Arylureas derived from colchicine: Enhancement of colchicine oncogene downregulation activity

Article abstract of DOI:10.1016/j.ejmech.2018.03.039

Our efforts to get therapeutically useful colchicine derivatives for the treatment of cancer have led us to synthetize and biologically evaluate twenty-seven N,N′-disubstituted ureas containing a colchicine moiety and an aryl fragment. The cytotoxicity of

Full text of DOI:10.1016/j.ejmech.2018.03.039

Accepted Manuscript
Arylureas derived from colchicine: Enhancement of colchicine oncogene
downregulation activity
Víctor Blasco, Ana C. Cuñat, Juan F. Sanz-Cervera, J. Alberto Marco, Eva Falomir,
Juan Murga, Miguel Carda
PII:
S0223-5234(18)30282-4
10.1016/j.ejmech.2018.03.039
EJMECH 10304
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 January 2018
Revised Date: 1 March 2018
Accepted Date: 13 March 2018
Please cite this article as: Ví. Blasco, A.C. Cuñat, J.F. Sanz-Cervera, J.A. Marco, E. Falomir, J. Murga,
M. Carda, Arylureas derived from colchicine: Enhancement of colchicine oncogene downregulation
activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.039.
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ACCEPTED MANUSCRIPT
Arylureas derived from colchicine: Enhancement of colchicine oncogene
downregulation activity
Víctor Blasco, Ana C. Cuñat, Juan F. Sanz-Cervera, J. Alberto Marco, Eva Falomir,
Juan Murga and Miguel Carda
Graphical Abstract
Twenty-seven N,N´-disubstituted ureas containing a colchicine moiety an and aryl
fragment have been synthetized and biologically evaluated. The cytotoxicity of the
compounds, their ability to inhibit the expression of oncogenes related to telomerase
activation and to the VEGF/VEGFR-2 autocrine process, such as c-MYC, hTERT and
VEGF and their capability to downregulate c-MYC and VEGFR-2 proteins and the
secretion of VEGF have been measured. In these biological evaluations, we have found
that the change of the acetyl group in colchicines for an N-arylurea unit causes a great
improvement in anticancer properties. The most promising derivatives were
compounds 6 (o-Cl) and 14 (o,o-di-F) as they were able to downregulate all the tested
targets at a concentration below their IC50 values. Thus, the arylurea unit enhances the
potential of colchicine as an anticancer agent.

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European Journal of Medicinal Chemistry
Manuscript draft
Title: Arylureas derived from colchicine: Enhancement of colchicine oncogene downregulation
activity
Article type: Original paper
Keywords: colchicine, urea derivatives, cytotoxicity, oncogenes downregulation
ABSTRACT
Our efforts to get therapeutically useful colchicine derivatives for the treatment of cancer have led us to
synthetize and biologically evaluate twenty-seven N,N´-disubstituted ureas containing a colchicine moiety an
and aryl fragment. The cytotoxicity of the compounds, their ability to inhibit the expression of oncogenes
related to telomerase activation and to the VEGF/VEGFR-2 autocrine process, such as c-MYC, hTERT and
VEGF and their capability to downregulate c-MYC and VEGFR-2 proteins and the secretion of VEGF have
been measured. In these biological evaluations, we have found that the change of the acetyl group in
colchicines for an N-arylurea unit causes a great improvement in anticancer properties. The most promising
derivatives were compounds 6 (o-Cl) and 14 (o,o-di-F) as they were able to downregulate all the tested
targets at a concentration below their IC50 values. Thus, the arylurea unit enhances the potential of colchicine
as an anticancer agent.

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Arylureas derived from colchicine: Enhancement of colchicine
oncogene downregulation activity
Víctor Blasco,a Ana C. Cuñat,a* Juan F. Sanz-Cervera, a J. Alberto Marco,a Eva Falomir,b* Juan
Murga,b and Miguel Cardab
aDpto. de Química Orgánica, Univ. de Valencia, E-46100 Burjassot, Valencia, Spain
bDpto. de Química Inorgánica y Orgánica, Univ. Jaume I, E-12071 Castellón, Spain
*Authors to whom correspondence should be addressed. E-Mail addresses: ana.cunat@uv.es;
efalomir@uji.es
ABSTRACT
Our efforts to get therapeutically useful colchicine derivatives for the treatment of cancer have led us to
synthetize and biologically evaluate twenty-seven N,N´-disubstituted ureas containing a colchicine moiety an
and aryl fragment. The cytotoxicity of the compounds, their ability to inhibit the expression of oncogenes
related to telomerase activation and to the VEGF/VEGFR-2 autocrine process, such as c-MYC, hTERT and
VEGF and their capability to downregulate c-MYC and VEGFR-2 proteins and the secretion of VEGF have
been measured. In these biological evaluations, we have found that the change of the acetyl group in
colchicines for an N-arylurea unit causes a great improvement in anticancer properties. The most promising
derivatives were compounds 6 (o-Cl) and 14 (o,o-di-F) as they were able to downregulate all the tested
targets at a concentration below their IC50 values. Thus, the arylurea unit enhances the potential of colchicine
as an anticancer agent.
1. Introduction
During eons Nature has been producing small-molecule products able to interact within living
organisms with macromolecular targets that contain structural domains similar to human proteins.
As a result of this natural selection process, a vast range of structures has arisen that have been
found to show optimal interactions with many biological targets [1]. Not surprisingly, natural
product research is a growing domain at the interface of chemistry and biology which has not
ceased to generate new chemical entities with high pharmacological interest [2]. In the last years,
we have been investigating a range of analogues of natural products [3] for their potential value in
anticancer therapy [4]. It is widely known that a range of both external and internal factors may
induce cancer. This has led to the development of various types of therapeutic approaches [5]. One
of them involves the use of cytotoxic drugs, which lead in many cases to cell apoptosis [6]. A
second type is based on the use of compounds with vascular-targeting properties, either through
inhibition of the formation of new blood vessels (antiangiogenic agents) or by means of destruction
of existing ones (antivascular agents) [7]. Tumor angiogenesis is a complex process and involves

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the tight interplay of many factors [8]. One of them is the vascular endothelial growth factor
(VEGF), a key regulator of angiogenesis that promotes endothelial cell survival, proliferation and
migration while increasing vascular permeability through the interaction with VEGFR-2, a
transmembrane protein kinase [9]. It is worth noting that overexpression in the production of VEGF
has been found to occur in various types of tumors [10]. In addition, though VEGFRs were initially
found on endothelial cells, recent studies have demonstrated that tumor cells of different origins
also express VEGFRs as well as the existence of autocrine VEGF/VEGFR-2 signalling pathways in
such cells. It is the presence of autocrine VEGF loops on tumor cells themselves that allows the
latter to mediate their own survival, invasiveness and migration through VEGF pathways. This has
converted VEGF and its receptor VEGFR-2 into key target molecules for cancer therapy [11,12].
A third therapeutic line follows the path of the chromosomal telomeres, the terminal zones of
chromosomes that, due to their special structure, are recognized as functional domains and thus
distinguished from random chromosomal breaks that would stimulate the onset of repair
mechanisms. The aforementioned structure is maintained by a special type of ribonucleoprotein
complex called telomerase, the expression of which is restricted or absent in normal human somatic
cells. Telomeres progressively shorten during cell lifespan [13]. For this reason, and since
telomerase has been detected in about 90% of all malignant tumors, drugs with ability to inhibit
telomerase activity are potentially useful weapons in the fight against cancer, aging and other
diseases related to premature telomere shortening [14].
Telomerase activity is positively associated with high levels of hTERT expression. A correlation
between hTERT and VEGF expression has been established suggesting the existence of an
autoregulatory loop between VEGF and hTERT. Further, expression of both hTERT and VEGF is
linked to tumour progression and shows a positive association with c-MYC, a transcription factor
that regulates cell proliferation, differentiation and promotes changes in the tumor
microenvironment [15].
The discovery and investigation of new drugs therefore constitutes an important goal in
chemistry and pharmacology. The emergence of resistances to existing drugs has led to a
continuous need of developing new bioactive compounds able to overcome such problems. Even
though first observed in the case of antibiotics, resistances have been reported to cancer therapies
with various types of cytotoxic [16], antiangiogenic [17] and telomerase inhibitory agents [18].
Our interest in natural product analogues has led us to prepare several structural classes and to
investigate their potential activity in the aforementioned types of biological activity. Among the
natural products that we have included in our research, colchicine has particularly called our
attention (see Figure 1). Surprisingly, even though it is one of the oldest drugs still currently
available, little is known about its precise mode of action. Beyond its present medicinal use (gout,

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among other diseases), numerous other conditions have been recently proposed for the use of this
drug. The marked toxicity of the compound, however, has prevented its use in cancer therapy [19].
Recently, we have published two reports on the biological properties of several colchicine
analogues [4,20]. The properties investigated included not only the cytotoxicity of the compounds
but also their ability to inhibit the expression of certain genes related to the angiogenesis process
[21] and to telomerase activation [22].
It is worth noting that protein kinases, such as VEGFR-2, have emerged as one of the most
successful families of drug targets [23]. It is thus not surprising that many thousands of inhibitors of
several hundreds of kinases have been identified to date [24]. In fact, one of these inhibitors,
sorafenib (see Figure 1), called our attention because of its activity against VEGFR-2, one of the
main topics of interest of our research. Sorafenib is an oral multikinase inhibitor with anticancer
activity against a wide spectrum of cancers and has been approved for the treatment of patients with
various types of cancer [25]. Sorafenib contains a N,N´-bisaryl urea moiety with two different arylic
fragments. Indeed, N-substituted urea derivatives of various structural types represent one highly
important class of biologically active agents [26].
Figure 1. Structure of the colchicine and sorafenib
In recent years, many reports have been published on synthetic and mechanistic aspects of the
aforementioned compound class, the evaluation of their various biological activities and the
establishment of structure-activity relationships (SAR) [27]. Urea bioisosteres have also been
investigated in SAR studies for their medicinal applications [28].
Focused on our research on novel natural product analogues with potential utility in cancer
therapy [4], and on the basis of the aforementioned aspects, we conceived the synthesis and
biological evaluation of a set of N,N´-disubstituted ureas containing one aryl fragment bound to one
the two nitrogen atoms and one colchicine moiety connected to the other nitrogen. Thus,
compounds 1-26 (see figure 2) were prepared and subsequently investigated for their biological
activity. For the sake of estimation of the importance of the aryl moiety, compound 27, which has
an N-cyclohexyl group, was also prepared.

