Synthesis and biological evaluation of new amino acid and dipeptide derivatives of neocryptolepine as anticancer agents

Article abstract of DOI:10.1021/jm300468t

The syntheses of neocryptolepine derivatives containing an amino acid or a dipeptide at the C-9 position and their evaluation for antitumor activity in vitro and in vivo are reported. To establish the influence of an amino acid or a peptide on the physicochemical properties of 5H-indolo[2,3-b]quinoline (DiMIQ), lipophilic and hemolytic properties were investigated. Most of the compounds displayed a high antiproliferative activity in vitro and strongly inhibited growth of tumor in mice compared to cyclophosphamide. The attachment of the hydrophilic amino acid or the peptide to the hydrophobic DiMIQ increased its hydrophilic properties and decreased its hemolytic activity. The glycylglycine conjugate (7a) was the most promising derivative. It strongly inhibited the growth of the tumor in mice (at dose 50 mg kg^-1 day^-1 it inhibited the tumor growth by 46-63% on days 11-16 and by 29-43% on days 18-23) and significantly decreased hemolytic activity and lowered the in vivo toxicity compared to DiMIQ.

Full text of DOI:10.1021/jm300468t

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of New Amino Acid and
Dipeptide Derivatives of Neocryptolepine as Anticancer Agents
,†
‡
‡
§
∥
́
Katarzyna Sidoryk,* Marta Switalska, Joanna Wietrzyk, Anna Jaromin, Magdalena Piętka-Ottlik,
Piotr Cmoch,†,⊥ Joanna Zagrodzka,† Wojciech Szczepek,† Łukasz Kaczmarek,†
and Wanda Peczynska-Czoch∥
́
†Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warszawa, Poland
‡Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Weigla Street, 53-114 Wrocław, Poland
§Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wrocław,
Poland
∥Division of Organic Technology, Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze Wyspian
skiego 27, 50-370
̇
Wrocław, Poland
⊥Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warszawa, Poland
S
* Supporting Information
ABSTRACT: The syntheses of neocryptolepine derivatives
containing an amino acid or a dipeptide at the C-9 position
and their evaluation for antitumor activity in vitro and in vivo
are reported. To establish the influence of an amino acid or a
peptide on the physicochemical properties of 5H-indolo[2,3-
b]quinoline (DiMIQ), lipophilic and hemolytic properties
were investigated. Most of the compounds displayed a high antiproliferative activity in vitro and strongly inhibited growth of
tumor in mice compared to cyclophosphamide. The attachment of the hydrophilic amino acid or the peptide to the hydrophobic
DiMIQ increased its hydrophilic properties and decreased its hemolytic activity. The glycylglycine conjugate (7a) was the most
promising derivative. It strongly inhibited the growth of the tumor in mice (at dose 50 mg kg−1 day−1 it inhibited the tumor
growth by 46−63% on days 11−16 and by 29−43% on days 18−23) and significantly decreased hemolytic activity and lowered
the in vivo toxicity compared to DiMIQ.
INTRODUCTION
■
It is well-known that natural indoloquinoline alkaloids like
neocryptolepine (5-methyl-5H-indolo[2,3-b]quinoline), crypto-
lepine (5-methyl-5H-indolo[3,2-b]quinoline), and isocryptole-
pine (5-methyl-5H-indolo[3,2-c]quinoline) display potent
cytotoxic activity against several tumor cell lines, and it is
believed that this is due to the interaction with the DNA.
However, despite intensive investigations of indoloquinolines
(hundreds of analogues in the past 30 years), the mechanism of
their action is still controversial.1
Figure 1. Structures of 5,11-dimethyl-5H-indolo[2,3-b]quinoline (I,
DiMIQ, IC50 = 1.14 μM against KB cell line) and 6,11-dimethyl-6H-
indolo[2,3-b]quinoline (II) (IC10 = 12.3 mM against KB cell line).
minoalkyl,6 glycosyl, aminoglycosyl,7 and alkylaminoalkyl8 were
synthesized. Although a lot of work has been done, the
potential of indoloquinolines as a drug is far from being
completely explored because all of the new derivatives of
indolo[2,3-b]quinoline, despite their high antitumor activity,
exhibited high toxicity or poor solubility in water. The studies
showed impaired bioavailability of 5H-indolo[2,3-b]quinoline
derivatives which brought necessity for further work on the
modification of the indoloquinoline ring structure.
It was found that 5,11-dimethyl-5H-indolo[2,3-b]quinoline
(I, DiMIQ), the analogue of neocryptolepine, is the most
promising lead compound for potential anticancer agents
among different derivatives of indoloquinolines.2 Contrary to
its 6,11-dimethyl-6H-analogue (II) (Figure 1), DiMIQ displays
potent antiproliferative activity in vitro.2 The activity of DIMIQ
derivatives results from the ability to intercalate the DNA and
create a drug−DNA−topoisomerase II complex.3,4 Unfortu-
nately, the solubility of DiMIQ in aqueous solutions, especially
at neutral pH, is very low and seriously limits the practical
application of this compound in the treatment of cancer.5 In the
hope to improve the bioavailability of the indoloquinoline
system, the new derivatives of 5H- and 6H-indolo[2,3-
b]quinolines substituted with methyl,4 methoxy, 6-dimethyla-
Therefore, there is still a need to look for new derivatives of
indolo[2,3-b]quinoline possessing a high anticancer activity
Received: December 30, 2011
Published: May 10, 2012
© 2012 American Chemical Society
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accompanied by a low general toxicity and a good solubility in
aqueous medium, improving their bioavailability.
As it was presented in our previous paper, the transformation
Scheme 1. Synthesis of Hydrochloride Amino Acid and
Dipeptide Derivatives of DiMIQ
a
of inactive 6,11-dimethyl-6H-indolo[2,3-b]quinoline (II, IC10
=
12.3 mM against oral carcinoma, KB cells) into its amino acid
derivatives results in a pronounced cytotoxicity of the obtained
conjugates.9 The derivatives substituted with glycylamide (IC50
= 3.32 μM) and with L-prolylamide (IC50 = 4.39 μM) exhibit
the highest antiproliferative activity, comparable to that of
DiMIQ, (IC50 = 1.14 μM). Compounds bearing L-histidylamide
and L-serinylamide also have a high antiproliferative activity,
and the values of IC50 for these compounds range from 5.96 to
10.77 μM.
