Converting potent indeno[1,2- b ]indole inhibitors of protein kinase CK2 into selective inhibitors of the breast cancer resistance protein ABCG2

Article abstract of DOI:10.1021/jm500943z

A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N⁵-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N⁵-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.

Full text of DOI:10.1021/jm500943z

Subscriber access provided by UNIV OF WEST FLORIDA
Article
Converting Potent Indeno[1,2-b]indole Inhibitors of Protein Kinase CK2
into Selective Inhibitors of the Breast Cancer Resistance Protein ABCG2
Gustavo Jabor Gozzi, Zouhair Bouaziz, Evelyn Winter, Nathalia Daflon-Yunes, Dagmar Aichele, Abdelhamid
Nacereddine, Christelle Marminon, Glaucio Valdameri, Wael Zeinyeh, Andre Bollacke, Jean Guillon,
Aline Lacoudre, Noel Pinaud, Silvia M. Cadena, Joachim Jose, Marc Le Borgne, and Attilio Di Pietro
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500943z • Publication Date (Web): 01 Oct 2014
Downloaded from http://pubs.acs.org on October 11, 2014
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 44
Journal of Medicinal Chemistry
1
2
Converting Potent Indeno[1,2-b]indole Inhibitors of Protein Kinase CK2 into Selective
3
4
5
6
Inhibitors of the Breast Cancer Resistance Protein ABCG2
7
8
9
Gustavo Jabor Gozzi,†, ‡,∇ Zouhair Bouaziz,§,∇ Evelyn Winter,†,║ Nathalia Daflon-Yunes,†
Dagmar Aichele,┴ Abdelhamid Nacereddine,§ Christelle Marminon,§ Glaucio Valdameri†,‡,
Waël Zeinyeh,§ Andre Bollacke,┴ Jean Guillon,# Aline Lacoudre,▲ Noël Pinaud,▲ Silvia M.
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
Cadena,‡ Joachim Jose,┴ Marc Le Borgne,§, and Attilio Di Pietro†, ,*

†Equipe Labellisée Ligue 2014, BMSSI UMR 5086 CNRS/Université Lyon 1, IBCP, 69367
Lyon, France
‡Department of Biochemistry and Molecular Biology, Federal University of Paraná, Curitiba,
Paraná, Brazil
§Université de Lyon, Université Lyon 1, Faculté de Pharmacie - ISPB, EA 4446 Biomolécules
Cancer et Chimiorésistances, SFR Santé Lyon-Est CNRS UMS3453 - INSERM US7, 8
avenue Rockefeller, F-69373, Lyon Cedex 8, France
║Department of Pharmaceutical Sciences, PGFAR, Federal University of Santa Catarina,
Florianopolis, Santa Catarina, Brazil
┴ Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfälische
Wilhelms-University Münster, Corrensstr. 48, 48149 Münster, Germany
#Université de Bordeaux, UFR des Sciences Pharmaceutiques, INSERM U869, Laboratoire
ARNA, 146 rue Léo Saignat, F-33076 Bordeaux Cedex, France
▲ISM - CNRS UMR 5255, Université de Bordeaux, 351 cours de la Libération, F-33405
Talence cedex, France
RUNNING TITLE: Converting CK2 inhibitors into ABCG2 inhibitors
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 44
1
2
3
4
5
6
ABSTRACT:
7
8
9
A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein
kinase II (CK2) inhibitors. The most potent inhibitors contained a N5-isopropyl substituent on
C-ring. The same series of compounds was found to also inhibit the breast cancer resistance
protein ABCG2 but with totally different structure-activity relationships: a N5-phenethyl
substituent was critical, and additional hydrophobic substituents at position 7 or 8 of D-ring or
a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best
ABCG2 inhibitors, such as 4c, 4h, 4i, 4j and 4k, behaved as very weak inhibitors of CK2,
whereas the most potent CK2 inhibitors, such as 4a, 4p and 4e, displayed limited interaction
with ABCG2. It was therefore possible to convert, through suited substitutions of the
indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2
inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a
very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported,
constitute promising candidates for further investigations.
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
ACS Paragon Plus Environment

Page 3 of 44
Journal of Medicinal Chemistry
1
2
INTRODUCTION
3
4
5
6
7
8
Human protein kinase casein kinase II (CK2) is a highly pleiotropic serine/threonine protein
kinase discovered in 1954.1 Overexpression of CK2 and its elevated activity are closely
related to many human cancers, including breast,2 lung,3 pancreas,4 and prostate.5 Recent
studies also confirmed CK2 as a key target in leukemia,6 and glioblastoma.7 One of the most
remarkable features of CK2 is the existence of several forms: (i) catalytic CK2α subunit and
its isoforms CK2α’ and CK2α”, (ii) regulatory CK2β subunit, and (iii) heterotetramer
composed of two catalytic subunits and two regulatory subunits.8 Both CK2 heterotetrameric
holoenzyme and its isolated catalytic subunits are constitutively active. Recent structural
insights of CK2α, CK2β and CK2 (quaternary structure of CK2) gave further knowledge to
perform tailor-made compounds.9-11 Thus CK2 is becoming an important drug target for the
21st century as mentioned by E.G. Krebs in 1999.12 Intensive drug discovery chemistry is
currently leading to design and synthesis of small molecule CK2 inhibitors targeting ATP-
binding pocket or exosites at the CK2α/ CK2β interface.13 For example we developed diverse
indeno[1,2-b]indole- and pyrrolo[1,2-a]quinoxaline-based scaffolds14,15 as ATP-competitive
inhibitors of CK2. Tetracyclic indeno[1,2-b]indole derivatives offer great opportunities to
functionalize A-, C- and/or D-rings and so access inhibitors with submicromolar IC50 and
antiproliferative activity against cancer cell lines.16
9
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
Protein kinase inhibitors were previously shown to interact with multidrug ABC
transporters involved in cancer cells resistance to chemotherapy. Protein kinase C inhibitors
were able to inhibit P-glycoprotein/ABCB1 and homologs from yeast and protozoan
parasites.17 A number of tyrosine kinase inhibitors were found to strongly inhibit the breast
cancer resistance protein ABCG2: canertinib (CI1033),18 imatinib,19 gefitinib,20 N-[4-[(3-
bromophenyl)amino]-6-quinazolinyl]-2-butynamide (EKI-785),21 nilotinib and dasatinib,22
vandetinib, pelitinib and neratinib,23 erlotinib,24 sorafenib,25 sunitinib26 and linsitinib.27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 44
1
2
3
4
5
Bisindolylmaleimides and indolocarbazoles28 as well as dimethoxyaurones29, which inhibit
various serine/threonine kinases, were also found to inhibit ABCG2.
6
ABCG2 is overexpressed in many types of tumors,30 and is recognized to play a role in
their multidrug resistance by catalyzing the efflux of anticancer drugs. Potent, selective and
nontoxic inhibitors might constitute a good therapeutic strategy to improve anticancer drugs
efficiency by increasing their bioavailability, and then sensitize tumor growth to their
cytotoxicity.31,32 New inhibitors should therefore be investigated. Our aim was to check the
capacity of the newly-synthesized indenoindole inhibitors of CK2 to interact with ABCG2
and inhibit its drug-efflux activity. It is shown that they indeed inhibit ABCG2-mediated
mitoxantrone transport but importantly the structure-activity relationships are totally different
from those governing CK2 inhibition.
7
8
9
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
CHEMISTRY
Tetrahydroindeno[1,2-b]indole-9,10-diones 4 were synthesized according to a previously
reported method,14,33 with a modification of the reagent for the deoxygenation of the vic-
dihydroxyindeno[1,2-b]indole-9,10-diones 3. Usually transition from 3 to 4 is performed with
tetramethylthionylamide (TMTA), nevertheless we observed better yields with the
tetraethylthionylamide (TETA).34 The latter was prepared by slowly addition of thionyl
chloride to 4 equivalents of diethylamine in dry ether at – 40 °C.
The synthetic route started with the preparation of the corresponding enaminones 2, readily
available in very good yields by reacting substituted cyclohexane-1,3-diones 1 with primary
amines. Subsequent condensation of 2 with 2,2-dihydroxyindane-1,3-diones (ninhydrins)
afforded dihydroxyindeno[1,2-b]indole-9,10-diones 3 (Scheme 1).
This protocol provided a useful way to modify the substituents R1, R2 and R3 in order to
establish structure-activity relationships (CK2 inhibition, ABCG2 inhibition, and
ACS Paragon Plus Environment

