Synthesis and preliminary evaluation of novel alkyl diamine linked bivalent β-carbolines as angiogenesis inhibitors

Article abstract of DOI:10.1039/c6md00360e

A series of novel bivalent β-carbolines linked with an alkyl diamine spacer at the C-3 position were synthesized and evaluated as potent angiogenesis inhibitors. The results demonstrated that most bivalent β-carbolines displayed significant anti-proliferative effects against EA.HY926 human umbilical vein cell lines. Compound 8z was found to be the most potent anti-proliferative agent with an IC₅₀ value of 1.10 μM against EA.HY926 cell lines. Preliminary investigations on the mechanisms of action revealed that compound 8z could dramatically inhibit EA.HY926 cell migration and tube formation in a dose-dependent manner. Moreover, compound 8z exhibited significant angiogenesis inhibitory effects in CAM assay, and the anti-angiogenic potency was comparable with that of the reference drug Endostar. These molecules might serve as candidates for further development into vascular-targeting antitumor drugs.

Full text of DOI:10.1039/c6md00360e

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CONCISE ARTICLE
Synthesis and preliminary evaluation of novel alkyl diamine linked
bivalent β-carbolines as angiogenesis inhibitors†
Liang Guo,b ,c Wei Chen,c Wenxi Fan,c Qin Ma,c Rongqin Sun,a Guang Shao*a and Rihui Cao*a
Received 00th January 20xx,
Accepted 00th January 20xx
A series of novel bivalent β-carbolines linked with a spacer of alkyl diamine at the C-3 position was synthesized and
evaluated as potent angiogenesis inhibitors. The results demonstrated that most bivalent β-carbolines displayed significant
anti-proliferative effects against human umbilical vein cell lines EA.HY926. Compound 8z was found to be the most potent
anti-proliferative agent with IC50 value of 1.10 μM against EA.HY926 cell lines. Preliminary investigations on mechanisms of
action revealed that compound 8z could dramatically inhibit EA.HY926 cells migration and tube formation in a dose-
dependent manner. Moreover, compound 8z exhibited significant angiogenesis inhibitory effects in CAM assay, and the
anti-angiogenetic potency was comparable with the reference drug Endostar. These molecules might be served as
candidates for further development of vascular targeting antitumor drugs.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
Previous investigations demonstrated that dimerization of
various intercalating agents by an appropriate linker could lead
Angiogenesis is a complicated physiological process involving
endothelial cell activation, invasion, migration, proliferation,
tube formation, and finally capillary network formation, which
is also vital for solid tumor growth, invasion and metastasis.1
Inhibiting angiogenesis is a proven strategy for the treatment
of solid tumors.2 So far, more than 300 angiogenesis inhibitors
have been developed, and there are 80 antiangiogenic drugs
currently in clinical trials.3
to a dramatic increase in the DNA binding affinity.24-28 Such
compounds can bind to DNA by bis-intercalation mode, which
produces binding sites that are at least twice as potent as
monointercalation sites and causes much more pronounced
structural changes of DNA.26 Consequently, bivalent β-
carbolines were expected to exhibit more potent antitumor
efficacies than monomers and exert tumor cell killing effects
by DNA bis-intercalation mode. Inspired by this information,
recently, our group reported the synthesis, in vitro evaluation,
in vivo efficacies and structure-activity relationships for the
new bivalent β-carbolines linked at the N-9 and C-3 position
The β-carboline alkaloids represent a large group of natural
and synthetic products associated with a broad spectrum of
biochemical effects and pharmaceutical functions.4 Recently,
there have been intense research efforts in the design and
development of β-carbolines as a new class of antitumor
agents, and a large number of β-carboline derivatives have
been prepared in search of more potent antitumor agents and
the structure-activity relationships of β-carbolines have been
extensively investigated.5-12 β-Carbolines are initially
discovered to exert antitumor effects by intercalating into
DNA.13, 14 Recent reports and our group investigations revealed
that topoisomerase I and II (Top I and II),15, 16 cyclin-dependent
kinase (CDK),17-19 mitogen activated protein kinase-activated
with a spacer of three to ten methylene units, respectively
30
(
Fig.1).29,
As predicted, bivalent β-carbolines not only
exhibited enhanced antitumor activities but also displayed
dramatically reduced toxicities. However, these bivalent
N
N
CH3
(CH2
N
)
4
N
N
N
H3
C
N
N
N
protein kinase 2 (MK-2),20 polo-like kinase (PLK1),21 kinesin-like
N
23
protein Eg522 and I-Kappa-B kinase (IKK)
were also the
O
O
potential pharmacological targets of this class of compounds.
