b

19a

14a

15b

13b

17b

20b

14b

15a

16b

19b

13a

20a

16a

12a

10b

6.60

5.29

5.01

4.67

4.48

4.44

4.42

4.38

4.34

4.32

4.28

4.25

4.23

4.22

4.21

4.19

4.18

4.17

4.15

4.14

4.10

4.09

4.08

4.00

1.00

0.74

0.42

ꢀ0.10

0.10

ꢀ0.74

ꢀ0.69

0.42

0.84

ꢀ0.06

ꢀ0.02

0.57

0.58

0.73

0.52

0.57

0.77

0.75

0.47

ꢀ0.64

0.51

0.45

0.44

0.10

0.00

<0.0001

0.036

0.74

1.00

0.68

ꢀ0.33

0.14

ꢀ0.28

ꢀ0.90

0.12

0.84

0.48

0.46

0.83

0.62

0.96

0.59

0.95

0.89

0.53

ꢀ0.13

0.58

0.68

0.49

0.10

0.00

<0.0001

0.0145

0.0364

0.0409

0.0024

0.0037

0.0175

0.42

0.68

1.00

ꢀ0.71

ꢀ0.39

ꢀ0.26

ꢀ0.51

ꢀ0.36

0.40

<0.0001

0.33

0.49

0.79

ꢀ0.14

0.58

ꢀ0.16

ꢀ0.17

0.76

0.49

0.32

0.00

0.17

ꢀ0.08

ꢀ0.01

0.04

0.26

0.00

11a

11b

compounds 13b, 14b, 18b and 20b have potencies higher than or

comparable to cisplatin on Jurkat cells; both compounds 17a and

18a have higher potencies than etoposide and 18a has only

1.4 ꢁ lower potency than cisplatin on MDA-MB-231 cells; and

compound 21b, the most potent among all tested compounds on

A-549 cells, is about 2 ꢁ more potent than doxorubicin on these

cells. Similarly, the potency of compound 18a on A-549 cells was

found to be comparable to that of etoposide. These results also

are promising with regard to the possible future role of these com-

pounds in anticancer drug design and development.

Two-way ANOVA determined that overall variation of

(ꢀlog IC₅₀) values is caused by cell lines (13.87% of total variation,

p = 0.0002) and tested compounds (23.98% of total variation,

p = 0.0111). This ﬁnding indicates that cells from our panel respond

to tested compounds signiﬁcantly differently, and that there are

signiﬁcant differences between compounds in their potencies

that some non-speciﬁc cytotoxic effect is responsible for the activ-

ity of tested compounds.

Results of Fisher’s exact test demonstrate statistically signiﬁ-

cant association between cis:trans diastereoisomeric status of

tested compounds and their cytostatic/cytotoxic effects for

MCF-7 breast cancer cell line (p = 0.0052), but not for other em-

ployed cell lines (p > 0.05). In MCF-7 cells, all cis-diastereoisomers

were classiﬁed as inactive (IC₅₀> 90

trans-diastereoisomers demonstrated potency with IC₅₀< 90 lmol

mol ꢁ L^ꢀ1), while 6 out of 12

ꢁ L^ꢀ1. In contrast, a similar association was not found for MDA-

MB-231 cells that were also derived from human breast adenocar-

cinoma and have approximately the same overall chemosensitivity

as MCF-7 cells (average ꢀlog IC₅₀for all tested compounds except

for doxorubicin, etoposide and cisplatin on MCF-7 and MDA-

MB-231 cells is 4.09 and 4.12, respectively). The association

between cis:trans stereochemistry and activity classiﬁcation on

MCF-7 cells remains to be elucidated, and we speculate a possible

role for estrogen receptors that are present in MCF-7, but not in

MDA-MB-231 cells.

across cell lines. Friedman’s test for differences in mean (ꢀlog IC₅₀

)

values among cell lines across all synthesized compounds (not

including conventional anticancer agents) also determined signiﬁ-

cant differences among cell line responses (p < 0.0001; Friedman’s

statistics corrected for ties F = 35.227; rows = 25; columns = 6).

Dunn’s multiple comparisons post test identiﬁed signiﬁcant differ-

ences in responses between Jurkat and MCF-7 cells (p < 0.001),

Jurkat and MDA-MB-231 (p < 0.01), Jurkat and A-549 (p < 0.05),

and HeLa and MCF-7 cells (p < 0.05). As a result, statistical analysis

demonstrates signiﬁcantly higher overall sensitivity of Jurkat cells

to tested compounds compared to MCF-7, MDA-MB-231, and

A-549 cells, which is consistent with generally higher in vitro sen-

sitivity of hemopoietic cells compared to solid tumor cell lines.²¹

These differences may result from a number of factors, in addition

to the interaction with the target involved in the cytostatic/cyto-

toxic response. These factors include number of cell cycles during

the incubation period, drug uptake, efﬂux and metabolism.²²On

the other hand, few differences among cell lines would suggest,

Growth inhibitory proﬁles (ﬁngerprints) of compounds 12b,

15b, 16b, 17b and 19b against the panel of 6 cell lines are strongly

positively, and statistically signiﬁcantly correlated to the proﬁle of

etoposide (Table 3), an anticancer agent that acts via inhibition of

topoisomerase II.²³In general, growth inhibitory proﬁles of drugs

against diverse cancer cell lines correlate well with their mode of

action;²⁴therefore, our results suggest, that compounds 12b,

15b, 16b, 17b and 19b likely inhibit topoisomerase II as a part of

their anticancer mode of action. Interestingly, the proﬁle of

compound 17a, a diastereoisomer of compound 17b, is strongly

negatively and signiﬁcantly correlated with that of etoposide

(Fig. 4), which indicates a different mode of action for 17a. Thus,

the analysis of ﬁngerprints of the tested compounds reveals the

possibility of at least two different anticancer modes of action

within the set of synthesized 2-amino analogs of 1-methoxyspiro-

3704

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

Figure 4. Growth inhibitory proﬁles of compounds 12b, 15b, 16b, 17b, and 19b (empty bars), etoposide (gray bars) and 17a (black bars). Cell lines: I—Jurkat; II—MCF-7;

III—MDA-MB-231; IV—HeLa; V—CCRF-CEM; VI—A-549. Vertical axis: (ꢀlog IC₅₀) of a compound on a given cell line—averaged (ꢀlog IC₅₀) of that compound across all cell

lines.

brassinol methyl ether. In addition, our data demonstrate strong

positive and statistically signiﬁcant correlation of compound 12b

proﬁle with that of doxorubicin, whose mode of action also in-

cludes inhibition of topoisomerase II.²⁵There were no statistically

signiﬁcant correlations between the proﬁles of tested compounds

and cisplatin.

Hierarchical clustering applied to our chemosensitivity data is

presented in the form of a heat map with red, green and yellow col-

or for high, low and intermediate potency of a given compound and

cell line compared to the average potency of that compound across

all cell lines (Fig. 5). Drugs with the same modes of action or tar-

gets form clusters in this type of clustering²⁶and, consistent with

the proﬁle correlation analysis, our results display clustering of

compounds 12b, 16b, 17b, etoposide, and 19b. Overall, 3 large

clusters can be identiﬁed from the dendrogram: one cluster that

includes inactive compounds 11a and 11b, a second that contains

compounds 15a, 15b, 19a, 20a and 21b, and a third with all

remaining compounds. However, due to the complex pattern of

clustering within the third cluster, it is likely that it also includes

compounds that differ in their anticancer mode of action. Cluster-

ing of cell types is consistent with cell origin, and shows clear sep-

aration of leukemias from solid tumors and clustering of MCF-7

and MDA-MB-231 breast cancer cell lines.

2.2.2. Antimicrobial activity

Synthesized compounds expressed mild antibacterial activity

against selected bacterial strains. The most valuable ﬁnding is

the inhibition of Streptococcus pyogenes growth by compound

Figure 5. Heat map with hierarchical clustering for (ꢀlog IC₅₀)—averaged (ꢀlog IC₅₀) for a given compound across all cell lines. Values are visualized by color scheme with red

color for high, yellow color for intermediate and green color for low values.

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

3705

19b. In spite of its relatively low potency (diameter of inhibition

zone was 7 mm, diameter of the disc was 6 mm), the zone of inhi-

bition was clear and remained stable over the next two days. Sim-

ilarly, compounds 18a, 21a, 21b showed inhibitory activity against

Bacillus subtilis; however, the potencies of tested compounds were

also relatively low. Activity of tested compounds appears to be di-

rected against gram positive bacteria. Among all, the most potent

compound was 19b active against S. pyogenes. Compound 19b

could potentially serve as lead molecule for synthesis of more

effective agents against S. pyogenes, bacteria that causes suppura-

tive infections with sometimes serious and lethal complications,

including necrotizing fasciitis and streptococcal toxic shock-like

syndrome (STSS).

were then scored, clustered and visually analyzed with active mol-

ecules superposed. 3D QSAR models were then generated for the

most predictive hypotheses.

Figure 6 illustrates such models for all cell lines under study.

The blue cubes represent positive contribution to the activity

whereas the red cubes represent negative contribution decreasing

the activities. The presented models can be clustered into two

groups. In the ﬁrst group (Jurkat, CCRF-CEM and A-549) the substi-

tuted phenyl ring has a signiﬁcant impact on molecular activity,

assuming the Jurkat-related molecules being best aligned and

exhibiting simultaneously the best activities. These results reason-

ably correlate with the right part of Figure 5 illustrating heat map

clustering. The most active molecules for Jurkat have cis-conﬁgura-

tion with left oriented thiazoline ring. For CCRF-CEM model, both

the cis- and trans-isomers are equally present between the super-

posed molecules and the thiazoline fragment exhibits both the left

and the right orientation. The negative contribution of thiazoline to

the inhibition is still insigniﬁcant according to the number of red

boxes shown in this part of the molecule. This scenario is changed

for A-549 where only two active molecules are in cis-conﬁguration

and the majority of the active analogs are trans-isomers. In this cell

line, the importance of the thiazoline fragment is increasing in

both a positive, as well as a negative way.

The second group of cell lines (MDA-MB-231, HeLa, MCF-7) and

the molecules do not exhibit equivalent importance for the substi-

tution on the phenylamino moiety. The structures of investigated

compounds do not superpose so effectively and the MCF-7 3D

QSAR model is the only one where the right-oriented thiazoline

group might have a dominant effect on growth inhibition. Interest-

ingly, the protonization studies indicated that the thiazoline ring

might undergo conformational change from left to right orienta-

tion if the ammonium group is present in the molecule. According

to the 3D QSAR model, the substitution of the arylamino group is

equally disturbing as activity supporting for A-549, while for

MDA-MB-231 and MCF-7 cells the thiazoline fragment becomes

the most signiﬁcant part of the molecules improving their activity

proﬁle. The thiazoline part corresponds to the pharmacophore for

hydrophobic interactions and is probably involved in binding.

Despite the presence of a nitrogen atom in thiazoline ring, the

hydrogen-bond acceptor is not a part of the pharmacophore for

MCF-7 cells. This might be the reason why the studied molecules

provide the weakest activity proﬁle for the MCF-7 cell line in accor-

dance with Figure 5.

2.3. Structure–activity relationships

The Phase program of Schrodinger Inc.²⁷was used for pharma-

cophore modeling and 3D QSAR analysis of the studied com-

pounds. Although Phase is a relatively new method,²⁸according

to Evans and coworkers,²⁹it performed comparably or even better

than the well-established Catalyst HypoGen for their tested set of

compounds. We were aimed accordingly in Phase in order to gen-

erate pharmacophores for molecules synthesized in our laborato-

ries. The DFT B3LYP 6-31^**minimized structures were used as

input geometries for Phase to generate conformations and create

hypotheses according to activities in the second step. Hypotheses

3. Conclusion

Analogs of indole phytoalexin 1-methoxyspirobrassinol methyl

ether [(ꢀ)-8] that possess the substituted phenyl amino group in-

stead of the methoxy group in position 2 of indoline moiety have

been designed and synthesized as new prospective anticancer

compounds. Using the effective synthetic strategy based on tan-

dem electrophilic–nucleophilic 3,2-heterodifunctionalisation of in-

dole and following the Topliss operational scheme, a set of cis- and

trans-diastereoisomers of target compounds was synthesized. We

found that cis-isomers were thermodynamically more stable than

trans-isomers, which can be isomerized to the corresponding

cis-diastereoisomers in acidic medium. The observed signiﬁcant

differences in chemical shifts of the H-2 proton between cis- and

trans-diastereoisomers in ¹H NMR spectra can be used for the

reliable assignment of the diastereoisomeric structure of new

compounds without performing the NOE experiments.

Figure 6. Pharmacophore (pink circles correspond to proton-acceptor, light blue to

proton donor, green to hydrophobic groups and orange rings to aromatic fragments)

and 3D QSAR models (blue and red cubes) projected onto the superposed most

active compounds for the different cell lines. Blue cubes for the QSAR models

represent positive contribution to activity increase; red cubes determine negative

contribution decreasing the activity. The most active compounds were superim-

posed into the given pharmacophore models and their color-coding is shown

individually for each cell line. Cell lines: MCF7; MDA-MB-231 (MDA); HeLa; A-549;

CCRF-CEM (CEM); Jurkat.

We examined cytostatic/cytotoxic activity of synthesized com-

pounds against selected human solid tumor and leukemia cell lines

in vitro (Jurkat, MCF-7, MDA-MB-231, HeLa, CCRF-CEM and A-549).

