Synthesis and biological evaluation of new cytotoxic indazolo[4,3-gh] isoquinolinone derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.01.022

A series of indazolo[4,3-gh]isoquinolinones derivatives have been synthesized to decrease cardiotoxic side effects in comparison to Mitoxantrone. The antiproliferative effects of different side chains were investigated and tested on at least four different cell lines of cervix, ovarian, CNS, NSCLC (non-small-cell lung cancer) and colon carcinoma. In addition to antiproliferative activities, influence on cell cycle and intercalation behavior have been tested.

Full text of DOI:10.1016/j.bmcl.2013.01.022

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1846–1852
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of new cytotoxic indazolo[4,3-
gh]isoquinolinone derivatives
Manochehr Shahabi a, Eva Schirmer a, Karem Shanab a, Theerachart Leepasert a, Jana Ruzicka a,
Wolfgang Holzer a, Helmut Spreitzer a, , Babette Aicher b, Peter Schmidt b, Lars Blumenstein b,
⇑
Gilbert Müller b, Eckhard Günther b
a Department of Drug and Natural Product Synthesis, University of Vienna, Althanstraße 14, 1090 Vienna, Austria
b AeternaZentaris GmbH, Weismuellerstrasse 50, 60314 Frankfurt, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of indazolo[4,3-gh]isoquinolinones derivatives have been synthesized to decrease cardiotoxic
side effects in comparison to Mitoxantrone. The antiproliferative effects of different side chains were
investigated and tested on at least four different cell lines of cervix, ovarian, CNS, NSCLC (non-small-cell
lung cancer) and colon carcinoma. In addition to antiproliferative activities, influence on cell cycle and
intercalation behavior have been tested.
Received 28 September 2012
Revised 2 January 2013
Accepted 4 January 2013
Available online 23 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keyword:
Anticancer compounds
Mitoxantrone is used for fighting a wide range of cancerous dis-
eases.1 Unfortunately, its applicability is strongly limited due to
cardiotoxic adverse effects. The search for antitumor antibiotics
led to Pixantrone which was developed to reduce heart damage re-
lated to treatment while retaining efficacy. Pixantrone (BBR 2778)
is currently in phase III of clinical trials in patients with Non-Hodg-
kin’s lymphoma.2
chemical properties are reported. Docking studies are discussed to
rationalize the observed cytotoxic activities.
We report the synthesis of 23 different N-substituted com-
pounds (shown in Table 1) and their precursors derived from the
key structure shown in Figure 1.
For the synthesis of the tetracyclic nucleus 11 commercially
available 3-chloropyridine 2 was treated with lithium diisopro-
pylamide (LDA) and carbon dioxide to give 3-chloroisonicotinic
acid 3 which was converted into methyl ester 4 using diazometh-
ane. The key step for this synthesis lies in the nickel mediated
coupling of ester 4 and compound 7. This organo zinc bromide
7 was prepared by treating 6 with zinc dust in tetrahydrofuran
(THF). Benzylbromide 6 was obtained by reacting 5-fluoro-2-chlo-
rotoluene 5 with N-bromosuccinimide (NBS) in carbon tetrachlo-
ride. The addition of a solution of 7 in THF to methyl ester 4 in
the presence of bis[triphenylphosphine]nickel(II) chloride led to
coupled product 8. Basic hydrolysis of 8 afforded carboxylic acid
9, which was cyclized and oxidized to 10 by fuming sulfuric acid.
The incorporation of the pyrazole ring was accomplished by
reacting 10 with N,N-dimethylethylhydrazine to give 11 in good
yields (Scheme 1).
Compounds 12–17 were prepared by nucleophilic aromatic
substitution of chlorine of precursor 11 with an appropriate com-
mercially available amine (Scheme 2).