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Figure 2. Structure of the colchicine analogues investigated in this study
2. Synthesis
The urea derivatives were prepared from the trifluoroacetate salt of N-deacetyl colchicine 28 [29]
as depicted in Scheme 1. Thus, treatment of 28 with phenyl chloroformiate gave urethane 29 with
good yield. Subsequently, 29 was allowed to react with a range of aromatic amines to yield the
desired ureas via nucleophilic substitution of the phenoxy group [30]. However, acceptable yields
were observed only in the case of aniline derivatives substituted with electron-donating groups. The
only nonaromatic amine, cyclohexylamine, gave an excellent yield of the corresponding urea
derivative 27 (see scheme 1).
Scheme 1. Synthesis of colchicine derivatives from aniline substituted with electron-donating
groups and 27
Anilines with electron-withdrawing substituents reacted very slowly and with poor yields. Under
forcing conditions, products of nucleophilic attack at the tropolone ring were observed [31]. In
order to avoid this problem, we decided to invert the order of formation of the two C−N bonds, with
deacetyl colchicine being now the nucleophilic component of the reaction. For this purpose, the less
reactive anilines were transformed into their carbamates by means of treatment with phenyl
chloroformate. The latter were then allowed to react with 28 under basic conditions (pyridine,
80°C) to yield the desired urea derivatives with the yields indicated in scheme 2.

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Scheme 2. Synthesis of colchicine derivatives from aniline substituted with electron-withdrawing
groups
3. Biological results
3.1. Effect on the inhibition of cell proliferation
The capability of our derivatives 1-27 to inhibit cell proliferation was measured by means of
their IC50 values towards the cancer cell lines HT-29, MCF-7 and HeLa and towards one non-tumor
cell line HEK-293. The achieved results are shown in Table 1.
Table 1. IC50 values (nM) of synthetic compounds 1-27 in cancer cell lines HT-29, MCF-7 and
A549, and one non-cancer cell line HEK-293.a
Compound
HT-29
MCF-7
HeLa
HEK-293
13 ± 2
20 ± 2
12 ± 3
119 ± 8
25 ± 1
1200 ± 20
6 ± 1
14 ± 1
12 ± 2
32 ± 6
18 ± 1
1400 ± 40
6 ± 1
13 ± 3
Colchicine
11 ± 2
10 ± 5
1
26 ± 2
44 ± 1
2
15 ± 1
19 ± 1
3
1200 ± 30
4.7 ± 0.8
1200 ± 20
5 ± 1
4
5
0.80 ± 0.09 1.30 ± 0.07 1.20 ± 0.05
0.70 ± 0.04
11 ± 2
6
11 ± 1
11 ± 4
10 ± 1
3 ± 1
7
3.8 ± 0.5
4.3 ± 0,1
4 ± 1
4.4 ± 0.7
370 ± 98
13 ± 1
5 ± 1
8
8± 2
4.6 ± 0.5
14± 2
9
6.5 ± 0.9
10 ± 2
242 ± 31
304 ± 49
10
11
12
13
5 ± 1
10 ± 1
13 ± 1
130 ± 4
838 ± 116
240 ± 23
185 ± 8
218 ± 13
801 ± 30

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25 ± 9
40 ± 3
14 ± 4
42 ± 3
13 ± 2
10 ± 1
18 ± 1
22 ± 1
21 ± 9
46 ± 3
14
15
16
17
18
19
20
21
22
23
24
25
26
27
42 ± 5
65 ± 3
1.75 ± 0.07 1.20 ± 0.04 5 ± 2
1.7 ± 0.8
3.0 ± 0.8
220 ± 5
32 ± 5
0.71 ± 0.08 0.9 ± 0.03
3 ± 1
190 ± 6
31 ± 6
10 ± 1
200 ± 8
24 ± 6
20 ± 1
220 ± 3
42 ± 7
16 ± 3
1700 ± 20
20 ± 5
12 ± 3
16 ± 4
10 ± 1.5
1500 ± 500
92 ± 2
2600 ± 300 1800 ± 10
30 ± 6
14 ± 1
1.2 ± 0.2
11.1 ± 0.2
1.5 ± 0.7
17 ± 3
1.7 ± 0.9
9 ± 8
8 ± 2
10 ± 3
89 ± 8
7 ± 2
7 ± 3
37 ± 3
87 ± 16
98 ± 5
aIC50 values are expressed as the compound concentration (nM) that inhibits the cell growth by 50%. Data
are the average (±SD) of three experiments.
The cytotoxicity measurements show that a high activity in the nM range is observed for all the
derivatives, with the exception of compounds 4 (o-OMe) and 22 (m,m-diCF3), the IC50 values of
which fall in the µM range (4: IC50 = 1.2-1.4 µM).
Compounds with an ortho substituent in the phenyl ring are slightly more cytotoxic when
bearing an electron withdrawing group, as in the cases of 3 (o-CF3), 5 (o-F), 6 (o-Cl) or 7 (o-CN),
than when having electron donor groups, like 2 (o-Me) or 4 (o-OMe). The remaining compounds
display similar cytotoxicities, irrespective of their substitution pattern. Substrate 1, with an
unsubstituted phenyl ring, also exhibited a potent cytotoxic activity.
Compounds bearing two substituents on the phenyl ring also show IC50 values in the nM range.
Like 6, compounds 17 (o-Cl, p-F) and 18 (o-Cl, p-Me) display a chlorine atom in ortho position and
show the lowest IC50 values of all the series. Thus, it may be concluded that the presence of a
chlorine atom in ortho position increases the antitumoral activity of these derivatives. In contrast,
the presence of two chlorine atoms in ortho position (compound 15, o,o-diCl) slightly lowers the
activity with respect to the previous substrates.
Compound 19 (o-Cl, p-CF3), structurally related to 18 (o-Cl, p-Me), shows much less cytotoxic
character (19; IC50: HT-29, 190 nM; MCF-7, 200 nM; HeLa 220 nM). This decrease in activity is
even greater in compound 22 (m,m-diCF3), whose cytotoxicity falls in the µM range for all the
assayed cell lines (22; IC50 = 1.7-2.6 µM).

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Compounds bearing two electron-donor groups in ortho position show a marked reduction in
activity values, compounds 12 (o,o-diMe) and 13 (o,o-diEt) being representative examples. These
compounds show IC50 values higher by a factor of over 100 than the remaining analogues, in
agreement with the increase in cytotoxicity observed when electron-withdrawing groups are
attached to the phenyl ring.
Not surprisingly, all these colchicine derivatives are also highly active against the non-tumor cell
line HEK-293. However, it is worth mentioning that some of them have a considerable therapeutic
window, as in compounds 14 (o,o-di-F), 16 (o-Me, p-F), 18 (o-Cl, p-Me) and 23 (o-diCl, p-F).
These compounds display cytotoxic activities similar to those of the other derivatives, but they
show a greater selectivity towards the non-tumor cell line, which makes them potentially interesting
for further studies.
3.2. Effect on the apoptosis induction
The capacity of the synthetic compounds to induce apoptosis at dosis around their IC50 values
was evaluated by measuring the translocation of phosphatidylserine from the cytoplasmic to the
extracellular side of the plasma membrane. Thus, HT-29 cells were incubated for 48 h in the
presence of colchicine and the most representative compounds and then, the amount of annexin V
was measured by flow cytometry.
Table 2 shows the percentage of viable, apoptotic and necrotic cells detected by flow cytometry
after treatment with urea derivatives at dosis below their IC50 values. It can be abserved that except
for colchicine and compound 17, more than a half of the population was still viable.

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Table 2. Apoptotic effect of colchicine and their derivatives.a
Conc.
(nM)
Viable cells
(%)
Apoptotic cells
(%)
Necrotic cells
(%)
Compound
Control
Colchicine
82 ± 1
26 ± 1
75 ± 1
64 ± 1
96 ± 1
55 ± 3
58 ± 1
62 ± 6
64 ± 5
51 ± 1
67 ± 1
87 ± 1
13 ± 3
73 ± 14
22 ± 14
34 ± 7
4 ± 3
2 ± 6
40
25000
25
1 ± 1
Sunitinib
3 ± 1
2 (o-MeC6H5)
3 (o-CF3C6H5)
4 (o-MeOC6H5)
6 (o-ClC6H5)
9 (m-ClC6H5)
10 (p-MeOC6H5)
11 (p-FC6H5)
13 (o,o-diEtC6H5)
14 (o,o-diFC6H5)
16 (o-Me-p-
FC6H5)
2 ± 3
20
1 ± 1
1000
0.5
5
44 ± 3
41 ± 2
37 ± 8
35 ± 6
47 ± 1
32 ± 5
12 ± 3
26 ± 1
1 ± 2
1 ± 2
1 ± 1
5
1 ± 2
5
2 ± 1
500
15
1 ± 1
1 ± 2
15
5
73 ± 3
46 ± 3
66 ± 3
1 ± 4
1 ± 3
1 ± 1
17 (o-Cl-p-FC6H5)
18 (o-Cl-p-
53 ± 1
33 ± 3
2000
MeC6H5)
22 (m,m-
4 ± 1
20
1
96 ± 1
86 ± 2
1 ± 1
3 ± 1
diCF3C6H5)
24 (p- C6H5C6H5)
11 ± 1
a Data are the average (±SD) of three experiments.
3.3. Effect on the expression of hTERT, c-MYC and VEGF genes
In order to determine the potential utility in cancer therapy of our derivatives, we began to
investigate the effect they exert on the expression of the hTERT, c-MYC and VEGF genes on the
HT-29 cell line. First, a conventional PCR was carried out as a preliminary study to qualitatively
determine which compounds were able to modify the expression of these three genes in HT-29 cell
line after 48 h of treatment (data not shown). This preliminary test let us select 10 urea derivatives
and their minimal active concentration (see Figure 3) for a quantitative study of gene expression on
the same cell line. As it is shown in Figure 3, the majority of these selected derivatives have one
group on ortho position in the phenylurea subunit. In addition, the size of this ortho group is similar
to that of a methyl group, except for the case of compound 14, which bears two smaller F atoms in
both ortho positions.

