Searching for new derivatives of neocryptolepine: Synthesis, antiproliferative, antimicrobial and antifungal activities

Article abstract of DOI:10.1016/j.ejmech.2014.03.060

A series of novel amino acid and dipeptide derivatives of neocryptolepine were synthesized and tested for their antimicrobial, antifungal and antiproliferative activity in vitro against cancer cell lines (KB, A549, MCF-7, LoVo) and normal mice fibroblast cells (BALB/3T3). Biological evaluation revealed that almost all of the new compounds displayed high antiproliferative activity against the tested cells and moderate to potent antibacterial activities. Interestingly, these compounds were active against Candida albicans biofilms at doses significantly lower than those required against free-floating planktonic fungal cells. The most promising compounds are derivatives with glycine and l-proline as a substituent both at 2 and at 9 position of 5H-indolo[2,3-b]quinoline. In general, these new compounds (2a, 3a, 6a and 7a) showed the highest dual action against cancer lines and infectious pathogenic microbes in vitro.

Full text of DOI:10.1016/j.ejmech.2014.03.060

European Journal of Medicinal Chemistry 78 (2014) 304e313
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Searching for new derivatives of neocryptolepine: Synthesis,
antiproliferative, antimicrobial and antifungal activities
,
b
c
d
Katarzyna Sidoryk a , Anna Jaromin , Jessica A. Edward , Marta Switalska ,
ꢀ
*
Joanna Stefanska , Piotr Cmoch f, Joanna Zagrodzka a, Wojciech Szczepek a,
e
a,
ꢀ
g
d
b
c
ꢀ
Wanda Peczynska-Czoch , Joanna Wietrzyk , Arkadiusz Kozubek , Robert Zarnowski ,
David R. Andes c, qukasz Kaczmarek a
a Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warsaw, Poland
b Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wroclaw, Poland
c Department of Medicine, Section of Infectious Diseases, 4125 Microbial Sciences Building, 1550 Linden Dr., University of Wisconsin-Madison, Madison,
WI 53706, USA
d Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Weigla St., 53-114 Wroclaw, Poland
e Medical University of Warsaw, Department of Pharmaceutical Microbiology, 3 Oczki St., 02-007 Warsaw, Poland
f Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
g Division of Organic Technology, Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel amino acid and dipeptide derivatives of neocryptolepine were synthesized and tested
for their antimicrobial, antifungal and antiproliferative activity in vitro against cancer cell lines (KB, A549,
MCF-7, LoVo) and normal mice fibroblast cells (BALB/3T3). Biological evaluation revealed that almost all
of the new compounds displayed high antiproliferative activity against the tested cells and moderate to
potent antibacterial activities. Interestingly, these compounds were active against Candida albicans
biofilms at doses significantly lower than those required against free-floating planktonic fungal cells. The
Received 6 August 2013
Received in revised form
23 February 2014
Accepted 19 March 2014
Available online 20 March 2014
most promising compounds are derivatives with glycine and L-proline as a substituent both at 2 and at 9
Keywords:
position of 5H-indolo[2,3-b]quinoline. In general, these new compounds (2a, 3a, 6a and 7a) showed the
highest dual action against cancer lines and infectious pathogenic microbes in vitro.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Neocryptolepine
Antiproliferative activity
Antimicrobial activity
Biofilm
Amino acid
Peptide
NMR
1. Introduction
Numerous pharmacological activities have been demonstrated
for the extracts prepared either from entire plants or their indi-
Many drugs used in modern medicine are either directly iso-
lated from plants or synthetically modified from lead compounds of
natural origin. Among numerous currently exploited medicinal
African plant species, Cryptolepis sanguinolenta (Lindl.) Schltr.
(Periplocaceae) has recently received greater attention from an
immense number of researchers and pharmaceutical companies.
This stemmed twining and scrambling shrub, which contains an
orange-colored juice in the cut stem [1], has been used by some
traditional herbalists in the treatment of broad symptoms of fever,
urinary and upper respiratory tract infections, malaria, rheuma-
tism, and venereal diseases [2,3].
vidual fractions. Indeed, anti-malarial [4e8], anti-diabetic [9e11],
anti-thrombotic [12], anti-inflammatory activities [13,14], and
more recently described anti-androgenic and anti-spermatogenic
properties with potential anti-aphrodisiac activity [15] have been
experimentally determined. Simultaneously, the C. sanguinolenta
extracts have aroused considerable interest because of their anti-
microbial activities that might be potentially beneficial to human
health [16e21]. The active components found in this plant are
known to be the indoquinoline alkaloids, consisting mainly of the
indole and the quinoline moieties. The major alkaloid of the roots,
cryptolepine (5-methyl-5H-indolo[3,2-b]quinoline), is reported to
possess intricate biological effects, while neocryptolepine (5-
methyl-5H-indolo[2,3-b]quinoline, Fig. 1a) remains a minor alka-
loid of C. sanguinolenta.
* Corresponding author.
E-mail addresses: k.sidoryk@ifarm.eu, sidorykk@gmail.com (K. Sidoryk).
http://dx.doi.org/10.1016/j.ejmech.2014.03.060
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
305
Fig. 1. Structures of neocryptolepine (a) and 5,11-dimethyl-5H-indolo[2,3-b]quinoline
(DiMIQ, b).
One of the reasons for the growing frequency of nosocomial
Scheme 1. General method for the synthesis of amino acid and dipeptide derivatives
of 5,11-dimethyl-5H-indolo[2,3-b]quinoline substituted in position 2.
infections is the increasing use of immunosuppressive agents in
antitumor and transplant therapies, which leads to breakdown of
the barrier between the gut and bloodstream in humans. This
process quite often results in the formation of highly drug-resistant
microbial biofilms, which is definitely disadvantageous for human
health. The development of new anticancer drugs that would offer
the efficacy of antimicrobial prophylaxis in clinical settings is
therefore urgently needed, especially since the number of thera-
peutic options for cancer-accompanying infectious diseases re-
mains relatively limited. There is also increasing awareness of the
hazards related to the overuse of antibiotics and other toxic
chemical agents that lead to multidrug resistance in pathogenic
microorganisms. Overall, this has led to accelerated investigations
on naturally occurring products and their chemically modified
derivatives as new sources of anticancer agents that would have the
potential of antimicrobial lock therapy in clinical practice.
In our previous studies we reported the syntheses of neo-
cryptolepine derivatives containing an amino acid or a dipeptide at
the C-9 position and their evaluation for antitumor activity [22]. It
was shown that the derivatives of 5,11-dimethyl-5H-indolo[2,3-b]
quinoline, an analogue of neocryptolepine, with a substituted amino
acid or dipeptide chain in position 9, displayed high antiproliferative
activity in vitro and antitumor activity in vivo. The biological data
revealed that the attachment of an amino acid moiety or a short
peptide chain to 5,11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ,
Fig. 1b) significantly improved its physicochemical properties,
resulting in auspicious in vivo anticancer actions with relatively low
hemolytic levels in the host. Research on the relationship between
structures and antiproliferative activity of indolo[2,3-b]quinolines
revealed that not only the nature of the substituents but also the
position of the substituents in the core of indolo[2,3-b]quinolines
played the crucial role for the cytotoxic activity of these derivatives
[23,24]. For example, many of the alkylaminoalkyl derivatives of 5H-
indolo[2,3-b]quinoline substituted at position C-2 displayed a higher
antiproliferative activity than the same derivatives substituted at
position C-9 [23]. Compounds of this family, especially amino acid
derivatives of DiMIQ, displayed high anticancer activity, but their
antimicrobial and antifungal activity have not been investigated yet.
