The discovery of new cytotoxic pyrazolopyridine derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.09.056

A number of new 3,7-disubstituted pyrazolo[3,4-c]pyridines have been designed and synthesized from suitable 2-aminopyridines. The antiproliferative activity of the derivatives was determined against the pancreatic MIA PaCa-2 and ovarian SCOV3 cancer cell-lines. IC₅₀values of the most promising analogue 46 lie in the submicromolar or low micromolar range. Furthermore, compound 46 shows similar inhibitory activities against DU145, A2058 and PC-3 cancer cells, blocks the cell cycle at the G₀/G₁phase and induce apoptosis, as determined by the appearance of apoptotic nuclei.

Full text of DOI:10.1016/j.bmcl.2016.09.056

Accepted Manuscript
The discovery of new cytotoxic pyrazolopyridine derivatives
Vassiliki Giannouli, Nikolaos Lougiakis, Ioannis K. Kostakis, Nicole Pouli,
Panagiotis Marakos, Alexios-Leandros Skaltsounis, Sangkil Nam, Richard
Jove, David Horne, Roxane Tenta, Harris Pratsinis, Dimitris Kletsas
PII:
S0960-894X(16)30997-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.056
BMCL 24280
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 July 2016
22 September 2016
23 September 2016
Please cite this article as: Giannouli, V., Lougiakis, N., Kostakis, I.K., Pouli, N., Marakos, P., Skaltsounis, A-L.,
Nam, S., Jove, R., Horne, D., Tenta, R., Pratsinis, H., Kletsas, D., The discovery of new cytotoxic pyrazolopyridine
derivatives, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.09.056
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The discovery of new cytotoxic pyrazolopyridine derivatives
Vassiliki Giannouli,a Nikolaos Lougiakis,a Ioannis K. Kostakis,a Nicole Pouli,a* Panagiotis
Marakos,a Alexios-Leandros Skaltsounis,b Sangkil Nam,c Richard Jove,d David Horne,c
Roxane Tenta,e Harris Pratsinisf and Dimitris Kletsasf
a Division of Pharmaceutical Chemistry, Department of Pharmacy, National and Kapodistrian
University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
b
Division of Pharmacognosy & Natural Products Chemistry, Department of Pharmacy,
National and Kapodistrian University of Athens, Panepistimiopolis-Zografou, Athens 15771
Greece
c Beckman Research Institute, Comprehensive Cancer Center City of Hope, Los Angeles, USA
d NSU Cell Therapy Institute, Nova Southeastern University, Fort Lauderdale, FL, USA
e
Department of Science of Nutrition & Dietetics, Harokopio University,70 El. Venizelou Street,
Athens 17671, Greece
f
Laboratory of Cell Proliferation and Ageing, Institute of Biosciences & Applications, NCSR
‘‘Demokritos,’’ 15310 Athens, Greece
Abstract. A number of new 3,7-disubstituted pyrazolo[3,4-c]pyridines have been designed and
synthesized from suitable 2-aminopyridines. The antiproliferative activity of the derivatives was
determined against the pancreatic MIA PaCa-2 and ovarian SCOV3 cancer cell-lines. IC50 values
of the most promising analogue 46 lie in the submicromolar or low micromolar range.
Furthermore, compound 46 shows similar inhibitory activities against DU145, A2058 and PC-3
cancer cells, blocks the cell cycle at the G0/G1 phase and induce apoptosis, as determined by the
appearance of apoptotic nuclei.
Keywords pyrazolo[3,4-c]pyridine; antiproliferative activity; Suzuki coupling; deazapurines;
cell-cycle selectivity
In addition to their contribution in encoding of genetic information, the naturally occurring
purines adenosine and guanosine and their nucleotide derivatives play crucial roles in a number
of biological processes, mainly involved in cell metabolism and cell proliferation.1 Many cellular
proteins contain a purine recognition pocket for their interaction with their corresponding energy
intermediates, substrates, allosteric modulators or cofactors, therefore, numerous purine
derivatives have been designed and developed to selectively inhibit these enzymes and
receptors.2 Depending on their substitution pattern and the wide variety of the substituents, these
compounds were found to exhibit a broad range of biological and pharmaceutical properties.3-6
Besides serving as privileged medicinal chemistry scaffolds, these derivatives also inspired
parallel development of alternative heterocyclic isosteres, in the attempt to explore the
therapeutic potential or improve the physicochemical and pharmacokinetic properties of the
corresponding purine derivatives.7-10 However, all these compounds must closely mimic the
overall shape of the endogenous natural products and retain certain hydrogen bond

