Selenopheno[2,3-: F] coumarins: Novel scaffolds with antimetastatic activity against melanoma and breast cancer

Article abstract of DOI:10.1039/c9nj01682a

The general aim of the current research is to find novel non-toxic small molecules with antimetastatic activity. Herein we present a series of novel selenopheno[2,3-f]coumarins with low cytotoxicity profiles against cancer cells with different natures (carcinomas, sarcomas, melanomas, neuroblastomas, and hepatomas). Based on their structure-activity relationships (SARs), few compounds selectively induce MMP inhibition and suppress angiogenesis in vivo up to 57% (10 μM). Certain derivatives are excellent agents for in vivo prevention of melanoma and breast cancer metastasis: up to 97% with no visual side effects. The present study provides scientific evidence that a selenophenocoumarin scaffold is a promising competitor for the development of drug candidates with metastasis-suppressing properties.

Full text of DOI:10.1039/c9nj01682a

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Selenopheno[2,3-f]coumarins: a novel scaffold with
antimetastatic activity against melanoma and breast cancer
Pavel Arsenyan,*a Jelena Vasiljeva,a Ilona Domracheva,a Iveta Kanepe-Lapsa,a Anita Gulbea
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
General aim of the current research is to find novel non-toxic small molecules with antimetastatic activity. Herein we
present series of novel selenopheno[2,3-f]coumarins with low cytotoxic profile against cancer cells with different nature
(carcinomas, sarcomas, melanoma, neuroblastoma, and hepatoma). Based on SAR, few compounds selectively induce
MMP inhibition and suppress angiogenesis in vivo up to 57% (10µM). Certain derivatives are excellent agents for in vivo
prevention of melanoma and breast cancer metastasis: up to 97% with no visual side effects. The present study provides
scientific evidence that selenophenocoumarin scaffold is a promising competitor for the development of drug candidates
with metastasis-suppressing properties.
because of high medical need, there is a clear trend towards search
for new antimetastatic drugs. These drugs must block the
development of the metastases via elimination of distant cancer
Introduction
Cancer is the second leading cause of death globally, and is
responsible for an estimated 9.6 million deaths in 2018 (World
Health Organization data). Globally, about 1 in 6 deaths is due to
cells, but also by normalization of tissue microenvironment. At
present, after the successful surgical removal of the primary
tumour, the likelihood of recurrence is very high, since the tumour
can spread and metastasize to surrounding tissues and organs.
Metastasis begins with local invasion of tumour cells from the
primary tumour into the surrounding tissue and cells. Entrance into
blood or lymphatic vessels occurs as tumours further propagate
throughout the body. [6, 7] The 2H-chromene system is a structural
feature of many natural compounds and pharmacological agents
possessing, among other properties, anti-cancer, [8] anti-HIV [9]
and antibacterial activity [10]. Coumarin derivatives as medicines
with substantial activity in vitro and in vivo have been discovered
(Psoralen, Angelicin, Xanthotoxin, Bergapten, Nodakenetin, etc).
Imperatorin shows the ability to inhibit tumour growth. [11, 12]
Osthole inhibits the migration and invasion of breast cancer cells
and effectively blocks matrix metalloproteinases promoter and
enzyme activity. [13, 14]
cancer. Tumour growth is
a complex multistage process.
Occurrence and progressive tumour growth is dependent on both
the properties of cancer cells, and on the state of immunological
reactivity. This determines the diversity of approaches of cancer
therapy using one or several basic methods: surgery, radiotherapy,
chemotherapy and immunotherapy. Their goal is to minimize the
mass of the tumour. Complications caused by metastases account
for 90% of deaths from cancer. [1] There are several ways of
metastases of malignant tumours: lymphogenous, haematogenous
and mixed (through lymphatic, haematogenous or through
seeding). Lymphogenous pathway occurs through the lymphatic
system, where metastases that penetrate the lymphatic system can
then enter the blood stream. Malignant tumours of internal organs
(oesophagus, stomach, colon, larynx, cervix) often metastasize to
the lymph nodes in this way. Haematogenous path causes the
tumour cells to penetrate the blood vessels, and through the blood
flow are transferred to other organs (e.g. lungs, liver, bone). Most
deaths associated with this pathway are due to increased risk of the
spread of tumour cells from the bloodstream after surgical
intervention. Malignant tumours of the lymphatic and
Recently we claimed 2H-selenopheno[3,2-h]chromenes as
antimetastatic agents in treatment of carcinomas and melanoma.
These derivatives exhibit low acute toxicity and almost completely
(98%) prevent metastases formation of mammary carcinoma 4T1 in
vivo and melanoma B16-F10 metastasis in lung by 82% without any
visual side effects. [15]
hematopoietic
tissues
–
sarcoma,
hypernephroma,
horionepitelioma, metastasize this way. However, most cancers
(breast, thyroid, lungs and bronchi, ovaries) can equally metastasize
by lymphogenous and haematogenous mechanisms. [2-5] Recently,
O
R'
O
Se
R
Br
R
Br
O
O
a.Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006, Riga, Latvia
pavel@osi.lv
Se
O
O
R'
WO2018015788 (A1)
This work
Electronic Supplementary Information (ESI) available: 1H and 13C NMR copies of
compounds are presented in the Supplementary Information]. See
DOI: 10.1039/x0xx00000x
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Unfortunately, the number of cancer diagnosis in past few compounds in the corresponding alcohol under acidic
View Article Online
DOI: 10.10739/C9NJ01682A
and 8 were
years has increased almost twofold. In clinical practice, the conditions for 4-5 days. As a result, derivatives
treatment of cancer involves a wide range of chemotherapy isolated in 92% and 81%, respectively. Next, hydroxy groups of
drugs. Besides, most of them exhibit various side effects, high 4b and were converted to triflates 9-11 in purpose to run
toxicity and moderate selectivity. Therefore, a new generation Sonogashira cross-coupling reactions. The introduction of C
of selective anticancer agents with low toxicity is one of the triple bond was performed at 60 using
oC
,
7
8

C
main tasks for medicinal chemistry and pharmaceutical bis(triphenylphosphino)palladium(II) chloride and copper(I)
industries. In continuation of our research, [16-19] herein we iodide as catalysts (Scheme 1, g). Thus, alkynyl coumarins 14a-
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would like to report our studies in the field of searching for
antimetastatic drug candidate in series of selenopheno[2,3- were obtained in higher yields (39-86%) from methyl 6-
f]coumarins.
d were isolated in 35-63% yields. Identical products 14b-d
iodocoumarin-3-carboxylate (13, Scheme 1, f). In this case, the
reaction was performed at lower temperature, however,
previously reported 14a [21] was obtained in 57% yield from
the corresponding bromide 5 (Scheme 1, g). Isopropyl 17a-b
and tert-butyl 18a-b esters of ethynyl coumarins were
synthesized according to f conditions in reverse sequence,
Results and discussion
Synthesis
6-Alkynylcoumarins 14a-d
,
15a-b
,
16a-b
,
17a-b, and 18a-b namely, alkynyl derivatives 12a-b were obtained from 5-
were synthesized in few steps using 5-hydroxy, 5-bromo or 5- bromo-2-hydroxybenzaldehyde
iodo salicylic aldehydes 1-3 as starting materials (Scheme 1). condensation reaction with disopropyl and di(tert)butyl
Methyl 6-hydroxy and 6-bromocoumarin-3-carboxylates 4a malonates (Scheme 1, h, 48-56%). 3-Acyl-6-ethynyl coumarins
2
(41-89%) followed by
and 4b were obtained by the reaction of
1
and
2
,
were easily prepared by the treatment of 12a,b with
correspondingly, with methyl malonate in the presence of few isobutyrylacetic acid methyl ester or methyl 3-oxoheptanoate
drops of piperidine. [20] Then methyl esters were converted to (Scheme 1, i) at r.t.
octyl and decyl analogues
7
and 8 by refluxing the initial
COOR1
HO
COOR1
O
R
R
b
a
O
from 4a
OH
1 (R=OH)
2 (R=Br)
3 (R=I)
O
O
O
4a (R=OH, R1=Me)
4b (R=Br, R1=Me)
5 (R=I, R1=i-Pr)
7 (R1=Oct)
8 (R1=Dec)
6 (R=I, R1=t-Bu)
c
R = Br
d
TfO
COOR1
c
e
R2
R1 = Me
R = Br
O
O
9 (R1=Me)
10 (R1=Oct)
11 (R1=Dec)
O
I
COOMe
OH
12a (R2=C(Me)2OH)
12b (R2=CH2N(CH2)5)
O
O
g
13
R2
COOR1
i
f
R2
O
h
O
O
R3
16a (R1=t-Bu, R2=C(Me)2OH)
16b (R1=t-Bu, R3=CH2N(CH2)5)
17a (R1=Oct, R2=C(Me)2OH)
17b (R1=Oct, R3=CH2N(CH2)5)
18a (R1=Dec, R2=C(Me)2OH)
18b (R1=Dec, R3=CH2N(CH2)5)
14a (R1=Me, R2=C(Me)2OH)
14b (R1=Me, R3=CH2N(CH2)5)
O
O
14c (R1=Me, R3=CH2N(CH2CH2)2O)
19a (R2=C(Me)2OH, R3=i-Pr)
14d (R1=Me, R3=CH2N(CH2CH2)NMe)
15a (R1=i-Pr, R2=C(Me)2OH)
19b (R2=CH2N(CH2)5, R3=i-Pr)
20a (R2=C(Me)2OH, R3=Bu)
20b (R2=CH2N(CH2)5, R3=Bu)
15b (R1=i-Pr, R3=CH2N(CH2)5)
2 | J. Name., 2012, 00, 1-3
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Scheme 1. The synthesis of ethynylcoumarins. Reagents and reaction conditions:
a
: R1OOCCH2COOR1 (2 equiv.), MeOH,
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piperidine (2-4 drops), 60 oC, 24 h.;
b: SiMe3Cl (4 mL), octanol or decanol (20 mL), reflux, 4-5 days.; c
: Tf2O D(3OIe: q10u.1iv0.3)9,/DCC9NMJ,01E6t832NA,
o
48 h.;
CuI (1.2 equiv.), DMEDA (1.0 equiv.), 1,4-dioxane, 120 oC, 48 h.;
mol%), CuI (10 mol%), DMF, Et3N, 40 °C, overnight. 9-11 (1 equiv.), terminal alkyne (1.5 equiv.), CuI (15 mol%), PdCl2 (10
mol%), and PPh3 (20 mol%) in DMF, Et3N, 60 oC for 18 h.
12a,b (1 equiv.), diisopropyl malonate (1.5 equiv.) or
di(tert)butylmalonate (1.5 equiv.), MeOH, 2 drops of piperidine, r.t., 24h. 12a,b (1 equiv.) and isobutyrylacetic acid methyl
d: terminal acetylene (1.5 equiv.), PdCl2(PPh3)2 (7mol%), CuI (7 mol%), THF/ET3N = 1/1, 60 C, overnight.;
e: KI (5 equiv.),
f:
5-6, 13 (1 equiv.), terminal alkyne (1.5 equiv.), PdCl2(PPh3)2 (10
g:
h:
i
:
ester (1.4 equiv.) or methyl 3-oxoheptanoate (1.4 equiv.) MeOH, 2 drops of piperidine, r.t., 24h.
