Synthesis and cytotoxic potency of novel tris(1-alkylindol-3-yl)methylium salts: Role of N-alkyl substituents

Article abstract of DOI:10.1016/j.bmc.2010.07.025

Novel derivatives of tris(indol-3-yl)methane and tris(indol-3-yl)methylium salts with the alkyl substituents at the N-atoms of the indole rings were synthesized. An easy substitution of indole rings in trisindolylmethanes for other indoles under the action of acids is demonstrated, and the mechanism of substitution is discussed. To obtain trisindolylmethylium salts, the environmentally safe method of oxidation of trisindolylmethanes with air oxygen in acidic conditions was developed. Tris(1-alkylindol-3-yl)methanes and tris(1-alkylindol-3-yl)methylium salts represent three-bladed molecular propellers whose physico-chemical and biological properties strongly depend on the N-alkyl substituent. The cytotoxicity of novel compounds increased with the number of C atoms in the alkyl chains, with optimal number n = 3-5 whereas the derivatives with longer side chains were less cytotoxic. The most potent novel compounds killed human tumor cells at nanomolar-to-submicromolar concentrations, being one order of magnitude more potent than the prototype antibiotic turbomycin A [tris(indol-3-yl)methylium salt]. Apoptosis in HCT116 colon carcinoma cell line induced by tris(1-pentyl-1H-indol-3-yl)methylium methanesulfonate was detectable at concentrations tolerable by normal blood lymphocytes. Thus, N-alkyl substituted tris(1-alkylindol-3-yl)methylium salts emerge as perspective anticancer drug candidates.

Full text of DOI:10.1016/j.bmc.2010.07.025

Bioorganic & Medicinal Chemistry 18 (2010) 6905–6913
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cytotoxic potency of novel tris(1-alkylindol-3-yl)methylium
salts: Role of N-alkyl substituents
Sergey N. Lavrenov a, Yuriy N. Luzikov a, Evgeniy E. Bykov a, Marina I. Reznikova a, Evgenia V. Stepanova b,
Valeria A. Glazunova b, Yulia L. Volodina b, Victor V. Tatarsky Jr. b, Alexander A. Shtil b,
Maria N. Preobrazhenskaya a,
*
a Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, 11B. Pirogovskaya Street, Moscow 119021, Russian Federation
b Blokhin Cancer Center, Russian Academy of Medical Sciences, 24 Kashirskoye shosse, Moscow 115478, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel derivatives of tris(indol-3-yl)methane and tris(indol-3-yl)methylium salts with the alkyl substitu-
ents at the N-atoms of the indole rings were synthesized. An easy substitution of indole rings in tris-
indolylmethanes for other indoles under the action of acids is demonstrated, and the mechanism of
substitution is discussed. To obtain trisindolylmethylium salts, the environmentally safe method of oxida-
tion of trisindolylmethanes with air oxygen in acidic conditions was developed. Tris(1-alkylindol-3-
yl)methanes and tris(1-alkylindol-3-yl)methylium salts represent three-bladed molecular propellers
whose physico-chemical and biological properties strongly depend on the N-alkyl substituent. The cyto-
toxicity of novel compounds increased with the number of C atoms in the alkyl chains, with optimal num-
ber n = 3–5 whereas the derivatives with longer side chains were less cytotoxic. The most potent novel
compounds killed human tumor cells at nanomolar-to-submicromolar concentrations, being one order
of magnitude more potent than the prototype antibiotic turbomycin A [tris(indol-3-yl)methylium salt].
Apoptosis in HCT116 colon carcinoma cell line induced by tris(1-pentyl-1H-indol-3-yl)methylium meth-
anesulfonate was detectable at concentrations tolerable by normal blood lymphocytes. Thus, N-alkyl
substituted tris(1-alkylindol-3-yl)methylium salts emerge as perspective anticancer drug candidates.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 15 December 2009
Revised 17 June 2010
Accepted 10 July 2010
Available online 23 July 2010
Key words:
Tris(1-alkyl-indol-3-yl)methane
Tris(1-alkyl-indol-3-yl)methylium salts
Antibiotic turbomycin
Three-bladed molecular propellers
Cytotoxicity
Apoptosis
Tumor cells
1. Introduction
synthesized by condensation of indol-3-carboxaldehyde (3) with
indoles (4) in the presence of acetic acid,4,5 La(OSO2CF3)3 or tri-
The antibiotic turbomycin A originally isolated as a product of
Saccharomyces cerevisiae fermentation is the salt of tris(indol-3-
yl)methylium.1 Eventually turbomycin A was obtained from soil
microorganisms by a metagenomic approach using a 24,546-mem-
ber DNA library expressed in Escherichia coli.2 Several clones pro-
duced the dark brown colonies from which the trisarylmethylium
cations designated turbomycin A [tris(indol-3-yl)methylium] and
flates of other lantanoids,6 acid-washed montmorillonite K10 clay,7
ammonium chloride8 and other low acidic catalysts (Scheme 1).
Oxidation of trisindolylmethanes to produce the salts of tris-
indolylmethylium has been performed using oxidizing agents such
as DDQ, chloranil1,2, trityl perchlorate9 or FeCl3 (Scheme 1).
10
2. Results and discussion
2.1. Chemistry
turbomycin
B [bis(indol-3-yl)phenyl)methylium], respectively,
were isolated (Fig. 1). Both these agents were capable of killing
Gram-negative and Gram-positive microorganisms.2
These results provided strong evidence that turbomycins com-
prise a perspective class of biologically active compounds. Given
the complex role of triphenylmethyl motif in anticancer com-
pounds,3 we set out to synthesize new N-alkyl substituted tris-
indolylmethylium cations and test their potency against cultured
human tumor cells.
To obtain the substituted trisindolylmethanes (2), we con-
densed N-alkylindol-3-carboxaldehydes (3) with corresponding
N-substituted indoles (4) in ethanol in the presence of La(OTf)3,
R
Y
The trisindolylmethylium salts (1) can be obtained by oxidation
of the corresponding trisindolylmethanes (2). These have been
R = Indol-3-yl.Turbomycin A
N
H
N
H
R = Phenyl.Turbomycin B
* Corresponding author. Tel.: +7499 245 3753; fax +7499 245 0295.