donor/acceptor binding motifs, in order to maintain the ability to substitute for them in various
biological processes. Changing the location of a nitrogen atom from position 9- to position 8- in
the purine ring leads to pyrazolopyrimidines, which were initially reported as adenosine receptor
antagonists, and were further found to exert potent anti-inflammatory,11 anti-viral12 and
anticancer properties.13-14 The closely related pyrazolopyridine core, which can be considered as
the bioisosteric deazapyrazolopyrimidine scaffold, can also serve as pharmacophore for the
discovery of biologically interesting compounds.15-16 The anticancer potential of several
pyrazolo[3,4-d]pyridines has been already studied,13 as well as their investigated molecular
mechanisms, such as inhibition of EGFR,17 IGF-1R18 and dual Src/Abl.19
In the course of our involvement in the synthesis and biological activity evaluation of
pyrazolo[3,4-c]pyridine derivatives20-22 we have initiated a project towards the study of the
impact of certain substitution patterns on this interesting scaffold. We thus present herein the
synthesis of some new 3,7-disubstituted pyrazolo[3,4-c]pyridines and their antiproliferative
activity against cancer cell lines.
The new compounds were prepared using as starting materials the commercial aminopicoline 1,
as well as the substituted aminopyridine 6 (Scheme 1). The later compound has not been
previously reported and was synthesized in four steps, according to the reactions depicted in
Scheme 1. Thus, the picoline 1 was first protected through conversion to the corresponding
o
carbamate 2, which was lithiated using 2.5 equiv. of n-butyllithium, in THF at -80 C and the
resulting 4-methylene anion attacked isopropylbromide to provide the pyridine 3. Compound 3
was then deprotected in acidic media and the resulting amine 4 was nitrated to give both
nitroderivatives 5 and 6,which were chromatographically separated and identified.
R
R
NH2
NH2
NO2
N
N
N
N
b
d
+
CH3
NO2
1 R= NH2
3 R= NHBoc
4 R= NH2
5
6
a
c
2 R= NHBoc
Scheme 1. Reagents and conditions. a) Boc2O, t-BuOH, r.t.; b) i) n-BuLi (2.5 eq.), THF dry, -80
˚C, then r.t., ii) isopropylbromide, -80 ˚C, then r.t.; c) HCl (5N), EtOH, r.t.; d) HNO3, H2SO4, 65
˚C.
The synthesis of the target 7-substituted pyrazolo[3,4-c]pyridines as well as the corresponding 3-
isopropyl analogues is depicted in Scheme 2. We used either 2-amino-5-nitro-4-picoline (7),
which was prepared from the picoline 1 following a previously reported methodology23, or the
homologous picoline 6. These aminopyridines were subjected to diazotization and the resulting