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Figure 3. Selected compounds and concentrations for a quantitative gene expression test
The expression of the c-MYC, hTERT and VEGF genes on the HT-29 cell line, quantitatively
determined by means of qPCR, is displayed in Table 3, in which the achieved values with
colchicine and sunitinib are also displayed. All values were normalized to the number of viable cells
that were present in each sample.
Table 3. Expression percentage for c-MYC, hTERT and VEGF genesa
c-Myc
hTERT VEGF
Compound
Conc.
gene exp.
37 ± 3
gene exp. gene exp.
25 nM
25 µM
20 nM
0.5 nM
5 nM
87 ± 6
68 ± 2
55 ± 2
46 ± 3
Colchicine
47 ± 2
107 ± 10
25 ± 2
104 ± 3
55 ± 4
52 ± 5
88 ± 9
65 ± 3
58 ± 7
85 ± 10
85 ± 9
Sunitinib
107 ± 13 101 ± 25
3
46 ± 5
90 ± 8
91 ± 4
70 ± 3
36 ± 5
68 ± 2
80 ± 8
80 ± 5
60 ± 3
30 ± 6
95 ± 5
70 ± 3
59 ± 7
38 ± 4
63 ± 3
64 ± 4
70 ± 6
72 ± 6
6
9
5 nM
10
11
14
16
17
18
24
5 nM
15 nM
15 nM
5 nM
0.5 nM
1 nM
aExpression percentage of VEGF, hTERT and c-Myc genes after 48 h of incubation with DMSO (control),
and selected compounds (at least three measurements were performed in each case). Gene expression was
normalized using β-ACTIN as endogenous gene and the number of living cells in each assay.
The tested urea derivatives inhibit the expression of the studied genes to a considerable degree,
even improving the results achieved by colchicine itself and sunitinib. Thus, colchicine was active
at a concentration of 25 nM (see Table 3) but it has no activity when tested at lower concentrations

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(results not shown). Sunitinib was also quite active inhibiting the expression of the three oncogenes
but at a dosis around 1000 times higher than colchicine.
As regards c-MYC gene expression, compound 6 (o-Cl) was the most active one and led to
downregulation in the expression of this gene to 25% at 0.5 nM dosis (below its IC50). p-Substituted
phenylureas 10 (p-OMe), 11 (o-CF3, p-F), 16 (o-Me, p-F) and 17 (o-Cl, p-F) performed also
remarkably well, downregulating gene expression to 55, 52, 68 and 58 % respectively.
As regards hTERT gene, compounds 6 (o-Cl) and 14 (o,o-diF) are the most active ones, as they
decrease gene expression to 46% and 36%, respectively. Compounds 11 (o-CF3, p-F), 16 (o-Me, p-
F) and 24 (p-Ph) are moderatively active with 70, 68 and 60 % of hTERT gene expression. In any
case, all these analogues improve the values that display both colchicine (87%) and sunitinib (68%).
The same result was observed for VEGF gene expression, compounds 6 (o-Cl) and 14 (o,o-diF)
being able to inhibit gene expression to 30% and 38% with respect to control. These two analogues
improve the values that display both colchicine (55%) and sunitinib (46%). Furthermore, p-
fluorophenylureas 11 (o-CF3, p-F), 16 (o-Me, p-F) and 17 (o-Cl, p-F) also deserve mention, with
VEGF expression values of 59%, 63% and 64%, respectively. The remainder of the assayed
derivatives show a certain degree of inhibition, although to a lesser extent that the previous ones.
Compound 6 (o-Cl) was the most outstanding compound, and improves to a high extent the
results obtained for colchicine and sunitinib. Indeed, it inhibits the three studied genes by an
average of 34% at 0.5 nM dosis (below its IC50).
3.4. Effect on c-MYC, VEGF and VEGFR-2 proteins
We next studied the action of colchicine derivatives on the c-MYC, VEGF and VEGFR-2
proteins. This study was performed by treating HT-29 cells with the selected compounds (see figure
3) for 48 hours. DMSO was used as a control, and colchicine and sunitinib as reference. Then, cell
medium was collected to measure the percentage of secreted VEGF with respect to the control
(untreated cells). On the other hand, cells were collected and lysated to measure the percentage of
total c-MYC and VEGFR-2 proteins inside cell referring to control. All the assays were performed
by ELISA (see methods and material section). The results are displayed in Table 4.
Table 4. Percentages of c-MYC and VEGFR-2 proteins inside cells and secreted VEGF a
c-Myc
protein protein
25 nM 63 ± 4 94 ± 2
100 ± 3 90 ± 9
VEGFR-2 VEGF
Compound
Conc.
secreted
40 ± 2
45 ± 3
Colchicine
Sunitinib
25 µM

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20 nM 99 ± 3
100 ± 3
38 ± 9
15 ± 2
95 ± 6
82 ± 5
34 ± 3
114± 11
44 ± 2
55 ± 8
11 ± 2
90 ± 4
67 ± 5
75 ± 6
72 ± 4
3
0.5 nM 51 ± 3
6
5 nM
5 nM
5 nM
87 ± 4
61 ± 6
9
10
11
14
16
17
18
24
105 ± 8 63 ± 6
35 ± 4
15 nM 110 ± 3 62 ± 5
5 nM 104 ± 8 15 ± 2
0.5 nM 112 ± 5 45 ± 7
1 nM 119 ± 3 74 ± 3
15 nM 42 ± 3
a Expression percentage of total c-MYC and VEGFR-2 detected proteins and secreted VEGF to the
medium after 48 h of incubation with DMSO (control) and selected compounds (at least three
measurements were performed in each case).
As regards the effect on total c-MYC protein, the most active compounds were 6 (o-Cl), 14 (o-
di-F) and colchicine itself, which caused reductions of the c-MYC protein to 51%, 42% and 63%,
respectively, when compared to untreated cells.
Regarding the effect on total VEGFR-2 in cells, a significant reduction in the detected values
for this protein is observed in HT-29 line with our set of derivatives. The most active derivatives
were those bearing a chlorine atom in the phenyl ring as in the case of 9 (m-Cl) and 17 (o-Cl, p-F),
which proved particularly active. Only 15% of VEGFR-2 protein was detected in both cases, in
relation to untreated cells. Compounds 6 (o-Cl), 14 (o,o-diF) and 18 (o-Cl, p-CH3), with 35, 38 and
45% of VEGFR-2 were also quite active in this biological target.
As regards the effect on VEGF protein secretion, compounds 6 and 14 were the most active
ones, as they reduced the percentage of the secreted VEGF to 34% and a 11% respectively.
Compound 10 (p-OMe), which exhibited similar activity to sunitinib and colchicine, was also quite
active (around 40% of secreted protein, see Table 4).
4. Discussion of the results and conclusions
Except for 4 and 22, all synthetized colchicine derivatives showed IC50 values in the nM range,
with activities in some cases higher than those of the natural product. Some of them substantially
improve the selectivity index against the studied tumoral cell lines, a point of interest for a possible
further drug development. Compound 14 for instance exhibited lower IC50 values in tumor cell lines
than in the non-tumor cell line HEK-293 (IC50: HT-29, 25 nM; MCF-7, 42 nM; HeLa, 18 nM;
HEK-293, 46 nM).

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Figure 4 represents the variation of hTERT gene expression, c-MYC detected protein and VEGF
secreted protein in function of the selected derivatives. It can be observed that when either hTERT
gene expression or when c-MYC protein disminishes around 40%, the secretion of VEGF protein to
cell medium also decreases. For example, with compounds 6 (o-Cl) and 14 (o,o-diF), which are able
to downregulate hTERT gene expression to 46 and 36%, respectively, and c-Myc protein to 51 and
42% respectively, secreted VEGF decreases to 34 and 11%, respectively. In the case of 10 (p-
OMe), the reduction of c-MYC protein is sufficient to downregulate VEGF secretion to 44%, and
with compound 24 (p-Ph), which downregulates to 60 % hTERT gene expression, secretion of
VEGF protein goes down to 72 %. These results demonstrate that there are remarkable differences
on the mode of action of these derivatives depending on the substitution pattern of the phenyl ring.
Figure 4. Variation of hTERT gene expression, c-MYC detected protein and VEGF secreted protein
in function of the selected derivatives
Figure 5 represents the variation of the levels of hTERT gene expression, c-MYC protein and
VEGFR-2 protein in function of the selected derivatives. It can be observed that only when the
compound causes a reduction of both hTERT gene expression and c-MYC protein around 40%, the
detection of VEGFR-2 decreases considerably. That is the case of compounds 6 (o-Cl) and 14 (o,o-
diF), which are able to downregulate the hTERT gene expression to 46 and 36 %, the c-Myc protein
to 51 and 42 % and the VEGFR-2 to 38 and 35 %, respectively. The cases of compounds 9 and 17,

14
ACCEPTED MANUSCRIPT
which are not very active against hTERT gene or c-MYC protein but are able to decrease the
detection of VEGFR-2 to 15%, are also worth mentioning. It may be conjectured that these
compounds are downregulating this latter target through a mechanism different from that of the
remaining derivatives.
Figure 5. Variation of hTERT gene expression, c-MYC detected protein and detected VEGFR-2
protein in function of the selected derivatives
Figure 6 shows the results obtained on hTERT, VEGF and c-MYC genes, c-MYC and VEGFR-2
proteins and secretion of the VEGF protein to the cell medium.