E-mail address: mnp@space.ru (M.N. Preobrazhenskaya).
Figure 1. Structure of turbomycin A and turbomycin B.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.025

6906
S. N. Lavrenov et al. / Bioorg. Med. Chem. 18 (2010) 6905–6913
RN
RN
O
b
a
+
N
R
N
R
N
R
N
R
N
N
R
R
4
3
2
1
R = H or Alkyl
Scheme 1. Standard synthesis of tris(indol-1-yl)methylium salts. Reagents and conditions: (a) ethanol, weak acidic catalyst (AcOH, La(OTf)3), reflux; (b) ethanol, DDQ, FeCl3
or trityl perchlorate.
Dy(OTf)3 or a mixture of acetic acid and acetic anhydride as the
catalysts. The yields of trisindolylmethanes were >95%. The proper-
ties of compounds 2b–i are presented in Table 1.
Chakrabarty and Sarkar have synthesized symmetrical and non-
symmetrical trisindolylmethanes from 3-formylindole and mono-
and dialkylindoles under the action of montmorillonite K10 clay.7
Table 1
Properties of tris(1-alkyl-indol-3-yl)methanes 2a–i
R
N
4
5
4a
6
3
7a
7
N
2
1
R
N
R
N-
Mass spectra
Combustion analysis
Mp (from
1H and 13C NMR Chemical shifts (ppm) and J (Hz)
substituent m/zb
ethanol) °C TLC Rf
(solvents)a
R = H 2a
Calcd: 361.16.
Calcd (C25H19N3): C, 83.08; H, 5.30; 244–246 0.50 (B) 1H (DMSO-d6): 6.11 (1H; s; CH); 6.89 (3H; t; J = 7.69; H-5(6)); 6.99 (3H; d;
Found: 361
N, 1.63. Found: C, 83.78; H,5.59; N,
11.72
J = 2.1; H-2); 7.05 (3H; t; J = 7.5; H-6(5)); 7.38 (3H; d; J = 8.1; H-7); 7.45
(3H; d; J = 7.9); 10.77 (3H; d; J = 2.1; N–H)
(M+)b
13C (DMSO-d6): 30.99 (3-CH); 111.43 (7-C); 118.01 (4-C); 118.32 (3-C);
119.36 (6-C); 120.70 (5-C); 123.26 (4a-C); 126.82 (2-C); 136.63 (7a-C)
R = methyl
2b
Calcd: 403.2.
Found: 403
(M+)b
Calcd (C28H25N3): C, 83.34; H, 6.24; 241–243 0.70 (B) 1H (Pyr/d5): 4.46 (9H; s; N–CH3); 6.51 (1H; s; CH); 6.98 (3H; s; H-2); 7.11
N, 10.41. Found: C, 83.45; H, 6.27;
N, 10.43
(3H; t; J = 7.6; H-6(5)); 7.28 (3H; t; J = 7.6; H-6(5)); 7.37 (3H; d; J = 8.0; H-
7); 7.79 (3H; d; J = 7.9; H-4)
13C (Pyr/d5): 31.60 (1-CH3); 32.26 (3-CH); 109.76 (7-C); 119.04 (4-C);
119.07 (3-C); 120.34 (6-C); 121.71 (5-C); 128.33 (4a-C); 128.40 (2-C);
138.21 (7a-C)
R = ethyl
2c
Calcd: 445.25.
Found: 445
(M+)b
Calcd (C31H31N3): C, 83.56; H, 7.01; 225–227 0.47 (B) 1H (Pyr/d5; 60 °C): 1.09 (9H; t; N–CH2CH3); 3.84 (6H; q; N–CH2CH3); 6.48
N, 9.43. Found: C, 83.67; H, 7.04; N,
9.43
(1H; s; CH); 7.06 (3H; s; H-2); 7.07 (3H; t; J = 7.5; H-6(5)); 7.23 (3H; t;
J = 7.5; H-6(5)); 7.36 (3H; d; J = 8.2; H-7); 7.74 (3H; d; J = 8.2; H-4)
13C (Pyr/d5): 15.37 (signal of N-ethyl group); 32.02 (3-CH); 40.78 (signal of
N-ethyl group); 109.89 (7-C); 118.96 (3-C); 118.99 (4-C); 120.59 (6-C);
121.59 (5-C); 126.83 (2-C); 128.54 (4a-C); 137.22 (7a-C)
R = propyl
2d
Calcd: 487.3.
Found: 487
(M+)b
Calcd (C34H37N3): C, 83.74; H, 7.65; 214–216 0.62 (A) 1H (Pyr/d5; 60 °C): 0.68 (9H; t; N–CH2CH2CH3); 1.57 (6H; m; N–
N, 8.62. Found: C, 83.56; H, 7.80; N,
8.72
CH2CH2CH3); 3.81 (6H; t; N–CH2CH2CH3); 6.50 (1H; s; CH); 7.07 (3H; s; H-
2); 7.09 (3H; t; J = 7.5; H-6(5)); 7.26 (3H; t; J = 7.5; H-6(5)); 7.40 (3H; d;
J = 8.2; H-7); 7.76 (3H; d; J = 8.2; H-4)
13C (Pyr/d5): 10.84; 23.22 (signals of N-propyl group); 31.61 (3-CH); 47.26
(signal of N-propyl group); 109.59 (7-C); 118.15 (3-C); 118.44 (4-C); 120.22
(6-C); 121.14 (5-C); 127.15 (2-C); 127.97 (4a-C); 137.07 (7a-C)
1H (Pyr/d5; 60 °C): 0.71 (9H; t; N–CH2CH2CH2CH3); 1.11 (6H; m; N–CH2CH2
CH2CH3);1.57 (6H; m; N–CH2CH2 CH2CH3); 3.81 (6H; t; N–CH2CH2CH2CH3);
6.50 (1H; s; CH); 7.07 (3H; s; H-2); 7.08 (3H; t; J = 7.5; H-6(5)); 7.25 (3H; t;
J = 7.5; H-6(5)); 7.40 (3H; d; J = 8.2; H-7); 7.78 (3H; d; J = 8.2; H-4)
13C (Pyr/d5): 13.67, 20.15, 31.97 (signals of N-butyl group); 32.48 (3-CH);
45.78 (signals of N-butyl group); 110.12 (7-C); 118.46 (3-C); 118.88 (4-C);
120.69 (6-C); 121.91 (5-C); 127.60 (2-C); 128.28 (4a-C); 137.38 (7a-C)
1H (Pyr/d5): 0.7–3.9 (37H, signals of N-pentyl group); 6.58 (1H; s; CH); 7.19
(6H; m; H-2 + H-6(5)); 7.33 (3H; t; J = 9.1; H-6(5)); 7.50 (3H; d; J = 9.5; H-
7); 7.84 (3H; d; J = 8.2; H-4)
R = n-butyl Calcd: 529.8.