Page 5 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
cytotoxicity). Structural variations on the A-, C- and/or D-ring of the indeno[1,2-b]indole
scaffold have been carried out thanks to ninhydrins, primary amines and cyclohexane-1,3-
diones, respectively.
9
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
Scheme 1a
aReagents and conditions: (a) Toluene, reflux; (b) MeOH, rt; (c) (NEt2)2SO (TETA), DMF,
AcOH, rt.
The 1- and 4-hydroxylated indenoindoles 4m and 4n (Scheme 2) were prepared from
enaminone 2a and 4-hydroxyninhydrin. The latter was readily prepared in two steps. Firstly,
the commercially available dihydrocoumarin reacted with AlCl3 to afford the 4-
hydroxyindan-1-one35 which was then oxidized with SeO2 in dioxane under microwave
irradiation (unpublished data).
Condensation of 4-hydroxyninhydrin with enaminon 2a led to the trihydroxylated
derivatives 3m and 3n as a mixture of two regioisomers (ratio 3m/3n: 78/22) which could not
be separated under the classical conditions. Deoxygenation procedure of the mixture using
TETA was carried out to afford the corresponding hydroxyindenoindoles 4m and 4n. At this
step, the two regioisomers were easily separated by chromatography column and identified by
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 44
1
2
3
NMR.
4
5
6
7
8
O-Prenylation of the 4-hydroxy derivative 4n was performed with prenyl bromide in
presence of K2CO3 in acetone at reflux to provide derivative 4p with 60% yield. The reaction
with the isomer 4m was more difficult, probably due to a hydrogen bond between the H of the
phenol on the C-1 and the O of ketone C-10 that forms a pseudo 6-membered ring more
stable, so more difficult to break. Therefore, O-prenylation of the 1-hydroxy derivative 4m
was carried out by means of K2CO3 in dimethylacetamide (DMA) at 80 °C to provide
derivative 4o with 27% yield.
9
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
Scheme 2a
OH
OH
O
O
O
O
O
OH
O
1
10
O
c
1
HO N
N
OH
O
O
N
OH
OH
4
3m
4m
O
1
4o
b
+
+
a
HN
O
O
O
O
O
O
OH
d
2a
4
4
HO
OH
N
N
N
O
OH
4n
4p
3n
aReagents and conditions: (a) MeOH, rt; (b) (NEt2)2SO (TETA), DMF, AcOH, rt; (c) K2CO3,
DMA, 80°C, 24h; (d) K2CO3, acetone, reflux, 8h.
Assignment of regiochemistry was established for each regioisomer by NOESY.
Compounds 4q and 4r were obtained by alkylation of 5,6,7,8-tetrahydroindeno[1,2-b]indole-
ACS Paragon Plus Environment

Page 7 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
9,10-dione 4a with CH3I and PhCH2Br, respectively, in the presence of a solution of lithium
cyclohexylisopropylamide (LCIA)36 prepared from cyclohexylisopropylamine and n-BuLi in
dry THF at – 40 °C (Scheme 3). Attempts to improve the yield of these alkylations by
modification of the reaction conditions (nature and amount of the base, amount of the
alkylating agent, temperature) proved to be unsuccessful. Moreover, methylation provided the
monomethylated compound 4q with 10% yield and only traces of the dimethylated derivative
when benzylation afforded only the disubstituted compound 4r with 35% yield, the
monobenzylated product was not detected. In either case, a large amount of degradation
products was observed.
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
Scheme 3a
O
O
O
O
R3a
R3b
a
N
N
4a
4q: R3a = Me, R3b = H
4r: R3a = R3b = CH2Ph
aReagents and conditions: (a) Cyclohexylisopropylamine, n-BuLi, THF, CH3I or PhCH2Br, -
40 °C.
All together, 18 indeno[1,2-b]indole-9,10-dione derivatives have been synthesized, and
their different substituents are shown in Figure 1.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 44
1
2
3
4
5
6
7
8
9
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
Figure 1. Substitution patterns of 4a-r. Note: compounds 4a-d and 4f-h are described in
reference 33, and compound 4e in reference 37.
ACS Paragon Plus Environment

Page 9 of 44
Journal of Medicinal Chemistry
1
2
3
The 3D structure of 4c was determined by X-ray crystallography (Figure 2) and confirmed
4
5
6
1
that expected on the basis of IR and H NMR data. The key bond lengths and angles of this
7
8
9
indeno[1,2-b]indole-9,10-dione 4c are very similar to those given in the literature for other
substituted indenoindole derivatives.38,39 The 7,8-dihydro-6H-indeno[1,2-b]indole-9,10-dione
system of 4c is not planar; a derivation of the C12 and C13 atoms was noticed at 0.1935 (3) Å
and 0.4429 (3) Å, respectively, from the plane defined by the heterotetracyclic system. The
non-planarity of this tetracyclic sytem is an important structural characteristic in contrast to
planar polycyclic aromatic compounds. The double bonds C1=O1 and C11=O2 of the
heterocyclic system of 4c are confirmed by their respective lengths of 1.218 (3) and 1.221 (3)
Å.
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
Figure 2. View of the crystal structure of 4c with our numbering scheme, displacement
ellipsoids are drawn at the 30% probability level. Note: the numbering used here is specific to
the crystallographic studies, and therefore different from that shown in Table 1 and used all
along the text.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 44
1
2
3
4
5
6
BIOLOGICAL EVALUATION, SARs AND DISCUSSION
7
8
9
The synthesized indeno[1,2-b]indoles were screened for their inhibition capacity, firstly
against protein kinase CK2, and then against ABCG2, as summarized here below.
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
Inhibition of human CK2 holoenzyme. Comparison of the results in Table 1 allowed us
to draw the following structure-activity relationships: i) compound 4a, with a N5-isopropyl-
substituted C-ring, produced a complete inhibition at 10 µM, with a good potency (IC50
=
0.36 µM); ii) a 14-fold increased potency was observed with 4p substituted at position 4 of A-
ring with O-3,3-dimethylallyl (“prenyl”), which was higher than most of the reference
inhibitors and approached the extremely potent silmitasertib (CX-494540); by contrast, the
same prenyl substituent at position 1 in 4o dramatically altered the inhibition capacity to 44%
at 10 µM; iii) a 2-fold increased potency was produced by a methyl substituent at position 7
of D-ring in 4e (IC50 = 0.17 µM), whereas a strong negative effect was observed at vicinal
position 8 with either the same substitution in 4q or a very hydrophobic and steric substitution
by two benzyl groups in 4r (IC50
≥
9.2 µM); iv) replacing the N5-isopropyl by either a benzyl
in 4b, a phenethyl in 4c or a phenpropyl in 4d produced a marked alteration of inhibition (
≥
17 fold) by comparison to 4a; a partial recovery in potency was produced when substituting
with a methoxy the phenethyl at position ortho in 4j, by 5.6-fold relatively to 4c, while the
effects were lower at either position para in 4l or meta in 4k; v) finally, a negative
contribution of phenyl substitution at position 7 was observed when comparing 4g to 4b, and
4i to 4c. The concentration dependence of the inhibition of CK2 activity is illustrated in
Figure 3 for the three best indenoindole inhibitors and the most potent reference inhibitor.
ACS Paragon Plus Environment

