Synthesis and cytotoxic properties of tryptamine derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.06.070

Abstract The cyclopropyliminium and subsequent Grandberg rearrangements of cyclopropylketone hydrozones lead to the formation of tryptamines, which were additionally substituted at either the aromatic ring atoms or the amino group. The products were tested for their cytotoxic properties against HepG2, Jurkat and HEK293 cell lines using MTT assay. The highest activity as well as the highest selectivity was found amongst the compounds derived with one benzyl substituent at the amino group. The flow cytometry technique revealed cell-type specificity in terms of the mechanism of viability inhibition. Thus, the compounds were found to induce mainly apoptosis in HEK293 and HepG2 cells, while Jurkat cells displayed late apoptotic and necrotic responses. The apoptosis pathway is most likely to include mitochondrial damage.

Full text of DOI:10.1016/j.bmcl.2015.06.070

Accepted Manuscript
Synthesis and cytotoxic properties of tryptamine derivatives
Rinat F. Salikov, Aleksandr Yu. Belyy, Nailya S. Khusnutdinova, Yulia V.
Vakhitova, Yury V. Tomilov
PII:
S0960-894X(15)00663-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.070
BMCL 22860
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 May 2015
16 June 2015
19 June 2015
Please cite this article as: Salikov, R.F., Belyy, A.Y., Khusnutdinova, N.S., Vakhitova, Y.V., Tomilov, Y.V.,
Synthesis and cytotoxic properties of tryptamine derivatives, Bioorganic & Medicinal Chemistry Letters (2015),
doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.070
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Synthesis and cytotoxic properties of tryptamine derivatives
a
Rinat F. Salikov, Aleksandr Yu. Belyy, Nailya S. Khusnutdinova, Yulia V. Vakhitova,
a,b
c
c
a
Yury V. Tomilov *
a
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47 Leninsky
prospect, 119991 Moscow, Russian Federation, Tel./fax: +7-499-135-63-90; e-mail:
tom@ioc.ac.ru
b
Higher Chemical College, Russian Academy of Sciences, Moscow, Russian Federation
c
Institute of Biochemistry and Genetics Ufa Science Centre of the Russian Academy of Sciences,
Ufa, Russian Federation
A B S T R A C T
The cyclopropyliminium and subsequent Grandberg rearrangements of cyclopropylketone
hydrozones lead to the formation of tryptamines, which were additionally substituted at either
the aromatic ring atoms or the amino group. The products were tested for their cytotoxic
properties against HepG2, Jurkat and HEK293 cell lines using MTT assay. The highest activity
as well as the highest selectivity was found amongst the compounds derived with one benzyl
substituent at the amino group. The flow cytometry technique revealed cell-type specificity in
terms of the mechanism of viability inhibition. Thus, the compounds were found to induce
mainly apoptosis in HEK293 and HepG2 cells, while Jurkat cells displayed late apoptotic and
necrotic responses. The apoptosis pathway is most likely to include mitochondrial damage.
Keywords: tryptamine derivatives, cytotoxicity, apoptosis, mitochondrial damage pathway
Tryptamine derivatives are commonly known as 5-HT agonists used for the treatment of
1
migraine and obesity. Nevertheless, tryptamine itself is neurotoxic and is thought to cause
2
3
-4
5-7
At the same time, tryptamines including melatonin have
8
neurodegenerative diseases.
demonstrated antitumor activity with high pharmacological indices. Recently, 4,6-dichloro-2-
9
methyl-3-aminoethylindole (DCAI) was found to inhibit the Ras oncoprotein. Taken together,
these suggest that tryptamine derivatives possess high potential for the treatment of tumors. Here
we present our preliminary studies of potential anticancer activities of tryptamine derivatives.
1
Previously, we have found that cyclopropyl methyl ketone hydrazones 1a–d (Scheme 1)
0
1
1-15
16-17
undergo cyclopropyliminium
and subsequent Grandberg
rearrangements into a mixture
of tryptamines 2a–d and tetrahydropyridazines 3a–d.
1
Scheme 1. The rearrangement of cyclopropyl ketone arylhydrazones. a: R = H, R = R = Me; b: R =
2
3
1
2
3
1
2
3
1
2
3
2
Br, R = R = Me; c: R = NO , R = R = Me; d: R = Br, R = cyclopropyl, R = 3-chloropropyl.
1