pyridinones were treated with phosphorous oxychloride to result in the chlorides 1024 and 11.
The 5-nitro group of the afore mentioned chlorides was reduced, using tin (II) chloride in HCl
and the amines thus prepared, 12 and 13, were acetylated to provide the acetamides 14 and 15.
Each acetamide was then treated with isoamyl nitrite in benzene at reflux in the presence of
acetic anhydride and potassium acetate,25-26 to result upon rearrangement of the intermediate N-
nitroso compound in 1-acetyl-5-chloropyrazolo[3,4-c]pyridine or 1-acetyl-5-chloro-3-
isopropylpyrazolo[3,4-c]pyridine. The acetyl group was cleaved upon treatment with methanolic
ammonia to result in derivatives 1626 and 17. The pyrazole NH of these derivatives was protected
using the 4-methoxybenzyl group, upon reaction with 4-methoxybenzylchloride in the presence
of NaH. In the case of 17, we have obtained selectively the N1-substituted isomer, due to the
presence of the bulky 3-isopropyl group. On the contrary, concerning compound 16, we isolated
both N1 and N2 regio-isomers which were separated and identified using NOE spectroscopic
data.27
1
R
Cl
NH2
H
N
N
N
N
N
a
c
e
N
Cl
R
R
R
R
1
R
NO2
7 R= CH3
NO2
1
1
8 R= CH3, R = OH
12 R= CH3, R = NH2
16 R= H
17 R= CH(CH3)2
6 R= CH2CH(CH3)2
1
1
b
9 R= CH2CH(CH3)2, R = OH
d
13 R= CH2CH(CH3)2, R = NH2
1
1
10 R= CH3, R = Cl
14 R= CH3, R = NHAc
b
d
1
1
f
11 R= CH2CH(CH3)2, R = Cl
15 R= CH2CH(CH3)2, R = NHAc
OCH3
O
OCH3
OCH3
N
OCH3
Ar
Cl
NH
N
N
N
N
N
g
i
N
h
N
N
N
N
N
Cl
Cl
Cl
Cl
R
R
R
R
24 R= H, Ar= C6H5
22 R= H
23 R= CH(CH3)2
20 R= H
21 R= CH(CH3)2
18 R= H
19 R= CH(CH3)2
25 R= CH(CH3)2, Ar= C6H5
26 R= H, Ar= 3,4,5-(CH3O)3C6H2
27 R= CH(CH3)2, Ar= 3,4,5-(CH3O)3C6H2
j
Ar
Ar
NH
NH
H
N
H
N
N
N
k
N
N
Cl
R
R
32 R= H, Ar= C6H5
33 R= CH(CH3)2, Ar= C6H5
28 R= H, Ar= C6H5
29 R= CH(CH3)2, Ar= C6H5
34 R= H, Ar= 3,4,5-(CH3O)3C6H2
30 R= H, Ar= 3,4,5-(CH3O)3C6H2
31 R= CH(CH3)2, Ar= 3,4,5-(CH3O)3C6H2
35 R= CH(CH3)2, Ar= 3,4,5-(CH3O)3C6H2
.
Scheme 2. Reagents and conditions. a) NaNO2, H2SO4, H2O; b) POCl3, 100 °C; c) SnCl2 2H2O,
HCl(c.), 55 °C for 12 or r.t. for 13; d) Ac2O, CH2Cl2, r.t.; e) i) AcOK, Ac2O, isoamyl nitrite,