In this paper, we describe synthesis of the new derivatives of
neocryptolepine substituted with either an amino acid or a
dipeptide chain at the C-2 position. These compounds were eval-
uated for their antiproliferative activity in vitro against normal
(BALB/3T3) and several types of cancer cell lines (KB, MCF-7, A549
and LoVo), as well as further investigated as potential antimicro-
bials against the most common pathogenic bacteria and fungi that
cause cancer- and transplant-associated infections in humans.
quinolin-2-ylamine (1) with 1.3 molar equivalent of Na-tert-buty-
loxycarbonyl-amino acid. The coupling was achieved using 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU) as a coupling activator in the presence of N-hydrox-
ybenzotriazole, and N,N-diisopropylethylamine [25]. The starting
material 1 was not commercially available and was synthesized as
described previously [26e29]. In all cases the crude compounds 2e
4 were treated with water and CHCl3, and the organic layer was
separated, and washed successively with NaHCO3 aq solution and
NaCl aq solution. The extract was dried over anhydrous MgSO4,
filtered and evaporated to dryness. The products 2e4 were then
purified by flash chromatography with a mixture of chloroform and
methanol. The pure fractions were crystallized from ethyl acetate
(compounds 2 and 3) or from diethyl ether (compound 4). The yield
of coupling reaction and the purification of compounds 2e4 ranged
from 63 to 67%. In the next step Boc groups were removed by 2.2 M
HCl in methanol. The deprotection gave the appropriate hydro-
chlorides 2ae4a as the final products with good yields ranging
from 85 to 95%. The peptide derivative of 1 was synthesized using
the “step by step” method. N-(5,11-dimethyl-5H-indolo[2,3-b]qui-
nolin-2-yl)glycylamide dihydrochloride (2a) was coupled with Boc-
Na-Gly by the TBTU method in DMF. The crude product 5 was then
separated by extraction and purified by flash chromatography with
a mixture of chloroform and methanol. The pure fraction was
evaporated and the product was crystallized from the ethyl acetate
and the yield after purification was 84%. The Boc removal of com-
pound 5 with hydrogen chloride in methanol gave the hydrochlo-
ride 5a with a good yield of 91%. The purity of the obtained final
products (2ae5a) was assessed by the analytical C18 RP-HPLC
method using acetonitrile-water as a mobile phase. The purity of
all synthesized analogues was in the range 95e98%.
The structures and the yields of all the new amino acid and
dipeptide derivatives of 1 are displayed in Table 1. The formation of
compounds 2e5 and 2ae5a was confirmed by the elementary
analysis and ESI-MS spectra. To confirm structures of the synthe-
sized compounds 1D and 2D NMR experiments (see Experimental
section) were performed and 1H NMR and 13C NMR characteris-
tics of these compounds are presented in the Experimental Section.
The 1H NMR spectra of compounds 2, 4 and 5 showed a charac-
teristic signal of nine protons of tert-butyl group as a singlet in the
range 1.40e1.52 ppm. In the case of compound 3, due to the pres-
ence of two rotamers [22], there are two singlets at
d 1.55 and
1.70 ppm (ca. 6.8 H and ca. 2.2 H, respectively). The aliphatic pro-
tons signals from the amino acid moiety were observed for all the
compounds in 1H NMR spectra. It is important, that the measure-
ments of NMR had to be performed in a mixture of DMSO and D2O
(solubility of the most of new compounds in organic solvents was
poor), and it led to a disappearance of several proton signals in the
1H NMR spectra of compounds 2ae5a. It was caused by their ex-
change with an excess of D2O. In case of N-Boc protected
2. Results and discussion
2.1. Synthesis
The target compounds depicted in Scheme 1 were obtained by
reacting the starting material 5,11-dimethyl-5H-indolo[2.3-b]

306
K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
Table 1
The yields (in %) obtained for the amino acid and dipeptide derivatives of 5,11-dimethyl-5H-indolo[2,3-b]quinoline substituted in position 2.
Compound
Substituent in position 2
Structure
Yields (%)
2
Boc-Gly
67
2a
Gly
95
3
Boc-
L
-Pro
63
3a
L-Pro
90
4
Boc-
L
-His
65
4a
L-His
85
5
Boc-Gly-Gly
84
91
5a
Gly-Gly

K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
307
compounds 2e5, NH protons are usually non-exchangeable and
that is why they were visible in 1H NMR spectra as sharp signals.
There was also one exception to the above-mentioned description.
range 3299e3426 cmꢀ1, is characteristic of NeH amide stretching.
The second one, located at 1669e1693 cme1 and broad, is a su-
perposition of two strong C]O absorption bands of amide and
carbamate. Finally, the last moderate band at 1365e1367 cme1 can
be attributed to CeH aliphatic rocking. After Boc deprotection the
obtained final products 2ae5a showed the presence of the same
bands in the ranges 3331e3457 cme1, 1681e1702 cme1 and 1361e
1369 cme1, respectively. However, the lack of carbamate carbonyl
resulted in narrowing of the amide carbonyl band at 1681e
1702 cme1. The remaining strong absorption bands, present in both
series of compounds, are characteristic of their heteroaromatic
nucleus.
In compound 4, which is a -histidine derivative of 5,11-dimethyl-
L
5H-indolo[2,3-b]quinoline, proton of the imidazole ring takes part
in an exchange process between both imidazole nitrogen atoms.
Therefore in 1H NMR spectrum no presence of this proton was
observed.
The attachment of the amino acids to 5,11-dimethyl-5H-indolo
[2,3-b]quinoline clearly evidenced from the 13C NMR spectra of
these compounds which showed the characteristic carbonyl signal
from amide above 160 ppm. In all cases, 13C NMR spectra showed
one carbonyl signal (C-10) at
d
166.4 ppm for 2a, 169.1 ppm for 3a,
170.8 ppm for 4, and 170.0 ppm for 4a. In the dipeptide derivatives
two carbonyl signals were observed: at
168.5 ppm (C-10), and at
170.0 ppm (C-30) for compound 5, and at 168.6 ppm (C-10), and
170.2 ppm (C-30) for compound 5a. Moreover, in 13C NMR
2.2. Biological studies
d
d
d
2.2.1. Antiproliferative activity in vitro
at
d
The synthesis and antiproliferative activity of amino acid and
dipeptide derivatives of 5,11-dimethyl-5H-indolo[2,3-b]quinoline,
substituted in position 9, were described previously [22]. The
structures of these compounds are summarized in Table 2.
All the synthesized compounds 2ae5a were evaluated for their
antiproliferative activity in vitro against the KB cells (cervix
spectra of Boc derivatives 4 and 5 the carbonyl signals from Boc
group were observed at 155.3 ppm, and 156.0 ppm, respectively.
d
They disappeared after deprotection.
For comparison purposes the 1H/13C NMR chemical shifts of 2-
(2a) and 9-(6a) glycine containing neocryptolepine derivatives are
presented in Table 1 in the Supplementary Material. To indentify
the structures of both isomeric indoloquinoline 2a and 6a, the re-
sults of 1D/2D NMR experiments were carefully analyzed. At first, to
distinguish both 1H NMR spin systems (ABX and AA0BB0) in isomeric
compounds 2a and 6a, NOE effects were used. The irradiation of the
methyl group at ca. 2.82 ppm in case of both isomeric compounds
(2a and 6a) leads to NOE signal enhancements at 7.95 and 8.33 ppm
for compound 2a or 8.03 and 8.23 ppm for compound 6a. Multi-
plicity of these signals and values of J (HeH) enable to differentiate
both X protons (at 8.03 and 8.33 ppm) of the spin system ABX in
isomeric 2a and 6a. Additionally, using results of 1He13C g-HSQC/g-
HMBC experiments for both 2a and 6a, complete 1H/13C NMR signal
assignment was achieved. Analysis of these NMR results
completely confirmed the structure of both isomeric neo-
cryptolepines 2a and 6a. Similar procedure was performed for all
other compounds 3e5 and 3ae5a leading to a complete confir-
Table 2
Structures of amino acid and dipeptide derivatives of 5,11-dimethyl-5H-indolo[2,3-
b]quinoline, substituted in position 9.