Aziridines 21–23 were prepared from mesylates 18–20, which in
turn were obtained by treatment of corresponding alcohols 41–43
with mesyl chloride/triethylamine. Alcohols 41–43 were synthe-
sized by introducing the appropriate O-protected hydroxyl amine
Based on the work of Krapcho et al.3 we developed a series of
new cytotoxic indazolo[4,3-gh]isoquinolinones to eliminate car-
diotoxicity. The new derivatives, lacking hydroxy substitution
and bearing a nitrogen atom in the anthrapyrazole chromophore,
should be less susceptible to biological reduction to anionic radi-
cals which are responsible for cardiotoxicity, as for inhibition of
topoisomerase II enzyme.4 A wider range of activity and reduced
side effects were the aims of the reported study.
In this letter, we present another series of novel cytotoxic com-
pounds with different side chains attached on the key structure
shown in Figure 1.
As previously reported by the authors,5–7 the position of the
nitrogen(s) in annelated rings are also essential for cytotoxic activ-
ity. All of the synthesized indazolo[4,3-gh]isoquinolinones were
tested for cytotoxicity against a panel of five cancer cell lines.
In addition to the antiproliferative effects, we also investigated
DNA intercalation behavior and the influence on cell cycle arrest.
Furthermore, data on metabolic stability, pH stability and physico-
⇑
Corresponding author. Tel.: +43 1427755621; fax: +43 142779556.
E-mail address: helmut.spreitzer@univie.ac.at (H. Spreitzer).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.022

M. Shahabi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1846–1852
1847
H
N
Cl
Cl
O
Cl
O
LDA / CO2
diazomethane
N
OH
N
N
NH2
OH
OH
O
O
HN
HN
OH
O
HN
OCH3
Ni(PPh3)2Cl
N
2
3
4
Cl
Cl
Cl
OH
O
HN
CH3
NBS
Zn / ZHF
N
H
NH2
ZnBr
Br
Mitoxantrone
BBR 2778 (Pixantrone)
benzoylperoxide
F
7
F
5
6
H3C
F
N
CH3
F
F
O
F
NaOH / H2O
fmg. H2SO4
N
N
N
N
N
N
O
OH
Cl
O
10
Cl
Cl
O
O
H3C
8
9
N
CH3
O
HN
R
N
N
Figure 1. Structures of Mitoxantrone, Pixantrone and novel indazolo[4,3-
gh]isoquinolinones.
NEt3
N
H2N
N
N
H
O
Cl
11
35–37 by nucleophilic aromatic substitution of chlorine in precur-
sor 11 and subsequent cleavage t-butyldiphenylsilyl (TBDPS) pro-
tecting group by tetrabutylammonium fluoride (Scheme 3).
O-TBDPS-protected aminohexynol 44 and aminohexenols 45
and 46 were reacted with 11 to give 47, 48, and 49 respectively,
which were then treated with TBAF to liberate alcohols 24, 50,
and 51. Aziridines 26, 28, and 29 were prepared from mesylates
25, 27, and 52, which were obtained by reacting mesyl chloride
with compounds 24, 50, and 51 (Table 2).
Compound 54 was prepared from 11 and O-TBDPS-protected
aminoethylthioethanol 53 in pyridine. Deprotection was carried
out with TBAF in tetrahydrofuran to afford target compound 30
(Scheme 4).
Scheme 1.
CH3
N
H2
N
H2
N
H3C
NH
N
CH3
15
12
N
N
H2N
S
H2
N
S
CH3
N
13
14
16
17
H2
N
H2
N
N
N
NH
O
Cl
11
Compound 31 was prepared straightforwardly by nucleophilic
aromatic substitution of chlorine of precursor 11 with 2-(2-amino-
ethylthio)-N,N-dimethyl-ethanamine 558 (Scheme 5).
Scheme 2.
The first step of the synthesis of 32 and 33 was the reaction of
precursor 11 with side chains 5615 and 5715 in pyridine to afford
compounds 58 and 59, which were subsequently deprotected by
treatment with TBAF to give alcohols 60 and 61. After mesylation
of alcohols 60 and 61, chlorides 32 and 33 were obtained by treat-
ing mesylates 62 and 63, with lithium chloride in N,N-DMF
(Scheme 6).
