N6-dipeptide derivatives of N12-ribosyl-indolo[2,3-a]carbazole

Article abstract of DOI:10.1134/S106816201401004X

N6-derivatives of N12-indolo[2,3-a]pirrolo[3,4-c]carbazole-5,7-dione are synthesized as potential antitumor agents. The pyrrol N6 atom of these compounds is included into the dipeptide residue of the general formula >N6-(CH ₂) _n -CO-Ala/βAla-OMe (n = 2 or 3). These compounds are synthesized by the reaction in DMF at 130 C between 13-methyl-12-(2,3,4-tri- O-acetyl-β-D-ribopyranosyl)indolo[2,3-a]furano[3,4-c]carbazole-5,7-dione and dipeptides containing the free N-terminal amino group. The nitrogen atom of the peptide amino group replaces O6 in the furan ring and is embedded as the N6 imide nitrogen atom of pyrrol. The ability of the compounds obtained to inhibit the growth of the SKOV3 human ovarian carcinoma cells was studied. The only derivative containing the >N6-(CH₂)₃-CO-L-Ala-OMe radical showed the cytotoxic activity with an inhibitory concentration of IC₅₀ = 8 μM.

Full text of DOI:10.1134/S106816201401004X

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 1, pp. 9–15. © Pleiades Publishing, Ltd., 2014.
Original Russian Text © O.V. Goryunova, G.M. Zakharchuk, O.S. Zhukova, L.V. Fetisova, N.E. Kuzmina, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 1,
pp. 12–19.
N
6ꢀDipeptide Derivatives of N12ꢀRibosylꢀindolo[2,3ꢀa]carbazole
1
O. V. Goryunovaa, , G. M. Zakharchuka, O. S. Zhukovaa, L. V. Fetisovaa, and N. E. Kuzminab
a Blokhin Russian Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe sh. 24, Moscow, 115478 Russia
b Scientific Center for Medical Application Products, Ministry of Health of the Russian Federation, Moscow, 127051 Russia
Received, March 18, 2013; in final form, April 29, 2013
Abstract
tial antitumor agents. The pyrrol
general formula 6ꢀ(CH2)nꢀCOꢀAla/
reaction in DMF at 130°C between 13ꢀmethylꢀ12ꢀ(2,3,4ꢀtriꢀ
]furano[3,4ꢀ ]carbazoleꢀ5,7ꢀdione and dipeptides containing the free
gen atom of the peptide amino group replaces O6 in the furan ring and is embedded as the
atom of pyrrol. The ability of the compounds obtained to inhibit the growth of the SKOV3 human ovarian
—N
6ꢀderivatives of
N
12ꢀindolo[2,3ꢀ
atom of these compounds is included into the dipeptide residue of the
AlaꢀOMe ( = 2 or 3). These compounds are synthesized by the
ꢀacetylꢀ ꢀDꢀribopyranosyl)indolo[2,3ꢀ
ꢀterminal amino group. The nitroꢀ
6 imide nitrogen
a]pirrolo[3,4ꢀc]carbazoleꢀ5,7ꢀdione are synthesized as potenꢀ
N6
>N
β
n
O
β
a
c
N
N
carcinoma cells was studied. The only derivative containing the
showed the cytotoxic activity with an inhibitory concentration of IC50 = 8
>
N
6ꢀ(CH2)3ꢀCOꢀ
μM.
LꢀAlaꢀOMe radical
Keywords: indolo[2,3ꢀa]carbazole, Nꢀriboside, dipeptide, synthesis, cytotoxic activity
DOI: 10.1134/S106816201401004X
1
INTRODUCTION
Z
5
7
Molecules containing the indolocarbazole heteroꢀ
cycle have a wide spectrum of biological action includꢀ
ing antiviral, antibacterial, antiparasitic and antiꢀ
inflammatory, immunomodulatory effects, etc. [1]. Antiꢀ
N
O
O
6
8
4
9
3
12
13
N
10
2
tumor activity is mostly expressed in
Nꢀglycosides of
N
11
1
indolo[2,3ꢀ ]pyrrolo[3,4ꢀ ]carbazoleꢀ5,7ꢀdione (IPC),
а
с
X
Y
as for example, in the microbiologically prepared derivꢀ
ative of antibiotic rebeccamycin, becatecarine, and its
synthetic analogue edocarine, which are the subject of
clinical trials being held abroad through phases II–III
[2–4]. To date, the search for new compounds of the
IPC class as potential anticancer agents has not stopped
[5–7]. The activity of the IPC derivatives is due to their
interaction at the cellular level with protein kinases A
and C, cyclinꢀdependent kinases, topoisomerases I and
II, and DNA, i.e., with cellular elements, which proꢀ
vide the entry of growth signals into cells and the initiaꢀ
Derivatives of IPC. X, Y, and Z
are different substituents at nitrogen atoms
N
12, N13, and N6
The IPC derivatives containing substituents at the
6 atom of the pyrrol part of the heterocycle are the
most diverse in structure and influence on the cytoꢀ
toxic activity [10–12]. The introduction in IPC of the
N
hydrocarbon residue bound to the
atom of aglycone through the ꢀglycosyl bond signifiꢀ
cantly improves the solubility of IPC. The influence of
the structure of the hydrocarbon residue of ꢀglycoꢀ
N12 indole nitrogen
N
N
tion of mitosis or apoptosis [8, 9]. The characteristic sides of IPC on their cytotoxicity is studied on a panel
of 60 types of human tumor cells. The ꢀpentosyl
derivatives were found to be more active than the
galactosyl derivative [13]. The IPC derivatives where
substituent X is the ꢀAla residue bound to the 12
nitrogen atom through the –(CH2)n– hydrocarbon
bridge ( = 3–5, Y = Me, Z = H) were synthesized [14,
N
effect of these compounds on tumor cells primarily
depends on the nature of substituents at the 12, 13,
and 6 nitrogen atoms of the heterocycle.