Compound
Substituent in position 9
Structure
DiMIQ
e
6a
7a
Gly
mation of their structures. Moreover, comparison of the 1H/13
C
NMR data for compounds 4 and 4a is in a complete accordance with
results presented by Bednarek and co-workers [30].
L-Pro
Additionally, we performed a gradient selected 1He15N HMBC
experiment for the most of the synthesized compounds. The 15N
NMR chemical shifts were presented in Experimental Section. Most
of the 15N results were obtained for these samples where the sol-
ubility was relatively high (compounds 3a, 4 and 5). In case of 2a, 4a
and 5a only N-5 signals were observed. Not all of the expected
correlations were obtained. Especially the measurements of hy-
drochlorides 2a and 5a were difficult because of their very low
solubility in a DMSO-D2O mixture. The 15N NMR chemical shifts
obtained for protected compounds, which existed in the neutral
form, are in agreement with data published by Bednarek and co-
workers [30]. Formation of hydrochlorides (2a, 3a, and 5a) is con-
nected with protonation of the nitrogen atom of the hetero-
aromatic core, most probably N-6. Comparison of the NMR data
8a
L-His
9a
Gly-Gly
(
13C/15N NMR chemical shifts) between neutral compounds and
their hydrochlorides leads to several deshielding/shielding effects.
Most significant are shielding increase of C5a and C6a cores (by ca.
8 and ca.14 ppm, respectively) as well as deshielding of nitrogen N5
by ca. 10 ppm.
All synthesized compounds were also characterized by FT-IR
spectra. Compared to the substrate, amine 1, the Boc protected
amino acid derivatives 2e5 revealed two additional strong ab-
sorption bands and one moderate absorption band coming from
the introduced amino acid fragments. The first one, lying in the
10a
Gly-L-Pro

308
K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
Table 3
In vitro antiproliferative activities of the hydrochloride amino acid and peptide derivative of DiMIQ against human cancer cell lines and normal mice fibroblasts. IC50
compound concentration leading to 50% inhibition of cell proliferation; DiMIQ e referential compound; DOX e doxorubicin hydrochloride.
e
Compd.
Subst.
IC50 [mM]
BALB/
3T3
A549
MCF-7
LoVo
KB
DiMIQ
DOX
2a
3a
4a
5.77 ꢁ 0.93
1.08 ꢁ 0.03
0.42 ꢁ 0.02
0.54 ꢁ 0.11
e
2.19 ꢁ 0.48
0.33 ꢁ 0.10
0.08 ꢁ 0.04
0.12 ꢁ 0.05
e
1.54 ꢁ 0.52
0.44 ꢁ 0.16
0.46 ꢁ 0.09
0.62 ꢁ 0.05
e
0.20 ꢁ 0.40
0.11 ꢁ 0.03
0.06 ꢁ 0.01
0.07 ꢁ 0.02
e
1.14 ꢁ 0.61
0.84 ꢁ 0.03
0.42 ꢁ 0.11
0.47 ꢁ 0.17
3.88 ꢁ 0.41
1.54 ꢁ 0.01
Gly
L
L
-Pro
-His
5a
Gly-Gly
2.11 ꢁ 0.20
1.44 ꢁ 0.53
4.11 ꢁ 0.44
1.17 ꢁ 0.38
carcinoma). The results of the studies on the antiproliferative ac-
tivity of the amino acid and peptide derivatives of DiMIQ are
summarized in Table 3. The tested compounds showed diverse
activity against the KB cell line. The IC50 values of the amino acid
activity against the non-mutated normal BALB/3T3 cell line was
noticeably lower (IC50 0.54 M).
m
Our experimental data presented in the current work showed
no significant differences in determined toxicity levels between the
tested C-2, and C-9 substituted amino acid and dipeptide DIMIQ
derivatives. However, the tested C-9 derivatives displayed no dif-
ferences in antiproliferative activity against normal and cancer cell
lines whereas the C-2 derivatives displayed notable alterations in
antiproliferative levels against both types of tested cell lines. This
exciting finding indicates the presence of a distinct mechanism of
action for this particular group of DiMIQ derivatives, which is
determined by the type of substituent introduced and its structural
localization. Indeed, derivatives substituted with an amino acid in
position 2 and 9 had higher antiproliferative activities than de-
rivatives containing peptides in their structures.
derivatives 2a and 3a (values in the range 0.42e0.47
lower than IC50 of the reference compounds, DiMIQ and doxoru-
bicin hydrochloride (DOX), which were 1.14 M and 0.84 M,
respectively. The compounds 4a, substituted with -histidine, and
5a, substituted with glycylglycine, had lower activity than the
reference compounds and their IC50 values were 3.88 M and
1.54 M, respectively. Furthermore, the antiproliferative activity of
mM) were
m
m
L
m
m
the derivative that contained glycine at position C-2 (2a) was
higher than the antiproliferative activity of the corresponding de-
rivative substituted with the same amino acid in position C-9 (IC50
0.67
pound derivatives either with
0.42 M [22]) or -histidine (IC50 3.88
m
M [22]). No significant differences in activities of the com-
-proline (IC50 0.47 M, vs. IC50
M, vs. IC50 3.46 M [22])
L
m
m
L
m
m
2.2.2. Antimicrobial and antibiofilm activity in vitro
were observed in this study. Strikingly, the activity of dipeptide
derivatives of DiMIQ, which in fact contains glycylglycine in its
skeleton at position C-2, was higher than the activity of the anal-
Antimicrobial resistance has become an increasingly serious
public health problem in a wide range of infectious diseases,
especially in those associated with cancer and transplant therapies.
It is, therefore, imperative to design novel and improve already
existing antimicrobials that lead towards more efficient treatment
of diseases caused by microbial pathogens. Strikingly, about 80% of
all human-related infections involve microbial biofilms, which
constitute loosely organized structured communities that are
significantly more difficult to culture and more resistant to control
strategies and host defenses than their planktonic counterparts.
The title compounds were tested for their in vitro antibacterial
and antifungal activity by the twofold serial agar dilution method
to determine their minimal inhibitory concentration (MIC) under
standardized conditions according to CLSI guidelines [31,32].
The results of antimicrobial activity of the compounds are
summarized in Table 4. The tested compounds were active
ogous dipeptide in position C-9 (IC50 1.54 mM, vs. IC50 4.65 mM [22]).
The selected compounds 2a, 3a and 5a were subjected to further
studies of their antiproliferative activity against the following cell
lines: non-small cell lung cancer A549, breast cancer MCF-7, colon
cancer LoVo, and the normal mice fibroblasts BALB/3T3. As outlined
in Table 2, the majority of the prepared compounds showed high
antiproliferative activity against all the tested cell lines. Interest-
ingly, the glycine derivative of DiMIQ (2a) had the highest activity
against A549 (IC50 0.08
this activity was higher than the activity observed against the
normal BALB/3T3 cell line (IC50 0.42 M). The -proline derivative of
DiMIQ (3a) was also active in the tested cell line systems with the
mM) and LoVo (IC50 0.06 mM) cell line, and
m
L
highest activity against LoVo cell line (IC50 0.07 mM), whereas its
Table 4
In vitro antimicrobial activities of the amino acid and dipeptide derivatives of 5H-indolo[2,3-b]quinoline substituted in position 2 and 9 (na) e compounds having MIC value
>300 g/mL. Cipr. e Ciprofloxacin, Fluc. e Fluconazole.
m
Tested strain
MIC (mg/mL)
Gly (2a) Pro (3a) His (4a) Gly-Gly (5a) Gly (6a) Pro (7a) His (8a) Gly-Gly (9a) Gly-Pro (10a) DiMIQ Cipr.