In a Grignard reaction O-TBDPS-protected butynol 649 was con-
verted into 65 which served as starting material for two different
Table 1
Structures of novel indazolo[4,3-gh]isoquinolinones 12–34
H3C
18: R1 = H; R2
=
=
27: R1 = H; R2
=
CH3
N
OMs
12: R1 = H; R2
=
N
OMs
CH3
19: R1 = H; R2
20: R1 = H; R2
N
N
OMs
N
N
28: R1 = H; R2
29: R1 = H; R2
30: R1 = H; R2
=
=
=
N
=
=
=
13: R1 = H; R2
14: R1 = H; R2
=
=
S
N
OMs
CH3
21: R1 = H; R2
22: R1 = H; R2
N
O
N
R1
R2
OH
S
S
N
CH3
N
23: R1 = H; R2
24: R1 = H; R2
=
=
31: R1 = H; R2
32: R1 = H; R2
=
=
N
CH3
15: R1 = H; R2
16: R1 = H; R2
17: R1 = H; R2
=
=
=
NH
OH
OMs
N
25: R1 = H; R2
26: R1 = H; R2
=
=
S
N((CH2)2Cl)2
33: R1 = H; R2
=
N
N((CH2)2Cl)2
34: R1 = CH3; R2 = CH3
NH

1848
M. Shahabi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1846–1852
N(CH3)2
pyridine
N
N
pyridine
n
n
Si Cl
HO
NH2
N
TBDPSO
NH2
n= 2,4,6
n=2: 35
36
37
n=4:
n=6:
O
Cl
11
N(CH3)2
N(CH3)2
N
N
N
N
HN
TEA
MsCl, TEA
OH
TBAF
N
N
18-20
21-23
n=2: 21
n=2: 18
O
HN
22
19
n=4:
n=6: 23
n=4:
n=2: 38
n=2: 41
42
O
HN
OTBDPS
n
n=6: 20
39
n=4:
n=6: 40
n=4:
n=6: 43
n
Scheme 3.
Table 2
Synthesis of target compounds 24–29
N(CH3)2
47: R2
48: R2
49: R2
=
=
=
R1 = H
OTBDPS
OTBDPS
H2
N
N
N
OTBDPS
N(CH3)2
44
N
pyridine
H2
N
47, 48, 49
N
N
OTBDPS
OH
OTBDPS
45
46
11
H2N
O
Cl
N
50: R2
51: R2
=
=
OTBDPS
HN
OH
O
N
R1
R2
TBAF
MsCl, TEA
25, 27, 52
26, 28, 29
24, 50, 51
TEA
52: R2
=
OMs
N(CH3)2
1. EtMgBr/THF
TBDPSCl
OH
OTBDPS
MsO
O
DMF, imidazole
N
N
2.
64
N
TBAF
S
pyridine
HO
30
OTBDPS
H2N
OTBDPS
11
S
MsCl
OTBDPS
OTBDPS
65
66
53
O
HN
phthalimide
Ph3P
NaN3
DMSO
54
N3
O
DIAD
NH3
EtOH
Scheme 4.
OTBDPS
68
N
Ph3P
THF
H2O
i-propanole
O
OTBDPS
67
NaBH4, H2O
pyridine
H2N
N
11
31
H2N
S
H2N
OTBDPS
Lindlar catalyst
EtOH
55
OTBDPS
45
44
Scheme 5.
Scheme 7.
N(CH3)2
H2N
N(CH3)2
TBAF
N((CH2)2OTBDPS)2
N
N
N
N
pyridine
N
N
N
n
56
n=4:
n=6: 57
58
n=4:
O
11
Cl
O
HN
n=6: 59
n
N(CH3)2
N(CH3)2
N((CH2)2OTBDPS)2
N
N
N
N
LiCl, DMF
MsCl, TEA
N
32 33
,
32
n=4:
n=6: 33
O
HN
N((CH2)2OH)2
O
62
HN
N((CH2)2OMs)2
60
n=4:
n=4:
n=6: 61
n
n=6: 63
n
Scheme 6.

M. Shahabi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1846–1852
1849
O
was mono O-protected by reaction with t-butyldiphenylsilyl chlo-
ride to afford compound 70.13 After mesylation of the remaining
hydroxyl group of 70, the resulting product 71 was reacted with
sodium azide in DMSO, followed by reduction of the intermediate
azide to afford amine 46 (Scheme 8).