Calcd (C37H43N3): C, 83.89; H, 8.18; 211–213
N, 7.93. Found: C, 83.68; H, 8.20; N,
8.03
2e
Found: 529.5
(M+)b
R = n-
pentyl
2f
Calcd: 571.8.
Found: 571.6
(M+)b
Calcd (C40H49N3): C, 84.01; H, 8.64; 193–195
N, 7.35. Found: C, 83.81; H, 8.86; N,
7.56
13C (Pyr/d5): 14.06, 22.45, 29.11, 30.18 (signals of N-pentyl group); 32.05
(3-CH); 46.08 (signal of N-pentyl group); 110.15 (7-C); 118.48 (3-C); 118.96
(4-C); 120.73 (6-C); 121.64 (5-C); 127.65 (2-C); 128.35 (4a-C); 137.41 (7a-
C)
R = n-he[yl Calcd: 613.44.
2g
Calcd (C43H55N3): C, 84.13; H, 9.03; 168–170 0.85 (A) 1H (Pyr/d5): 0.7–3.9 (39H, signals of N-hexyl group); 6.58 (1H; s; CH); 7.19
Found: 612.997 N, 6.84. Found: C, 83.91; H, 9.24; N,
(6H; m; H-2 + H-6(5)); 7.33 (3H; t; J = 9.1; H-6(5)); 7.50 (3H; d; J = 9.5; H-
7); 7.84 (3H; d; J = 8.2; H-4)
(M+)c
6.93

S. N. Lavrenov et al. / Bioorg. Med. Chem. 18 (2010) 6905–6913
6907
Table 1 (continued)
N-
Mass spectra
Combustion analysis
Mp (from
1H and 13C NMR Chemical shifts (ppm) and J (Hz)
substituent m/zb
ethanol) °C TLC Rf
(solvents)a
13C (Pyr/d5): 14.09, 22.69, 26.63, 30.41, 31.47 (signals of N-hexyl group);
31.96 (3-CH); 46.09 (signal of N-hexyl group); 110.11 (7-C); 118.47 (3-C);
118.93 (4-C); 120.68 (6-C); 121.60 (5-C); 127.62 (2-C); 128.32 (4a-C);
137.37 (7a-C)
R = n-decyl Calcd: 782.2.
2i
Calcd (C55H79N3): C, 84.45; H,
10.18; N, 5.37. Found: C, 84.25; H,
10.10; N, 5.17
156–158
1H (Pyr/d5): 0.94–4.01 (63H, signals of N-decyl group); 6.57 (1H; s;
CH);7.19 (6H; m; H-2 + H-6(5)); 7.31 (3H; t; J = 9.1; H-6(5)); 7.49 (3H; d;
J = 9.5; H-7); 7.83 (3H; d; J = 8.2; H-4)
Found: (M+)
781.5b
13C (Pyr/d5): 14.20, 22.91, 27.17, 29.50, 29.52, 29.78, 29.79, 30.58, 32.11
(signals of N-decyl group); 32.24 (3-CH); 46.30 (signal of N-decyl group);
110.17 (7-C); 118.85 (3-C); 119.03 (4-C); 120.75 (6-C); 121.70 (5-C); 127.63
(2-C); 128.56 (4a-C); 137.67 (7a-C)
R = benzyl
2h
Calcd: 631.8.
Found: (M+)
631.3b
Calcd (C46H37N3): C, 87.45; H, 5.90; 236–238
N, 6.65. Found: C, 87.17; H, 6.05; N,
6.71
1H (Pyr/d5): 5.22 (6H; s; signals of N–CH2Ph); 6.56 (1H; s; CH); 7.12–7.80
(30H; m; signals of N–CH2Ph and indole rings)
13C (Pyr/d5): 32.34 (3-CH); 49.97 (CH2 of Bn group); 110.42; 119.27;
119.30; 120.77; 121.98; 127.07; 127.55; 128.18; 128.69; 128.87; 137.90;
138.75
7 R = Pr;
Pr; Et
Calcd: 473.3.
Found: (M+)
473.1
Calcd (C33H35N3): C, 83.68; H, 7.45;
N, 8.87. Found: C, 83.35; H, 7.55; N,
8.67
1H (Pyr/d5): 0.66 (6H; t; N–CH2CH2CH3); 1.13 (3H; t; N–CH2CH3); 1.56 (4H;
m; N–CH2CH2CH3); 3.91 (4H; t; N–CH2CH2CH3); 3.98 (2H; q; N–CH2CH3);
6.17 (1H; s; CH); 6.90 (3H; t; J = 7.5; H-6(5)); 6.97 (2H; s; H-2); 6.99(1H; s;
H-2); 7.08 (3H; t; J = 7.5; H-6(5)); 7.36 (3H; d; J = 8.2; H-7); 7.49 (3H; d;
J = 8.2; H-4)
13C (Pyr/d5): 12.67, 19.15, 30.95 (signal of N-ethyl and N-propyl group);
31.85 (3-CH); 44.83, 45.65 (signal of N-ethyl and N-propyl group); 109.55
(7-C); 118.73 (3-C); 118.81 (4-C); 120.48 (6-C); 121.92 (5-C); 126.86 (2-C);
128.44 (4a-C); 137.15 (7a-C)
8 R = Pr;
Et; Et
Calcd: 459.27.