These results encourage us to continue the SAR study of
amino acid or peptide derivatives of DiMIQ (I). It is well-
known that attachment of an amino acid or a peptide to the
drug can increase its selectivity.10 Several L- and D- amino acids
were introduced into the anthraquinone skeleton, and
numerous derivatives were synthesized for the evaluation of
the anticancer activity. For example, such conjugates as
conjugates of doxorubicin, daunorubicin, and mitoxantrone
with an amino acid or peptides show more selective antitumor
activity than the drugs themselves.11−20 The attachment of an
amino acid or a peptide to the drug can also increase the
solubility in water of the new compound.21,22
In light of these facts and in the hope to obtain some novel
compounds with a significant anticancer activity, this work
reports the synthesis of a novel series of amino acid and
dipeptide derivatives of DiMIQ (I) and the testing of these
compounds for their in vitro and in vivo activity. At first the
antiproliferative activity in vitro against KB cells was screened
for all the new obtained compounds. The most promising
compounds, selected on the basis of this screening, were tested
for their antiproliferative activity against non-small-cell lung
cancer (A549), breast cancer (MCF-7), and colon cancer
(LoVo). In addition to the above cancer cell lines, compounds
were also screened for normal cell lines. The hemolysis of
human erythrocytes was also investigated in order to obtain
additional data on potential toxic effects of the tested
compounds. On the basis of the above studies, four compounds
were selected for the in vivo tests. Their physicochemical
properties (pKa and log P) were also examined.
a
Reagents and conditions: (a) Boc-AA, TBTU, HOBt, DIPEA, DMF,
rt, 6−24 h; (b) 2.2 M HCl/methanol, rt, 2 h.
gave the best yields in a relatively short reaction time.9 All the
coupling reactions were performed in DMF (because of a very
poor solubility of substrate 1 in the majority of organic solvents
like DCM or THF) at room temperature for 2−24 h. In all
cases Boc-Nα-L-amino acid or Boc-Nα-D-amino acids derivatives
of 1, compounds 2−6, were separated by extraction and
purified by column chromatography on silica gel, and the yield
after the purification was between 55% and 67%. In the next
step Boc groups were removed by 2.2 M HCl in methanol. The
deprotection gave the corresponding hydrochlorides 2a−6a as
the final products with good yields ranging from 80% to 95%.
The purity of the obtained final products was proved by
elemental analysis and the C-18 RP-HPLC method using
acetonitrile−water as the mobile phase.
The peptide derivatives of 1 were synthesized using the “step
by step” method. The appropriate amino acid derivatives of 1
(N-(5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-yl)glycylamide
dihydrochloride (2a) and N-(5,11-dimethyl-5H-indolo[2,3-
b]quinolin-9-yl)-L-prolylamide dihydrochloride (3a)) were
coupled with the Boc-Nα-L-amino acids by the TBTU method
in DMF. The crude products, compounds 7−11, were then
separated by extraction and purified by column chromatog-
raphy. The coupling yields of the amino acid derivatives of
DiMIQ with the next Boc-amino acid were good and ranged
from 80% to 88%. The synthetic yield for the second coupling
was found to be better than the initial coupling. The superiority
in the later coupling may be due to the improved solubility in
DMF. The Boc-removal of compounds 7−11 with hydrogen
chloride in methanol gave the hydrochloride peptide derivatives
of DiMIQ, compounds 7a−11a, with good yields ranging from
91% to 98%. The hydrochlorides of the deprotected derivatives
of DiMIQ were purified by recrystallization. The structures and
the yields of all the new peptide derivatives of 1 are displayed in
Table 2.
RESULTS AND DISCUSSION
■
Chemistry. This paper presents the synthesis of the amino
acid derivatives of 5,11-dimethyl-5H-indolo[2,3-b]quinoline (I)
by the reaction of its 9-aminoderivative (1) with selected amino
acids such as glycine, L-proline, D-proline, L-histidine, and D-
histidine. The peptide derivatives of 5,11-dimethyl-5H-indolo-
[2,3-b]quinoline were also synthesized. The derivatives of 5,11-
dimethyl-5H-indolo[2,3-b]quinoline with such dipeptides as
Gly-Gly, Gly-L-Pro, L-Pro-Gly, L-Pro-L-Pro, and L-His-Gly were
obtained by a “step by step” method. The synthesis of the
amino acid and peptide conjugates of 1 is outlined in Scheme 1.
The structures of the new compounds and the yields are
summarized in Tables 1 and 2. The starting 1 was not
commercially available and was synthesized as described
previously.4,6,23 Compound 1 was then coupled with the
commercially available N-Boc protected L- and D-amino acids.
The coupling was achieved using the 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) meth-
od.24 The activation method was selected for the coupling
based on our recent results which showed that this method
The examination of the solubility of the resulting conjugates
was important, since DiMIQ has a very limited solubility in
water. We examined the solubility in water of the obtained
hydrochloride amino acids and the peptide derivatives of 1
(2a−11a) in Tables 1 and 2, according to European
Pharmacopoeia, General Notices.25 It turned out that attaching
of the amino acid or the peptide moiety to the sparingly soluble
reference compound DiMIQ (hydrochloride of DiMIQ was
tested and 10 mg needed 1 mL of water to completely dissolve,
so it was sparingly soluble) substantially increased the solubility
in water of the resulting conjugate, and compounds 2a−11a
were very soluble or freely soluble in water (for example, for 2a,
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Table 1. Overall Coupling and Deprotection Yields (%) Obtained for All the Derivatives of DiMIQ and Solubility of the
a
Obtained the Hydrochloride Amino Acid Derivatives of DiMIQ
a
The symbol “*/” indicates data according to European Pharmacopoeia.25
10 mg was completely dissolved in 0.01 mL of water and it was
very soluble; for 9a, 10 mg was completely dissolved in 0.1 mL
of water and it was freely soluble). This better solubility results
from the hydrophilic nature of the amino acid and the peptide
part of the conjugates occurring in the hydrochloride form.
When tested in vivo, these novel analogues with improved
water solubility might play critical role in their bioavailability.
Antiproliferative Activity in Vitro. All the synthesized
compounds 2a−11a were evaluated for their antiproliferative
activity in vitro against the KB cells (oral carcinoma cell). The
results of the studies on the antiproliferative activity of the
amino acid and peptide derivatives of DiMIQ are summarized
in Table 3. The tested compounds showed diverse activity
against the KB cells. The IC50 values of the amino acid
derivatives 2a, 3a, and 4a (values between 0.42 and 0.73 μM)
and the dipeptide derivatives 10a and 11a (value 0.56 and 0.7,
respectively) were lower than IC50 of the reference compounds
DiMIQ (IC50 = 1.14 μM) and doxorubicin hydrochloride
(DOX, IC50 = 0.84 μM). The maximal potency was achieved
the other hand, compounds 5a, 6a, 7a, 8a, 9a appeared to be
less active than DiMIQ (and also DOX). It is interesting that
the antiproliferative activity of the dipeptide derivatives of
DiMIQ was generally lower than the activity of the
unsubstituted DiMIQ. The only exception was the dipeptide
derivative, where the dipeptide moiety was attached to the
indoloquinoline skeleton by the L-proline linker. For example,
the IC50 value for compound 9a is only 3.73 μM, but the IC50
for the compound 10a is 0.56 μM. This different antiprolifer-
ative activity among the amino acid and the dipeptide
derivatives to DiMIQ probably results from their different
mechanisms of action and suggests that the kind of the amino
acid linked with DiMIQ plays an important role in the
antiproliferative activity of the tested compounds.