TBDPSCl
LiAlH4
THF
HO
HO
HO
OH
OH
DMF, imidazole
69
O
MsCl
MsO
OTBDPS
OTBDPS
2,20-Thiodiethanol was mono-protected as t-butyldiphenylsilyl
ether 7214 and subsequently reacted with thionyl chloride to afford
compound 73. Amine 53 was prepared from 73 with sodium azide,
followed by treatment with triphenylphosphine and water
(Scheme 9).
Amine 55 was straightforwardly prepared by alkylation of
2-(dimethylamino)ethanethiol with 2-bromoethan-amine hydro-
chloride (Scheme 10).
70
71
1. NaN3
DMSO
H2N
OTBDPS
2. Ph3P
46
THF, H2O
Scheme 8.
Compound 7416 was prepared from 5-phenylpentanoic acid
by nitration in the para-position of the phenyl ring. Subsequent
esterification led to compound 75,17 which was hydrogenated
under pressure catalyzed by palladium on carbon to afford
amine 77. Utilizing the same method amine 76 was synthesized
from (E)-ethyl 3-(4-nitro-phenyl)acrylate. Following reaction
steps were carried out in the same manner for both amines
7618 and 7718 starting with dihydroxyethylation of the nitrogen
atoms with ethylenoxide. Both hydroxyl groups of reaction prod-
ucts 7819 and 7919 were then protected (80, 81)20 with t-butyl-
diphenylsilyl chloride and the ester moieties reduced to alcohols
82 and 83. Mesylation of the hydroxyl groups of alcohols 8220
and 8320 and immediate treatment with lithium bromide gave
bromides 8421 and 8521 which upon treatment with tetraethy-
lammounium cyanide were converted into nitriles 8622 and
87.22 Final reduction of the nitrile moieties with lithium alumi-
num hydride afforded the desired amine products 56 and 57
(Scheme 11).
Indazolo[4,3-gh]isoquinolinones 12–34 were analysed regard-
ing their cytotoxic/antiproliferative activity against different can-
cer cell lines, namely KB/HeLa (cervical carcinoma), SKOV-3
(ovarian carcinoma), SF-268 (CNS, glioma), NCI-H460 (non-small
cell lung carcinoma (NSCLC)), and of RKOp27 (colon adenocarci-
noma).23 The concentration of the compound that inhibits 50%
(EC50) of cellular viabilty after 48 h was calculated by nonlinear
regression (GraphPad Prism™) using the data of at least two
independent cytotoxicity assays.24 Results of the cytotoxicity as-
says are shown in detail in Table 3.
TBDPSCl
DMF, imidazole
SOCl2
OTBDPS
HO
OH
HO
S
S
72
1. NaN3
DMSO
Cl
OTBDPS
H2N
OTBDPS
S
73
S
2. Ph3P
THF, H2O
53
Scheme 9.
+
NH3 Cl-
Br
N
H2N
N
SH
S
DMF, tBuO-K+
55
Scheme 10.
pathways to obtain intermediate 44. In the first pathway amine 44
was introduced by Gabriel synthesis via intermediate 67. In the
second pathway alcohol 6510 was reacted with mesyl chloride to
give 6611 which was either reacted with ammonia in ethanol to
give amine 44 directly or with sodium azide and subsequent
reduction of 68–44. Finally, O-TBDPS-protected (Z)-hexenamine
45 was obtained by partial reduction using a lindar catalyst
(Scheme 7).
Attempts to prepare 46 from 44 by partial reduction with so-
dium/liquid ammonia or lithium aluminum hydride failed. Thus,
another four-step approach was carried out starting with reduction
of 2-butene-1,4-dicarboxylic acid to give diole 69.12 This product
NO2
O
O
HO
n=4
n
O
O
Pd/C
H2
HNO3
3.5 bar
NO2
NO2
Pd/C
NH2
O
O
O
EtOH
TsOH
H2
3.5 bar
H2O, AcOH
HO
O
O
n
n
n
n=4: 74
n=4: 75
n=2: 76
77
n=4:
N((CH2)2OH)2
TBDPSCl
N((CH2)2OTBDPS)2
1. MsCl
N((CH2)2OTBDPS)2
LiAlH4
O
O
pyridine
THF
TEA
O
HO
O
n
n
n
DMAP
2. LiBr
n=2: 78
79
n=2: 82
n=2: 80
81
83
n=4:
n=4:
n=4:
N((CH2)2OTBDPS)2
Et4NH4+CN-
N((CH2)2OTBDPS)2
N((CH2)2OTBDPS)2
LiAlH4
H2N
Br
n=2: 84
NC
n
n
n
n=2: 56
57
n=2: 86
87
85
n=4:
n=4:
n=4:
Scheme 11.