Found: (M+)
459.1
Calcd (C32H33N3): C, 83.62; H, 7.24;
N, 9.14. Found: C, 83.32; H, 7.54; N,
9.11
1H (Pyr/d5): 0.91 (3H; t; N–CH2CH2CH3); 1.11 (6H; t; N–CH2CH3); 1.58 (2H;
m; N–CH2CH2CH3); 3.92 (2H; t; N–CH2CH2CH3); 4.05 (4H; q; N–CH2CH3);
6.21 (1H; s; CH); 6.92 (3H; t; J = 7.5; H-6(5)); 6.99 (2H; s; H-2); 6.99(1H; s;
H-2); 7.08 (3H; t; J = 7.5; H-6(5)); 7.36 (3H; d; J = 8.2; H-7); 7.49 (3H; d;
J = 8.2; H-4)
13C (Pyr/d5): 11.82; 15.39; 23.24 (signal of N-ethyl and N-propyl group);
31.82 (3-CH); 44.78, 45.59 (signal of N-ethyl and N-propyl group); 109.79
(7-C); 118.85 (3-C); 118.89 (4-C); 120.46 (6-C); 121.59 (5-C); 126.78 (2-C);
128.48 (4a-C); 137.19 (7a-C)
a
b
c
Analytical TLC was performed on Kieselgel F254 plates (Merck) in systems: petroleum ether–EtOAc 5:1 (A); 1:1 (B).
Electron-impact mass spectra.
Matrix-assisted laser desorption/ionization (MALDI) mass spectra.
The authors suggested that the formation of symmetrical and
non-symmetrical trisindolylmethanes proceeded via bis(indol-3-
yl)methanol (5) which is transformed into Z-3-(indol-3-yl)methy-
lene-3H-indolium (urorosein) (6). The latter compound interacts
with indole molecules to produce a mixture of trisindolylmethanes
(Scheme 2).7
with an excess of 1-alkylindole also leads to a mixture of symmet-
rical and non-symmetrical trisindolylmethanes (Scheme 3).
Table 1-SM (Supplementary data) shows the ratios of tris-
indolylmethanes formed by interaction of N-substituted 3-formyl-
indole (3) with N0-substituted indole (4) or in the reaction of
tris(1-alkylindol-3-yl)methane (2) with N0-alkylindole (4). The
data demonstrate that isolation of a symmetrical trisindolylme-
thane (2) depends not only on the reaction conditions but also
We found that the reflux of tris(1-alkyl-indol-3-yl)methanes in
ethanol in the presence of a catalyst (La(OTf)3, Dy(OTf)3 or NH4Cl)
O
OH
5
+
N
R
N
R
N
R
N
H
N
R
N
R
6
4
3
R = H or Alkyl
RN
RN
and others
+
N
N
R
N
N
H
R
R
2
1
Scheme 2. Interaction of indolcarboxaldehyde with N-alkylindoles under the action of montmorillonite K10 clay.7

6908
S. N. Lavrenov et al. / Bioorg. Med. Chem. 18 (2010) 6905–6913
R
N
+
N
R'
H
N
N
R
R
4
2
R'
R
R'
R
N
N
N
N
+
+
+
H
H
H
H
N
R
N
R
N
R
N
R'
N
R
N
R'
N
R'
N
R'
2
7
8
2
Scheme 3. Interaction of tris(1-alkylindol-3-yl)methanes with indoles in the presence of acidic catalysts. R = CnH2n + 1, R1 = Cn + 1H2(n + 1) + 1; Compounds 2 and 4: R, R1 = H (a);
CH3 (b); C2H5 (c); n-C3H7 (d); n-C4H9 (e); n-C5H11 (f); n-C6H13 (g); CH2C6H5 (h); C10H21 (i). Compounds 7 and 8: R = C2H5, R0 = n-C3H7, R0 = R = C2H5, n-C3H7.
on the methods of isolation and purification of the reaction
products. Figure 2 presents HPLC chromatograms of the products
formed in ethanol solution (A) by refluxing tris(1-ethylindol-3-
yl)methane (2c) with an excess of 1-propylindole (4d) and (B) by
refluxing tris(1-propylindol-3-yl)methane (2d) with an excess of
1-ethylindole (4c).
In the interaction of trisindolylmethane with indole urorosein
cannot be obtained since it is formed by dehydration of the inter-
mediate bis(indol-3-yl)methanol. One can suggest that the substi-
tution reaction starts with protonation of trisindolylmethane
followed by substitution of the indole ring with N-alkyl indole
(Schemes 4a and b). Protonation of one of indole rings in
mAU
229nm,4nm (1.00)
200
100
0
20.0
22.5
22.5
25.0
25.0
min
min
mAU
229nm4nm (1.00)
250
0
20.0
Figure 2. HPLC of the reaction products. (A) Trisindolylmethanes formed by the interaction of tris(1-ethylindol-3-yl)methane (2c) with 1-propylindole (4d). rt
26.13 min—tris(1-propylindol-3-yl)methane (2d); rt 19.55 min—tris(1-ethylindol-3-yl)methane (2c); rt 21.12 min—bis(1-ethylindol-3-yl)(1-propylindol-3-yl)methane (7)
and 23.58 min—bis(1-propylindol-3-yl)(1-ethylindol-3-yl)methane (7). (B) Trisindolylmethanes formed by the interaction of tris(1-propylindol-3-yl)methane (2d) with
1-ethylindole (4c).
#
HN
HN
HN
NH
H
NR
N
R
C
NH
C
H
H
H
-
N
HN
H
HN
HN
R = -CH3
Scheme 4a. The suggested SN1-type mechanism of nucleophilic substitution in trisindolylmethane.

S. N. Lavrenov et al. / Bioorg. Med. Chem. 18 (2010) 6905–6913
6909
#
HN
HN
NH
NH
H
RN
N
R
H
RN
C
NH
C
H
H
-
N
H
HN
HN
NH
R = -CH3
Scheme 4b. The suggested SN2-type of mechanism of nucleophilic substitution in trisindolylmethane.
trisindolylmethane leads to the removal of the protonated indole
and formation of bisindolylmethyl cation (urorosein) attacked by
a new indole molecule. These events resemble the reaction of sub-
stitution of a substituted phenyl ring for an unsubstituted one in
triphenylmethanes under the action of super acids. The mecha-
nism of substitution of a substituted phenyl ring in triphenylme-
thanes for a phenyl ring in the presence of strong acids has been
interpreted as trans-alkylation of substituted triphenylmethanes.11
Quantum-chemical study using semi-empirical methods AM1
and DFT B3LYP/6-31G(d)13,14 showed that the calculated activation
R
R
N
N
a
Y
H
N
R
N
R
N
R
N
R
2a-i
1a-i
Scheme 5. Synthesis of tris(indol-3-yl)methylium salts (1a–i). R = H (a); CH3 (b);
C2H5 (c); n-C3H7 (d); n-C4H9 (e); n-C5H11 (f); n-C6H13 (g); CH2C6H5 (h); n-C10H21 (i).