Nꢀ
N
N
N
L
N
Abbreviations: GABA,
centration; ТEА, triethylamine; IPC, indolo[2,3ꢀ
]carbazoleꢀ5,7ꢀdione.
Corresponding author: phone: +7 (495)ꢀ612ꢀ8616; eꢀmail:
o.goryunova@mail.ru
γ
ꢀaminobutyric acid; IC, inhibition conꢀ
а]pyrrolo[3,4ꢀ
n
с
1
15]. These compounds show the inhibitory activity for
protein kinase C: the IC50 values were 38 nM and 9.6 nM
9

10
GORYUNOVA et al.
when
n
= 3 or 4, respectively. The authors believe that
RESULTS AND DISCUSSION
Dipeptide derivatives of ꢀriboside of IPC (IVа–е
were synthesized according to [16] by the interaction
of 13ꢀmethylꢀ12ꢀ(2,3,4ꢀtriꢀ ꢀacetylꢀ ꢀDꢀribopyraꢀ
]furano[3,4ꢀ ]carbazoleꢀ5,7ꢀdione
with dipeptides НCl H2Nꢀ(CH2)nꢀ
ꢀribopyranosyl residue and the COAla/ AlaꢀOMe ( = 2 or 3) (IIa–e) in DMF at
(CH2)nꢀCOꢀAla/ AlaꢀOMe = 2 or 3) 130°C (Schemes 1 and 2). In this reaction, the nitroꢀ
fragment at the 12 and 6 nitrogen atoms, respecꢀ gen atom of the ꢀterminal amino group of the pepꢀ
tively. The resulting compounds were tested for their tide replaces oxygen O6 in the furan ring of the heteroꢀ
ability to inhibit the growth of SKOV3 human ovarian cycle and is embedded as the 6 imide nitrogen atom
the inhibitory properties of these compounds are due
to the ability of alanine to suppress the activity of the
regulatory domain of protein kinase C.
N
)
О
β
In order to search for new cytotoxic drugs based on nosyl)indolo[2,3ꢀ
indolocarbazole, we synthesized in this work the IPC III
derivatives containing
dipeptide
а
с
(
)
·
β
β
n
>
N6ꢀ
β
(n
N
N
N
N
carcinoma cells.
of pyrrol.
+
HCl
·
H Nꢀ[C H ]ꢀ
2
BocꢀHNꢀ(CH )nꢀCOOH
2
COOMe
2
4
n
n
= 2 BocꢀβAla
= 3 BocꢀGABA
[C2H4] = CH2CH2
[C H ] = ( )ꢀСH(Me)
)ꢀСH(Me)
β
L
AlaꢀOMe
ꢀAlaꢀOMe
S
R
2
4
[C H ]= (
D
ꢀAlaꢀOMe
2
4
(i)
(ii)
(
Ia–e), R = Boc
(
IIa–e), R = HCl
IIa
AlaꢀOMe (Ib), (IIb
AlaꢀOMe (Ic), (IIc
AlaꢀOMe (Id), (IId
AlaꢀOMe (Ie), (IIe
(i) (CH3)2CHꢀCH2ꢀOꢀCOCl, THF, DMF, TEA, –10
(ii) HCl, 1,4ꢀdioxane
⋅ H
Rꢀ
Rꢀ
Rꢀ
β
β
β
Alaꢀ
AlaꢀL
AlaꢀD
β
AlaꢀOMe (Ia),
(
)
ꢀ
ꢀ
)
)
RꢀGABAꢀL
RꢀGABAꢀD
ꢀ
ꢀ
)
)
°
C;
.
Scheme 1. Synthesis of dipeptides.
Z
O
O
O
N
O
O
(i) TEA, DMF
°C
130
+ HCl
IIa–e
⋅ H2NꢀZ
(II) K CO ,
3
2
MeOH
N
N
(
)
N
N
OAc
CH3
OR
O
O
CH3
OAc
OAc
III
OR
(
)
OR
(
IVa–e) R = Ac;
Va – e ) R = H:
Z =
ꢀ(CH ) ꢀCOꢀ
β
AlaꢀOMe (а),
ꢀ(CH ) ꢀCOꢀLꢀAlaꢀOМе (b),
2 2
(
2 2
ꢀ(CH ) ꢀCOꢀDꢀAlaꢀOМе (c),
2 2
ꢀ(CH ) ꢀCOꢀLꢀAlaꢀOМе (d),
2 3
ꢀ(CH ) ꢀCOꢀDꢀAlaꢀOМе (e).
2 3
Scheme 2. Synthesis of N6ꢀdipeptide derivatives of N12ꢀribopyranosylꢀIPC.