Page 11 of 44
Journal of Medicinal Chemistry
1
2
3
Table 1. Inhibition of Human CK2 Holoenzyme
4
5
CK2 inhibition
6
7
Indenoindoles
% at 10 µMa IC50 (µM)
8
9
4a
99
34
59
62
94
16
9
0.36b
(~15)c
7.0b
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
4b
4c
4d
6.0b
4e
0.17b
(~35)c
(~55)c
2.5b
4f
4g
4h
66
35
87
63
69
44
100
52
48
4i
(~15)c
1.4b
4j
4k
5.1b
4l
4.1b
4o
(~12)c
0.025b
9.2b
4p
4q
4r
(~11)c
Reference inhibitors
Silmitasertibd
Ellagic acid
TBBe
100
95
0.0037b
0.04b
99
0.06b
Emodin
99
0.58b
aThe percent inhibition of CK2 activity was determined for each compound at a fixed
b
concentration of 10 µM. For the best compounds producing at least 50% inhibition at 10
c
µM, the concentration was varied to precisely determine the IC50 values. For the other, less
potent compounds, a rough estimation was obtained from the experimental inhibition
d
produced at 10 µM. Silmitasertib = 5-[(3-chlorophenyl)amino]benzo[c][2,6] naphthyridine-
8-carboxylic acid. eTBB = 4,5,6,7-tetrabromobenzotriazole.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 44
1
2
3
4
5
6
7
8
9
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
Figure 3. Concentration dependence for the most potent inhibitors of CK2 activity.
Inhibition of MCF-7 cell proliferation by the CK2 inhibitor 4p. The antiproliferative
effect of prenyl-substituted indeno[1,2-b]indole 4p, which appeared to be the most potent
CK2 inhibitor with an IC50 value of 25 nM, was evaluated in MCF-7 breast cancer cells.41 For
this purpose, a commercially-available EdU-click assay was applied, resulting in the
incorporation of a TAMRA fluorophore into the nucleic acid of cells performing DNA
replication and preparing cell proliferation.42 Proliferating cells can be recognized by a violet
fluorescence of their nuclei (Figure 4B), and the number of such cells are counted with and
without inhibitor and set into relation of the corresponding number of control cells.
ACS Paragon Plus Environment

Page 13 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
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
Figure 4. Fluorescence images of MCF-7 cells treated with different concentrations of the
inhibitor 4p for 24 h. Cell nuclei were double-stained by 1 (blue fluorescence), able to cross
the membrane of all cells, and by EdU-assay using 5-TAMRA-PEG3-azide as a coupled
fluorophore (violet fluorescence). Cell proliferation was monitored by EdU-assay. Panel A:
Hoechst-staining of treated MCF-7 cells. Panel B: Overlay of the fluorescence images of 1
stained cells and TAMRA-labeled proliferating cells. The cells which are emitting only blue
fluorescence are not proliferating, in contrast to those emitting an additional violet
fluorescence.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 44
1
2
3
4
5
6
7
8
9
As shown in Figure 5, incubation of MCF-7 cells with 20 µM 4p for 24 h resulted in a
dramatic reduction of the proliferation rate. Only 14% of the cells were still able to synthesize
DNA, a ratio that was further decreased after 48 h to 1.3 % (not shown here). When 4p was
used at 100 µM, cell proliferation was completely inhibited even after 24-h exposure. Staining
of MCF-7 cells with 1 (Hoechst 33258, 4-{6-[6-(4-methylpiperazin-1-yl)-1H-1,3-
benzodiazol-2-yl]-1H-1,3-benzodiazol-2-yl}phenol)43 indicated that the cells were markedly
damaged after treatment with 4p (Figure 4A). Already at 24 h of treatment, the entire number
of MCF-7 cells underwent cell death and the total number of detectable cells was strongly
reduced, down to 10%. The remaining cells showed characteristic features of apoptosis, such
as reduction of nuclear size and condensation of chromatin (Figure 4A). In contrast, 1 staining
did not indicate any sign of apoptosis after treatment with 20 µM 4p. The nuclear morphology
of cells under these conditions was similar to DMSO-treated control cells. This clearly
indicated that the prenyl-substituted indeno[1,2-b]indole 4p has a distinct antiproliferative
effect at 20 µM concentration against MCF-7 breast cancer cells.
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
Figure 5. Antiproliferative effect of the inhibitor 4p on MCF-7 cells. Results are shown as
percent proliferating cells relatively to control cells (with 1% DMSO), and represent the mean
ACS Paragon Plus Environment

Page 15 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
(±
SD) of four assays.
Inhibition of ABCG2-Mediated Drug Efflux. The same 16 indeno[1,2-b]indole
7
8
9
derivatives were assayed for their ability to inhibit ABCG2-mediated mitoxantrone efflux
from transfected HEK293 cells. Table 2 shows that the worst CK2 inhibitors appear to be the
best ABCG2 inhibitors. Indeed, the N5-phenethyl substituted compounds produced a complete
inhibition, with strong efficiency at submicromolar concentrations, as exemplified by 4c (IC50
= 0.43 µM). A 2-fold further increase in potency was observed by methyl substitution at
position 7 in 4h, or by methoxy substitution at phenethyl position ortho in 4j whereas an
opposite, negative, effect was produced at phenethyl position para in 4l and an intermediate
one at phenethyl position meta in 4k.
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
Table 2. Inhibition of Mitoxantrone Efflux in ABCG2-Transfected Cells
ABCG2 inhibition
ABCG2/CK2d
Cytotoxicity
Indenoindoles
4a
% at 10 µMa IC50 (µM)
IG50 (µM)e
TRf
15
18
7
3
~35c
~30c
~0.01
~0.5
nd
nd
4b
4c
4d
4e
100 14
83 25
65 17
0.43 0.01b
0.79 0.13b
4.1 1.1b
16
8
30.7 9.5
> 100
nd
71
> 127
~0.04
4f
33 14
~15c
~2
nd
4g
4h
4i
68 10
100 21
83 13
106 22
78 14
0.96 0.09b
0.23 0.02b
0.49 0.22b
0.21 0.07b
0.31 0.09b
0.70 0.04b
3.0 0.5b
~30
11
~30
7
16.9 6.5
> 100
18
> 435
96
20.1 4.9
27.2 0.7
12.7 3.1
68.5 0.2
nd
4j
130
41
4k
4l
16
6
131
6
98
4o
74 12
60 26
~4
4p
1.6 0.7b
0.02
31.0 8.4
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 44
1
2
3
4
5
4q
40 28
90 20
20 14b
~0.46
~18
nd
4r
0.61 0.1b
6.0 1.0
10
6
7
8
9
Reference inhibitorsg
Chromone 1i
Ko143j
98
7
0.13 0.09
0.09 0.01
96
32
6
3
106
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Reference substratesh
Mitoxantrone
SN-38k
0.009 0.005
0.005 0.001
aThe percent inhibition of ABCG2-mediated mitoxantrone efflux was determined for each
b
compound at a fixed concentration of 10 µM. For the best compounds producing at least
50% inhibition at 10 µM, the concentration was varied to precisely determine the IC50 values.
cFor the other, less potent compounds, a rough estimation was obtained from the
d
experimental inhibition produced at 10 µM. The ABCG2/CK2 ratio, indicating the
inhibitory efficiency of compounds toward ABCG2 relatively to CK2, was calculated by the
e
ratio between the IC50(CK2) values from Table 2 and the IC50(ABCG2) values. The IG50
values of compounds cytotoxicity were determined after 72 h with MTT cell survival tests.
f
nd, not determined. The TR ratio was calculated upon dividing the IG50 values of
cytotoxicity by corresponding IC50 values of ABCG2 inhibition. gOther known inhibitors and
hcytotoxic substrates have been added for comparison with indenoindoles. Chromone 1 = 5-
i
[(4-bromobenzyl)oxy]-N-[2-(5-methoxy-1H-indol-3-yl)ethyl]-4-oxo-4H-chromene-2-
carboxamide.
jKo143
=
(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-
methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic
acid
1,1-
k
dimethylethyl ester. SN-38 = 7-ethyl-10-hydroxycamptothecin, active metabolite of the
topoisomerase I inhibitor irinotecan.
The concentration dependence of the inhibition of ABCG2-mediated mitoxantrone efflux
ACS Paragon Plus Environment