Compounds 13b, 14b, 18b and 20b have potencies higher than or

comparable to cisplatin on Jurkat cells, compounds 17a and 18a

3706

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

have higher potencies than etoposide on MDA-MB-231 cells, and

compound 21b, that was found to be the most potent among all

tested compounds on A-549 cells, is about 2 ꢁ more potent than

doxorubicin on A-549 cells. These results are promising with re-

gard to the possible future role of these compounds in anticancer

drug development. The pharmacophore/3D QSAR modeling high-

lighted the molecular fragments of the lead molecules responsible

for activities on different cell lines. More targeted molecular design

on selected lead molecules taking into account all experience com-

ing from the 3D/pharmacophore modeling will be the subject of

our future studies.

tography on aluminum oxide 90 neutral, 0.063–0.200 mm, activity

I, Merck (15 g, diethyl ether/n-hexane 1:4) to give products 10a

(0.013 g, 12%, containing 25% of 10b as inseparable impurity) and

10b (0.029 g, 26%). trans-1-Methoxy-2-methylamino-2⁰-(meth-

ylsulfanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrothiazole} (10a). Colorless

oil, R_f= 0.11 (diethyl ether/n-hexane, 1:4). ¹H NMR (CDCl₃) d 2.55

(broad singlet, 1H, NH), 2.56 (s, 3H, SCH₃), 2.69 (s, 3H, N–CH₃),

3.90 (d, J = 15.5 Hz, 1H, H-4⁰), 3.93 (s, 3H, OCH₃), 4.44 (s, 1H, H-

2), 4.73 (d, J = 15.5 Hz, 1H, H-4⁰), 6.95 (d, J = 7.9 Hz, 1H, ArH), 7.01

(ddd, J = 1.0, 7.5, 7.5 Hz, 1H, ArH), 7.24 (ddd, J = 1.2, 7.6, 7.6 Hz,

1H, ArH), 7.30 (dd, J = 1.1, 7.5 Hz, 1H, ArH). ¹³C NMR (CDCl₃) d

15.3, 35.3, 64.1, 69.5, 69.8, 94.8, 113.0, 123.8, 123.9, 129.6, 129.8,

149.7, 165.4. cis-1-Methoxy-2-methylamino-2⁰-(methylsulfanyl)-

spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrothiazole} (10b). Colorless oil,

R_f= 0.19 (diethyl ether/n-hexane, 1:4). IR (CHCl₃) m_max3573,

4. Experimental

4.1. Chemistry

3100, 2307, 1700, 1393, 1113, 980, 787 cm^ꢀ^{1 1}H NMR (DMSO-

d₆) d 2.39 (broad singlet, 1H, NH), 2.52 (s, 3H, SCH₃), 2.63 (broad

singlet, 3H, N–CH₃), 3.86 (s, 3H, OCH₃), 4.08 (broad singlet, 1H,

H-2), 4.27 (d, J = 15.7 Hz, 1H, H-4⁰_b), 4.61 (d, J = 15.7 Hz, 1H, H-4_a⁰),

6.94 (d, J = 7.6 Hz, 1H, H-7), 7.00 (dd, J = 7.3, 7.4 Hz, 1H, H-5),

7.22–7.27 (m, 2H, H-4, H-6). ¹³C NMR (DMSO-d₆) d 14.5 (SCH₃),

34.1 (NCH₃), 63.1 (OCH₃), 70.5 (C-3), 72.7 (C-4⁰), 91.6 (C-2), 112.2

(C-7), 122.7 (C-4), 123.2 (C-5), 129.3 (C-6), 129.7 (C-3a), 149.1

(C-7a), 161.7 (C-2⁰). NOESY correlations: N–CH₃/H-2; NH/N–CH₃;

H-2/H-4⁰, N–CH₃; H-4⁰/H-2, H-4⁰; H-4⁰/H-4⁰, H-4; H-7/H-6; H-5/

Melting points were determined on a Kofﬂer micro melting

point apparatus and are uncorrected. IR spectra were recorded on

an IR-75 spectrometer (Zeiss Jena). ¹H NMR (400 MHz) and ¹³C

NMR (100 MHz) spectra were measured on a Varian Mercury Plus

spectrometer. Chemical shifts (d) are reported in ppm downﬁeld

from TMS as internal standard and coupling constants (J) are given

in hertz. Microanalyses were performed with a Perkin–Elmer,

Model 2400 analyzer. The EI mass spectra were recorded on a

GC–MS Trio 1000 (Fisons Instruments) spectrometer at ionization

energy of 70 eV, whereas MALDI-TOF mass spectra of 10b, 17a

and 17b were measured on a MALDI IV (Shimadzu, Kratos Analyt-

ical). The samples were ionized with a N₂-laser (k = 337 nm). The

progress of chemical reactions was monitored by thin layer chro-

matography, using Macherey-Nagel plates Alugram^ÒSil G/UV254.

Preparative column chromatography was performed on Kieselgel

60 Merck Type 9385 (0.040–0.063 mm) and aluminum oxide 90

neutral, Merck (0.063–0.200 mm, activity I).

H-4, H-6; H-4/H-4⁰_a, H-5; H-6/H-5, H-7. MALDI-TOF MS, m/z (rela-

tive intensity): 295.3 M⁺(84%), 264.7 (100%), 199.3 (26%), 160.7

(24%). Anal. Calcd for C₁₃H₁₇N₃OS₂requires: C, 52.85; H, 5.80; N,

14.22. Found: C, 52.71; H, 5.59; N, 14.02.

4.1.3. trans- and cis-1-Methoxy-2-phenoxy-2⁰-(methylsulfanyl)-

spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazoles} (11a and 11b)

a stirred solution of 1-methoxybrassinin (2, 0.08 g,

0.3 mmol) with powdered molecular sieves (3Å) in anhydrous

dichloromethane (5 mL) was added freshly prepared solution of

bromine (0.76 mL, 0.33 mmol, stock solution prepared by dissolv-

ing of 0.04 mL of Br₂in 1.76 mL of anhydrous dichloromethane).

After stirring for 5 min, the solution of phenol (0.056 g, 0.6 mmol),

triethylamine (0.306 g, 0.421 mL, 3 mmol) in dry dichloromethane

(5 mL) with powdered molecular sieves (3 Å) was added. Stirring

was continued for another 20 min, the reaction mixture diluted

with dichloromethane (25 mL), washed with 1 M HCl (6 mL) and

brine (15 mL). Organic layer was dried over anhydrous Na₂SO₄

and the residue obtained after evaporation of solvent was sub-

jected to chromatography on silica gel (23 g, hexane/acetone

5:1), affording the products 11a (0.022 g, 20%) and 11b (0.073 g,

68%). trans-1-Methoxy-2-phenoxy-2⁰-(methylsulfanyl)spiro{indo-

line-3,5⁰-[4⁰,5⁰]dihydrothiazole} (11a). Colorless oil; R_f= 0.45 (hex-

ane/acetone, 5:1). ¹H NMR (CDCl₃) d 2.52 (s, 3H, SCH₃), 3.87 (s, 3H,

OCH₃), 4.01 (d, J = 15.4 Hz, 1H, H-4⁰), 5.18 (d, J = 15.4 Hz, 1H, H-4⁰),

4.1.1. Improved procedure for the preparation of 1-

methoxybrassinin (2)

Crude product of (1-methoxyindole-3-yl)methylamine freshly

prepared by the reduction of 1-methoxyindole-3-caboxaldehyde

oxime (0.570 g, 3 mmol) with NaBH₃CN in the presence of TiCl₃

was dissolved in methanol (24 mL). Triethylamine (1.25 mL,

0.91 g, 9 mmol) and carbon disulﬁde (0.54 mL, 0.685 g, 9 mmol)

were added and mixture was stirred for 5 min at room tempera-

ture. Then iodomethane (0.56 mL, 1.277 g, 9 mmol) was added

and stirring was continued for 15 min. The residue obtained after

evaporation of the solvent was dissolved in dichloromethane, small

amount of silica gel was added, dichloromethane evaporated and

product preabsorbed on silica gel was chromatographed on silica

gel (45 g, hexane–acetone 5:1). Yield 0.61 g, 76%. Spectral data of

the obtained 1-methoxybrassinin (2) were identical with those de-

scribed previously.¹⁸

5.78 (s, 1H, H-2), 6.99 (dd, J = 7.8, 0.8 Hz, 1H, H-7), 7.06 (ddd,

J = 1.1, 7.6 Hz, 1H, H-5), 7.06 (dddd, J = 1.1, 1.2, 7.3, 7.3 Hz, 1H, H-

4⁰⁰), 7.18 (dd, J = 1.1, 8.7 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 7.29 (ddd, J = 1.2, 7.7,

7.7 Hz, 1H, H-6), 7.32 (dd, J = 7.3, 8.6 Hz, 2H, H-3⁰⁰, H-5⁰⁰), 7.35

(dd, J = 1.2, 7.6 Hz, 1H, H-4). ¹³C NMR (CDCl₃) d 15.1 (SCH₃), 64.0

(OCH₃), 69.3 (C-3), 70.2 (C-4⁰), 107.3 (C-2), 113.0 (C-7), 117.8 (C-

2⁰⁰,C-6⁰⁰), 122.9 (C-4⁰⁰), 123.9 (C-4), 124.0 (C-5), 127.0 (C-3a),

129.6 (C-3⁰⁰, C-5⁰⁰), 129.9 (C-6), 147.3 (C-7a), 1₀58.2 (C-1⁰⁰)₀, 163.5

(C-2⁰). NOESY correlations: OCH₃/H-7; H-4⁰_a/H-4_b, H-4; H-4_b/H-4⁰_a;

H-2/H-2⁰⁰, H-6⁰⁰; H-7/OCH₃, H-6; H-5/H-6, H-4; H-4⁰⁰/H-3⁰⁰, H-5⁰⁰;

H-2⁰⁰, H-6⁰⁰/H-2, H-3⁰⁰, H-5⁰⁰; H-6/H-7, H-5; H-3⁰⁰, H-5⁰⁰/H-4⁰⁰, H-2⁰⁰,

H-6⁰⁰; H-4/H-4⁰_a, H-5. EIMS m/z (relative intensity): 359 M⁺(5%),

265 (100%), 251 (76%), 161 (35%), 160 (25%), 117 (52%). Anal. Calcd

for C₁₈H₁₈N₂O₂S₂requires: C, 60.31; H, 5.06; N, 7.81. Found: C,

60.03; H, 5.40; N, 7.53. cis-1-Methoxy-2-phenoxy-2⁰-(methylsulfa-

nyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrothiazole} (11b). Colorless oil;

4.1.2. trans- and cis-1-Methoxy-2-methylamino-2⁰-(methylsulf-

anyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazoles} (10a and 10b)

stirred solution of 1-methoxybrassinin (2, 0.1 g,

0.375 mmol) with powder molecular sieves (3 Å) in anhydrous

dichloromethane (4 mL) at room temperature was added freshly

prepared solution of bromine (0.96 mL, 0.413 mmol, stock solution

prepared by dissolving of 0.04 mL of Br₂in 1.76 mL of anhydrous

dichloromethane). After stirring for 5 min, a mixture of methyl-

amine hydrochloride (0.051 g, 0.75 mmol, dried at 75 °C for

90 min) and triethylamine (0.721 g, 1.0 mL, 7.125 mmol) in anhy-

drous dichloromethane (5 mL) was added. After being stirred for

5 min, the reaction mixture was diluted with dichloromethane

(10 mL), successively washed with 1 M HCl (10 mL), water

(20 mL), and brine (20 mL), and dried over Na₂SO₄. The residue ob-

tained after solvent removal in vacuum was submitted to chroma-

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

3707

R_f= 0,35 (hexane/acetone, 5:1). ¹H NMR (CDCl₃) d 2.54 (s, 3H,

IR (CHCl₃) m_max3400, 2927, 1700, 1593, 1460, 1400, 1120, 987,

933, 733, 573 cm^ꢀ^{1 1}H NMR (acetone-d₆) d 2.51 (s, 3H, SCH₃),

SCH₃), 3.88 (s, 3H, OCH₃), 4.31 (d, J = 15.3 Hz, 1H, H-4⁰), 4.44 (d,

J = 15.3 Hz, 1H, H-4⁰_a), 5.49 (s, 1H, H-2), 7.00 (dd, J = 1.0, 8.1 Hz,

1H, H-7), 7.06 (ddd, J = 1.0, 7.6, 7.6 Hz, 1H, H-5), 7.07 (dddd,

J = 1.1, 1.1, 7.3, 7.3 Hz, 1H, H-4⁰⁰), 7.18 (dd J = 1.0, 8.7 Hz, 2H, H-

2⁰⁰, H-6⁰⁰), 7.31 (dd, J = 1.2, 7.4 Hz, 1H, H-4), 7.31 (ddd, J = 1.1, 7.6,

8.0 Hz, 1H, H-6), 7.32 (dd, J = 7.3, 8.7 Hz, 1H, H-3⁰⁰, H-5⁰⁰). ¹³C

NMR (CDCl₃) d 15.1 (SCH₃), 63.9 (OCH₃), 70.7 (C-3), 72.6 (C-4⁰),

103.3 (C-2), 112.7 (C-7), 118.0 (C-2⁰⁰, C-6⁰⁰), 123.1 (C-4⁰⁰), 123.2

(C-4), 124.0 (C-5), 127.8 (C-3a), 129.6 (C-3⁰⁰, C-5⁰⁰), 130.0 (C-6),

147.5 (C-7a), 158.3₀(C-1⁰⁰), 166.31 ₀(C-2⁰). NOESY ₀correlations:

OCH₃/H-7; H-4_a⁰/H-4_b, H-4; H-4_b⁰/H-4_a, H-2; H-2/H-4_b, H-2⁰⁰, H-6⁰⁰;

H-7/OCH₃, H-6; H-5/H-6, H-4; H-4⁰⁰/H-3⁰⁰, H-5⁰⁰; H-2⁰⁰, H-6⁰⁰/H-2,

H-3⁰⁰, H-5⁰⁰; H-4/H-4_a⁰, H-5; H-6/H-7, H-5; H-3⁰⁰, H-5⁰⁰/H-4⁰⁰, H-2⁰⁰,

H-6⁰⁰. EIMS m/z (relative intensity): 359 M⁺(5%), 265 (100%), 251

(76%), 161 (35%), 160 (25%), 117 (52%). Anal. Calcd for

C₁₈H₁₈N₂O₂S₂requires: C, 60.31; H, 5.06; N, 7.81. Found: C,

60.60; H, 4.88; N, 8.05.