15
ACCEPTED MANUSCRIPT
Figure 6. Summary of biological results of some urea derivatives and reference products
It can be observed that compounds 6 (o-Cl) and 14 (o,o-diF) were the most active ones on all the
tested targets. Compound 6 exhibited similar IC50 values for all the tested cell lines (ranging 0.70-
1.30 nM) while compound 14 exhibited lower IC50 values for tumoral cell lines than for HEK-293
(see Tables 1 and 3). It thus seems that compound 14 could be more selective than 6 towards
tumoral cells and therefore less nocive for non-tumor cells. In any case, both derivatives are very
active in downregulating all the tested targets at dosis 0.5 nM for 6 and 15 nM for 14, which are
below their IC50 values (see Table 5).
Table 5. Summary of the results obtained in the biological assays of the most active urea derivatives
IC50
c-Myc hTERT VEGF
c-Myc
VEGFR-2 VEGF
IC50
Comp.
protein
protein
38 %
35 %
94 %
secreted
34 %
HEK-293
HT-29
gen.
25 %
88 %
37 %
gen.
46 %
36 %
87 %
gen.
30 %
38 %
55 %
0.80 nM 0.70 nM
51 %
42 %
63 %
6
25 nM
13 nM
46 nM
13 nM
11 %
14
40 %
Colch.
In summary, it can be concluded that compounds 6 and 14 may be good candidates for further in
vivo assays against cancer. Thus, the arylurea unit enhances the potential of colchicine as an anticancer
agent.

16
ACCEPTED MANUSCRIPT
5. Materials and methods
5.1. Chemical Procedures.
Conversion of deacetyl colchicine trifluoroacetate salt 28 into uretane 29. Compound 28 (472
mg, ca. 1 mmol) was dissolved under Ar in dry CH2Cl2 (5 mL), cooled in an ice bath and treated
with a solution of phenyl chloroformiate (190 µL, 1.5 mmol) and pyridine (200 µL, 2.5 mmol) in
dry CH2Cl2 (1 mL). The mixture was then stirred for 1 h at 0 ºC and then for 2 h at room
temperature. Work-up (extraction with CH2Cl2) and column chromatography on silica gel (elution
with hexane-EtOAc 1:4) afforded 29 (420 mg, 88%).
Conversion of 29 into urea derivatives 2, 9, 10, 12, 13 and 27. Uretane 29 (48 mg, 0.1 mmol) was
dissolved under Ar in dry CH2Cl2 (0.5 mL) and treated with a solution of triethyl amine (21 µL,
0.15 mmol) and the appropriate amine (0.1 mmol) in dry CH2Cl2 (0.5 mL). The reaction mixture
was then stirred for 24-96 h at room temperature (TLC monitoring). Work-up (extraction with
CH2Cl2), was followed by column chromatography of the oily residue on silica gel (elution with
EtOAc to EtOAc-MeOH 98:2) to afford the desired urea derivatives. For individual yields, see the
synthetic Scheme.
Conversion of 28 into urea derivatives 1, 3-8, 11 and 14-26. The appropriate aromatic amine (1
mmol) was dissolved under Ar in dry CH2Cl2 (3 mL), cooled in an ice bath and treated with a
solution of phenyl chloroformiate (190 µL, 1.5 mmol) and pyridine (200 µL, 2.5 mmol) in dry
CH2Cl2 (1 mL). The mixture was then stirred for 2 h at room temperature and then worked up. The
residue was subjected to column chromatography on silica gel (elution with hexane-EtOAc 9:1) to
afford the corresponding urethane.
The urethane from above (1 mmol) and compound 28 (472 mg, 1 mmol) were dissolved under Ar in
dry pyridine (3 mL) and heated at 80 ºC for 12-16 h (TLC monitoring). Work-up (extraction with
CH2Cl2) was followed by column chromatography of the oily residue on silica gel (elution with
EtOAc to EtOAc-MeOH 98:2) to afford the desired urea derivatives. For individual yields, see the
synthetic Scheme.
(S)-1-Phenyl-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)urea (1):
off-white solid, mp 198-199°C; [α]D −44; 1H NMR (300 MHz) δ 8.25 (1H, br s, NH), 8.00 (1H, br
s, NH), 7.42 (1H, d, J = 10.8 Hz), 7.25 (2H, br d, J ~ 8 Hz), 7.12 (3H, br t, J ~ 7.5 Hz), 6.94 (1H, d,
J = 10.8 Hz), 6.86 (1H, t, J = 7.2 Hz), 6.53 (1H, s), 4.80 (1H, m), 3.96 (6H, s), 3.90 (3H, s), 3.67
(3H, s), 2.50-2.35 (3H, br m), 1.85-1.75 (1H, br m); 13C NMR (75 MHz) δ 180.0, 164.1, 155.2,
154.6, 153.7, 151.3, 141.7, 139.5, 137.8, 134.6, 125.6 (C), 135.8, 131.8, 128.7 (x 2), 122.3, 119.7 (x
2), 113.5, 107.7, 52.4 (CH), 38.3, 30.4 (CH2), 61.8, 61.5, 56.5, 56.2 (CH3); IR νmax 3330 br, 1698,

17
ACCEPTED MANUSCRIPT
1614, 1590, 1544, 1488, 1251, 1091 cm−1; HR ESMS m/z 477.2018 (M+H+). Calcd. for
C27H29N2O6, 477.2025.
(S)-1-(1,2,3,10-Tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)-3-(o-tolyl)urea
1
(2): off-white solid, mp 169-170 °C; [α]D −15; H NMR (300 MHz) δ 7.85 (1H, br s, NH), 7.50
(1H, dd, J = 8, 1.5 Hz), 7.37 (1H, d, J = 10.8 Hz), 7.30 (1H, br s, NH), 7.05-6.95 (3H, br m), 6.90-
6.80 (2H, br m), 6.52 (1H, s), 4.67 (1H, m), 3.94 (3H, s), 3.92 (3H, s), 3.88 (3H, s), 3.65 (3H, s),
2.50-2.35 (3H, br m), 2.06 (3H, s), 1.80-1.70 (1H, br m); 13C NMR (75 MHz) δ 180.0, 164.1, 155.3,
154.3, 153.7, 151.3, 141.7, 137.4, 137.0, 134.5, 129.5, 125.6 (C), 135.8, 131.2, 130.4, 126.6, 123.8,
123.1, 113.3, 107.5, 52.8 (CH), 37.7, 30.3 (CH2), 61.8, 61.5, 56.5, 56.2, 17.8 (CH3); IR νmax 3340
br, 1698, 1614, 1589, 1538, 1488, 1456, 1249, 1091 cm−1; HR ESMS m/z 491.2167 (M+H+). Calcd.
for C28H31N2O6, 491.2182.
(S)-1-(1,2,3,10-Tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)-3-(2-
1
trifluoromethyl-phenyl)urea (3): off-white solid, mp 203-204 °C; [α]D −77; H NMR (300 MHz)
δ 7.90 (1H, br s, NH), 7.85 (1H, br d, J ~ 6 Hz, NH), 7.79 (1H, d, J = 8 Hz), 7.52 (1H, br s), 7.44
(1H, d, J = 10.8 Hz), 7.38 (1H, br d, J ~ 7.8 Hz), 7.22 (1H, br t, J ~ 7.8 Hz), 7.00-6.90 (2H, m), 6.53
(1H, s), 4.68 (1H, m), 4.00 (3H, s), 3.94 (3H, s), 3.90 (3H, s), 3.67 (3H, s), 2.50-2.35 (3H, br m),
1.85-1.75 (1H, br m); 13C NMR (75 MHz) δ 180.0 (C=O), 164.3, 154.7 (C=O), 154.2, 153.6, 151.3,
3
141.7, 137.3, 137.0 (center point of quadruplet with JC−F ~ 1.6 Hz), 134.5, 125.5, 124.0 (center
1
2
point of quadruplet with JC−F ~ 273 Hz), 120.0 (center point of quadruplet with JC−F ~ 28.5 Hz)
(C), 135.9, 132.4, 131.1, 125.6 (center point of quadruplet with 3JC−F ~ 5.5 Hz), 124.5, 122.5, 113.5,
107.4, 53.0 (CH), 37.4, 30.2 (CH2), 61.7, 61.4, 56.5, 56.2 (CH3); 19F NMR (282 MHz) δ −61.5; IR
νmax 3300 br, 1704, 1616, 1583, 1544, 1456, 1322, 1249, 1093 cm−1; HR ESMS m/z 545.1893
(M+H+). Calcd. for C28H28F3N2O6, 545.1899.
(S)-1-(2-Methoxyphenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)
1
urea (4): off-white solid, mp 180-181 °C; [α]D −28; H NMR (300 MHz) δ 7.80 (1H, br s, NH),
7.76 (1H, dd, J = 7.8, 1.5 Hz), 7.35 (1H, d, J = 10.8 Hz), 7.31 (1H, d, J = 8 Hz), 6.90 (1H, d, J =
10.8 Hz), 6.80 (1H, td, J = 7.8, 1.5 Hz), 6.70 (1H, td, J = 7.8, 1.5 Hz), 6.64 (1H, dd, J = 7.8, 1.5
Hz), 6.52 (1H, br d, J ~ 6 Hz, NH), 6.50 (1H, s), 4.67 (1H, m), 4.01 (3H, s), 3.95 (3H, s), 3.90 (3H,
13
s), 3.69 (3H, s), 3.57 (3H, s), 2.45-2.30 (3H, br m), 1.80-1.70 (1H, br m); C NMR (75 MHz) δ
179.9, 164.2, 154.3, 153.7, 153.4, 151.3, 148.1, 141.8, 137.1, 134.6, 128.6, 125.7 (C), 135.5, 131.4,
122.1, 120.8, 119.2, 113.0, 110.0, 107.5, 52.8 (CH), 37.6, 30.2 (CH2), 61.7, 61.5, 56.6, 56.3, 55.7
(CH3); IR νmax 3340 br, 1694, 1588, 1536, 1460, 1242, 1089 cm−1; HR ESMS m/z 507.2129
(M+H+). Calcd. for C28H31N2O7, 507.2131.
(S)-1-(2-Fluorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)
1
urea (5): off-white solid, mp 189-190 °C; [α]D −71; H NMR (300 MHz) δ 7.95 (1H, br s, NH),
7.87 (1H, br d, J ~ 1.5 Hz), 7.82 (1H, td, J = 8, 1.5 Hz), 7.55 (1H, br d, J ~ 3 Hz, NH), 7.40 (1H, d,