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11
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R2
R2
R2
O
O
O
Se
O
O
Se
b or c
a
R3
Br
Br
R3
OH
O
O
O
O
14a-d, 15a-b, 16a-b, 17a-b,
18a-b, 19a-b, 20a-b
36-39
21-35
OH
OH
OH
O
N
MeN
N
N
O
O
Se
O
O
O
O
Se
Se
O
O
Se
Br
Br
Br
Br
Br
Br
OMe
OMe
OMe
OMe
O
O
O
O
23 (65%)
24 (62%)
22 (60%)
21 [21]
OH
N
N
O
O
Se
O
Se
O
Se
O
O
Se
Br
Br
Br
O
O
O
O
O
O
O
O
O
O
28 (42%)
26 (58%)
27 (36%)
25 (38%)
OH
N
N
O
O
Se
O
Se
O
Se
O
Se
Br
Br
Br
OOct
OOct
ODec
ODec
O
O
O
O
O
O
O
30 (32%)
29 (92%)
32 (45%)
31 (89%)
OH
OH
N
O
O
Se
O
O
Se
O
O
Se
Br
Br
Br
O
O
O
35 (68%)
34 (66%)
33 (41%)
OH
O
N
MeN
N
N
O
O
Se
O
O
O
Se
Se
O
O
Se
Br
Br
Br
Br
OH
OH
OH
OH
O
O
O
O
O
38 (92%)
39 (99%)
37 (88%)
36 (85%)
o
Scheme 2. The synthesis of selenophenocoumarins. Reagents and reaction conditions:
a: SeO2 (2 equiv.), HBr, 1,4-dioxane, 0 C
to r.t., 24-48 h. : NaOH (6 equiv.), water, r.t.;
b
c
: HepG2 (human hepatocellular carcinoma) cells lysate, 37 oC.
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DOI: 10.1039/C9NJ01682A
MMP, in vitro and in vivo angiogenesis
Next, we used previously developed method for the synthesis inhibition
of selenophene moiety, which is based on treatment of It is well known that the metastatic process is a multi-step
ethynyl-substituted arenes and hetarenes [16] with in situ phenomenon by which cells in the primary tumour invade
prepared selenium(IV) bromide.
A solution of ethynyl surrounding tissues, penetrate blood and lymphatic circulation
coumarins 14–20 in 1,4-dioxane was treated with SeBr4, and propagate into the organ parenchyma, proliferating to
formed in situ from SeO2 and concentrated hydrobromic acid form metastatic colonies at secondary sites. Cancer invasion
(48%) at r.t. Selenophenocoumarins 21-35 were prepared in takes place when the cancer cells respond and migrate
excellent or satisfactory yields (32–95%). In the reaction of 6- towards gradients of stimuli, for example, growth factors
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(3-hydroxy-3-methylbut-1-ynyl)-3-pentanoyl-2H-chromen-2-
require proteolysis of basement membrane (BM), and
one (19a) with SeBr4, besides cyclization reaction, bromination extracellular matrix (ECM) proteins. It creates a path for
of pentanoyl substituent proceeded at α-CH2 position with the migration. Matrix metalloproteinases (MMPs) are a group of
formation of bromo-substituted compound, which can be enzymes responsible for the proteolysis of BM and ECM
easily debrominated through refluxing with equimolar amount proteins. The expression level of MMPs appears to correlate
of N-acetyl guanidine and sodium acetate yielding 35. Methyl with the invasiveness of cancer cells. [24] MMPs, also
selenopheno[2,3-f]coumarin
carboxylates
21-24
were designated matrixins, hydrolyse components of the
hydrolyzed to their corresponding acids 36-39 by stirring 21-24 extracellular matrix. These proteinases play important role in
in methanol with an excess of aqueous 2N sodium hydroxide different biological processes, such as embryogenesis, normal
solution at r.t. overnight. It should be noted that this step is tissue remodelling, wound healing, angiogenesis, as well as in
slow; besides, temperature elevation led to destruction of diseases such as atheroma, arthritis, cancer, and tissue
coumarin cycle. However, hydrolysis of methyl ester by ulceration. [25] Despite their promising abilities [e.g.
incubation with HepG2 (human hepatocellular carcinoma) cells marimastat (BB-2516)], most rationally designed MMP
lysate at 37 oC was complete in 4h.
inhibitors show a broad-spectrum MMP inhibition. Besides,
cipemastat (Ro 32-3555), MMP-1 selective inhibitor, have
performed poorly in clinical trials. Development of selective
and non-toxic MMP inhibitors still lies in uncovered areas of
Biological evaluation
Cytotoxicity
In vitro cytotoxicity caused by novel selenium analogues was anti-cancer medicine.
tested on 10 monolayer tumour cell lines: MDA-MB-435s Novel selenopheno[f]coumarins 21-39 passed cytotoxicity test
(human melanoma), MCF-7 (human breast adenocarcinoma, showing medium toxicity. Our next goal was to study their
estrogen-positive), MES-SA (human uterus sarcoma), HT-1080 MMP inhibition ability. Inhibition of matrix metalloproteinase
(human connective tissue fibrosarcoma), A549 (human lung enzymes by 21-39 was detected on MMP Inhibitor Fluorimetric
carcinoma), SH-SY5Y (human neuroblastoma), CCL-8 (mouse Profiling kit (Biomol, USA) using NNGH (N-isobutyl-N-4-
sarcoma), 3T3 (Mouse Swiss Albino embryo fibroblasts), MH- methoxyphenylsulfonyl)glycyl-hydroxamic acid) as a standard
22A (mouse hepatoma), HepG2 (human hepatocellular (Table 2). It was found that selenopheno[f]coumarins 21-23,
carcinoma), B16-F10 (mouse melanoma). Using the NIH cell 36 and 37 inhibit up to 75% of MMP2 activity. It occurred that
line, the borderline concentration which is relevant to the MMP8 is sensitive to methyl ester of piperidylmethyl
highest tolerated dose, was determined for each compound. derivative 22, while hydrolysis of ester group led to a
The basal cytotoxicity was used to predict starting doses for in remarkable increase of MMP13 inhibition (61%). Derivatives
vivo acute oral LD50 values in rodents. [22, 23] The results of 21-23, 36 and 37 were chosen for angiogenesis inhibition
these experiments are summarized in Table 1. In the search for studies in vitro and in vivo according to MMPs inhibition data.
a non-toxic antimetastatic drug candidate only derivatives with In vitro experiments were performed on Matrigel (BD
slight cytotoxic effect can be chosen for the further studies. In Biosciences) human umbilical vein endothelial cell line
general, all novel selenopheno[f]coumarins 21
exceptions, in vitro showed medium to low ability to suppress showed good activity in inhibiting angiogenesis process in vitro
cancer cells’ growth. Derivatization of coumarin carboxylic (49-100% in 20 concentration), except for
group (31) with decyl chain led to a remarkable increase of morpholylmethyl derivative 23. Then in vivo studies of
antiproliferative activity on metastatic mouse melanoma B16- angiogenesis were conducted in mice treated with 21 22 36
F10 cell line (IC50=8 M). In addition, human neuroblastoma and 37. The angiogenesis in vivo model was based on the use
-39, with few (HUVECs) tube formation model (Table 2). Chosen compounds
M
,
,

SH-SY5Y and B16-F10 were sensitive to 3-bromo-2- of Matrigel implants in Balb/c AnNCrl mice (20 g, 4 weeks of
(morpholinomethyl)-7-oxo-7H-selenopheno[2,3-f]chromene-8- age) and VEGF-induced vessel formation inside the implants.
carboxylic acid (38) (IC50=3÷8

M).
Derivative 21 suppresses angiogenesis by 57%, however its
carboxylic acid 36 showed loss in activity (-10%).
Table 1. In vitro cytotoxicity caused by selenopheno[f]coumarins was tested on monolayer tumour cell lines: MDA-MB-435s (human melanoma), MCF-7
(human breast adenocarcinoma, estrogen-positive), MES-SA (human uterus sarcoma), HT-1080 (human connective tissue fibrosarcoma), A549 (human lung
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carcinoma), SH-SY5Y (human neuroblastoma), CCL-8 (mouse sarcoma), 3T3 (Mouse Swiss Albino embryo fibroblasts), MH-22A (mouse hepatoma), HepG2
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(human hepatocellular carcinoma), B16-F10 (mouse melanoma).
DOI: 10.1039/C9NJ01682A
Nr.
Estimated LD50
Cytotoxicity IC50, M
6
7
M/kg
mg/kg
8
9
21
22
23
24
176±6
110±5
ne
219±6
52±2
ne
ne
46±2
ne
108±6
91±3
ne
153±6
83±2
>206
81±2
50±2
>206
72±2
43±6
ne
153±6
79±2
ne
86±2
72±4
ne
164±2
136±6
ne
108±20
20±1
63±10
3200
1900
>4200
1421
918
>2000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
48±2
40±2
36±2
44±5
66±3
38±2
36±2
70±2
52±4
74±2
60±1
1700
847
25
27
28
29
31
32
33
36
37
38
39
98±2
105±16
ne
76±11
19±2
>154
126±5
ne
ne
59±6
58±10
ne
65±2
30±2
ne
120±11
70±4
ne
32±6
35±6
110±11
64±4
ne
54±2
132±8
ne
62±1
45±4
>190
59±4
81±7
154±8
76±4
232±5
12±1
8±1
108±12
51±16
190±8
79±13
42±11
46±12
76±1
86±2
117±2
ne
35±2
120±9
84±8
>190
163±14
107±16
ne
51±3
111±4
ne
100±6
32±4
>154
10±1
>232
ne
79±8
43±10
>195
48±7
8±1
>154
51±10
84±16
138±22
3±0
1800
1000
5000
1000
1000
3000
5800
2500
>4000
2700
1600
839
476
2467
587
97±16
137±27
133±20
107±25
ne
46±2
26±2
>154
13±2
193±2
109±9
>197
42±2
70±4
37±4
154±6
34±2
232±12
198±3
167±5
63±2
142±13
137±5
39±5
113±8
139±5
198±6
130±2
67±3
68±6
60±2
154±5
46±6
70±3
89±2
>197
50±2
671
2061
2644
1075
>2000
1371
833
ne
228±3
63±5
ne
85±4
ne
49±5
118±2
71±5
38±2
36±1
>192
ne- no effect
Table 2. Matrix metalloproteinases, in vitro and in vivo angiogenesis inhibition caused by 21 – 39
Inhibition of MMP subtypes, %
Nr.
Angiogenesis inhibition, %
in vitro,**
in vivo,***
NNGH*
21
22
23
24
25
27
28
29
31
32
33
36
37
38
39
100
100
100
7.2
100
100
100
100
100
100
23±3
21±2
16±2
17±1
13±1
16±3
22±5
21±2
22±1
20±1
12±5
21±3
8±1
43±3
56±3
45±3
11±2
11±5
0±1
2±4
3±3
7±2
10±2
3±2
14±3
16±2
9±1
14±2
14±3
11±3
15±2
0±7
17±1
30±4
13±2
20±2
0±2
16±8
18±1
23±2
24±6
25±4
4±1
19±6
22±2
18±3
18±3
3±2
15±2
24±3
15±2
15±2
9±4
13±8
17±4
20±2
20±1
22±1
13±3
19±3
21±1
13±1
7±2
20±1
19±2
19±2
16±3
5±2
13±3
14±2
7±2
11±3
3±1
25±3
27±3
20±3
19±2
12±7
14±3
11±5
15±2
22±3
14±6
12±4
28±2
33±2
22±1
15±5
92±11
49±11
ne
57±19
-51±39
21±3
12±1
15±4
11±4
21±4
16±2
26±3
12±2
21±3
22±2
11±2
14±1
8±1
9±4
8±3
8±5
10±4
8±2
12±8
14±2
14±6
15±3
19±4
10±1
19±1
61±5
4±2
11±2
15±5
16±2
6±7
17±3
14±3
11±3
8±1
12±2
12±3
13±3
9±2
17±1
15±4
13±2
9±2
17±4
16±1
16±2
10±2
19±5
17±2
12±3
13±4
44±3
75±4
7±1
18±2
18±2
16±3
15±4
100±1
73±11
-10±7
-37±5
14±1
9±1
17±1
4±1
* N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (1.3 μM);
** In vitro inhibition of HUVEC tube formation (compound dose is 20 M);
*** In vivo inhibition of vessel formation into Matrigel implant (compound dose is 10 M).
ne- no effect.