We selected three amino acid derivatives (2a, 3a, 4a) and
four dipeptide derivatives (7a, 9a, 10a, and 11a) for further
study of the antiproliferative activity against the following cell
lines: A549 (non-small-cell lung cancer), MCF-7 (breast
cancer), LoVo (colon cancer), and normal mice fibroblasts
(BALB/3T3). As seen in Table 4, the majority of the prepared
compounds showed high cytotoxicity against all the cell lines.
The most potent ones were derivatives 3a, 4a, 10a, and 11a
(Table 4). The IC50 values for these derivatives (value between
with the glycine (2a, IC50 = 0.67) and the L-proline (3a, IC50
=
0.42) side chain, but no significant differences in the cytotoxic
activity between the L-amino acid and the D-amino acid
derivatives (3a, IC50 = 0.42; 4a, IC50 = 0.73) were observed. On
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Table 2. Overall Coupling and Deprotection Yields (%) Obtained for All the Derivatives of DiMIQ and Solubility of the
a
Obtained the Dihydrochloride Dipeptide Derivatives of DiMIQ
a
The symbol “*/” indicates data according to European Pharmacopoeia.25
0.6 and 0.96 μM) were greater than the IC50 of the reference
compound DiMIQ (IC50 between 0.2 and 2.19 μM), and some
of these differences were statistically significant. Good activity,
comparable to that of DiMIQ, is shown by compounds 2a, 7a,
and 9a with IC50 ranging from 0.73 to 2.7 μM.
All the tested derivatives were cytotoxic against the normal
mouse fibroblast (BALB/3T3). The antiproliferative activity of
compounds 7a and 9a was comparable to that of the reference
DiMIQ (IC50 of 1.71−3.96 μM vs 5.77 μM), but it was lower
than against the A549 and LoVo tumors cell lines. The
antiproliferative activity of the remaining compounds against
BALB/3T3 (IC50 of 0.36−0.56 μM) was 5 times higher than
the cytotoxicity of DiMIQ and comparable to the activity
against cancer cells.
Antineoplastic Effect in Vivo. The collected study results
allowed us to select derivatives for in vivo studies. We have
chosen cyclophosphamide as a positive control because it is in
clinical use for the treatment of malignant diseases for over 40
years and it is one of the most useful anticancer agents, used in
the treatment of, for example, breast, lung, and ovary cancer.
The evaluation of the antitumor activity in vivo was done for
the following four conjugates of DiMIQ: with glycine (2a), L-
proline (3a), and the peptides glycylglycine (7a), and glycyl-L-
proline (9a). All the compounds were dissolved in water and
investigated in the mouse Lewis lung cancer model (LLC)
inoculated subcutaneously. We selected these compounds
because 2a and 3a had the highest activity against the KB
cell line and a very good activity against A549, MCF 7, and the
LoVo cell line. Compound 3a showed a higher activity in vitro
against all the tested cell lines, in particular the cell lung cancer
(A549). However, both compounds 2a and 3a showed a potent
cytotoxic activity against the normal mouse fibroblast (BALB/
3T). On the basis of the obtained data, it was quite clear that
compounds 7a and 9a, a little less active against KB but highly
active against the lung cancer cell line (A549) and against the
colon cancer cell line (LoVo) and moderately cytotoxic against
the normal mouse fibroblast (BALB/3T), were attractive
candidates for a further assessment in vivo as antitumor agents.
The results are summarized in Figures 2−5 and Tables 5 and
6. Mice received tested compounds in doses 15 and 30 mg kg−1
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observed the antitumor properties of the dipeptide derivative
7a (Figure 4b). At dose 50 mg kg−1 day−1 it inhibited the tumor
growth by 46−63% on days 11−16 and 29−43% on days 18−
23. The lower dose of this derivative inhibited the tumor
growth by about 22−37% (Table 5). The derivative 7a at the
dose 50 mg kg−1 day−1 has antitumor activity similar to that of
cyclophosphamide at the beginning of the experiment (days
11−16), but at the end of the experiment when the antitumor
effect of CY was decreased (20−30% TGI), the activity of 7a
and also 3a derivatives was still high (29−43% TGI). The
dipeptide derivative 9a did not reveal antitumor activity at dose
25 mg kg−1 day−1 (Figure 5b). At the highest dose used, it
inhibited the tumor growth by 33−36% on days 14, 19, and 21
(Table 6), and the effect was similar to the anticancer activity of
100 mg/kg CY. In the groups of mice receiving the tested
compounds some body weight loss, however not exceeding
15%, was observed (Figures 2a, 3a, 4a, and 5a).
Table 3. In Vitro Cytotoxic Activities of the Hydrochloride
Amino Acid and Dipeptide Derivatives of DiMIQ against KB
Cell Line
a
compd
amino acid
IC50 [μM]
DiMIQ
DOX
2a
1.14 0.61
0.84 0.03
0.67 0.93
0.42 0.16
0.73 0.24
3.46 0.68
4.85 0.93
4.65 0.75
3.64 1.4
3.73 1.0
0.56 0.24
0.7 0.21
Gly
3a
L-Pro
4a
D-Pro
5a
L-His
6a
D-His
7a
Gly-Gly
L-His-Gly
L-Pro-Gly
L-Pro-L-Pro
Gly-L-Pro
8a
9a
10a
11a
a
IC50: compound concentration leading to 50% inhibition of cell
Results demonstrated that the attachment of amino acid or
peptide moiety to the indoloquinoline skeleton enhances its
solubility in water, but there is no close dependency between
solubility and anticancer activity of the “hybrid” compounds. It
seems that the presence of the amino acid or peptide moiety
may also have an influence on the mechanism of biological
action of the “hybrid“. The high anticancer activity of
compounds 3a, which has L-proline as a substituent, particularly
proved that the kind of amino acid linked to DiMIQ plays a
crucial role in the anticancer activity. We know that the
mechanism of action of neocryptolepine depends on the
topoisomerase II inhibition and on the DNA intercalation, but
in this case we cannot exclude that the inhibitions of targets
other than this system can contribute to the anticancer activity
of the presented compounds. The exact mechanism of action of
these compounds has not been established; however, it will be a
subject of our further studies.