18
ACCEPTED MANUSCRIPT
J = 10.8 Hz), 6.95 (1H, d, J = 10.8 Hz), 6.85-6.75 (3H, br m), 6.47 (1H, s), 4.70 (1H, m), 4.00 (3H,
13
s), 3.93 (3H, s), 3.89 (3H, s), 3.67 (3H, s), 2.40-2.30 (3H, br m), 1.85-1.75 (1H, br m); C NMR
1
(75 MHz) δ 180.0, 164.3, 154.4, 154.2, 153.7, 152.5 (center point of doublet with JC−F ~ 242 Hz),
2
151.3, 141.7, 137.5, 134.6, 127.8 (doublet with JC−F ~ 10.2 Hz), 125.6 (C), 136.1, 131.3, 124.1
3
3
(center point of doublet with JC−F ~ 3.3 Hz), 122.2 (doublet with JC−F ~ 7.5 Hz), 121.3, 114.5
2
(doublet with JC−F ~ 19.5 Hz), 113.6, 107.5, 52.8 (CH), 37.7, 30.2 (CH2), 61.8, 61.5, 56.6, 56.2
19
(CH3); F NMR (282 MHz) δ −131.3; IR νmax 3320 br, 1704, 1620, 1588, 1544, 1249 cm−1; HR
ESMS m/z 495.1936 (M+H+). Calcd. for C27H28FN2O6, 495.1931.
(S)-1-(2-Chlorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)
urea (6): off-white solid, mp 190-191 °C; [α]D +3; 1H NMR (300 MHz) δ 8.00 (1H, br s), 7.87 (1H,
dd, J = 8, 1.5 Hz), 7.85 (1H, br d, J ~ 6 Hz, NH), 7.70 (1H, br s, NH), 7.42 (1H, d, J = 10.8 Hz),
7.03 (1H, dd, J = 8, 1.5 Hz), 6.96 (1H, d, J = 10.8 Hz), 6.95 (1H, td, J = 8, 1.5 Hz), 6.72 (1H, td, J =
8, 1.5 Hz), 6.45 (1H, s), 4.67 (1H, m), 4.03 (3H, s), 3.95 (3H, s), 3.89 (3H, s), 3.68 (3H, s), 2.40-
13
2.30 (3H, br m), 1.85-1.80 (1H, br m); C NMR (75 MHz) δ 180.0, 164.3, 154.3, 154.2, 153.7,
151.3, 141.7, 137.5, 136.1, 134.5, 125.6 (C), 136.1, 131.2, 128.7, 127.2, 122.5, 121.0, 113.6, 107.4,
52.9 (CH), 37.4, 30.2 (CH2), 61.8, 61.5, 56.7, 56.2 (CH3); IR νmax 3320 br, 1702, 1614, 1586, 1536,
1253 cm−1; HR ESMS m/z 511.1635 (M+H+). Calcd. for C27H2835ClN2O6, 511.1636.
(S)-1-(2-Cyanophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)
1
urea (7): off-white solid, mp 177-178 °C; [α]D −54; H NMR (400 MHz) δ 8.03 (2H, br d, J 8.5
Hz), 7.95 (1H, br s, NH), 7.80 (1H, br s, NH), 7.40 (2H, m), 7.25 (1H, br t, J 8 Hz), 6.95 (1H, br
d, J 10.7 Hz), 6.89 (1H, br t, J 7.5 Hz), 6.55 (1H, s), 4.70 (1H, m), 4.00 (3H, br s), 3.96 (3H, s),
3.93 (3H, s), 3.70 (3H, s), 2.55-2.35 (3H, br m), 1.95 (1H, br m); 13C NMR (75 MHz) δ 180.1,
164.3, 153.8, 153.5, 153.2, 151.2, 142.6, 141.5, 137.2, 134.5, 125.6, 116.9, 100.4 (C), 135.9, 133.7,
132.5, 131.5, 121.6, 119.9, 113.5, 107.3, 52.9 (CH), 37.2, 30.1 (CH2), 61.7, 61.4, 56.5, 56.1 (CH3);
IR νmax 3300 br, 1704, 1586, 1534, 1249, 1231 cm−1; HR ESMS m/z 502.1982 (M+H+). Calcd. for
C28H28N3O6, 502.1978.
(S)-1-(1,2,3,10-Tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)-3-(2-
trifluoromethoxy-phenyl)urea (8): off-white solid, mp 186-187 °C; [α]D −58; 1H NMR (500 MHz)
δ 7.95 (1H, br s), 7.91 (1H, dd, J = 8.3, 1.5 Hz), 7.70 (1H, br s, NH), 7.65 (1H, br d, J ~5.5 Hz,
NH), 7.42 (1H, d, J = 10.8 Hz), 7.00 (1H, dt, J = 8.3, 1.5 Hz), 6.95 (1H, d, J = 10.8 Hz), 6.92 (1H,
td, J = 8.3, 1.5 Hz), 6.77 (1H, td, J = 8.3, 1.5 Hz), 6.52 (1H, s), 4.70 (1H, m), 4.00 (3H, br s), 3.95
(3H, s), 3.91 (3H, s), 3.67 (3H, s), 2.50-2.35 (3H, br m), 1.80 (1H, m); 13C NMR (125 MHz) δ
180.2, 164.4, 154.2, 154.1, 153.7, 151.3, 141.8, 137.8, 137.4, 134.5, 132.0, 125.7, 120.6 (center
point of quadruplet with 1JC−F ~ 257 Hz) (C), 136.0, 131.1, 127.0, 121.7, 121.1, 119.8, 113.5, 107.4,
19
53.0 (CH), 37.5, 30.2 (CH2), 61.7, 61.6, 56.6, 56.3 (CH3); F NMR (282 MHz) δ −58.2; IR νmax

19
ACCEPTED MANUSCRIPT
3330 br, 1702, 1614, 1542, 1247 cm−1; HR ESMS m/z 561.1834 (M+H+). Calcd. for C28H28F3N2O7,
561.1848.
(S)-1-(3-Chlorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)
1
urea (9): off-white solid, mp 179-180 °C; [α]D −26; H NMR (300 MHz) δ 8.60 (1H, br s, NH),
8.05 (1H, s), 7.50 (1H, d, J = 10.8 Hz), 7.35 (1H, t, J = 2 Hz), 7.30-7.20 (2H, m), 7.10-7.00 (2H,
m), 6.85 (1H, dd, J = 8, 2 Hz), 6.56 (1H, s), 4.88 (1H, m), 4.02 (3H, br s), 3.97 (3H, s), 3.93 (3H, s),
3.71 (3H, s), 2.60-2.35 (3H, br m), 1.85 (1H, br m); 13C NMR (75 MHz) δ 180.0, 164.1, 155.0,
154.8, 153.9, 151.3, 141.8, 141.2, 138.2, 134.6, 134.2, 125.5 (C), 136.1, 132.0, 129.7, 121.9, 119.1,
117.1, 113.8, 107.7, 52.3 (CH), 38.7, 30.5 (CH2), 61.9, 61.5, 56.6, 56.3 (CH3); IR νmax 3330 br,
1701, 1589, 1541, 1484, 1250 cm−1; HR ESMS m/z 511.1622 (M+H+). Calcd. for C27H2835ClN2O6,
511.1636.
(S)-1-(4-Methoxyphenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)
urea (10): off-white solid, mp 179-180 °C; [α]D −13; 1H NMR (300 MHz) δ 7.97 (1H, s), 7.85 (1H,
br s, NH), 7.40 (1H, d, J = 10.8 Hz), 7.15 (2H, apparent d, J ~ 9 Hz), 6.95 (1H, br d, J ~ 8 Hz), 6.92
(1H, d, J = 10.8 Hz), 6.70 (2H, apparent d, J ~ 9 Hz), 6.55 (1H, s), 4.80 (1H, m), 3.95 (3H, s), 3.94
(3H, s), 3.90 (3H, s), 3.69 (3H, s), 3.67 (3H, s), 2.50 (1H, m), 2.45-2.35 (2H, br m), 1.80 (1H, br m);
13C NMR (75 MHz) δ 179.9, 164.0, 155.7, 155.6, 154.5, 153.7, 151.3, 141.7, 137.7, 134.6, 132.2,
125.6 (C), 135.7, 131.7, 122.7 (x 2), 114.1 (x 2), 113.3, 107.6, 52.5 (CH), 38.2, 30.4 (CH2), 61.8,
61.5, 56.5, 56.2, 55.5 (CH3); IR νmax 3320 br, 1689, 1613, 1588, 1544, 1510, 1225 cm−1; HR ESMS
m/z 507.2145 (M+H+). Calcd. for C28H31N2O7, 507.2131.
(S)-1-(4-Fluorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)
1
urea (11): off-white solid, mp 177-178 °C; [α]D −23; H NMR (500 MHz) δ 8.20 (1H, br s, NH),
8.05 (1H, s), 7.45 (1H, d, J = 10.8 Hz), 7.18 (2H, dd, J = 9, 4.8 Hz), 7.10 (1H, br d, J ~ 8 Hz, NH),
6.96 (1H, d, J = 10.8 Hz), 6.82 (2H, t, J = 9 Hz), 6.55 (1H, s), 4.82 (1H, m), 3.96 (6H, s), 3.92 (3H,
13
s), 3.69 (3H, s), 2.52 (1H, m), 2.45-2.35 (2H, br m), 1.85-1.80 (1H, br m); C NMR (125 MHz) δ
180.0, 164.1, 158.6 (center point of doublet with 1JC−F ~ 240 Hz), 155.3, 154.8, 153.8, 151.3, 141.8,
3
138.0, 135.3, 134.6, 125.5 (C), 136.0, 131.7, 121.7 (x 2, doublet with JC−F ~ 7.5 Hz), 115.3 (x 2,
doublet with 2JC−F ~ 22 Hz), 113.6, 107.6, 52.5 (CH), 38.4, 30.4 (CH2), 61.9, 61.5, 56.5, 56.3 (CH3);
19F NMR (282 MHz) δ −121.6; IR νmax 3320 br, 1698, 1614, 1588, 1544, 1508, 1249, 1089 cm−1;
HR ESMS m/z 495.1912 (M+H+). Calcd. for C27H28FN2O6, 495.1931.
(S)-1-(2,6-Dimethylphenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl) urea (12): off-white solid, mp 231-233 °C; [α]D −20; H NMR (400 MHz, 50 °C)† δ 7.30-7.25
(2H, m), 7.15 (3H, br m), 6.76 (1H, d, J = 10.8 Hz), 6.52 (1H, s), 5.80 (1H, br s, NH), 4.80 (1H, br
s, NH), 4.65 (1H, m), 3.95 (6H, s), 3.91 (3H, s), 3.68 (3H, s), 2.50-2.35 (2H, br m), 2.32 (6H, br s),
2.20-2.10 (1H, m), 1.60-1.50 (1H, br m); 13C NMR (125 MHz)‡ δ 179.6, 164.1, 155.7, 153.6, 151.5,
141.8, 134.4, 129.0 (br), 125.6 (C), 135.1, 131.1, 112.2, 107.5, 52.7 (CH), 37.8 (br), 30.3 (CH2),

