The synthesis and anticancer activity of analogs of the indole phytoalexins brassinin, 1-methoxyspirobrassinol methyl ether and cyclobrassinin This work is dedicated to the memory of Associated Professor Peter Kutschy (1953-2012)

Article abstract of DOI:10.1016/j.bmc.2013.08.020

An effective synthesis of analogs of the indole phytoalexin cyclobrassinin with NR₁R₂ group instead of SCH₃ was developed starting from indole-3-carboxaldehyde. The target compounds were prepared by spirocyclization of 1-Boc-thioureas with the formation of isolable spiroindoline intermediates, followed by the trifluoroacetic acid-induced cascade reaction consisting of methanol elimination, deprotection and rearrangement of the iminium ion. The structures of novel products were elucided by the ¹H and ¹³C NMR spectroscopy, including HMBC, HSQC, COSY, NOESY and DEPT measurements. Several newly synthesized compounds demonstrated significant antiproliferative/cytotoxic activity against human leukemia and solid tumor cell lines, as well as remarkable selectivity of these effects against cancer cells relative to the non-malignant HUVEC cells.

Full text of DOI:10.1016/j.bmc.2013.08.020

Accepted Manuscript
The synthesis and anticancer activity of analogs of the indole phytoalexins bras‐
sinin, 1-methoxyspirobrassinol methyl ether and cyclobrassinin
Mariana Budovská, Martina Pilátová, Lenka Varinská, Ján Mojžiš, Roman
Mezencev
PII:
S0968-0896(13)00702-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.020
BMC 11036
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
29 April 2013
6 August 2013
8 August 2013
Please cite this article as: Budovská, M., Pilátová, M., Varinská, L., Mojžiš, J., Mezencev, R., The synthesis and
anticancer activity of analogs of the indole phytoalexins brassinin, 1-methoxyspirobrassinol methyl ether and
cyclobrassinin, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.08.020
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The synthesis and anticancer activity of analogs of the indole phytoalexins brassinin, 1-
methoxyspirobrassinol methyl ether and cyclobrassinin.
Mariana Budovskáa*, Martina Pilátováb, Lenka Varinskáb, Ján Mojžišb and Roman Mezencevc
aDepartment of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science, P. J.
Šafárik University, Moyzesova 11, 040 01, Košice, Slovak Republic
bDepartment of Pharmacology, Faculty of Medicine, P. J. Šafárik University, SNP 1, 040 66
Košice, Slovak Republic
cGeorgia Institute of Technology, College of Science, School of Biology, 310 Ferst Drive,
Atlanta, GA 30332, USA
*Corresponding author. Tel.: +421552341196;
E-mail address: mariana.budovska@upjs.sk
This work is dedicated to the memory of Associated Professor Peter Kutschy (1953-2012).
Abstract
An effective synthesis of analogs of the indole phytoalexin cyclobrassinin with NR1R2 group
instead of SCH3 was developed starting from indole-3-carboxaldehyde. The target compounds
were prepared by spirocyclization of 1-Boc-thioureas with the formation of isolable
spiroindoline intermediates, followed by the trifluoroacetic acid-induced cascade reaction
consisting of methanol elimination, deprotection and rearrangement of the iminium ion. The
1
structures of novel products were elucided by the H and 13C NMR spectroscopy, including
HMBC, HSQC, COSY, NOESY and DEPT measurements. Several newly synthesized
compounds demonstrated significant antiproliferative/cytotoxic activity against human
leukemia and solid tumor cell lines, as well as remarkable selectivity of these effects against
cancer cells relative to the non-malignant HUVEC cells.
Keywords: cyclobrassinin, phytoalexins, indoles, antiproliferative activity, spirocyclization
1. Introduction
Phytoalexins are low molecular weight secondary metabolites with antimicrobial properties,
produced by plants after their exposure to physical, chemical or biological stress. While these
compounds are not present in healthy plant tissues, their accumulation is an induced defense
response associated with plant disease resistance.1,2 A specific group of these natural products
is represented by indole phytoalexins from economically important plants of the family
Cruciferae.2,3
Basic structures of indole phytoalexins are characterized by an indole ring variably substituted
at positions 2 and/or 3 with nitrogen and sulfur-containing substituents. For example,
brassinin
methoxyspirobrassinin
(1),
1-methoxybrassinin
(2),
(S)-(−)-spirobrassinin
(5),
(3),
(R)-(+)-1-
(4), 1-methoxyspirobrassinol
trans-(2R,3R)-(−)-1-
methoxyspirobrassinol methyl ether (6), cyclobrassinin (7), sinalbin B (8), and rutalexin (9,
Fig. 1) are typical representatives of naturally occurring cruciferous phytoalexins of
dithiocarbamate, spiroindoline[3,5']thiazolidine, and thiazino[6,5-b]indole type.2,3
Due to their limited availability from plant materials, biological activity of many indole
phytoalexins, including spiroindoline phytoalexins and cyclobrassinin could not have been
investigated before synthetic strategies for these compounds have been developed. Among
these synthetic strategies, Hugerschoff's cyclization of brassinin (1) with pyridinium
1

tribromide4, N-bromosuccinimide5, dioxane dibromide6 or phenyltrimethylammonium
tribromide7 has been shown to yield natural cyclobrassinin (7). Likewise, the cyclization of
isobrassinin with phenyltrimethylammonium tribromide gave isocyclobrassinin, a regioisomer
of natural indole phytoalexin cyclobrassinin.8 Furthermore, a dioxane dibromide (DDB)-
mediated spirocyclization of 1-methoxybrassinin (2) was developed as a synthetic route to 1-
methoxyspiroindoline phytoalexins.6 In this reaction, water or methanol were originally used
as nucleophiles for the synthesis of diastereoisomers of 1-methoxyspirobrassinol (5) and 1-
methoxyspirobrassinol methyl ether (natural trans-[( )-6a] and unnatural cis-[( )-6b]) from
1-methoxybrassinin (2).6 More recently, the DDB-mediated spirocyclization of 1-Boc- and 1-
methoxybrassinin (2) in the presence of amines afforded corresponding 2-aminoanalogs of 1-
Boc or 1-methoxyspirobrassinol.9-11
Figure 1. Structures of selected indole phytoalexins
Besides their antibacterial, antifungal3 and antiprotozoal12 properties, the indole phytoalexins
and their derivatives have also been shown to display anticancer properties (reviewed in
refs13, 14). Specifically, brassinin and spirobrassinin reportedly inhibited growth and viability
of murine leukemia L1210 and melanoma B16 cells in vitro,15 as well as the formation of
7,12-dimethylbenz[a]anthracene (DMBA)-induced precancerous lesions in mouse mammary
gland organ model.16 In addition to these antiproliferative and anticarcinogenic properties,
brassinin was found to inhibit indoleamine 2,3-dioxygenase (IDO, EC 1.13.11.42), a
tryptophan-catabolizing enzyme that promotes tumor escape via mechanisms of immune
tolerance.17 The role of the inhibition of IDO in the antitumor mechanism of 5-bromobrassinin
(a bioavailable analog of brassinin) was confirmed in vivo, where this compound suppressed
growth of B16-F10 melanoma xenografts in C57BL/6 mice but not in athymic NCr-nu/nu and
IDO knock-out mice.18 Similarly, cyclobrassinin16,19 and 1-methoxyspirobrassinol methyl
ether20 have also been previously reported to exert anticancer properties through
antiproliferative/cytotoxic and/or cancer chemopreventive activity.
Since these natural compounds and their analogs can serve as lead compounds for the
development of new biologically active agents, research into their synthesis is warranted and
in this paper we report on the synthesis and anticancer activity of new analogs of the indole
phytoalexin cyclobrassinin with NR1R2 group instead of SCH3 (10). Retrosynthesis of these
analogs (Fig. 2) led via thioureas 12 to indole-3-carboxaldehyde (13), a suitable starting
compound.
Figure 2. Retrosynthetic analysis
2. Results and discussion
2.1 Chemistry
The key intermediate, 1-[(tert-butoxycarbonyl)indol-3-yl]methylamine (15), was prepared by
nickel chloride-catalyzed reduction of 1-(tert-butoxycarbonyl)indol-3-carboxaldehyde oxime
(14) with sodium borohydride as previously described.21 Unstable oily amine 15 was used as
a crude product immediately in the next reaction.
N-[1-(tert-butoxycarbonyl)indol-3-yl]methyl-N’-phenylthiourea (17) and N’-(substituted
phenyl)thioureas 18-25 were synthesized via the reaction of amine 15 with commercially
available phenyl isothiocyanate or substituted phenyl isothiocyanates, respectively, in
dioxane-chloroform (2:1 V/V) with 5 equivalents of triethylamine as a base (Scheme 1). After
12-hour reaction, the products were isolated and purified by column chromatography on silica
2