In vivo studies
catalogue, CCL-6475) is a highly metastatic murine melanoma
There are various in vivo experimental models for studying cell line from a C57BL/6J mouse. The cells are adherent to an
growth and metastasis of different tumours after epithelial morphology. 4T1 is a transplantable tumour cell line
transplantation. The injection site and the tumour tropism in that is highly tumorigenic and invasive. Unlike most tumour
the selected cell line were mainly determined by the primary models, it can spontaneously metastasize from the primary
and secondary metastases and their growth. Pulmonary tumour in the mammary gland to multiple distant sites
metastases model is widely used to assess the treatment of including lymph nodes, blood, liver, lungs, brain, and bones
various in vivo tumour models. B16F10 Mice melanoma and (American Type Culture Collection (ATCC) catalogue no CRL-
4T1 mammary carcinoma serve as models for highly metastatic 2539, 2004).
melanoma and stage IV breast cancer. B16F10 (ATCC
This journal is © The Royal Society of Chemistry 20xx
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9
Due to many of obvious reasons, those animal studies in Figure 2C. The tumours in the control animals develop a
could not be conducted for all selenopheno[f]coumarins; 167% tumour spleen weight increase. Notably, spleen volume
View Article Online
DOI: 10.1039/C9NJ01682A
derivatives 21
compounds, as they had already demonstrated MMP inhibiting observed in treatment with 21
activity (see above). One of the most aggressive forms of skin Additionally, we tested two most active compounds 21 and
, 22, 23, 36, 37 were picked as representative is significantly smaller and only 73% weight increase is
.
cancer – melanoma B16-F10 model was selected for 37 for their ability to induce haemolysis of rat erythrocytes
intravenous administration in the tail vein. [26] (Figure 1D). Widely used detergent Triton x100 was used as
C57BL/6JOlaHsd 6-weeks old female mice in cohorts of 6-8 positive control. According to received results, both
animals were injected with 100,000 B16-F10 melanoma cells compounds are not harmful to red blood cells in
10
11
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16
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60
via the tail vein and then treated with 21
and 20 mg/kg doses s.c. according to the following scheme: were haemolysed at extra high concentration (>300
Day 1, 7, 8, 9, 10, 11, 14, 15, 16th. Total dose reached 120 case of selenophenocoumarin 21. To determine the possible
mg/kg. On day 18 experiments were terminated (Table 3). mechanism of action of 21 and 37, which causes the death of
Indeed, after the treatment with 21 (dose 5 mg/kg) in cohorts tumour cells, we tested the effect of 21 and 37 on cell
of 7 mice, 4 animals had no signs of metastasis nodules in apoptosis. Determination of apoptotic and necrotic cells was
lungs with overall metastasis inhibition by 85%. Therapeutic performed using AO/EB (Figure 1, E-H). An increase in number
dose increase to 20 mg/kg led to impressive 96% of metastasis of apoptotic cells was observed in case of 21 at a dose of 100
prevention (p=0.002). In vitro hydrolysis of ester group in μM after only 24h. The ratio of apoptotic cells reached 21%,
derivative 21, which was detected during incubation of 21 in while the number of necrotic cells did not increase. In the case
HepG2 cells lysate at 37 oC for 4h, resulted in slight decrease of of compound 37, the appearance of apoptotic cells plateaued
the antimetastatic activity till 85% (compound 36) Again, no at 27% after 6h.
,
22
,
23
,
36
,
37 in 5 concentrations less than 50

M. Moreover, only 10% of cells
M) in the
signs of metastatic nodules were observed in two mice lungs
out of six in cohort. Experiments with 22 and 23 in 5 mg/kg
dose resulted with modest effect of inhibition (44% and 17%,
Conclusions
correspondingly). However, hydrolysis of the ester group in
derivative 22 to carboxylic acid (37) considerably improved
selenopheno[2,3-f]coumarins that are variously modified in
prevention of metastasis development. Compound 37
suppresses metastasis by 60% in 5 mg/kg dose. Notably, 4
elaborated and series of 14 new derivatives was obtained.
mice out of 8 had no metastasis nodules at all. Increase of
dose to 20 mg/kg led to almost complete prevention of
metastasis formation (97%, p=0.002).
To sum up, methods for the preparation of
position 3 of coumarin ring and selenophene moiety were
Based on structure-activity relationship data it can be
concluded that these compounds exhibit medium to low in
vitro cytotoxicity. Notably, few compounds suppress
Next, compound 21 was studied in BALB/c female mice
angiogenesis in vivo up to 57% (10µM). Exploring the
cohorts, consisting of five animals each, with innoculated 4T1
intracellular processes in tumour cells allows us to conclude
tumours (Figure 1B). Compound 21 in 20 mg/kg dose
that their low toxicities and potent antiproliferating activities
depend on ability to induce apoptosis. The most impressive
data received from in vivo experiments confirming scientific
(administration: s.c. according to the following scheme: day 1,
4, 7, 9, 11 and 14th; total dose 120 mg/kg dose) exhibited
ability to suppress 4T1 metastasis up to 97 % after 18 days.
evidence that selenophenocoumarin scaffold is promising
Described effect was observed for each mouse that
trend for the development of drug candidates with metastasis-
participated in the experiment. Those results were particularly
suppressing properties. It was found that certain derivatives
are excellent for in vivo prevention of melanoma and breast
cancer metastasis development up to 97% with no visual side
encouraging, bearing in mind that this compound did not
induce any major side effects either: all animals treated with
this compound stayed healthy and active, weight loss was
effects. Our next task will relate to determination of
virtually non-existent. Transplantation of breast cancer tumour
mechanism of action.
cells to mice developed splenomegaly, which is associated with
the induction of tumour leukemoid reaction and massive
granulocytic infiltrates of the red pulp. [27–29] The results of
spleen weight changes in the animals with tumour are shown
6 | J. Name., 2012, 00, 1-3
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60
Figure 1. A: Melanoma B16F10 antimetastatic activity caused by 21
– 23, 36 and 37 (5 and 20 mg/kg doses s.c. according to the
following scheme: Day 1, 7, 8, 9, 10, 11 and 14th; total dose 120 mg/kg, the number of macroscopic lung metastases was
determined 18 days later); B and C: inhibition of 4T1 metastasis in lung as well as spleen weight in BALB/c mice (number of lung
metastasis nodules at day 18 is shown for each mouse); D: haemolysis of rat erythrocytes caused by 21 and 37; E–F: the effects
of 21 and 37 on morphology of B16-F1. Cells were stained by acridine orange/ethidium bromide and observed
under fluorescence microscope: (E) Control; (F) in the presence of 21 after 6h incubation; (G) in the presence of 21
after 24h incubation; (H) in the presence of 37 after 6h incubation (×200); I and J: Number of apoptotic and necrotic
cells after incubation with 21 and 37
.
Table 3. The number of individual metastatic nodules in whole lung assessed at endpoint. N=6-8 mice per group. Points
represent individual animals and bars represent the mean number of metastatic nodules (B16F10) and percent of total lung area
occupied with metastases (4T1).
N1
N2
N3
N4 N5
N6
N7
N8
Average ± SD
Inhibition %
TTEST
B16F10
Control
5
50
0
34
0
8
50
40
2
68
0
50
0
75
43±26
6.4±15
1.7±3.2
24±30
35±37
12±20
6.3±7.0
17±30
1.2±2.0
21, 5 mg/kg
21, 20 mg/kg
22, 5 mg/kg
23, 5 mg/kg
36, 5 mg/kg
36, 20 mg/kg
37, 5 mg/kg
37, 20 mg/kg
3
2
85
96
44
17
72
85
60
97
0.006
0.002
0.202
0.668
0.023
0.006
0.091
0.002
0
0
0
8
0
10
85
0
50
40
0
30
3
85
0
2
0
5
0
0
9
0
80
28
4
40
50
0
0
5
11
3
5
18
2
0
80
2
0
0
0
50
0
5
0
0
0
4T1
Control
42
24
76
26
61
46±22
1±1
21, 20 mg/kg
1
3
0
2
0
97
0.002
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DOI: 10.1039/C9NJ01682A
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ARTICLE
9
Tert-butyl 6-iodo-coumarin-3-carboxylate (6
). Yield: 86%. 1H
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NMR (400 MHz, DMSO-d6) δ (ppm): 1.53 (9H, s, CH3), 7.25 (1H,
d, J = 8.8 Hz, 8-CH), 7.98 (1H, dd, J = 8.8, 2.0 Hz, 7-CH), 8.29
(1H, d, J = 2.0 Hz, 5-CH), 8.59 (1H, s, 4-CH).
Experimental
General remarks
General procedure for the re-esterification of methyl 6-
hydroxycoumarin-3-carboxylate (4) to the corresponding octyl
Unless otherwise stated, all reagents were purchased from
commercial suppliers and used without further purification.
Thin layer chromatography (TLC) was performed using MERCK
Silica gel 60 F254 plates and visualized by UV (254 nm)
fluorescence. ZEOCHEM silica gel (ZEOprep 60/35-70 microns –
SI23501) was used for column chromatography. 1H and 13C
NMR spectra were recorded on a Varian 400 Mercury
spectrometer at 400.0 and 100.58 MHz correspondingly at 298
K in CDCl3 or DMSO-d6. The melting points were determined
on a “Digital melting point analyser” (Fisher), and the results
are given without correction.
and decyl esters 7-8: Methyl 6-hydroxycoumarin-3-carboxylate
4a (5 g, 22.72 mmol) was suspended in octanol or decanol (20
mL), then chlorotrimethylsilane (4 mL) was added and vial was
closed. The reaction mixture was heated at 120 oC for 4-5 days.
Then the mixture was cooled and petroleum ether (100 mL)
was added. Precipitates were filtered off, washed with
petroleum ether and dried to give pure esters 7 and 8.
1
Octyl 6-hydroxycoumarin-3-carboxylate (7) Yield: 92%. H NMR
(400 MHz, CDCl3/TMS) δ (ppm): 0.86 (3H, t, J = 6.8 Hz, CH3),
1.20-1.45 (10H, m, CH2), 1.76 (2H, qui, J = 14.0, 6.8 Hz, CH2),
4.33 (2H, t, J = 6.8 Hz, OCH2), 7.12 (1H, d, J = 2.4 Hz, 8-CH),
7.21-7.27 (2H, m, 5,7-CH), 8.46 (1H, s, 4-CH). 13C NMR (100.6
MHz, CDCl3/TMS) δ (ppm): 14.0, 22.6, 25.9, 28.5, 29.1, 29.2,
31.7, 66.3, 113.7, 117.7, 118.0, 118.3, 123.3, 148.7, 149.2,
153.2, 157.7, 163.3.
Synthesis of initial compounds
General procedure for the preparation of methyl 6-hydroxy- or
6-bromo-coumarin-3-carboxylate (4a-b) [20]: to 2,5-
dihydroxybenzaldehyde (4 g, 29 mmol) or 5-bromo-2-
hydroxybenzaldehyde (4 g, 19.9 mmol) and dimethyl malonate
(5.78 g, 43 mmol) solution in dry methanol (20 mL) 3 drops of
piperidine was added. The reaction mixture was heated at 60
oC for 24h. The mixture was then cooled to -15 oC and
precipitates were filtered off, washed with cold methanol (50
mL) and dried to give pure methyl 6-hydroxycoumarin-3-
carboxylate 4a in 84% yield or 6-bromocoumarin-3-carboxylate
Decyl 6-hydroxycoumarin-3-carboxylate (8) Yield: 81%. 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 0.82 (3H, t, J = 6.6 Hz,
CH3), 1.21-1.41 (14H, m, CH2), 1.66 (2H, qui, J = 14.0, 6.6 Hz,
CH2), 4.22 (2H, t, J = 6.6 Hz, OCH2), 7.13-7.29 (3H, m, 5,7,8-CH),
8.63 (1H, s, 4-CH). 13C NMR (100.6 MHz, DMSO-d6) δ (ppm):
13.8, 22.0, 25.2, 27.9, 28.5, 28.6, 28.81, 28.83, 31.2, 65.0,
113.7, 117.7, 118.2, 118.3, 122.5, 147.8, 148.3, 153.9, 156.2,
162.8.