3.72 (s, 3H, OCH₃), 4.33 (d, J = 15.8 Hz, 1H, H-4⁰_b), 4.72 (d,

J = 15.8 Hz, 1H, H-4⁰_a), 5.19 (d, J = 11.0 Hz, 1H, H-2), 5.32 (d,

J = 11.0 Hz, 1H, NH), 6.73 (dddd, J = 1.1, 1.1, 7.4, 7.4 Hz, 1H, H-4⁰⁰),

6.98 (dd, J = 1.1, 8.3 Hz, 1H, H-7), 7.05 (dd, J = 1.1, 8.6 Hz, 2H, H-

2⁰⁰, H-6⁰⁰), 7.06 (ddd, J = 1.1, 7.4, 7.6 Hz, 1H, H-5), 7.17 (dd, J = 7.3,

8.6 Hz, 2H, H-3⁰⁰, H-5⁰⁰), 7.30 (ddd, J = 1.2, 7.4, 7.5 Hz, 1H, H-6),

7.31 (d, J = 7.5 Hz, 1H, H-4). ¹³C NMR (acetone-d₆) d 15.0 (SCH₃),

64.2 (OCH₃), 72.1 (C-3), 73.4 (C-4⁰), 86.4 (C-2), 113.8 (C-7), 115.3

(C-2⁰⁰, C-6⁰⁰), 119.4 (C-4⁰⁰), 123.6 (C-4), 124.5 (C-5), 129.9 (C-3⁰⁰, C-

5⁰⁰), 130.3 (C-3a), 130.5 (C-6), 147.7 (C-1⁰⁰), 149.8 (C-7a), 163.0

(C-2⁰). NOESY correlations: OCH₃/H-7; H-4_b⁰/H-4_a⁰, H-2; H-4_a⁰/H-4⁰_b,

H-4; H-2/H-4_b⁰, NH, H-2⁰⁰, H-6⁰⁰; NH/H-2, H-2⁰⁰, H-6⁰⁰; H-4⁰⁰/H-3⁰⁰,

H-5⁰⁰; H-7/OCH₃, H-6; H-2⁰⁰, H-6⁰⁰/H-2, NH, H-3⁰⁰, H-5⁰⁰; H-5/H-6,

H-4; H-3⁰⁰, H-5⁰⁰/H-4⁰⁰, H-2⁰⁰, H-6⁰⁰; H-6/H-7, H-5; H-4/H-4⁰_a, H-5.

EIMS m/z (relative intensity) 358 M⁺(1%), 326 (100%), 265 (12%),

233 (12%), 161 (20%), 104 (26%), 77 (47%). MALDI-TOF MS, m/z

(relative intensity): 396.7 [M+K]⁺(9%), 380.7 [M+Na]⁺(20%),

357.9 [M+H]⁺(16%), 326.4 (50%), 283.3 (26%), 265.8 (100%),

222.2 (42%), 171.5 (33%), 161.6 (28%), 108.3 (50%). Anal. Calcd for

C₁₈H₁₉N₃OS₂requires: C, 60.47; H, 5.36; N, 11.75. Found: C,

60.71; H, 5.65; N, 11.99.

4.1.4. General procedure for the synthesis of trans- and cis-

diastereoisomers of 2-(substituted phenyl)amino analogs of 1-

methoxyspirobrassinol methyl ether (12a–21a and 12b–21b)

stirred solution of 1-methoxybrassinin (2, 0.1 g,

0.375 mmol) in anhydrous dichloromethane (4 mL) at room tem-

perature was added freshly prepared solution of bromine

(0.96 mL, 0.413 mmol, stock solution prepared by dissolving of

0.04 mL of Br₂in 1.76 mL of anhydrous dichloromethane). After

stirring for 5 min, a solution of corresponding substituted aniline

4.1.4.2. trans- and cis-1-Methoxy-2-(4-methylphenylamino)-2⁰-

(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole}

(13a and 13b). Following the general procedure products 13a and

13b were obtained using 0.362 g (3.375 mmol) of 4-methylaniline

and separated on silica gel (20 g, ethyl acetate/n-hexane 1:8).

trans-1-Methoxy-2-(4-methylphenylamino)-2⁰-(methylsulphanyl)-

spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole} (13a). Yield: 0.035 g (25%);

colorless crystals, mp 155–157 °C (acetone–hexane); R_f= 0.18

(ethyl acetate/n-hexane, 1:8). IR (CHCl₃) m_max3420, 3010, 1610,

(0.75–3.375 mmol)

and

triethylamine

(0.721 g,

1.0 mL,

7.125 mmol) in anhydrous dichloromethane (5 mL) was added.

After being stirred for 5 min, the reaction mixture was diluted with

dichloromethane (10 mL), successively washed with 1 M HCl

(10 mL), water (20 mL), and brine (20 mL), and dried over Na₂SO₄.

The residue obtained after solvent removal in vacuum was submit-

ted to chromatography on silica gel to give products 12a–21a and

12b–21b.

1560, 1510, 1460, 1400, 1290, 1105, 995, 960, 805, 620 cm^ꢀ^{1 1}H

NMR (acetone-d₆) d 2.20 (s, 3H, CH₃), 2.48 (s, 3H, SCH₃), 3.78 (s,

3H, OCH₃), 3.88 (d, J = 15.6 Hz, 1H, H-4⁰), 4.91 (d, J = 15.6 Hz, 1H,

H-4⁰_b), 5.31 (d, J = 10.2 Hz, 1H, H-2), 5.45 (d, J = 10.2 Hz, 1H, NH),

6.87 (d, J = 8.4 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 6.97 (d, J = 8.4 Hz, 2H, H-3⁰⁰, H-

5⁰⁰), 6.99 (d, J = 7.8 Hz, 1H, H-7), 7.05 (ddd, J = 1.0, 7.5, 7.5 Hz, 1H,

H-5), 7.28 (ddd, J = 1.2, 7.6, 7.8 Hz, 1H, H-6), 7.34 (dd, J = 1.1,

7.6 Hz, 1H, H-4). ¹³C NMR (acetone-d₆) d 15.0 (SCH₃), 20.5 (CH₃),

64.2 (OCH₃), 70.5 (C-3), 70.9 (C-4⁰), 90.7 (C-2), 114.0 (C-7), 115.4

(C-2⁰⁰, C-6⁰⁰), 124.4 (C-5), 124.6 (C-4), 127.9 (C-4⁰⁰), 129.0 (C-3a),

130.3 (C-6), 130.3 (C-3⁰⁰, C-5⁰⁰), 146.2 (C-1⁰⁰), 150.1 (C-7a), 163.6

(C-2⁰). NOESY correlations: CH₃/H-3⁰⁰, H-5⁰⁰; H-4⁰_a/H-4_b⁰, H-4; H-4⁰_b/

H-4_a⁰, NH; H-2/NH, H-2⁰⁰, H-6⁰⁰; NH/H-4⁰_b, H-2, H-2⁰⁰, H-6⁰⁰; H-2⁰⁰,

H-6⁰⁰/NH, H-2, H-3⁰⁰, H-5⁰⁰; H-3⁰⁰, H-5⁰⁰/CH₃, H-2⁰⁰, H-6⁰⁰; H-7/H-6;

H-5/H-6, H-4; H-6/H-7, H-5; H-4/H-5. EIMS m/z (relative intensity)

372 M⁺(1%), 340 (100%), 265 (22%), 233 (24%), 161 (25%), 117

(18%), 91 (32%). Anal. Calcd for C₁₉H₂₁N₃OS₂requires: C, 61.42;

H, 5.70; N, 11.31. Found: C, 61.73; H, 5.47; N, 11.56. cis-1-Meth-

oxy-2-(4-methylphenylamino)-2⁰-(methylsulphanyl)spiro{indoline-3,

5⁰-[4⁰,5⁰]dihydrotiazole} (13b). Yield: 0.059 g (42%); colorless oil;

R_f= 0.38 (ethyl acetate/n-hexane, 1:8). IR (CHCl₃) m_max3400,

3010, 2930, 1605, 1570, 1510, 1460, 1400, 1240, 1095, 1040, 985,

4.1.4.1. trans- and cis-1-Methoxy-2-phenylamino-2⁰-(methyl-

sulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole} (12a and

12b). Following the general procedure products 12a and 12b were

obtained using 0.314 g (0.31 mL, 3.375 mmol) of aniline and

separated on silica gel (20 g, diethyl ether/n-hexane 1:3). trans-1-

Methoxy-2-phenylamino-2⁰-(methylsulphanyl)spiro{indoline-3,5⁰-

[4⁰,5⁰]dihydrotiazole} (12a). Yield: 0.030 g (22%); colorless crystals;

mp 131–132 °C (acetone–hexane); R_f= 0.14 (diethyl ether/n-hex-

ane, 1:3). IR (CHCl₃) m_max3410, 3010, 2930, 1600, 1400, 1100,

990, 940, 620 cm^ꢀ^{1 1}H NMR (acetone-d₆) d 2.47 (s, 3H, SCH₃),

3.79 (s, 3H, OCH₃), 3.89 (d, J = 15.6 Hz, 1H, H-4⁰_a), 4.92 (d,

J = 15.6 Hz, 1H, H-4⁰_b), 5.38 (d, J = 10.2 Hz, 1H, H-2), 5.64

(d, J = 10.2 Hz, 1H, NH), 6.71 (dd, J = 7.3, 7.3 Hz, 1H, H-4⁰⁰), 6.97

(d, J = 7.9 Hz, 2H, H-2⁰⁰,H-6⁰⁰), 6.99 (d, J = 7.7 Hz, 1H, H-7), 7.06

(dd, J = 7.5, 7.6 Hz, 1H, H-5), 7.15 (dd, J = 7.3, 8.0 Hz, 2H, H-3⁰⁰, H-

5⁰⁰), 7.29 (dd, J = 7.6, 7.7 Hz, 1H, H-6), 7.35 (d, J = 7.5 Hz, 1H, H-4).

¹³C NMR (acetone-d₆) d 14.9 (SCH₃), 64.2 (OCH₃), 70.5 (C-3), 70.9

(C-4⁰), 90.2 (C-2), 114.1 (C-7), 115.2 (C-2⁰⁰, C-6⁰⁰), 119.1 (C-4⁰⁰),

124.4 (C-5), 124.6 (C-4), 129.0 (C-3a), 129.8 (C-3⁰⁰, C-5⁰⁰), 130.3

(C-6), 148.5 (C-₀1⁰⁰), 1₀50.0 (C-7a), 163.7 (C-2⁰). NOESY correlations:

H-4⁰_a/H-4⁰_b; H-4_b/H-4_a, NH; H-2/NH, H-2⁰⁰, H-6⁰⁰; NH/H-4⁰_b, H-2, H-

2⁰⁰, H-6⁰⁰; H-4⁰⁰/H-2⁰⁰, H-6⁰⁰, H-3⁰⁰, H-5⁰⁰; H-2⁰⁰, H-6⁰⁰/H-2, NH, H-4⁰⁰,

H-3⁰⁰, H-5⁰⁰; H-5/H-6; H-3⁰⁰, H-5⁰⁰/H-4⁰⁰, H-2⁰⁰, H-6⁰⁰; H-6/H-5. EIMS

m/z (relative intensity): 358 M⁺(1%), 326 (100%), 265 (6%), 233

(13%), 161 (18%), 117 (17%), 104 (26%), 77 (68%) Anal. Calcd for

C₁₈H₁₉N₃OS₂requires: C, 60.47; H, 5.36; N, 11.75. Found: C,

60.29; H, 5.48; N, 11.93. cis-1-Methoxy-2-phenylamino-2⁰-(methyl-

sulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole} (12b). Yield:

0.062 g (46%); yellow oil; R_f= 0.37 (diethyl ether/n-hexane, 1:3).