gel and crystallization from dichloromethane/hexane. Thioureas 17-25 were obtained in 44-
52% yields after two reaction steps starting from oxime 14.
The starting point in the synthesis of thioureas 26-30 was the preparation of [1-(tert-
butoxycarbonyl)indol-3-yl]methyl isothiocyanate (16) by treatment of amine 15 with
thiophosgene following previously reported procedure.21 Brassinin derivatives 26-30 were
synthesized by the reaction of isothiocyanate 16 with 2 equivalents of corresponding amines,
in dioxane (Scheme 1). Starting isothiocyanate 16 was consumed in reaction within 20
minutes except for the synthesis of thiourea 26, in which case the reaction required 24 hours.
The reactions afforded products that could be efficiently purified by crystallization from
dichloromethane/hexane in 73-83% yields. Using this approach, thiourea 18 was prepared
from 4-bromoaniline in 77% yield; however, the reaction required 24 hours. For this reason,
thioureas 17-25 were synthesized via the reactions of amine 15 with corresponding
isothiocyanates in order to reduce the reaction time and the number of reaction steps.
Scheme 1.
In the next step we investigated the bromine-mediated spirocyclization of thioureas 17-30
with water or methanol as nucleophiles, which produced derivatives of natural spiroindoline
phytoalexins 1-methoxyspirobrassinol methyl ether 31-44 and 1-methoxyspirobrassinol 45-
46. The reactions were performed under the spirocyclization conditions in dry
dichloromethane using bromine (1.1 eq.) in the presence of methanol (10%) or water (10%).
The reaction mixture was stirred for 15 min at room temperature and triethylamine was added
thereafter to trap HBr liberated during the reaction. As previously suggested, the reaction
likely starts at the thiocarbamoyl functionality with the formation of sulfenyl bromide A, in
which the electrophilic sulfur attacks the 3-position of indole to provide a 1-Boc-
spiroindoleninium intermediate B.6 The iminium ion B, after its reaction with corresponding
nucleophiles, afforded a diastereomeric mixture of products ( )-31a-( )-46b (Scheme 2). As
shown in Table 1, the spirocyclization of 17-30 led to the preferential formation of trans-
diastereoisomers ( )-31a-( )-46a. This preference was also observed in the spirocyclization
reactions of 1-Boc- and 1-methoxycarbonylbrassinin using bromine in the presence of water
that always afforded trans-diastereoisomers as the major products.6,22
Individual diastereoisomers ( )-31a,( )-31b-( )-44a,( )-44b were separated by
chromatography on silica gel. A suitable eluent for the separation of diastereoisomers was the
mixture of dichloromethane/ethyl acetate/ammonia in various ratios. The major trans-
diastereoisomers ( )-31a-( )-44a were obtained in 43-71% yields and cis-diastereoisomers
( )-31b-( )-44b were isolated as minor products in 8-17% yield (Table 1). cis-Isomer ( )-35b
was obtained as a mixture with trans-diastereoisomer ( )-35a in a 50:50 ratio. cis-Product
( )-37b was isolated as a mixture with trans-diastereoisomer ( )-37a in a 76:24 ratio.
Diastereoisomers ( )-35a, ( )-35b and ( )-37a, ( )-37b had very similar Rf values in various
eluents, which prevented their separation. Synthesized compounds ( )-31a-( )-44b are stable
substances that did not undergo isomerization during the TLC.
Spirocyclization of thiourea 17 in dichloromethane with water as a nucleophile yielded the
mixture of trans- a cis-diastereoisomers ( )-45a and ( )-45b in a 67:33 ratio (Scheme 2,
Table 1). Diastereoisomers ( )-45a and ( )-45b were not separable by column
chromatography on silica gel, in spite of large differences in their Rf values, due to their
isomerization during the separation on silica gel. After removal of side products by column
chromatography, the mixture of diastereoisomers ( )-45a, ( )-45b was obtained in 67:33 ratio
favoring trans-isomer. In this case, only the trans-isomer ( )-45a was obtained as a pure
substance by crystallization from acetone/hexane in 29% yield. Small amount of cis-
diastereoisomer ( )-45b remained in mother liquor and it was not isolated. We assume that
3

compound 45 exists in solution as a mixture of diastereoisomers ( )-45a and ( )-45b in a
67:33 ratio at equilibrium owing to its unstable hemiaminal structure analogous to 1-
methoxyspirobrassinol (5).23 The less soluble trans-diastereoisomer ( )-45a crystallized, and
the concentration of cis-diastereoisomer ( )-45b in the solution has subsequently decreased
due to its isomerization. The isomerization of diastereoisomers was also observed and
confirmed on the TLC plates, when the mixture of diastereoisomers ( )-45a, ( )-45b was
spotted on the TLC and the plate was developed. After 1 hour, the plate was turned 90
degrees and developed in the perpendicular direction. From the original spot of cis-
diastereoisomer ( )-45b, two spots corresponding to trans- and cis-diastereoisomers ( )-45a,
( )-45b emerged after the 2D-TLC. In contrast, the original spot of trans-diastereoisomer ( )-
45a has not developed into multiple spots after 2D-TLC.
N´-methyl-N´-phenyl derivative of spiroindoline phytoalexin 1-methoxyspirobrassinol 46,
consisted of stable and well separable trans-( )-46a (57%) and cis-( )-46b (16%)
diastereoisomers (Scheme 2, Table 1).
1
In all cases, the ratio of diastereoisomers was determined from the analysis of H NMR
spectra of crude reaction products by integration of separated signals corresponding to the H-
2, Ha and Hb protons. In trans-diastereoisomer, the sulfur of the thiazoline ring and the 2-
methoxy or 2-hydroxy group are located on the opposite sides of the indoline ring, while in
the cis-diastereoisomer the sulfur and 2-methoxy or 2-hydroxy groups are on the same side.
Scheme 2.
Table 1. Yields and ratios of trans- and cis-diastereoisomers ( )-31a-( )-46b produced via
Scheme 2
The structures of diastereoisomers ( )-31a-( )-46b were confirmed by NMR methods,
including 2D HSQC, HMBC, COSY and NOESY experiments. In the NOESY spectra of cis-
diastereoisomers (Table 2), the interaction between Hb and H-2 protons was expected, while
in the trans-diastereoisomers, the expected interaction involved Hb and OCH3 or OH protons.
Derivatives ( )-31b-( )-46b displayed in their NOESY spectra the cross peak between H-2
and Hb protons, which supports their cis-diastereoisomeric structure (Table 2). While the
spectra of compounds ( )-31a-( )-46a did not show the interaction between Hb and OCH3 or
OH protons, which would be consistent with their trans-configuration, the interactions
between H-2 and Hb protons were not observed as well. For this reason, these products were
assigned trans-distereoisomeric structures.
1
Examination of the H NMR spectra of spiroindoline[3,5']thiazolidine-type compounds
revealed a significant difference in the chemical shifts between the H-2 protons of trans- and
cis-diastereoisomers. In these compounds, the δ(H-2)trans appeared at lower field compared to
1
δ(H-2)cis in deuterated chloroform, dimethysulfoxide or acetone.6,11,22 Examination of H
NMR spectra of derivatives 31a–46b confirmed this relationship (Table 2). 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. As a result, the chemical
shifts of H-2 protons can be used for the assignment of diastereoisomeric structure of 1-
methoxyspirobrassinol methyl ether analogs without need to examine the Nuclear Overhauser
Effects (NOE).
Table 2. Chemical shifts of H-2 protons of cis- and trans-diastereoisomers ( )-31a-( )-46b
Natural cyclobrassinin (7) was prepared in a high yield by oxidative Hugerschoff´s
bromocyclization of brassinin (1) using phenyltrimethylammonium tribromide.7 However,
4

attempts to cyclize phenylthiourea with unsubstituted indole nitrogen into thiazino[6,5-
b]indole 48 using various bromination reagents (e.g. NBS, Br2, DDB,
phenyltrimethylammonium tribromide) failed and resulted in decomposition of starting
compound. After several unsuccessful attempts to prepare an analog of cyclobrassinin 48 via
direct cyclization of thiourea unsubstituted on the indole nitrogen, we have developed another
synthetic route, which exploited transformation of easily available spirocyclic derivatives 31-
44 into analogs of cyclobrassinin 48-61 in the presence of 20 equivalents of trifluoroacetic
acid (TFA). Spiroindoline intermediates 31-44 undergo trifluoroacetic acid-induced cascade
of reactions that includes elimination of methanol, removal of the Boc group and
rearrangement of iminium ion C, and results in the formation of analogs of cyclobrassinin 48-
61. The reaction cascade has run smoothly in dry dichloromethane at room temperature for
1.5 hour and the sole reaction products 48-61 were obtained in 60-78% yields (from thioureas
17-30 after 2 reaction steps, Scheme 3). Thiazino[6,5-b]indole 48 was also prepared by TFA-
induced rearrangement from analog of spirobrassinol 45 in the same yield and with the same
reaction time from corresponding analog of methyl ether 31.
We also attempted to synthesize an analog of cyclobrassinin 48 without isolation of
intermediate spiro compound 31 by direct addition of TFA to the reaction mixture. This
method did not afford compound 48 and the reaction mixture after 24 hours contained only
the spiro compound 31, probably due to the backward addition of methanol on the iminium
ion, since the reaction mixture contained excess of methanol.
Scheme 3.
2.2 Antiproliferative/cytotoxic activity
The antiproliferative/cytotoxic activities of newly synthesized compounds and conventional
anticancer agents doxorubicin, etoposide and cisplatin were examined on the panel of 6
human cancer cell lines Jurkat (acute T-lymphoblastic leukemia), MCF-7 (mammary gland
adenocarcinoma), MDA-MB-231 (mammary gland adenocarcinoma), HeLa (cervical
adenocarcinoma), CCRF-CEM (acute T-lymphoblastic leukemia), and A-549 (non-small cell
lung cancer). The potency of these compounds was determined using MTT (Thiazolyl Blue
Tetrazolium Bromide) assay after 72 h incubation of cells and presented in tables 3-5 as IC50
values (concentration of a given compound that decreased amount of viable cells to 50%
relative to untreated control cells). To allow comparisons, table 3 also includes IC50 values for
brassinin (1), 1-methoxybrassinin (2) and 1-Boc-brassinin (62), whose syntheses were
published previously.11,21
Dithiocarbamate compound Boc-brassinin (62) displays higher potency of
antiproliferative/cytotoxic activity than its corresponding thiourea analog 27 against all tested
cancer cell lines suggesting that thiourea derivatives of Boc-substituted indole phytoalexins
are less potent than their corresponding dithiocarbamate bioisosteres. Nevertheless, several
newly synthesized compounds display comparable or higher in vitro potency than traditional
anticancer agents cisplatin and etoposide (Table 3). Compound 17 exhibited remarkable
potency against MCF-7 breast cancer cell line with an IC50 value in sub-micromolar range,
comparable to that of doxorubicin. Overall, compounds 17-30 displayed higher potency on
leukemia than on solid tumor cell lines (median IC50 ~14 µM and ~26 µM respectively) and
the estrogen receptor (ER)-positive MCF-7 cell line displayed higher or comparable
susceptibility to these compounds than the ER-negative breast cancer cell line MDA-MB-231.
Preliminary QSAR analysis in the series of thiourea derivatives suggests that the lack of
activity of methyl 27 and morpholine-4-yl 30 derivatives on Jurkat cells may be related to the
low overall lipophilicity (log P) of these two compounds. Furthermore, potency in the series
5