1
4b in 81% yield. H NMR (4a) (400 MHz, DMSO-d6) δ (ppm):
3.82 (3H, s, CH3), 7.15-7.29 (3H, m, Ar), 8.69 (1H, s, 5-CH), 9.89
(1H, s, OH).
General procedure for the preparation of isopropyl
5 or tert-
General procedure for the preparation of alkyl 6-
butyl 6-iodo-coumarin-3-carboxylates (6): to a solution of 2-
hydroxy-5-iodo-benzaldehyde (2 g, 8 mmol) and di-isopropyl
malonate (3.01 g, 16 mmol) or di-tert-butyl malonate (3.45 g,
16 mmol) in dry methanol (12 mL) 3 drops of piperidine was
added. The reaction mixture was stirred at r.t for 24h. The
mixture was then cooled to -15 oC and precipitates were
filtered off, washed with cold methanol (50 mL) and dried to
(((trifluoromethyl)sulfonyl)oxy)coumarin-3-carboxylates 9-11
:
solution of alkyl 6-hydroxycoumarin-3-carboxylate ( 7-8) (45
4
,
mmol) in dry dichloromethane (100 mL) and Et3N (10 mL) was
o
cooled to 0 C, and then trifluoromethanesulfonic anhydride
(15.11 mL, 90 mmol) was added dropwise. After 24h of stirring
at rt mixture was cooled to 0 oC and additional portion of
trifluoromethanesulfonic anhydride (7.5 mL, 45 mmol) was
added dropwise and reaction was continued for 24h. Then
mixture was cooled to 0 oC and 1N HCl (100 mL) and
dichloromethane (100 mL) were added. Organic phase was
separated and washed with brine (100 mL), dried over sodium
sulphate. After removal of the solvent under reduced pressure,
the crude product 9-11 was purified by flash chromatography
on silica gel using the mixture of petroleum
ether/dichloromethane as an eluent.
give pure isopropyl 6-iodo-coumarin-3-carboxylate (
butyl 6-iodo-coumarin-3-carboxylate ( ) in 82-86% yield.
5) or tert-
6
Isopropyl 6-iodo-coumarin-3-carboxylate (5
). Yield: 82%. 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 1.38 (6H, d, J = 6.2 Hz,
CH3), 5.23-5.30 (1H, m, CH), 7.28 (1H, d, J = 8.6 Hz, 8-CH), 7.63-
7.66 (2H, m, 5,7-CH), 8.37 (1H, s, 4-CH).
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15 min through a dry 1,4-dioxane solution (15 mLV)ieowfAcrtoicplepOenrli(nIe)
6-(((trifluoromethyl)sulfonyl)oxy)coumarin-3- iodide (4.04 g, 21.20 mmol), grounded potassium iodide (14.67
DOI: 10.1039/C9NJ01682A
Methyl
1
carboxylate (9). Yield: 56%. H NMR (400 MHz, CDCl3/TMS) δ g, 88.35 mmol). Then subsequently DMEDA (1.9 mL, 17.67
(ppm): 3.91 (3H, s, CH3), 7.42 (1H, d, J = 9.0 Hz, 8-CH), 7.52 (1H, mmol) and after 5 min a solution of methyl 6-bromocoumarin-
dd, J = 2.8 Hz, 9.0 Hz, 7-CH), 7.58 (1H, d, J = 2.8 Hz, 5-CH), 8.53 3-carboxylate (4b) (5.00 g, 17.67 mmol) in 1,4-dioxane (15 mL)
o
(1H, s, 4-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 52.9, were added. The reaction mixture was heated at 120 C for 48
118.6, 118.8, 119.7, 120.1, 121.6, 126.9, 145.1, 147.2, 153.9, h. The mixture was then cooled to rt, poured into ethyl acetate
155.4, 162.7.
(200 ml); the organic layer was washed with saturated
ammonium chloride solution (2x100 mL), brine (100 mL),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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Octyl 6-(((trifluoromethyl)sulfonyl)oxy)coumarin-3-carboxylate water (100 mL) and dried over sodium sulphate. After removal
(10) Yield: 68% Foam. 1H NMR (400 MHz, CDCl3/TMS) δ (ppm): of the volatiles under reduced pressure, the crude product was
1
0.86 (3H, t, J = 6.8 Hz, CH3), 1.22-1.45 (10H, m, CH2), 1.76 (2H, purified by washing with diethyl ether. Yield: 3.4 g (58%). H
qui, J = 14.0, 6.8 Hz, CH2), 4.34 (2H, t, J = 6.8 Hz, OCH2), 7.42- NMR (400 MHz, DMSO-d6) δ (ppm): 3.83 (3H, s, CH3), 7.24 (1H,
7.45 (1H, m, 8-CH), 7.51-7.57 (2H, m, 5,7-CH), 8.46 (1H, s, 4- d, J = 8.6 Hz, 8-CH), 7.87 (1H, dd, J = 8.6, 2.0 Hz, 7-CH), 8.28
CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 14.0, 22.6, (1H, d, J = 2.0 Hz, 5-CH), 8.69 (1H, s, 4-CH). 13C NMR (100.6
25.8, 28.5, 29.0, 29.1, 31.7, 66.4, 117.0, 118.7, 118.8, 120.3, MHz, DMSO-d6) δ (ppm): 52.5, 88.3, 116.2, 118.3, 119.5, 119.9,
121.6, 126.8, 145.1, 146.5, 153.9, 155.4, 162.3.
136.6, 137.9, 147.5, 155.4, 162.8.
Decyl 6-(((trifluoromethyl)sulfonyl)oxy)coumarin-3-carboxylate General procedures for the preparation of methyl (14a-d),
1
(11) Yield: 71% H NMR (400 MHz, CDCl3/TMS) δ (ppm): 0.87 isopropyl (15a-b), tert-butyl (16a-b) octyl (17a-b), and decyl
(3H, t, J = 6.8 Hz, CH3), 1.23-1.46 (14H, m, CH2), 1.77 (2H, qui, J
= 13.9, 6.8 Hz, CH2), 4.35 (2H, t, J = 6.6 Hz, OCH2), 7.43-7.46
(
18a-b) 6-(alkynyl)-coumarin-3-carboxylates.
(1H, m, 8-CH), 7.51-7.56 (2H, m, 5,7-CH), 8.46 (1H, s, 4-CH). 13
C
f: A solution of 6-iodocoumarin 5-6, or 13 (2.0 mmol) and
NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 14.1, 22.6, 25.8, 28.5, terminal alkyne (3.0 mmol) in dry DMF (10 mL) was added to a
29.2, 29.3, 29.4, 29.5, 31.9, 66.5, 118.7, 118.9, 120.4, 121.5, solution of bis(triphenylphosphine)palladium(II) chloride (0.07
126.8, 145.2, 146.5, 153.9, 155.4, 162.3.
g, 0.1 mmol) and copper(I) iodide (0.019 g, 0.1 mmol) in dry
DMF (2 mL) and Et3N (2 mL). Then reaction mixture was
General procedure for the preparation of 2-hydroxy-5-(alkynyl)- heated at 40 °C overnight. After consumption of initial 5-6, or
benzaldehydes 12a-b): solution of 5-bromo-2- 13 the mixture was poured into ethyl acetate (100 mL). The
(
A
hydroxybenzaldehyde 4b (1 g, 5 mmol) and terminal alkyne organic layer was washed with water and dried over sodium
(7.5 mmol) in dry THF (8 mL) was added to a solution of sulphate. After removal of the solvent under reduced pressure
bis(triphenylphosphine)palladium(II) chloride (0.245 g, 0.35 the crude product was purified by flash chromatography on
mmol) and copper(I) iodide (0.67 g, 0.35 mmol) in mixture of silica gel using mixture of DCM/MeOH as eluent for 14b-d
,
THF/Et3N (7 mL/15 mL). The reaction mixture obtained was 15b
,
16b; and DCM/EtOAc for 16a
.
heated to 60 °C overnight. The mixture was then poured into
ethyl acetate (150 mL); the organic layer was washed with
g: Argon gas was bubbled through a mixture of copper(I)
water and dried over sodium sulphate. After removal of the iodide (32 mg, 0.17 mmol), palladium(II) chloride (20 mg, 0.11
solvent under reduced pressure, the crude product was mmol) and triphenylphosphine (58 mg, 0.22 mmol) in
purified by flash chromatography on silica gel.
DMF/Et3N (8mL/4mL) for 10 min at 40 oC. Then solution of
corresponding 6-(((trifluoromethyl)sulfonyl)oxy)coumarin-3-
2-Hydroxy-(hydroxy-3-methylbutyn-1-yl)-benzaldehyde (12a) carboxylate (9-11) (1.11 mmol) followed by terminal alkyne in
1
Yield: 89%. H NMR (400 MHz, CDCl3/TMS) δ (ppm): 1.62 (6H, dry DMF (12 mL) were added. The reaction mixture was
o
s, CH3), 1.97 (1H, s, OH), 6.94 (1H, d, J = 8.6 Hz, 3-CH), 7.56 (1H, heated at 60 C for 18 h. The mixture was then cooled to rt,
dd, J = 8.6, 2.1 Hz, 4-CH), 7.65 (1H, d, J = 2.1 Hz, 6-CH), 9.86 poured into ethyl acetate (200 ml); the organic layer was
(1H, s, CHO), 11.08 (1H, s, OH).
washed with saturated ammonium chloride solution (1x100
mL), brine (3x100 mL), water (100 mL) and dried over sodium
(12b) sulphate. After removal of the volatiles under reduced
2-Hydroxy-(3-piperid-1-yl-prop-1-ynyl)-benzaldehyde
1
Yield: 41%. H NMR (400 Hz, CDCl3/TMS) δ (ppm): 1.44-1.48 pressure, the crude product was purified by flash
(2H, m, CH2), 1.61-1.67 (4H, m, CH2), 2.52-2.60 (4H, m, CH2), chromatography on silica gel using mixture of DCM/MeOH for
3.45 (2H, s, CH2), 6.92 (1H, d, J = 8.6 Hz, 3-CH), 7.55 (1H, dd, J = 14b-d, 15b, 16b, DCM/EtOAc for 16a, petroleum ether/EtOAc
8.6, 2.1 Hz, 4-CH), 7.64 (1H, d, J = 2.1 Hz, 6-CH), 9.84 (1H, s, for 17a, 18a, petroleum ether/DCM/EtOAc 17b, 18b – petrol
CHO). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 23.9, 25.9, ether/DCM/EtOAc (10/1/2) for 17b and 18b
.
48.4, 53.5, 83.1, 84.6, 115.2, 117.9, 120.3, 136.9, 139.9, 161.2,
196.0.
h: To a solution of 2-hydroxy-(alkynyl)-benzaldehyde 12a,b
(2.94 mmol) and diisopropyl malonate (0.83 g, 4.41 mmol) or
General procedure for the preparation of methyl 6- di(tert)butylmalonate in dry methanol (10 mL) 2 drops of
iodocoumarin-3-carboxylate (13): Argon gas was bubbled for piperidine was added. The reaction mixture was stirred at rt
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for 24h, the mixture was then cooled to -15 oC and precipitates 65.5, 79.9, 83.1, 94.9, 116.8, 117.8, 119.6, 120.4, V1ie3w2A.1rti,cl1e3O6nl.i7ne,
were filtered off, washed with cold methanol (25 mL) and 146.6, 154.3, 156.3, 161.7.
DOI: 10.1039/C9NJ01682A
dried to give pure products 15a,b and 16a,b in 48-56% yields.
Tert-butyl ester of 6-(3-piperid-1-yl-prop-1-ynyl)coumarin-3-
Methyl ester of 6-(3-hydroxy-3-methylbutyn-1-yl)coumarin-3- carboxylic acid (16b) Yield: 48% (f), 55% (h
). 1H NMR (400 MHz,
carboxylic acid (14a) [Ref. 21]. Yield: 38% (
f
), 57% (
g
).