The initial
zole (III) was synthesized by the method developed in hydrochlorides of methyl esters of
the chemical synthesis laboratory of the Blokhin Rusꢀ ꢀAla (Scheme 1) [20]. Characteristics of the syntheꢀ
sian Cancer Research Center [17]. Peptides (Ia–e sized peptides are presented in Table 1. The removal of
Оꢀacetylated
βꢀriboside of furanocarbaꢀ from the Bocꢀderivatives of
β
Ala and GABA [19] and
β
Ala and or
Lꢀ
D
)
were synthesized by the method of mixed anhydrides the Bocꢀprotecting group of peptides (Ia–e) was carꢀ
with the use of isobutylchloroformiate [18] starting ried out by HCl in dioxane [21]. Hydrochlorides of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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2014

N
6ꢀDIPEPTIDE DERIVATIVES
11
Table 1. Yields and data of 1H NMR spectra (
δ
, ppm) of synthesized dipeptides
Chemical shifts , ppm in DMSOꢀd6
7.93 (br.s*,1H, NHCO), 6.7 (br.s, 1H, NHꢀВос), 3.26 (q, 2H, CONHꢀCH2),
Code
Yield, %
δ
(
(
(
(
(
Ia
Ib
Ic
Id
)
)
)
)
54
3.12 (q, 2H, CH2ꢀNHꢀВос), 2.45 (t, 2H, CH2ꢀCOOCH3), 2.20, (t, 2H, CH2ꢀCONH),
3.6 (s, 3H, OCH3), 1.35 (s, 9H, ꢀBu)
t
8.25 (br.d, 1|H, NHCO), 6.72 (br.s, 1H, NHꢀВос), 4.25 (m, 1H,
CH2ꢀNHꢀВос ), 2.25 (t, 2H, CH2ꢀCONH), 1.25 (d, 3H, ꢀCH3), 3.6 (s, 3H, OCH3),
1.35 (s, 9H, ꢀBu)
αꢀCH), 3.1 (q, 2H,
67
55
71
47
α
t
8.30 (br.d, 1H, NHCO), 6.72 (br.s, 1H, NHꢀВос), 4.25 (m, 1H,
NHꢀВос ), 2.25 (t, 2H, CH2ꢀCONH), 1.25 (d, 3H, ꢀCH3), 3.6 (s, 3H, OCH3), 1.35 (s,
9H, ꢀBu)
αꢀCH), 3.1 (q, 2H, CH2ꢀ
α
t
8.25 (br.d, 1H, NHCO), 6.77 (br.s, 1H, CHꢀВос), 4.25 (m, 1H,
CH2ꢀNHꢀВоc), 2.07 (t, 2H, CH2ꢀCONH), 1.57 (m, 2H, CH2), 1.25 (d, 3H,
3.6 (s, 3H, OCH3), 1.35 (s, 9H, ꢀBu)
α
ꢀCH), 2.90 (q, 2H,
α
ꢀCH3),
t
8.25 (br.d, 1H, NHCO), 6.77 (br.s, 1H, NHꢀВос), 4.25 (m, 1H,
CH2ꢀNHꢀВоc), 2.07 (t, 2H, CH2ꢀCONH), 1.57 (m, 2H, CH2), 1.25 (d, 3H,
3.6 (s, 3H, OCH3), 1.35 (s, 9H, ꢀBu)
α
ꢀCH), 2.90 (q, 2H,
Ie)
α
ꢀCH3),
t
Abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br.s, broad signal.
Table 2. Characteristics of
N6ꢀZꢀderivatives of 13ꢀmethylꢀ12ꢀ(2,3,4ꢀtriꢀORꢀβꢀDꢀribopyranosyl)indolo[2,3ꢀa]pyrroꢀ
lo[3,4ꢀ
c
]carbazoleꢀ5,7ꢀdione
Retention
time*, min
Code
Z
R
Yield, %
Molecular weight, m/z
3ꢀ(>
methyl ester
N6ꢀyl)propanoylꢀβꢀalanine,
(
IVa
)
Ac
Ac
Ac
Ac
Ac
H
83
87
3ꢀ(>N
6ꢀyl)propanoylꢀLꢀalanine,
755 (M+
652 (M+ ꢀNHC2H4COOCH3 – H+)
)
(
IVb
)
)
2.7
2.9
methyl ester
3ꢀ(>N6ꢀyl)propanoylꢀDꢀalanine,
(
IVc
70
methyl ester
4ꢀ(>N6ꢀyl)butanoylꢀLꢀalanine,
(
IVd
)
51
methyl ester
769 (M2+
666 [(M 2+ ꢀNHC2H4COOCH3 – H+)+]
)
4ꢀ(>N6ꢀyl)butanoylꢀDꢀalanine,
(
IVe)
69
methyl ester
3ꢀ(>N6ꢀyl)propanoylꢀβꢀalanine,
(
Va )
quant.
81
methyl ester
3ꢀ(>N
6ꢀyl)propanoylꢀLꢀalanine,
629 (M+
526 (M+ ꢀNHC2H4COOCH3 – H+)
)
(
Vb)
H
2.3
methyl ester
3ꢀ(>
methyl ester
N6ꢀyl)propanoylꢀDꢀalanine,
(
Vc)
H
85
4ꢀ(>
methyl ester
N6ꢀyl)butanoylꢀLꢀalanine,
(
Vd)
H
67
643 (M 2+
540 (M+ ꢀNHC2H4COOCH3 – H+)
)
2.45
4ꢀ(>
methyl ester
N6ꢀyl)butanoylꢀDꢀalanine,
(Ve)
H
quant.