1850
M. Shahabi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1846–1852
Table 3
In vitro cytotoxicity of compounds 12–34 towards different cell lines
a
Cells (origin)/compound
EC50 (lM)
KB/HeLa (cervix)
SKOV-3 (ovarian)
SF-268 (CNS)
NCI-H460b (NSCLC)
RKOp27 (colon)
RKOp27IND (colon)
12
13
14
15
16
17
18
19
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
0.15
0.36 0.00 (2)
1.36 0.33 (2)
1.41 0.30 (2)
1.52 0.11 (2)
3.22 0.22 (2)d
1.65 0.18 (2)
0.55
Not tested
Not tested
Not tested
Not tested
Not tested
Not tested
0.39
0.16 0.01 (2)
0.68 0.05 (2)
0.99 0.12 (2)
1.32 0.02 (2)
1.47 0.12 (2)
3.02 0.02 (2)d
0.25
0.37
2.94
2.68
0.46
4.76d
3.77
1.33
1.51
3.12d
1.33
4.84d
4.81d
1.83
0.17
1.18
0.51
3.17d
No inhib.c
No inhib.c
1.37
1.07
0.82
No inhib.c
1.93
20
1.67
3.61d
21
0.07
0.39
0.69
0.08
0.08
1.05
22
0.08
0.53
0.42
0.07
1.00
1.02
23
24
25
26
0.34
1.39
0.39
0.14
0.77
0.76
0.20
0.13
1.63
0.47
0.07
1.28
1.21 0.00 (2)
0.73 0.07 (2)
0.31 0.10 (2)
5.38
3.45d
0.68 0.14 (2)
0.411 0.08 (2)
0.06 0.00 (2)
2.18
3.23d
0.98
0.75
0.24
0.35
2.61
27
0.75
3.07d
0.63
28
29
30
31
32
33
34
0.43 0.21 (3)
1.22
0.96
1.77 1.11 (3)
1.71 0.53 (3)
2.99
0.66
0.59 0.37 (3)
0.69 0.10 (2)
0.15 0.03 (2)
0.31 0.05 (2)
3.76d
0.29 0.15 (3)
0.72
0.55
1.20 0.49
2.78
1.06
3.22d
0.40 0.00 (2)
0.70 0.00 (2)
No inhib.c
0.75
2.47
2.37
0.92
1.19
1.79
1.63
4.17d
No inhib.c
2.19
No inhib.c
No inhib.c
1.34
No inhib.c
0.77 0.07 (2)
0.12 0.01 (3)
No inhib.c
2.63
No inhib.c
3.06d
0.76 0.20
2.08 0.30 (2)
0.12 0.04 (3)
Mitoxantrone
0.36 0.14 (5)
0.32 0.21 (3)
0.09 0.01 (3)
a
b
c
EC50 values determined in replicates are given as mean values standard deviation with number of replicates given in round brackets.
EC50(NCI-H460) denotes the activity for NCI-H460 cell line, which is employed to compare compound activities.
No inhib. = no inhibition, that is <30% inhibition in the highest concentration (3.16
lM) during EC50 determination.
d
‘Moderate’ activity describes compound activity, that showed at least 30% inhibition in the highest concentration (3.16
lM) during EC50 determination, but since a full
dose–response curve is not available, compound activity has been described as EC50 >3 lM.
Table 4
consistently shift their respective peaks for 6–8 nm (shown in
Fig. 2).