Determination of pKa and Octanol−Water Partition
Coefficients for Compounds 2a, 3a, 7a, and 9a. The pKa
values of 2a, 3a, 7a, and 9a were deduced from their UV spectra
taken in buffers of various pH values in a range of 2−10 at 37
°C at a fixed wavelength. The final concentration of the tested
compounds was 5 μM. Under such conditions, the linearity of
the absorbance−compound concentration relationship was
preserved. The pKa values of 2a, 3a, 7a, and 9a were found
to range from 7.2 to 7.5 with the formation of the isosbestic
point at 280 nm (Table 7). This indicates that they are partially
protonated under physiological conditions (pH 7.4).
proliferation. DiMIQ: reference compound. DOX: doxorubicin
hydrochloride.
day−1 (2a and 3a) or in doses 25 and 50 mg kg−1 day−1 (7a and
9a) intraperitoneally (ip). One single dose (100 mg/kg) of
cyclophosphamide (CY) was used as a positive control
reference cytostatic. The doses of the 2a, 3a, 7a, and 9a agents
used in the animal experiments were selected on the basis of
the previous toxicity evaluation (Figures S1−S4 and Table S1
of Supporting Information) which was conducted in the range
of doses from 5 to 50 mg/kg. The compounds 7a and 9a did
not reveal any toxicity up to the highest dose; however,
derivatives 2a and 3a were highly toxic at this dose.
Compound 3a possessed the best antitumor properties
(Figure 3b). At the dose 30 mg kg−1 day−1 it inhibited the
tumor growth (TGI) by 60−74% on days 11−16 and by 38−
50% on days 18−23. The antitumor properties were much
higher than antitumor properties of a single dose of
cyclophosphamide (100 mg/kg) whose TGI reached a
maximum 30−49.8% on days 11−16. At the lower dose (15
mg kg−1 day−1) of 3a the maximal inhibition of the tumor
growth reached 48% (Table 5). Compound 2a had also
antitumor properties (Figure 2b). At the highest dose used, it
inhibited the tumor growth by 50−55% on days 11−16 and by
14−20% on days 18−23. The lower dose gave a similar effect
(Table 5). The antitumor effects of 2a were similar to the
influence of cyclophosphamide on tumor growth. We also
Table 4. In Vitro Cytotoxic Activities of the Hydrochloride Amino Acid and Peptide Derivative of DiMIQ against Human
a
Cancer Cell Lines and Normal Mice Fibroblasts
IC50 [μM]
compd
amino acid
BALB/3T3
A549
MCF-7
LOVO
DiMIQ
DOX
2a
5.77 0.93
1.08 0.03
0.55 0.01
0.56 0.07
0.36 0.04
1.71 0.34
3.96 0.51
0.48 0.11
0.45 0.12
2.19 0.48
0.33 0.10
1.31 0.11
0.48 0.32
0.32 0.10
0.90 0.03
1.15 0.20
0.54 0.13
0.86 0.08
1.54 0.52
0.44 0.16
1.45 0.39
0.82 0.05
0.60 0.07
2.70 0.54
1.43 0.65
0.96 0.01
1.13 0.13
0.20 0.40
0.11 0.03
0.88 0.14
0.81 0.12
0.62 0.21
0.73 0.13
0.98 0.02
0,36 0,05
0.44 0.12
Gly
3a
L-Pro
4a
D-Pro
7a
Gly-Gly
L-Pro-Gly
L-Pro-L-Pro
Gly-L-Pro
9a
10a
11a
a
IC50: compound concentration leading to 50% inhibition of cell proliferation. DiMIQ: reference compound. DOX: doxorubicin hydrochloride. (∗)
p < 0.05 in comparison to DIMIQ, Mann−Whitney U test, Statistica 7.0.
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Figure 2. Effect of 2a on the mice body weight (a) and the growth of the mouse Lewis lung cancer LLC inoculated sc (median) (b). Schedule was as
follows: therapy with ip administration of 2a in dose 15 mg kg−1 day−1 from day 9 to day 13 and in dose 30 mg kg−1 day−1 from day 9 to day 12.
Cyclophosphamide (CY) in single dose 100 mg/kg was used as a cytostatic control. For 2a, 30 mg kg−1 day−1, the difference on day 16 was of
statistical significance in comparison to control group: Kruskal−Wallis multiple comparison test, nonparametric, p < 0.05.
Table 5. Effect of Amino Acid (2a, 3a) and Dipeptide (7a) Derivatives of DiMIQ on Inhibition of Mouse Lewis Lung Cancer
a
LLC Inoculated sc
TGI [%]
treatment
dose [mg kg−1 day−1
]
day 11
day 14
day 16
day 18
day 21
day 23
CY
100
15
30
15
30
25
50
49.79
42.4
36.8
31.94
23.85
55.28
47.73
75.82
27.25
52.19
12.46
0
21.26
26.1
13.53
0
30.42
25.78
19.71
24.65
41.1
2a
47.43
54.66
42.61
73.96
31.76
62.61
50.1
14.27
8.38
3a
7a
41.98
61.21
37.41
46.89
49.96
22.41
38.26
37.88
28
35.82
29.32
42.94
a
TGI% was calculated for median. Schedule: therapy with ip administration of 2a or 3a at dose 15 mg kg−1 day−1 from day 9 to day 13 and at dose
30 mg kg−1 day−1 from day 9 to day 12; 7a at doses 25 and 50 mg kg−1 day−1 from day 9 to day 13. Cyclophosphamide (CY) at single dose 100 mg/
kg was used as a cytostatic control.
a
Table 6. Effect of Dipeptide (9a) Derivative of DiMIQ on Inhibition of the Mouse Lewis Lung Cancer LLC Inoculated sc
TGI [%]
treatment
dose [mg kg−1 day−1
]
day 12
day 14
day 17
day 19
day 21
day 24
CY
100
25
24.13
12.93
16.29
48.01
11.6
37.02
0
40.01
3.1
26.99
0
0
9a
0
50
36.52
18.53
33.11
32.46
8.19
a
TGI%: calculated for median. Schedule: therapy with ip administration of 9a at doses 25 and 50 mg kg−1 day−1 from day 10 to day 14.
Cyclophosphamide (CY) at single dose 100 mg/kg was used as a cytostatic control.
The hydrophobic parameter (log P) of 2a, 3a, 7a, and 9a was
estimated under physiological as well as in acidic and basic
conditions. The determined partition coefficients covered a
range of over 4.0 log units (Figure 6). The log P values for all
the compounds were negative at pH 2.5 and positive at pH 9.0.