Reagents and conditions: (a) butanol, O2, activated charcoal, H+Yꢀ, stirring 48 h.
barriers (
mol) are preferred for SN1-type mechanism (Scheme 4a) than for
SN2-type mechanism (Scheme 4b) where
E#AM1 = 51.8 kcal/mol
and
E#B3LYP/6-31G(d) = 80.2 kcal/mol. E# means activation barrier
D D
E#AM1 = 16.4 kcal/mol and E#B3LYP/6-31G(d) = 16.1 kcal/
D
D
D
perature was 1:2. The 1H NMR spectra (low field area) of tris(1-
decylindol-3-yl)methylium methanesulfonate at 22 °C (A) and
100 °C (B) are shown (Fig. 3). NMR spectra of compounds 1a–1h
are broadened at room temperature and the area of rapid dynamics
was reached at the temperature about 100 °C. This process is inde-
pendent of the length of the N-alkyl substituent, demonstrating
that the dynamic equilibrium is not dependent on steric hindrance.
13C NMR spectrum of 1c measured at room temperature and at
140 °C is presented in Supplementary data.
energy. The quantum chemical computation by density functional
method B3LYP/6-31G(d) with considered influence of the solvent
(method of overlaying spheres DPCM) showed that
ciation reaction of the protonated trisindolylmethane in ethanol as
a solvent (Schemes 4a and b) was by 5 kcal/mol smaller than E for
DE of the disso-
D
this reaction in a gas phase. The calculated effect of solvatation is
an additional argument in favor of SN1-type mechanism of the
investigated reaction.
The reaction of indole substitution in trisindolylmethanes is
reversible and all four possible symmetrical and non-symmetrical
trisindolylmethanes were formed. Presumably, the reversibility of
the substitution reaction is due to similar values of QMal and Fukui
index of HOMO for C-3 atom of 1-alkylindoles (see Table 2 in Sup-
plementary data).
Introduction of three indole rings into trisindolylmethanes gen-
erates three-bladed molecular propellers that consist of three rigid
aromatic rings (blades) connected to the central atom (hub) in the
propeller axis. The indole rings are rapidly twisted and adopt clock-
wise and counterclockwise arrangement. Due to unsymmetry with-
in each indole ring, this gives rise to stereoisomeric conformations.12
The NMR spectra of novel tris(indol-3-yl)methanes (Table 1) were
well resolved at room temperature. The width of signals did not
change with the increase of temperature, indicating that the indole
rings undergo free rotation (flips) at room temperature.
To obtain trisindolylmethylium salts, we developed an environ-
mentally safe method of oxidation of trisindolylmethanes with air
oxygen in acidic conditions. Trisindolylmethanes were dissolved in
butanol with the addition of an acid depending on the salt we
aimed at (i.e., methanesulfonic or acetic acid). The solutions were
stirred after the addition of activated charcoal to enhance the con-
tact of the compound with air oxygen. These procedures resulted
in trisindolylmethylium salts in good yields (Scheme 5). All tris(in-
dol-3-yl)methylium salts 1a–i were water soluble red or orange
solids. The properties of novel tris(indol-3-yl)methylium methane
sulfonates are summarized in Table 2.
Four isomeric propeller conformations (A, A0, B, and B0) are pos-
sible for a molecule of this type including the stereoisomers that
isomerize by flip mechanism (Scheme 6). The transition state of
the one ring flip of trisindolylcarbonium ions is energetically fa-
vored due to resonance stabilization. The quantum chemical calcu-
lations using semi-empirical AM1 and DFT methods showed that
the delocalized form of trisindolylmethylium cation (B) is more
stable than the indolenine form (Scheme 7) by 1.4 kcal/mol.
2.2. Biological testing
We tested compounds 1a–i for the ability to induce death of
cultured human colon carcinoma HCT116 and leukemia K562 cell
lines. In these experiments the cells were treated with each com-
pound for 72 h followed by determination of cell viability using
an MTT-test. Mean IC50 decreased progressively with the increase
of the number of C atoms in the alkyl chain: from 3.2
substituted derivative 1b) down to 0.05–0.15
l
M
M (for Me
(n-pentyl
l
containing compound 1f). The compounds 1g–i with longer sub-
stituents at the N atom of the indole ring were less potent than
1f (Table 3). The cytotoxicity of tris(1-alkylindol-3-yl)methylium
methanesulfonates for K562 cells was generally higher than for
HCT116 cell line; still, the dependence of the activity on the length
of the alkyl chain was the same for both cell lines (Table 3). Turbo-
mycin A induced tumor cell death at micromolar concentrations
whereas some novel compounds were one order of magnitude
more potent.
In the NMR spectra of trisindolylmethylium salts the two sets of
signals detectable at room temperature merged with the increase
of temperature. At P100 °C only one set of signals was observed
(Table 2). The ratio of intensities of two C2-H signals at room tem-
Thus, introduction of alkyl substituents at the N atoms of in-
doles produced the derivatives with more pronounced cytotoxicity
compared with turbomycin A. Gradual increase of the cytotoxic

6910
S. N. Lavrenov et al. / Bioorg. Med. Chem. 18 (2010) 6905–6913
Table 2
Tris(1-alkylindol-3-yl)methylium methanesulfonates 1a–i
R
N
+
N
R
N
R
N-Substituent
MW of methylium salt/
cation calculated, found
by mass spectra**
HPLC data: (HPLC
system), purity,%
1H NMR Chemical shifts (ppm) and J (Hz)
1b R = methyl
1c R = ethyl
Calcd: 497.18/402.2.