* Chromatography conditions: Inertsil 3
acetonitrile; 0–100% gradient B for 6 min at a flow rate of 250
µ
m ODSꢀ3 column (50
×
2.0 mm); solution A, 0.05% TFA in water; solution B, 0.05% TFA in
µL/min.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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12
GORYUNOVA et al.
Table 3. Data of 1H NMR spectra (
δ
, ppm) of synthesized dipeptide derivatives of
N
ꢀribopyranoside of IPC
Peptide residue
Heterocyclic aglicone
Hydrocarbon residue
6.19 (d, 1H, H1'), 5.44 (dd, 1H,
H2'), 5.63 (t, 1H, H3'), 5.31 (m,
1H, H4'), 4.38 (dd, 1H, H5'a),
4.16 (t, 1H, H5'b), 2.07 (s, 3H, 4'
COCH3), 2.01 (s, 3H, 3' COCH3),
1.01 (s, 3H, 2' COCH3)
9.10 (d*, 1H, H8), 9.02 (d, 1H,
H4), 7.90 (d, 1H, H11), 7.67 (d,
1H, H1), 7.74 (m, 1H, H2),
7.62(1H, H10), 7.55–7.45 (m, 2H,
H3, H9), 4.32 (s, 3H, NCH3)
(
III
)
_
CDCl3
6.06 (d, 1H, H1'), 5.51 (s, 1H, H3'),
6.20 (br.s, 1H, NHCO), 4.04 (t,
2H, N6ꢀCH2), 3.49 (s, 3H,
COOCH3), 3.42 (m, 2H, CONHꢀ
CH2), 2.61 (t, 2H, CH2ꢀCONH),
2.44 (t, 2H, CH2ꢀCOOCH3)
9.19 (d, 1H, H8), 9.06 (d, 1H, H4), 5.32 (dd, 1H, H2'), 5.19 (m, 1H,
7.75 (d, 1H, H11), 7.58 (t, 1H, H1), H4'), 4.24 (m, 1H, H5'a), 4.03 (t,
CDCl3 7.49 (m, 2H, H2,H10), 7.38 (q, 2H, 1H, H5'b), 1.95 (s, 3H, 4' COCH3),
(
IVa)
H3, H9), 4.14 (s, 3H, NCH3)
1.89 (s, 3H, 3' COCH3), 0.91 (s,
3H, 2' COCH3)
6.05 (d, 1H, H1'), 5.50 (s, 1H, H3'),
9.15 (d, 1H, H8), 9.03 (d, 1H, H4), 5.31 (dd, 1H, H2'), 5.18 (m, 1H,
7.72 (d, 1H, H11), 7.57 (t, 1H, H1), H4'), 4.24 (dd, 1H, H5'a), 4.01 (m,
6.27 (d, 1H, NHCO), 4.50 (m, 1H,
α
ꢀCH), 4.05 (m, 2H,
N
6ꢀCH2),
(
IVb
)
3.55 (s, 3H, COOCH3), 2.68 (t,
2H, CH2ꢀCONH), 1.27(d, 3H,
CDCl3 7.40 (m, 4H, H2, H3, H9, H10), 1H, H5'b), 1.94 (s, 3H, 4' COCH3),
4.13 (s, 3H, NCH3)
1.89 (s, 3H, 3' COCH3), 0.91 (s,
3H, 2' COCH3)
α
ꢀCH3)
6.05 (d, 1H, H1'), 5.50 (s, 1H, H3'),
9.15 (d, 1H, H8), br.s 9.04 (d, 1H, 5.31 (dd, 1H, H2'), 5.18 (m, 1H,
H4), 7.72 (d, 1H, H11), 7.58 (t, 1H, H4'), 4.24 (dd, 1H, H5'a), 4.01 (m,
6.27 (d, 1H, NHCO), 4.49 (m, 1H,
ꢀCH), 4.05 (m, 2H, 6ꢀCH2),
3.55 (s, 3H, COOCH3), 2.68 (t,
α
N
(
IVc
CDCl3 H1), 7.46 (m, 4H, H2, H3, H9,
H10), 4.13 (s, 3H, NCH3)
)
1H, H5'b), 1.95 (s, 3H, 4' COCH3),
1.89 (s, 3H, 3' COCH3), 0.89 (s,
3H, 2' COCH3)
2H, CH2ꢀCONH), 1.27 (d, 3H,
ꢀCH3)
α
6.62 (d, 1H, NHCO), 4.51 (m, 1H,
6.07 (d, 1H, H1'), 5.51 (s, 1H, H3'),
α
ꢀCH), 3.64 (m, 3H, COOCH3),
3.84 (m, 2H, 6ꢀCH2ꢀCH2ꢀCH2ꢀ
CONH), 2.24 (t, 2H, 6ꢀCH2ꢀ
CH2ꢀCH2ꢀCONH), 2,04 (m, 2H,
6ꢀCH2ꢀCH2ꢀCH2ꢀCONH), 1.36
(d, 3H, ꢀCH3)
9.16 (d, 1H, H8), 9.06 (d, 1H, H4), 5.33 (dd, 1H, H2'), 5.19 (m, 1H,
7.75 (d, 1H, H11), 7.53 (t, 1H, H1), H4'), 4.24 (dd, 1H, H5'a), 4.03 (t,
CDCl3 7.48 (m, 2H, H2,H10), 7.38 (q, 2H, 1H, H5'b), 1.95 (s, 3H, 4' COCH3),
N
(
IVd)
N
H3, H9), 4.14 (s, 3H, NCH3)
1.89 (s, 3H, 3' COCH3), 0.91 (s,
3H, 2' COOCH3)
N
α
6.62 (d, 1H, NHCO), 4.50 (m, 1H,