20
ACCEPTED MANUSCRIPT
61.8, 61.5, 56.3, 56.2, 18.5 (x 2) (CH3); IR νmax 3330 br, 1687, 1614, 1588, 1544, 1488, 1460, 1249,
1091 cm−1; HR ESMS m/z 505.2359 (M+H+). Calcd. for C29H33N2O6, 505.2338.
†Several 1H signals were very broadened at room temperature due to hindered rotation.
‡A number of 13C signals did not emerge from the background due to hindered rotation.
(S)-1-(2,6-Diethylphenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl) urea (13): off-white solid, mp 245-247 °C; [α]D −68; H NMR (400 MHz, 50 °C)† δ 7.35-7.15
(5H, br m), 6.75 (1H, d, J = 10.7 Hz), 6.51 (1H, s), 5.70 (1H, br s, NH), 4.70-4.60 (2H, m), 3.96
(3H, s), 3.95 (3H, s), 3.91 (3H, s), 3.68 (3H, s), 2.70 (4H, br m), 2.50-2.30 (2H, br m), 2.10-2.00
(1H, m), 1.60-1.50 (1H, br m), 1.20 (6H, t, J = 7.5 Hz); 13C NMR (125 MHz)‡ δ 179.6, 164.1,
156.3, 153.6, 151.4, 141.8, 134.4, 132.8 (br), 125.8 (C), 135.0, 131.1 (br), 112.2 (br), 107.5, 52.7
(CH), 37.8 (br), 30.3, 24.9 (x 2) (CH2), 61.6, 61.5, 56.3, 56.2, 15.0 (x 2) (CH3); IR νmax 3340 br,
1687, 1614, 1588, 1547, 1247, 1091 cm−1; HR ESMS m/z 533.2667 (M+H+). Calcd. for
C31H37N2O6, 533.2651.
†Several 1H signals were very broadened at room temperature due to hindered rotation.
‡A number of 13C signals did not emerge from the background due to hindered rotation.
(S)-1-(2,6-Difluorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl) urea (14): off-white solid, mp 190-192 °C; [α]D −36; H NMR (500 MHz) δ 8.25 (1H, br s,
NH), 8.15 (1H, s), 7.40 (1H, d, J = 10.8 Hz), 7.30 (1H, br d, J ~ 8.8 Hz, NH), 7.05 (1H, m), 6.87
(1H, d, J = 10.8 Hz), 6.85 (2H, t, J = 8 Hz), 6.55 (1H, s), 4.93 (1H, m), 3.93 (3H, s), 3.91 (3H, s),
3.72 (3H, s), 3.64 (3H, s), 2.55-2.40 (3H, br m), 1.95-1.85 (1H, br m); 13C NMR (125 MHz) δ
1
3
180.0, 163.8, 158.3 (x 2, center point of double doublet with JC−F ~ 248 Hz and JC−F ~ 5 Hz),
154.9, 154.0, 153.7, 151.3, 141.7, 137.9, 134.7, 125.6, 116.1 (triplet with 2JC−F ~ 16 Hz) (C), 135.5,
3
2
132.1, 125.9 (triplet with JC−F ~ 9 Hz), 113.3, 111.5 (x 2, double doublet with JC−F ~ 19 Hz and
4JC−F ~ 5 Hz), 107.7, 52.6 (CH), 39.0, 30.5 (CH2), 61.8, 61.4, 56.3, 56.2 (CH3); 19F NMR (282
MHz) δ −119.1; IR νmax 3300 br, 1704, 1614, 1588, 1536, 1462, 1244, 1091 cm−1; HR ESMS m/z
513.1828 (M+H+). Calcd. for C27H27F2N2O6, 513.1837.
(S)-1-(2,6-Dichlorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl) urea (15): off-white solid, mp 176-178 °C; [α]D −82; H NMR (500 MHz) δ 8.25 (1H, br s,
NH), 8.17 (1H, br s), 7.37 (1H, d, J = 10.8 Hz), 7.28 (2H, d, J = 8 Hz), 7.05 (1H, t, J = 8 Hz), 6.97
(1H, br d, J ~ 8 Hz, NH), 6.82 (2H, d, J = 10.8 Hz), 6.55 (1H, s), 4.87 (1H, m), 3.91 (3H, s), 3.90
13
(3H, s), 3.65 (3H, br s), 3.62 (3H, s), 2.55-2.40 (3H, br m), 1.95-1.90 (1H, br m); C NMR (125
MHz) δ 180.0, 163.7, 154.8, 154.0, 153.7, 151.3, 141.7, 137.7, 134.8 (x 2), 134.7, 134.0, 125.6 (C),
135.3, 132.0, 128.3 (x 2), 127.6, 113.0, 107.7, 52.5 (CH), 38.9, 30.5 (CH2), 61.8, 61.5, 56.2, 56.1
(CH3); IR νmax 3320 br, 1698, 1657, 1614, 1580, 1547, 1460, 1432, 1249, 1091 cm−1; HR ESMS
m/z 545.1229 (M+H+). Calcd. for C27H2735Cl2N2O6, 545.1246.

21
ACCEPTED MANUSCRIPT
(S)-1-(4-Fluoro-2-methylphenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
1
tetrahydrobenzo[a]heptalen-7-yl)urea (16): off-white solid, mp 182-184 °C; [α]D −6; H NMR
(500 MHz) δ 7.95 (1H, br s, NH), 7.45-7.35 (3H, br m), 7.20 (1H, br d, J ~ 7 Hz, NH), 6.94 (1H, d,
J = 11 Hz), 6.75-6.65 (2H, br m), 6.54 (1H, s), 4.70 (1H, m), 3.95 (6H, s), 3.91 (3H, s), 3.66 (3H, s),
2.55-2.35 (3H, br m), 2.05 (3H, s), 1.80-1.70 (1H, br m); 13C NMR (125 MHz) δ 180.0, 164.2,
159.4 (center point of doublet with 1JC−F ~ 243 Hz), 155.5, 154.7, 153.8, 151.3, 141.8, 137.7, 134.5,
132.8 (doublet with 4JC−F ~ 3 Hz), 132.5 (doublet with 3JC−F ~ 8 Hz), 125.6 (C), 136.1, 131.2, 125.1
3
2
2
(doublet with JC−F ~ 8 Hz), 116.6 (doublet with JC−F ~ 22 Hz), 113.6, 113.0 (doublet with JC−F
~
22 Hz), 107.5, 53.0 (CH), 37.8, 30.3 (CH2), 61.8, 61.5, 56.5, 56.3, 18.0 (CH3); 19F NMR (282 MHz)
δ −120.2; IR νmax 3320 br, 1698, 1614, 1588, 1544, 1490, 1249, 1091 cm−1; HR ESMS m/z
509.2083 (M+H+). Calcd. for C28H30FN2O6, 509.2088.
(S)-1-(2-Chloro-4-fluorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
1
tetrahydrobenzo[a]heptalen-7-yl)urea (17): off-white solid, mp 181-183 °C; [α]D +17; H NMR
(300 MHz) δ 8.00 (1H, s), 7.95 (1H, br d, J ~ 6.5 Hz, NH), 7.80 (1H, dd, J = 9.2, 5.7 Hz), 7.70 (1H,
br s, NH), 7.44 (1H, d, J = 10.8 Hz), 6.98 (1H, d, J = 10.8 Hz), 6.77 (1H, dd, J = 8.2, 3 Hz), 6.67
(1H, ddd, J = 9.2, 8.2, 3 Hz), 6.51 (1H, s), 4.66 (1H, m), 4.03 (3H, s), 3.95 (3H, s), 3.90 (3H, s),
3.67 (3H, s), 2.50-2.30 (3H, br m), 1.90-1.80 (1H, br m); 13C NMR (75 MHz) δ 180.0, 164.3, 157.3
(center point of doublet with 1JC−F ~ 243 Hz), 154.6, 154.4, 153.8, 151.3, 141.7, 137.7, 134.5, 132.6
4
3
(doublet with JC−F ~ 3 Hz), 125.5, 123.0 (doublet with JC−F ~ 10.5 Hz) (C), 136.3, 131.2, 122.1
3
2
2
(doublet with JC−F ~ 7.5 Hz), 115.6 (doublet with JC−F ~ 25 Hz), 113.9 (doublet with JC−F ~ 22
Hz), 113.8, 107.4, 53.0 (CH), 37.4, 30.1 (CH2), 61.8, 61.5, 56.7, 56.2 (CH3); 19F NMR (282 MHz) δ
−120.6; IR νmax 3300 br, 1700, 1614, 1588, 1531, 1486, 1251 cm−1; HR ESMS m/z 529.1529
(M+H+). Calcd. for C27H2735ClFN2O6, 529.1541.
(S)-1-(2-Chloro-4-methylphenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
1
tetrahydrobenzo[a]heptalen-7-yl) urea (18): off-white solid, mp 194-196 °C; [α]D −33; H NMR
(300 MHz) δ 7.95 (1H, br s, NH), 7.70 (1H, d, J =8.3 Hz), 7.50 (2H, m), 7.40 (1H, d, J = 10.8 Hz),
6.94 (1H, d, J = 10.8 Hz), 6.88 (1H, br d, J ~ 2 Hz), 6.77 (1H, br dd, J ~ 8.3, 2 Hz), 6.50 (1H, s),
4.67 (1H, m), 4.00 (3H, s), 3.95 (3H, s), 3.90 (3H, br s), 3.68 (3H, s), 2.45-2.30 (3H, br m), 2.15
13
(3H, s), 1.85-1.80 (1H, m); C NMR (75 MHz) δ 180.0, 164.2, 154.4, 154.1, 153.7, 151.3, 141.7,
137.4, 134.6, 133.5, 132.5, 125.6, 122.7 (C), 135.9, 131.3, 129.1, 127.9, 121.4, 113.5, 107.5, 53.0
(CH), 37.5, 30.2 (CH2), 61.9, 61.5, 56.6, 56.2 (CH3); IR νmax 3300 br, 1704, 1687, 1562, 1544,
1253, 1095 cm−1; HR ESMS m/z 525.1789 (M+H+). Calcd. for C28H3035ClN2O6, 525.1792.
(S)-1-(2-Chloro-4-trifluoromethylphenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
1
tetrahydrobenzo[a] heptalen-7-yl) urea (19): off-white solid, mp 174-176 °C; [α]D −10; H NMR
(500 MHz) δ 8.35 (1H, br s, NH), 8.03 (1H, br s), 7.95 (1H, br d, J ~ 8.8 Hz), 7.90 (1H, br s, NH),
7.48 (1H, d, J = 10.8 Hz), 7.25 (1H, s), 7.04 (1H, d, J = 10.7 Hz), 7.02 (1H, dd, J = 8.8, 2 Hz), 6.52