DNA intercalation for selected compounds demonstrated by the
shift of the most blue shifted respective peaks
Comparison of the activities of indazolo isoquinolinones against
RKOp27 with or without p27 induced cell cycle arrest suggest, that
progression through cell cycle might be required for the mode of
action for some, but not all compounds analysed. In order to exam-
ine the compound effects on cell cycle by FACS analysis, exponen-
tially dividing KB/HeLa cells were treated with different
concentrations of selected compounds for 48 h and compared to
untreated controls.26
Compound
DNA—intercalation
22
24
26
30
34
12
23
15
508 ? 514 nm
505 ? 512 nm
505 ? 512 nm
505 ? 513 nm
491 ? 499 nm
502 ? 510 nm
509 ? 515 nm
No intercalation
492 ? 502 nm
662 ? 681 nm
Results from these cell cycle studies are rather inhomoge-
Acridine
Mitoxantrone
neous. This is exemplified by compounds 26 (
23 (hexane spacer, 0.195 M), 28 (cis
0.594 M) and 29 (trans -hexene spacer, 0.691
in Table 5.
v
v
-hexinyl spacer),
-hexene spacer,
M) as depicted
l
l
v
l
A first comparison between the tested indazolo isoquinolinones
and reference compound Mitoxantrone reveals excellent antipro-
liferative activity. Within this report, EC50(NCI-H460) activity val-
ues will be used to compare compounds. Based on this activities,
an improvement by a factor of two with regard to Mitoxantrone
Aziridine 26, showing the best NCI-H460 activity within the ser-
ies, is not at all inducing cell cycle arrest in KB/HeLa cells, while the
other C6 spaced aziridines do so with single digit micromolar EC50
values.
Moreover, inverse dose-dependent S-phase arrest is observed
for compounds 23, 28 (see Fig. 3) and 29, which is a rarely observed
dose depency feature.
is observed for 26 and 22 (EC50(NCI-H460): 0.062 and 0.071
vs 0.122 M). Compound 30, 12 and 23 show a similar antiprolif-
erative activity (EC50(NCI-H460): 0.151 M, 0.159 and
0.195 M, respectively).
lM
l
l
lM
Further experiments will be necessary to explain these unusual
effects.
l
Due to the similarity of the indazolo isoquinolinones and ref-
In conclusion, a series of indazolo[4,3-gh]isoquinolinones were
synthesized and tested for antiproliferative activity against at least
four cancer cell lines. Compared to reference compound Mitoxan-
trone, aziridine 23 turned out to show an increase in activity by
a factor of 2.
Cell cycle arrest studies gave complex results, including
an induction of S-phase arrest with an inversed dose
dependency.
erence compound Mitoxantrone, DNA intercalation25 as a possi-
ble mode of action was evaluated for
compounds.
a set of selected
Using Acridine and Mitoxantrone as positive controls, the
tested indazolo isoquinolinones clearly show DNA intercalation
(see Table 4). Mitoxantrone moves the most blue shifted peak
(i.e., at 662 nm) for 19 nm. The tested indazolo isoquinolinones

M. Shahabi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1846–1852
1851
Mitoxantrone Intercalation
1.2
1
0.8
0.6
0.4
0.2
0
400
450
500
550
600
650
700
750
800
-0.2
Wavelength [nm]
Mitoxantrone (125µM)
Mitoxantrone (125µM) + DNA
Cpd. 26 Interkalation
1.2
1
0.8
0.6
0.4
0.2
0
300
350
400
450
Wavelength [nm]
500
550
600
-0.2
Cpd. 26 (125µM)
Cpd. 26 (125µM) + DNA
Figure 2. UV spectra showing DNA intercalation of reference compound Mitoxantrone and aziridine 26.
Table 5
Compound 26
Cell cycle arrest results for aziridines 23, 26, 28 and 29
100%
Compound Cell cycle
26
23
28
29
No cell cycle arrest
50%
0%
Increase of S-Phase below 4
Increase of S-Phase below 6
Increase of S-Phase below 7
lM with inverse dose dependency
lM with inverse dose dependency
lM with inverse dose dependency
11
10
9
8
7
6
5
4
3
2
1
0
µ M
Supplementary data
G1 Phase S Phase G2M Phase
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.01.