940, 805, 610 cm^ꢀ^{1 1}H NMR (CDCl₃) d 2.25 (s, 3H, CH₃), 2.53 (s,

3H, SCH₃), 3.75 (s, 3H, OCH₃), 4.22 (d, J = 15.6 Hz, 1H, H-4⁰), 4.66

(d, J = 10.6 Hz, 1H, NH), 4.68 (d, J = 15.6 Hz, 1H, H-4⁰_a), 4.95 (d,

J = 10.6 Hz, 1H, H-2), 6.83 (d, J = 8.2 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 6.96 (dd,

J = 0.6, 7.8 Hz, 1H, H-7), 7.01 (d, J = 8.4 Hz, 2H, H-3⁰⁰, H-5⁰⁰), 7.03

(ddd, J = 0.9, 7.6, 7.6 Hz, 1H, H-5), 7.25 (d, J = 7.1 Hz, 1H, H-4),

7.28 (dd, J = 7.7, 7.6 Hz, 1H, H-6). ¹³C NMR (CDCl₃) d 15.1 (SCH₃),

20.4 (CH₃); 64.0 (OCH₃), 70.9 (C-3), 72.8 (C-4⁰), 86.0 (C-2), 113.0

(C-7), 114.6 (C-2⁰⁰, C-6⁰⁰), 122.7 (C-4), 123.8 (C-5), 128.3 (C-4⁰⁰),

129.1 (C-3a), 129.8 (C-3⁰⁰,C-5⁰⁰), 129.8 (C-6), 143.5 (C-1⁰⁰), 148.7

(C-7a), 163.4 (C-2⁰). NOESY correlations: CH₃/H-3⁰⁰, H-5⁰⁰; OCH₃/

3708

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

H-7; H-4_b⁰/H-4⁰_a, H-2; NH/H-2, H-2⁰⁰, H-6⁰⁰; H-4⁰_a/H-_b⁰, H-4; H-2/H-4⁰_b,

NH, H-2⁰⁰, H-6⁰⁰; H-2⁰⁰, H-6⁰⁰/NH, H-2, H-3⁰⁰, H-5⁰⁰; H-7/OCH₃, H-6; H-

3⁰⁰, H-5⁰⁰/CH₃, H-2⁰⁰, H-6⁰⁰; H-5/H-6, H-4; H-6/H-7, H-5; H-4/H-4⁰_a, H-

5. EIMS m/z (relative intensity) 372 M⁺(1%), 341 (100%), 265 (14%),

233 (22%), 161 (45%), 117 (18%), 91 (31%). Anal. Calcd for

C₁₉H₂₁N₃OS₂requires: C, 61.42; H, 5.70; N, 11.31. Found: C,

61.71; H, 5.83; N, 11.62.

(C-2⁰). NOESY correlations: 4⁰⁰-CH₃/H-5⁰⁰; 3⁰⁰-CH₃/H-2⁰⁰; OCH₃/H-7;

H-4⁰_a/H-4_b⁰, H-4; H-4_b⁰/H-4_a⁰, NH; H-2/NH, H-6⁰⁰, H-2⁰⁰; NH/H-4⁰_b, H-

2, H-6⁰⁰, H-2⁰⁰; H-6⁰⁰/H-2, NH, H-2⁰⁰, H-5⁰⁰; H-2⁰⁰/3⁰⁰-CH₃, H-2, NH,

H-6⁰⁰; H-5⁰⁰/4⁰⁰-CH₃, H-6⁰⁰; H-7/OCH₃, H-6; H-5/H-6, H-4; H-6/H-7,

H-5; H-4/H-4⁰_a, H-5. EIMS m/z (relative intensity) 386 M⁺(1%),

355 (100%), 266 (8%), 233 (11%), 161 (21%), 121 (21%), 117 (24%),

77 (26%). Anal. Calcd for C₂₀H₂₃N₃OS₂requires: C, 62.30; H, 6.01;

N, 10.90. Found: C, 62.63; H, 6.28; N, 10.59. cis-1-Methoxy-2-(3,

4-dimethylphenylamino)-2⁰-(methylsulphanyl)spiro{indoline-3,5⁰-

[4⁰,5⁰]dihydrotiazole} (14b). Yield: 0.048 g (33%); yellow oil;

R_f= 0.50 (ethyl acetate/n-hexane (1:5). IR (CHCl₃) m_max3014,

2935, 1700, 1614, 1570, 1500, 1456, 1400, 1300, 1256, 1191,

4.1.4.3. cis-1-Methoxy-2-(4-methylphenylamino)-2⁰-(methylsul-

phanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole} (13b) by isom-

erisation of trans-diastereoisomer 13a. Procedure A. To a stirred

solution of 1-methoxybrassinin 2 (0.031 g, 0.1164 mmol) in anhy-

drous dichloromethane (1.3 mL) was added the solution of bro-

mine (0.3 mL, 0.128 mmol, stock solution prepared by dissolving

of 0.04 mL of Br₂in 1.76 mL of anhydrous dichloromethane). After

stirring at room temperature for 5 min a solution of 4-methylani-

line (0.112 g, 1.048 mmol) and triethylamine (0.224 g, 0.31 mL,

2.212 mmol) in anhydrous dichloromethane (1.5 mL) was added.

After being stirred for 10 min, the reaction mixture was diluted

with dichloromethane (5 mL), successively washed with of 1 M

HCl (5 mL), water (10 mL) and brine (10 mL), and the obtained

dichloromethane solution was dried over Na₂SO_4.The residue ob-

tained after the evaporation of solvent was dissolved in anhydrous

dichloromethane (3.7 mL) and solution of triﬂuoroacetic acid

(0.43 mL, 0.011 mmol, stock solution prepared by dissolving

0.01 mL TFA in 5 mL anhydrous dichloromethane) was added. After

stirring for 24 h the solvent was evaporated and the residue

subjected to the ﬂash chromatography on silica gel (7 g, ethyl

acetate/n-hexane 1:8), affording 13b (29 mg, 67%).

Procedure B. To a stirred solution of 1-methoxybrassinin 2

(0.031 g, 0.1164 mmol) in anhydrous dichloromethane (1.3 mL)

was added the solution of bromine (0.3 mL, 0.128 mmol, stock solu-

tion prepared by dissolving of 0.04 mL of Br₂in 1.76 mL of anhydrous

dichloromethane). After stirring at room temperature for 5 min a

solution of p-toluidine (0.025 g, 0.233 mmol) in anhydrous dichloro-

methane (1.5 mL) was added. After 1 h the triethylamine (0.224 g,

0.31 mL, 2.212 mmol) was added and stirring was continued for

another 5 min. The reaction mixture was then diluted with dichloro-

methane (5 mL), successively washed with 1 M HCl (5 mL), water

(10 mL), and brine (10 mL). The obtained dichloromethane solution

was driedover Na₂SO₄and the residue obtainedafter theevaporation

of solvent was subjected to the ﬂash chromatography on silica gel

(7 g, Merck 230–400 mesh), affording separated 13b (28 mg, 65%).

1128, 1091, 1049, 998, 735 cm^ꢀ^{1 1}H NMR (acetone-d₆) d 2.13 (s,

3H, 4⁰⁰-CH₃), 2.17 (s, 3H, 3⁰⁰-CH₃), 2.51 (s, 3H, SCH₃), 3.72 (s, 3H,

OCH₃), 4.30 (d, J = = 15.8 Hz, 1H, H-4⁰), 4.70 (d, J = 15.8 Hz, 1H,

H-4⁰_a), 5.04 (d, J = 11.0 Hz, 1H, NH), 5.12 (d, J = 11.0 Hz, 1H, H-2),

6.77 (dd, J = 2.4, 8.1 Hz, 1H, H-6⁰⁰), 6.84 (d, J = 2.2 Hz, 1H, H-2⁰⁰),

6.92 (d, J = 8.1 Hz, 1H, H-5⁰⁰), 6.97 (d, J = 8.0 Hz, 1H, H-7), 7.04

(ddd, J = 0.9, 7.5, 7.6 Hz, 1H, H-5), 7.29 (dd, J = 1.1, 7.6 Hz, 1H, H-

4), 7.29 (ddd, J = 1.1, 7.5, 7.9 Hz, 1H, H-6). ¹³C NMR (acetone-d₆) d

15.0 (SCH₃), 18.8 (4⁰⁰-CH₃), 20.0 (3⁰⁰-CH₃), 64.2 (OCH₃), 72.1 (C-3),

73.4 (C-4⁰), 86.9 (C-2), 112.8 (C-6⁰⁰), 113.7 (C-7), 117.1 (C-2⁰⁰),

123.6 (C-4), 124.4 (C-5), 127.0 (C-4⁰⁰), 130.3 (C-3a), 130.4 (C-6),

130.9 (C-5⁰⁰), 137.6 (C-3⁰⁰), 145.6 (C-1⁰⁰), 149.8 (C-7a), 162.9 (C-2⁰).

NOESY correlations: ₀4⁰⁰-CH₃/H-5⁰⁰; 3⁰⁰-CH₃-/H-2⁰⁰; OCH₃/H-7;

H-4⁰_b/H-4⁰_a, H-2; H-4_a/H-4_b⁰, H-4; NH/H-2, H-2⁰⁰; H-2/H-4⁰_b, NH,

H-6⁰⁰, H-2⁰⁰; H-6⁰⁰/H-2, H-5⁰⁰; H-2⁰⁰/3⁰⁰-CH₃, NH, H-2; H-5⁰⁰/4⁰⁰-CH₃,

H-6⁰⁰; H-7/H-6; H-5/H-4, H-6; H-4/H-4⁰_a, H-5; H-6/H-7, H-5. EIMS

m/z (relative intensity) 386 M⁺(1%), 355 (100%), 265 (14%), 233

(15%), 117 (23%), 77 (24%). Anal. Calcd for C₂₀H₂₃N₃OS₂requires:

C, 62.30; H, 6.01; N, 10.90. Found: C, 62.65; H, 5.86; N, 10.67.

4.1.4.5. trans- and cis-1-Methoxy-2-(4-methoxyphenylamino)-

2⁰-(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole}

(15a and 15b). Following the general procedure products 15a and

15b were obtained using 0.092 g (0.75 mmol) of 4-methoxylaniline

and separated on silica gel (20 g, ethyl acetate/n-hexane 1:5).

trans-1-Methoxy-2-(4-methoxyphenylamino)-2⁰-(methylsulphanyl)-

spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole} (15a). Yield: 0.026 g (18%);

white crystals; mp 143–145 °C (acetone–hexane); R_f= 0.13 (ethyl

acetate/n-hexane, 1:5). IR (CHCl₃) m_max3000, 2300, 1614, 1514,

1456, 1400, 1314, 1235, 1100, 998, 949, 814, 614 cm^ꢀ^{1 1}H NMR

(CDCl₃) d 2.50 (s, 3H, SCH₃), 3.76 (s, 3H, 4⁰⁰-OCH₃), 3.80 (s, 3H, N–

OCH₃), 3.95 (d, J = 15.7 Hz, 1H, H-4⁰), 3.97 (d, J = 9.9 Hz, 1H, NH),

4.1.4.4. trans- and cis-1-Methoxy-2-(3,4-dimethylphenylami-

no)-2⁰-(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiaz-

ole} (14a and 14b). Following the general procedure products 14a

and 14b were obtained using 0. 091 g (0.75 mmol) of 3,4-dimeth-

ylaniline and separated on silica gel (20 g, ethyl acetate/n-hexane

1:5). trans-1-Methoxy-2-(3,4-dimethylphenylamino)-2⁰-(methylsul-

phanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole} (14a). Yield: 0.034 g

(24%); white crystals; mp 134–135 °C (dichloromethane–hexane);

4.81 (d, J = 15.7 Hz, 1H, H-4⁰_b), 5.15 (d, J = 9.9 Hz, 1H, H-2), 6.79

(d, J = 9.0 Hz, 2H, H-3⁰⁰, H-5⁰⁰), 6.82 (d, J = 9.0 Hz, 2H, H-2⁰⁰, H-6⁰⁰),

6.98 (d, J = 7.8 Hz, 1H, H-7), 7.04 (dd, J = 7.5, 7.5 Hz, 1H, H-5),

7.27 (dd, J = 7.7, 7.8 Hz, 1H, H-6), 7.35 (d, J = 7.5 Hz, 1H, H-4). ¹³C

NMR (CDCl₃) d 15.1 (SCH₃), 55.7 (4⁰⁰-OCH₃), 64.0 (N–OCH₃), 69.4

(C-3), 69.8 (C-4⁰), 91.6 (C-2), 113.4 (C-7), 114.7 (C-3⁰⁰, C-5⁰⁰), 116.2

(C-2⁰⁰, C-6⁰⁰), 123.7 (C-5), 123.8 (C-4), 127.0 (C-3a), 129.6 (C-6),

140.6 (C-1⁰⁰), 149.2 (C-7a), 153.2 (C-4⁰⁰), 166.1 (C-2⁰). NOESY corre-

lations: 4⁰⁰-OCH₃/H-3⁰⁰, H-5⁰⁰; N-OCH₃/H-7; H-4_a⁰/H-4⁰_b, H-4; NH/

H-4_b⁰, H-2, H-2⁰⁰,H-6⁰⁰; H-4_b⁰/H-4_a⁰, NH; H-2/NH, H-2⁰⁰, H-6⁰⁰; H-3⁰⁰,

H-5⁰⁰/4⁰⁰-OCH₃, H-2⁰⁰, H-6⁰⁰; H-2⁰⁰, H-6⁰⁰/NH, H-2, H-3⁰⁰, H-5⁰⁰; H-7/

R_f= 0.32 (ethyl acetate/n-hexane (1:5). IR (CHCl₃)