Page 17 of 44
Journal of Medicinal Chemistry
1
2
3
is illustrated for selected inhibitors in Figure 6.
4
5
6
7
8
9
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
Figure 6. Concentration dependence for the most potent indenoindole inhibitors of ABCG2
transport activity.
Increasing the length of the N5-substituent into phenpropyl in 4d induced a moderate
alteration (2 fold), whereas a dramatic loss of potency was produced by shortening into either
benzyl in 4b or isopropyl in 4a (approximately 70-100 fold). A main part of this loss in
potency could be recovered through hydrophobic substitution by two benzyl groups at
position 8 in 4r versus 4a, while a partial recovery was observed at vicinal position 7 with
either a phenyl group in 4g versus 4b or a methyl group in 4e versus 4a. A partial recovery of
inhibition potency was observed with prenyl substitution not only at position 4 in 4p, but also
at position 1 in 4o, by comparison to 4a. The best indenoindole inhibitors were nearly as
potent as ABCG2 reference inhibitors, 2 (chromone 1) and 3 (Ko143), assayed under the
same conditions.44 In some cases, the maximal inhibition, as observed at 10 µM, was not
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 44
1
2
3
4
5
6
7
8
9
complete (with maximal values of 60-83%) for hydrophobic derivatives such as 4d, 4g, 4i and
4p (with LogP values of 4.9-6.2) relative to a complete inhibition produced by 4c and 4j with
a LogP of 4.4, may be partly related to lower water solubility, as suggested for other ABCG2
inhibitors.45 However, the fact that 4j, 4k and 4l (with the same LogP of 4.4) gave a variable
maximal inhibition indicates that other parameters are critical for inhibition potency, in
agreement with known polyspecificity of multidrug transporters, where the number and
positions of hydrophobic substituents can modulate the orientation of binding and subsequent
inhibition.
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
Studies of the intermolecular interactions between ABCG2 and diverse ligands have
revealed three important chemical features: hydrogen-bond acceptor (HBA), hydrophobic part
(HP) and aromatic ring system (ARS).46,47 According to different possible combinations (0-3
HBA + 0-3 HP + 0-2 ARS), various inhibition pharmacophores were designed. Interestingly,
a given ligand may adopt different orientations to form its complex with ABCG2. Our lead
compound 4h is mainly characterized by 2 HBAs, 1 HP and 3 ARSs and, according to
different pharmacophore models, could indeed interact in variable orientations with the
protein target. This point is closely related with the promiscuous nature of polyspecific
multidrug transporters such as ABCG2.
Comparison in Table 2 of the relative ability of each compound to inhibit CK2 and
ABCG2 indicates that the best CK2 inhibitors, with a N5-isopropyl substituent were about two
orders of magnitude more selective for CK2 in 4a, 4e and 4p whereas the best ABCG2
inhibitors, with a N5-phenethyl substituent, 4c, 4h, 4i, 4j, 4k and 4l, were around one order of
magnitude more selective for ABCG2. These differences are well illustrated in Figure 7A,
also displaying that 4d, with a longer N5-substituent, and both 4r and 4g, with aromatic
substituents on D-ring, were good ABCG2 inhibitors. By contrast, 4b, 4f, 4o and 4q were
only weak inhibitors of both CK2 and ABCG2, whereas no indenoindole derivative was
ACS Paragon Plus Environment

Page 19 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
found to potently inhibit both CK2 and ABCG2. Overall, three orders of magnitude in
selectivity were separating the best ABCG2 inhibitors from the best CK2 inhibitors despite
they belong to the same indeno[1,2-b]indole family of compounds, for example 4c and 4a
which only differ from the N5-substitution.
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
Finally, Table 2 shows that among the best ABCG2 inhibitors, some displayed a very low
cytotoxicity characterized by IG50 values higher than 100 µM, for 4h and 4d, or of 69 µM for
4l, whereas other derivatives appeared more cytotoxic, such as 4r (IG50 = 6 µM), and others
were moderately cytotoxic such as 4k, 4g, 4j and 4c (IG50 = 13-31 µM). A similar low or
moderate cytotoxicity was observed with the reference inhibitors 2 and 3, by difference with
known cytotoxic substrates such as mitoxantrone and SN-3818 (IG50 = 5-9 nM). For
indenoindole inhibitors, comparison between cytotoxicity and potency of ABCG2 inhibition
showed a very high therapeutic ratio, >435, for 4h, compatible with future in vivo assays in
animal models; three other derivatives, 4j, 4l and 4d, with a therapeutic ratio around 100,
might also be used. Some of the compounds displaying a moderate cytotoxicity, such as 4j
and 4h, when assayed under 72-h cell survival MTT tests in both control HEK293 cells and
ABCG2-transfected cells indicated no significant difference in IG50 values (not shown here);
this indicated the absence of apparent ABCG2-mediated cross resistance, which suggested the
absence of transport of the inhibitors.
Diverse structural modifications were performed on indeno[1,2-b]indoles to design new
CK2 inhibitors. The N5-isopropyl derivative 4a was progressively modified,14,16,37 and the
introduction of a prenyl group on position 4 of A-ring was extremely favorable (CK2+++,
IC50 = 0.025 ꢀM) (Figure 7A). Its analogue 4o, with shifted substitution at position 1, gave
significant information by (i) the emergence of ABCG2 inhibitory activity (ABCG2+) and (ii)
the loss of CK2 inhibitory activity (CK2-). New pharmacomodulations allowed us to access to
nine potent ABCG2 inhibitors (4g, 4d, 4l, 4r, 4i, 4c, 4k, 4j, and 4h) exhibiting submicromolar
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 44
1
2
3
4
IC50 values (ABCG2+++).
5
6
7
8
9
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
A
B
CK2 ---
ABCG2
+
O
O
1
A
4
CK2 --
ABCG2 ++
8
7
D
C
CK2
ABCG2 ++
+
N
CK2 +++
ABCG2
+
n
ortho
CK2 +++
ABCG2 +++
Figure 7. Relationships between CK2 inhibitors and ABCG2 inhibitors. Panel A: graphical
representation as a function of relative IC50 values. Panel B: structural modifications of
indenoindoles for exploring CK2 or ABCG2 inhibitors.
The main structure-activity relationships of indeno[1,2-b]indoles for either CK2 inhibitors
or ABCG2 inhibitors are schematically represented in Figure 7B. Quite interestingly, the
different critical substitutions concerned the same positions, but generally with opposite
effects of substituents, as following: 1) at N5 position, the preferred substituent was isopropyl
(20-fold over phenethyl) for CK2 inhibition, whereas phenethyl was the best (70 to 100-fold
over benzyl and isopropyl) for ABCG2 inhibition; 2) at position 8, hydrophobic substitutions
by a methyl and a set of two benzyl groups were highly negative (30-fold versus H) for CK2
inhibition whereas they were highly positive (up to 60-fold) for ABCG2 inhibition; 3) at
ACS Paragon Plus Environment