of substituted phenylthiourea derivatives on Jurkat cells tends to increase with increasing
values of Hammett’s electronic descriptor ꢀ suggesting a positive role of electron-
withdrawing substituents on the benzene ring in the anticancer potency of evaluated
compounds (data not shown).
Table 3. Antiproliferative activities of brassinin (1) and its analogs 2, 17-30, 62
Several new analogs of 1-methoxyspirobrassinol methyl ether 31-44 or 1-
methoxyspirobrassinol 44-45 demonstrated higher antiproliferative/cytotoxic potency than
natural phytoalexin ( )-6a on at least one evaluated cancer cell line (Table 4).
In addition, compounds (±)-38b, ( )-40a, ( )-42a and ( )-46a are significantly more potent on
Jurkat cells than anticancer agent cisplatin, whose IC50 value on this cell line is at least 2 ×
higher. Similarly, compounds ( )-40a and ( )-46a have significantly higher potencies than
etoposide on MCF-7 cells.
Table
4 also includes IC50 values for trans- and cis-diastereoisomers of 1-
methoxyspirobrassininol methyl ether [(±)-6a) and (±)-6b)], 1-methoxyspirobrassininol [(±)-5],
1-Boc-spirobrassininol methyl ether [(±)-63a) and (±)-63b)] and 1-Boc-spirobrassininol [(±)-
47a) and (±)-47b)] synthesized and reported previously.6,24
Table 4. Antiproliferative activities of 1-methoxyspirobrassinol methyl ether (6) and its
analogs 31-47
The replacement of 2-methylthio group in natural indole phytoalexin cyclobrassinin (7) with
RNH- moiety generally increased anticancer potency relative to the parental compound 7
(Table 5). The lower potency of compound 57 relative to compound 48 and other analogs
implies the importance of NH-group on the anticancer activity in this series of compounds.
Similarly, the presence of NH-group appears to be important for anticancer activity in the
series of brassinine-derived thioureas (compound 26 vs 17). Compounds 58 and 59 are about
2 times more potent than cisplatin on Jurkat cells and compounds 49-54 are at least 2 ×more
potent than etoposide on MDA-MB-231 cells.
Table 5. Antiproliferative activities of cyclobrassinin (7) and its analogs 48-61
The cytotoxic effect of synthesized compounds 48-56 was also examined on human
nonmalignant endothelial cells HUVEC (human umbilical vein endothelial cells, Table 6).
Selectivity index (SI) of a given compound is a measure of its selective toxicity for cancer cell
lines relative to nonmalignant cells. In this report, the selectivity index (SI) of a tested
compound is calculated as its IC50 for HUVEC cells divided by its IC50 for individual cancer
cell lines. Higher SI values for a given compound are indicative of higher resistance of non-
transformed cells relative to cancel cell lines. The results demonstrate that all derivatives 48-
54, except for chloro-analog 51, have higher selectivity index than cyclobrassinin (7) for all
tested cancer cell lines. The highest selectivity index was found on CCRF-CEM cells for 4-
fluorophenyl analog 50 (SI = 24.7) and 4-methylphenyl analog 53 (SI = 20), but the
compound 50 displayed lower selectivity of toxicity on other cell lines in comparison with 4-
methylphenyl analog 53. The values of SI for compound 51 suggest that this compound is
equally or more toxic on HUVEC cells than on tested cancer cell lines. Overall, compound 53
possesses high SI values across all cell lines (SI≥3, Table 6) and, at the same time, this
compound displays reasonably good potency in low micromolar range for 3 cancer cell lines
(Jurkat, MDA-MB-231 and CCRF-CEM, Table 5).
6

We have determined SI values for anticancer agents cisplatin, etoposide and doxorubicin and
added these data to the table 6. For the reference, the values of selectivity index (SI) for
anticancer agents paclitaxel and combretastatin A-4 on MCF-7 are also presented in table 6.25
Table 6. Selectivity index of cyclobrassinin (7) and its analogs 48-56
3. Conclusion
This paper reports on the synthesis of analogs of cyclobrassinin with NR1R2 group instead of
SCH3 48-61. Their synthesis was accomplished by bromine-mediated spirocyclization of 1-
Boc-thioureas 17-30 to spiroindoline intermediates 31-44, which subsequently underwent
trifluoroacetic acid-induced cascade of reactions, which included methanol elimination,
deprotection, and rearrangement, and lead to the formation of target analogs of cyclobrassinin
48-61. Some of these newly synthesized compounds exhibited significant
antiproliferative/cytotoxic activity against human cancer cell lines Jurkat (acute T-
lymphoblastic leukemia), MCF-7 (mammary gland adenocarcinoma), MDA-MB-231
(mammary gland adenocarcinoma), HeLa (cervical adenocarcinoma), CCRF-CEM (acute T-
lymphoblastic leukemia), or A-549 (non-small cell lung cancer). Substance 17, which was
found to be the most potent among all tested compounds on the MCF-7 cells, displays very
close potency to that of doxorubicin on these cells. Replacement of the 2-methylthio moiety
of cyclobrassinin with Ar-NH group resulted in the considerable increase of potency relative
to parental compound.
4. Experimental
4.1 Chemistry
Melting points were determined on a Koffler micro melting point apparatus and are
not corrected. IR spectra were recorded on an IR-75 spectrometer (Zess Jena). 1H NMR (400
MHz) and 13C NMR (100 MHz) spectra were measured on a Varian Mercury Plus
spectrometer. Chemical shifts (ꢁ) are reported in ppm downfield from TMS as an internal
standard and coupling constans (J) are given in Hertz. Microanalyses were performed with a
Perkin-Elmer, Model 2400 analyzer. The EI mass spectra were recorded on a GS-MS Trio
1000 (Fisons Instruments) spectrometer at ionization energy of 70 eV, whereas MALDI-TOF
mass spectra of 17, 26, ( )-31a, ( )-31b, ( )-40a, ( )-40b, ( )-45a, ( )-46a and ( )-46b were
measured on a MALDI IV (Shimazu, Kratos Analytical). The samples were ionized with a
N2-laser (ꢂ = 337 nm). The progress of chemical reactions was monitored by thin layer
chromatography (TLC), using Macherey-Nagel plates Alugram® Sil G/UV254. Preparative
column chromatography was performed on Kieselgel 60 Merck Type 9385 (0.040-0.063).
4.1.1 General procedure for the synthesis of thioureas 17-25
Crude product of 1-[(tert-butoxycarbonyl)indol-3-yl]methylamine (15) freshly
prepared by the reduction of 1-(tert-butoxycarbonyl)indole-3-carboxaldehyde oxime21 (14;
0.780 g, 3 mmol) with NaBH4 (1.11 g, 30 mmol) in the presence NiCl2.6H2O (0.710 g, 3
mmol) was dissolved in dioxane/chloroform (20 mL/10 mL). Triethylamine (2.10 mL, 1.53 g,
15 mmol) and corresponding phenyl isothiocyanate or substituted phenyl isothiocyanate (3
mmol) were added and mixture was stirred for 12 h at room temperature. The residue
obtained after evaporation of the solvent was dissolved in acetone, small amount of silica gel
was added, acetone evaporated and product preabsorbed on silica gel was chromatographed
7

on silica gel (20 g, n-hexane/acetone 3:1). The obtained compounds were further crystallized
from dichloromethane/n-hexane to afford pure products 17-25.
4.1.1.1 N-[1-(tert-Butoxycarbonyl)indol-3-yl]methyl-N’-phenylthiourea (17). Following
the general procedure, product 17 was obtained using 0.406 g (0.359 mL, 3 mmol) of phenyl
isothiocyanate. Yield: 0.546 (47%); white crystals; mp 130-132 °C (dichloromethane/n-
1
hexane); Rf = 0.31 (n-hexane/acetone 3:1). IR (CHCl3) ꢃmax 3400, 1727, 1520 cm-1. H NMR
(CDCl3): ꢁ 8.16 (bs, 2H, NH-Ph, H-7), 7.62 (d, J = 7.7 Hz, 1H, H-4), 7.52 (s, 1H, H-2), 7.35-
7.31 (m, 3H, H-6, H-3´, H-5´), 7.27-7.20 (m, 2H, H-4´, H-5), 7.16-7.14 (m, 2H, H-2´, H-6´),
6.16 (bs, 1H, NH-CH2), 4.99 (d, J = 4.6 Hz, 2H, CH2), 1.66 [s, 9H, C(CH3)3]. 13C NMR
(CDCl3): ꢁ 180.5 (C=S), 149.5 (C=O), 135.9 (C-1´), 135.7 (C-7a), 130.3 (C-3´, C-5´), 129.2
(C-3a), 127.4 (C-4´), 125.2 (C-2´, C-6´), 124.9 (C-6), 124.4 (C-2), 123.0 (C-5), 119.3 (C-4),
116.4 (C-3), 115.4 (C-7), 84.0 [C(CH3)3], 41.2 (CH2), 28.2 [C(CH3)3]. MALDI-TOF MS, m/z
(relative intenzity): 420.20 [M+K]+ (20%), 404.20 [M+Na]+ (55%), 381.75 [M+H]+ (55%).
Anal. Calcd for C21H23N3O2S requires: C, 66.12; H, 6.08; N, 11.01. Found: C, 65.89; H, 6.16;
N, 11.24.
4.1.2 General procedure for the synthesis of thioureas 26-30
To a stirred solution of corresponding amine (4 mmol) in dioxane (20 mL) was added
triethylamine (2.68 mL, 2.02 g, 20 mmol) and mixture was stirred for 5 min at room
temperature. Then [1-(tert-butoxycarbonyl)indol-3-yl]methyl isothiocyanate21 (16; 0.576 g, 2
mmol) was added and stirring was continued for 20 min. The residue obtained after
evaporation of the solvent was dissolved in dichloromethane (30 mL), washed with 1M HCl
(30 mL), water (30 mL) and dried over Na2SO4. The obtained compounds were further
crystallized from dichloromethane/n-hexane to afford pure product 26-30.
4.1.2.1 N-[1-(tert-Butoxycarbonyl)indol-3-yl]methyl-N’-methyl-N’-phenylthiourea (26).
Following the general procedure, product 26 was obtained using 0.429 g (0.432 mL, 4 mmol)
of N-methylaniline. Yield: 0.649 g (82%); white crystals; mp 138-142 °C (dichloromethane/n-
1
hexane); Rf = 0.34 (n-hexane/ethyl acetate 5:1). IR (CHCl3) ꢃmax 3410, 1730, 1513 cm-1. H
NMR (CDCl3): ꢁ 8.08 (d, J = 7.7 Hz, 1H, H-7), 7.48-7.47 (m, 1H, H-4), 7.41-7.36 (m, 3H, H-
2, H-3´, H-5´), 7.32-7.28 (m, 2H, H-6, H-4´), 7.22-7.17 (m, 3H, H-5, H-2´, H-6´), 5.49 (bs,
1H, NH), 4.92 (d, J = 4.9 Hz, 2H, CH2), 3.71 (s, 3H, CH3), 1.64 [s, 9H, C(CH3)3]. 13C NMR
(CDCl3): ꢁ 182.1 (C=S), 149.7 (C=O), 142.9 (C-1´), 135.8 (C-7a), 130.8 (C-3´, C-5´), 129.4
(C-3a), 128.4 (C-4´), 127.2 (C-2´, C-6´), 124.9 (C-6), 124.2 (C-2), 122.9 (C-5), 119.0 (C-4),
117.2 (C-3), 115.5 (C-7), 84.0 [C(CH3)3], 43.7 (CH3), 41.8 (CH2), 28.4 [C(CH3)3]. MALDI-
TOF MS, m/z (relative intenzity): 418.94 [M+Na]+ (26%), 396.09 [M+H]+ (68%). Anal.
Calcd for C22H25N3O2S requires: C, 66.81; H, 6.37; N, 10.62. Found: C, 67.02; H, 6.21; N,
10.77.
4.1.3 General procedure for the synthesis of trans- and cis-diastereoisomers of analogs of
1-methoxyspirobrassinol methyl ether 31a-44a and 31b-44b
To a stirred solution of corresponding thiourea (0.6 mmol) in a mixture of anhydrous
dichloromethane/methanol (9 mL/1 mL) at room temperature was added freshly prepared
solution of bromine (1.53 mL, 0.66 mmol; the stock solution prepared by dissolving of 0.04
mL of bromine in 1.76 mL of anhydrous dichloromethane). After stirring for 15 min,
triethylamine (0.133 g, 0.190 mL, 1.32 mmol) was added. After stirring for 5 min, the
reaction mixture was diluted with dichloromethane (30 mL), washed with brine (2 × 30 mL)
and dried over Na2SO4. The residue obtained after evaporation of the solvent was subjected to
chromatography on silica gel to give products 31a-44b.
8