O
6
O
N
N
N
N
C4H9
C4H9
Fig.1 The chemical structure of the representative reported bivalent β-carbolines.
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Scheme 1 Synthesis of compounds 2-7. Reagents and conditions: (i) acetic acid, R1CHO, reflux, 3 h; (ii) ethanol, SOCl2, reflux, 4 h; (iii) xylene, S, reflux, 8 h; (iv) DMF, NaH, alkyl
halide, stirred at RT; (v) THF, LiBH4, stirred at RT; (vi) CH3CN, MnO2, reflux, 2 h.
bivalent β-carbolines had limited utility for cancer therapy
because of their poor water solubility. To circumvent the
Chemistry
water solubility problem and find congeners more active as
potential antitumor agents, more recently, we described the
The synthetic route for the preparation of monovalent and
synthesis and cytotoxic potencies of novel bivalent β-
bivalent β-carbolines is outlined in Scheme 1-2. 1-Substituted-
carbolines linked with piperazine group (Figure 1), and these
1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids
2
was
compounds showed significantly enhanced antitumor activities
and improved water solubility.31 Further investigation on
mechanisms of action of these compounds disclosed that
bivalent β-carbolines had no effect on DNA and topoisomerase
I, but exhibited significant inhibitory effect on the vessel
growth in chicken chorioallantoic membrane (CAM) assay.31
In a continuing effort to develop novel bivalent β-carbolines
endowed with better angiogenesis inhibitory effects, in the
present investigation, we designed and synthesized a series of
alkyl diamine linked bivalent β-carbolines as potent
angiogenesis inhibitors. These compounds were expected to
exhibit significantly enhanced anti-angiogenetic activity due to
improved water solubility. We report herein the preparation of
novel bivalent β-carbolines and their biological evaluation as
angiogenesis inhibitors.
prepared by the condensation of L-tryptophan with the
corresponding aldehydes via well-known Pictet-Spengler
condensation32, 33 in acetic acid solution. Esterification of
2
with ethanol in the presence of SOCl2 by heating provided the
corresponding
ethyl
1-substituted-1,2,3,4-tetrahydro-β-
, and followed by dehydrogenation
carboline-3-carboxylates
3
with sulfur in refluxing xylene to afford fully unsaturated β-
carbolines
The N9 of
4
4
(
Scheme 1).9, 34
was alkylated by the action of sodium hydride in
anhydrous DMF followed by the addition of the appropriate
alkylating and arylating agents to obtain intermediates . The
ester group in position-3 of was reduced to its corresponding
alcohols by LiBH4 in dry THF to provide compounds , and
further oxidized by MnO2 in CH3CN to give 3-carboxaldehydes
7 (Scheme 1)
5
5
6
.
2 | J. Name., 2012, 00, 1-3
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Scheme 2 Synthesis of the bivalent β-carbolines 8a-ae. Reagents and conditions: (i) sym-diamine, NaBH3CN, stirred at RT.
The reaction of compounds
7 with the corresponding diamines absorbance Reader, USA). IC50 values were calculated using
to form schiff bases took place readily at room temperature in percentage of growth versus untreated control. Combretastain
good yield. The crude schiff bases without further purification A4 phosphate (CA4P) and Endostar were used as positive
were directly reduced with NaBH3CN in anhydrous methanol controls and DMSO was used as the solution for drugs. Final
to give the target bivalent β-carbolines
8 in 18-70% yield concentration of DMSO in the growth medium was 2% (v/v) or
(Scheme 2). The chemical structures of all new bivalent β- lower, concentration without effect on cell replication. In all of
carbolines were characterized by EI-MS, 1H NMR, 13C NMR and these experiments, three replicate wells were used to
HRMS.
determine each point.
Experimental Section
Anti-proliferative assay
Cell migration assay
Cell migration assay were carried out using 24-well plate and
wound-healing method according to the procedures described
by Queiroz.35 Briefly, EA.HY926 cells were seeded in a 24-well
plate pre-coated with 0.1% gelatin and were allowed to grow
Cell proliferative assay were carried out using 96 microtitre
plate cultures and MTT staining. The human umbilical
vein cell line EA.HY926 was obtained from Shanghai Institute
of Biochemistry and Cell Biology, Chinese Academy of Science.