22
ACCEPTED MANUSCRIPT
(1H, s), 4.66 (1H, m), 4.06 (3H, br s), 3.96 (3H, s), 3.90 (3H, s), 3.69 (3H, s), 2.50-2.35 (3H, br m),
13
1.90-1.85 (1H, m); C NMR (75 MHz) δ 180.2, 164.3, 154.6, 153.9, 153.6, 151.3, 141.8, 139.3,
2
137.9, 134.4, 125.4, 123.8 (quadruplet with JC−F ~ 33 Hz), 123.6 (center point of quadruplet with
1JC−F ~ 268 Hz), 121.2 (C), 136.6, 131.1, 125.7 (quadruplet with 3JC−F ~ 3.5 Hz), 124.3 (quadruplet
with 3JC−F ~ 3.5 Hz), 119.2, 114.2, 107.4, 53.1 (CH), 37.1, 30.1 (CH2), 61.8, 61.5, 56.8, 56.2 (CH3);
19F NMR (282 MHz) δ −62.6; IR νmax 3300 br, 1709, 1614, 1588, 1531, 1322, 1251 cm−1; HR
ESMS m/z 579.1489 (M+H+). Calcd. for C28H2735ClF3N2O6, 579.1509.
(S)-1-(2,4-Dichlorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl) urea (20): off-white solid, mp 178-180 °C; [α]D +25; H NMR (500 MHz) δ 8.10 (1H, br s,
NH), 7.98 (1H, br s), 7.83 (1H, d, J = 8.8 Hz), 7.70 (1H, br s, NH), 7.45 (1H, d, J = 10.8 Hz), 7.05-
6.95 (2H, m), 6.86 (1H, dd, J = 8.8, 2 Hz), 6.50 (1H, s), 4.65 (1H, m), 4.04 (3H, br s), 3.95 (3H, s),
13
3.90 (3H, s), 3.66 (3H, s), 2.50-2.30 (3H, br m), 1.85-1.75 (1H, m); C NMR (125 MHz) δ 180.1,
164.3, 154.6, 153.9, 153.8, 151.3, 141.7, 137.8, 135.0, 134.5, 126.5, 125.5, 122.3 (C), 136.4, 131.2,
128.2, 127.2, 121.0, 114.0, 107.5, 53.0 (CH), 37.2, 30.2 (CH2), 61.8, 61.5, 56.7, 56.3 (CH3); IR νmax
3300 br, 1704, 1614, 1581, 1544, 1519, 1249 cm−1; HR ESMS m/z 545.1217 (M+H+). Calcd. for
C27H2735Cl2N2O6, 545.1246.
(S)-1-(3,5-Difluorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl) urea (21): off-white solid, mp 204-206 °C; [α]D −24; H NMR (500 MHz) δ 8.80 (1H, br s,
NH), 8.02 (1H, br s), 7.51 (1H, d, J = 10.8 Hz), 7.25 (1H, br s, NH), 7.05 (1H, d, J = 10.8 Hz), 6.95
3
3
(2H, br d, JHF ~ 9 Hz), 6.56 (1H, s), 6.30 (1H, br t, JHF ~ 9 Hz), 4.87 (1H, m), 4.05 (3H, s), 3.97
(3H, s), 3.91 (3H, s), 3.72 (3H, s), 2.55-2.35 (3H, br m), 1.90-1.80 (1H, br m); 13C NMR (125 MHz)
1
3
δ 180.0, 164.2, 163.2 (x 2, center point of double doublet with JC−F ~ 242 and JC−F ~ 15 Hz),
155.0, 154.4, 153.9, 151.3, 142.3 (triplet with 3JC−F ~ 13 Hz), 141.8, 138.3, 134.5, 125.5 (C), 136.3,
131.8, 114.1, 107.7, 101.4 (x 2, double doublet with 2JC−F ~ 22 and 4JC−F ~ 7 Hz), 96.8 (triplet with
19
2JC−F ~ 25 Hz), 52.2 (CH), 38.6, 30.4 (CH2), 61.8, 61.5, 56.5, 56.2 (CH3); F NMR (282 MHz) δ
−111.1; IR νmax 3320 br, 1704, 1613, 1544, 1249 cm−1; HR ESMS m/z 513.1820 (M+H+). Calcd. for
C27H27F2N2O6, 513.1837.
(S)-1-[3,5-Bis(trifluoromethyl)phenyl]-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
1
tetrahydrobenzo[a] heptalen-7-yl)urea (22): off-white solid, mp 205-207 °C; [α]D −31; H NMR
(500 MHz) δ 9.25 (1H, br s, NH), 8.10 (1H, s), 7.85 (2H, s), 7.56 (1H, d, J = 10.8 Hz), 7.35 (2H,
m), 7.08 (1H, d, J = 10.8 Hz), 6.58 (1H, s), 4.96 (1H, m), 4.02 (3H, br s), 3.99 (3H, s), 3.93 (3H, s),
3.75 (3H, s), 2.55-2.40 (3H, br m), 1.95-1.90 (1H, br m); 13C NMR (125 MHz) δ 180.0, 164.1,
155.2, 154.5, 154.1, 151.4, 142.0, 141.7, 138.5, 134.5, 131.8 (x 2, center point of quadruplet with
2JC−F ~ 33 Hz), 125.4, 123.5 (x 2, center point of quadruplet with 1JC−F ~ 270 Hz) (C), 136.6, 132.1,
3
3
118.1 (x 2, quadruplet with JC−F ~ 4 Hz), 114.8 (septuplet with JC−F ~ 3.5 Hz), 114.3, 107.7, 52.2
19
(CH), 38.9, 30.5 (CH2), 61.9, 61.5, 56.6, 56.3 (CH3); F NMR (282 MHz) δ −63.4; IR νmax 3320

23
ACCEPTED MANUSCRIPT
br, 1704, 1616, 1586, 1547, 1387, 1251, 1125, 1093 cm−1; HR ESMS m/z 613.1751 (M+H+). Calcd.
for C29H27F6N2O6, 613.1773.
(S)-1-(2,6-Dichloro-4-fluorophenyl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
tetrahydrobenzo[a] heptalen-7-yl) urea (23): off-white solid, mp 187-189 °C; [α]D −72; 1H NMR
(500 MHz) δ 8.35 (1H, br s, NH), 8.21 (1H, br s), 7.40 (1H, d, J = 10.7 Hz), 7.15 (1H, br d, J ~ 8
3
Hz), 7.05 (2H, d, JHF = 8 Hz), 6.84 (1H, d, J = 10.7 Hz), 6.55 (1H, s), 4.87 (1H, m), 3.91 (3H, s),
13
3.90 (3H, s), 3.64 (3H, s), 3.62 (3H, s), 2.55-2.40 (3H, br m), 1.95-1.90 (1H, m); C NMR (125
1
MHz) δ 180.0, 163.6, 159.8 (center point of doublet with JC−F ~ 250 Hz), 155.1, 154.3, 153.7,
3
4
151.3, 141.7, 138.0, 135.6 (x 2, doublet with JC−F ~ 12 Hz), 134.7, 130.7 (doublet with JC−F ~ 4
Hz), 125.5 (C), 135.4, 132.0, 115.8 (x 2, doublet with 2JC−F ~ 25 Hz), 113.1, 107.7, 52.5 (CH), 39.0,
19
30.5 (CH2), 61.8, 61.5, 56.3, 56.1 (CH3); F NMR (282 MHz) δ −114.5; IR νmax 3300 br, 1698,
1581, 1544, 1462, 1249, 1091 cm−1; HR ESMS m/z 563.1151 (M+H+). Calcd. for
C27H2635Cl2FN2O6, 563.1152.
(S)-1-([1,1'-Biphenyl]-4-yl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl) urea (24): off-white solid, mp 197-199 °C; [α]D +25; H NMR (300 MHz) δ 8.25 (1H, br s,
NH), 8.00 (1H, br s), 7.45-7.35 (3H, m), 7.30-7.15 (8H, br m), 6.92 (1H, d, J = 11 Hz), 6.47 (1H, s),
4.65 (1H, m), 3.93 (3H, br s), 3.89 (3H, s), 3.83 (3H, s), 3.65 (3H, s), 2.45-2.30 (3H, br m), 1.85-
1.75 (1H, m); 13C NMR (75 MHz+) δ 180.0, 164.1, 155.1, 154.7, 153.7, 151.3, 141.7, 141.0, 138.8,
137.9, 135.0, 134.6, 125.5 (C), 136.0, 131.6, 128.6 (x 2), 127.3 (x 2), 126.7 (x 2), 126.6, 120.0 (x
2), 113.7, 107.5, 52.5 (CH), 38.2, 30.4 (CH2), 61.9, 61.5, 56.5, 56.2 (CH3); IR νmax 3300 br, 1700,
1613, 1590, 1544, 1536, 1488, 1251, 1093 cm−1; HR ESMS m/z 553.2335 (M+H+). Calcd. for
C33H33N2O6, 553.2338.
(S)-1-(Naphthalen-1-yl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl)urea (25): off-white solid, mp 180-182 °C; [α]D −1; 1H NMR (500 MHz) δ 8.25 (1H, br s, NH),
8.10 (1H, br s), 8.00 (1H, br s), 7.75 (1H, d, J = 8 Hz), 7.60 (1H, d, J = 7.5 Hz), 7.57 (1H, d, J = 8
Hz), 7.39 (1H, d, J = 10.8 Hz), 7.40-7.25 (3H, br m), 7.05 (1H, br s, NH), 6.80 (1H, d, J = 10.8 Hz),
6.55 (1H, s), 4.88 (1H, m), 3.92 (3H, br s), 3.90 (3H, s), 3.65 (3H, s), 3.57 (3H, s), 2.55-2.35 (3H, br
m), 1.85-1.75 (1H, m); 13C NMR (125 MHz) δ 180.0, 164.1, 156.0, 154.3, 153.7, 151.3, 141.7,
137.6, 134.6, 134.3, 134.0, 128.7, 125.6 (C), 135.7, 131.8, 128.2, 126.0, 125.9, 125.8, 125.0, 122.3,
121.7, 113.3, 107.6, 52.8 (CH), 38.5, 30.4 (CH2), 61.8, 61.5, 56.3, 56.2 (CH3); IR νmax 3300 br,
1698, 1544, 1536, 1249 cm−1; HR ESMS m/z 527.2176 (M+H+). Calcd. for C31H31N2O6, 527.2182.
(S)-1-(Naphthalen-2-yl)-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl)urea (26): off-white solid, mp 182-184 °C; [α]D −38; 1H NMR (400 MHz) δ 8.50 (1H, br s, NH),
8.15 (1H, br s), 7.85 (1H, br s), 7.56 (2H, br t, J ~ 8.5 Hz), 7.50 (1H, m), 7.46 (1H, d, J = 10.8 Hz),
7.30-7.20 (4H, br m), 6.96 (1H, d, J = 10.8 Hz), 6.53 (1H, s), 4.90 (1H, m), 3.96 (6H, s), 3.90 (3H,
s), 3.74 (3H, s), 2.50-2.35 (3H, br m), 1.90-1.80 (1H, m); 13C NMR (75 MHz) δ 180.0, 164.1,