Found: 402.51 [M+]
33.32 (1A) 22.10
(2A) 95%
1H (DMSO-d6; 90 °C): 2.44 (3H; s; CH3SO3); 4.09 (9H; s; N–CH3); 7.01 (3H; d; J = 8.0; 7(4)-H);
7.10 (3H; t; J = 8.0; 5(6)-H); 7.41 (3H; t; J = 7.9; 6(5)-H; 7.78 (3H; d; J = 8.2; 4(7)-H); 8.39 (3H; s;
2-H).
Calcd: 539.22/444.24.
Found: 444.6 [M+]
39.18 (1A) 26.05
(2A) 97%
1H (DMSO-d6; 110 °C): 1.61 (9H; t, N–CH2CH3); 2.37 (3H; s; CH3SO3); 4.53 (6H; q, N–CH2CH3);
7.04 (3H; d; J = 8.0; 7(4)-H); 7.13 (3H; t; J = 8.0; 5(6)-H); 7.42 (3H; t; J = 8.2; 6(5)-H); 7.85 (3H;
d; J = 8.2; 4(7)-H); 8.43 (3H; s; 2-H).
1d R = n-propyl
1e R = n-butyl
Calcd: 581.27/486.29.
Found: 486.68 [M+]
28.37(1A) 29.69
(2A) 95%
1H (DMSO-d6; 90 °C): 1.01 (9H; m; N–CH2CH2CH3); 2.01 (6H; m; N–CH2CH2CH3); 2.42 (3H;s;
CH3SO3); 4.45 (6H; m; N–CH2CH2CH3); 7.01 (3H; d; J = 7.9; 7(4)-H); 7.12 (3H; t; J = 7.8; 6(5)-H);
7.41 (3H; t; J = 7.9; 5(6)-H); 7.85 (3H; d; J = 8.1; 4(7)-H); 8.41 (3H; s; 2-H).
1H (DMSO-d6; 50 °C): 0.94 (9H; m; N–CH2CH2CH2CH3); 1.37 (6H; m; N–CH2CH2CH2CH3); 1.92
(6H; m; N–CH2CH2CH2CH3); 2.31 (3H; s; CH3SO3); 4.45 (6H; m; N–CH2CH2CH2CH3); 6.78 (3H; d;
J = 7.9; 7(4)-H); 7.11 (3H; t; J = 7.9; 5(6)-H); 7.40 (3H; t; J = 7.9; 6(5)-H); 7.86 (3H; d; J = 7.9;
4(7)-H); 8.43 (3H; s; 2-H).
Calcd: 623.32/528.34.
Found: 528.64 [M+]
12.98 (1C) 7.78
(2B) 95%
1f R = n-pentyl
1g R = n-hexyl
1h R = benzyl
1i R = n-decyl
Calcd: 665.37/570.38.
Found: 570.38 [M+]
18.68 (1C) 10.57
(2B) 96%
1H (DMSO-d6; 90 °C): 0.96, 1.35, 1.44, 1.92 (27H; 4m; N–CH2–C4H9); 2.33 (3H; s; CH3SO3); 4.46
(6H; m; N–CH2–C4H9); 7.03 (3H; d; J = 8.0; 7(4)-H); 7.11 (3H; t; J = 8.0; 5(6)-H); 7.43 (3H; t;
J = 7.9; 6(5)-H); 7.83 (3H; d; J = 8.0; 4(7)-H); 8.41 (3H; s; 2-H).
Calcd: 707.41/612.43.
Found: 612.92 [M+]
17.05 (2B) 5.65 (3)
95%
1H (DMSO-d6; 90 °C): 0.98, 1.35, 1.44, 1.61, 1.95 (33H; 5m; N–CH2–C5H11); 2.33 (3H; s; CH3SO3);
4.46 (6H; m; N–CH2–C5H11) 7.02 (3H; d; J = 8.0; 7(4)-H); 7.10 (3H; t; J = 8.0; 5(6)-H); 7.43 (3H; t;
J = 7.8; 6(5)-H); 7.84 (3H; d; J = 8.0; 4(7)-H); 8.42 (3H; s; 2-H).
Calcd: 725.27/630.29.
Found: 630.41 [M+]
10.33 (1C) 6.33
(2B) 96%
1H (DMSO-d6; 90 °C): 2.34 (3H; s; CH3SO3); 5.72 (6H; s; N–CH2Ph); 7.04 (3H; d; J = 8.0; 7(4)-H);
7.11 (3H; t; J = 8.0; 5(6)-H); 7.42 (18H; m; N–CH2Ph + 6(5)-H); 7.79 (3H; d; J = 8.2; 4(7)-H); 8.57
(3H; s; 2-H)
Calcd: 875.6/780.62.
Found: 780.83 [M+]
7.78 (3) 95%
1H (DMSO-d6; 90 °C): 0.84, 1.29, 1.94, 2.09 (57H; 4m; N–CH2–C9H19); 2.32 (3H; s; CH3SO3); 4.45
(6H; m; N–CH2–C9H19); 7.02 (3H; d; J = 8.0; 7(4)-H); 7.10 (3H; t; J = 8.0; 5(6)-H); 7.41 (3H; t;
J = 7.9; 6(5)-H); 7.84 (3H; d; J = 8.0; 4(7)-H); 8.38 (3H; s; 2-H).
For systems 1A–C: (A) 0.2% HCO2NH4, pH 4.5; (B) acetonitrile. The proportion of acetonitrile varied for system 1A from 30% to 90% for 30 min and then 90% for 15 min, for
system 1B: 10–40% for 30 min, for system 1C: 70–90% for 10 min and then 90% for 20 min. The systems 2A, 2B: (A) 0.01 M H3PO4, pH 2.6; (B) acetonitrile. The proportion of
acetonitrile varied for system 2A from 10% to 90% for 30 min and then 90% for 10 min, for system 2B: 65–90% for 25 min, for system 3: 95% CH3OH and 5% acetonitrile for
15 min. For all systems the flow rate was 1 mL/min.
Figure 3. Low field areas of 1H NMR spectra of 1i in DMSO-d6. A, 20 °C; B 100 °C.