Fluc.
S. aureus NCTC 4163
S. aureus ATCC 25923
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
B. subtilis ATCC 6633
B. cereus ATCC 11778
E. hirae ATCC 10541
M. luteus ATCC 9341
M. luteus ATCC 10240
C. albicans ATCC 10231
C. albicans ATCC 90028
C. albicans SN250
12.5
12.5
12.5
12.5
12.5
12.5
50
100
6.25
12.5
100
100
15.62
12.5
50
12.5
50
12.5
6.25
25
100
6.25
6.25
100
50
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
250
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>500
>200
50
50
50
100
25
12.5
50
100
6.25
6.25
25
50
15.62
25
12.5
50
12.5
50
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
125
>200
>200
>200
>200
>200
100
>200
>200
200
200
200
200
200
>200
>200
>200
100
100
100
>200
>200
50
50
200
100
200
100
100
100
50
100
200
50
0.25
0.5
0.25
0.5
0.25
<0.125
1
4
2
1
e
e
e
e
e
e
e
e
e
e
e
1
12.5
12.5
50
100
6.25
6.25
200
200
200
200
50
12.5
12.5
12.5
12.5
200
200
200
e
1
e
31.25
50
e
C. parapsilosis ATCC 22019 100
>200
>200
200
e
2

K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
309
against Gram-positive bacteria and Candida sp. All examined
derivatives were inactive against Gram-negative rods (data not
shown). The observed inactivity phenomenon could be deter-
mined by multiple yet undefined factors, including (i) a putative
inability of the tested compounds to cross over the Gram-
negative cell wall structure; (ii) the lack of putative target re-
ceptors for DiMIQ derivatives in Gram-negative bacteria; or (iii)
putative interactions with intracellular bacterial molecules that
result in complex formation and drug deactivation. Compounds
2a, 3a, 6a and 7a showed higher activity against tested Gram-
positive cocci in comparison with the reference compound
DiMIQ. For most bacterial strains tested the MIC values of com-
The current medical treatment options for C. albicans biofilm-
related infections are rather rare due to the intrinsic increased
tolerance to antimicrobial drugs. Our striking findings showed the
tested of neocryptolepine derivatives to be potent agents against C.
albicans in vitro biofilms, which were active at doses significantly
lower than those required against free-floating planktonic fungal
cells. Since the examined neocryptolepine derivatives also
possessed anticancer activities, there is a great potential of anti-
fungal lock therapy for the prevention and treatment of biofilm-
related infections in immunocompromised patients. This strategy
lies within a novel trend recently observed in the antimicrobial
research area towards the identification of natural products, plants,
and their extracts that could be potentially used in other medical
fields due to their numerous biological (e.g. anticancer) activities.
Combining both anticancer and antimicrobial activities in one drug
seems to be beneficial as a means to enhance efficacy in a variety of
clinical settings. There are several foreseeable advantages to this
anticancer/antimicrobial strategy that include a widened spectrum
and potency of drug activity, more rapid treatment effects, and
lowered dosing of drugs required for biofilm control and
eradication.
Utilizing neocryptolepine derivatives as agents with different
mechanisms of action may be a hallmark in future medical thera-
pies in numerous medical disciplines, but it is also obvious that one
has to be cautious of some combinations that may be antagonistic
or clinically indifferent with additive side effects. Our results pre-
sented in this study demonstrate the proof-of-concept for the
application of these neocryptolepine derivatives as anticancer and
antimicrobial agents, and their further testing is warranted as a
novel cancer/transplant-associated anti-Candida therapy. It is also
apparent that the dual anticancer/antimicrobial action described
here is an in vitro concept that is difficult to translate into clinical
studies, which are definitely needed, but expensive and hard to
perform.
pounds 2a, 3a, 6a and 7a were between 6.25 and 12.5 mg/mL. An
opposite observation was made with reference to the screened
fungal Candida species. In this case DiMIQ showed significantly
higher antifungal activity (MIC 12.5
compounds.
mg/mL) than other tested
We selected Candida albicans SN250 strain, which appeared
slightly more susceptible to the tested of neocryptolepine de-
rivatives when compared to MIC values measured for the other
fungal isolates examined in this study. First of all doses of the tested
neocryptolepine derivatives required to inhibit growth were defi-
nitely lower for biofilms than for planktonic cell cultures that
contained similar numbers of metabolically active cells. The con-
centrations of -proline and glycine derivatives of 5H-indolo[2,3-b]
L
quinoline substituted in position either 2 or 9 needed to decrease
the burden of mature biofilms by 50% were lower for biofilm cell
inhibition than for planktonic cell inhibition (Fig. 2). -Histidine and
L
both types of dipeptide derivatives of 5,11-dimethyl-5H-indolo[2,3-
b]quinoline substituted in position either 2 or 9 were considerably
less active against C. albicans biofilms and their resulting doses
required to obstruct the burden of mature biofilm cells were
significantly higher. It is also noteworthy that compounds 2a, 6a,
and 7a were most active in the tested in vitro fungal biofilm model,
but levels of their antifungal action were still comparable to anti-
fungal action of DiMIQ, which was used as a reference compound in
this study.
Nevertheless, our study provides evidence of dual action of the
tested neocryptolepine derivatives against cancer lines and infec-
tious pathogenic microbes in vitro, which might be the first step in
Fig. 2. Percentage reduction of SN250 biofilms after 24 h growth and 24 h treatment with amino acid and dipeptide derivatives at C-2 and C-9 position of 5,11-dimethyl-5H-indolo
[2,3-b]quinoline.

310
K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
establishing complex and efficient concurrent anticancer and
antimicrobial therapy.
consisting of A (H2O þ 0.1% TFA, v/v) and B (acetonitrile þ 0.1% TFA,
v/v) was used with a linear gradient from 20% B to 80% B for 20 min
(2a, 3a, 4a, 5a). All key compounds were proven by HPLC method to
show ꢄ95% purity.
3. Conclusion
In conclusion, we synthesized a set of neocryptolepine de-
rivatives substituted with an amino acid or a dipeptide either at C-2
or C-9 position that were further evaluated for antiproliferative and
antimicrobial/antifungal activities. The examined chemicals exer-
ted antiproliferative activity against selected cancer cell lines and
possessed antimicrobial activity against pathogenic Gram-positive
bacteria. Next, this investigation showed the tested neo-
cryptolepine derivatives to be potent agents against C. albicans
in vitro biofilms. Interestingly, some of these compounds were
active against C. albicans biofilms at doses significantly lower than
those required against free-floating planktonic fungal cells as
shown in Table 4.
Our study provides evidence of dual action of the tested indo-
loquinoline derivatives against cancer cell lines and infectious
pathogenic microbes in vitro, and demonstrates the proof-of-
concept for the application of these derivatives as anticancer and
antimicrobial/antifungal agents. Our findings also showed a great
potential of antifungal lock therapy, which might be the first step in
establishing complex and efficient concurrent anticancer and
antimicrobial strategies for the prevention and treatment of
biofilm-related infections in humans.