4.1.3.1
trans
and
cis-( )-1-(tert-Butoxykarbonyl)-2-methoxy-2‘-
[( )-31a and ( )-31b].
(phenylamino)spiro{indoline-3,5‘-[4‘,5‘]-dihydrothiazol}
Following the general procedure, products ( )-31a and ( )-31b were obtained using 0.229 g
(0.6 mmol) of thiourea 17 and separated on silica gel (75 g, dichlormethane/ethyl
acetate/ammonia
20:4:0.1).
trans-( )-1-(tert-Butoxykarbonyl)-2-methoxy-2‘-
(phenylamino)spiro{indoline-3,5‘-[4‘,5‘]-dihydrothiazol} [( )-31a]. Yield: 0.106 g (43%);
white crystals; mp 172-175 °C (dichloromethane/n-hexane); Rf = 0.50 (dichlormethane/ethyl
acetate/ammonia 20:4:0.1). IR (CHCl3) ꢃmax 3420, 1700, 1633 cm-1. 1H NMR (CDCl3): ꢁ 7.76
(bs, 1H, H-7), 7.39 (d, J = 7.5 Hz, 1H, H-4), 7.28-7.24 (m, 3H, H-6, H-3´´, H-5´´), 7.15-7.13
(m, 2H, H-2´´, H-6´´), 7.06-7.01 (m, 2H, H-5, H-4´´), 5.59 (s, 1H, H-2), 4.36 (d, J = 12.0 Hz,
13
1H, H-4´b), 4.09 (d, J = 12.0 Hz, 1H, H-4´a), 3.56 (s, 3H, OCH3), 1.58 [s, 9H, C(CH3)3]. C
NMR (CDCl3): ꢁ 159.3 (C=N), 152.8 (C=O), 145.9 (C-1´´), 141.7 (C-7a), 130.1 (C-6, C-3a),
129.3 (C-3´´, C-5´´), 123.8 (C-5), 123.7 (C-4), 123.6 (C-4´´), 121.2 (C-2´´, C-6´´), 116.2 (C-
7), 98.8 (C-2), 82.5 [C(CH3)3], 65.5 (C-3), 58.4 (OCH3), 54.1 (CH2), 28.6 [C(CH3)3]. NOESY
correlations: H-4´a/H-4; H-4´a/H-4´b; H-2/OCH3. MALDI-TOF MS, m/z (relative intenzity):
412.19 [M+H]+ (100%). Anal. Calcd for C22H25N3O3S requires: C, 64.21; H, 6.12; N, 10.21.
Found: C, 64.58; H, 5.87; N, 10.49. cis-( )-1-(tert-Butoxykarbonyl)-2-methoxy-2‘-
(phenylamino)spiro{indoline-3,5‘-[4‘,5‘]-dihydrothiazol} [( )-31b]. Yield: 0.032 g (13%);
colourless oil; Rf = 0.60 (dichlormethane/ethyl acetate/ammonia 20:4:0.1). 1H NMR (CDCl3):
ꢁ 7.78 (bs, 1H, H-7), 7.42 (d, J = 7.4 Hz, 1H, H-4), 7.33-7.26 (m, 5H, H-6, H-3´´, H-5´´, H-
2´´, H-6´´ ), 7.08-7.03 (m, 2H, H-5, H-4´´), 5.29 (s, 1H, H-2), 3.97 (d, J = 12.1 Hz, 1H, H-
4´b), 3.67 (d, J = 12.1 Hz, 1H, H-4´a), 3.55 (s, 3H, OCH3), 1.59 [s, 9H, C(CH3)3]. 13C NMR
(CDCl3): ꢁ 159.4 (C=N), 154.5 (C=O), 145.5 (C-1´´), 140.8 (C-7a), 129.8 (C-6, C-3a), 129.4
(C-3´´, C-5´´), 124.1 (C-5), 123.8 (C-4), 123.6 (C-4´´), 120.6 (C-2´´, C-6´´), 115.8 (C-7), 95.4
(C-2), 82.5 [C(CH3)3], 64.8 (C-3), 60.6 (CH2), 58.4 (OCH3), 28.6 [C(CH3)3]. NOESY
correlations: H-4´a/H-4´b; H-2/H-4´b. MALDI-TOF MS, m/z (relative intenzity): identical
with spectrum of ( )-31a. Anal. Calcd for C22H25N3O3S requires: C, 64.21; H, 6.12; N, 10.21.
Found: C, 64.48; H, 5.97; N, 10.39.
4.1.4 General procedure for the synthesis of trans- and cis-diastereoisomers of analogs of
1-methoxyspirobrassinol 45a,46a and 45b,46b
To a stirred solution of corresponding thiourea (0.4 mmol) in a mixture
dichloromethane/water (6.3 mL/0.7 mL) at room temperature was added freshly prepared
solution of bromine (1.0 mL, 0.44 mmol; the stock solution prepared by dissolving of 0.04
mL of bromine in 1.76 mL of anhydrous dichloromethane). After stirring for 15 min,
triethylamine (0.087 g, 0.12 mL, 0.88 mmol) was added. After stirring for 5 min, the reaction
mixture was diluted with dichloromethane (20 mL), washed with brine (2 × 20 mL) and dried
over Na2SO4. The residue obtained after evaporation of the solvent was subjected to
chromatography on silica gel to give products 45a-46b.
4.1.4.1
trans
and
cis-( )-1-(tert-Butoxykarbonyl)-2-hydroxy-2‘-
[( )-45a and ( )-45b].
(phenylamino)spiro{indoline-3,5‘-[4‘,5‘]-dihydrothiazol}
Following the general procedure, products ( )-45a and ( )-45b were obtained using 0.153 g
(0.4 mmol) of thiourea 17 and isolated on silica gel (30 g, n-hexane/ethyl acetate 1:1) as a
mixture in a 67:33 ratio. The pure product ( )-45a was obtained by crystallization from
acetone/n-hexane.
trans-( )-1-(tert-Butoxykarbonyl)-2-hydroxy-2‘-(phenylamino)spiro
{indoline-3,5‘-[4‘,5‘]-dihydrothiazol} [( )-45a]. Yield: 0.046 g (29%); white crystals; mp
165-167 °C (acetone/n-hexane); Rf = 0.40 (n-hexane/ethyl acetate 1:1). IR (CHCl3) ꢃmax 3466,
1
1680, 1633 cm-1. H NMR (CDCl3): ꢁ 7.71 (bs, 1H, H-7), 7.52-7.41 (m, 1H, H-4), 7.36-7.21
9