S. N. Lavrenov et al. / Bioorg. Med. Chem. 18 (2010) 6905–6913
6911
RN
NR
NR
RN
NR
RN
N
R
N
N
R
N
R
N
R
N
R
R
Scheme 6. Isomeric propeller conformations of tris(1-alkylindol-3-l)methylium cations.
0.8
R
R
N
N
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Y
N
R
N
R
N
R
N
R
Scheme 7. Resonance stabilization of tris(1-alkylindol-3-yl)cation.
potency was documented if the length of the alkyl N-substituent
changed from one C atom to a longer chain substituents, with
n = 3–5 showing the highest potency. Further elongation of the al-
kyl chains was associated with lower cytotoxicity. Compounds 1
c–g were 2–3 orders of magnitude more potent than N-acetylated
triindolylmetanes whose cytotoxicity has been reported to be at
decimolar concentrations or higher.5
Figure 4. Cytotoxicity of compound 1f. IC50 after a 72 h continuous exposure
To investigate the cytotoxicity of tris(indol-3-yl)methylium salts
in more detail, we selected 1f as the most potent derivative. At sub-
micromolar concentrations 1f killed human leukemia, melanoma,
breast carcinoma and ovarian carcinoma cell lines (Fig. 4). The
Mel Kor cell line derived from disseminated skin melanoma15 was
the most sensitive to 1f. Importantly, 1f was less cytotoxic for
peripheral blood lymphocytes (PBL) than for tested tumor cell lines
(p <0.05 for all tumor cell lines compared with PBL; Fig. 4). These
data suggest that tris(indol-3-yl)methylium salts might be prefer-
entially toxic for proliferating cells. Furthermore, 1f caused a
programmed death as determined by characteristic traits of apop-
tosis, that is, the increase of Annexin V-positive cells and the loss
of DNA integrity. The latter was determined as the increased per-
centage of hypodiploid (sub-G1) nuclei (bar diagram) and internu-
cleosomal DNA fragmentation (Fig. 5). Apoptosis induced by 1f was
p53-independent since the drug was equally potent for HCT116
(p53+/+) cell line and for its isogenic subline HCT116p53KO with
deleted p53 gene16 (data not shown). Altogether, these results
strongly suggest that tris(indol-3-yl)methylium salts are perspec-
tive for further development as anticancer drug candidates.
(MTT-test, mean S.D. of three replicates).
yl)methylium salts. The N-substituted alkyl derivatives of tris(in-
dol-3-yl)methylium salts with 2–6 carbon atoms in the side chain
demonstrated a higher cytotoxicity for cultured human tumor cell
lines than turbomycin A. The most potent compound tris(1-pentyl-
1H-indol-3-yl)methylium methanesulfonate caused apoptosis of
cultured tumor cells at concentrations that were less toxic for nor-
mal lymphocytes. These results expand the biological properties of
turbomycins beyond antimicrobial activities, and demonstrate for
the first time that tris(1-alkylindol-3-yl)methylium salts are prom-
ising chemical class for the search of anticancer drug candidates.
4. Experimental
4.1. Chemistry
4.1.1. Tris(1-ethyl-1H-indol-3-yl)methane (2c) (method A)
To a mixture of 1-ethylindole 4c (2.9 g, 20 mmol) and 1-ethyl-
3-formylindole 3c (1.73 g, 10 mmol) in ethanol (50 mL) 50 mg
La(OTf)3 was added. After 3 h of refluxing the mixture was cooled
to room temperature, the precipitate was filtered off, washed with
50 mL of ethanol and dried to give 2c (4.25 g, 95% yield) as color-
less crystals.
3. Conclusion
A novel environmentally safe and simple method of oxidation of
tris(indol-3-yl)methanes yielded the corresponding tris(indol-3-
Table 3
Cytotoxicity (IC50 values,
l
M) of compounds 1a–i for human tumor cell linesa
Cell line
Compound
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
Me
Et
Pr
Butyl
Pentyl
Hex
Bn
Decyl
Colon carcinoma HCT116
Leukemia R562
2.3 0.2
1.6 0.4
3.2 0.3
3.2 0.2
1.3 0.3
0.2 0.1
1.0 0.3
0.2 0.1
0.3 0.1
0.09 0.04
0.15 0.04
0.05 0.03
0.3 0.1
0.074 0.03
15.0 2.2
9.3 2.1
16.4 2.3
12.4 2.1
a
IC50 (lM), MTT-test after 72 h incubation. Data are mean SD of 3–5 measurements.

6912
S. N. Lavrenov et al. / Bioorg. Med. Chem. 18 (2010) 6905–6913
Figure 5. Apoptotic death of HCT116 cells treated with compound 1f. (A) Cells were treated with 1 lM of 1f for 24–48 h followed by flow cytometry-assisted analysis of
Annexin V-FITC reactivity and DNA integrity. (B) Internucleosomal DNA fragmentation detected by electrophoresis in 1% agarose gel. The experiments were repeated three
times with essentially the same results.
Tris(1H-indol-3-yl)methane (2a); tris(1-methyl-1H-indol-3-yl)
methane (2b); tris(1-propyl-1H-indol-3-yl)methane (2d); tris
(1-butyl-1H-indol-3-yl)methane (2e); tris(1-pentyl-1H-indole-3-
yl) methane (2f); tris(1-hexyl-1H-indol-3-yl)methane (2g); tris
(1-benzyl-1H-indol-3-yl)methane (2h); tris(1-decyl-1H-indol-3-yl)
methane (2i) were also obtained by the same method using corre-
sponding indoles 4a–i and 3-formylindoles 3a–i.
mixture of 7, 8, 2c and 2d (HPLC data, Table 2). The individual 7
and 8 were purified by preparative TLC and mixture 10:1 of diox-
ane/petroleum ether as eluent.