022.
Compound 23
100%
50%
0%
References and notes
1. Vuimo, T. A.; Kulikova, E. V.; Sinauridze, E. I.; Yurkevich, A. M.; Kravchenko, S.
K.; Ataullakhanov, F. I. In New Research on Biotechnology and Medicine; Egorov,
A. M., Zaikov, G., Eds.; Nova Science Publishers: New York, 2006; p 87.
2. Pettengell, R.; Coiffier, B.; Narayanan, G.; de Men-doza, F. H.; Digumarti, R.;
Gomez, H.; Zinzani, P. L.; Schiller, G.; Rizzieri, D.; Boland, G.; Cernohous, P.;
Wang, L.; Kuepfer, C.; Gorbatchevsky, I.; Singer, J. W. Lancet Oncol. 2012, 13,
696.
6
5
4
3
2
1
0.8 0.6 0.4 0.2 0.1
0
[µM]
G1 Phase S Phase
G2M Phase
3. Krapcho, A. P.; Gallagher, C. E.; Hammach, A.; Hacker, M. P.; Menta, E.; Oliva, A.;
Di Domenico, R.; Da Re, G.; Lotto, A.; Spinelli, S. J. Heterocycl. Chem. 1998, 35,
895.
4. Tomillero, A.; Moral, M. A. Methods Find. Exp. Clin. Pharmacol. 2009, 31, 661.
5. Shanab, K.; Schirmer, E.; Knafl, H.; Wulz, E.; Holzer, W.; Spreitzer, H.; Schmidt,
P.; Aicher, B.; Mueller, G.; Guenther, E. Bioorg. Med. Chem. Lett. 2010, 20, 3950.
6. Pongprom, N.; Bachitsch, H.; Bauchinger, A.; Ettefagh, H.; Haider, T.; Hofer, M.;
Knafl, H.; Slanz, R.; Waismeyer, M.; Wieser, F.; Spreitzer, H. Monatsh. Chem.
2010, 141, 53.
7. Shanab, K.; Pongprom, N.; Wulz, E.; Holzer, W.; Spreitzer, H.; Schmidt, P.;
Aicher, B.; Müller, G.; Günther, E. Bioorg. Med. Chem. Lett. 2007, 17, 6091.
8. Ohno, M.; Miyamoto, M.; Hoshi, K.; Takeda, T.; Yamada, N.; Ohtake, A. J. Med.
Chem. 2005, 48, 5279.
9. Labelle, M.; Falgueyret, J.-P.; Riendeau, D.; Rokach, J. Tetrahedron 1990, 46,
6301.
Figure 3. Cell cycle arrest results for aziridines 26 and 23. While no cell cycle arrest
is observed by treatment with up to 11 M of aziridine 26, compound 23 induces S-
phase arrest with an inverse-shaped dose–response, peaking between 1 and 0.2 M,
and flanked by transition to normal cell cycle distribution at concentrations above
and below this concentration range. Similar data are shown in the supporting
materials section.
l
l
10. Collins, C. J.; Hanack, M.; Stutz, H.; Auchter, G.; Schoberth, W. J. Org. Chem.
1983, 48, 5260.
11. Delorme, D.; Girard, Y.; Rokach, J. J. Org. Chem. 1989, 54, 3635.
12. Eya, B. K.; Otsuka, T.; Kubo, I.; Wood, D. L. Tetrahedron 1990, 46, 2695.
13. Braddock, D. C.; Bhuva, R.; Millan, D. S.; Perez-Fuertes, Y.; Roberts, C. A.;
Sheppard, R. N.; Solanki, S.; Stokes, E. S. E.; White, A. J. P. Org. Lett. 2007, 9, 445.

1852
M. Shahabi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1846–1852
14. Zacharie, B.; Gagnon; Attardo, G.; Connolly, T. P.; St-Denis, Y.; Penney, C. L. J.
Med. Chem. 1997, 40, 2883.