4.1.2. General procedure for synthesis of compounds 2, 3 and 4
To a solution of Na-protected
-amino acid (1 mM), TBTU (1 mM)
L
and HOBt (1 mM) in DMF (3 mL), DIPEA (1.5 mM) was added and
the mixture was stirred for 15 min at room temperature. Then the
solution of 5,11-dimethyl-5H-indolo[2.3-b]quinolin-2-ylamine (1)
(0.76 mM) in 2 mL DMF was added and the reaction mixture was
stirred at room temperature for 6e24 h (TLC monitoring). After the
reaction was completed the solvent was evaporated under reduced
pressure at ca. 40 ꢂC. The resulting oil was treated with water and
CHCl3, and the organic layer was separated, and washed succes-
sively with NaHCO3 aq solution and NaCl aq solution. The extract
was dried over anhydrous MgSO4, filtered and evaporated to
dryness.
4.1.3. Na-tert-Butyloxycarbonyl-N-(5,11-dimethyl-5H-indolo[2,3-b]
quinolin-2-yl)glycylamide (2)
Compound 2 was obtained as a yellow solid from Boc-Gly-OH
and 1. The crude product 2 was crystallized from ethyl acetate to
afford orange crystal; yield 213 mg (67%); m.p. (ꢂC): 135e137; IR:
3426 (NeHamide), 2977, 1688 (C]Oamideþcarbamate), 1631 (C]Car-
omatic), 1583 (C]Caromatic), 1560, 1489 (ring stretching), 1466 (ring
stretching), 1448 (ring stretching), 1366 (CeHalifatic), 1240 (CeHar-
4. Experimental section
omatic), 1169, 759 (CeHaromatic), 741 (CeHaromatic) cmꢀ1 1H NMR
;
(CDCl3): 8.58 (1H, m, NH), 8.43 (1H, d, J ¼ 2.2 Hz), 8.14 (1H, d,
J ¼ 7.7 Hz), 7.74 (2H, m), 7.60 (1H, d, J ¼ 9.5 Hz), 7.53 (1H, brd t, J ca
7.7 Hz), 7.24 (1H, brd t, J ca 7.3 Hz), 5.32 (1H, m, NH), 4.27 (3H, s),
3.99 (2H, d, J ¼ 6.2 Hz), 3.05 (3H, s), 1.52 (9H, s); ESI-MS: 837.5
(2MþH)þ, 419.2 (MþH)þ; Anal. Calcd. for C24H26N4O3 ꢃ 2H2O
[454.52]: C 64.42, H 6.65, N 12.33 Found: C 64.08, H 6.61, N 12.46.
4.1. Chemistry
4.1.1. General
Melting points (m.p.) were determined using a Kofler-type
apparatus and were uncorrected. The IR spectra were recorded
with a PerkineElmer 1640 FTIR BX spectrophotometer in KBr pel-
lets. The 1H and 13C/15N NMR spectra of all compounds studied
were measured in CDCl3, DMSO-D6 or a mixture of DMSO-D6/D2O
using Varian-NMR-vnmrs500, Varian-NMR-vnmrs600 and Varian
Gemini 200 spectrometers at a temperature of 298 K. Standard
experimental conditions and standard Varian programs were used.
To assign the structures under consideration the following 1D and
2D experiments were employed: the 1D selective NOESY, and 2D
gradient selected COSY, 1He13C HSQC and 1He13C/1He15N HMBC.
The 1H and 13C NMR chemical shifts are given relative to the DMSO
signal at 2.5 ppm (1H NMR spectra) and 39.5 ppm (13C NMR
spectra), whereas nitromethane, whose chemical shift of the 15N
nucleus is 0.0 ppm, was used as a calibration standard for nitrogen
15N NMR spectra. Concentration of all solutions used for mea-
surements was about 10e20 mg of compounds in 0.6e0.8 cm3 of
solvent. The ESI-MS 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 Kie-
selgel 60 F254. The chemicals and solvent were purchased from
Fluka Company. Column chromatography was performed on Merck
silica gel 60 (230e400 mesh). Measurement of optical rotation was
performed using a Jasco Digital Polarimeter P-2000; measurements
of optical rotation were made at 589 nm at 20 ꢂC. HPLC experiments
were carried out on a: Waters HPLC system (Waters Assoc., Milford,
MA, USA) consisting of a Multisolvent Delivery System 600E, a
Photodiode Array Detector 2996, a Rheodyne Model 7725i injector
and Chromatography Manager Empower 2 software for PC com-
4.1.4. N-(5,11-Dimethyl-5H-indolo[2,3-b]quinolin-2-yl)glycylamide
dihydrochloride (2a)
Product 2 (400 mg, 0.95 mM) was treated with 2.2 M HCl/
CH3OH (13 mM, 6 mL) and stirred for 2 h (TLC monitoring). The
precipitated salt 2a was collected by filtration and recrystallized
from ethyl acetate; yield 400 mg (95%); m.p. (ꢂC): 265; IR: 3401 (Ne
Hamide), 2951, 1699 (C]Oamide), 1640 (C]Caromatic), 1615 (C]Car-
omatic), 1579 (C]Caromatic), 1537, 1499 (ring stretching), 1463 (ring
stretching), 1361 (CeHalifatic), 1323, 1251 (CeHaromatic), 1198, 747
(CeHaromatic) cmꢀ1; 1H NMR (DMSO þ D2O): 8.33 (1H, d, J ¼ 2.3 Hz,
H-1), 7.96 (1H, m, H-4), 7.95 (1H, m, H-10), 7.86 (1H, dd, J ¼ 2.3,
J ¼ 9.3 Hz, H-3), 7.29 (1H, m, H-8), 7.28 (1H, m, H-7), 7.19 (1H, m, H-
9), 4.06 (3H, s, 5-CH3), 3.82 (2H, s, H-20), 2.83 (3H, s, 11-CH3); 13C
NMR (DMSO þ D2O): 166.4 (C-10), 148.9 (C-11), 146.5 (C-5a), 140.3
(C-6a), 136.2 (C-2), 132.7 (C-4a), 130.9 (C-8), 127.0 (C-3), 124.9 (C-
10), 124.8 (C-9), 124.3 (C-11a), 120.9 (C-10a), 120.7 (C-10b), 118.6 (C-
4), 116.0 (C-1), 113.5 (C-7), 42.2 (C-20), 37.2 (5-CH3), 17.1 (11-CH3);
15N NMR (DMSO þ D2O): ꢀ243.6 (N-5); ESI-MS: 319.3 (MþH)þ;
Anal. Calcd. for C19H18N4O ꢃ 3H2O ꢃ 2HCl [445.34]: C 51.24, H 5.88,
N 12.58, Cl 15.92 Found: C 51.95, H 5.90, N 12.55, Cl 15.90; HPLC:
97.1%.
4.1.5. Na-tert-Butyloxycarbonyl-N-(5,11-dimethyl-5H-indolo[2,3-b]
quinolin-2-yl)-L-prolylamide (3)
Compound 3 was obtained as a red solid from Boc-L-Pro-OH and
1. The crude product 3 was purified by chromatography on a silica
gel column with chloroform-methanol 8:1 (v/v) and crystallized
from ethyl acetate/diethyl ether to afford an orange solid;
yield 220 mg (63%); m.p. (ꢂC): 198e200; IR: 3302 (NeHamide), 2974,
1669 (C]Oamideþcarbamate), 1632 (C]Caromatic), 1575 (C]Caromatic),
putations; and a Luna C-18 column (Phenomenex, USA) with 5
particles, 250 ꢃ 4.6 mm. Detection was performed using UV at
¼ 275 nm; the compound concentration was about 2.0 mg/mL,
and the injection volume was 20 L. Method: the mobile phase
mm
l
m

K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
311
1557, 1489 (ring stretching), 1467 (ring stretching), 1446 (ring
acetate; yield 170 mg (85%); m.p. (ꢂC): 270; IR: 3401 (NeHamide),
3004, 1702 (C]Oamide), 1638 (C]Caromatic), 1616 (C]Caromatic), 1585
(C]Caromatic), 1465 (ring stretching), 1362 (CeHalifatic), 1248 (Ce
stretching), 1365 (CeHalifatic), 1238 (CeHaromatic), 1162, 759, 741 (Ce
H
aromatic) cmꢀ1; 1H NMR (CDCl3) (two rotamers [22]): 9.97 (1H, m,
NH), 8.44 (1H, n m), 8.13 (1H, brd d, J ¼ 7.8 Hz), 7.81 (1H, dd,
J ¼ 9.3 Hz, J ¼ 2.1 Hz), 7.72 (1H, brd d, J ¼ 7.8 Hz), 7.52 (2H, brd t, J ca
7.6 Hz), 7.22 (1H, brd t, J ¼ 7.6 Hz), 4.57 (1H, m), 4.20 (3H, s), 3.50
(2H, m), 3.06 (3H, s), 2.50 (1H, m), 2.02 (3H, m), 1.70 and 1.55 (9H
[1:3.16], s and s); ESI-MS: 917.6 (2MþH)þ, 459.3 (MþH)þ; Anal.