At low pH values, the tested compounds occurred in the form
of salts, and because of a better water solubility their log P
values became negative (values ranged between −1.3 and
−1.9), similar to that of DiMIQ whose log P is −1.0 at pH 2.2.4
Nevertheless, we observed distinct differences in more basic
conditions, at pH 7.4, because the log P values of compounds
2a and 3a were positive (0.6 and 1.4 respectively), but at the
same condition the log P values of compounds 7a and 9a were
negative with values −0.2 and −0.1, respectively. These
compounds appeared to be much more hydrophilic in contrast
to the unsubstituted DiMIQ, which is more hydrophobic (log P
of 2.0 in pH 5.0). The presence of the hydrophilic amino acid
chain or even more hydrophilic peptide chain like Gly-Gly or
Gly-L-Pro in conjugates with DiMIQ increased their hydro-
philic properties, and it corresponded with the good biological
profile of these compounds, for example, the anticancer activity
in vivo. It is possible that the hydrophilic compounds react
mainly with the cytosol soluble targets like glutathione, leading
to its depletion, causing oxidative stress and triggering cell
death.26
DiMIQ, 2a, 3a, 7a, and 9a Induced Isotonic Hemolysis
of Human Erythrocytes. The effect of the tested compounds
on human erythrocytes to determine whether they are able to
interact with the natural membranes and lead to the disruption
of red blood cells was also estimated. The effects of the studied
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Figure 3. Effect of 3a on the mice body weight (a) and the growth of the mouse Lewis lung cancer LLC inoculated sc (median) (b). Schedule was as
follows: therapy with sc administration of 3a in dose 15 mg kg−1 day−1 from day 9 to day 13 and in dose 30 mg kg−1 day−1 from day 9 to day 12.
Cyclophosphamide (CY) in single dose 100 mg/kg was used as a cytostatic control. For 3a, 30 mg kg−1 day−1, differences in days 14, 16, and 23 were
of statistical significance in comparison to control group: Kruskal−Wallis multiple comparison test, nonparametric, p < 0.05.
Figure 4. Effect of 7a on the mice body weight (a) and the growth of the mouse Lewis lung cancer LLC inoculated sc (median) (b). Schedule was as
follows: therapy with sc administration of 7a in doses 25 and 50 mg kg−1 day−1 from day 9 to day 13. Cyclophosphamide (CY) in single dose 100
mg/kg was used as a cytostatic control. For 7a, 50 mg kg−1 day−1, differences in days 16, 18, and 23 were of statistical significance in comparison to
control group: Kruskal−Wallis multiple comparison test, nonparametric, p < 0.05.
Figure 5. Effect of 9a on the mice body weight (a) and the growth of the mouse Lewis lung cancer LLC inoculated sc (median) (b). Schedule was a
follows: therapy with sc administration of 9a at doses 25 and 50 mg kg−1 day−1 from day 9 to day 13. Cyclophosphamide (CY) at single dose 100
mg/kg was used as a cytostatic control.
compounds were assayed as a function of the concentration. As
shown in Figure 7, all the compounds present in the
erythrocyte suspension caused the disruption of red cells in
isotonic conditions (pH 7.4, hematocrit of 50%). Among the
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solubility in water of these conjugates. These novel neo-
cryptolepine derivatives were obtained by the reaction of the
key intermediate 5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-
amine (1) with the appropriate amino acid to give products
with a moderate to good yield. Attaching the following amino
acid to the amino acid derivatives of DiMIQ gave the dipeptide
derivatives of DiMIQ. All the new hydrochloride amino acid
and peptide derivatives of DiMIQ were tested for their
solubility in water, and all of the new compounds showed
very good or free solubility in water. During this study the new
amino acid and dipeptide derivatives of DiMIQ were tested for
their antiproliferative activity against tumor cells. As a result,
four of the new compounds were selected for the investigation
of their anticancer activity in vivo, and their physicochemical
properties (pKa and log P) were also examined. The hemolysis
of human erythrocytes was also investigated in order to obtain
additional data on potential toxic effects of the tested
compounds.
Table 7. Isosbestic Points and pKa of 2a, 3a, 7a, and 9a
a
a
compd
isosbestic point [nm]
pKa
λ [nm]
2a
3a
7a
9a
280
280
280
278
7.2
7.2
7.5
7.3
290
290
290
290
a
Determination of pKa was performed at indicated wavelength.
Summing up, we arrived at the following conclusions: (1) All
the amino acid and peptide derivatives of 5H-indolo[2,3-
b]quinoline displayed good antiproliferative activity against the
KB cells. (2) The majority of the new derivatives displayed
good antiproliferative activity against A549, MCF-7, LoVo. (3)
The cytotoxic activity of the new compounds against the
normal mice fibroblasts was mixed. (4) The amino acids and
peptide derivatives of 5H-indolo[2,3-b]quinoline showed a high
antitumor activity in vivo. (5) The conjugate 5H-indolo[2,3-
b]quinoline with peptide glycylglycine (7a) was the most
promising derivative because it strongly inhibited the growth of
the tumor in mice and it did not show any disadvantageous
effects. (6) The attachment of the hydrophilic amino acid or
the peptide chain to the hydrophobic 5H-indolo[2,3-b]-
quinoline increased its hydrophilic properties. (7) The
attachment of the amino acid or the peptide chain to 5H-
indolo[2,3-b]quinoline significantly decreased its hemolytic
activity compared to DiMIQ, which is correlated with the low
in vivo toxicity (especially for compounds 7a, and 9a).
Figure 6. Octanol−water partition coefficients (log P) of compounds
2a, 3a, 7a, 9a. The log P was defined as log(Coctanol/Cwater). The
amount of the compound in the water phase was determined
spectrophotometrically, and the amount in the octanol phase was
obtained by subtracting the supernatant concentration from the total
concentration. Presented values are the mean of two independent
experiments done in triplicate.
Our results clearly show that the attachment of an amino acid
or a peptide chain to DiMIQ significantly improves its
physicochemical properties, although the antiproliferative
activity in vitro remains similar to that of DiMIQ. On the
basis of our studies, concerning the encouraging anticancer
activity in vivo and low hemolytic activity, we choose
compound 7a as our new lead compound. These results are
very promising and the detailed studies of the mechanisms of
action, the pharmacokinetics, and the tissue distribution of
these new amino acid and dipeptide derivatives of neo-
cryptolepine will be the subject of the next investigations.
Nevertheless, the problem of low solubility, high toxicity, and
maybe the drug selectivity has been at least partially solved by
synthesizing the salts of the amino acid and peptide derivatives
of DiMIQ. Overall, the neocryptolepine derivatives containing
the amino acid or the peptide side chains have been confirmed
as promising lead compounds for the development of new,
highly potent, and selective agents against cancer.
Figure 7. DiMIQ, 2a, 3a, 7a, 9a induced isotonic hemolysis of human
erythrocytes (pH 7.4, hematocrit of 50%, incubation time of 30 min at
37 °C). Data are given as the mean
SD of three individual
determinations: (∗) p < 0.05 in comparison to DIMIQ, Mann−
Whitney U test, Statistica 7.0.
newly synthesized agents, 2a exhibits the strongest hemolytic
behavior (42.5% at 0.3 mM). On the other hand the hemolytic
activity of all the tested compounds is lower than the hemolytic
activity of DiMIQ. Moreover, as shown earlier, compounds 7a
and 9a seemed to be the most promising ones because of the
lowest hemolytic activity (only 9.97% at 0.3 mM) which is
correlated with the low in vivo toxicity of these derivatives.