2356, 1700, 1600, 1570, 1500, 1456, 1398, 1300, 1228, 1191, 1114,

1091, 1049, 991, 735 cm^ꢀ^{1 1}H NMR (acetone-d₆) d 2.12 (s, 3H, 4⁰⁰-

m_max3014, 2935,

CH₃), 2.16 (s, 3H, 3⁰⁰-CH₃), 2.48 (s, 3H, SCH₃), 3.78 (s, 3H, OCH₃),

3.88 (d, J = 15.6 Hz, 1H, H-4_a⁰), 4.91 (d, J = 15.6 Hz, 1H, H-4⁰_b), 5.31

(d, J = 10.2 Hz, 1H, H-2), 5.36 (d, J = 10.2 Hz, 1H, NH), 6.70 (dd,

J = 2.5, 8.1 Hz, 1H, H-6⁰⁰), 6.77 (d, J = 2.3 Hz, 1H, H-2⁰⁰), 6.90 (d,

J = 8.1 Hz, 1H, H-5⁰⁰), 6.98 (ddd, J = 0.6, 0.9, 7.8 Hz, 1H, H-7), 7.05

(ddd, J = 1.1, 7.6, 7.6 Hz, 1H, H-5), 7.28 (ddd, J = 1.2, 7.6, 7.7 Hz,

1H, H-6), 7.34 (ddd, J = 0.6, 1.2, 7.6 Hz, 1H, H-4). ¹³C NMR (ace-

tone-d₆) d 14.3 (SCH₃), 18.2 (4⁰⁰-CH₃), 19.5 (3⁰⁰-CH₃), 63.6 (OCH₃),

69.8 (C-3), 70.3 (C-4⁰), 90.0 (C-2), 112.1 (C-6⁰⁰), 113.4 (C-7), 116.3

(C-2⁰⁰), 123.7 (C-5), 124.0 (C-4), 126.0 (C-4⁰⁰), 128.4 (C-3a), 129.6

(C-6), 130.2 (C-5⁰⁰), 136.8 (C-3⁰⁰), 145.8 (C-1⁰⁰), 149.4 (C-7a), 163.0

N-OCH₃, H-6; H-5/H-6, H-4; H-6/H-7, H-5; H-4/H-4⁰, H-5. EIMS

m/z (relative intensity) 387 M⁺(1%), 356 (100%), 265 (28%), 233

(14%), 161 (57%), 134 (28%), 122 (22%), 117 (31%), 108 (33%). Anal.

Calcd for C₁₉H₂₁N₃O₂S₂requires: C, 58.89; H, 5.46; N, 10.84. Found:

C, 58.61; H, 5.75; N, 10.49.

cis-1-Methoxy-2-(4-methoxyphenylamino)-2⁰-(methylsulphanyl)-

spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole} (15b). Yield: 0.030 g (21%);

white crystals; mp 178–180 °C (acetone–hexane); R_f= 0.24 (ethyl

acetate/n-hexane, 1:5). IR (CHCl₃) m_max3400, 3014, 2935, 1607,

1570, 1514, 1463, 1400, 1307, 1235, 1177, 1100, 1049, 998, 949,

828, 621 cm^ꢀ^{1 1}H NMR (acetone-d₆) d 2.52 (s, 3H, SCH₃), 3.72 (s,

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

3709

3H, 4⁰⁰-OCH₃), 3.72 (s, 3H, N–OCH₃), 4.32 (d, J = 15.8 Hz, 1H, H-4⁰_b),

4.71 (d, J = 15.8 Hz, 1H, H-4⁰_a), 5.00 (d, J = 11.1 Hz, 1H, NH), 5.07 (d,

J = 11.1 Hz, 1H, H-2), 6.79 (d, J = 9.0 Hz, 2H, H-3⁰⁰, H-5⁰⁰), 6.97 (dd,

J = 1.1, 8.1 Hz, 1H, H-7), 7.00 (d, J = 9.0 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 7.05

(ddd, J = 1.1, 7.6, 7.8 Hz, 1H, H-5), 7.28 (dd, J = 1.2, 7.8 Hz, 1H, H-

4), 7.29 (ddd, J = 1.2, 7.5, 7.8 Hz,1H, H-6). ¹³C NMR ¹H NMR (ace-

tone-d₆) d 15.0 (SCH₃), 55.7 (4⁰⁰-OCH₃), 64.2 (N–OCH₃), 72.1 (C-3),

73.5 (C-4⁰), 87.9 (C-2), 113.8 (C-7), 115.3 (C-2⁰⁰, C-6⁰⁰), 116.8 (C-3⁰⁰,

C-5⁰⁰), 123.6 (C-4), 124.4 (C-5), 130.4 (C-6), 130.4 (C-3a), 141.4

(C-1⁰⁰), 149.9 (C-7a), 153.9 (C-4⁰⁰), 163.0 (C-2⁰). NOESY correlations:

4⁰⁰-OCH₃/H-3⁰⁰, H-5⁰⁰; H-4_b⁰/H-4_a⁰, H-2; H-4_a⁰/H-4_b⁰, H-4; NH/H-2, H-

2⁰⁰, H-6⁰⁰; H-2/H-4_b⁰, NH, H-2⁰⁰, H-6⁰⁰; H-3⁰⁰, H-5⁰⁰/4⁰⁰-OCH₃, H-2⁰⁰, H-

6⁰⁰; H-7/H-6; H-2⁰⁰, H-6⁰⁰/NH, H-2, H-3⁰⁰, H-5⁰⁰; H-5/H-4, H-6; H-4/

H-4⁰_a, H-5; H-6/H-7, H-5. EIMS m/z (relative intensity) 387 M⁺

(1%), 356 (100%), 265 (22%), 233 (11%), 161 (62%), 134 (27%), 122

(28%), 117 (34%), 108 (31%). Anal. Calcd for C₁₉H₂₁N₃O₂S₂requires:

C, 58.89; H, 5.46; N, 10.84. Found: C, 59.06; H, 5.31; N, 11.07.

intensity) 391 M⁺(1%), 360 (100%), 265 (10%), 233 (15%), 161

(26%), 150 (24%), 138 (25%), 127 (25%), 117 (45%). Anal. Calcd for

C₁₈H₁₈ClN₃OS₂requires: C, 55.16; H, 4.63; N, 10.72. Found: C,

55.39; H, 4.36; N, 10.98.

4.1.4.7. trans- and cis-1-Methoxy-2-(4-chlorophenylamino)-2⁰-

(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole}

(17a and 17b). Following the general procedure products 17a and

17b were obtained using 0.431 g (3.375 mmol) of 4-chloroaniline

and separated on silica gel (20 g, ethyl acetate/cyclohexane 1:5).

trans-1-Methoxy-2-(4-chlorophenylamino)-2’-(methylsulphanyl)spiro-

{indoline-3,5’-[4’,5’]dihydrotiazole} (17a). Yield: 0.024 g (16%); white

crystals; mp 162–164 °C (dichloromethane–hexane); R_f= 0.39 (ethyl

acetate/cyclohexane, 1:5). IR (CHCl₃) m_max3400, 3000, 2362, 1600,

1563, 1491, 1456, 1400, 1284, 1177, 1098, 1050, 998, 942, 805,

620 cm^ꢀ^{1 1}H NMR (CDCl₃) d 2.49 (s, 3H, SCH₃), 3.80 (s, 3H, OCH₃),

3.94 (d, J = 15.7 Hz, 1H, H-4⁰_a), 4.22 (d, J = 9.9 Hz, 1H, NH), 4.78 (d,

J = 15.7 Hz, 1H, H-4⁰_b), 5.19 (d, J = 9.9 Hz, 1H, H-2), 6.79 (d, J = 8.8 Hz,

2H, H-2⁰⁰, H-6⁰⁰), 6.99 (d, J = 7.8 Hz, 1H, H-7), 7.06 (ddd, J = 1.0, 7.5,

7.6 Hz, 1H, H-5), 7.15 (d, J = 8.8 Hz, 2H, H-3⁰⁰, H-5⁰⁰), 7.29 (ddd,

J = 1.2, 7.5, 7.8 Hz, 1H, H-6), 7.36 (dd, J = 1.2, 7.6 Hz, 1H, H-4). ¹³C

NMR (CDCl₃) d 15.1 (SCH₃), 64.0 (OCH₃), 69.3 (C-3), 69.7 (C-4⁰), 90.5

(C-2), 113.4 (C-7), 115.7 (C-2⁰⁰, C-6⁰⁰), 123.8 (C-4), 123.9 (C-5), 123.9

(C-4⁰⁰), 126.5 (C-3a), 129.1 (C-3⁰⁰, C-5⁰⁰), 129.8 (C-6), 145.2 (C-1⁰⁰₀),

149.1 (C-7a), 166.4 (C-2⁰). NOESY correlations: OCH₃/H-7; H-4_a/

H-4⁰_b, H-4; NH/H-4_b⁰, H-2, H-2⁰⁰, H-6⁰⁰; H-4_b⁰/H-4⁰_a, NH; H-2/NH, H-2⁰⁰,

H-6⁰⁰; H-2⁰⁰, H-6⁰⁰/NH, H-2, H-3⁰⁰, H-5⁰⁰; H-7/H-6; H-5/H-6, H-4; H-3⁰⁰,

H-5⁰⁰/H-2⁰⁰, H-6⁰⁰; H-6/H-7, H-5; H-4/H-⁰_a, H-5. MALDI-TOF MS: 430.1

[M+K]⁺(6%), 413.7 [M+Na]⁺(10%), 392.8 [M+H]⁺(4%), 262.0 (10%),

283.9 (19%), 266.2 (13%), 200.3 (36%), 156.6 (19%), 138.6 (30%). Anal.

CalcdforC₁₈H₁₈ClN₃OS₂requires:C, 55.16;H,4.63;N,10.72.Found:C,

55.19; H, 4.41; N, 10.60. cis-1-methoxy-2-(4-chlorophenylamino)-

4.1.4.6. trans- and cis-1-Methoxy-2-(2-chlorophenylamino)-2⁰-

(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole}

(16a and 16b). Following the general procedure products 16a and

16b were obtained using 0.096 g (0.08 mL, 0.75 mmol) of 2-chloro-

aniline and separated on silica gel (20 g, ethyl acetate/n-hexane

1:8). Fractions of 16a contained small amount of 2-chloroaniline

as an impurity which was removed by repeated chromatography

on silica gel (5 g, dichloromethane/n-hexane 1:1). trans-1-Meth-

oxy-2-(2-chlorophenylamino)-2’-(methylsulphanyl)spiro{indoline-3,5’-

[4’,5’]dihydrotiazole} (16a). Yield: 0.052 g (35%); yellow oil;

R_f= 0.44 (ethyl acetate/n-hexane (1:8). IR (CHCl₃) m_max3360,

3167, 1733, 1587, 1453, 1120, 1033, 940, 740 cm^ꢀ1

¹H NMR

(CDCl₃) 2.48 (s, 3H, SCH₃), 3.83 (s, 3H, OCH₃), 3.96 (d,

J = 15.7 Hz, 1H, H-4⁰_a), 4.89 (d, J = 15.7 Hz, 1H, H-4⁰_b), 5.06 (d,

J = 9.2 Hz, 1H, NH), 5.27 (d, J = 9.2 Hz, 1H, H-2), 6.70–6.74 (m, 1H,

H-4⁰⁰), 7.00 (dd, J = 0.8, 7.8 Hz, 1H, H-7), 7.07 (ddd, J = 1.0, 7.6,

7.6 Hz, 1H, H-5), 7.14–7.16 (m, 2H, H-5⁰⁰, H-6⁰⁰), 7.24–7.29 (m, 1H,

H-3⁰⁰), 7.29 (ddd, J = 1.2, 7.6, 7.8 Hz, 1H, H-6), 7.38 (dd, J = 1.2,

7.6 Hz, 1H, H-4). ¹³C NMR (CDCl₃) d 15.0 (SCH₃), 64.3 (OCH₃),

69.7 (C-3), 70.0 (C-4⁰), 89.7 (C-2), 113.4 (C-7), 113.7 (C-6⁰⁰), 119.0

(C-4⁰⁰), 120.0 (C-2⁰⁰), 123.9 (C-4), 124.0 (C-5), 126.8 (C-3a), 127.8

(C-5⁰⁰), 129.2 (C-3⁰⁰), 129.7 (C-6), 142.6 (C-1⁰⁰), 149.0 (C-7a), 165.₀6

(C-2⁰). NOESY correlations: OCH₃/H-7; H-4_a⁰/H-4_b⁰, H-4; H-4_b⁰/H-4_a,

NH; NH/H-4_b⁰, H-2, H-6⁰⁰; H-2/NH, H-6⁰⁰; H-4⁰⁰/H-5⁰⁰, H-3⁰⁰; H-7/

OCH₃, H-6; H-5/H-6, H-4; H-5⁰⁰/H-4⁰⁰; H-6⁰⁰/NH, H-2; H-3⁰⁰/H-4⁰⁰;

H-6/H-7, H-5; H-4/H-4⁰_a, H-5. EIMS m/z (relative intensity) 391

M⁺(1%), 360 (100%), 265 (13%), 233 (20%), 161 (39%), 150 (30%),

138 (28%), 127 (32%), 117 (48%). Anal. Calcd for C₁₈H₁₈ClN₃OS₂re-

quires: C, 55.16; H, 4.63; N, 10.72. Found: C, 54.88; H, 4.50; N,

10.84. cis-1-Methoxy-2-(2-chlorophenylamino)-2’-(methylsulpha-

nyl)spiro{indoline-3,5’-[4’,5’]dihydrotiazole} (16b). Yield: 0.036 g

(25%); yellow oil; R_f= 0.26 (ethyl acetate/n-hexane (1:8). IR

(CHCl3) m_max3407, 3173, 1580, 1507, 1453, 1387, 1113,

2’-(methylsulphanyl)spiro{indoline-3,5’-[4’,5’]dihydrotiazole}

(17b).