Calcd. for C27H30N4O3 ꢃ 2H2O [494.58]: C 65.57, H 6.93, N 11.33
Found: C 65.83, H 6.53, N 11.19.
H
aromatic), 1195, 832, 756 (CeHaromatic) cmꢀ1; 1H NMR (unprotected
neutral form for NMR measurements in DMSO-D6): 8.67 (1H, m, H-
1), 8.23 (1H, m, H-10), 8.13 (1H, m, H-3), 7.96 (1H, m, H-4), 7.61 (1H,
m, H-60), 7.58 (1H, m, H-7), 7.48 (1H, m, H-8), 7.18 (1H, m, H-9), 6.92
(1H, m, H-80), 4.27 (3H, s, 5-CH3), 3.82 (1H, m, H-20), 3.09 (3H, s, 11-
CH3), 3.08 (1H, m, H-3’A), 2.85 (1H, m, H-3’B); 13C NMR (unpro-
tected neutral form for NMR measurements in DMSO-D6): 172.0
(C-10), 155.1 (C-6a), 154.4 (C-5a), 139.7 (C-11), 134.9 (C-60), 133.1 (C-
2), 132.8 (C-4a), 128.0 (C-8), 124.7 (C-10b), 124.4 (C-10a), 123.3 (C-
10), 123.1 (C-3), 120.7 (C-11a), 119.0 (C-9), 117.1 (C-7), 115.6 (C-4),
114.8 (C-1), 55.4 (C-20), 32.7 (5-CH3), 31.7 (C-30), 15.2 (11-CH3); 15N
NMR (DMSO): ꢀ254.9 (N-5); ESI-MS: 399.2 (MþH)þ, 200.3
(Mþ2H)þþ; Anal. Calcd. for C23H22N6O ꢃ 5H2O ꢃ 4HCl [670.84]: C
41.18, H 5.56, N 12.53, Cl 26.42 Found: C 41.76, H 5.06, N 12.54, Cl
4.1.6. N-(5,11-Dimethyl-5H-indolo[2,3-b]quinolin-2-yl)-L-
prolylamide dihydrochloride (3a)
Product 3 (180 mg, 0.32 mM) was treated with 2.2 M HCl/CH3OH
(13 mM, 6 mL) and stirred for 2 h (TLC monitoring). The precipi-
tated salt 3a was collected by filtration and recrystallized from ethyl
acetate; yield 134 mg (90%); m.p. (ꢂC): 270; IR: 3457 (NeHamide),
2950, 1698 (C]Oamide), 1643 (C]Caromatic), 1617 (C]Caromatic), 1579
(C]Caromatic), 1499 (ring stretching), 1459 (ring stretching), 1369
26.45; HPLC: 98.4%; ½a D20
¼ 17.4 (c ¼ 0.1, H2O).
ꢅ
(CeHalifatic), 1254 (CeHaromatic), 1202, 747 (CeHaromatic) cmꢀ1
;
1H
4.1.9. Na-tert-Butyloxycarbonyl-N-(5,11-dimethyl-5H-indolo[2,3-b]
quinolin-2-yl)glycylglycylamide (5)
NMR (DMSO þ D2O): 8.32 (1H, d, J ¼ 2.4 Hz, H-1), 7.94 (1H, d,
J ¼ 9.3 Hz, H-4), 7.92 (1H, d, J ¼ 8.0 Hz, H-10), 7.89 (1H, dd,
J ¼ 2.4 Hz, J ¼ 9.3 Hz, H-3), 7.27 (1H, m, H-8), 7.25 (1H, m, H-7), 7.16
(1H, m, H-9), 4.40 (1H, dd, J ¼ 7.0 Hz, J ¼ 9.0 Hz, H-20), 4.04 (3H, s, 5-
CH3), 3.36 (2H, m, H-50), 2.83 (3H, s, 11-CH3), 2.49 (1H, m, H-3’A),
2.06 (1H, m, H-3’B), 2.03 (2H, m, H-40); 13C NMR (DMSO þ D2O):
169.1 (C-10), 149.2 (C-11), 146.8 (C-5a), 140.4 (C-6a), 136.1 (C-2),
133.1 (C-4a), 131.1 (C-8), 127.6 (C-3), 124.98 (C-10), 124.95 (C-9),
124.5 (C-11a), 121.0 (C-10a), 121.0 (C-10b), 118.7 (C-4), 117.0 (C-1),
113.7 (C-7), 61.6 (C-20), 47.8 (C-50), 37.2 (5-CH3), 31.1 (C-30), 25.2 (C-
40), 17.2 (11-CH3); 15N NMR (DMSO þ D2O): ꢀ243.8 (N-5), ꢀ251.2
(2-NH), ꢀ328.0 (N-60); ESI-MS: 359.3 (MþH)þ; Anal. Calcd. for
To a solution of Boc-Gly (122 mg, 0.7 mM) in DMF (7 mL), TBTU
(224 mg, 0.7 mM), HOBt (107 mg, 0.7 mM) and DIPEA (0.35 mL,
2 mM) were added at RT and the solution was stirred for 15 min.
Then the N-(5,11-dimethyl-5H-indolo[2,3-b]quinolin-2-yl)glycyla-
mide dihydrochloride (2a) (200 mg, 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 ca. 40 ꢂC. The resulting oil was treated with water
(10 mL) and chloroform (40 mL) and stirred for 5 min. The organic
layer was separated, and washed successively with NaHCO3 aq
solution (30 mL) and NaCl aq solution (20 mL). The extract was
dried over anhydrous MgSO4, filtered and evaporated to dryness.
The crude product was purified by flash chromatography and
crystallization from ethyl acetate to afford compound 5 as orange
crystal, yield 200 mg (84%); m.p. (ꢂC): 185e186; IR: 3350 (Ne
C
22H22N4O ꢃ 2H2O ꢃ 2HCl [467.39]: C 56.53, H 6.04, N 11.99, Cl
15.17 Found: C 56.65, H 6.10, N 11.89, Cl 15.16; HPLC: 97.1%;
a 2D0
¼ ꢀ2.12 (c ¼ 0.1, H2O).
½
ꢅ
4.1.7. Na-tert-Butyloxycarbonyl-N-(5,11-dimethyl-5H-indolo[2,3-b]
quinolin-2-yl)-L-histidylamide (4)
H
amide), 2979, 1685 (C]Oamideþcarbamate), 1640 (C]Caromatic), 1578
(C]Caromatic), 1500 (ring stretching), 1464 (ring stretching), 1367
(CeHalifatic), 1250 (CeHaromatic), 1170, 750 (CeHaromatic) cmꢀ1 1H
Compound 4 was obtained as a red oil from Boc-L-His-OH and 1.