Although the method is limited by the commercial and synthetic availability of
arylhydrazines, it can be extended to other derivatives using functional group modifications.
Thus, the presence of a nitro group in compound 2c allows to obtain the corresponding amino-
substituted tryptamine derivative. The amino-group acylation in 2c leads to the formation of 4,
which is further subjected to reduction with hydrazine hydrate and Raney-nickel followed by
conversion of the amine into hydrochloride to give the desired tryptamine 5 (Scheme 2).
3 2 2 2 4 2
Scheme 2. Reagents and conditions: (a) AcCl, NEt , CH Cl ; (b) N H /H O, Raney-Ni, EtOH; (c)
HCl/H O, EtOH.
2
In order to study a broader range of substituted aromatic derivatives of tryptamine, we
accomplished the substitution of a phenyl group for a bromine in 2b by the Suzuki coupling
reaction. The use of the tryptamine hydrochloride in the reaction gave a complex mixture of
products, while N-Boc-protected tryptamine 6 demonstrated higher selectivity to give the desired
5
-phenyltryptamine 7 after deprotection. The Boc-derivative 6 was also subjected to benzylation
to form 8, deprotection of which gave tryptammonium salt 9 substituted at the indole nitrogen
atom (Scheme 3).
2 3 2 2 2 2 3 2 3
Scheme 3. Reagents and conditions: (a) Boc O, NEt , CH Cl ; (b) PhB(OH) , PdCl (PPh ) , NaHCO ,
H
2
O, toluene, EtOH; (c) HCl/H O, EtOH; (d) NaH, DMF; (e) BnBr, DMF; (f) HCl, 1,4-dioxane.
2
Tryptamines substituted at the amino-group attracted our attention for two reasons. First,
tertiary and secondary amines are thought to be more resistant to enzyme degradation, e.g. by
1
monoamine oxidase. And secondly, in some cases secondary metabolites of tertiary amines,
8
such as those formed by removal of alkyl groups, demonstrate biological activity similar to the
1
parent compound which leads to extension of the drug action.
9
2

Thus, we have synthesized mono- (10a–d), dibenzyl- and dimethyltryptamines (11a–e) with
various substituents in the benzyl fragment via the reaction of 2b with corresponding aldehydes
in different ratios in the presence of sodium cyanoborohydride (Scheme 4).
Scheme 4. Synthesis of 5-bromotryptamines substituted at the amino nitrogen. Reagents and
conditions: (a) NaBH
3
CN, RCHO (1.1 eq. for 10a–d, 4 eq. for 11a–d, 6 eq. for 11e), AcOH, AcONa,
CO , H O; (c) HCl, 1,4-dioxane, MeOH.
MeOH (or MeOH/THF for 10c and 11c); (b) Na
2
3
2
All the synthesized tryptamines have then been tested on their ability to affect the viability of
the HEK293 (human embryonic kidney cells), HepG2 (hepatocellular carcinoma cells) and
Jurkat cells (human T- cell lymphoblast-like cell line) in order to obtain a preliminary biological
profile of this series of compounds that will be useful for future design and development of other
derivatives. The cytotoxicity of the compounds was evaluated by MTT assay. As shown in Table
1, tryptamine derivatives with different substituents at the aromatic ring (2a–d, 5, 7) show
moderate or low cytotoxic effect in tested cell lines, except for compounds 2d with an IC50 value
3