Yield: 0.042 g (29%); bright yellow oil; R_f= 0.39 (ethyl acetate/cyclo-

hexane, 1:5). IR (CHCl₃) m_max3400, 3000, 2935, 2300, 1598, 1498,

1456, 1400, 1300, 1235, 1177, 1091, 1042, 982, 942, 814, 614 cm^ꢀ1

¹H NMR (CDCl₃) d 2.54 (s, 3H, SCH₃), 3.73 (s, 3H, OCH₃), 4.22 (d,

J = 15.7 Hz, 1H, H-4⁰_b), 4.70 (d, J = 15.7 Hz, 1H, H-4⁰_a), 4.81 (d,

J = 10.7 Hz, 1H, NH), 4.92 (d, J = 10.7 Hz, 1H, H-2), 6.86 (d, J = 8.8 Hz,

2H, H-2⁰⁰, H-6⁰⁰), 6.97 (d, J = 7.8 Hz, 1H, H-7), 7.05 (ddd, J = 0.8, 7.5,

7.6 Hz, 1H, H-5), 7.16 (d, J = 8.8 Hz, 2H, H-3⁰⁰,H-5⁰⁰), 7.27 (d,

J = 7.7 Hz, 1H, H-4), 7.30 (dd, J = 7.7, 7.7 Hz, 1H, H-6). ¹³C NMR (CDCl₃)

d 15.1 (SCH₃), 64.1 (OCH₃), 70.7 (C-3), 73.0 (C-4⁰), 85.5 (C-2), 113.1

(C-7), 115.6 (C-2⁰⁰, C-6⁰⁰), 122.8 (C-4), 123.7 (C-4⁰⁰), 124.0 (C-5), 128.7

(C-3a), 129.2 (C-3⁰⁰, C-5⁰⁰), 130.0 (C-6), 144.5 (C-1⁰⁰), 148.6 (C-7a),

163.4 (C-2⁰). NOESY correlations: OCH₃/H-7; H-4⁰_b/H-4_a⁰, H-2; H-4⁰_a/

H-4⁰_b, H-4; NH/H-2, H-2⁰⁰, H-6⁰⁰; H-2/NH, H-2⁰⁰, H-6⁰⁰; H-2⁰⁰, H-6⁰⁰/NH,

H-2, H-3⁰⁰, H₀-5⁰⁰; H-7/OCH₃, H-6; H-5/H-4, H-6; H-3⁰⁰, H-5⁰⁰/H-2⁰⁰, H-

6⁰⁰; H-4/H-4_a, H-5; H-6/H-7, H-5. MALDI-TOF MS: 430.1 [M+K]⁺

(8%), 413.5 [M+Na]⁺(12%), 392 [M+H]⁺(13%), 361.8 (20%), 285.7

(25%), 265.9 (36%), 200.3 (39%), 161.5 (20%), 138.6 (15%). Anal. Calcd

for C₁₈H₁₈ClN₃OS₂requires: C, 55.16; H, 4.63; N, 10.72. Found: C,

54.87; H, 4.90; N, 10.56.

933 cm^ꢀ^{1 1}H NMR (CDCl3) d 2.53 (s, 3H, SCH₃), 3.74 (s, 3H,

OCH₃), 4.25 (d, J = 15.7 Hz, 1H, H-4⁰), 4.71 (d, J = 15.7 Hz, 1H,

H-4⁰_a), 4.98 (d, J = 10.3 Hz, 1H, H-2), 5.67 (d, J = 10.3 Hz, 1H, NH),

6.73 (ddd, J = 1.8, 6.9, 7.9 Hz, 1H, H-4⁰⁰), 6.98 (d, J = 7.7 Hz, 1H, H-

7), 7.06 (ddd, J = 1.1, 7.7, 7.8 Hz, 1H, H-5), 7.13 (ddd, J = 1.3, 6.9,

8.3 Hz, 1H, H-5⁰⁰), 7.17 (dd, J = 1.8, 8.3 Hz, 1H, H-6⁰⁰), 7.29 (dd,

J = 1.3, 7.9 Hz, 1H, H-3⁰⁰), 7.30 (dd, J = 1.3, 7.8 Hz, 1H, H-4), 7.30

(ddd, J = 1.2, 7.6, 7.8 Hz, 1H, H-6). ¹³C NMR (CDCl₃) d 15.2 (SCH₃),

64.1 (OCH₃), 70.7 (C-3), 73.2 (C-4⁰), 85.3 (C-2), 113.0 (C-7), 113.5

(C-6⁰⁰), 119.1 (C-4⁰⁰), 120.3 (C-2⁰⁰), 122.9 (C-4), 124.0 (C-5), 127.8

(C-5⁰⁰), 128.5 (C-3a), 129.4 (C-3⁰⁰), 130.0 (C-6), 142.1 (C-1⁰⁰), 148.₀8

(C-7a), 163.9 (C-2⁰). NOESY correlations: OCH₃/H-7; H-4_b⁰/H-4_a,

H-2; H-4⁰_a/H-4⁰_b, H-4; H-2/H-4_b⁰, NH, H-6⁰⁰; NH/H-2; H-4⁰⁰/H-5⁰⁰, H-

3⁰⁰; H-7/OCH₃, H-6; H-5/H-4, H-6; H-5⁰⁰/H-4⁰⁰, H-6⁰⁰; H-6⁰⁰/H-2, H-

5⁰⁰; H-3⁰⁰/H-4⁰⁰; H-4/H-4⁰_a, H-5; H-6/H-7, H-5. EIMS m/z (relative

4.1.4.8. trans- and cis-1-methoxy-2-(3,4-dichlorophenylami-

no)-2⁰-(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiaz-

ole} (18a and 18b). Following the general procedure products 18a

and 18b were obtained using 0.122 g (0.75 mmol) of 3,4-dichloro-

aniline and separated on silica gel (20 g, ethyl acetate/n-hexane

1:8). Fractions of 18a contained small amount of 3,4-dichloroani-

line as an impurity which was removed by repeated chromatogra-

phy on silica gel (5 g, dichloromethane).

trans-1-Methoxy-2-(3,4-dichlorophenylamino)-2’-(methylsulphanyl)-

spiro{indoline-3,5’-[4’,5’]dihydrotiazole} (18a). Yield: 0.045 g (28%);

white crystals; mp 156–158 °C (dichloromethane–hexane);

R_f= 0.3 (ethyl acetate/n-hexane, 1:8). IR (CHCl₃) m_max3400, 3014,

3710

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

2935, 1700, 1591, 1556, 1456, 1398, 1356, 1300, 1121, 1091, 991,

406 (100%), 404 (94%), 265 (41%), 233 (35%), 218 (26%), 161

(68%), 150 (44%), 117 (62%). Anal. Calcd for C₁₈H₁₈BrN₃OS₂re-

quires: C, 49.54; H, 4.16; N, 9.63. Found: C, 49.24; H, 4.35; N, 9.32.

cis-1-Methoxy-2-(4-bromophenylamino)-2’-(methylsulphanyl)-

spiro{indoline-3,5’-[4’,5’]dihydrotiazole} (19b). Yield: 0.070 g (43%);

bright yellow oil; R_f= 0.35 (ethyl acetate/cyclohexane, 1:8). IR

(CHCl₃) m_max3380, 2973, 2933, 1573, 1486, 1400, 1300, 1226,

942 cm^ꢀ^{1 1}H NMR (acetone-d₆) d 2.47 (s, 3H, SCH₃), 3.82 (s, 3H,

OCH₃), 3.89 (d, J = 15.6 Hz, 1H, H-4⁰), 4.87 (d, J = 15.6 Hz, 1H,

H-4⁰_b), 5.41 (d, J = 10.3 Hz, 1H, H-2), 6.13 (d, J = 10.3 Hz, 1H, NH),

6.98 (dd, J = 2.7, 8.8 Hz, 1H, H-6⁰⁰), 7.01 (d, J = 7.8 Hz, 1H, H-7),

7.07 (ddd, J = 1.0, 7.5, 7.5 Hz, 1H, H-5), 7.20 (d, J = 2.7 Hz, 1H, H-

2⁰⁰), 7.30 (d, J = 8.6 Hz, 1H, H-5⁰⁰), 7.30 (ddd, J = 1.2, 7.6, 7.5 Hz,

1H, H-6), 7.36 (d, J = 7.6 Hz, 1H, H-4). ¹³C NMR (acetone-d₆) d

15.0 (SCH₃), 64.4 (OCH₃), 70.5 (C-3), 70.7 (C-4⁰), 89.5 (C-2), 114.1

(C-7), 115.1 (C-6⁰⁰), 116.3 (C-2⁰⁰), 120.6 (C-4⁰⁰), 124.6 (C-5), 124.6

(C-4), 128.4 (C-3a), 130.4 (C-6), 131.4 (C-5⁰⁰), 132.8 (C-3⁰⁰), 148.6

(C-1⁰⁰), 149.9 (C-7a), 163.9 (C-2⁰). NOESY correlations: OCH₃/H-7;

H-4⁰_a/H-4_b⁰, H-4; H-4_b⁰/H-4_a⁰, NH; H-2/NH, H-2⁰⁰, H-6⁰⁰; NH/H-4⁰_b, H-

2, H-2⁰⁰, H-6⁰⁰; H-6⁰⁰/H-2, NH, H-5⁰⁰; H-5/H-6, H-4; H-4/H-4⁰_a, H-5.

EIMS m/z (relative intensity) 426 M⁺(1%), 396 (46%), 394 (61%),

265 (25%), 233 (28%), 163 (25%), 161 (100%), 150 (29%), 145

(21%), 117 (52%), 90 (20%), 72 (32%). Anal. Calcd for

C₁₈H₁₇Cl₂N₃OS₂requires: C, 50.70; H, 4.02; N, 9.86. Found: C,

51.02; H, 3.79; N, 9.53. cis-1-Methoxy-2-(3,4-dichlorophenylami-

no)-2’-(methylsulphanyl)spiro{indoline-3,5’-[4’,5’]dihydrotiazole} (18b).

Yield: 0.049 g (31%); yellow oil; R_f= 0.36 (ethyl acetate/n-hexane,

1:8). IR (CHCl₃) m_max3400, 3000, 2827, 1733, 1593, 1493, 1387,

947, 930, 800, 733 cm^ꢀ^{1 1}H NMR (CDCl₃) d 2.53 (s, 3H, SCH₃),

3.73 (s, 3H, OCH₃), 4.22 (d, J = 15.7 Hz, 1H, H-4⁰_b), 4.69 (d,

J = 15.7 Hz, 1H, H-4⁰_a), 4.82 (d, J = 10.7 Hz, 1H, NH), 4.92

(d, J = 10.7 Hz, 1H, H-2), 6.81 (d, J = 8.9 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 6.97

(d, J = 7.8 Hz, 1H, H-7), 7.05 (ddd, J = 1.1, 7.6, 7.6 Hz, 1H, H-5),

7.26 (d, J = 7.6 Hz, 1H, H-4), 7.29 (d, J = 8.9 Hz, 2H, H-3⁰⁰, H-5⁰⁰),

7.29 (ddd, J = 1.2, 7.5, 7.8 Hz, 1H, H-6). ¹³C NMR (CDCl₃) d 15.4

(SCH₃), 64.3 (OCH₃), 71.0 (C-3), 73.3 (C-4⁰), 85.7 (C-2), 111.1 (C-

4⁰⁰), 113.3 (C-7), 116.4 (C-2⁰⁰, C-6⁰⁰), 123.1 (C-4), 124.3 (C-5), 129.0

(C-3a), 130.2 (C-6), 132.3 (C-3⁰⁰,C-5⁰⁰), 145.2 (C-1⁰⁰), 148.9 (C-7a),

163₀.6 (C-2⁰). NOESY correlations: OCH₃/H-7; H-4_b⁰/H-4⁰_a, H-2;

H-4_a/H-4_b⁰, H-4; NH/H-2, H-2⁰⁰, H-6⁰⁰; H-2/H-4⁰_b, NH, H-2⁰⁰, H-6⁰⁰;

H-2⁰⁰, H-6⁰⁰/NH, H-2, H-3⁰⁰, H-5⁰⁰; H-7/OCH₃, H-6; H-5/H-4, H-6;

H-4/H-4_a⁰, H-5; H-3⁰⁰, H-5⁰⁰/H-2⁰⁰, H-6⁰⁰; H-6/H-7, H-5. EIMS m/z

(relative intensity) 435 M⁺(1%), 436 M⁺(1%), 406 (100%), 404

(87%), 265 (28%), 233 (20%), 161 (38%), 150 (28%), 117 (51%), 91

(25%), 76 (22%). Anal. Calcd for C₁₈H₁₈BrN₃OS₂requires: C, 49.54;

H, 4.16; N, 9.63. Found: C, 49.29; H, 4.41; N, 9.43.