The crude product 4 was purified by chromatography on a silica gel
column with chloroform-methanol 6:1 (v/v) and crystallized
from diethyl ether to afford a red solid; yield 245 mg (65%); m.p. (ꢂC):
166e168; IR: 3299 (NeHamide), 2978, 1693 (C]Oamideþcarbamate),
1633 (C]Caromatic), 1574 (C]Caromatic), 1489 (ring stretching), 1448
(ring stretching), 1366 (CeHalifatic), 1250 (CeHaromatic), 1168, 744 (Ce
;
NMR (DMSO): 10.5 (1H, m, 2-NH), 8.87 (1H, d, J ¼ 2.2 Hz, H-1), 8.40
(1H, d, J ¼ 8 Hz, H-10), 8.34 (1H, d, J ¼ 9.4 Hz, H-4), 8.30 (1H, t,
J ¼ 5.8 Hz, 20-NH), 8.21 (1H, dd, J ¼ 2.2 Hz, J ¼ 9.4 Hz, H-3), 7.70 (1H,
d, J ¼ 8 Hz, H-7), 7.65 (1H, m, H-8), 7.46 (1H, m, H-9), 7.13 (1H, t,
J ¼ 6.1 Hz, 40-NH), 4.40 (3H, s, 5-CH3), 4.01 (2H, d, J ¼ 5.8 Hz, H-20),
3.67 (2H, d, J ¼ 6.1 Hz, H-40), 3.20 (3H, s, 11-CH3), 1.41 (9H, s, CH3,
13
H
aromatic) cmꢀ1; 1H NMR (DMSO): 10.35 (1H, 2-NH), 8.60 (1H, d, H-1),
Boc); C NMR (DMSO): 170.0 (C-30), 168.5 (C-10), 156.0 (CO, Boc),
8.19 (1H, m, H-10), 8.06 (1H, m, H-3), 7.92 (1H, m, H-4), 7.67 (1H, m,
H-60) 7.57 (1H, m, H-7), 7.47 (1H, m, H-8), 7.18 (1H, m, H-9), 7.15 (1H,
m, 20-NH), 6.89 (1H, m, H-80), 4.43 (1H, m, H-20), 4.25 (3H, s, 5-CH3),
3.03 (3H, s,11-CH3), 3.02 (1H, m, H-3’A), 2.94 (1H, m, H-3’B),1.40 (9H,
s, CH3, Boc); 13C NMR (DMSO): 170.8 (C-10), 155.3 (CO, Boc),154.1 (C-
6a),153.8 (C-5a),140.1 (C-11),134.8 (C-60),133.53 (C-2),133.53 (C-40),
132.5 (C-4a), 128.0 (C-8), 124.3 (C-10b), 124.1 (C-10a), 123.3 (C-10),
123.2 (C-3),120.7 (C-11a),119.2 (C-9),116.72 (C-7),116.72 (C-80),115.6
(C-4), 114.7 (C-1), 78.2 (C, Boc), 55.3 (C-20), 32.8 (5-CH3), 29.7 (C-30),
28.2 (CH3, Boc), 15.2 (11-CH3); 15N NMR (DMSO þ D2O): ꢀ254.2 (N-
5), ꢀ251.3 (2-NH), ꢀ289.7 (20-NH); ESI-MS: 997.6 (2MþH)þ, 499.3
(MþH)þ; Anal. Calcd. for C28H30N6O3 ꢃ 3H2O [552.62]: C 60.86, H
6.57, N 18.06 Found: C 60.21, H 6.05, N 18.45.
147.1 (C-11), 146.4 (C-5a), 140.5 (C-6a), 136.3 (C-2), 131.5 (C-4a),
129.4 (C-8), 125.3 (C-3), 124.1 (C-10), 123.3 (C-11a), 123.0 (C-9),
120.5 (C-10a),120.1 (C-10b),117.7 (C-4),113.8 (C-1),112.8 (C-7), 78.2
(C, Boc), 43.3 (C-40), 42.8 (C-20), 36.2 (5-CH3), 28.2 (CH3, Boc), 16.0
(11-CH3); 15N NMR (DMSO): ꢀ241.8 (N-5), ꢀ251.2 (2-NH), ꢀ276.0
(20-NH), ꢀ301.8 (40-NH); ESI-MS: 951.5 (2MþH)þ, 498.3 (MþNa)þ,
476.3 (MþH)þ; Anal. Calcd. for C26H29N5O4 ꢃ 2H2O [511.57]: C
61.04, H 6.50, N 13.68 Found: C 61.00, H 6.49, N 13.50.
4.1.10. N-(5,11-Dimethyl-5H-indolo[2,3-b]quinolin-2-yl)
glycylglycylamide dihydrochloride (5a)
Product 5 (150 mg, 0.31 mM) was treated with 2.2 M HCl/CH3OH
(13 mM, 6 mL) and stirred for 2 h (TLC monitoring). The precipi-
tated salt 5a was collected by filtration and recrystallized from ethyl
acetate; yield 134 mg (91%); m.p. (ꢂC): 210e212; IR: 3331 (Ne
4.1.8. N-(5,11-Dimethyl-5H-indolo[2,3-b]quinolin-2-yl)-L-
histidylamide tetrahydrochloride (4a)
Hamide), 3134, 1790 (weak), 1681 (C]Oamide), 1644 (C]Caromatic),
Product 4 (100 mg, 0.2 mM) was treated with 2.2 M HCl/CH3OH
(13 mM, 6 mL) and stirred for 12 h (TLC monitoring). The precipi-
tated salt 4a was collected by filtration and recrystallized from ethyl
1619 (C]Caromatic), 1572 (C]Caromatic), 1533, 1504 (ring stretching),
1432 (ring stretching), 1366 (CeHalifatic), 1198, 1136, 795, 759 (Ce
H
aromatic), 705 cmꢀ1; 1H NMR (DMSO þ D2O): 8.32 (1H, d, J ¼ 2 Hz,

312
K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
H-1), 7.98 (1H, d, J ¼ 8 Hz, H-10), 7.93 (1H, d, J ¼ 9.5 Hz, H-4), 7.83
(1H, dd, J ¼ 2.5, J ¼ 9.5 Hz, H-3), 7.34 (1H, m, H-8), 7.32 (1H, m, H-7),
7.23 (1H, m, H-9), 4.06 (3H, s, 5-CH3), 4.00 (2H, s, H-20), 3.77 (2H, s,
density (at 540 nm) in a computer-interfaced, 96-well microtiter
plate reader Synergy H4 photometer (BioTek Instruments, USA).
The background optical density was measured in the wells filled
with culture medium, without the cells. Each compound in a given
concentration was tested in triplicate in each experiment, which
was repeated 3e5 times.
The results of cytotoxic activity in vitro were expressed as IC50 e
the concentration of compound (in mg/mL and mM) that inhibits the
proliferation rate of the tumor cells by 50% as compared to control
untreated cells.