24
ACCEPTED MANUSCRIPT
155.3, 154.6, 153.7, 151.3, 141.7, 137.9, 137.1, 134.6, 134.2, 129.7, 125.5 (C), 135.9, 131.8, 128.3,
127.4, 127.3, 125.9, 123.8, 120.5, 115.3, 113.6, 107.5, 52.4 (CH), 38.3, 30.4 (CH2), 61.8, 61.5,
56.5, 56.2 (CH3); IR νmax 3300 br, 1698, 1544, 1249 cm−1; HR ESMS m/z 527.2179 (M+H+). Calcd.
for C31H31N2O6, 527.2182.
(S)-1-Cyclohexyl-3-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
1
yl)urea (27): off-white solid, mp 168-170 °C; [α]D −54; H NMR (500 MHz) δ 7.70 (1H, s), 7.35
(1H, d, J = 10.7 Hz), 6.87 (1H, d, J = 10.7 Hz), 6.52 (1H, s), 5.95 (1H, br d, J ~ 7 Hz, NH), 5.00
(1H, br d, J ~ 8 Hz, NH), 4.60 (1H, m), 4.00 (3H, s), 3.93 (3H, s), 3.90 (3H, s), 3.66 (3H, s), 3.45
(1H, m), 2.48 (1H, m), 2.40-2.25 (2H, br m), 1.90-1.50 (6H, br m), 1.30-1.20 (2H, m), 1.10-0.95
13
(3H, br m); C NMR (125 MHz) δ 180.0, 164.1, 156.5, 153.9, 153.6, 151.3, 141.7, 137.1, 134.6,
125.7 (C), 135.4, 131.3, 112.9, 107.5, 52.7, 48.8 (CH), 37.9, 34.1, 33.9, 30.3, 25.7, 25.1, 25.0
(CH2), 61.8, 61.5, 56.5, 56.3 (CH3); IR νmax 3320 br, 1650. 1614, 1547, 1488, 1249, 1093 cm−1; HR
ESMS m/z 483.2486 (M+H+). Calcd. for C27H35N2O6, 483.2495.
5.2. Biological studies. Materials and methods
5.2.1. Cell culture
Cell culture media were purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS)
was a product of Harlan-Seralab (Belton, U.K.). Supplements and other chemicals not listed in this
section were obtained from Sigma Chemical Co. (St. Louis, MO). Plastics for cell culture were
supplied by Thermo Scientific BioLite. All tested compounds were dissolved in DMSO at a
concentration of 20 µM and stored at -20°C until use.
HT-29, MCF-7, HeLa and HEK-293 cell lines were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) containing glucose (1g/L), glutamine (2 mM), penicillin (50 IU/mL),
streptomycin (50 µg/mL), and amphotericin B (1.25 µg/mL), supplemented with 10% FBS.
5.2.2 MTT assay
A total of 5 × 103 HT-29, MCF-7, HeLa or HEK-293 cells in a total volume of 100 µL of their
respective growth media were incubated with serial dilutions of the tested compounds. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) dye reduction
assay in 96-well microplates was used, as previously described [26]. After 2 days of incubation (37
°C, 5% CO2 in a humid atmosphere), 10 µL of MTT (5 mg/mL in phosphate-buffered saline, PBS)
was added to each well, and the plate was incubated for a further 3 h (37 °C). The supernatant was
discarded and replaced by 100 µL of DMSO to dissolve formazan crystals. The absorbance was
then read at 490 nm by spectrophotometry. For all concentrations of compound, cell viability was
expressed as the percentage of the ratio between the mean absorbance of treated cells and the mean

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absorbance of untreated cells. Three independent experiments were performed, and the IC50 values
(i.e., concentration half inhibiting cell proliferation) were graphically determined.
5.2.3 Apoptosis assay
Apoptosis was determined by quantifying FITC-Annexin V translocation by means of flow
cytometry. HT-29 cells were incubated with compounds for 48 h and then stained following
instructions of BD Apoptosis DetectionTM Kit. Analysis was performed with a BD AccuriTM C6
flow cytometer.
5.2.4. RT-qPCR Assay
HT-29 cells at 70–80% confluence were collected and 1.5 x 105 cells were placed in a six well
plate in 1.5 mL of medium. After 24h, cells were incubated with the corresponding compounds for
48 h. Cells were collected and the total cellular RNA from HT-29 cells was isolated using Ambion
RNA extraction Kit according to the manufacturer’s instructions. The cDNA was synthesized by
MMLV-RT with 1–21 µg of extracted RNA and oligo(dT)15 according to the manufacturer’s
instructions.
Genes were amplified by use of a thermal cycler and StepOnePlus ™ Taqman ® probes. TaqMan ®
Gene Expression Master Mix Fast containing the appropriate buffer for the amplification
conditions, dNTPs, thermostable DNA polymerase enzyme and a passive reference probe was used.
To amplify each of the genes the predesigned primers were used and sold by Life Technologies
TaqMan ® Gene Expression Assays, Hs99999903-m1 (β-actin), Hs00972646-m1 (hTERT),
Hs00153408-m1 (c-Myc) and Hs009900055-m1 (VEGF-A).
5.2.5. Detection of total c-Myc and VEGFR-2 proteins by ELISA
HT-29 cells at 70–80% confluence were collected and 1.5 x 105 cells were placed in a six well
plate in 1.5 mL of medium. After 24h, cells were incubated with the corresponding compounds for
48 h. Cells were collected, washed twice in PBS and then cells were lysated with 200 µL of lysis
buffer. Buffer we used for the cell lysis consists of 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton™ X-100, 10%
glycerol, 0.1% SDS and 0.5% deoxycholate. Immediately before use, we added PMSF (0.3 M
stock in DMSO) to 1 mM and 50 µL/mL protease inhibitor cocktail (e.g., Sigma Cat. No. P-2714).
Protein concentrations of the cell lysates were determined by Bradford method and then we carried
out to a concentration of 200 µg/mL. Afterwards, we performed ELISA test according to the
manufacturer’s instructions. For determining total c-Myc, we used InvitrogenTM NovexTM c-Myc

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(Total) Human ELISA Kit de ThermoFisher® and total VEGFR-2 we used Sigma Human VEGFR-
2 ELISA test (ref. RAB0328).
5.2.6. Detection of secreted VEGF by ELISA
HT-29 cells at 70–80% confluence were collected and 1.5 x 105 cells were placed in a six well
plate in 1.5 mL of medium. After 24h, cells were incubated with the corresponding compounds for
48 h and then, cell culture media were collected. Protein concentrations of the cell media were
determined by Bradford method and then we carried out to a concentration of 200 µg/mL.
Afterwards, we performed ELISA test according to the manufacturer’s instructions. We used
InvitrogenTM NovexTM VEGF Human ELISA Kit de ThermoFisher.
Acknowledgments
This research has been funded by the Ministerio de Economía y Competitividad (project
CTQ2014-52949-P), by the Universitat Jaume I (project PI-1B2015-75) and by the Conselleria
d’Educaciò, Investigaciò, Cultura i Sport de la Generalitat Valenciana (project PROMETEO
2013/027). V. B. thanks the Conselleria d’Educaciò, Investigaciò, Cultura i Esport de la Generalitat
Valenciana for financial support. We thank Dr. Artur Bladé for a generous gift of colchicine.
Supplementary Information
Additional figures, graphics, experimental data, physical, IR, NMR and mass spectrometry data
of all new synthetic compounds are provided in the Supplementary Information.
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