6.07 (d, 1H, H1'), 5.51 (s, 1H, H3'),
α
ꢀCH), 3.64 (m, 3H, COOCH3),
3.84 (m, 2H, 6ꢀCH2ꢀCH2ꢀCH2ꢀ
CONH), 2.24 (t, 2H, 6ꢀCH2ꢀ
CH2ꢀCH2ꢀCONH), 2,04 (m, 2H,
6ꢀCH2ꢀCH2ꢀCH2ꢀCONH), 1.37
(d, 3H, ꢀCH3)
9.15 (d, 1H, H8), 9.07 (d, 1H, H4), 5.33 (dd, 1H, H2'), 5.18 (m, 1H,
7.76 (d, 1H, H11), 7.57 (t, 1H, H1), H4'), 4.26 (m, 1H, H5'a), 4.03 (t,
CDCl3 7.48 (m, 2H, H2,H10), 7.38 (q, 2H, 1H, H5'b), 1.94 (s, 3H, 4' COCH3),
N
(IVe)
N
H3, H9), 4.13 (s, 3H, NCH3)
1.90 (s, 3H, 3' COCH3), 0.91 (s,
3H, 2' COCH3)
N
α
9.12 (t, 2H, H4,H8), 7.93 (d, 1H,
H11), 7 .83 (d, 1H, H1), 7.68 (t,
1H, H2), 7.57 (t, 1H, H10),
7.44 (m, 2H, H3, H9), 4.19 (s, 3H, 3.92 (m, 1H, H5'a)
NCH3)
8.17 (br.s, 1H, NHCO), 3.97 (m,
5.99 (d, 1H, H1'), 4.41 (br.s, 1H, 2H, 6ꢀCH2), 3.50 (s, 3H,
H3'), 4.03 (m, 3H, H2', H4', H5'b), COOCH3), 3.26 (t, 2H, CONHꢀ
N
(
Va
)
DMSOꢀd6
CH2), 2.45 (m, 4H, CH2ꢀCONH,
CH2ꢀCOOCH3)
9.13 (d, 1H, H8), 9.11 (d, 1H, H4),
7.94 (d, 1H, H11), 7 .84 (d, 1H,
8.50 (d, 1H, NHCO), 4.26 (m, 1H,
αꢀCH), 3.91(t, 2H, N6ꢀCH2), 3.52
5.99 (d, 1H, H1'), 4.39 (d, 1H,
(
Vb
)
H1), 7.69 (t, 1H, H2), 7.58 (t, 1H, H3'), 4.05 (m, 2H, H2', H4'), 3.95 (s, 3H, COOCH3), 2.61 (t, 2H,
DMSOꢀd6
H10), 7.45 (m, 2H, H3, H9), 4.20 (m, 2H, H5'a, H5'b)
(s, 3H, NCH3)
CH2ꢀCONH), 1.23 (d, 3H,
ꢀCH3)
α
8.51 (d, 1H, NHCO), 4.26 (m, 1H,
9.12 (t, 2H, H4,H8), 7.94 (d, 1H,
6.00 (d, 1H, H1'), 4.36 (d, 1H,
H3'), 4.05 (m, 2H, H2', H4'),
3.95 (m, 2H, H5'a, H5'b)
α
ꢀCH), 3.91 (t, 2H, 6ꢀCH2), 3.52
(s, 3H, COOCH3), 2.63 (t, 2H,
CH2ꢀCONH), 1.22 (d, 3H,
ꢀCH3)
N
(
Vc
DMSOꢀd6 H2), 7.58 (t, 1H, H10), 7.45 (m,
2H, H3, H9), 4.20 (s, 3H, NCH3)
)
H11), 7.84 (d, 1H, H1), 7.69 (t, 1H,
α
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
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2014

N
6ꢀDIPEPTIDE DERIVATIVES
Hydrocarbon residue
13
Table 3. (Contd.)
Heterocyclic aglicone
Peptide residue
8.29 (d, 1H, NHCO), 4.32 (m, 1H,
9.19 (d, 1H, H8), 9.17 (d, 1H, H4),
7.96 (d, 1H, H11), 7 .83 (d, 1H,
α
ꢀCH), 3.80 (t, 2H, 6ꢀCH2ꢀ
CH2ꢀCH2ꢀCONH), 3.65 (s, 3H,
H1), 7.71 (t, 1H, H2), 7.57 (t, 1H, H3'), 4.05 (m, 2H, H2', H4'), 3.94 COOCH3), 2.29 (t, 2H, 6ꢀCH2ꢀ
H10), 7.47 (m, 2tH, H3, H9), 4.25 (m, 1H, H5'a), 3.98 (m, 1H, H5'b) CH2ꢀCH2ꢀCONH), 2.03 (m, 2H,
N
6.04 (d, 1H, H1'), 4.32 (m, 1H,
( )
Vd
N
DMSOꢀd6
(s, 3H, NCH3)
N
6ꢀCH2ꢀCH2ꢀCH2ꢀCONH),
1.27 (d, 3H, ꢀCH3)
α
8.30 (d, 1H, NHCO), 4.20 (1H,
ꢀCH), 3.74 (t, 2H, 6ꢀCH2ꢀ
CH2ꢀCH2ꢀCONH), 3.59 (s, 3H,
6ꢀCH2ꢀ
(m, 1H, H5'a), 3.89 (m, 1H, H5'b) CH2ꢀCH2ꢀCONH), 1.95 (t, 2H,
6ꢀCH2ꢀCH2ꢀCH2ꢀCONH),
1.18 (d, 3H, ꢀCH3)
9.13 (m, 2H, H4), 9.15 (d, 1H,
H8), 7.94 (d, 1H, H11), 7 .83 (d, 6.00 (d, 1H, H1'), 4.41 (m, 1H,
1H, H1), 7.69 (t, 1H, H2), 7.57 (t, H3'), 4.06 (m, 2H, H2', H4'), 3.96 COOCH3), 2.24 (t, 2H,
1H, H10), 7.50 (t, 2H, H3, H9),
4.20 (3H, NCH3)
α
N
( )
DMSOꢀd6
Ve
N
N
α
* dd, doublet of doublets; the remaining symbols see in Table 1.