CDCl3/TMS) δ (ppm): 1.42-1.49 (2H, m, piperidine), 1.59 (9H, s,
3CH3), 1.61-1.67 (4H, m, piperidine), 2.53-2.58 (4H, m,
Methyl ester of 6-(3-piperid-1-yl-prop-1-ynyl)coumarin-3- piperidine), 3.47 (2H, s, CH2), 7.24-7.27 (1H, m, 8-CH), 7.62-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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58
59
60
o
1
carboxylic acid (14b) Yield: 74% (f), 63% (g
), m.p. = 150 C. H 7.64 (2H, m, 5,7-CH), 8.31 (1H, s, 4-CH). 13C NMR (100.6 MHz,
NMR (400 MHz, CDCl3/TMS) δ (ppm): 1.44-1.47 (2H, m, CDCl3/TMS) δ (ppm): 23.8, 25.9, 28.0, 48.4, 53.6, 82.7, 83.0,
CH2CH2CH2), 1.61-1.67 (4H, m, NCH2CH2), 2.52-2.57 (4H, m, 86.5, 116.8, 117.8, 120.1, 120.3, 132.1, 136.9, 146.6, 154.2,
NCH2), 3.46 (2H, s, CH2), 3.94 (3H, s, CH3), 7.27 (1H, d, J = 9.2 156.3, 161.7.
Hz, 8-CH), 7.64-7.67 (2H, m, 5-CH, 7-CH), 8.47 (1H, s, 4-CH). 13
C
NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 23.8, 25.8, 48.4, 52.9, Octyl ester of 6-(3-hydroxy-3-methylbutyn-1-yl)coumarin-3-
53.6, 82.6, 86.8, 116.9, 117.7, 118.5, 120.4, 132.3, 137.4, carboxylic acid (17a) Yield: 47% (
g
). m.p. = 145-146 oC. 1H NMR
148.3, 154.4, 156.2, 163.4.
(400 MHz, CDCl3/TMS) δ (ppm): 0.88 (3H, t, J = 6.8 Hz, CH3),
1.25-1.46 (10H, m, 5CH2), 1.63 (6H, s, 2CH3), 1.77 (2H, qui, J =
Methyl ester of 6-(3-morphol-4-yl-prop-1-ynyl)coumarin-3- 13.8 Hz, CH2), 2.04 (1H, br s, OH), 4.34 (2H, t, J = 6.8 Hz, CH2),
o
1
carboxylic acid (14c) Yield: 86% (f), 54% (g), m.p. = 185 C. H 7.28 (1H, d, J = 8.4 Hz, 8-CH), 7.63-7.65 (2H, m, 5,7-CH), 8.42
NMR (400 MHz, CDCl3/TMS) δ (ppm): 2.64 (4H, t, J = 4.8 Hz, (1H, s, 4-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 14.1,
CH2NCH2), 3.51 (2H, s, CH2), 3.77 (4H, t, J = 4.8 Hz, CH2OCH2), 22.6, 25.9, 28.5, 29.1, 29.2, 31.4, 31.8, 65.6, 66.3, 79.9, 95.0,
3.95 (3H, s, CH3), 7.29 (1H, d, J = 9.2 Hz, 8-CH), 7.65-7.68 (2H, 116.9, 117.8, 119.1, 119.8, 132.3, 137.1, 147.6, 154.5, 156.1,
m, 5,7-CH), 8.48 (1H, s, 4-CH). 13C NMR (100.6 MHz, 162.9.
CDCl3/TMS) δ (ppm): 47.9, 52.5, 53.0, 66.8, 83.2, 85.7, 116.9,
117.7, 118.7, 120.1, 132.3, 137.4, 148.2, 154.5, 156.1, 163.4.
Octyl ester of 6-(3-piperid-1-yl-prop-1-ynyl)coumarin-3-
1
carboxylic acid (17b) Yield: 32% (
6-(3-(4-methylpiperaz-1-yl)-prop-1- CDCl3/TMS) δ (ppm): 0.87 (3H, t, J = 6.8 Hz, CH3), 1.24-1.49
ynyl)coumarin-3-carboxylic acid (14d) Yield: 39% ( ), 35% ( ), (12H, m, 5CH2, 2H-piperidine), 1.62-1.67 (4H, m, piperidine),
g). Foam. H NMR (400 MHz,
Methyl
ester
of
f
g
o
1
m.p. = 140 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm): 2.29 1.76 (2H, qui, J = 13.8 Hz, CH2), 2.54-2.58 (4H, m, piperidine),
(3H, s, NCH3), 2.45-2.55 (4H, m, CH2NCH3), 2.61-2.69 (4H, m, 3.47 (2H, s, CH2), 4.33 (2H, t, J = 6.8 Hz, CH2), 7.26-7.29 (1H, m,
NCH2CH2), 3.51 (2H, s, CH2), 3.92 (3H, s, CH3), 7.25 (1H, d, J = 8-CH), 7.64-7.67 (2H, m, 5,7-CH), 8.42 (1H, s, 4-CH). 13C NMR
9.2 Hz, 8-CH), 7.61-7.64 (2H, m, 5-CH, 7-CH), 8.44 (1H, s, 4-CH). (100.6 MHz, CDCl3/TMS) δ (ppm): 14.1, 22.6, 23.8, 25.9, 28.5,
13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 45.8, 47.5, 51.9, 29.1, 29.2, 31.7, 48.4, 53.6, 66.2, 82.7, 86.6, 116.9, 117.7,
52.9, 54.8, 83.0, 85.9, 116.9, 117.7, 118.5, 120.1, 132.4, 137.3, 119.0, 120.3, 132.3, 137.2, 147.7, 154.4, 156.2, 162.9.
148.2, 154.4, 156.1, 163.4.
Decyl ester of 6-(3-hydroxy-3-methylbutyn-1-yl)coumarin-3-
o
Isopropyl ester of 6-(3-hydroxy-3-methylbutyn-1-yl)coumarin-3- carboxylic acid (18a) Yield: 45% (
g
). m.p. = 87-88 C. 1H NMR
carboxylic acid (15a) Yield: 50% ( ), 56% (
f
h
). 1H NMR (400 MHz, (400 MHz, CDCl3/TMS) δ (ppm): 0.87 (3H, t, J = 6.8 Hz, CH3),
CDCl3/TMS) δ (ppm): 1.39 (6H, d, J = 6.2 Hz, CH3), 1.63 (6H, s, 1.25-1.44 (14H, m, 7CH2), 1.63 (6H, s, 2CH3), 1.76 (2H, qui, J =
CH3), 2.02 (1H, s, OH), 5.22-5.31 (1H, m, CH), 7.28 (1H, d, J = 13.7 Hz, CH2), 2.08 (1H, br s, OH), 4.34 (2H, t, J = 6.8 Hz, CH2),
8.6 Hz, 8-CH), 7.63 (1H, dd, J = 8.6, 1.9 Hz, 7-CH), 7.65 (1H, d, J 7.28 (1H, d, J = 8.4 Hz, 8-CH), 7.62-7.66 (2H, m, 5,7-CH), 8.42
= 1.9 Hz, 5-CH), 8.38 (1H, s, 4-CH).
(1H, s, 4-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 14.1,
22.6, 25.8, 28.5, 29.2, 29.3, 29.4, 29.5, 31.4, 31.8, 65.5, 66.3,
Isopropyl ester of 6-(3-piperid-1-yl-prop-1-ynyl)coumarin-3- 79.9, 95.0, 116.9, 117.8, 119.1, 119.7, 132.3, 137.1, 147.6,
carboxylic acid (15b) Yield: 44% ( ), 48% (
f
h
). 1H NMR (400 MHz, 154.5, 156.1, 162.9.
DMSO-d6) δ (ppm): 1.31 (6H, d, J = 6.2 Hz, CH3), 1.35-1.46 (1H,
m, piperidine), 1.70-1.86 (5H, m, piperidine), 2.95-3.06 (2H, m, Decyl ester of 6-(3-piperid-1-yl-prop-1-ynyl)coumarin-3-
o
1
piperidine), 3.48-3.55 (2H, m, piperidine), 4.32 (2H, s, CH2), carboxylic acid (18b) Yield: 40% (g). m.p. = 83-84 C. H NMR
5.08-5.15 (1H, m, CH), 7.49 (1H, d, J = 8.7 Hz, 8-CH), 7.86 (1H, (400 MHz, CDCl3/TMS) δ (ppm): 0.87 (3H, t, J = 6.8 Hz, CH3),
dd, J = 8.7, 2.0 Hz, 7-CH), 8.14 (1H, d, J = 2.0 Hz, 5-CH), 8.69 1.26-1.48 (16H, m, 7CH2, 2H-piperidine), 1.62-1.68 (4H, m,
(1H, s, 4-CH).
piperidine), 1.76 (2H, qui, J = 13.7 Hz, CH2), 2.53-2.59 (4H, m,
piperidine), 3.47 (2H, s, CH2), 4.33 (2H, t, J = 6.8 Hz, CH2), 7.27-
Tert-butyl ester of 6-(3-hydroxy-3-methylbutyn-1-yl)coumarin- 7.29 (1H, m, 8-CH), 7.64-7.67 (2H, m, 5,7-CH), 8.42 (1H, s, 4-
1
3-carboxylic acid (16a) Yield: 42% (f), 49% (h
). H NMR (400 CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 14.1, 22.6,
MHz, CDCl3/TMS) δ (ppm): 1.59 (9H, s, CH3), 1.62 (6H, s, CH3), 23.9, 25.8, 25.9, 28.5, 29.2, 29.3, 29.4, 29.5, 31.8, 48.4, 53.6,
7.23-7.26 (1H, m, 8-CH), 7.59-7.62 (m, 2H, 5,7-CH), 8.29 (s, 1H, 66.2, 82.7, 86.7, 116.9, 117.8, 119.0, 120.3, 132.3, 137.2,
4-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 28.0, 31.3, 147.6, 154.4, 156.2, 162.9.
10 | J. Name., 2012, 00, 1-3
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ethyl acetate. The organic phase was washed with brine, dried
: General procedure for the preparation of alkynyl ketones over anhydrous Na2SO4, filtered, concentrated, and the
19a,b and 20a,b. To 2-hydroxy-(alkynyl)-benzaldehyde 12a,b residue was purified by flash chromatography on silica gel.
(2.94 mmol) and isobutyrylacetic acid methyl ester (0.59 g,
4.11 mmol) or methyl 3-oxoheptanoate (0.65 g, 4.11 mmol) Methyl 3-bromo-2-(2-hydroxypropan-2-yl)-7-oxo-7H-
View Article Online
DOI: 10.1039/C9NJ01682A
i
solution in dry methanol (10 mL) 2 drops of piperidine was selenopheno[2,3-f]chromene-8-carboxylate (21)
[Ref. 21]
added. The reaction mixture was stirred at rt for 24h. The HRMS (ESI) calcd for C16H13BrNO4Se [M+H] 444.9184, found
mixture was then cooled to -15 oC and precipitates were 444.9194. HPLC: 96.2% (RT=8.34 min, Apollo C18-8
filtered off, washed with cold methanol (25 mL) and dried to (4.6x150mm), mobile phase 5→95% Acetonitrile+0.1%H3PO4, 1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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42
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47
48
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51
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53
54
55
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58
59
60
give pure products 19a,b and 20a,b in 46-61% yield.
mL/min, 40 oC).