Page 21 of 44
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
vicinal position 7, a hydrophobic phenyl was negative (3-fold versus H) for CK2 inhibition
but quite positive (up to 30-fold) for ABCG2 inhibition. Position 7 for methyl substitution and
phenethyl position ortho for methoxy substitution, produced similar effects on both CK2 and
ABCG2, namely a 2 to 5-fold increase in potency. Prenyl substitution produced similar
positive effects at position 4 (up to 20-fold increase in potency); however, it was highly
selective for CK2 inhibition relatively to position 1, by contrast to ABCG2 inhibition where a
positive contribution was also observed. Finally, the ABCG2 inhibitory site appeared more
hydrophobic and able to bind bigger indeno[1,2-b]indole derivatives than the CK2 inhibitory
site. This fully agrees with the fact that the ABCG2 binding site is expected to be located in
close proximity to the hydrophobic drug-binding sites, as for previously characterized for
various heterocyclic inhibitors,31,32 as recently reviewed,48,49 by contrast to the CK2 inhibitory
site which has been shown to widely overlap the hydrophilic ATP-binding site.16
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
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on an Electrothermal 9200 capillary
apparatus. The IR spectra were recorded on a Perkin Elmer Spectrum Two IR Spectrometer.
The 1H and 13C NMR spectra were recorded at 400 MHz on a Bruker DRX 400 spectrometer.
Chemical shifts are expressed in ppm (δ) downfield from internal tetramethylsilane and
coupling constants J are reported in hertz (Hz). The following abbreviations are used: s:
singlet; bs: broad singlet; d: doublet; t: triplet; dd: doubled doublet; dt: doubled triplet; q:
quartet; m: multiplet; Cquat: quaternary carbons. The mass spectra were performed by direct
ionization (EI or CI) on a ThermoFinnigan MAT 95 XL apparatus. Chromatographic
separations were performed on silica gel columns by column chromatography (Kieselgel 300–
400 mesh). All reactions were monitored by TLC on GF254 plates that were visualized under
a UV lamp (254 nm). Evaporation of solvent was performed in vacuum with rotating
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 44
1
2
3
4
5
evaporator. The purity of the final compounds (greater than 95%) was determined by
uHPLC/MS on an Agilent 1290 system using a Agilent 1290 Infinity ZORBAX Eclipse Plus
6
7
8
C18 column (2.1 x 50 mm, 1.8 ꢀm particle size) with a gradient mobile phase of H2O/CH3CN
9
(90:10, v/v) with 0.1% of formic acid to H2O/CH3CN (10:90, v/v) with 0.1% of formic acid at
a flow rate of 0.5 mL/min, with UV monitoring at the wavelength of 254 nm with a runtime
of 10 min.
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
General Procedure for the Synthesis of Compounds 2. A solution containing 10.62
mmol of primary amine and 10.62 mmol of the corresponding cyclohexane-1,3-dione
dissolved in 60 mL of toluene was refluxed in a Dean-Stark trap until the separation of H2O
had finished. The solvent was then evaporated under vacuum and the resultant residue was
taken up in EtOAc to give a yellow solid that was isolated by filtration. Further purification
was then accomplished by silica gel column chromatography with CH2Cl2/acetone (1:3, v/v)
as the eluent.
5-Phenyl-3-(2-phenylethylamino)cyclohex-2-enone (2i). Yellow solid. Yield 75%. mp 117
1
°C. IR (
ν
cm-1): 3240, 1698, 1660, 1601. H NMR (CDCl3, 400 MHz)
δ: 7.33-7.20 (m, 10H,
Harom), 5.30 (s, 1H, H-2), 5.00 (s, 1H, NH), 3.44-3.31 (m, 3H, H-5 and CH2), 2.94 (t, 2H, J =
7.0 Hz, CH2), 2.70-2.40 (m, 4H, 2 CH2). 13C NMR + DEPT (CDCl3, 100 MHz)
δ: 196.60
(C=O), 163.52 (Cquat), 143.41 (Cquat), 138.35 (Cquat), 129.11 (2 CH), 129.02 (2 CH),
128.93 (2 CH), 127.22 (CH), 127.12 (CH), 127.02 (2 CH), 96.97 (CH), 44.24 (CH2), 43.79
(CH2), 40.24 (CH), 37.57 (CH2), 34.62 (CH2). HRMS calcd for C20H22NO [M + H]+
292.1696, found 292.1692.
3-[2-(2-Methoxyphenyl)ethylamino]cyclohex-2-enone (2j). Yellow solid. Yield 90%. mp
105 °C. IR (ν δ: 7.22 (dt, 1H, J1
cm-1): 3253, 1600, 1569, 1533. 1H NMR (CDCl3, 400 MHz)
= 1.7 Hz, J2 = 8.2 Hz, Harom), 7.10 (dd, 1H, J1 = 1.6 Hz, J2 = 7.3 Hz, Harom), 6.92-6.86 (m,
2H, Harom), 5.15 (s, 1H, H-2), 5.1 (s, 1H, NH), 3.84 (s, 3H, OMe), 3.29 (m, 2H, CH2), 2.89
ACS Paragon Plus Environment