CONCLUSION
■
EXPERIMENTAL SECTION
■
On the basis of the previous reports indicating that the amino
acids Gly, L-Pro, and L-His are a significant requirement for the
antiproliferative activity of conjugates with 6H-indolo[2,3-
b]quinoline (II), a series of DiMIQ analogues containing the
amino acid and the dipeptide chains was synthesized with the
initial objective to increase the selectivity of the action and the
General. Melting points were determined using a Kofler type
apparatus and were uncorrected. The IR spectra were recorded with a
Perkin-Elmer 1640 FTIR BX spectrophotometer in KBr pellets. The
1H and 13C NMR spectra were measured using Varian-NMR-
vnmrs500, Varian-NMR-vnmrs600, and Varian Gemini 200 spec-
trometers. The 1H and 13C NMR spectral data are given in relation to
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the TMS and DSS signal at 0.0 ppm for DMSO and D2O solutions,
respectively. The ESI mass spectra were recorded on a PE Biosystems
Mariner mass spectrometer. Progress of the reaction was monitored by
thin layer chromatography (TLC) with Merck DC-Alufolien Kieselgel
60 F254. The chemicals and solvent were purchased from Fluka
Company. Column chromatography was performed on Merck silica
gel 60 (230−400 mesh). Measurement of optical rotation is performed
using a Jasco digital polarimeter P-2000, and measurements of optical
rotation are made at 589 nm at 20 °C. HPLC experiments were carried
out on a Waters HPLC system (Waters Associates, Milford, MA, U.S.)
consisting of a multisolvent delivery system 600E, a photodiode array
detector 2996, a Rheodyne model 7725i injector, chromatography
manager Empower 2 software for PC coputations, and Luna C-18
column (Phenomenex, U.S.) with 5 μm particles, 250 mm × 4.6 mm.
The detection was performed by using UV at λ = 275 nm. The
compound concentration was about 2.0 mg/mL, and the injection
volume was 20 μL. Method 1 was as follows: mobile phase consisting
of A (H2O + 0.1% TFA, v/v) and B (acetonitrile + 0.1% TFA, v/v)
was used with a linear gradient from 20% B to 100% B for 20 min (2a)
and from 30% B to 100% B for 20 min (3a, 7a). Method 2 was as
follows: mobile phase consisting of A (H2O + 0.05% TFA, v/v) and B
(acetonitrile) was used with a linear gradient from 10% B to 80% B for
20 min (9a). All key compounds were proven by HPLC method to
have ≥95% purity.
General Procedure for the Synthesis Compounds 2 and 3.
To a solution of Nα-protected L-amino acid (0.50 mM), TBTU (1
mM), and HOBt (1 mM) in DMF (3 mL) was added DIPEA (1.5
mM). The mixture was stirred for 15 min at room temperature. Then a
solution of 5,11-dimethyl-5H-indolo[2.3-b]quinolin-9-ylamine (1)
(0.76 mM) in 2 mL of DMF was added, and the reaction mixture
was stirred at room temperature for 6−24 h (TLC monitoring). After
the reaction was completed, the solvent was evaporated under reduced
pressure at ∼40 °C. The resulting oil was treated with water and
CHCl3, and the organic layer was separated and washed successively
with aqueous NaHCO3 solution and aqueous NaCl solution. The
extract was dried over anhydrous MgSO4, filtered, and evaporated to
dryness.
d, J = 8.1 Hz), 7.63 (1H, m), 7.49 (1H, d, J = 1.8 Hz), 6.74 (1H, d, J =
8.3 Hz), 6.47 (1H, dd, J = 1.8 and 8.3 Hz), 3.78 (2H, m), 3.74 (3H, s),
2.51 (3H, s). 13C NMR (150.83 MHz, D2O) [ppm]: 166.9, 151.4,
147.0, 136.9, 136.72, 136.70, 135.1, 129.1, 128.8, 124.6, 122.8, 121.2,
119.6, 118.6, 114.7, 114.3, 43.6, 38.2, 18.2. ESI-MS: calcd for (M +
H)+, 319.3; found, 319.3. Anal. Calcd for C19H18N4O·2H2O·2HCl: C
53.40, H 5.66, N 13.11, Cl 16.59. Found: C 53.54, H 5.67, N 13.07, Cl
16.58. Purity (HPLC): 99.3%.
N-(5,11-Dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-L-prolyla-
mide Dihydrochloride (3a). Compound 3 (115 mg, 0.25 mM) was
treated with 2.2 M HCl/CH3OH (2 mL) with stirring for 3 h (TLC
monitoring). When TLC showed that the Boc group was completely
removed, the precipitated salt 3a was collected by filtration and
recrystallized from ethyl acetate/diethyl ether. Yield 97 mg (87%). Mp
(°C): 255. IR: 3411, 2943, 1675, 1640, 1575, 1477, 1269, 764 cm−1.
1H NMR (500 MHz, D2O) [ppm]: 8.34 (1H, d, J = 8.5 Hz), 8.08 (1H,
s), 8.07 (1H, m), 8.00 (1H, d, J = 8.5 Hz), 7.79 (1H, m), 7.29 (1H, d, J
= 8.0 Hz), 7.17 (1H, d, J = 8.0 Hz), 4.50 (1H, dd, J = 6.5 and 8.5 Hz),
4.12 (3H, s), 3.58 (2H, m), 2.95 (3H, s), 2.66 (1H, m), 2.23 (3H, m).
13C NMR (125.68 MHz, D2O) [ppm]: 169.8, 151.8, 148.3, 138.4,
137.3, 136.4, 134.9, 129.1, 128.5, 125.1, 124.2, 122.4, 120.7, 118.7,
117.0, 114.9, 62.7, 49.2, 38.1, 32.6, 26.4, 18.2. ESI-MS: calcd for (M +
H)+, 359.3; found, 359.3. Anal. Calcd for C22H22N4O·2H2O·2HCl: C
56.53, H 6.04, N 11.99 Cl 15.17. Found: C 56.97, H 6.27, N 12.05, Cl
15.20. Purity (HPLC): 98.4%. [α]2D0 −12.3 (c 0.1, H2O)
General Procedure for the Synthesis 7 and 9. To a solution of
Nα-protected amino acid (0.8 mM) in DMF (6 mL) were added
TBTU (0.8 mM), HOBt (0.8 mM), and DIPEA (0.4 mL, 2.2 mM) at
room temperature, and the solution was stirred for 15 min. Next 2a
(0.5 mM) was added to the solution, and the reaction mixture was
stirred for 24 h. After the reaction was completed the solvent was
evaporated under reduced pressure at ∼40 °C. The resulting oil was
treated with water and CHCl3, and the organic layer was separated and
washed successively with aquoeus NaHCO3 solution and aqueous
NaCl solution. The extract was dried over anhydrous MgSO4, filtered,
and evaporated to dryness.