1127, 987, 580 cm^ꢀ^{1 1}H NMR (acetone-d₆) d 2.51 (s, 3H, SCH₃),

3.75 (s, 3H, OCH₃), 4.39 (d, J = 15.9 Hz, 1H, H-4⁰), 4.71 (d, J =

15.9 Hz, 1H, H-4⁰_a), 5.24 (d, J = 10.9 Hz, 1H, H-2), 5.81 (d,

J = 10.9 Hz, 1H, NH), 7.00 (dd, J = 0.9, 7.8 Hz, 1H, H-7), 7.07 (ddd,

J = 1.0, 7.6, 7.7 Hz, 1H, H-5), 7.08 (dd, J = 2.7, 8.8 Hz, 1H, H-6⁰⁰),

7.31 (ddd, J = 1.2, 7.6, 7.6 Hz, 1H, H-6), 7.31 (d, J = 8.8 Hz, 1H,

H-5⁰⁰), 7.33 (d, J = 2.7 Hz, 1H, H-2⁰⁰), 7.33 (dd, J = 1.0, 7.6 Hz, 1H,

H-4). ¹³C NMR d (ppm): 15.0 (SCH₃), 64.3 (OCH₃), 71.8 (C-3), 73.4

(C-4⁰), 85.9 (C-2), 113.8 (C-7), 115.4 (C-6⁰⁰), 116.7 (C-2⁰⁰), 120.9

(C-4⁰⁰), 123.8 (C-4), 124.7 (C-5), 129.9 (C-3a), 130.6 (C-6), 131.4

(C-5⁰⁰), 132.9 (C-3⁰⁰), 148.1 (C-1⁰⁰), 149.8 (C-7a), 163.1 (C-2⁰). NOESY

correlations: OCH₃/H-7; H-4⁰/H-4⁰, H-2; H-4⁰/H-4⁰, H-4; H-2/H-4⁰,

4.1.4.10. trans- and cis-1-Methoxy-2-(4-nitrophenylamino)-2⁰-

(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole}

(20a and 20b). Following the general procedure products 20a and

20b were obtained using 0.077 g (0.563 mmol) of 4-nitroaniline

and separated on silica gel (20 g, ethyl acetate/n-hexane 1:2).

Fractions of 20a contained small amount of 4-nitroaniline as an

impurity which was removed by repeated chromatography on sil-

ica gel (5 g, dichloromethane). trans-1-Methoxy-2-(4-nitrophenyla-

mino)-2’-(methylsulphanyl)spiro{indoline-3,5’-[4’,5’]dihydrotiazole}

(20a). Yield: 0.020 g (13%); yellow crystals; mp 184–186 °C (ethyl

acetate-hexane); R_f= 0.40 (ethyl acetate/n-hexane, 1:2). IR (CHCl₃)

m_max3400, 3133, 3013, 1700, 1587, 1313, 1160, 1107, 933,

NH, H-6⁰⁰, H-2⁰⁰; NH/H-2, H-6⁰⁰, H-2⁰⁰; H-7/OCH₃, H-6; H-5/H-6, H-4;

H-6⁰⁰/H-2, NH,₀H-5⁰⁰, H-2⁰⁰; H-6/H-7, H-5; H-5⁰⁰/H-6⁰⁰; H-2⁰⁰/H-2, NH,

H-6⁰⁰; H-4/H-4_a, H-5. EIMS m/z (relative intensity) 426 M⁺(1%), 396

(68%), 394 (100%), 265 (27%), 233 (23%), 161 (85%), 150 (31%), 145

(29%), 117 (60%), 91 (36%), 72 (41%). Anal. Calcd for

C₁₈H₁₇Cl₂N₃OS₂requires: C, 50.70; H, 4.02; N, 9.86. Found: C,

50.49; H, 4.35; N, 10.17.

827 cm^ꢀ^{1 1}H NMR (CDCl₃) d 2.47 (s, 3H, SCH₃), 3.82 (s, 3H,

OCH₃), 3.95 (d, J = 15.8 Hz, 1H, H-4⁰), 4.77 (d, J = 15.8 Hz, 1H,

H-4⁰_b), 5.03 (d, J = 9.9 Hz, 1H, NH), 5.35 (d, J = 9.9 Hz, 1H, H-2),

6.88 (d, J = 9.2 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 7.02 (d, J = 7.9 Hz, 1H, H-7),

7.10 (ddd, J = 1.0, 7.5, 7.6 Hz, 1H, H-5), 7.32 (ddd, J = 1.2, 7.6,

7.8 Hz, 1H, H-6), 7.39 (d, J = 7.6 Hz, 1H, H-4), 8.12 (d, J = 9.2 Hz,

2H, H-3⁰⁰, H-5⁰⁰). ¹³C NMR (CDCl₃) d 15.3 (SCH₃), 64.5 (OCH₃), 69.8

(C-4⁰), 69.9 (C-3), 89.1 (C-2), 113.2 (C-2⁰⁰, C-6⁰⁰), 113.9 (C-7), 124.1

(C-4), 124.6 (C-5), 126.1 (C-3a), 126.4 (C-3⁰⁰, C-5⁰⁰), 130.3 (C-6),

139.9 (C-4⁰⁰), 149.2 (C-7a), 152.4 (C-1⁰⁰), 166.9 (C-2⁰). NOESY corre-

lations: OCH₃/H-7; H-4⁰/H-4⁰, H-4; H-4⁰/H-4⁰, NH; NH/H-4⁰, H-2,

4.1.4.9. trans- and cis-1-Methoxy-2-(4-bromophenylamino)-2⁰-

(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihydrotiazole}

(19a and 19b). Following the general procedure products 19a and

19b were obtained using 0.581 g (3.375 mmol) of 4-bromoaniline

and separated on silica gel (20 g, ethyl acetate/cyclohexane 1:8).

trans-1-Methoxy-2-(4-bromophenylamino)-2’-(methylsulphanyl)

spiro{indoline-3,5’-[4’,5’]dihydrotiazole} (19a). Yield: 0.035 g (21%);

white crystals; mp 177–179 °C (acetone–hexane); R_f= 0.15 (ethyl

acetate/cyclohexane, 1:8). IR (CHCl₃) m_max3410, 3010, 1610,

H-2⁰⁰, H-6⁰⁰; H-2/NH, H-2⁰⁰, H-6⁰⁰; H-2⁰⁰, H-6⁰⁰/NH, H-2, H-3⁰⁰, H-5⁰⁰;

H-7/OCH₃; H-5/H-6, H-4; H-6/H-5; H-4/H-⁰_a, H-5; H-3⁰⁰, H-5⁰⁰/H-

2⁰⁰, H-6⁰⁰. EIMS m/z (relative intensity) 403 M⁺(1%), 371 (100%),

323 (14%), 265 (13%), 233 (13%), 160 (26%), 149 (22%), 117 (30%).

Anal. Calcd for C₁₈H₁₈N₄O₃S₂requires: C, 53.71; H, 4.51; N, 13.92.

Found: C, 53.99; H, 4.35; N, 13.74.

1480, 1450, 1390, 1090, 985, 935, 805, 610 cm^ꢀ1

¹H NMR (Ace-

2.48 (s, 3H, SCH₃), 3.80 (s, 3H, OCH₃), 3.90 (d,

tone-d₆)

J = 15.7 Hz, 1H, H-4⁰_a), 4.89 (d, J = 15.7 Hz, 1H, H-4⁰_b), 5.36

(d, J = 10.3 Hz, 1H, H-2), 5.81 (d, J = 10.3 Hz, 1H, NH), 6.95 (d,

J = 8.9 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 7.00 (dd, J = 0.9, 7.8 Hz, 1H, H-7), 7.06

(ddd, J = 1.1, 7.4, 7.4 Hz, 1H, H-5), 7.28 (d, J = 8.9 Hz, 2H, H-3⁰⁰, H-

5⁰⁰), 7.29 (ddd, J = 1.1, 7.6, 7.7 Hz, 1H, H-6), 7.35 (dd, J = 1.1,

cis-1-Methoxy-2-(4-nitrophenylamino)-2’-(methylsulphanyl)-

spiro{indoline-3,5’-[4’,5’]dihydrotiazole} (20b). Yield: 0.035 g (23%);

bright yellow oil; R_f= 0.63 (ethyl acetate/n-hexane, 1:2). IR (CHCl₃)

m_max3391, 3014, 2363, 1714, 1598, 1500, 1400, 1321, 1249, 1184,

7.4 Hz, 1H, H-4). ¹³C NMR (acetone-d₆)

d 15.0 (SCH₃), 64.3

(OCH₃), 70.6 (C-3), 70.9 (C-4⁰), 90.0 (C-2), 110.2 (C-4⁰⁰), 114.2 (C-

7), 117.1 (C-2⁰⁰, C-6⁰⁰), 124.6 (C-5), 124.6 (C-4), 128.8 (C-3a), 130.4

(C-6), 132.5 (C-3⁰⁰, C-5⁰⁰), 147.9 (C-1⁰⁰), 150.1 (C-7a), 164.0 (C-2⁰).

NOESY correlations: H-4⁰_a/H-4_b⁰; H-4_b⁰/H-4⁰_a, NH; H-2/NH, H-2⁰⁰,

H-6⁰⁰; NH/H-4⁰_b, H-2, H-2⁰⁰, H-6⁰⁰; H-2⁰⁰, H-6⁰⁰/H-2, NH, H-3⁰⁰, H-5⁰⁰;

H-7/H-6; H-5/H-4, H-6; H-3⁰⁰, H-5⁰⁰/H-2⁰⁰, H-6⁰⁰; H-6/ H-7, H-5; H-

4/H-5. EIMS m/z (relative intensity) 435 M⁺(1%), 436 M⁺(1%),

1114, 1049, 991, 942, 742 cm^ꢀ^{1 1}H NMR (CDCl₃) d 2.54 (s, 3H,

SCH₃), 3.74 (s, 3H, OCH₃), 4.25 (d, J = 15.8 Hz, 1H, H-4⁰), 4.73

(d, J = 15.8 Hz, 1H, H-4⁰_a), 5.04 (d, J = 10.5 Hz, 1H, H-2), 5.52

(d, J = 10.5 Hz, 1H, NH), 6.95 (d, J = 9.2 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 7.00

(d, J = 7.9 Hz, 1H, H-7), 7.10 (ddd, J = 0.7, 7.6, 7.6 Hz, 1H, H-5),

7.30 (d, J = 7.9 Hz, 1H, H-4), 7.34 (ddd, J = 0.9, 7.6, 7.8 Hz, 1H, H-

6), 8.14 (d, J = 9.2 Hz, 2H, H-3⁰⁰, H-5⁰⁰). ¹³C NMR (CDCl₃) d 15.2

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

3711

(SCH₃), 64.2 (OCH₃), 70.7 (C-3), 73.1 (C-4⁰), 83.9 (C-2), 113.2 (C-7),

4.2. Molecular modeling computational protocols

113.2 (C-2⁰⁰, C-6⁰⁰), 123.0 (C-4), 124.4 (C-5), 126.2 (C-3⁰⁰, C-5⁰⁰), 128.0

(C-3a), 130.3 (C-6), 139.8 (C-4⁰⁰), 148.5 (C-7a), 151.5 (C-1⁰⁰)₀, 163.4

(C-2⁰). NOESY correlations: OCH₃/H-7; H-4_b⁰/H-4_a⁰, H-2; H-4_a/H-4⁰_b,

H-4; H-2/H-4_b⁰, NH, H-2⁰⁰, H-6⁰⁰; NH/H-2, H-2⁰⁰, H-6⁰⁰; H-2⁰⁰, H-6⁰⁰/

H-2, NH, H-3⁰⁰, H-5⁰⁰; H-7/OCH₃, H-6; H-5/H-6, H-4; H-6/H-7, H-5;

H-4/H-5, H-3⁰⁰, H-5⁰⁰/H-2⁰⁰, H-6⁰⁰. EIMS m/z (relative intensity) 403

M⁺(1%), 371 (100%), 265 (19%), 233 (16%), 161 (35%), 160 (30%),

149 (56%), 117 (56%). Anal. Calcd for C₁₈H₁₈N₄O₃S₂requires: C,

53.71; H, 4.51; N, 13.92. Found: C, 53.95; H, 4.83; N, 14.12.