Cells were grown in RPMI-1640 medium containing 10% (v/v)
fetal calf serum and 100 μM penicillin and 100 μM
to 100% confluence. Cell culture were injured by a 10 μL tip,
cells were washed twice with PBS, and then incubated with
fresh medium containing VEGF with or without compound 8z
at different concentrations for 24 h. DMSO was used as the
solution for drugs. Final concentration of DMSO in the medium
was 2% (v/v). Endostar was used as positive control. Cell
migration to the damaged area was then visualized and
photographed on a phase contrast microscope. Inhibition
percentage was expressed as percentage of the control
(100%).
streptomycin. Cultures were propagated at 37
℃ in a humified
atmosphere containing 5% CO2, Cells in logarithmic phase
were diluted to a density of 50,000 cells/mL in culture medium
and treated with various concentrations of monovalent 7a-t
and bivalent β-carbolines 8a-ae in 96-well plates for 48 h in
final volumes of 200 μL. Then 20 μL MTT (5 mg/mL) was added
to each well, and the cells were incubated for additional 4 h.
After carefully removing the medium, the precipitates were
dissolved in 150 μL of DMSO, shaken mechanically for 2 min,
and then absorbance values at a wavelength of 570nm were
taken on a spectrophotometer (Bio-Rad iMark microplate
Tube formation assay
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Tube formation assay were generally performed as described concentration of 0.3, 3.0 and 30 µM/disc, respectively.
by Queiroz’s group.35 Matrigel@ basement membrane matrix Endostar was used as positive control. Discs containing the
Table 1 Inhibitory effect of monovalent and bivalent β- vehicle only (DMSO) were used as negative control. A small
carbolines on cell proliferation
window opening was made in the shell, and the discs were
directly applied onto the CAM. The square opening was
covered with sterilized surgical tape and the embryos were
incubated for 48 h at 37°C. The CAMs were photographed
under a dissecting microscope and blood vessels in each CAM
were counted. The results are presented as a mean percentage
of inhibition to the control ± SD, (n = 3).
Compd
IC50 (μM)
Compd
IC50 (μM)
7a
7b
7c
7d
7e
7f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8h
8i
3.18
11.1
9.87
>20
8j
8k
8l
2.38
1.40
13.5
2.03
2.47
1.61
>20
8m
8n
8o
8p
8q
8r
7g
7h
7i
Statistical analyses
All experiments were performed at least in three independent
experiments. Data were expressed as mean±SEM. The
significance of the differences between the means observed
was evaluated using the one-way ANOVA followed by
Bonferroni test. p<0.05 was considered statistically significant.
7j
7k
7l
8s
8t
8.09
>20
7m
7n
7o
7p
8u
8v
8w
4.85
4.32
3.63
Results and discussion
7q
7r
>100
>100
>100
>100
2.76
1.85
1.28
1.69
11.8
2.56
15.3
8x
8y
10.3
2.94
1.10
4.25
3.88
4.40
13.4
1.69
In vitro anti-proliferative effects
The synthesized monovalent and bivalent β-carbolines were
evaluated for the anti-proliferative effects in vitro by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
assay against human umbilical vein cell lines EA.HY926 and
compared with the reference drugs CA4P and Endostar. In
order to enhance the solubility in aqueous solution, all bivalent
β-carbolines were prepared in the form of hydrochloride salt
before use. As predicted, the hydrochloride salt of all bivalent
β-carbolines had good water-solubility (more than 10 mg/ml).
7s
7t
8z
8aa
8ab
8ac
8ad
8ae
8a
8b
8c
8d
8e
8f
CA4P
6.40
1.76
8g
Endostar
The results were summarized in Table 1
.