peptides (IIa–e) were isolated in quantitative yields
βꢀconfiguration of the Nꢀribopyranosyl fragment of
1
and used without the additional purification at the the molecules remains intact. The H NMR data of
coupling stage with furanocarbazole (III). The dipeptides (Ia–e, in DMSOꢀd6) and acetylribosides of
removal of the acetyl protecting group from the hydroꢀ IPC (IVa–e, in CDCl3) are presented in Tables 1 and
carbon residue of dipeptide derivatives (IVa–e) was 3. Since the spectra were registered in different solꢀ
performed by potash in absolute methanol followed by vents, the insertion of the dipeptides in the heterocycle
the treatment with cationite Dowex 50 (H+) [17]. The was accompanied by the downfield shifting of signals
results are presented in Tables 2 and 3.
of the methylene protons of the –(CH2)2–CO– and
(CH2)3–CO– groups, i.e., fragments of the first
amino acid ( Ala or GABA, respectively). The shift
was the more significant, the closer to the heterocycle
were located the methylene groups. In the case of the
remote amino acid, the downfield shift by 0.1 ppm was
observed only for the doublet of the methyl group at
⎯
The purification of the resulting products was carꢀ
ried out by the standard methods of organic chemistry
including preparative TLC on silica gel in the chosen
solvent systems. The purity of the compounds was
tested at all stages of the synthesis by analytical TLC
on silufol, and additionally by reverseꢀphase HPLC
β
the asymmetric center of Lꢀ and
Dꢀalanine for the
for peptide derivatives of IPC (IVa–e) and (Va – e
(Table 2).
)
GABA derivatives (IVd, e). The doublet chemical shift
of the proton at the nitrogen atom of the peptide bond
in derivatives (IVd, e) was 6.62 ppm, while this doublet
was shifted to the higher field by 0.2–0.3 ppm in the
The structure of the products was confirmed by
1H NMR and mass spectrometry (Tables 1–3). The
NMR spectra were interpreted by the values of chemꢀ
ical shifts of signals and their multiplicity and using
spectral library data of the ACD/Iꢀlabs program [22].
βAla derivatives (IVa–c). As expected [17], the proton
signals of the hydrocarbon residue of deacetylated
ribopyranosides (Va – e ) are shifted to the higher field
in DMSOꢀd6. The shifts were 1.1–1.2 ppm for the 2',
3', and 4' protons and did not exceed 0.27 ppm for the
germinal protons 5'a and 5'b. The position of the anoꢀ
meric proton and the protons of the amino acid resiꢀ
dues did not significantly change, unlike the signal of
the –NH– proton of the peptide bond, which shifted
downfield in the region of 8.3 ppm. It should be noted
that the position of the signals of both the protons and
the methyl groups at the asymmetric center differed by
0.1 ppm only for the GABA derivatives containing
1
The H NMR spectrum of acetylriboside of furaꢀ
nocarbazole (III) was consistent with literature data
[17]. In the spectra of peptide derivatives of acetyl
riboside of IPC (IVa–e), signals of the H8, H4, H11,
H1, H2, H10, H3, and H9 protons of the heterocyclic
part of the molecule were observed in the region of
9.19–7.35 ppm (towards the high field). The form and
the order of the proton signals of aglycone were the
same as for initial furanocarbazol (III); however the
proton signals of aglycone and hydrocarbon cycle were
shifted to the high field by not more than 0.2 ppm. The
exception was the downfield signals of the H4 and H8
optic isomers
LꢀAla (Vd) and DꢀAla (Ve).