The 2D molecular structures drawn and saved in MDL mol

format were imported into Accelrys Discovery Studio³⁰in order

to convert them into 3D form and add hydrogens. The ‘clean

geometry’ option of the program was used to remove the un-

wanted short contacts. The 3D structures thus obtained were

saved in MDL mol format, consecutively. The geometries obtained

in this way were imported into ^MAESTRO(Schrodinger, LLC) and

submitted from there for ab initio minimization runs using the

JAGUAR program²⁰on DFT level with 6–31^**or cc-pVTZ-pp (mole-

cules with Br) basis sets. The geometries of the trans- and cis-dia-

stereoisomers were optimized. Different ring conformers were

generated taking into account the nonplanar geometry of the

ﬁve-membered rings. Conformational alterations resulting from

the sp³character of nitrogen also were considered, resulting thus

into multiple conformations for the particular molecules under

study. A comprehensive set of molecular parameters (in addition

4.1.4.11. trans- and cis-1-Methoxy-2-(4-triﬂuoromethylphe-

nylamino)-2⁰-(methylsulphanyl)spiro{indoline-3,5⁰-[4⁰,5⁰]dihy-

drotiazole} (21a and 21b). Following the general procedure

products 21a and 21b were obtained using 0.121 g (0.094 mL,

0.75 mmol) of 4-triﬂuoromethylaniline and separated on silica

gel (20 g, ethyl acetate/n-hexane 1:5). Fractions of 21a contained

small amount of 4-triﬂuoromethylaniline as an impurity which

was removed by repeated chromatography on silica gel (5 g,

dichloromethane/n-hexane 1:1). trans-1-Methoxy-2-(4-triﬂuorom-

ethylphenylamino)-2’-(methylsulphanyl)spiro{indoline-3,5’-[4’,5’]-

dihydrotiazole} (21a). Yield: 0.037 g (23%); white crystals; mp

114–116 °C (acetone–hexane); R_f= 0.13 (ethyl acetate/n-hexane

(1:5). IR (CHCl₃) m_max3414, 2970, 1700, 1607, 1556, 1528, 1456,

to the standard Lipinski’s parameters) was obtained from ^QIKPROP

(Schrodinger, LLC) calculations. The geometries generated in the

above mentioned way were used for pharmacophore modeling

using the ^PHASE(Schrodinger LLD) program. The experimental

IC₅₀were used in this part of the molecular modeling allowing

the evaluation of generated pharmacophores and consequent 3D

QSAR models.

1400, 1314, 1256, 1163, 1114, 1070, 991, 942, 735 cm^ꢀ1

¹H NMR

(CDCl₃) 2.47 (s, 3H, SCH₃), 3.80 (s, 3H, OCH₃), 3.95 (d,

J = 15.8 Hz,1H, H-4⁰_a), 4.53 (d, J = 9.9 Hz, 1H, NH), 4.78

(d, J = 15.8 Hz, 1H, H-4⁰_b), 5.30 (d, J = 9.9 Hz, 1H, H-2), 6.90 (d,

J = 8.5 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 7.00 (d, J = 7.8 Hz, 1H, H-7), 7.07 (ddd,

J = 1.0, 7.5, 7.6 Hz, 1H, H-5), 7.30 (ddd, J = 1.1, 7.6, 7.8 Hz, 1H, H-

6), 7.38 (d, J = 7.6 Hz, 1H, H-4), 7.44 (d, J = 8.5 Hz, 2H, H-3⁰⁰, H-5⁰⁰).

¹³C NMR (CDCl₃) d 15.3 (SCH₃), 64.4 (OCH₃), 69.7 (C-3), 69.9 (C-

4⁰), 89.8 (C-2), 113.8 (C-7), 113.9 (C-2⁰⁰, C-6⁰⁰), 121.1 (q,

J = 32.7 Hz, C-4⁰⁰), 124.1 (C-4), 124.3 (C-5), 124.9 (q, J = 270.6 Hz,

CF₃), 126.5 (C-3a), 126.9 (C-3⁰⁰, C-5⁰⁰), 130.1 (C-6), 149.3 (C-7a),

149.5 (C-1⁰⁰), 166.9 (C-2⁰). NOESY correlations: OCH₃/H-7; H-4⁰_a/

H-4⁰_b, H-4; NH/H-4⁰_b, H-2, H-2⁰⁰, H-6⁰⁰; H-4_b⁰/H-4_a⁰, NH; H-2/NH, H-

2⁰⁰,H-6⁰⁰; H-2⁰⁰, H-6⁰⁰/NH, H-2, H-3⁰⁰, H-5⁰⁰; H-7/OCH₃, H-6; H-5/H-

6, H-4; H-6/H-7, H-5; H-4/H-4⁰_a, H-5; H-3⁰⁰; H-5⁰⁰/H-2⁰⁰, H-6⁰⁰. EIMS

m/z (relative intensity) 426 M⁺(1%), 395 (100%), 265 (11%), 233

(11%), 161 (25%), 150 (22%), 117 (29%). Anal. Calcd for

C₁₉H₁₈F₃N₃OS₂requires: C, 53.63; H, 4.26; N, 9.88. Found: C,

53.91; H, 4.63; N, 9.57. cis-1-Methoxy-2-(4-triﬂuoromethylphenyla-

mino)-2’-(methylsulphanyl)spiro{indoline-3,5’-[4’,5’]dihydrotiazole}

(21b). Yield: 0.070 g (44%); yellow oil; R_f= 0.28 (ethyl acetate/n-

hexane (1:5). IR (CHCl₃) m_max3391, 2984, 2928, 1700, 1614,

1570, 1528, 1500, 1456, 1400, 1314, 1256, 1191, 1163, 1114,

4.3. Anticancer activity

4.3.1. Tumor cell lines

Jurkat (human T-cell acute lymphoblastic leukemia), HeLa (hu-

man cervical adenocarcinoma) MCF-7 (human breast adenocarci-

noma, estrogen receptor-positive), MDA-MB-231 (human breast

adenocarcinoma, estrogen receptor-negative) and A-549 cell lines

(human lung adenocarcinoma) were kindly provided by Dr. M.

Hajdúch (Olomouc, Czech Republic). CCRF-CEM cell line (human

T-cell acute lymphoblastic leukemia) was obtained from the Ger-

man Collection of Microorganisms and Cell Cultures (Braun-

schweig, Germany). The cells were routinely maintained in RPMI

1640 medium with L-Glutamine and HEPES (Jurkat, HeLa and

CCR-CEM) or Dulbecco’s modiﬁed Eagle’s medium with Gluta-

max-I (MCF-7, MDA-MB-231 and A-549) supplemented with 10%

fetal calf serum, penicillin (100 IU ꢁ mL^ꢀ1) and streptomycin

(100

g ꢁ mL^ꢀ1) (all from Invitrogen, USA), in humidiﬁed air with

5% CO₂at 37 °C. Before each cytotoxicity assay, cell viability was

determined by the trypan blue exclusion method and found to be

greater than 95%.

1070, 991, 942, 828, 735 cm^ꢀ^{1 1}H NMR (CDCl₃) d 2.53 (s, 3H,

SCH₃), 3.74 (s, 3H, OCH₃), 4.23 (d, J = 15.7 Hz, 1H, H-4⁰), 4.71 (d,

4.3.2. Cytotoxicity assay

J = 15.7 Hz, 1H, H-4⁰_a), 5.01 (d, J = 10.6 Hz, 1H, H-2), 5.16 (d,

J = 10.6 Hz, 1H, NH), 6.97 (d, J = 8.4 Hz, 2H, H-2⁰⁰, H-6⁰⁰), 6.98 (d,

J = 7.7 Hz, 1H, H-7), 7.07 (ddd, J = 1.1, 7.6, 7.6 Hz, 1H, H-5), 7.28

(d, J = 7.2 Hz, 1H, H-4), 7.31 (ddd, J = 1.2, 7.6, 7.7 Hz, 1H, H-6),

7.45 (d, J = 8.4 Hz, 2H, H-3⁰⁰, H-5⁰⁰). ¹³C NMR (CDCl₃) d 15.1

(SCH₃), 64.1 (OCH₃), 70.8 (C-3), 73.1 (C-4⁰), 84.6 (C-2), 113.2 (C-

7), 113.7 (C-2⁰⁰, C-6⁰⁰), 121.1 (q, J = 32.7 Hz, C-4⁰⁰), 122.9 (C-4),

124.1 (C-5), 124.7 (q, J = 270.5 Hz, CF₃), 126.7 (C-3⁰⁰, C-5⁰⁰), 128.5

(C-3a), 130.1 (C-6), 148.6 (C-1⁰⁰), 148.6 (C-7a), 163.4 (C-2⁰). NOESY

correlations: OCH₃/H-7; H-4⁰/H-4⁰, H-2; H-4⁰/H-4⁰, H-4; H-2/NH,

The cytostatic/cytotoxic effects of compounds was studied

using the colorimetric microculture assay with the MTT end-

point.³²Brieﬂy, 3 ꢁ 10³(A-549, MCF-7, MDA-MB-231), 5 ꢁ 10³

(HeLa) or 1 ꢁ 10⁴(Jurkat and CEM) cells were plated per well in

96-well polystyrene microplates (Sarstedt, Germany) in the culture

medium containing tested chemicals at ﬁnal concentrations of

1 ꢁ 10^ꢀ4, 5 ꢁ 10^ꢀ5, 1 ꢁ 10^ꢀ5and 1 ꢁ 10^ꢀ6mol ꢁ L^ꢀ1. After 72 h of

incubation, 10

L of MTT (5 mg ꢁ mL^ꢀ1) (Sigma, Germany) were

added in each well. After an additional 4 h, during which insoluble

formazan was produced, 100 lL of 10% sodium dodecylsulphate

H-4⁰_b, H-2⁰⁰, H-6⁰⁰; NH/H-2, H-2⁰⁰, H-6⁰⁰; H-2⁰⁰, H-6⁰⁰^b/H-3⁰⁰, H-5⁰⁰; H-

were added in each well and another 12 h were allowed for the dis-

solution of formazan. Absorbance was measured at 540 nm using

the automated MRX microplate reader (Dynatech Laboratories,

UK). The blank-corrected absorbance of the control wells was ta-

ken as 100% and the results were expressed as a percentage of

the control.

7/OCH₃, H-6; H-5/H-4, H-6; H-6/H-5, H-7; H-4/H-4⁰, H-5. EIMS

m/z (relative intensity) 426 M⁺(2%), 395 (100%), 265 (10%), 233

(9%), 161 (29%), 150 (31%), 145 (28%), 117 (32%). Anal. Calcd for

C₁₉H₁₈F₃N₃OS₂requires: C, 53.63; H, 4.26; N, 9.88. Found: C,

53.89; H, 4.02; N, 9.63.

3712

P. Kutschy et al. / Bioorg. Med. Chem. 17 (2009) 3698–3712

4.4. Data processing and analysis

6 mm) were layered onto the inoculated Petri dishes and soaked

with tested substances or antibiotics dissolved in DMSO (20 g/

2.5 l/disk). Microorganisms were cultured with tested substances

IC₅₀values (concentrations of tested agents that inhibited

growth of cell cultures to 50% of the untreated control) were deter-

mined by GraphPad Prism for Windows version 5.01 (GraphPad

Software, Inc.). Values of IC₅₀were not determined for a given com-

pound and cell line if the growth inhibition was less than 50% at

for 24 h at 37 °C. The antimicrobial activity was evaluated as a

diameter of inhibition zone around the disk with tested compound.

All experiments involved clinically used antimicrobial substances

(amphotericin purchased from Sigma, USA and cefotaxime from

the highest tested concentration 100

respective IC₅₀values were recorded in Table 2 as 100

mol ꢁ L^ꢀ1. In such cases,

Lek, a.s. Lublana, Slovenia) and solvent (DMSO) as control.

mol ꢁ L^ꢀ1

IC₅₀values were analyzed using an approach similar to that em-

Acknowledgments

ployed with the ^COMPAREalgorithm.²⁴Speciﬁcally, growth inhibitory

proﬁles were created for each compound using their (ꢀlog IC₅₀

)

This work was supported by the Slovak Research and Develop-

ment Agency under the Contract No. APVV-0514-06 and Slovak

Grant Agency for Science, Grant No. 1/3553/06. This work was also

supported by the State NMR Programme No. 2003SP200280203.

Authors thank Dr. DeEtte Walker from Georgia Institute of Tech-

nology, School of Biology, Atlanta, GA, USA, for reviewing the

manuscript.

values for speciﬁc cell lines after subtraction of their mean

(ꢀlog IC₅₀) values across all cell lines. Similarities of their proﬁles

with proﬁles of the conventional anticancer agents doxorubicin,

etoposide and cisplatin were subsequently determined by Spotﬁre

DecisionSite 9.1 (TIBCO Software, Palo Alto, CA, USA), and their

strength was expressed as a correlation coefﬁcient. Classiﬁcation

of tested compounds according to their activity patterns against

cancer cell lines was accomplished by hierarchical clustering sim-

ilar to previously reported procedure.²⁶Clustering algorithm was

applied to (ꢀlog IC₅₀) values for each compound after subtraction

of mean (ꢀlog IC₅₀) for that compound across all tested cell lines

using Spotﬁre DecisionSite 9.1. The following clustering parame-

ters were applied: WPGMA method (Weighted Pair-Group Method

using Arithmetic averages), correlation (similarity measure), and

average value (ordering function).

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) or inactive

mers and classiﬁed active (IC₅₀< 90

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(IC₅₀> 90

9489.

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4.6. Antimicrobial activity

Bacterial strains used in the present study were obtained from

the Czech Collection of Microorganisms (CCM, Czech Republic)

and involved Pseudomonas aeruginosa (CCM 162/78, ATCC 27853),

Enterococcus faecalis (CCM 4224), Escherichia coli (CCM 326/71,

ATCC 10418), Staphylococcus aureus (CCM 4223, ATCC 29213), S.

pyogenes (CCM 4425) and B. subtilis (CCM 4062). Candida albicans

(ATCC 60193) was obtained from the American Type Culture Col-

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isms on blood agar was used in order to prepare the inoculum

(1.5 ꢁ 10⁸CFU/ml; 0.5 McFarland). The experiments were carried

out in Petri dishes of 80 mm diameter. Inoculum was prepared in

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