Page 23 of 44
Journal of Medicinal Chemistry
1
2
3
13
(t, 2H, J = 6.8 Hz, CH2), 2.29-2.24 (m, 4H, 2 CH2), 1.93 (m, 2H, CH2). C NMR + DEPT
4
5
6
(CDCl3, 100 MHz) : 197.43 (C=O), 164.50 (Cquat), 157.64 (Cquat), 130.85 (CH), 128.46
δ
7
8
9
(CH), 127.21 (Cquat), 121.21 (CH), 110.78 (CH), 96.98 (CH), 55.57 (CH3), 43.59 (CH2),
36.71 (CH2), 30.14 (CH2), 29.59 (CH2), 22.28 (CH2). HRMS calcd for C15H20NO2 [M + H]+
246.1489, found 246.1489.
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
3-[2-(3-Methoxyphenyl)ethylamino]cyclohex-2-enone (2k). Beige solid. Yield 80%. mp 94
1
°C. IR (
ν
cm-1): 3256, 1601, 1570, 1531. H NMR (CDCl3, 400 MHz)
δ: 7.22 (t, 1H, J = 7.9
Hz, Harom), 6.79-6.75 (m, 2H, Harom), 6.71 (m, 1H, Harom), 5.17 (s, 1H, H-2), 4.73 (s, 1H,
NH), 3.79 (s, 3H, OMe), 3.44 (m, 2H, CH2), 2.85 (t, 2H, J = 7.0 Hz, CH2), 2.31-2.26 (m, 4H,
13
2 CH2), 1.93 (m, 2H, CH2). C NMR + DEPT (CDCl3, 100 MHz)
δ
: 197.63 (C=O), 164.36
(Cquat), 160.20 (Cquat), 140.04 (Cquat), 130.11 (CH), 121.22 (CH), 114.77 (CH), 112.24
(CH), 97.36 (CH), 55.51 (CH3), 43.91 (CH2), 36.75 (CH2), 34.67 (CH2), 30.11 (CH2), 22.60
(CH2). HRMS calcd for C15H20NO2 [M + H]+ 246.1489, found 246.1494.
3-[2-(4-Methoxyphenyl)ethylamino]cyclohex-2-enone (2l). Yellow solid. Yield 90%. mp
1
89 °C. IR (
ν
cm-1): 3247, 1596, 1531, 1511. H NMR (CDCl3, 400 MHz)
δ: 7.08 (d, 2H, J =
8.6 Hz, Harom), 6.84 (d, 2H, J = 8.6 Hz, Harom), 5.16 (s, 1H, H-2), 4.79 (s, 1H, NH), 3.78 (s,
3H, OMe), 3.30 (m, 2H, CH2), 2.82 (t, 2H, J = 7.0 Hz, CH2), 2.30-2.26 (m, 4H, 2 CH2), 1.93
(m, 2H, CH2). 13C NMR + DEPT (CDCl3, 100 MHz)
δ: 197.54 (C=O), 164.43 (Cquat),
158.74 (Cquat), 130.37 (Cquat), 129.88 (2 CH), 114.47 (2 CH), 97.28 (CH), 55.57 (CH3),
44.24 (CH2), 36.75 (CH2), 30.77 (CH2), 30.08 (CH2), 22.26 (CH2). HRMS calcd for
C15H20NO2 [M + H]+ 246.1489, found 246.1483.
General Procedure for the Synthesis of Compounds 3. A solution containing 6.86 mmol
of enaminone 2 and 6.86 mmol of ninhydrin dissolved in 25 mL of MeOH was stirred at room
temperature for 8 h. Generally, a precipitate of compound 3 was formed. It was recovered and
washed with MeOH. A second quantity was obtained from the filtrate by purification by silica
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 44
1
2
3
gel column chromatography with CH2Cl2/acetone (1:3, v/v) as the eluent.
4
5
4b,9b-Dihydroxy-7-phenyl-5-(2-phenylethyl)-4b,5,6,7,8,9b-hexahydroindeno[1,2-b]indole-
6
7
8
9,10-dione (3i). White solid. Yield 84%. mp 128 °C. IR (
ν
cm-1): 3471, 1714, 1603, 1521. 1H
9
NMR (CDCl3, 400 MHz) : 7.95-7.87 (m, 2H, Harom), 7.77 (m, 1H, Harom), 7.57 (m, 1H,
δ
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
Harom), 7.36-7.20 (m, 8H, Harom), 7.09-7.03 (m, 2H, Harom), 4.11 (m, 1H, NCH2), 3.82 (m,
1H, NCH2), 3.25 (m, 1H, CH2Ph), 3.11 (m, 1H, H-7), 2.99 (m, 1H, CH2Ph), 2.61-2.11 (m, 4H,
CH2-6 and CH2-8). 13C NMR + DEPT (CDCl3, 100 MHz)
δ: 197.78 (d, C=O), 191.10 (d,
C=O), 166.84 (d, Cquat), 148.51 (d, Cquat), 143.01 (d, Cquat), 138.70 (d, Cquat), 136.10 (d,
CH), 135.52 (d, Cquat), 130.80 (d, CH), 129.51 (d, 2 CH), 129.20 (d, 2 CH), 128.99 (d, 2
CH), 127.32 (d, 2 CH), 127.12 (d, 2 CH), 125.12 (d, CH), 124.52 (d, CH), 105.37 (d, Cquat),
96.87 (d, Cquat), 83.12 (d, Cquat), 44.88 (d, CH2), 44.11 (d, CH2), 40.42 (d, CH), 37.83 (s,
CH2), 30.84 (d, CH2). HRMS calcd for C29H26NO4 [M + H]+ 452.1856, found 452.1840.
4b,9b-Dihydroxy-5-[2-(2-methoxyphenyl)ethyl]-4b,5,6,7,8,9b-hexahydroindeno[1,2-
b]indole-9,10-dione (3j). Yellow solid. Yield 93%. mp 114 °C. IR (
1601, 1524. 1H NMR (CDCl3, 400 MHz)
ν
cm-1): 3465, 1716, 1656,
δ
: 7.92 (d, 1H, J = 7.8 Hz, Harom), 7.85 (d, 1H, J =
7.6 Hz, Harom), 7.73 (m, 1H, Harom), 7.54 (m, 1H, Harom), 7.26 (m, 1H, Harom), 7.16 (dd,
1H, J1 = 1.7 Hz, J2 = 7.4 Hz, Harom), 6.94 (m, 1H, Harom), 6.89 (d, 1H, J = 8.2 Hz, Harom),
4.88 (bs, 1H, OH), 3.98 (m, 1H, NCH2), 3.77 (m, 1H, NCH2), 3.13 (m, 1H, CH2Ph), 2.98 (m,
1H, CH2Ph), 2.38 (bs, 1H, OH), 2.22 (m, 2H, CH2), 2.16 (m, 2H, CH2), 1.84 (m, 1H, CH2),
13
1.73 (m, 1H, CH2). C NMR + DEPT (CDCl3, 100 MHz)
δ: 198.11 (C=O), 192.39 (C=O),
167.26 (Cquat), 157.84 (Cquat), 148.42 (Cquat), 136.05 (CH), 135.44 (Cquat), 131.28 (CH),
130.75 (CH), 128.83 (CH), 126.71 (Cquat), 125.13 (CH), 124.43 (CH), 121.31 (CH), 110.81
(CH), 105.53 (Cquat), 96.22 (Cquat), 83.01 (Cquat), 55.84 (CH3), 43.29 (CH2), 36.64 (CH2),
32.90 (CH2), 23.16 (CH2), 21.88 (CH2). HRMS calcd for C24H24NO5 [M + H]+ 406.1649,
found 406.1641.
ACS Paragon Plus Environment