of 9.7 µM in Jurkat cells and 7 with an IC50 of 15.1 µM in HepG2 cell line. Benzylation of the
indole nitrogen (compound 9) resulted in increased activity by an order of magnitude compared
with its close structural analogue, 2b. Compounds 11a–e containing two substituents at the
amino group were found to be less potent against HEK293, HepG2 and Jurkat cells, whereas
derivatives 10a–d with only one N-benzyl group demonstrated higher activity as compared to the
parent compound 2b, with IC50 values in the range of 4.3 to 19.3 µM against all the cell lines
tested. Of note, HepG2 and Jurkat cells showed more sensitivity to the action of the N-
monobenzyl derivatives (10a–d) compared to HEK293 cells.
a
Table 1. IC50 (µM) values of tryptamines against HEK293, HepG2 and Jurkat cells lines
1
R
2
3
4
5
IC50
HEK293
IC50
HepG2
IC50
Jurkat
ID
R
R
R
R
b
2
a
H
Br
H
H
H
H
H
H
H
H
H
Me
Me
Me
H
H
H
H
H
H
–
–
–
2b
–
126.1 ± 8.5
70.4 ± 3.5
28.1 ± 2.8
–
–
–
2
c
NO
Br
2
2
H
81.4 ± 8.0
40.1 ± 9.0
–
2d
H
(CH
2
)
3
Cl
9.7 ± 2.2
–
5
NH
Ph
Ac
H
Me
Me
Me
7
9
45.5 ± 4.6
15.1 ± 1.8
8.1 ± 0.9
34.3 ± 2.7
8.9 ± 1.5
Br
H
Bn 15.5 ± 0.9
1
1
1
0a Br
Bn
H
H
H
Me
Me
Me
Me
H
H
H
H
17.2 ± 1.9
10.6 ± 0.1
19.3 ± 4.0
8.8 ± 3.0
6.4 ± 0.1 9.0 ± 1.3
p=0.0002) (p=0.0002)
c
d
(
(
(
(
4.8 ± 0.1
p=0.0002)
6.4 ± 0.1
(p=0.01)
0b Br
2 6 4
CH C H OMe
c
d
4
.5 ± 0.3
7.8 ± 1.4
p=0.0003) (p=0.0006)
2 6 2 2 2
0c Br CH C H (O CH )OMe
c
d
d
4.3 ± 0.05
p=0.0002) (p=0.0002)
5.2 ± 0.3
1
0d Br
CH
2
C
6
H
4
Cl
H
c
1
1a Br
1b Br
Bn
Bn
Me
Me
Me
Me
Me
H
H
H
H
H
18.9 ± 5.0
27.1 ± 3.0
34.1 ± 0.5
12.1 ± 3.0
53.3 ± 2.3
24.2 ± 6.0
1
CH
2
C
6
H
4
OMe OMe
CH )OMe CH CH )OMe
2 6 4
Cl CH C H Cl
CH
2
C
6
H
4
1
1c Br CH
1d Br
1e Br
2
6
C H
2
(O
2
2
2
C
6
H
2
(O
2
2
153.3 ± 3.0 60.1 ± 9.0 156.7 ± 15.0
29.3 ± 1.8 75.0 ± 10.0 55.0 ± 3.8
1
CH
2
C
6
H
4
1
Me
Me
–
82.4 ± 6.0 113.0 ± 12.0
a
–
cells were incubated with compounds for 48 h. Data are presented as means ± SEM of two
independent experiments, performed in triplicate.
b
–
–
–
not active (IC50 is above 150 µM)
indicate comparison of HepG2 with HEK293 cells using one-way ANOVA with Tukey’s post test
indicate comparison of Jurkat with HEK293 cells using one-way ANOVA with Tukey’s post test
c
d
4

To elucidate the mechanisms responsible for antiproliferative activity of N-
monobenzyltryptamines, we investigated the effect of compound 10c on cell cycle and apoptosis.
HEK293, HepG2 and Jurkat cells were treated with 10c for 48 h and the cell cycle distribution
was detected by flow cytometry and propidium iodide staining (Table 2). Apoptosis was
quantified by double staining of cells with Annexin-V/PI (Fig. 1; Table S, see Supporting
information) to distinguish healthy cells (Annexin−/PI−) from early apoptotic cells
(
Annexin+/PI−), late apoptotic cells (Annexin+/PI+),) and necrotic cells (Annexin−/PI+). As
shown in Table 2, though compound 10c proved to be the potent inducer of the hypodiploid cell
population (sub-G1 phase), no substantial differences were evidenced in the cell cycle
distributions of the treated HEK293 and HepG2 cells. Notably, effect of 10c on apoptosis
induction in HepG2 cells is more pronounced compared to that in other cell types, which is
consistent with higher cytotoxicity of the compound towards hepatocellular carcinoma cells. It is
also noteworthy, that an increase of apoptosis in Jurkat is accompanied by reduction of cells in
G1, S and G2/M phases.
a
Table 2. Effect of 10c on cell cycle distribution of HEK293, HepG2 and Jurkat cells
Cell cycle phase (% of cells)
Cell line
Sub-G1
0.9 ± 0.08
2.6 ± 0.08
0.6 ± 0.07
5.8 ± 0.05
4.7 ± 0.002
30.9 ± 1.7
G1
S
G2/M
Control
HEK293
Control
HepG2
Control
Jurkat
43.5 ± 0.9
40.9 ± 0.08
36.8 ± 1.5
38.6 ± 0.5
49.7 ± 0.5
19.8 ± 2.8
12.5 ± 0.7
12.4 ± 0.03
8.0 ± 0.3
19.7 ± 0.05
18.9 ± 0.6
26.2 ± 1.6
27.0 ± 0.05
15.3 ± 0.1
7.1 ± 0.5
7.2 ± 0.02
17.5 ± 0.02
10.5 ± 0.4
a
–
Human HEK293, HepG2 and Jurkat cells were treated with 10c at IC50 value for 48 h.
All values presented as mean ± SEM from two independent experiments, performed in triplicate.
Annexin V/PI staining has demonstrated (Figure 1) the occurrence of both types of cell
death (apoptosis and necrosis) in HEK293, HepG2 and Jurkat cells following the treatment with
10c.
5