6-(3-Hydroxy-3-methylbut-1-ynyl)-3-isobutyryl-2H-chromen-2-
Methyl 3-bromo-7-oxo-2-(piperidin-1-ylmethyl)-7H-
1
one (19a) Yield: 45%. H NMR (400 MHz, CDCl3/TMS) δ (ppm): selenopheno[2,3-f]chromene-8-carboxylate (22) Yield: 60%,
o
1
1.17 (6H, d, J = 6.8 Hz, CH3), 1.62 (6H, s, CH3), 1.97 (1H, s, OH), m.p. = 160 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm): 1.47-
3.75-3.87 (1H, m, CH), 7.29 (1H, d, J = 8.6 Hz, 8-CH), 7.63 (1H, 1.50 (2H, m, piperidine), 1.59-1.64 (4H, m, piperidine), 2.56-
dd, J =8.6, 1.9 Hz, 7-CH), 7.66 (1H, d, J = 1.9 Hz, 5-CH), 8.34 (1H, 2.61 (4H, m, piperidine), 3.76 (2H, s, CH2), 3.97 (3H, s, CH3),
s, 4-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 18.2, 31.3, 7.33 (1H, d, J = 9.0 Hz, 5-CH), 7.91 (1H, d, J = 9.0 Hz, 4-CH), 8.58
38.6, 65.5, 79.8, 95.1, 116.7, 118.2, 119.9, 125.3, 132.7, 136.9, (1H, s, 9-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 23.9,
146.7, 154.4, 158.3, 202.1.
26.0, 52.9, 55.1, 59.2, 104.7, 114.7, 114.9, 117.4, 129.7, 138.1,
139.9, 146.4, 147.4, 153.4, 156.5, 163.5. LC-MS m/z 484 [M+1].
3-Isobutyryl-6-(3-(piperidin-1-yl)prop-1-ynyl)-2H-chromen-2one HRMS (ESI) calcd for C19H18BrNO4Se [M+H] 483.9657, found
(19b) Yield: 43%. H NMR (400 MHz, DMSO-d6) δ (ppm): 1.09 483.9661.
1
(6H, d, J = 6.8 Hz, CH3), 1.32-1.41 (2H, m, CH2), 1.50-1.56 (2H,
m, CH2), 2.46-2.52 (4H, m, CH2), 3.48 (2H, s, CH2), 3.57-3.68 Methyl 3-bromo-2-(morpholinomethyl)-7-oxo-7H-
(1H, m, CH), 7.45 (1H, d, J = 8.6 Hz, 8-CH), 7.74 (1H, dd, J = 8.6, selenopheno[2,3-f]chromene-8-carboxylate (23) Yield: 65%,
o
1
1.9 Hz, 7-CH), 8.03 (1H, d, J = 1.9 Hz, 5-CH), 8.58 (1H, s, 4-CH). m.p. = 197-199 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm):
13C NMR (100.6 MHz, DMSO-d6) δ (ppm): 17.9, 23.5, 25.4, 37.7, 2.68 (4H, t, J = 4.6 Hz CH2N), 3.76 (4H, t, J = 4.6 Hz OCH2), 3.86
47.4, 52.6, 82.5, 86.9, 116.7, 118.4, 119.0, 125.2, 132.9, 136.5, (2H, s, CH2), 2.56-2.61 (4H, m, piperidine), 3.99 (3H, s, CH3),
146.8, 153.8, 157.7, 201.8.
7.39 (1H, d, J = 8.9 Hz, 5-CH), 7.98 (1H, d, J = 8.9 Hz, 4-CH), 8.63
(1H, s, 9-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 53.0,
54.0, 58.9, 67.0, 106.0, 115.0, 117.7, 130.0, 138.0, 139.9,
6-(3-Hydroxy-3-methylbut-1-ynyl)-3-pentanoyl-2H-chromen-2-
one (20a) Yield: 61%. 1H NMR (400 MHz, CDCl3/TMS) δ 145.0, 146.4, 153.7, 156.5, 163.5. LC-MS m/z 486 [M]. HRMS
(ppm): 0.94 (3H, t, J = 7.4 Hz, CH3), 1.35-1.44 (2H, m, CH2), 1.63 (ESI) calcd for C18H16BrNO5Se [M+H] 485.9450, found
(6H, s, CH3), 1.63-1.71 (2H, m, CH2), 2.01 (1H, s, OH), 3.12 (2H, 485.9460.
t, J = 7.4 Hz, CH2), 7.30 (1H, d, J = 8.6 Hz, 8-CH), 7.64 (1H, dd, J
= 8.6, 1.9 Hz, 7-CH), 7.69 (1H, d, J = 1.9 Hz, 5-CH), 8.39 (1H, s, Methyl 3-bromo-2-((4-methylpiperazin-1-yl)methyl)-7oxo-7H-
4-CH).
selenopheno[2,3-f]chromene-8-carboxylate (24) Yield: 62%,
o
1
m.p. = 174-176 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm):
3-Pentanoyl-6-(3-(piperidin-1-yl)prop-1-ynyl)-2H-chromen-2-
2.33 (3H, s, CH3), 2.41-2.77 (8H, m, piperazine), 3.85 (2H, s,
one (20b) Yield: 34%. 1H NMR (400 MHz, CDCl3/TMS) δ (ppm): CH2), 3.99 (3H, s, CH3), 7.39 (1H, d, J = 8.9 Hz, 5-CH), 7.98 (1H,
0.93 (3H, t, J = 7.2 Hz, CH2), 1.33-1.43 (2H, m, CH2-piperidine), d, J = 8.9 Hz, 4-CH), 8.64 (1H, s, 9-CH). 13C NMR (100.6 MHz,
1.42-1.50 (2H, m, CH2), 1.61-1.69 (6H, m, CH2, CH2-piperidine), CDCl3/TMS) δ (ppm): 45.9, 53.0, 53.6, 55.1, 58.4, 105.5, 114.9,
2.53-2.58 (4H, m, CH2- piperidine), 3.11 (2H, t, J = 7.2 Hz, CH2), 115.0, 117.6, 130.0, 138.1, 139.9, 146.1, 146.5, 153.6, 156.5,
3.47 (2H, s, CH2), 7.28 (1H, d, J = 8.6 Hz, 8-CH), 7.65 (1H, dd, J = 163.6. LC-MS m/z 499 [M+1]. HRMS (ESI) calcd for
8.6, 1.9 Hz, 7-CH), 7.69 (1H, d, J = 1.9 Hz, 5-CH), 8.39 (1H, s, 4- C19H19BrN2O4Se [M+H] 498.9766, found 498.9779.
CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 13.9, 22.3,
23.8, 25.8, 25.9, 42.2, 48.3, 53.6, 82.7, 86.6, 116.7, 118.2, Isopropyl 3-bromo-2-(2-hydroxypropan-2-yl)-7-oxo-7H-
120.4, 125.3, 132.9, 137.1, 146.5, 154.4, 158.6, 197.9.
selenopheno[2,3-f]chromene-8-carboxylate (25) Yield: 38%,
o
1
m.p. = 194-196 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm):
1.42 (6H, d, J = 6.2 Hz, CH3CHCH3), 1.85 (6H, s, CH3), 2.78 (1H,
Synthesis of selenopheno[2,3-f]coumarins (21-35)
General procedure for the preparation of selenopheno[2,3- br s, OH), 5.26-5.32 (1H, m, CH), 7.37 (1H, d, J = 8.9 Hz, 5-CH),
f]coumarins (21-35): 6-Alkynylcoumarin 14-20 (1.0 mmol) in 7.97 (1H, d, J = 8.9 Hz, 4-CH), 8.52 (1H, s, 9-CH). 13C NMR (100.6
dioxane was added to a solution of in situ prepared SeBr4 from MHz, CDCl3/TMS) δ (ppm): 21.8, 29.1, 70.1, 74.3, 101.4, 114.6,
o
selenium dioxide (0.22 g, 2.0 mmol) in HBr (2 mL) at 0 C. The 115.0, 118.6, 130.3, 138.3, 139.1, 145.3, 153.6, 154.7, 156.5,
resulting mixture was stirred at rt for 24–48h. After the 162.5. LC-MS m/z 472 [M]. HRMS (ESI) calcd for C18H17BrO5Se
consumption of substrate (LC-MS monitoring), the reaction [M+H] 472.9497, found 472.9517. HPLC: 99.4% (RT=10.93 min,
mixture was basified with aqueous Na2CO3 and extracted with
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Apollo C18-8 (4.6x150mm), mobile phase 5→95% 1H NMR (400 MHz, CDCl3/TMS) δ (ppm): 0.85-0.90 (1H, m,
View Article Online
Acetonitrile+0.1%H3PO4, 1 mL/min, 40 oC).
piperidine), 0.88 (3H, t, J = 6.6 Hz, CH3D),OI1:.1205.1-013.95/4C9(N1J20H16,8m2A,
CH2), 1.60-1.65 (4H, m, piperidine), 1.76-1.84 (2H, m, CH2),
2.58-2.62 (4H, m, piperidine), 3.79 (2H, br s, CH2), 4.37 (2H, t, J
Isopropyl 3-bromo-7-oxo-2-piperidin-1-ylmethyl)-7H-
selenopheno[2,3-f]chromene-8-carboxylate (26) Yield: 58%, = 6.6 Hz, CH2), 7.38 (1H, d, J = 8.8 Hz, 5-CH), 7.97 (1H, d, J = 8.8
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m.p. = 174-176 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm): Hz, 4-CH), 8.59 (1H, s, 9-CH). 13C NMR (100.6 MHz, CDCl3/TMS)
1.42 (6H, d, J = 6.2 Hz, CH3CHCH3), 1.47-1.53 (2H, m, δ (ppm): 14.1, 22.6, 24.0, 25.9, 26.1, 28.6, 29.17, 29.20, 31.8,
piperidine), 1.60-1.66 (4H, m, piperidine), 2.58-2.61 (4H, m, 55.1, 59.3, 66.3, 72.4, 114.8, 115.0, 118.1, 129.7, 145.9, 153.5,
piperidine), 3.78 (2H, s, CH2), 5.26-5.32 (1H, m, CH), 7.37 (1H, 156.5, 163.1. LC-MS m/z 582 [M+1]. HRMS (ESI) calcd for
d, J = 8.8 Hz, 5-CH), 7.95 (1H, d, J = 8.8 Hz, 4-CH), 8.52 (1H, s, 9- C26H32BrO4Se [M+H] 582.0753, found 582.0773.
CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 21.8, 24.0,
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26.1, 55.1, 59.3, 69.9, 104.9, 114.8, 115.0, 118.5, 129.5, 138.1, Decyl 3-bromo-2-(2-hydroxypropan-2-yl)-7-oxo-7H-
139.8, 145.4, 147.0, 153.4, 156.5, 162.4. LC-MS m/z 512 [M+1]. selenopheno[2,3-f]chromene-8-carboxylate (31) Yield: 89%,
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HRMS (ESI) calcd for C21H22BrNO4Se [M+H] 511.9970, found m.p. = 105-106 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm):
511.9982. HPLC: 97.9% (RT=3.28 min, Apollo C18-8 0.87 (3H, t, J = 6.8 Hz, CH3), 1.26-1.48 (14H, m, CH2), 1.76-1.83
(4.6x150mm), mobile phase 5→95% Acetonitrile+0.1%H3PO4, 1 (2H, m, CH2), 1.86 (6H, s, CH3), 2.66 (1H, br s, OH), 4.38 (2H, t, J
mL/min, 40 oC).
= 6.8 Hz, CH2), 7.40 (1H, d, J = 8.8 Hz, 5-CH), 7.99 (1H, d, J = 8.8
Hz, 4-CH), 8.59 (1H, s, 9-CH). 13C NMR (100.6 MHz, CDCl3/TMS)
δ (ppm): 14.1, 22.6, 25.8, 28.5, 29.0, 29.2, 29.3, 29.48, 29.51,
Tert-butyl 3-bromo-2-(2-hydroxypropan-2-yl)-7-oxo-7H-
selenopheno[2,3-f]chromene-8-carboxylate (27) Yield: 36%, 31.0, 31.8, 66.4, 74.3, 101.4, 114.6, 114.9, 118.1, 130.4, 138.3,
foam. 1H NMR (400 MHz, CDCl3/TMS) δ (ppm): 1.62 (9H, s, 139.1, 145.7, 153.6, 154.9, 156.5, 163.2. LC-MS m/z 571 [M+1].