Page 25 of 44
Journal of Medicinal Chemistry
1
2
3
4b,9b-Dihydroxy-5-[2-(3-methoxyphenyl)ethyl]-4b,5,6,7,8,9b-hexahydroindeno[1,2-
4
5
6
b]indole-9,10-dione (3k). White solid. Yield 94% yield. mp 95 °C. IR (ν
cm-1): 3471, 1713,
7
8
1
1655, 1599, 1521. H NMR (DMSO-d6, 400 MHz)
δ: 8.04 (d, 1H, J = 7.8 Hz, Harom), 7.84
9
(m, 1H, Harom), 7.74 (d, 1H, J =7.5 Hz, Harom), 7.62 (m, 1H, Harom), 7.29 (m, 1H, Harom),
6.95-6.92 (m, 2H, Harom), 6.90 (bs, 1H, OH), 6.85 (dd, 1H, J1 = 2.6 Hz, J2 = 8.2 Hz, Harom),
5.79 (bs, 1H, OH), 4.01 (m, 1H, NCH2), 3.75 (m, 1H, NCH2), 3.40 (s, 3H, OMe), 2.97 (m,
2H, CH2Ph), 2.24 (m, 2H, CH2), 2.05 (m, 2H, CH2), 1.73 (m, 1H, CH2), 1.65 (m, 1H, CH2).
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
13C NMR + DEPT (DMSO-d6, 100 MHz)
δ: 198.58 (C=O), 189.68 (C=O), 166.22 (Cquat),
160.36 (Cquat), 149.19 (Cquat), 141.49 (Cquat), 136.53 (CH), 135.73 (Cquat), 131.20 (CH),
130.51 (CH), 125.74 (CH), 124.11 (CH), 122.19 (CH), 115.52 (CH), 113.00 (CH), 105.71
(Cquat), 96.60 (Cquat), 84.54 (Cquat), 55.99 (CH3), 44.37 (CH2), 37.93 (CH2), 37.84 (CH2),
23.24 (CH2), 22.33 (CH2). HRMS calcd for C24H24NO5 [M + H]+ 406.1649, found 406.1658.
4b,9b-Dihydroxy-5-[(2-(4-methoxyphenyl)ethyl]-4b,5,6,7,8,9b-hexahydroindeno[1,2-
b]indole-9,10-dione (3l). Yellow solid. Yield 97%. mp 116 °C. IR (ν
cm-1): 3489, 1713, 1660,
1
1605, 1577, 1539, 1509. H NMR (CDCl3, 400 MHz)
δ: 7.89-7.84 (m, 2H, Harom), 7.73 (m,
1H, Harom), 7.54 (m, 1H, Harom), 7.13-7.10 (m, 2H, Harom), 6.89-6.85 (m, 2H, Harom),
5.53 (bs, 1H, OH), 5.14 (bs, 1H, OH), 3.99 (m, 1H, NCH2), 3.80 (s, 3H, OMe), 3.72 (m, 1H,
NCH2), 2.99 (m, 2H, CH2Ph), 2.23 (m, 2H, CH2), 2.12 (m, 2H, CH2), 1.87 (m, 1H, CH2), 1.71
(m, 1H, CH2). 13C NMR + DEPT (CDCl3, 100 MHz)
δ: 197.88 (C=O), 192.44 (C=O), 167.17
(Cquat), 158.92 (Cquat), 148.53 (Cquat), 136.04 (CH), 135.49 (Cquat), 130.73 (CH), 130.62
(Cquat), 130.32 (2 CH), 125.16 (CH), 124.43 (CH), 114.60 (2 CH), 105.64 (Cquat), 96.22
(Cquat), 83.19 (Cquat), 55.68 (CH3), 45.02 (CH2), 36.97 (CH2), 36.59 (CH2), 23.35 (CH2),
21.82 (CH2). HRMS calcd for C24H24NO5 [M + H]+ 406.1649, found 406.1638.
General Procedure for the Synthesis of Compounds 4i-n. A solution containing 4.43
mmol of 3 and 8.86 mmol (2 equiv) of TETA dissolved in 15 mL of DMF and 2.5 mL of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 44
1
2
3
4
5
6
7
8
9
AcOH was stirred at room temperature for 22 h. The solution was then poured into 300 mL of
ice and water and stirred for 1 h. The resulting precipitate was filtered and washed with water
and dried to give a first quantity of 4. The filtrate was evaporated and the residue was diluted
with H2O. The solution was basified with NaHCO3 and extracted with CH2Cl2. The organic
phase was dried over anhydrous Na2SO4 and evaporated in vacuum to give a second quantity
of 4 which was purified by silica gel column chromatography with CH2Cl2/acetone (95:5, v/v)
as the eluent.
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
7-Phenyl-5-(2-phenylethyl)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4i). Orange
1
solid. Yield 95%. mp 228 °C. IR (
ν
cm-1): 1698, 1660, 1603, 1520. H NMR (CDCl3, 400
MHz)
δ: 7.47 (d, 1H, J = 6.8 Hz, Harom), 7.33-7.22 (m, 7H, Harom), 7.14 (m, 1H, Harom),
7.07 (d, 2H, J = 7.0 Hz, Harom), 6.98-6.93 (m, 3H, Harom), 4.15 (m, 2H, NCH2), 3.08- 3.00
(m, 3H, H-7 and CH2Ph), 2.60 (dd, 1H, J1 = 4.0 Hz, J2 = 16.4 Hz, H-6 or H-8), 2.51 (dd, 1H,
J1 = 12.6 Hz, J2 = 16.4 Hz, H-6 or H-8), 2.25 (dd, 1H, J1 = 4.8 Hz, J2 = 16.4 Hz, H-6 or H-8),
13
2.10 (dd, 1H, J1 = 11.6 Hz, J2 = 16.4 Hz, H-6 or H-8). C NMR + DEPT (CDCl3, 100 MHz)
δ: 191.47 (C=O), 184.55 (C=O), 152.81 (Cquat), 150.27 (Cquat), 142.97 (Cquat), 139.04
(Cquat), 137.01 (Cquat), 135.09 (Cquat), 132.75 (CH), 129.43 (2 CH), 129.30 (2 CH), 128.99
(2 CH), 128.73 (CH), 127.76 (CH), 127.38 (CH), 127.01 (2 CH), 124.16 (CH), 120.39
(Cquat), 117.42 (CH), 117.16 (Cquat), 47.76 (CH2), 44.90 (CH2), 41.64 (CH), 37.22 (CH2),
29.94 (CH2). HRMS calcd for C29H24NO2 [M + H]+ 418.1802, found 418.1791.
5-[2-(2-Methoxyphenyl)ethyl]-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione
(4j).
Orange solid. Yield 83%. mp 198 °C. IR (
400 MHz)
ν
cm-1): 1698, 1664, 1605, 1531. 1H NMR (CDCl3,
δ
: 7.40 (d, 1H, J = 7.0 Hz, Harom), 7.22-7.17 (m, 2H, Harom), 7.08 (m, 1H,
Harom), 6.98 (d, 1H, J = 7.1 Hz, Harom), 6.90 (d, 1H, J = 6.4 Hz, Harom), 6.83-6.79 (m, 2H,
Harom), 4.12 (t, 2H, J = 6.9 Hz, NCH2), 3.79 (s, 3H, OMe), 3.06 (t, 2H, J = 6.9 Hz, CH2Ph),
2.34 (m, 2H, CH2-6 or CH2-8), 2.27 (m, 2H, CH2-6 or CH2-8), 1.90 (m, 2H, CH2-7). 13C
ACS Paragon Plus Environment