15. Abd El Samii, Z. K. M.; Al Ashmaney, M. J.; Mellor, J. M. J. Chem Soc., Perkin Trans.
1 1988, 2517.
and were grown for 24 h at 37 °C/5% CO2. Cell numbers were adapted for each
cell line to generate signals in the linear detection range under the
experimental conditions applied. After 45 h of compound incubation at
37 °C/5% CO2 15 ll of the Resazurin detection reagent (0.2 mg/mL in DPBS
16. Denny, W. A.; Cain, B. F. J. Med. Chem. 1978, 21, 430.
17. Becker, H. G. O.; Berger, W.; Domschke G. Organikum, 21. Auflage, Deutscher
Verlag der Wissenschaften, Weinheim, 2001. p. 475.
18. Street, L. J.; Baker, R.; Castro, J.; Chambers, M. S.; Guiblin, A. R.; Hobbs, S. C.;
Matassa, V. G.; Reeve, A. J.; Beer, M. S. J. Med. Chem. 1993, 36, 1529.
19. Kupczyk-Subotkowska, L.; Siahaan, T. J.; Basile, A. S.; Friedman, H. S.; Higgins,
P. E.; Song, D.; Gallo, J. M. J. Med. Chem. 1997, 40, 1726.
(Gibco, 14190), steril filtered) was added for additional 3 h and after a total of
48 h of compound incubation cellular metabolic activity was quantified by
measurement of fluorescence at 590 nm. Non-treated cells and blank controls
w/o cells were set as reference values.
MS EXCEL was used for formating and analysis of data. All data were calculated
as % efficacy compared to the mean of the respective negative (non-treated
cells) and positive control wells (blank) on each assay plate. EC50 values
(Table 3) were calculated by using non-linear regression software GraphPad
Prism.
20. Pichler, A. Ph.D. Thesis, University of Vienna, 1996.
21. Wissner, A.; Carroll, M. L.; Johnson, B. D.; Kerwar, S. S.; Pickett, W. C.; Schaub, R.
E.; Torley, L. W.; Trova, M. P.; Kohler, C. A. J. Med. Chem. 1992, 35, 4779.
22. Kobler, H.; Schuster, K. H.; Simchen, G. Liebigs Ann. Chem. 1978, 1946.
23. Schmidt, M.; Lu, Y.; Liu, B.; Fang, M.; Mendelsohn, J.; Fan, Z. Oncogene 2000, 19,
2423.
24. Assessment of cytotoxic/antiproliferative activity was conducted with five
diverse tumor cell lines, in which inducible expression of the cell cycle
inhibitor p27Kip1 leads to cell arrest (see Ref. 23).
25. Stock solution of reference and test compounds up to 5 mM concentration in
5% (v:v) DMSO or methanol were prepared. The compound solution was
diluted stepwise in reaction buffer (5 mM NaH2PO4, 5 mM Na2HPO4, 70 mM
NaCl, pH7) and a compound concentration resulting in absorbance [Abs max]
of approx. 1–2 between 230 and 600 nm using a Spectramax 190 plus reader
(Molecular Devices, Sunnyvale, CA) was determined. Finally, curves for these
compound concentrations were compared with or without 2 mg/ml (w:v) calf
thymus DNA (Sigma–Aldrich Corp., St. Louis, MO).
26. Flow cytometry analysis was done at FACSCalibur™ cytometer (Becton
Dickinson, Heidelberg, GER). For cell cycle analysis, KB/HeLa cells (ATCC
CCL17) were exposed to test compounds for 24 h @ 37 °C. Cell preparation was
done with CycleTEST™ PLUS kit. The number of cells in G2/M phase was
calculated by cell cycle analysis software (Mod Fit LT; VERITY) and EC50-values
were calculated by nonlinear regression.
Measurement of the cellular cytotoxic/antiproliferative activity is based on the
dye Resazurin (Sigma, cat. no. R7017), which exhibits fluorescence change in
the appropriate oxidation-reduction range relating to cellular metabolic
reduction (Nociari et al., J. Immunol. Methods 1998, 213, 157) yielding
fluorescence signal at 590 nm. The increase of fluorescence at 590 nM is an
indicator of cellular viability/cell number.
a
The cells were seeded in the respective growth medium recommended by the
supplier (media and reagents purchased from Gibco-BRL) in 125 ll per 96 well