Nα-tert-Butyloxycarbonyl-N-(5,11-dimethyl-5H-indolo[2,3-
b]quinolin-9-yl)glycylglycylamide (7). Compound 7 was obtained
from Boc-Gly and 2a. The crude products were purified by
crystallization from ethyl acetate to afford compound 7 as an orange
crystal. Yield 231 mg (86%). Mp (°C): 240. IR: 3383, 2976, 2930,
Nα-tert-Butyloxycarbonyl-N-(5,11-dimethyl-5H-indolo[2,3-
b]quinolin-9-yl)glycylamide (2). Compound 2 was obtained as a
red solid from Boc-Gly-OH and 1. The crude product was
recrystallized from ethyl acetate. Yield 206 mg (65%). Mp (°C):
212−215. IR: 3278, 2976, 1688, 1651, 1569, 1523, 1461, 1249, 1167
cm−1. 1H NMR (500 MHz, DMSO-d6) [ppm]: 9.89 (1H, s), 8.56 (1H,
s), 8.29 (1H, d, J = 7.5), 7.92 (1H, d, J = 9.0 Hz), 7.83 (1H, m), 7.59
(1H, d, J = 9.0 Hz), 7.50 (1H, m), 7.49 (1H, d, J = 8.0 Hz), 4.23 (3H,
s), 3.76 (2H, d, J = 6.0 Hz), 3.07 (3H, s), 1.40 (9H, s). ESI-MS: calcd
for (M + H)+, 419.2; found, 419.2. Anal. Calcd for C24H26N4O3: C
68.88, H 6.26, N 13.39. Found: C 68.34, H 6.42, N 13.07.
1
1684, 1639, 1534, 1482, 1367, 1228, 1169, 760 cm−1. H NMR (200
MHz, CD3OD) [ppm]: 8.83 (1H, br s), 8.63 (1H, d, J = 9.5 Hz), 8.32
(1H, d, J = 8.8 Hz), 8.13 (1H, m), 7.86 (1H, m), 7.76 (1H, d, J = 8.8
Hz), 7.62 (1H, d, J = 8.8 Hz), 4.45 (3H, s), 4.09 (2H, s), 3.82 (2H, s),
3.33 (3H, s), 1.47 (9H, s). ESI-MS: calcd for (M + H)+, 476.3; found,
476.3. Anal. Calcd for C26H29N5O4·H2O: C 63.27, H 6.33, N 14.19.
Found: C 63.31, H 6.31, N 14.12.
Nα-tert-Butyloxycarbonyl-N-(5,11-dimethyl-5H-indolo[2,3-
b]quinolin-9-yl)-L-prolylamide (3). Compound 3 was obtained as
an orange solid from Boc-L-Pro and 1. The crude product 3 was
purified by chromatography on silica gel column with CHCl3/
CH3OH, 9:1 (v/v), and crystallized from diethyl ether. Yield 187 mg
(45%). Mp (°C): 210−212. IR: 3311, 2975, 1673, 1635, 1525, 1460,
Nα-tert-Butyloxycarbonyl-N-(5,11-dimethyl-5H-indolo[2,3-
b]quinolin-9-yl)-L-prolylglycylamide (9). Compound 9 was
obtained from Boc-L-Pro and 2a. The crude products were purified
by crystallization from ethyl acetate to afford compound 9 as an orange
crystal. Yield 186 mg (84%). Mp (°C): 225−227. IR: 3436, 3302,
2973, 1703, 1675, 1565, 1168, 747 cm−1. 1H NMR (200 MHz,
DMSO-d6) (two rotamers) [ppm]: 10.28 and 9.85 (1H, s and s), 8.77
and 8.73 (1H, s and s), 8.53 (1H, d, J = 8.4 Hz), 8.23 (1H, d, J = 8.4
Hz), 8.05 (1H, m), 7.98 (1H, d, J = 8.4 Hz), 7.74 (1H, m), 7.67 (1H,
d, J = 9.0 Hz), 4.39 (3H, s), 4.19 (1H, m), 3.94 (2H, m), 3.60 (2H,
m), 3.23 (3H, s), 2.16 (1H, m), 1.86 (3H, m), 1.41 and 1.37 (9H, s
and s). ESI-MS: calcd for (2M + H)+ 1031.6, (M + H)+ 516.3; found,
1031.6, 516.3. Anal. Calcd for C29H33N5O4·3H2O: C 61.14, H 6.90, N
12.29. Found: C 59.84, H 6.87, N 12.29.
1
1166, 748 cm−1. H NMR (500 MHz, DMSO-d6) (two rotamers)
[ppm]: 9.98 (1H, s), 8.67 and 8.64 (1H, s and s), 8.33 (1H, d, J = 8.0
Hz), 7.96 (1H, d, J = 8.5 Hz), 7.86 (1H, m), 7.61 (1H, dd, J = 1.0 and
8.5 Hz), 7.53 (2H, m), 4.29 (1H, m), 4.27 (3H, s), 3.47 (1H, m), 3.38
(1H, m), 3.11 (3H, s), 2.25 (1H, m), 1.96 (2H, m), 1.83 (1H, m), 1.43
and 1.33 (9H, s and s). ESI-MS: calcd for (M + H)+, 459.3; found,
459.3. Anal. Calcd for C27H30N4O3·2H2O: C 65.57, H 6.93, N 11.33.
Found: C 65.44, H 6.76, N 11.43.
N-(5,11-Dimethyl-5H-indolo[2,3-b]quinolin-9-yl)glycylamide
Dihydrochloride (2a). Compound 2 (206 mg, 0.49 mM) was treated
with 2.2 M HCl/CH3OH (3 mL) with stirring for 2 h (TLC
monitoring). When TLC showed that the Boc group was completely
removed, the precipitated salt 2a was collected by filtration and
recrystallized from ethyl acetate. Yield 153 mg (80%). Mp (°C): 270.