As shown in Table 1, all monovalent β-carbolines 7a-t failed to
exhibit anti-proliferative activities at the concentration of 100
(growth factor reduced) (Corning, USA) was thawed at 4
pipetted into pre-chilled 24-well plates (200μL matrigel/well)
and incubated at 37 for 30 min. Then EA.HY926 cells (5
104
℃,
μM against EA.HY926 cell lines. Bivalent β-carbolines 8b
, 8c,
8d, 8m, 8q, 8z and 8ae exhibited significant anti-proliferative
℃
×
effects with IC50 value of 1.85, 1.28, 1.69, 1.40, 1.61, 1.10 and
1.69 μM against EA.HY926 cell lines, respectively. Similarly,
cells/mL) were added to matrigel coated plates, followed by
addition of various concentrations of the selected compound
8z with VEGF (60 ng/mL). Endostar was used as positive
compounds 8a, 8f, 8h, 8l, 8o, 8p, 8u-w, 8y and 8aa-ac also
displayed good anti-proliferative activities with IC50 value of
lower than 5.0 μM against EA.HY926 cell lines. While
control. After 24 h of incubation with 5% CO2 at 37
number of capillary-like tube formation of each well was
photographed using a phase-contrast microscope at 100
℃, the
compounds 8e
anti-proliferative potencies with IC50 values range from 8.03 to
15.3 μM. Unfortunately, compounds 8k 8r and 8t had no anti-
, 8g, 8i, 8j, 8n, 8s and 8ad only showed medium
×
magnifications. Tube formation was quantitated by manual
counting the number of branch points. DMSO was used as the
solution for drugs. Final concentration of DMSO in the
medium was 2% (v/v). All experiments were done in triplicate.
Inhibition percentage was expressed as percentage of control
(100%).
,
proliferative effects at the concentration of 20 μM.
Particularly, compound 8z was found to be the most potent
inhibitory agent with IC50 value of 1.10 μM against EA.HY926
cell lines, and the inhibitory potency of compound 8z was
more potent than positive control drug Endostar (1.76 μM)
and CA4P (6.40 μM).
CAM assay in vivo
Firstly, we examined the influence of the spacer length of
bivalent β-carbolines on anti-proliferative effects. The data
Antiangiogenic activity of the selected compound 8z was
investigated in vivo using chicken chorioallantoic membrane (
CAM) assay.31 Five day-old fertilized eggs were obtained from
local hatchery. 5 mL of albumin was aspirated and the eggs
were incubated horizontally to allow the CAM to detach from
the shell. Compound 8z was prepared in 1.2% agarose discs at
collected in Table 1 displayed that compounds 8e
8n 8r 8t and 8ad with a spacer of three methylene units only
had moderate or no anti-proliferative effects against EA.HY926
cell lines, while their congeners 8a-d 8f 8h 8l-m 8o-q 8s 8u-
8y- ac and 8ae with a spacer of four to six methylene units
, 8g, 8i, 8j,
,
,
,
,
,
,
,
,
w
,
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exhibited good to significant anti-proliferative potencies carboline nucleus and with a spacer of four methylene units,
against EA.HY926 cell lines. In addition, compound 8b with a compounds 8c (9-isopropy) and 8d (9-butyl) showed more
A
A
B
B
Fig.3 The effect of inhibiting tube formation of EA.HY926 cells by compound 8z. Data
were presented as the percentage of control, as mean ± SEM of three independent
experiment. **p<0.05 versus control.
Fig.2 The effect of inhibiting EA.HY926 cells migration by compound 8z. Data were
presented as the percentage of control, as mean
± SEM of three independent
experiment. **p<0.05 versus control.
potent anti-proliferative effects than compounds 8a (9-
methyl), 8f (9-isobutyl) and 8h (9-benzyl). Similarly,
compounds 8q and 8z having a methyl group in position-9 of β-
carboline nucleus, respectively, showed stronger anti-
proliferative potencies than their congeners 8s and 8ab
bearing a benzyl group in position-9 of β-carboline ring. These
data suggested that the introduction substituent into position-
9 of β-carboline ring facilitated their anti-proliferative effects,
and the short alkyl substituent was the optimal group giving
rise to potent anti-proliferative agents.
spacer of six methylene units displayed more potent anti-
proliferative activities than compound 8a with a spacer of four
methylene units. Similarly, compound 8m and 8z with a spacer
of five methylene units showed more potent anti-proliferative
activities than compound 8l and 8y with a spacer of four
methylene units. Interestingly, we also observed that
compound 8v and 8ab with a spacer of five methylene units
demonstrated more potent anti-proliferative effects than
compound 8w and 8ac with a spacer of six methylene units.
These results indicated that the length of the spacer affected
anti-proliferative effects and five methylene units might be
more favorable.
Thirdly, we investigated the influence of various substituents
in position-1 of β-carboline ring on anti-proliferative effects in
these bivalent compounds. Compound 8a exhibited good anti-
proliferative activities with IC50 value of 2.76 μM against
EA.HY926 cell lines, replacement of the methyl group in
position-1 of compound 8d by an isopropyl and 4-
methoxyphenyl group to yield compounds 8p and 8y displayed
similar anti-proliferative activities against EA.HY926 cell lines.