CH3), 1.85 (6H, s, CH3), 2.78 (1H, br s, OH), 7.32 (1H, d, J = 8.8 HRMS (ESI) calcd for C25H31BrO5Se [M+H] 571.0593, found
Hz, 5-CH), 7.92 (1H, d, J = 8.8 Hz, 4-CH), 8.44 (1H, s, 9-CH). 13
C
571.0589. HPLC: 97.7% (RT=12.17 min, Apollo C18-8
NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 28.1, 29.0, 74.2, 83.2, (4.6x150mm), mobile phase 5→95% Acetonitrile+0.1%H3PO4, 1
101.3, 114.6, 114.9, 119.5, 130.0, 138.1, 139.0, 144.7, 153.4, mL/min, 40 oC).
154.8, 156.7, 162. LC-MS m/z 487 [M+1]. HRMS (ESI) calcd for
C19H19BrO5Se [M+H] 486.9654, found 486.9492.
Decyl 3-bromo-7-oxo-2-piperidin-1-ylmethyl)-7H-
selenopheno[2,3-f]chromene-8-carboxylate hydrochloride (32)
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Tert-butyl 3-bromo-7-oxo-2-piperidin-1-ylmethyl)-7H-
Yield: 45%, m.p. = 149-150 C. H NMR (400 MHz, CDCl3/TMS)
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selenopheno[2,3-f]chromene-8-carboxylate (28) Yield: 42%. H δ (ppm): 0.85 (3H, t, J = 7.0 Hz, CH3), 1.19-1.52 (16H, m, CH2),
NMR (400 MHz, CDCl3/TMS) δ (ppm): 1.46-1.51 (2H, m, 1.76-1.83 (2H, m, CH2), 1.90-1.97 (2H, m, piperidine), 2.15-2.25
piperidine), 1.59-1.65 (4H, m, piperidine), 1.63 (9H, s, CH3), (2H, m, piperidine), 2.77-2.82 (2H, m, piperidine), 3.57-3.62
2.56-2.60 (4H, m, piperidine), 3.77 (2H, s, CH2), 7.35 (1H, d, J = (2H, m, piperidine), 4.36 (2H, t, J = 7.0 Hz, CH2), 4.63 (2H, br s,
8.8 Hz, 5-CH), 7.92 (1H, d, J = 8.8 Hz, 4-CH), 8.45 (1H, s, 9-CH). CH2), 7.42 (1H, d, J = 8.8 Hz, 5-CH), 8.03 (1H, d, J = 8.8 Hz, 4-
13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 24.0, 26.0, 28.1, CH), 8.57 (1H, s, 9-CH), 12.6 (0.5H, br s, HCl). 13C NMR (100.6
55.1, 59.2, 83.1, 114.7, 115.0, 119.5, 129.3, 137.9, 139.6, MHz, CDCl3/TMS) δ (ppm): 14.0, 22.0, 22.6, 22.9, 25.8, 28.5,
144.8, 146.8, 156.6, 162.0. LC-MS m/z 526 [M+1]. HRMS (ESI) 29.19, 29.24, 29.45, 29.49, 31.8, 52.8, 56.2, 66.4, 114.9, 115.9,
calcd for C22H24BrNO4Se [M+H] 526.0127, found 526.0143. 116.0, 118.8, 128.9, 131.0, 135.4, 142.9, 144.7, 154.6, 155.9,
HPLC: 98.3% (RT=9.70 min, Apollo C18-8 (4.6x150mm), mobile 162.1. LC-MS m/z 610 [M+1]. HRMS (ESI) calcd for
phase 5→95% Acetonitrile+0.1%H3PO4, 1 mL/min, 40 oC).
C28H36BrO4Se [M+H] 610.1066, found 610.1075.
Octyl
3-bromo-2-(2-hydroxypropan-2-yl)-7-oxo-7H- 3-Bromo-2-(2-hydroxypropan-2-yl)-8-isobutyryl-7H-
selenopheno[2,3-f]chromene-8-carboxylate (29) Yield: 92%, selenopheno[2,3-f]chromen-7-one (33) Yield: 41%, m.p. > 200
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m.p. = 119-120 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm): oC. 1H NMR (400 MHz, CDCl3/TMS) δ (ppm): 1.21 (6H, d, J = 6.8
0.88 (3H, t, J = 6.8 Hz, CH3), 1.25-1.48 (10H, m, CH2), 1.76-1.83 Hz, CH3), 1.85 (6H, s, CH3), 2.71 (1H, br s, OH), 3.85-3.93 (1H,
(2H, m, CH2), 1.85 (6H, s, CH3), 2.72 (1H, br s, OH), 4.37 (2H, t, J m, CH), 7.42 (1H, d, J = 8.9 Hz, 5-CH), 7.99 (1H, d, J = 8.9 Hz, 4-
= 6.8 Hz, CH2), 7.39 (1H, d, J = 9.0 Hz, 5-CH), 7.99 (1H, d, J = 9.0 CH), 8.56 (1H, s, 9-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ
Hz, 4-CH), 8.58 (1H, s, 9-CH). 13C NMR (100.6 MHz, CDCl3/TMS) (ppm): 18.3, 29.1, 38.6, 74.3, 101.4, 114.9, 115.2, 124.3, 130.3,
δ (ppm): 14.1, 22.6, 25.9, 28.5, 29.10, 29.16, 29.19, 31.8, 66.4, 138.9, 139.2, 145.0, 153.7, 154.8, 158.7, 202.2. LC-MS m/z 457
74.3, 101.5, 114.6, 115.0, 118.2, 130.5, 138.4, 139.1, 145.8, [M+1]. HRMS (ESI) calcd for C18H17BrO4Se [M+H] 456.9548,
153.7, 154.7, 156.5, 163.2. LC-MS m/z 543 [M+1]. HRMS (ESI) found 456.9561.
calcd for C23H27BrO5Se [M+H] 543.0280, found 543.0286. HPLC:
96.4% (RT=9.93 min, Apollo C18-8 (4.6x150mm), mobile phase 3-Bromo-8-isobutyryl-2-(piperidin-1-ylmethyl)-7H-
5→95% Acetonitrile+0.1%H3PO4, 1 mL/min, 40 oC).
selenopheno[2,3-f]chromen-7-one (34) Yield: 66%, m.p. = 160-
162 oC. 1H NMR (400 MHz, CDCl3/TMS) δ (ppm): 1.20 (6H, d, J =
6.8 Hz, CH3), 1.47-1.51 (2H, m, piperidine), 1.59-1.65 (4H, m,
Octyl 3-bromo-7-oxo-2-piperidin-1-ylmethyl)-7H-
selenopheno[2,3-f]chromene-8-carboxylate (30) Yield: 32%, oil. piperidine), 2.58-2.62 (4H, m, piperidine), 3.77 (2H, s, CH2),
12 | J. Name., 2012, 00, 1-3
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3.84-3.91 (1H, m, CH), 7.36 (1H, d, J = 8.8 Hz, 5-CH), 7.93 (1H,
View Article Online
d, J = 8.8 Hz, 4-CH), 8.51 (1H, s, 9-CH). 13C NMR (100.6 MHz, 3-bromo-2-(piperidin-1-ylmethyl)-7-oxo-7DHO-sI:e1l0e.m10o39p/hCe9nNoJ[0216,38-2A
CDCl3/TMS) δ (ppm): 18.3, 23.9, 26.1, 38.5, 55.1, 59.2, 114.5, f]chromene-8-carboxylic acid (37) Yield: 88%, m.p. > 200 C. 1H
o
115.5, 124.2, 129.4, 138.1, 140.4, 145.0, 153.4, 158.7, 202.1. NMR (400 MHz, DMSO-d6) δ (ppm): 1.48-1.63 (2H, m,
LC-MS m/z 496 [M+1]. HRMS (ESI) calcd for C21H22BrNO3Se piperidine), 1.76-1.79 (4H, m, piperidine), 2.99-3.20 (4H, m,
[M+H] 495.9948, found 496.0017. HPLC: 96.4% (RT=3.93 min, piperidine), 4.56 (2H, br s, CH2), 7.59 (1H, d, J = 8.8 Hz, 5-CH),
Apollo C18-8 (4.6x150mm), mobile phase 5→95% 8.06 (1H, d, J = 8.8 Hz, 4-CH), 8.74 (1H, s, 9-CH). 13C NMR
Acetonitrile+0.1%H3PO4, 1 mL/min, 40 oC).
(100.6 MHz, DMSO-d6) δ (ppm): 21.5, 22.8, 51.9, 114.8, 115.6,
119.3, 130.1, 135.5, 144.4, 153.5, 156.4, 163.8. LC-MS m/z 470
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3-Bromo-2-(2-hydroxypropan-2-yl)-8-(2-bromo-pentanoyl)-7H- [M+1]. HRMS (ESI) calcd for C18H16BrNO4Se [M+H] 469.9501,
selenopheno[2,3-f]chromene-7-one (35-Br) Yield: 95%, m.p. = found 469.9512.
178-180 C. H NMR (400 MHz, CDCl3/TMS) δ (ppm): 1.02 (3H,
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t, J = 7.4 Hz, CH3), 1.44-1.69 (2H, m, CH2), 1.86 (6H, s, CH3), 3-bromo-2-(morpholinomethyl)-7-oxo-7H-selemopheno[2,3-
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2.00-2.09 (1H, m, CH2), 2.14-2.23 (1H, m, CH2), 2.62 (1H, br s, f]chromene-8-carboxylic acid (38) Yield: 92%, m.p. > 200 C. 1H
OH), 5.82-5.86 (1H, m, CH), 7.44 (1H, d, J = 8.9 Hz, 5-CH), 8.03 NMR (400 MHz, DMSO-d6) δ (ppm): 3.40-3.62 (4H, m,
(1H, d, J = 8.9 Hz, 4-CH), 8.69 (1H, s, 9-CH). 13C NMR (100.6 morpholine), 3.72-4.04 (4H, m, morpholine), 4.75 (2H, br s,
MHz, CDCl3/TMS) δ (ppm): 13.6, 20.7, 29.10, 29.11, 34.7, 51.4, CH2), 7.59 (1H, d, J = 8.8 Hz, 5-CH), 8.07 (1H, d, J = 8.8 Hz, 4-
74.4, 101.4, 115.0, 115.2, 122.5, 130.9, 139.4, 146.3, 153.9, CH), 8.76 (1H, s, 9-CH). LC-MS m/z 472 [M+1]. HRMS (ESI) calcd
155.1, 158.5, 191.2. LC-MS m/z 550 [M+1].
for C17H14BrNO5Se [M+H] 471.9293, found 471.9304.
3-Bromo-2-(2-hydroxypropan-2-yl)-8-pentanoyl-7H-
3-bromo-2-((4-methylpiperazin-1-yl)methyl)-7-oxo-7H-
selenopheno[2,3-f]chromene-7-one (35)
: The bromide 35-Br selemopheno[2,3-f]chromene-8-carboxylic acid (39) Yield: 99%,
(0.2 g, 0.36 mmol), N-acetylguanidine (0.036 g, 0.36 mmol) and m.p. > 200 oC. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.75 (3H,
sodium acetate (0.030 g, 0.36 mmol) were refluxed in s, CH3), 2.86-2.97 (2H, m, piperazine), 3.07-3.26 (4H, m,
anhydrous ethanol for 12 h and cooled to rt. Reaction mixture piperazine), 3.42-3.45 (2H, m, piperazine), 4.17 (2H, br s, CH2),
was then evaporated and the residue was purified by flash 7.58 (1H, d, J = 8.8 Hz, 5-CH), 8.03 (1H, d, J = 8.8 Hz, 4-CH), 8.69
chromatography on silica gel (CH2Cl2/EtOAc = 1/1). Yield: 68%. (1H, s, 9-CH). LC-MS m/z 485 [M+1]. HRMS (ESI) calcd for
1H NMR (400 MHz, CDCl3/TMS) δ (ppm): 0.95 (3H, t, J = 7.2 Hz, C18H17BrN2O4Se [M+H] 484.9610, found 484.9660. HPLC: 96.9%
CH3), 1.36-1.47 (2H, m, CH2), 1.65-1.73 (2H, m, CH2), 1.86 (6H, (RT=7.26 min, Apollo C18-8 (4.6x150mm), mobile phase
s, CH3), 2.93 (1H, br s, OH), 3.16 (2H, t, J = 7.2 Hz, CH2), 7.39 5→95% Acetonitrile+0.1%H3PO4, 1 mL/min, 40 oC).