N-(5,11-Dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-
glycylglycylamide Dihydrochloride (7a). Compound 7 (124.1 mg,
0.26 mM) was treated with 2.2 M HCl/CH3OH (3 mL) with stirring
for 1 h (TLC monitoring). When TLC showed that Boc group was
completely removed, the precipitated salt 7a was collected by filtration
and recrystallized from ethyl acetate. Yield 110 mg (94%). Mp (°C):
252−254. IR: 3330, 3100, 1788, 1682, 1645, 1430, 1200, 1135, 795
1
IR: 3418, 2957, 1682, 1642, 1573, 1476, 1240 cm−1. H NMR (600
1
MHz, D2O) [ppm]: 8.00 (1H, d, J = 8.1 Hz), 7.91 (1H, m), 7.70 (1H,
cm−1. H NMR (500 MHz, DMSO-d6 with small addition of H2O)
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[ppm]: 10.56 (1H, s), 8.92 (1H, t, J = 5.4 Hz), 8.79 (1H, s), 8.58 (1H,
d, J = 8.2 Hz), 8.32 (1H, d, J = 8.8 Hz), 8.27 (3H, br s), 8.12 (1H, m),
7.83 (1H, m), 7.76 (1H, d, J = 8.6 Hz), 7.63 (1H, d, J = 8.6 Hz), 4.48
(3H, s), 4.08 (2H, d, J = 6.0 Hz), 3.71 (2H, s), 3.24 (3H, s). 13C NMR
(125.68 MHz, DMSO-d6 with small addition of H2O) [ppm]: 167.4,
166.7, 148.3, 146.9, 136.4, 135.4, 134.4, 133.4, 126.9, 125.6, 122.7,
120.9, 120.6, 119.6, 116.9, 114.1, 112.9, 42.9, 40.4, 36.6, 16.0. ESI-MS:
calcd for (M + H)+ 376.3, (M + 2H)2+ 188.8; found, 376.3, 188.8.
Anal. Calcd for C21H21N5O2·2HCl·6H2O: C 45.33, H 6.34, N 12.59,
Cl 12.74. Found: C 45.30, H 6.24, N 12.45, Cl 12.98. Purity (HPLC):
98.6%.
(3) Pognan, F.; Saucier, J.-M.; Paoletti, C.; Kaczmarek, Ł.; Nantka-
Namirski, P.; Mordarski, M.; Peczynska-Czoch, W. A carboline
́
derivative as a novel mammalian DNA topoisomerase II targeting
agent. Biochem. Pharmacol. 1992, 44, 2149−2155.
́ ́
(4) Peczynska-Czoch, W.; Pognan, F.; Kaczmarek, Ł.; Boratynski, J.
Synthesis and structures−activity relationship of methyl-substituted
indolo[2,3-b]quinolines: novel cytotoxic, DNA topoisomerase II
inhibitors. J. Med. Chem. 1994, 37, 3503−3510.
(5) Jaromin, A.; Kozubek, A.; Suchoszek-Lukaniuk, K.; Malicka-
́
Blaszkiewicz, M.; Peczynska-Czoch, W.; Kaczmarek, L. Liposomal
formulation of DIMIQ, potential antitumor indolo[2,3-b]quinoline
agent and its cytotoxicity on hepatoma Morris 5123 cells. Drug
Delivery 2008, 15, 49−56.
N-(5,11-Dimethyl-5H-indolo[2,3-b]quinolin-9-yl)-L-prolylgly-
cylamide Dihydrochloride (9a). The compound 9 (159 mg, 0.31
mM) was treated with 2.2 M HCl/CH3OH (2 mL) with stirring for 2
h (TLC monitoring). When TLC showed that Boc group was
completely removed, the precipitated salt 9a was collected by filtration
and recrystallized from ethyl acetate. Yield 139 mg (92%). Mp (°C):
(6) Kaczmarek, Ł.; Łuniewski, W.; Zagrodzki, B.; Godlewska, J.;
Osiadacz, J.; Wietrzyk, J.; Opolski, A.; Peczynska-Czoch, W. Synthesis
́
of 6-substituted 6H-indolo[2,3-b]quinolines as novel cytotoxic agents
and topoisomerase II inhibitors. Acta Pol. Pharm. 2002, 59, 199−207.
(7) Godlewska, J.; Badowska-Rosłonek, K.; Ramza, J.; Kaczmarek, Ł.;
1
267. IR: 3410, 3300, 2927, 1686, 1649, 1482, 1225, 766 cm−1. H
NMR (500 MHz, DMSO-d6 with small addition of H2O) [ppm]:
10.47 (1H, s), 8.91 (1H, t, J = 5.8 Hz), 8.74 (1H, d, J = 1.7 Hz), 8.60
(1H, d, J = 8.6 Hz), 8.32 (1H, d, J = 8.9 Hz), 8.12 (1H, m), 7.85 (1H,
m), 7.76 (1H, dd, J = 1.8 and 8.8 Hz), 7.67 (1H, d, J = 8.8 Hz), 4.39
(3H, s), 4.26 (1H, m), 4.05 (2H, d, J = 5.6 Hz), 3.26 (3H, s), 3.22
(2H, m), 2.36 (1H, m), 1.92 (3H, m). 13C NMR (125.68 MHz,
DMSO-d6 with small addition of H2O) [ppm]: 169.1, 167.6, 149.2,
147.3, 136.3, 135.8, 134.8, 133.8, 127.3, 126.1, 123.3, 121.6, 121.1,
120.0, 117.3, 114.9, 113.3, 59.5, 46.1, 43.1, 36.7, 29.9, 23.9, 16.3. ESI-
MS: calcd for (M + H)+ 416.2, (M + H)++ 208.6; found, 416.2, 208.6.
Anal. Calcd for C24H25N5O2·2HCl·2H2O: C 54.96, H 5.96, N 13.35,
Cl 13.52. Found: C 54.15, H 5.63, N 13.39, Cl 14.01. Purity (HPLC):
98.2%. [α]2D0 −16.96 (c 0.1, H2O).
́
Peczynska-Czoch, W.; Opolski, A. New saccharide derivatives of
indolo[2,3-b]quinoline as cytotoxic compounds and topoisomerase II
inhibitors. Radiol. Oncol. 2004, 38, 137−144.
(8) Godlewska, J.; Łuniewski, W.; Zagrodzki, B.; Kaczmarek, Ł.;
Bielawska-Pohl, A.; Dus, D.; Wietrzyk, J.; Opolski, A.; Siwko, M.;
́
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for compounds 4, 5, 6, 8, 10, 11, 4a,
1
5a, 6a, 8a, 10a, 11a; H NMR, 13C NMR, IR, MS, elemental
analysis, purity (HPLC), and toxicity evaluation of compounds
2a, 3a, 7a, and 9a; biological procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +48-22-456-3915. Fax: +48-22-456-3838. E-mail: k.
sidoryk@ifarm.eu.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
Boc, tert-butyloxycarbonyl; DIPEA, N,N-diisopropylethylamine;
DCM, dichloromethane; DMF, dimethylformamide; DMSO,
dimethylsulfoxide; DSS, 4,4-dimethyl-4-silapentane-1-sulfonic
acid; HCl, hydrochloride acid; HOBt, N-hydroxybenzotriazole
monohydrate; HPLC, high performance liquid chromatogra-
phy; IR, infrared; MS, mass spectrometry; NMR, nuclear
magnetic resonance; SAR, structure−activity relationship;
TBTU, O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate; TEA, triethylamine; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; TMS, trimethylsilyl
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