13
H-40), 2.82 (3H, s, 11-CH3); C NMR (DMSO þ D2O): 170.2 (C-30),
168.6 (C-10), 149.0 (C-11), 146.7 (C-5a), 140.5 (C-6a), 136.5 (C-2),
132.7 (C-4a), 131.1 (C-8), 127.3 (C-3), 125.1 (C-10), 125.0 (C-9), 124.4
(C-11a), 121.1 (C-10a), 120.9 (C-10b), 118.5 (C-4), 116.2 (C-1), 113.7
(C-7), 44.1 (C-20), 41.6 (C-40), 37.2 (5-CH3), 17.1 (11-CH3); 15N NMR
(DMSO þ D2O): ꢀ243.6 (N-5); ESI-MS: 376.1 (MþH)þ; Anal. Calcd.
for C21H21N5O2 ꢃ 2HCl ꢃ 2H2O [484.38]: C 52.07, H 5.62, N 14.46, Cl
14.64 Found: C 52.14, H 5.98, N 14.39, Cl 14.60; HPLC: 95.1%.
4.2.2. Antimicrobial assay
4.2. Biological assays
Minimal inhibitory concentration (MIC) was tested by the
twofold serial dilution method using MuellereHinton II agar me-
dium (Becton Dickinson) for bacteria and RPMI agar (Sigma) for
Candida, according to CLSI guidelines [31,32]. Concentrations of
4.2.1. Antiproliferative assay
4.2.1.1. Cell lines. Established in vitro, human cancer cell lines KB
(cervix carcinoma), A549 (non-small cell lung cancer), MCF-7
(breast cancer), LoVo (colon) and normal mice fibroblasts (BALB/
3T3) were used. All lines were obtained from American Type Cul-
ture Collection (Rockville, Maryland, USA) with the exception of
LoVo by courtesy of Prof. E. Borowski (Technical University of
tested agents in solid medium ranged from 3125 to 200 mg/mL. The
final inoculum of all studied organisms was 104 CFU mLꢀ1 (colony
forming units per mL), except the final inoculum for Enterococcus
hirae ATCC 10541, which was 105 CFU mLꢀ1. Minimal inhibitory
concentrations were read after 18 h of incubation at 35 ꢂC. Micro-
organisms used in this study were as follows: Gram-positive cocci:
Staphylococcus aureus NCTC 4163, S. aureus ATCC 25923, S. aureus
ATCC 6538, S. aureus ATCC 29213, Staphylococcus epidermidis ATCC
12228, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, E.
hirae ATCC 10541, Micrococcus luteus ATCC 9341, M. luteus ATCC
10240; Gram-negative rods: Escherichia coli ATCC 10538, E. coli
ATCC 25922, E. coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseu-
domonas aeruginosa ATCC 15442, P. aeruginosa NCTC 6749, P. aer-
uginosa ATCC 27863, Bordetella bronchiseptica ATCC 4617.
ꢀ
Gdansk, Poland). All the cell lines were maintained in the Institute
of Immunology and Experimental Therapy, Wroclaw, Poland.
KB, A549, MCF-7 and LoVo cells were cultured in a mixture of
RPMI 1640 and Opti-MEM (1:1) medium (Gibco, UK) supplemented
with 2 mM L-glutamine and 1.0 mM sodium pyruvate (KB and
LoVo), 5% fetal bovine serum (all from SigmaeAldrich Germany).
The culture of MCF-7 cells was supplemented with 0.01 mg/mL
insulin (SigmaeAldrich Germany). BALB/3T3 cells were cultured in
Dulbecco medium (IIET, Wroclaw) supplemented with 2 mM L-
glutamine and 1.0 mM sodium pyruvate, 10% fetal bovine serum. All
culture medium was supplemented with 100 units/mL penicillin
C. albicans ATCC 10231, C. albicans ATCC 90028, and Candida
parapsilosis ATCC 90028 were used for testing antifungal activities
of the compounds. Microorganisms were obtained from the
collection of the Department of Pharmaceutical Microbiology,
Medical University of Warsaw, Poland.
(SigmaeAldrich, Germany), and 100
mg/mL streptomycin (Polfa,
Tarchomin S.A., Poland). All cell lines were grown at 37 ꢂC with 5%
CO2 humidified atmosphere.
4.2.1.2. In vitro antiproliferative assay. Test solutions of the com-
pounds tested (1 mg/mL) were prepared by dissolving the sub-
4.2.3. Antifungal susceptibility testing in in vitro C. albicans biofilms
Experiments designed to assess the impact of the selected
neocryptolepine derivatives on C. albicans biofilm cell viability
were performed in a 96-well format as previously described [34].
stances in 100
medium. Afterwards, the tested compounds were diluted in culture
medium to reach the final concentrations of 10, 1, 0.1 and 0.01 g/
mL. Results were converted into M concentrations.
ml of water completed with 900 ml of tissue culture
m
For the antifungal susceptibility assay, 100 ml of the inoculum
m
(106 cells/mL in RPMI medium-MOPS) was added to each well of a
96-well flat-bottom plate. After 24-h incubation at 37 ꢂC, the wells
were washed with phosphate-buffered NaCl (PBS) three times to
remove any nonadherent cells. Fresh medium and the tested
chemicals were added followed by additional periods of 24-h long
incubation. The concentrations of the tested chemicals ranged from
Twenty-four hours before addition of the tested agents, the cells
were plated in 96-well plates (Sarstedt, USA) at a density of
104 cells per well in 100
ml of culture medium. The cells were
cultured in RPMI 1640 and Opti-MEM (1:1) medium supplemented
with 2 mM glutamine, penicillin (100 U/mL) (both from Sigmae
Aldrich, Germany), streptomycin (100
mg/mL) (Polfa, Tarchomin,
2.4 mg/mL up to 625.0 m
g/mL. After 24 h of incubation at 30 ꢂC, an
Poland) and 5% fetal bovine serum (Gibco, USA). The cell cultures
were maintained at 37 ꢂC in a humid atmosphere saturated with 5%
CO2.
XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-
5-carboxanilide] reduction assay was performed and endpoints
were determined spectrophotometrically at 492 nm as a measure
of cell metabolic activity. Briefly, 90
ml of XTT (1 mg/mL) and 10 ml of
4.2.1.3. SRB assay. The details of this technique were described by
Skehan [33]. The antiproliferative assay was performed after 72-
h exposure of the cultured cells to varying concentrations (from
phenazine methosulfate (320 g/mL) were added to each well and
m
the plate was incubated at 37 ꢂC for 30 min. Absorbance at 490 nm
was measured using an automated plate reader. Each of the above
growth assays was performed in triplicate for each of the per-
formed experiments.
0.01 to 10 mg/mL) of the tested agents. The cells attached to the
plastic were fixed by gently layering cold 50% TCA (trichloroacetic
acid, POCh, Gliwice, Poland) on the top of the culture medium in
each well. The plates were incubated at 4 ꢂC for 1 h and then
washed five times with tap water. The cellular material fixed with
TCA was stained with 0.4% sulforhodamine B (SigmaeAldrich,
Germany) dissolved in 1% acetic acid (POCh, Gliwice, Poland) for
30 min. Unbound dye was removed by rinsing (4ꢃ) with 1% acetic
acid. The protein-bound dye was extracted with 10 mM unbuffered
Tris base (SigmaeAldrich, Germany) for determination of optical
Abbreviations
Boc, tert-butyloxycarbonyl group; DIPEA, N,N-diisopropylethyl-
amine; 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 chromatography;

K. Sidoryk et al. / European Journal of Medicinal Chemistry 78 (2014) 304e313
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IR, infrared spectroscopy; MS, mass spectrometry; NMR, nuclear
magnetic resonance; SAR, structure activity relationship;
TBTU, O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium tet-
rafluoroborate; TEA, triethylamine; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; TMS, trimethylsilyl; NeHamide, NeHamide stretch-
ing; C]Oamideþcarbamate and C]Oamide, C]O stretching, amide
I band; C]Caromatic, C]Caromatic stretching; ring stretching, het-
erocyclic compounds ring stretching; CeHalifatic
,
CeHalifatic
rocking; CeHaromatic (ca 1250 cmꢀ1), CeHaromatic bending, in-plane;
CeHaromatic (ca 750 cmꢀ1), CeHaromatic bending, out-of-plane; brd,
broadened; n, narrow
Acknowledgments
We extend our thanks to Jeniel Nett, MD, PhD, for fruitful dis-
cussions on the topic.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.060.
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