(m, 4H, H-6, H-3´´, H-5´´, H-4´´), 7.11-7.02 (m, 3H, H-2´´, H-6´´, H-5), 5.97 (s, 1H, H-2),
5.18 (s, 1H, OH), 4.55 (d, J = 12.1 Hz, 1H, H-4´b), 4.06 (d, J = 12.1 Hz, 1H, H-4´a), 1.58 [s,
13
9H, C(CH3)3]. C NMR (CDCl3): ꢁ 161.7 (C=N), 153.2 (C=O), 145.8 (C-1´´), 141.3 (C-7a),
132.0 (C-3a), 130.2 (C-6), 129.2 (C-3´´, C-5´´), 124.2 (C-4), 123.7 (C-5), 121.8 (C-4´´), 121.3
(C-2´´, C-6´´), 115.2 (C-7), 91.4 (C-2), 83.2 [C(CH3)3], 68.4 (C-3), 54.4 (CH2), 28.6
[C(CH3)3]. NOESY correlations: H-4´a/H-4; H-4´a/H-4´b. MALDI-TOF MS, m/z (relative
intenzity): 398.15 [M+H]+ (100%). Anal. Calcd for C21H23N3O3S requires: C, 63.46; H, 5.83;
N, 10.57. Found: C, 63.21; H, 5.70; N, 10.73. cis-( )-1-(tert-Butoxykarbonyl)-2-hydroxy-2‘-
(phenylamino)spiro{indoline-3,5‘-[4‘,5‘]-dihydrothiazol} [( )-45b]. Product ( )-45b isolated
with ( )-46a as a mixture in a 33:67 ratio. Colourless oil; Rf = 0.60 (n-hexane/ethyl acetate
1:1). 1H NMR (CDCl3): ꢁ 7.71 (bs, 1H, H-7), 7.52-7.41 (m, 1H, H-4), 7.36-7.21 (m, 4H, H-6,
H-3´´, H-5´´, H-4´´), 7.11-7.02 (m, 3H, H-2´´, H-6´´, H-5), 5.72 (s, 1H, H-2), 5.18 (s, 1H,
OH), 3.95 (d, J = 12.1 Hz, 1H, H-4´b), 3.73 (d, J = 12.1 Hz, 1H, H-4´a), 1.60 [s, 9H, C(CH3)3].
13C NMR (CDCl3): ꢁ 161.5 (C=N), 153.2 (C=O), 145.6 (C-1´´), 141.0 (C-7a), 132.2 (C-3a),
130.0 (C-6), 129.4 (C-3´´, C-5´´), 124.6 (C-4), 123.6 (C-5), 121.7 (C-4´´), 121.2 (C-2´´, C-
6´´), 115.1 (C-7), 87.8 (C-2), 83.2 [C(CH3)3], 65.5 (CH2), 64.4 (C-3), 28.6 [C(CH3)3].
NOESY correlations: H-4´a/H-4´b; H-2/H-4´b. Anal. Calcd for C21H23N3O3S requires: C,
63.46; H, 5.83; N, 10.57. Found: C, 63.37; H, 5.76; N, 10.63.
4.1.4.2
trans
and
cis-( )-1-(tert-Butoxykarbonyl)-2-hydroxy-2‘-(N-
methylphenylamino)spiro{indoline-3,5‘-[4‘,5‘]-dihydrothiazol} [( )-46a and ( )-46b].
Following the general procedure, products ( )-46a and ( )-46b were obtained using 0.158 g
(0.4 mmol) of thiourea 26 and separated on silica gel (40 g, n-hexane/ethyl acetate 2:1). trans-
( )-1-(tert-Butoxykarbonyl)-2-hydroxy-2‘-(N-methylphenylamino)spiro{indoline-3,5‘-[4‘,5‘]-
dihydrothiazol} [( )-46a]. Yield: 0.094 g (57%); white crystals; mp 168-172 °C
(dichloromethane/n-hexane); Rf = 0.25 (n-hexane/ethyl acetate 2:1). IR (CHCl3) ꢃmax 3466,
1
1680, 1633 cm-1. H NMR (CDCl3): ꢁ 7.71 (bs, 1H, H-7), 7.44-7.42 (m, 1H, H-4), 7.33-7.29
(m, 2H, H-3´´, H-5´´), 7.25-7.19 (m, 4H, H-6, H-4´´, H-2´´, H-6´´), 7.02 (ddd, J = 7.6 Hz, J =
7.6 Hz, J = 1.0 Hz, 1H, H-5), 5.92 (s, 1H, H-2), 4.88 (d, J = 14.3 Hz, 1H, H-4´b), 4.27 (d, J =
14.3 Hz, 1H, H-4´a), 3.44 (s, 3H, CH3), 1.58 [s, 9H, C(CH3)3]. 13C NMR (CDCl3): ꢁ 161.3
(C=N), 153.3 (C=O), 146.0 (C-1´´), 140.7 (C-7a), 131.2 (C-3a), 129.7 (C-6), 129.2 (C-3´´, C-
5´´), 127.1 (C-4´´), 126.5 (C-2´´, C-6´´), 124.1 (C-4), 123.6 (C-5), 114.8 (C-7), 92.1 (C-2),
82.8 [C(CH3)3], 68.8 (C-3), 64.2 (CH2), 40.3 (CH3), 28.6 [C(CH3)3]. NOESY correlations: H-
4´a/H-4; H-4´a/H-4´b. MALDI-TOF MS, m/z (relative intenzity): 412.08 [M+H]+ (100%).
Anal. Calcd for C22H25N3O3S requires: C, 64.21; H, 6.12; N, 10.21. Found: C, 64.45; H, 5.91;
N,
10.33.
cis-( )-1-(tert-Butoxykarbonyl)-2-hydroxy-2‘-(N-methylphenylamino)-
spiro{indoline-3,5‘-[4‘,5‘]-dihydrothiazol} [( )-46b]. Yield: 0.026 g (16%); white crystals;
mp 151-154 °C (dichloromethane/n-hexane); Rf = 0.55 (n-hexane/ethyl acetate 2:1). IR
1
(CHCl3) ꢃmax 3466, 1680, 1633 cm-1. H NMR (CDCl3): ꢁ 7.62 (bs, 1H, H-7), 7.42-7.33 (m,
5H, H-4, H-3´´, H-5´´, H-2´´, H-6´´), 7.28-7.21 (m, 2H, H-4´´, H-6), 7.02 (ddd, J = 7.5 Hz, J =
7.5 Hz, J = 1.0 Hz, 1H, H-5), 5.63 (s, 1H, H-2), 4.23 (d, J = 13.9 Hz, 1H, H-4´b), 3.93 (d, J =
13.9 Hz, 1H, H-4´a), 3.44 (s, 3H, CH3), 1.59 [s, 9H, C(CH3)3]. 13C NMR (CDCl3): ꢁ 162.2
(C=N), 152.6 (C=O), 145.9 (C-1´´), 139.9 (C-7a), 131.0 (C-3a), 129.6 (C-6), 129.4 (C-3´´, C-
5´´), 127.2 (C-4´´), 126.5 (C-2´´, C-6´´), 124.2 (C-4), 123.5 (C-5), 115.1 (C-7), 88.5 (C-2),
82.7 [C(CH3)3], 73.0 (C-3), 72.4 (CH2), 40.6 (CH3), 28.6 [C(CH3)3]. NOESY correlations: H-
4´a/H-4´b; H-2/H-4´b. MALDI-TOF MS, m/z (relative intenzity): 412.19 [M+H]+ (100%).
Anal. Calcd for C22H25N3O3S requires: C, 64.21; H, 6.12; N, 10.21. Found: C, 64.32; H, 5.96;
N, 10.45.
4.1.5 General procedure for the synthesis of analogs of cyclobrassinin 48-61
10

To a stirred solution of corresponding thiourea (0.6 mmol) in a mixture anhydrous
dichloromethane/methanol (9 mL/1 mL) at room temperature was added freshly prepared
solution of bromine (1.53 mL, 0.66 mmol; the stock solution prepared by dissolving of 0.04
mL of bromine in 1.76 mL of anhydrous dichloromethane). After stirring for 15 min,
triethylamine (0.133 g, 0.190 mL, 1.3 mmol) was added. After stirring for 5 min, the reaction
mixture was diluted with dichloromethane (30 mL), washed with brine (2 × 30 mL) and dried
over Na2SO4. The residue obtained after evaporation of the solvent was dissolved in
dichlomethane (3 mL). Trifluoroacetic acid (0.900 mL, 1.36 g, 12 mmol) was added and
mixture was stirred for 1.5 h at room temperature. The reaction mixture was poured into
solution of NaHCO3 (1.02 g, 12 mmol in 6 mL of water) cooled at 0 °C. Reaction mixture
was diluted with dichloromethane (15 mL), the organic layer was removed and the aqueous
layer was extracted with dichloromethane (15 mL). The organic layer was dried over
anhydrous Na2SO4. The residue obtained after evaporation of the solvent was further
crystallized from acetone/n-hexane to afford pure products 48-61.
4.1.5.1 2-Phenylamino-4H-1,3-thiazino[6,5-b]indol (48)
Following the general procedure, product 48 was obtained using 0.229 g (0.6 mmol) of
thiourea 17. Yield: 0.112 g (67%); yellow crystals; mp 134-136 °C (acetone/n-hexane); Rf =
0.68 (n-hexane/ethyl acetate 1:1). IR (KBr) ꢃmax 3439, 3359, 2922, 1631, 1596 cm-1. 1H NMR
(DMSO-d6): ꢁ 11.39 (bs, 1H, NH-indol), 9.05 (bs, 1H, NH-Ph), 7.57 (bs, 2H, H-2´, H-6´),
7.45 (d, J = 7.6 Hz, 1H, H-5), 7.32 (d, J = 7.6 Hz, 1H, H-8), 7.26-7.22 (m, 2H, H-3´, H-5´),
7.07-6.98 (m, 2H, H-7, H-6), 6.94 (t, J = 7.3 Hz, 1H, H-4´), 4.85 (s, 2H, CH2). 13C NMR
(DMSO-d6): ꢁ 143.1 (C=N), 136.2 (C-8a), 130.3 (C-1´), 128.6 (C-3´, C-5´), 124.7 (C-4b),
123.6 (C-9a), 121.7 (C-4´), 120.8 (C-7), 119.1 (C-6, C-2´, C-6´), 116.8 (C-5), 110.8 (C-8),
101.6 (C-4a); 44.8 (CH2). EIMS m/z (relative intenzity): 279 [M]+ (1%), 118 (100%), 91
(50%), 77 (32%), 51 (40%). Anal. Calcd for C16H13N3S requires: C, 68.79; H, 4.69; N, 15.04.
Found: C, 68.51; H, 4.98; N, 14.84.
Experimental procedures and spectral data for all new compounds are presented in
Supplementary Data.
4.2. Antiproliferative activity
4.2.1. Cell culture
Tumor cell lines
Jurkat (acute T-lymphoblastic leukemia), HeLa (cervical adenocarcinoma), MCF-7
(mammary gland adenocarcinoma), MDA-MB-231 (mammary gland adenocarcinoma), A-
549 (non-small cell lung cancer) and CCRF-CEM cell line (acute T-lymphoblastic leukemia)
cells were maintained in RPMI 1640 medium supplemented with Glutamax-I or in
Dulbecco’s modified Eagle’s medium with Glutamax-I and glucose. Both of these media
were supplemented with 10% (V/V) fetal calf serum, penicillin (100 IU × mL-1) and
streptomycin (100 ꢄg ×mL-1) (all from Invitrogen, UK), in humidified air with 5% CO2 at
37 °C. Before each cytotoxicity assay, cell viability was determined by the trypan blue
(Invitrogen) exclusion method and found to begreater than 95%.
Human umbilical vein endothelial cells
Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and characterized
as previously described.26,27 Cells were cultured on gelatin-coated dishes in cM199 (= M199
medium supplemented with 10% heat-inactivated human serum, 10% heat-inactivated
newborn calf serum, 3.75 µg/ mL crude endothelial cell growth factor (ECGF), 5 U/mL
11