4.2. Biological testing
Unless specified otherwise, the cell lines were from American
Type Culture Collection, and chemicals were purchased from Sig-
ma-Aldrich. The HCT116 human colon carcinoma (wild type
p53), its isogenic subline HCT116p53KO with deletion of both al-
leles of the p53 gene (generated in B. Vogelstein lab, Johns Hopkins
University, Baltimore, MD; gift of B. Kopnin), leukemia K562 and
Jurkat, melanoma FEMX (gift of M. Krasil’nikov) and Mel Kor,
breast carcinoma MCF-7 and SK-BR-3, as well as ovarian carcinoma
SKOV-3 cell lines were cultured in Dulbecco modified Eagle’s med-
ium supplemented with 5% fetal calf serum (HyClone, Logan, UT),
4.1.2. Tris(1-ethyl-1H-indol-3-yl)methane (2c) (method B)
A solution of 1-ethylindole 4c (2.9 g, 20 mmol) and 1-ethyl-3-
formylindole 3c (1.73 g, 10 mmol) in 50 mL of AcOH/Ac2O (10:1)
mixture was refluxed for 2 h. Then the mixture was diluted with
10% aqueous NaHCO3, the precipitate was filtered off, washed with
50 ml of ethanol and dried to give 2c (4.25 g, 95% yield) as colorless
crystals. Using substituted indoles 4b–i and corresponding
3-formylindoles 3b–i, compounds 2b–i were obtained.
2 mM L-glutamine, 100 U/mL penicillin, and 100 lg/mL streptomy-
4.1.3. Tris(1-ethyl-1H-indol-3-yl)methylium methanesulfonate
(1c)
cin at 37 °C, 5% CO2. Cells in logarithmic phase of growth were used
in all experiments. Human lymphocytes were freshly isolated from
peripheral blood of four healthy donors and pooled before the
experiments. Novel compounds were dissolved in DMSO as
10 mM stock solutions followed by serial dilutions in water imme-
diately before experiments.
To a suspension of 2c (4.45 g, 10 mmol) in 100 mL of butanol,
0.5 g of activated charcoal Norit A and methanesulfonic acid
(1.43 mL, 20 mmol) were added. After 24 h of stirring with an excess
of air, the mixture was filtered, butanol solution was washed twice
with water and evaporated in vacuo. Dry residue was pounded with
ether, filtered and washed with ether to give 1c (4.31 g, 80% yield) as
amorphous dark red powder.
Tris(1H-indole-3-yl)methylium methanesulfonate (1a); tris(1-
methyl-1H-indol-3-yl) methylium methanesulfonate (1b); tris
(1-propyl-1H-indol-3-yl)methylium methanesulfonate (1d); tris
(1-butyl-1H-indol-3-yl)methylium methanesulfonate (1e); tris(1-
pentyl-1H-indol-3-yl)methylium methanesulfonate (1f); tris
(1-hexyl-1H-indol-3-yl)methylium methanesulfonate (1g); tris(1-
benzyl-1H-indol-3-yl)methylium methanesulfonate (1h); tris
(1-decyl-1H-indol-3-yl) methylium methanesulfonate (1i) were
obtained by similar method from corresponding 3a–i.
The cytotoxic potency of novel compounds was determined in a
formazan conversion assay (MTT-test). Cells (5 ꢁ 103 in 190
lL of
culture medium) were plated into a 96-well plate (Costar) and
treated with 0.1% DMSO (vehicle control) or with increasing con-
centrations of tested compounds (each dose in triplicate) for
72 h. After the completion of drug exposure, 50 lg of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were
added into each well for an additional 2 h. Formazan was dissolved
in DMSO, and the absorbance at k = 540 nm was measured. Cell
viability at a given drug concentration was calculated as the per-
centage of absorbance in wells with drug-treated cells to that of
vehicle control cells (100%). The IC50 (50% inhibitory concentration)
was defined as the concentration of the compound that inhibited
cell viability by 50%.
4.1.4. 3,30-((1-Ethyl-1H-indol-3-yl)methylene)bis(1-propyl-1H-
indole) (7) and 3,30-((1-propyl-1H-indol-3-yl)methylene)bis
(1-ethyl-1H-indole) (8)
A mixture of 4d (0.19 g, 1.2 mmol), 3c (0.1 g, 0.6 mmol) and
NH4Cl (0.033 g, 0.6 mmol) was heated at 110 °C for 5 h. Then
5 mL water was added to dissolve NH4Cl, the mixture was filtered
and 30 mL of ethanol was added to the residue what represents the
To analyze apoptosis, the HCT116 cells were plated on 6-well
plates (5 ꢁ 104 cells in 3 ml of culture medium) overnight. Then
compound 1f was added up to the final concentration 1 lM, and
cells were incubated for another 24–48 h at 37 °C, 5%CO2. The con-
trol wells contained no drug. After the completion of drug exposure
cells were washed with cold saline, harvested and divided into

S. N. Lavrenov et al. / Bioorg. Med. Chem. 18 (2010) 6905–6913
6913
three portions. One aliquot of cells was stained with Annexin V-
fluorescein isothiocyanate (FITC) as recommended by the manu-
facturer (Molecular Probes). The second portion was lysed in the
charges and Fukui indexes of HOMO for 1-alkylindoles; Figure1-
SM. 13C NMR spectrum of 1c measured at room temperature and
at 110 °C. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2010.07.025.
solution containing 0.1% sodium citrate, 0.3% NP-40, 50
lg/ml
RNAse A and 10 g/ml propidium iodide and analyzed by flow
l
cytometry on a FACSCanto II (Becton Dickinson) on FL2. The per-
centage of hypodiploid nuclei (a sub-G1 peak on histograms of cell
cycle distribution) was calculated using FACSDiva software. The
third portion of cells was lysed in the buffer (350 mM NaCl,
20 mM Tris–HCl pH 7.4, 2 mM MgCl2, 1 mM dithiotreitol, 0.5%
NP-40) for 30 min on ice. DNA was isolated with phenol–chloro-
form (1:1 v/v) mixture, precipitated with 0.3 M sodium acetate
and ethanol, and resolved by electrophoresis in a 1% agarose gel.
Statistical analysis was performed using Student’s t-test.
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Supplementary data
Chemistry; General; Table 1-SM. Reaction products formed by
the interaction of N-substituted 3-formylindole (3) with N0-substi-
tuted indole (4) or by the interaction of tris(1-alkylindol-3-yl)
methane (2) with N0-substituted indole (4); Table 2-SM. Malliken
16. Bunz, F.; Dutriaux, A.; Lengauer, C.; Waldman, T.; Zhou, S.; Brown, J.; Sedivy, J.;
Kinzler, K.; Vogelstein, B. Science 1998, 282, 1497.