(1H, d, J = 8.8 Hz, 5-CH), 7.98 (1H, d, J = 8.8 Hz, 4-CH), 8.57 (1H,
s, 9-CH). 13C NMR (100.6 MHz, CDCl3/TMS) δ (ppm): 13.9, 22.3, In Vitro cytotoxicity assay: Monolayer tumour cell line: MDA-
25.9, 29.0, 42.3, 74.3, 101.3, 114.8, 115.1, 124.2, 130.4, 139.0, MB-435s (human melanoma), MCF-7 (human breast
139.2, 144.6, 153.7, 155.0, 159.0, 198.1. LC-MS m/z 471 [M+1]. adenocarcinoma, oestrogen-positive), MES-SA (human uterus
HRMS (ESI) calcd for C21H24BrNO4Se [M] 470.9705, found sarcoma), HT-1080 (human connective tissue fibrosarcoma),
470.9716.
A549 (human lung carcinoma), SH-SY5Y (human
neuroblastoma), CCL-8 (mouse sarcoma), 3T3 (Mouse Swiss
Hydrolysis
carboxylates:
of
methyl
Methyl
selenopheno[2,3-f]chromene-8- Albino embryo fibroblasts), MH-22A (mouse hepatoma),
selenopheno[2,3-f]chromene-8- HepG2 (human hepatocellular carcinoma), B16-F10 (mouse
carboxylate (0.4 mmol) was suspended in methanol (30 mL). melanoma). All cell lines from ATCC. The cells were seeded in
Then sodium hydroxide (4 mmol) dissolved in water (10 mL) 96-well plates in DMEM and cultivated for 72 h while exposed
was added. After the consumption of the initial compound (12- to different concentrations of the compounds. Cell viability
24 h), reaction mixture was acidified with 2N HCl till pH=3 and was measured via MTT assay [28]. Briefly, after incubation
evaporated. The residue was purified by washing solids with with the compounds, the culture medium was removed, and
water or flash chromatography on silica gel.
fresh medium containing 0.2-mg/ml MTT was added. After
incubation (3 h, 37°C, 5% CO2), the MTT-containing medium
3-bromo-2-(2-hydroxypropan-2-yl)-7-oxo-7H-selemopheno[2,3- was removed, and 200 μL of dimethyl sulfoxide (DMSO) was
o
f]chromene-8-carboxylic acid (36) Yield: 85%, m.p. > 200 C. 1H immediately added to each sample. The samples were
NMR (400 MHz, DMSO-d6) δ (ppm): 1.68 (6H, s, CH3), 6.50 (1H, assessed at 540 nm on a Tecan Infinite1000 microplate reader.
br s, OH), 7.51 (1H, d, J = 8.8 Hz, 5-CH), 7.93 (1H, d, J = 8.8 Hz, The half-maximal inhibitory concentration (IC50) of each
4-CH), 8.69 (1H, s, 9-CH). 13C NMR (100.6 MHz, DMSO-d6) δ compound was calculated using Graph Pad Prism® 3.0.
(ppm): 28.4, 72.2, 99.4, 114.6, 114.9, 119.0, 129.2, 137.8,
138.5, 144.6, 152.6, 156.4, 157.8, 163.8. 77Se-NMR (DMSO-d6, Basal cytotoxicity test [22]: The Neutral Red Uptake (NRU)
76.36 MHz) δ (ppm): 570.3. LC-MS m/z 431 [M+1]. HRMS (ESI) assay was performed according to the standard protocol of
calcd for C15H11BrO5Se [M+H] 430.9028, found 430.9026. HPLC: Stokes modified by the NICEATM-ECVAM validation study. The
97.2% (RT=6.71 min, Apollo C18-8 (4.6x150mm), mobile phase NRU cytotoxicity assay procedure is based on the ability of
5→95% Acetonitrile+0.1%H3PO4, 1 mL/min, 40 oC).
viable cells to incorporate and bind Neutral Red, a supravital
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dye. Balb/c 3T3 (Mouse Swiss Albino embryo fibroblast) cells labelled with calcein-AM (Molecular PrVoiebweAsrt)icle Oanlninde
(9000 cells/well) were placed into 96-well plates for 24 h in photographed at ×4 magnification DuOsI:in10g.103a9n/C9NinJv0e16rt8e2dA
Dulbecco's modified Eagle's (DMEM) medium containing 5% fluorescence microscope (Nicon) calculating the length of
fetal bovine serum. The cells were then exposed to the test capillary tubes.
compounds over a range of eight concentrations (1000, 316,
100, 31, 10, 3, 1 µg/ml) for 24 h. Untreated cells were used as In vitro haemolytic assay: Haemolytic effect of the compounds
a control. After 24 h, the medium was removed from all plates. on rat erythrocytes was evaluated by using washed
Then, 250 μl of Neutral Red solution were added (0.05 mg/ml). erythrocytes. [30] Blood samples from the rats were collected
Plates were incubated for 3 h and then cells were washed from Charles foster strain, (each weighing 130-180 g) in
three times with PBS. The dye within viable cells was released citrated tubes. The cells were then washed three times with
by extraction with a mixture of acetic acid, ethanol and water phosphate-buffered saline (150 mM NaCl, 1.9 mM NaH2PO4,
(1:50:49). Absorbance of Neutral Red was measured using a and 8.1 mM Na2HPO4, pH 7.4) and 2% erythrocyte suspension
spectrophotometer multiplate reader (TECAN, Infinite M1000) was prepared. The haemolytic activities of the compounds
at 540 nm. The optical density (OD) was calculated using the were tested in 96-well plates. PBS solution was used as
following formula: ODtreated cells x 100/ODcontrol cells. The IC50 negative control, 1% solution of Triton X-100 in PBS was a
values were calculated using the computer program Graph Pad positive control. Compounds were tested in various
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Prism® 3.0.
concentrations (from 5 to 300 µg/mL). After an incubation for
1h at 37oC, the plate was centrifuged at 1500 rpm for five
Estimation of LD50 from IC50 values [23]: Data from the in vitro minutes and 50 µl of supernatant was transferred to a fresh
NRU tests were used to estimate the starting dose for acute 96-well plate. The supernatant was used to measure the
oral systemic toxicity tests in rodents. The in vivo starting dose absorbance of the liberated haemoglobin at 405 nm. The
is an estimated LD50 value calculated by inserting the in vitro average value was calculated from triplicate assay. The OD of
IC50 value into a regression formula: log LD50 (mM/kg) = 0.439 cells exposed to 1% Triton X-100 represented 100% lysis; the
log IC50 (mM) + 0.621. The value is recalculated to mg/kg and OD of cells incubated in PBS represented 0%.
compounds are evaluated in accordance with four toxicity
categories: category 1: LD50 ≤ 5 mg/kg (highly toxic); category Hydrolysis of selenophenocoumarin methyl esters with HepG2
2: 5 < LD50 ≤ 50 mg/kg (moderately toxic); category 3: 50 < LD50 cells lysate: Human hepatocellular carcinoma HepG2 cells were
≤ 300 mg/kg (slightly toxic); category 4: 300 < LD50 ≤ 2000 seeded into six-well plates at a density of 2x106 cells/ml and
mg/kg (practically non-toxic). Using this alternative in vitro grown in DMEM with 10% FBS. After cells reached 80 to 90%
method allows comparisons of possible toxicity of new of confluence, the medium was replaced with serum-free
compounds and to select compounds for further study, thus culture medium with 21 or 37 (100 M) and exposed for 6 h.
vastly reducing the number of animal experiments.
Then the medium was separated and cells were sonicated. The
existence of compounds and its metabolites in the cells lysate
MMP assay: Inhibitors of matrix metalloproteinase enzymes and conditioned medium was analysed using UPLC-MS.
were detected with the use of MMP Inhibitor Fluorimetric
Profiling kit (Biomol, USA) accordingly to manufacturer’s In vivo experiments: Six-week-old female C57BL/6JOlaHsd and
instructions. MMP activity assays were performed in 96-well BALB/cOlaHsd mice were purchased from the Envigo (Lab
plates using the recombinant human MMP-1–10, 12, 13 and Animal, Nederland). The mice were housed five to a cage, and
14 catalytic domains and OmniMMPTM fluorogenic substrate under standard conditions (21–23°C, 12 h light: dark cycle), fed
Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2. The test compounds ad libitum (R3 diet, Lactamin AB, Kimstad, Sweden) and
(20µM) were dissolved in DMSO. The compound NNGH (N- observed daily. The experimental procedures involving
isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid) experimental animals were carried out in accordance with the
was used as a prototypic control inhibitor. The rate of guidelines of the European Community, local laws and policies,
substrate hydrolysis was determined by fluorescence intensity and were approved by the Latvian Animal Protection Ethical
measuring for 10 minutes at 37 oC temperature using a Committee, the Food and Veterinary Service, Riga, Latvia.
fluorescence plate reader (Tecan infinite M1000, Austria) with
excitation at 328 nm and emission at 420 nm.
In vivo angiogenesis assay: The matrigel plug angiogenesis
assay is a simple in vivo technique to detect the newly formed
In vitro angiogenesis assay: HUVEC cells were grown in blood vessels in the transplanted gel plugs in mice (5 animals,
Vascular Cell Basal medium with 2% fetal bovine serum, bovine BALB/c). The antiangiogenic potential of compounds was
brain extract, hydrocortisone, human endothelial growth assessed using a modified Matrigel plug assay as described in
factor, and gentamicin/amphotericin B (ATCC) in incubator Ref. 40. The female Balb/c mice were anaesthetized, placed on
with 5% CO2 and at 37°C. Matrigel was dispensed into 96-well a heated pad (37°C) and 0.5ꢀml of ice-cold Matrigel
plates (50 µL/well). Plates were incubated at 37°C for 1 hour to supplemented with 10ꢀµg/ml of basic fibroblast growth factor
allow polymerization of matrigel. HUVEC cultures were added (bFGF) and 100ꢀng/ml of vascular endothelial growth factor
into each matrigel-coated well at a final concentration of (VEGF) without or with compounds in dose 10 µM were
10,000 cells/well. After 8 h incubation at 37°C cells were injected subcutaneously into the flanks of each mouse. Control
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mice received Matrigel without bFGF and VEGF. After 7 days, approved by the Latvian Animal Protection EthicaVlieCwoAmrtimcleitOtneliene,
mice were euthanized and the matrigel implant was recovered the Food and Veterinary Service, Riga, Latvia.
DOI: 10.1039/C9NJ01682A
and frozen gel were cut with cryostat. Sections cut and stained
with haematoxylin and eosin was studied by light microscopy.
Results were expressed as percentage±SD of the vessel area to
Notes and references
the total Matrigel area.
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(Sigma). C57BL/6 mice were injected with 10,000 B16-F10
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hr later with subcutaneous injections (s.c.) of compounds
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Acknowledgements
The financial support of this work provided by European
Regional Development Fund Project No. 1.1.1.1/16/A/294 is
gratefully acknowledged.
The experimental procedures involving experimental animals
were carried out in accordance with the guidelines of the
European Community, local laws and policies, and were
Sciences.
Available:
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Selenopheno[2,3-f]coumarins: a novel scaffold with antimetastatic
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Pavel Arsenyan*, Jelena Vasiljeva, Ilona Domracheva, Iveta Kanepe-Lapsa, Anita Gulbe