Similarly, compound 8b and 8q bearing a methyl and phenyl
group in position-1, respectively, also exhibited significant anti-
proliferative activities against EA.HY926 cell lines. However,
replacement of the methyl group in position-1 of compound
Secondly, we evaluated the effect of the substituent in
position-9 of the β-carboline ring on anti-proliferative
potencies. Compound 8k had no substituent in position-9 of
the β-carboline ring failed to exhibit anti-proliferative effects
against EA.HY926 cell lines, while compound 8m bearing an n-
butyl group in position-9 of β-carboline ring displayed
significant anti-proliferative effects. Similarly, compound 8x
with no substituent in position-9 of the β-carboline nucleus
showed weaker anti-proliferative potencies than compound 8y
having a methyl group in position-9 of the β-carboline ring. Of
all compounds bearing a methyl group in position-1 of β-
8d by an isopropyl group (8l) and a 4-chlorophenyl (8u
)
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resulted in slight decline in the activity against EA.HY926 cell significantly decreased the capillary-like tubules in
a
lines. These results implied that substituent in position-1 of β-
concentration-dependent manner (Fig.3B).
A
B
Fig.4 Inhibitory effects of compound 8z on angiogenesis of CAM. Data were presented
as the percentage of control, as mean ± SEM of three independent experiment.
**p<0.05 versus control.
carboline nucleus is not crucial to anti-proliferative effect of
this class of compounds.
Inhibitory effect of bivalent β-carbolines on cell migration
Cell migration is an essential feature for vascular endothelial cells in
angiogenesis. Logically, we investigated the inhibitory effect on the
EA.HY926s chemotactic motility of the most potent compound 8z
by wound-healing assay. As shown in Fig.2A, compound 8z could
inhibit VEGF-induced EA.HY926s migration in a dose-dependent
manner ranging from 0.3 μM to 30 μM. Treatment with compound
8z significantly inhibited EA.HY926 cells migration at concentration
of 30 μM and the inhibitory potency of compound 8z was more
potent than positive drug Endostar (Fig.2B).
Inhibitory effect of bivalent β-carbolines on tube formation
We also evaluated the ability of the selected compound 8z in a
tube formation assay. EA.HY926 cells plated on a Matrigel
coated plate could formed capillary-like tubules with
multicentric junctions in cell cultured in the absence of
compound (control). After 24
concentrations (0.3-30 μM) of compound 8z, the capillary-like
tubes were interrupted in different levels Fig.3A).
h treatment in different
(
Quantitative image analysis showed that compound 8z
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The most potent compound 8z was selected to evaluate the
angiogenetic activity by CAM assay. The inhibitory effects of
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compound 8z on angiogenesis of CAM are shown in Fig.4A
.
675-682.
The anti-angiogenetic activities of compounds 8z was
semiquantitatively analyzed using Graph Pad Prism 5.0 (shown
in Fig.4B). The result showed that compound 8z (p < 0.05)
could inhibit the angiogenesis of CAM. The anti-angiogenetic
activity of compound 8z was comparable to Endostar in vivo
CAM assay at the same dose (30 μM).
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In this work, a series of novel alkyl diamine linked bivalent β-
carbolines with linkers of different lengths and incorporating
various substituents into position-1 and 9 was synthesized and
evaluated as angiogenesis inhibitors. Some bivalent β-
carbolines exhibited significant anti-proliferative effects
against EA.HY926 cell lines, and compound 8z was found to be
the most potent inhibitory agent with IC50 value of 1.10 μM
against EA.HY926 cell lines. Structure-activity relationships
analysis revealed that (1) the introduction substituent into
position-9 of β-carboline nucleus facilitated their anti-
proliferative effects, and the short alkyl substituent was the
optimal group giving rise to potent anti-proliferative agents,
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (21342010) and the Guangdong Natural
Science Foundation (s2013010012138 and 2016A030313349)
and Xinjiang Huashidan Pharmaceutical Co. Ltd.
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A table of contents entry
CH2NH(CH2)nNHCH2
N
N
N
R2
N
R2
R1
R1
CH2NH(CH2)5NHCH2
N
N
N
N
CH3
CH3
H3CO
OCH3
8z
A series of novel bivalent βꢀcarbolines was synthesized and evaluated as potent
angiogenesis inhibitors.