heparin, 100 IU/mL penicillin, and 100 µg/mL streptomycin) at 37°C under 5% CO2/95% air
atmosphere.
Twenty-four hours prior to experiments endothelial cell cultures were refreshed with medium
without crude endothelial cell growth factor and heparin.
4.2.2. Cytotoxicity assay
IC50 values (concentration of a given compound that decreased amount of viable cells to 50%
relative to untreated control cells) were determined by 4 parameter logistic non-linear
regression model using normalized concentration-response data obtained by MTT (Thiazolyl
Blue Tetrazolium Bromide) assay.
Pocedure A
The cytostatic/cytotoxic effects of compounds was studied using the colorimetric
microculture assay with the MTT endpoint.28 Briefly, 5 ×103 cells were plated per well in 96-
well polystyrene microplates (Sarstedt, Germany) in 100 ꢄL of the culture medium containing
tested chemicals at final concentrations of 10-6-10-4 mol ×L-1. After 72 h incubation, 10 ꢄL of
MTT (5 mg ×mL-1) (Sigma-Aldrich) was added into each well. After an additional 4 h at
37 °C, during which insoluble formazan was produced, 100 ꢄL of 10% (m/m) sodium
dodecylsulphate (SDS, Sigma-Aldrich) were added into each well and another 12 h were
allowed for the dissolution of formazan. The absorbance was measured at 540 nm and 630 nm
– reference wavelenght by the automated uQuantTM Universal Microplate Spectrophotometer
(Biotek Instruments Inc., Winooski, VT USA). The blank corrected absorbance of the control
wells was taken as 100% and the results were expressed as a percentage of the control.
Procedure B
The assay conditions were essentially the same as described previously.29 Briefly, aliquots of
80 ꢄL cell suspension (8000 cells/well) were added to 96-well microculture plates. Twenty-
four hours after seeding, 20 ꢄL aliquots of drug solutions were added to the HUVEC in
triplicate wells for 48 h. After 48 h of culturing, 10 ꢄL of the MTT solution (Sigma-Aldrich)
were added into each well and incubated for another 4 h. Then, the formazan crystals were
dissolved with 100 ꢄL of 10% SDS. The optical density (OD) was measured at 540 nm with
an EL-312 microplate reader (Biotek Instruments Inc., Winooski, VT USA) for wells with
treated cells and normalized to untreated control.
Acknowledgments
We would like to thank the Slovak Grant Agency for Science (Grant No. 1/0954/12 and No.
1/0406/10) for financial support of this work. This work was also supported by the State
NMR Programme No. 2003SP200280203.
References
1.
Purkayastha, R. P. In Handbook of Phytoalexin Metabolism and Action; Daniel, M.;
Purkayastha, R. P. Eds; Progress in phytoalexin research during the past 50 years.
Marcel Dekker: New York, 1995, pp 1-39.
2.
3.
Pedras, M. S. C.; Yaya, E. E.; Glawischnig, E. Nat. Prod. Rep. 2011, 28, 1381-1405.
Pedras, M. S. C.; Zheng, Q.; Sarma-Mamillapale, V. K. Nat. Prod. Commun. 2007, 2,
319-330.
12

4.
5.
Takasugi, M.; Monde, K.; Katsui, N.; Shirata, A. Bull. Chem. Soc. Jpn. 1988, 61, 285-
289.
Mehta, R. G.; Liu, J.; Constantinou, A.; Thomas, C. F.; Hawthorne, A.; You, M.;
Gerhäuser, C.; Pezzuto, J. M.; Moon, R. C.; Moriarty, R. M. Carcinogenesis 1995, 16,
399-404.
6.
Kutschy, P.; Suchý, M.; Monde, K.; Harada, N.; Marušková, R.; Ćurillová, Z.;
Dzurilla, M.; Miklošová, M.; Mezencev, R.; Mojžiš, J. Tetrahedron Lett. 2002, 43,
9489-9492.
7.
8.
Csomós, P.; Fodor, L.; Sohár, P.; Bernáth, G. Tetrahedron 2005, 61, 9257-9262.
Csomós, P.; Zupkó, I.; Réthy, B.; Fodor, L.; Falkay, G.; Bernáth, G. Bioorg. Med.
Chem. 2006, 16, 6273-6276.
9.
Mezencev, R; Kutschy, P; Salayová, A; Ćurillová, Z; Mojžiš, J; Pilátová, M;
McDonald, J. Chemotherapy 2008, 54, 372-378.
10.
11.
Mezencev, R; Kutschy, P; Salayová, A; Updegrove, T; McDonald, J. F. Neoplasma
2009; 56, 321-330.
Kutschy, P.; Salayová, A.; Ćurillová, Z.; Kožár, T.; Mezencev, R.; Mojžiš, J.; Pilátová,
M.; Balentová, E.; Pazdera, P.; Sabol, M.; Zburová, M. Bioorg. Med. Chem. 2009, 17,
3698-3712.
12.
Mezencev, R.; Galizzi, M.; Kutschy, P.; Docampo, R. Experimental Parasitol. 2009,
122, 66-69.
13.
14.
Mezencev, R.; Mojžiš, J.; Pilátová, M.; Kutschy, P. Neoplasma 2003, 50, 239-245.
Kutschy, P.; Mezencev, R. Indole Phytoalexins from Brassicaceae: Synthesis and
Anticancer Activity. In: Targets in Heterocyclic Systems – Chemistry and Properties;
Attanasi, O. A.; Spinelli, D., Eds.; Italian Society of Chemistry: Urbino, Italy, 2009;
Vol. 12, pp 120-148. ISBN: 978-88-86208-56-7 ISSN 1724-9449
Sabol, M.; Kutschy, P.; Siegfried, L.; Miroššay, A.; Suchý, M.; Hrbková, H.; Dzurilla,
M.; Marušková, R.; Starková, J.; Paulíková, E. Biologia 2000, 55, 701-707.
Mehta, R. G.; Liu, J.; Constantinou, A.; Hawthorne, M.; Pezzuto, J. M.; Moon, R. C.;
Moriarty, R. M. Anticancer Res. 1994, 14, 1209-1213.
Gaspari, P.; Banerjee, T.; Malachowski, W. P.; Muller, A. J.; Prendergast, J. D.;
DuHadaway, J.; Bennett, S.; Donovan, A. M. J. Med. Chem. 2006, 49, 684-692.
Banerjee, T.; Duhadaway, J. B; Gaspari, P.; Sutanto-Ward, E.; Munn, D. H.; Mellor,
A. L.; Malachowski, W. P.; Prendergast, G. C.; Muller, A. J. Oncogene 2008, 27,
2851-2857.
15.
16.
17.
18.
19.
20.
Tempete, C.; Devys, M.; Barbier, M. Z. Naturforsch 1991, 46c, 706-707.
Monde, K.; Taniguchi, T.; Miura, N.; Kutschy, P.; Ćurillová, Z.; Pilátová, M.; Mojžiš,
J. Bioorg. Med. Chem. 2005, 13, 5206-5212.
21.
22.
Kutschy, P.; Dzurilla, M.; Takasugi, M.; Török, M.; Achbergerová, I.; Homzová, R.;
Rácová, M. Tetrahedron 1998, 54, 3549-3566.
Budovská, M.; Kutschy, P.; Kožár, T.; Gondová, T.; Petrovaj, J. Tetrahedron 2013, 69,
1092-1104.
23.
24.
Monde, K.; Takasugi, M.; Shirata, A. Phytochemistry 1995, 39, 581-586.
Budovská, M.; Kutschy, P.; Ćurillová, Z. Diastereoselectivity of spirocyclization of 1-
methoxy and 1-acyl derivatives of indole phytoalexin brassinin. Unpublished results.
La Regina, G.; Sarkar, T.; Bai, R.; Edler, M. C; Saletti, R.; Coluccia, A.; Piscitelli, F.;
Minelli, L.; Gatti, V.; Mazzoccoli, C.; Palermo, V.; Mazzoni, C.; Falcone, C.;
Scovassi, A. I.; Giansanti, V.; Campiglia, P.; Porta, A.; Maresca, B.; Hamel, E.;
Brancale, A.; Novellino, E.; Silvestri, R. J. Med. Chem. 2009, 52, 7512-7527.
Defilippi, P.; van Hinsbergh, V. W. M.; Bertolotto, A.; Rossino, P.; Silengo, L.;
Tarone, G. J. Cell Biol. 1991, 114, 855-863.
25.
26.
13

27.
van Hinsbergh, V. W.; Sprengers, E. D.; Kooistra, E. A. Thromb. Haemost. 1987, 57,
148-153.
28.
29.
Mosmann, T. J. Immunol. Methods 1983, 65, 55.
Kaspers, G. J.; Veerman, A. J.; Pieters, R.; Broekema, G. J.; Huismans, D. R.;
Kazemier, K. M.; Loonen, A. H.; Rottier, M. A.; van Zantwijk, C. H.; Hählen, K.; van
Wering, E. R. Br. J. Cancer 1994, 70, 1047–1052.
14

Figure 1. Structures of selected indole phytoalexins
Figure 2. Retrosynthetic analysis
15