The mass spectra of the synthesized peptide derivꢀ
protons, which slightly shifted to the low field. The atives of IPC show the following peaks of molecular
signal of the anomeric proton of the hydrocarbon resꢀ ions (m/z
): М+ 755 for acetylribosides (IVa–c) conꢀ
idue (doublet) for all peptide derivatives (IVa–e) was taining the –(CH2)2–СО– fragment of aglicon; М2+
in the region of 6.1 ppm with the spinꢀspin interaction 769 for acetylribosides (IVd, e) containing the
constant J1'2' = 9.3–9.5 Hz. This confirms that the
⎯(CH2)3–СО– fragment of aglicon; М
+ 629 for correꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 1
2014

14
GORYUNOVA et al.
sponding deacetylated derivatives (Va – c );
М
2+ 643 for water (10 : 1 : 1) system. The Rf value of the resulting
derivatives (Vd, e). The fragmentation of the molecule dipeptides was 0.8. The residue was filtered, the filtrate
was accompanied by the cleavage of the second amino was evaporated, the residue was dissolved in a mixture
acid of the peptide to form peaks (m/z): 652 (М+ of ethylacetate (6 mL) with water (6 mL). The water fracꢀ
ꢀNHC2H4COOCH3 – H+) for compounds (IVa–c); tion was separated, washed with ethylacetate (2
×
6 mL);
2+ ꢀNHC2H4COOCH3 – H+)+] for compounds the organic extracts were combined, washed succesꢀ
IVd, e); 526 (
+ ꢀNHC2H4COOCH3 – H+) for comꢀ sively with 1 M HCl and a saturated solution of sodium
+ ꢀNHC2H4COOCH3 – H+) for bicarbonate, and dried with calcined magnesium sulꢀ
666 [(М
(
М
pounds (Va – c ); 540 (
compounds (Vd, e) (Table 2).
М
fate. The solvent was evaporated, the residue was crysꢀ
tallized from petroleum ether. Products were: Восꢀ
The cytotoxic activity of the prepared peptide
derivatives (Va – e ) was evaluated in vitro by their influꢀ
ence on the viability of the SKOV3 human ovarian carꢀ
cinoma cells using the MTT test [23, 24], which is
based on the ability of dehydrogenase of the living cells
to reduce colorless tetrasolium salt to blue formazan
crystals dissolved in DMSO. The results are presented
as mean values of four measurements relative to the
control. The compound was considered active if the
βAlaꢀ
Вос
β
AlaꢀOMe
(
Ia), Восꢀ
β
AlaꢀLꢀAlaꢀOMe
(
Ib)
,
ꢀ
βAlaꢀDꢀAlaꢀOMe
(Ic), ВосꢀGABAꢀLꢀAla
ꢀ
OMe (Id) or ВосꢀGABAꢀDꢀAlaꢀOMe (Ie). The
results are presented in Table 1.
Removal of Bocꢀprotective group of dipeptide. The
solution of 10% gaseous HCl in dry dioxane (5 mL)
was added to corresponding methyl ester of Bocꢀ
dipeptide (Ia–e) (100 mg), and the reaction mixture
was kept at room temperature with occasional shakꢀ
ing. The reaction was monitored by TLC on the silufol
plates in the above system. The Rf values were 0.2 for
hydrochlorides (IIa–c) and 0.15 for hydrochlorides
cell survival was 50% or less at 10
IC50 < 10 M). The measurement error did not
exceed 5%. According to this criterion, the only derivꢀ
ative containing the 6ꢀ(CH2)3ꢀCOꢀ ꢀAlaꢀOMe
radical (Vd) had IC50 = 8 M.
μM concentration
(
μ
>
N
μ
L
(
IId, e). The reaction mixture was evaporated, then
reevaporated with dry ether, and dried in a desiccator
over P2O5. Chromatographically pure dipeptide
hydrochlorides were obtained in quantitative yields as
EXPERIMENTAL
We used GABA, and ꢀalanine (Reanal, Hunꢀ
gary), ꢀalanine and TFA (Acros Organics, United
βꢀ
L
a pale yellow oil: HCl · Hꢀ
Hꢀ Alaꢀ ꢀAlaꢀOMe IIb), НCl · Hꢀ
OMe IIc), НCl · HꢀGABAꢀ ꢀAlaꢀOMe (IId) or HCl ·
HꢀGABAꢀ ꢀAlaꢀOMe (IIe).
βAlaꢀβAlaꢀOMe
(IIa), НCl
·
D
β
L
(
β
Alaꢀ ꢀAlaꢀ
D
States), isobutyl chloroformate (Merck, Germany),
acetonitrile (Aldrich, United States). Solvents of Rusꢀ
sian production were purified by standard methods.
1H NMR spectra were recorded at room temperature
under standard conditions on a 300 MHz Bruker DPX
spectrometer (Bruker, Germany) with the Bruker XW
software or on a Bruker AV 300 spectrometer (Bruker,
Germany). The internal standard was tetramethylsiꢀ
lane. The HPLCꢀMS analysis of the samples was carꢀ
ried out on a Shimadzu Analytical HPLC chromatoꢀ
graph (Japan) with the UV detection at two wave
lengths and on a PE SCIEX API150EX mass specꢀ
trometer equipped with a light scattering Sedex 75
ELSD detector. Preparative TLC was performed on
(
L
D
N
ꢀ{3ꢀ[13ꢀMethylꢀ12ꢀ(2,3,4ꢀtriꢀ
pyranosyl)indolo[2,3ꢀ ]pyrrolo[3,4ꢀ
dioneꢀ 6ꢀyl]propanoyl}, derivatives of methyl esters of
Ala (IVa), ꢀAla (IVb), DꢀAla (IVc) and ꢀ{4ꢀ[13ꢀ
methylꢀ12ꢀ(2,3,4ꢀtriꢀ ꢀacetylꢀ ꢀDꢀribopyranosyl)indoꢀ
lo[2,3ꢀ ]pyrrolo[3,4ꢀ ]carbazoleꢀ5,7ꢀdionꢀ 6ꢀyl]butaꢀ
O
ꢀacetylꢀ
βꢀDꢀriboꢀ
a
c
]carbazoleꢀ5,7ꢀ
N
β
L
N
O
c
β
a
N
noyl}, derivatives of methyl esters of LꢀAla (IVd) and
DꢀAla (IVe). TEA (0.024 mL, 0.18 mmol) was added
to the solution of 13ꢀmethylꢀ12ꢀ(2,3,4ꢀtriꢀ
Oꢀacetylꢀβꢀ
Dꢀribopyranosyl)indolo[2,3ꢀa]furano[3,4ꢀc] carbazoleꢀ
5,7ꢀdione (III) (55 mg, 0.082 mmol) and hydrochloꢀ
ride of corresponding peptide (IIa–e) in DMF
(1 mL), and the mixture was kept in an autoclave for
2.5 h at 130°C. The mixture was then evaporated, and
the residue was crystallized from water. The precipitate
was purified by preparative TLC on silica gel in a benꢀ
zeneꢀethylacetateꢀisopropanol (80 : 20 : 1) mixture.