Page 27 of 44
Journal of Medicinal Chemistry
1
2
3
4
NMR + DEPT (CDCl3, 100 MHz) δ: 192.63 (C=O), 184.63 (C=O), 157.73 (Cquat), 153.04
5
6
7
8
(Cquat), 150.91 (Cquat), 139.08 (Cquat), 135.25 (Cquat), 132.53 (CH), 131.05 (CH), 129.15
(CH), 128.49 (CH), 125.05 (Cquat), 123.92 (CH), 121.14 (CH), 120.06 (Cquat), 117.52
(Cquat), 117.36 (CH), 110.54 (CH), 55.57 (CH3), 46.08 (CH2), 38.05 (CH2), 32.16 (CH2),
23.28 (CH2), 21.74 (CH2). HRMS calcd for C24H22NO3 [M + H]+ 372.1594, found 372.1590.
9
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
5-[2-(3-Methoxyphenyl)ethyl]-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione
(4k).
1
Orange solid. Yield 60%. mp 176 °C. IR (
MHz)
ν
cm-1): 1696, 1661, 1594. H NMR (CDCl3, 400
δ
: 7.43 (dd, 1H, J1 = 0.6 Hz, J2 = 7.1 Hz, Harom), 7.22-7.08 (m, 3H, Harom), 6.88 (d,
1H, J =7.1 Hz, Harom), 6.75 (m, 1H, Harom), 6.57 (d, 1H, J =7.6 Hz, Harom), 6.49 (m, 1H,
Harom), 4.14 (t, 2H, J = 6.5 Hz, NCH2), 3.69 (s, 3H, OMe), 3.03 (t, 2H, J = 6.5 Hz, CH2Ph),
2.33 (m, 2H, CH2-6 or CH2-8), 2.09 (m, 2H, CH2-6 or CH2-8), 1.85 (m, 2H, CH2-7). 13C
NMR + DEPT (CDCl3, 100 MHz) δ: 192.58 (C=O), 184.54 (C=O), 160.25 (Cquat), 152.53
(Cquat), 151.04 (Cquat), 139.07 (Cquat), 138.50 (Cquat), 135.12 (Cquat), 132.63 (CH),
130.35 (CH), 128.65 (CH), 124.14 (CH), 121.45 (CH), 120.47 (Cquat), 117.60 (Cquat),
117.27 (CH), 114.86 (CH), 113.01 (CH), 55.54 (CH3), 47.71 (CH2), 38.02 (CH2), 37.29
(CH2), 23.22 (CH2), 21.90 (CH2). HRMS calcd for C24H22NO3 [M + H]+ 372.1594, found
372.1601.
5-[2-(4-Methoxyphenyl)ethyl]-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione
(4l).
Orange solid. Yield 87%. mp 181 °C. IR (
400 MHz)
ν
cm-1): 1697, 1660, 1605, 1511. 1H NMR (CDCl3,
δ
: 7.44 (d, 1H, J = 7.1 Hz, Harom), 7.20 (m, 1H, Harom), 7.11 (m, 1H, Harom),
6.90-6.87 (m, 3H, Harom), 6.79-6.76 (m, 2H, Harom), 4.12 (t, 2H, J = 6.5 Hz, NCH2), 3.73
(s, 3H, OMe), 3.01 (t, 2H, J = 6.5 Hz, CH2Ph), 2.33 (m, 2H, CH2-6 or CH2-8), 2.10 (m, 2H,
CH2-6 or CH2-8), 1.85 (m, 2H, CH2-7). 13C NMR + DEPT (CDCl3, 100 MHz)
δ: 192.65
(C=O), 184.60 (C=O), 159.31 (Cquat), 152.54 (Cquat), 150.96 (Cquat), 139.11 (Cquat),
135.17 (Cquat), 132.61 (CH), 130.31 (2 CH), 128.88 (Cquat), 128.65 (CH), 124.16 (CH),
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 44
1
2
3
4
5
6
7
8
120.52 (Cquat), 117.62 (Cquat), 117.28 (CH), 114.65 (2 CH), 55.67 (CH3), 47.96 (CH2),
38.04 (CH2), 36.39 (CH2), 23.26 (CH2), 21.95 (CH2). HRMS calcd for C24H21NNaO3 [M +
Na]+ 394.1414, found 394.1403.
9
1-Hydroxy-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4m). Orange
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
1
solid. Yield 81%. mp = 167 °C. IR (
400 MHz)
ν
cm-1): 3214, 1696, 1672, 1623. H NMR (DMSO-d6,
δ
: 9.74 (s, 1H, OH), 7.22 (m, 1H, H-3), 6.91 (d, 1H, J = 7.3 Hz, H-4), 6.71 (d, 1H,
J = 8.5 Hz, H-2), 4.74 (m, 1H, CHMe2), 2.93 (t, 2H, J = 5.6 Hz, CH2-6 or CH2-8), 2.35 (t, 2H,
J = 5.8 Hz, CH2-6 or CH2-8), 2.06 (m 2H, CH2-7), 1.54 (d, 6H, J = 9.3 Hz, CHMe2). 13C
NMR + DEPT (DMSO-d6, 100 MHz) δ: 192.25 (C=O), 185.44 (C=O), 156.24 (2 Cquat),
151.19 (Cquat), 150.40 (Cquat), 136.57 (Cquat), 135.64 (CH), 120.62 (CH), 120.14 (Cquat),
117.56 (Cquat), 113.15 (CH), 50.02 (CH), 38.58 (2 CH2), 23.75 (CH2), 22.15 (2 CH3). HRMS
calcd for C18H17NNaO3 [M + Na]+ 318.1101, found 318.1094.
4-Hydroxy-5-isopropyl-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-dione (4n). Pink solid.
1
Yield 19%. mp = 309 °C. IR (
ν
cm-1): 2642, 1698, 1596. H RMN (DMSO-d6, 400 MHz)
δ:
10.58 (s, 1H, OH), 7.08 (m, 1H, H-2), 6.92 (d, 1H, J = 8.3 Hz, H-1 or H-3), 6.89 (d, 1H, J =
6.8 Hz, H-1 or H-3), 5.91 (m, 1H, CHMe2), 2.93 (t, 2H, J = 5.7 Hz, CH2-6), 2.38 (t, 2H, J =
13
6.1 Hz, CH2-8), 2.07 (m, 2H, CH2-7), 1.56 (d, 6H, J = 6.8 Hz, CHMe2). C NMR + DEPT
(DMSO-d6, 100 MHz) δ: 191.33 (C=O), 183.21 (C=O), 149.75 (Cquat), 147.94 (Cquat),
140.20 (Cquat), 131.60 (Cquat), 131.51 (Cquat), 130.00 (CH), 128.64 (Cquat), 123.40 (CH),
118.61 (Cquat), 115.10 (CH), 50.66 (CH), 41.42 (CH2), 37.68 (2 CH2), 21.60 (2 CH3). HRMS
calcd for C18H18NO3 [M + H]+ 296.1281, found 296.1279.
5-Isopropyl-1-(3-methylbut-2-enyloxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-
dione (4o). A mixture of 1-hydroxyindenoindole 4m (1 mmol), K2CO3 (3 mmol), and prenyl
bromide (1.5 mmol) in DMA was heated at 80 °C for 24 h. The mixture was then poured into
H2O and extracted with CH2Cl2. The organic layers were washed with H2O, dried over
ACS Paragon Plus Environment

Page 29 of 44
Journal of Medicinal Chemistry
1
2
3
Na2SO4, filtered and concentrated. The residue was purified by flash chromatography using
4
5
6
acetone/CH2Cl2 (2:10, v/v) as the eluent to provide compound 4o. Orange solid. Yield 27%.
7
8
1
mp = 212 °C. IR (
ν
cm-1): 1698, 1659, 1588, 1503. H NMR (CDCl3, 400 MHz)
δ: 7.17 (m,
9
1H, H-3), 6.77 (d, 1H, J = 7.3 Hz, H-2 or H-4), 6.73 (d, 1H, J = 8.6 Hz, H-2 or H-4), 5.49 (m,
1H, Me2C=CH), 4.67 (d, 2H, J = 6.5 Hz, OCH2), 4.61 (m, 1H, CHMe2), 2.83 (t, 2H, J = 6.0
Hz, CH2-6 or CH2-8), 2.46 (t, 2H, J = 5.8 Hz, CH2-6 or CH2-8), 2.14 (m, 2H, CH2-7), 1.73 (d,
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
13
3H, J = 1.0 Hz, Me2C=CH), 1.71 (s, 3H, Me2C=CH), 1.62 (d, 6H, J = 7.1 Hz, CHMe2). C
NMR + DEPT (CDCl3, 100 MHz) δ: 192.30 (C=O), 183.33 (C=O), 157.24 (Cquat), 149.61
(Cquat), 148.91 (Cquat), 138.06 (Cquat), 137.99 (Cquat), 134.10 (CH), 123.87 (Cquat),
121.64 (Cquat), 120.20 (CH), 117.90 (Cquat), 117.46 (CH), 112.71 (CH), 66.90 (CH2), 49.60
(CH), 38.18 (CH2), 26.07 (CH3), 23.95 (CH2), 23.61 (CH2), 22.13 (2 CH3), 18.67 (CH3).
HRMS calcd for C23H25NNaO3 [M + Na]+, 386.1727, found 386.1711.
5-Isopropyl-4-(3-methylbut-2-enyloxy)-5,6,7,8-tetrahydroindeno[1,2-b]indole-9,10-
dione (4p). A solution of 4-hydroxyindenoindole 4n (0.677 mmol, 1 equiv), K2CO3 (2.03
mmol, 3 equiv) and prenyl bromide (2.03 mmol, 3 equiv) in acetone (20 mL) was refluxed for
8 h. The mixture was then filtered and concentrated. The residue was purified by flash
chromatography using acetone/CH2Cl2 (2:10, v/v) as the eluent to provide compound (4p).
1
Red solid. Yield 60%. mp = 167 °C. IR (
d6, 400 MHz)
ν
cm-1): 1694, 1660, 1642, 1598. H RMN (DMSO-
δ
: 7.20-7.18 (m, 2H, H-1 and H-3), 6.98 (m, 1H, H-2), 5.84 (bs, 1H,
Me2C=CH), 5.52 (m, 1H, CHMe2), 4.68 (d, 2H, J = 7.1 Hz, OCH2), 2.98 (t, 2H, J = 5.87 Hz,
CH2-6 or CH2-8), 2.37 (t, 2H, J = 5.74 Hz, CH2-6 or CH2-8), 2.06 (m, 2H, CH2-7), 1.82 (s,
13
3H, Me2C=CH), 1.77 (d, 3H, J = 0.9 Hz, Me2C=CH), 1.49 (d, 6H, J = 7.1 Hz, CHMe2). C
NMR + DEPT (DMSO-d6, 100 MHz) δ: 192.30 (C=O), 184.03 (C=O), 154.19 (Cquat),
151.07 (Cquat), 150.01 (Cquat), 140.61 (Cquat), 140.28 (Cquat), 131.35 (CH), 122.29
(Cquat), 120.86 (CH), 119.57 (CH), 119.12 (Cquat), 118.66 (Cquat), 117.10 (CH), 66.13
ACS Paragon Plus Environment