Table 1. Yields and ratios of trans- and cis-diastereoisomers ( )-31a-( )-46b produced via
Scheme 2
Ratioa
trans : cis
76:24
73:27
76:24
73:27
78:22
74:26
74:26
72:28
71:29
78:22
76:24
74:26
68:32
73:27
67:33
76:24
Yield (%)
trans : cis
43:13
52:15
46:13
51:15
46:21b
51:14
59:14c
43:15
54:16
71:13
68:8
Product
R1
R2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
(±)-31a, (±)-31b
(±)-32a, (±)-32b
(±)-33a, (±)-33b
(±)-34a, (±)-34b
(±)-35a, (±)-35b
(±)-36a, (±)-36b
(±)-37a, (±)-37b
(±)-38a, (±)-38b
(±)-39a, (±)-39b
(±)-40a, (±)-40b
(±)-41a, (±)-41b
(±)-42a, (±)-42b
(±)-43a, (±)-43b
(±)-44a, (±)-44b
(±)-45a, (±)-45b
(±)-46a, (±)-46b
C6H5NH
4-Br-C6H4NH
4-F-C6H4NH
4-Cl-C6H4NH
4-CH3O-C6H4NH
4-CH3-C6H4NH
4-NO2-C6H4NH
4-CF3-C6H4NH
3,5-bis-CF3-C6H3NH
C6H5 N(CH3)
CH3NH
CyclohexylNH
Piperidine-1-yl
Morpholine-4-yl
C6H5NH
42:13
49:17
42:14
29d
H
C6H5N(CH3)
57:16
aThe ratios of diastereoisomers ( )-31a-( )-46b were determined by integration of separated signals
corresponding to H-2, Ha and Hb protons in the 1H NMR spectrum of the crude product mixture.
bcis-Product ( )-35b was obtained as a mixture with trans-diastereoisomer ( )-35a in a 50:50 ratio.
ccis-Product ( )-37b was obtained as a mixture with trans-diastereoisomer ( )-37a in a 76:24 ratio.
dcis-Product was not isolated.
16

Table 2. Chemical shifts of H-2 protons of cis- and trans-diastereoisomers ( )-31a-( )-46b
3´
N
Hb
2´ R1
R1
Ha
Ha
N
H4
H4
Hb
S1´
H2
OR2
S
3a
3
OR2
H2
N
1
N
7a
trans- Boc
Boc
cis-
NOESY
1H NMR
ꢀ(H-2) ppm
5.63c
5.49c
5.59d
5.29d
5.52d
5.33d
5.58d
5.31d
5.57d
5.31d
5.57d
5.29d
5.56d
5.30d
5.60d
5.35d
5.62d
5.32d
5.62d
5.35d
5.41c
5.23c
5.50d
5.27d
5.49d
5.31d
5.50d
5.23d
5.52d
5.25d
5.97d
5.72d
5.92d
5.63d
Compound R1
R2
Isomer
(±)-47a
transa
cisa
SCH3
H
(±)-47b
(±)-31a
(±)-31b
(±)-32a
(±)-32b
(±)-33a
(±)-33b
(±)-34a
(±)-34b
(±)-35a
(±)-35b
(±)-36a
(±)-36b
(±)-37a
(±)-37b
(±)-38a
(±)-38b
(±)-39a
(±)-39b
(±)-40a
(±)-40b
(±)-41a
(±)-41b
(±)-42a
(±)-42b
(±)-43a
(±)-43b
(±)-44a
(±)-44b
(±)-45a
(±)-45b
(±)-46a
(±)-46b
trans
cisb
C6H5NH
CH3
trans
cisb
4-Br-C6H4NH
4-F-C6H4NH
4-Cl-C6H4NH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
trans
cisb
trans
cisb
trans
cisb
4-CH3O-C6H4NH
4-CH3-C6H4NH
4-NO2-C6H4NH
4-CF3-C6H4NH
3,5-bis-CF3-C6H3NH
C6H5N(CH3)
trans
cisb
trans
cisb
trans
cisb
trans
cisb
CH3
CH3
trans
cisb
trans
cisb
CH3NH
CH3
CH3
CH3
CH3
H
trans
cisb
CyclohexylNH
Piperidine-1-yl
Morpholine-4-yl
C6H5NH
trans
cisb
trans
cisb
trans
cisb
trans
cisb
C6H5N(CH3)
H
aThe structures were determined by NOE experiments.6
bThe structures were determined by NOESY experiments.
cDMSO-d6
dCDCl3
17

Table 3. Antiproliferative activities of brassinin (1) and its analogs 2, 17-30, 62
S
R1
NH
N
R2
Compound
R1
R2
Cell line, IC50 SD (µmol × L-1)
Jurkat
MCF-7
MDA-MB-231
HeLa
CCRF-
CEM
A-549
a
1
SCH3
H
>100
>100
>100
>100
>100
90.2 9.8
63.5 2.8
>100
b
2
SCH3
OCH3
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
37.5 6.2
17.8 3.1
3.1 2.1
4.3 1.3
19.8 5.2
4.2 1.3
30.0 5.3
20.0 4.1
3.0 1.6
3.0 1.5
3.6 3.6
>100
>100
>100
>100
c
62
SCH3
23.0 2.8
0.72 1.3
8.5 2.5
27.6 7.1
8.6 2.2
92.0 8.4
30.0 3.8
9.3 2.6
3.5 2.7
3.5 2.1
>100
21.4 3.2
28.0 4.1
13.0 2.2
22.2 6.4
17.1 4.3
>100
16.9 5.7 19.6 6.3
20.4 2.1 90.7 6.7
21.4 3.4
6.6 2.8
6.8 3.3
24.3 4.2
7.6 2.1
>100
17
C6H5NH
18
4-Br-C6H4NH
4-F-C6H4NH
4-Cl-C6H4NH
4-CH3O-C6H4NH
4-CH3-C6H4NH
4-NO2-C6H4NH
4-CF3-C6H4NH
3,5-bis-CF3-C6H3NH
C6H5N(CH3)
CH3NH
4.5 5.1
10.7 2.3 12.0 3.8
4.0 2.2 3.8 1.8
3.8 2.3
19
20
21
34.0 7.2 22.0 4.4
29.0 4.3 16.0 2.9
22
37.4 6.2
8.6 2.8
4.5 1.6
4.1 3.2
>100
31.3 5.3
11.5 3.3
4.0 3.2
3.6 2.7
>100
23
14 3.2
4.0 2.1
6.6 3.7
>100
3.8 2.1
3.5 2.1
2.9 2.5
>100
24
25
26
27
>100
>100
>100
>100
>100
>100
28
CyclohexylNH
Piperidine-1-yl
Morpholine-4-yl
11.0 6.2
37.6 8.7
95.8 12.1
21.5 8.7
>100
37.5 7.3
>100
8.0 3.7
7.0 3.2
33.5 7.3
60.1 8.5
>100
29
36.8 7.6 37.0 7.8
80.0 7.3 >100
30
>100
>100
Doxorubicin
Cisplatin
0.078 0.02 0.5 0.024
0.2 0.8
14.7 2.7
21.2 4.2
0.2 0.06 0.09 0.04
1.9 0.7
12.2 2.8
14.3 4.2
12 1.8
1.2 1.5
11.4 2.4
10.9 2.1
7.7 2.3
3.9 2.3
4.4 1.6
1.1 2.7
VP-16
(Etoposide)
aSynthesized according the published precedure.21
bSynthesized according the published precedure.11
cSynthesized according the published precedure.21
The potency of compounds was determined using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay after 72
h incubation of cells and presented as IC50 (concentration of a given compound that decreased amount of viable
cells to 50% relative to untreated control cells, see Experimental section 4.2.2, procedure A).
18