Figure 1. Induction of apoptosis/necrosis in HEK293, HepG2 and Jurkat cells after 48 h incubation
with compound 10c at IC50 value.
In general, incubation with compound led to an increase of early, late apoptotic and
necrotic cells and to reduction of viable cells in all tested cell lines. Nevertheless, our data
revealed some cell-type specific differences between the percentages of viable, early, late
apoptotic and necrotic cells in HEK293, HepG2 and Jurkat cells after 10c exposure. In particular,
as shown in Fig. 1 and Table S (see Supporting information) an increase in both the necrosis as
well as late stages of apoptosis, were profound in Jurkat cells compared with those in HEK293
(
p=0.0003) and HepG2 cells (p=0.0003). Simultaneously, significant decline of the viable and
early apoptotic cells was observed in leukemia cells. Noteworthy, HepG2 cells exhibited a
significant increase in the percentage of the early apoptotic cells (p=0.007 vs. HEK293;
p=0.0002 vs. Jurkat) with a corresponding decrease in the late apoptotic and necrotic cells when
compared with HEK293 (p=0.0004 for late apoptosis), and more remarkable, with Jurkat cells
(
p=0.0003 for late apoptosis and necrosis). Thus, 10c elicited the cytotoxic activity, probably, via
induction of apoptosis in immortalized non-cancerous human kidney cells and hepatocellular
carcinoma line. Unlike HEK293 and HepG2, Jurkat cells displayed mainly late apoptosis and
necrotic response to 10c.
Alterations of mitochondrial functions such as loss of mitochondrial membrane potential
(
∆Ψm) have been found to play a major role in the apoptotic process induced by anticancer
drugs. Exposure of many cultured cell lines of different origins to cytotoxic agents can cause
dissipation of mitochondrial membrane potential, release of cytochrome C, AIF (apoptosis
inducing factor) and subsequent triggering the caspase-dependent and caspase-independent
2
0
signaling cascades contributing to cell death.
In order to determine whether 10c-induced apoptosis was dependent upon mitochondrial
dysfunction, cells (HEK293, HepG2 and Jurkat) were treated with the compound and a reduction
in the ∆Ψm was evaluated by JC-1 assay. As shown in figure 1, all three cell lines demonstrated
6

a significant decrease in mitochondrial membrane potential (by 75 % in HEK293 cells, 79 % in
HepG2 cells and 80 % in Jurkat cells as compared with control cells), indicating a mitochondrial
damage and an early stage of cell death. These results are consistent with the proliferation and
apoptosis assays suggesting that the decrease of cell viability is accompanied by the apoptosis
induction and loss of ∆Ψm.
Figure 2. Effect of compound 10c on mitochondrial membrane potential in HEK293, HepG2 and
Jurkat cells. Cells were incubated with 10c at IC50 value or 0.1% DMSO (control group) for
2
4 h. The results are means ± SEM of two independent experiments carried out in triplicate.
Several reports have shown that tryptamines possess the antiproliferative activities towards
4
various cancer cells, but the mechanism of their action remains unclear. For instance, it has
-6
been demonstrated that high concentrations of tryptamine (0.1–1 mM) and 5-methoxytryptamine
4
induced autophagy in HT22 and SK-N-SH nerve cell lines and in primary cultures of astrocytes .
9
Maurer et al. have identified the small-molecule ligand, capable to bind to a distinct pocket in
Ras protein and to inhibit SOS-mediated nucleotide exchange. The reported Ras-binding small
molecule - 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI) is the close structural analog of
the parent compound 2b.
Taken together, these data imply that compounds, probably, elicited the distinct signaling
pathways leading to apoptosis or necrosis depending on cell types. Although the precise
mechanism of the mode of action for the evaluated compounds remains unclear, most likely, 10c
could cause the DNA damage, which triggers apoptosis through mitochondrial pathway.
Therefore, structural optimization of the N-benzyltryptamines with prominent selectivity and
availability to cancerous cells, and detailed characterization of their antiproliferative mechanisms
remains a challenging and important research field.
In conclusion, we have synthesized a series of tryptamines derived at the heterocyclic
fragment and amino group and studied their effect on viability of HEK293, HepG2 and Jurkat
cells.
We
have
also
investigated
the
effect
of
5-bromo-2-methyl-N-((7-
methoxybenzodioxolyl)methyl)tryptamine on cell cycle and apoptosis.
7

Acknowledgements
We are grateful to the Russian Scientific Fund (Grant 14-13-01054) for main support of this
research. The part of this work devoted to the biological evaluations was supported by the Grant
for the state support of leading scientific schools of the Russian Federation (№ 5923.2014.4).
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Graphical Abstract
Synthesis and cytotoxic properties of
tryptamine derivatives
Leave this area blank for abstract info.
Rinat F. Salikov, Aleksandr Yu. Belyy, Nailya. S. Khusnutdinova, YuliaV. Vakhitova, Yury V. Tomilov
9