The main yellowꢀcolored fraction was collected, the
product was eluted from silica gel with methanol, the
eluate was evaporated, the residue was dried in a desꢀ
iccator over P2O5. The results are presented in Table 2.
Kieselgel 60 P254 + 366 plates (Merck, Germany) (20
×
20 cm) with the layer thickness of 1 mm. Analytical
TLC was performed on silufol UV254 plates (Cheꢀ
mapol, Czech Republic).
Synthesis of protected peptides (Ia–e). TEA
(0.26 mL, 1.85 mmol) and isobutyl chloroformate
(0.24 mL, 1.85 mmol) were added to the solution of
Bocꢀ
drofuran (30 mL) cooled to
kept for 15 min at this temperature followed by the
addition of the solution of methyl ester of Ala, Lꢀ, or
ꢀAla hydrochloride (0.26 g, 1.85 mmol) in DMF ribosyl residue of
(1.9 mL) containing TEA (0.26 mL, 1.85 mmol). The cined K2CO3 (0.115 mmol) was added to the solution
reaction mixture was stirred for 30 min at 10 , then of 12ꢀtriꢀ ꢀacetylribopyranoside of the correspondꢀ
for 1 h at and kept overnight at room temperature. ing dipeptide derivative of IPC (IVa–e), and the mixꢀ
The completeness of the reaction was detected by TLC ture was stirred at room temperature for 1.5 h. The
on the silufol plates in an ꢀbutanol–acetic acid– reaction was monitored by TLC on the silufol plates in
β
Ala or BocꢀGABA (1.85 mmol) in dry tetrahyꢀ
⎯
12 . The mixture was
°С
β
Removal of
O
ꢀacetyl protective group from
N12ꢀ
D
N6ꢀdipeptide derivatives of IPC. Calꢀ
⎯
°
С
N
O
0°С
n
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 1
2014

N
6ꢀDIPEPTIDE DERIVATIVES
15
11. Bailly, C., Riou, J.F., Colson, P., Houssier, C., Rodꢀ
a chloroformꢀisopropanol mixture (19 : 1) using the Rf
riguesꢀPereira, E., and Prudhomme, M., Biochemistry
1997, vol. 36, pp. 3917–3929.
,
,
values 0.5 and 0.65 for deacetylated
Nꢀribopyranoꢀ
sides (Va – c ) and (Vd, e), respectively. The reaction
mixture was neutralized by the addition of cationite
Dowex 50 (H+), the resin was filtered, the filtrate was
evaporated, and the residue was purified by the preparꢀ
ative TLC on silica gel in a chloroformꢀethanol (19 : 1)
mixture (this TLC was repeated two or three times).
The main yellowꢀcolored fraction of corresponding
12. Prudhomme, M., Curr. Med. Chem. Anticancer Agents
2004, vol. 4, pp. 435–438.
13. Mel’nik, S.Ya., Eksperimental’naya onkologiya na
rubezhe vekov, Davydov, M.I., Baryshnikov, A.Yu., Ed.,
Moscow: izdatel’skaya gruppa RONTs im. N.N. Blokhina
RAMN, 2003, pp. 281–293.
N
ꢀribopyranoside (Vа–e) was collected, the product 14. Trostmann, U., Hartenstein, J., Barth, H., Reck, R.,
Schächtele, C., Rudolph, K., and Kölch, W., Patent
Germanii Ger. Offen DE 42 43 321 A1, 1994.
was eluted with methanol, and the eluate was evapoꢀ
rated. The yield of the product was evaluated after
additional drying in vacuum below 1 mm Hg over Р2О5
15. Trostmann, U., Schaechtele, C., Hartenstein, J.,
Rudolph, K., Barth, H., Reck, R., and Kolch, W., US
Patent No. 5750555, 1998.
at 60
°
С
for 3 h. The results are shown in Table. 2.
16. Smirnova, L.I., Goryunova, O.V., Ektova, L.V., Miniꢀ
ker, T.D., Plikhtyak, I.L., Ustinkina, S.V., and Vlasenꢀ
kova, N.K., Peptides, 2004. Proceedings of the 3rd Interꢀ
national and 28th European Peptide Symposium, Flegel, M.,
Fridkin, M., Gilon, C., Slaninova, J., Eds. Kenes
International, 2005, pp. 603–604.
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Translated by A. Levina
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 40
No. 1
2014