Table 4. Antiproliferative activities of 1-methoxyspirobrassinol methyl ether (6) and its
analogs 31-47
N
N
R1
R1
S
OR2
S
OR2
N
R3
N
R3
trans-
Compound Isomer
cis-
R1
R2
R3
Cell line, IC50 SD (µmol × L-1)
Jurkat
MCF-7
MDA-MB-
231
HeLa
CCRF-
CEM
A-549
a
(±)-6a
(±)-6b
trans
cis
SCH3
SCH3
SCH3
SCH3
CH3 OCH3 30.2 6.7 100 1.3
CH3 OCH3 44.0 6.8 65.0 7.4
100 7.2
100 9.8
48.9 8.2
100 7.3
100 16.4
100 14.8
70.5 9.8
96.3 9.3
29.2 14.2
34.0 9.3
33.0 7.3
76.0 13.2
30.0 8.3
34.0 8.6
26.0 7.3
16.5 5.3
31.3 5.2
31.4 4.4
30.5 4.3
33.0 7.3
24.6 7.3
17.9 7.4
29 8.2
a
53.2 11.3 100 18.7
b
b
(±)-63a
trans
cis
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
CH3 Boc
37.3 8.2 70.2 11.9 87.0 12.6
74.3 9.3
77.6 7.9
31.7 6.8
28.0 7.2
28.0 7.2
65.5 8.3
29.0 8.3
27.5 8.3
20.8 5.8
10.0 6.2
25.0 3.7
20.2 3.2
23.0 3.8
31.3 6.5
32.7 9.3
11.3 3.2
34.2 9.2
8.2 4.4
37.9 6.7
41.9 6.4
12.2 8.5
22.0 6.9
5.0 3.8
(±)-63b
(±)-31a
(±)-31b
(±)-32a
(±)-32b
(±)-33a
(±)-33b
(±)-34a
(±)-34b
(±)-35a
(±)-36a
(±)-36b
(±)-37a
(±)-38a
(±)-38b
(±)-39b
(±)-40a
(±)-40b
(±)-41a
(±)-41b
(±)-42a
(±)-42b
(±)-43a
(±)-43b
(±)-44a
(±)-44b
43.4 7.3 100 12.8
10.0 7.2 6.2 3.2
19.0 6.3 32.0 9.3
97.7 13.6
40.3 8.4
34.0 9.7
31.5 6.8
trans
cis
C6H5NH
C6H5NH
trans
cis
4-Br-C6H4NH
4-Br-C6H4NH
4-F-C6H4NH
4-F-C6H4NH
4-Cl-C6H4NH
4-Cl-C6H4NH
8.0 4.2
29.5 6.4
30.0 4.3 78.0 12.7 >100
50.0 7.9
27.0 7.9
14.5 6.8
26.5 6.2
19.0 4.9
24.7 4.3
14.4 2.3
19.0 2.9
18.4 7.4
7.2 5.4
trans
cis
19.0 6.2 30.0 8.1
15.0 6.2 30.5 7.4
23.5 6.7 36.5 8.3
30.0 9.4
32.5 9.2
42.0 7.9
20.5 6.3
38.3 7.4
35.6 5.6
35.5 7.2
37.5 7.3
28.6 7.2
21.3 4.8
38.6 5.3
31.7 7.6
27.5 6.2
>100
trans
cis
7.0 3.8
16.0 4.3
trans
trans
cis
4-CH3O-C6H4NH
4-CH3-C6H4NH
4-CH3-C6H4NH
4-NO2-C6H4NH
4-CF3-C6H4NH
4-CF3-C6H4NH
25.0 4.8 31.2 6.2
25.7 3.8 32.9 4.2
9.0 2.1
31.5 6.3
trans
trans
cis
20.2 3.3 33.0 5.2
9.3 5.3
5.6 4.2
26.5 6.3
11.1 6.3
6.7 5.2
cis
3,5-bis-CF3-C6H3NH
C6H5N(CH3)
C6H5N(CH3)
CH3NH
21.5 4.4 20.9 6.2
5.9 4.2 1.5 3
26.0 6.4 28.0 5.7
83.4 >100
42.0 7.9 >100
17.2 6.6
18.3 6.2
26.6 4.7
44.4
trans
cis
NT
32.0 7.2
>100
28.7 6.8
>100
trans
cis
CH3NH
>100
>100
53.0 4.3
5.1 3.8
>100
trans
cis
CyclohexylNH
CyclohexylNH
Piperidine-1-yl
Piperidine-1-yl
Morpholine-4-yl
Morpholine-4-yl
SCH3
5.2 3.6
27.5 4.3
26.1 6.3
41.4 5.4
61.3 6.1
69.0 4.2
69.3 11.7
>100
32.0 4.9
36.7 6.2
54.9 5.4
62.3 3.4
55.5 4.2
76.0 3.8
100 4.2
78.0 9.7
93.6 3.2
27.7 5.4
38.0 4.3
54.2 5.3
55.4 5.2
>100
27.0 4.9 37.2 5.2
24.9 4.3 52.6 3.8
33.1 4.7 64.4 3.7
31.3 2.7 70.1 5.3
34.5 3.2 69.5 4.8
100 7.1
29.1 4.7
26.1 4.6
30.7 4.1
51.4 3.2
35.9 2.7
100 6.3
30.6 6.2
27.1 5.3
trans
cis
trans
cis
>100
c
(±)-5
cis + trans
trans
cis
H
H
H
H
H
H
OCH3 100 5.3
100 2.7
82.8 7.2
95.0 4.9
64.0 12.3
>100
100 3.7
100 3.2
100 4.2
74.0 10.2
d
(±)-47a
(±)-47b
(±)-45a
(±)-46a
(±)-46b
SCH3
Boc
Boc
Boc
Boc
Boc
34.0 3.8 100 6.2
29.8 4.7 100 8.4
47.7 7.6 27.4 6.2
d
SCH3
trans
trans
cis
C6H5NH
72.4 14.3 33.0 6.4
C6H5N(CH3)
C6H5N(CH3)
5.3 4.1
2.7 3
>100
64.3 11.7 >100
21.6 6.7 30.3 7.4
32.4 5.2
22.3 8.3
22.8 4.8
28.7 7.2
aSynthesized according the published precedure.6
19

bSynthesized according the published precedure.24
cSynthesized according the published precedure.6
dSynthesized according the published precedure.6
NT-not tested
The potency of compounds was determined using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay after 72
h incubation of cells and presented as IC50 (concentration of a given compound that decreased amount of viable
cells to 50% relative to untreated control cells, see Experimental section 4.2.2, procedure A).
20

Table 5. Antiproliferative activities of cyclobrassinin (7) and its analogs 48-61
N
R
S
N
H
Compound
R
Cell line, IC50 SD (µmol × L-1)
Jurkat
MCF-7
MDA-MB-231 HeLa
CCRF-
CEM
A-549
a
7
SCH3
29.5 3.2
9.5 5.7
8.3 2.7
8.0 5.3
9.8 4.2
8.2 5.2
8.0 6.1
7.4 4.3
10 6.2
72.0 8.4
31.6 4.3
29.8 4.2
33.8 6.9
30 4.8
48.3 5.3
12.2 5.2
8.6 3.7
8.9 4.2
7.2 5.1
7.3 5.3
7.9 5.7
5.4 5.2
36.2 6.3
37.1 5.3
42.4 6.9
34.3 4.6
30.9 5.2
35.5 7.3
33.1 6.3
0.2 0.8
14.7 2.7
21.2 4.2
-
57.2 4.1
26.2 4.2
9.0 5.2
<10
70.3 6.3
34.1 3.8
32.0 6.7
29.0 7.2
7.4 3.3
30.1 6.7
31.2 7.2
32.0 6.7
40.8 7.1
70.1 8.4
91.5 10.2
43.4 7.8
55.3 6.3
42.2 8.3
62.9 11.4
1.9 0.7
12.2 2.8
14.3 4.2
-
48
C6H5NH
27.0 4.4
30.0 5.2
21.7 5.8
32.4 11.3
13.9 4.2
27.5 4.9
9.3 6.4
72.4 9.2
69.8 4.8
71.7 7.4
67.2 5.4
65.0 7.6
40.7 4.3
72.5 9.6
0.2 0.06
7.7 2.3
3.9 2.3
-
49
4-Br-C6H4NH
4-F-C6H4NH
4-Cl-C6H4NH
4-OCH3-C6H4NH
4-CH3-C6H4NH
4-NO2-C6H4NH
4-CF3-C6H4NH
50
1.7 2.8
29.8 8.4
<10
51
52
28.7 4.4
32.1 8.3
7.7 3.8
53
5.0 4.2
5.0 3.2
10 5.2
7.5 2.6
28.0 5.2
4.7 4.3
3.9 1.7
6.6 2.6
5.9 1.9
0.09 0.04
4.4 1.6
1.1 2.7
-
54
55
35.7 8.2
38.7 6.4
95.0 5.3
43.2 6.7
64.3 8.2
37.1 6.8
55.1 7.3
0.5 0.024
11.4 2.4
10.9 2.1
0.00325
56
3,5-bis-CF3-C6H3NH 7.5 4.8
57
C6H5N(CH3)
CH3NH
75.5 13.4
5.2 4.8
5.2 3.2
7.3 5.4
8.7 6.2
0.078 0.02
12 1.8
1.2 1.5
-
58
59
CyclohexylNH
Piperidine-1-yl
Morpholine-4-yl
60
61
Doxorubicin
Cisplatin
VP-16 (Etoposide)
Paclitaxel
Combretastatin A-4
-
0.002525
-
-
-
-
aSynthesized according the published precedure.7
The potency of compounds was determined using the MTT (Thiazolyl Blue Tetrazolium Bromide) assay after 72
h incubation of cells and presented as IC50 (concentration of a given compound that decreased amount of viable
cells to 50% relative to untreated control cells, see Experimental section 4.2.2, procedure A).
21

Table 6. Selectivity index of cyclobrassinin (7) and its analogs 48-56
Compound
IC50 (ꢁmol × L-1)
Selectivity index SIb
HUVECa
Jurkat
MCF-7
MDA-MB-
231
HeLa
CCRF-
CEM
A-549
7
57.9
1.9
>10
7.8
5.3
0.8
6.2
>12.5
7.8
6.9
6.6
1.03
0.42
1.04
-
0.8
1.2
>8
1.0
>3.7
2.2
1.9
0.3
3.7
>3.6
6.2
0.9
0.7
0.40
0.66
0.32
-
2.2
>11
>6.5
24.7
0.3
>5.1
>20
11.6
6.9
6.6
0,90
1.15
1.14
-
0.8
>2.9
2.0
1.4
1.1
1.7
>3.2
1.8
1.7
0.7
0.04
0.41
0.09
-
48
>100
>3
49
64.8
2.2
7.5
4.7
1.2
7.0
>12.6
10.7
1.9
1.3
0.40
0.34
0.06
-
50
42.0
1.2
51
8.5
0.3
52
51.0
1.8
53
>100
>3.1
7.5
54
58.0
55
68.5
1.9
56
49.6
1.3
Doxorubicin
Cisplatin
0.0807
5.06
0.16
0.44
0.11
2.325
2.025
VP-16 (Etoposide)
Paclitaxel
Combretastatin A-4
1.25
0.00725
0.00525
-
-
-
-
-
aHUVEC (human umbilical vein endothelial cells).
bSelectivity index (SI) represents selective toxicity for cancer cell lines in comparison with nonmalignant cells.
Selectivity index of a tested compound is calculated as its IC50 for HUVEC cells divided by its IC50 for
individual cancer cell lines.
IC50 values were determined by the MTT (Thiazolyl Blue Tetrazolium Bromide) assay (see Experimental section
4.2.2, procedure B).
22

FIG1

FIG2

Scheme1

Scheme2

Scheme3

The synthesis and anticancer activity of analogs of the indole phytoalexins brassinin, 1-
methoxyspirobrassinol methyl ether and cyclobrassinin.
N
S
N
NR1R2
NR1R2
N
NR1R2
Br2,
S
S
NH
NR1R2
S
N
H
+
CF3COOH,
CH2Cl2
N
OCH3
N
OCH3
Boc
N
Boc
CH2Cl2, MeOH
Boc
trans-( )-diastereoisomer cis-( )-diastereoisomer
Mariana Budovskáa*, Martina Pilátováb, Lenka Varinskáb, Ján Mojžišb and Roman Mezencevc
aDepartment of Organic Chemistry, Institute of Chemical Sciences, Faculty of Science, P. J.
Šafárik University, Moyzesova 11, 040 01, Košice, Slovak Republic
bDepartment of Pharmacology, Faculty of Medicine, P. J. Šafárik University, SNP 1, 040 66
Košice, Slovak Republic
cGeorgia Institute of Technology, College of Science, School of Biology, 310 Ferst Drive,
Atlanta, GA 30332, USA
23