benzene, reflux, ii) NH3(g.), MeOH, r.t.; f) i) NaH, DMF, r.t., ii) 4-methoxybenzyl chloride,
DMF, r.t.; g) m-CPBA, CHCl3, r.t.; h) POCl3, THF, r.t.; i) NaH, aniline (for 24,25) or 3,4,5-
trimethoxyaniline (for 26,27), DMF, 100 °C; j) trifluoroacetic acid, r.t.; k) Pd/C, H2, AcOK,
EtOH, 50 psi, r.t.
The protected derivatives 18 and 19 were converted to the corresponding N-oxides 2027 and 21,
using m-CPBA as oxidizing agent. The rearrangement of the N-oxides in the presence of
phosphorous oxychloride, resulted in the 5,7-dichloropyrazolopyridines 2227 and 23, which were
used for the nucleophilic substitution of the 7-chloro group using aniline or 3,4,5-
trimethoxyaniline, in order to provide compounds 24-27. These derivatives were then
subsequently deprotected to give the analogues 28-31, which upon catalytic dehalogenation
resulted in the target compounds 32-35.
The preparation of the corresponding 3-phenyl derivatives is reported in Scheme 3. They resulted
from the intermediate 5-chloropyrazolo[3,4-c]pyridine (16), which was easily iodinated upon
treatment with NIS and the resulting 3-iodide 36 was selectively protected at N1, using 4-
methoxybenzylchloride, providing 37.28 The iodide 37 was introduced to Suzuki coupling using
phenylboronic acid in the presence of Pd(PPh3)4 and was converted to the 3-phenyl derivative
38.27 Compound 38 was introduced to a reaction sequence analogous to the one mentioned in
Scheme 2 and provided in five steps the 3-phenyl analogues 45 and 46.
OCH3
O
OCH3
R
N
H
N
N
N
N
N
N
N
N
c
a
d
N
N
N
Cl
Cl
Cl
Cl
I
16
36 R= H
37 R= CH2C6H4(OCH3)-p
38
39
b
e
OCH3
OCH3
Ar
Ar
Ar
NH
NH
NH
Cl
H
N
H
N
N
N
N
N
N
N
g
h
f
N
N
N
N
Cl
Cl
Cl
45 Ar= C6H5
46 Ar= 3,4,5-(CH3O)3C6H2
43 Ar= C6H5
41 Ar= C6H5
42 Ar= 3,4,5-(CH3O)3C6H2
40
44 Ar= 3,4,5-(CH3O)3C6H2
Scheme 3. Reagents and conditions. a) NIS, MeOH, r.t.; b) i) NaH, DMF, r.t., ii) 4-
methoxybenzyl chloride, DMF, r.t.; c) phenylboronic acid, Pd(PPh3)4, NaHCO3,
toluene/ethanol/H2O (10/1/0.2), reflux; d) m-CPBA, CHCl3, r.t.; e) POCl3, THF, r.t.; f) i) NaH,

aniline (for 41) or 3,4,5-trimethoxyaniline (for 42), DMF, 100 °C; g) trifluoroacetic acid, r.t.; h)
Pd/C, H2, AcOK, EtOH, 50 psi, r.t.
The cytotoxic activity of the new derivatives was tested against two cancer cell lines, pancreatic
(MIA PaCa-2) and ovarian (SCOV3), as well as against normal human fibroblasts (WI-38). The
IC50 values of the derivatives are presented in Table 1.
Table 1. IC50 values of the derivatives. MTS assay for cell viability. Experiment in triplicate.
Ar
NH
H
N
N
N
R2
R1
IC50 (µM)
compd.
28
R1
H
R2
Cl
Ar
MIA PaCa-2
36.2 2.2
SCOV3
34.2 2.9
WI-38
>50
H3C
Cl
Cl
21.5 2.4
16.2 0.7
>50
38.7 1.0
>50
29
30
H3C
CH3O
H
13.1 0.7
CH3O
CH3O
CH3O
H3C
H3C
Cl
6.1
1
5.6 0.4
43.8 15.0
31
CH3O
CH3O
H
H
H
H
>50
>50
>50
>50
32
33
H3C
H3C
41.2 3.5
>50
48.3 4.1
CH3O
H
>50
>50
34
CH3O
CH3O

H3C
H3C
CH3O
H
10.0 0.6
22.9 1.7
>50
35
CH3O
CH3O
Cl
Cl
4.3 0.5
6.7 0.6
2.5 0.3
5.9 0.5
23.4 0.7
43
44
CH3O
21.9 12.0
CH3O
CH3O
H
H
9.0 0.9
3.8 0.3
22.1 1.4
24.7 4.3
14.9 0.7
45
46
CH3O
0.73 0.04
CH3O
CH3O
A number of interesting structure-activity relationships can be extracted from the study of these
series of derivatives. Compounds bearing only one substituent at position 7 (32 and 34) are
inactive against both cancer cell lines. On the contrary, their counterparts, possessing a 5-chloro
group (28 and 30) showed marginally interesting cytotoxicity with IC50 values ranging between
13 and 36 µM. This pattern is replicated in the case of the 3-isopropylpyrazolopyridine
analogues, however, in this case, the compounds substituted at position 7 with the (3,4,5-
trimethoxyphenyl)amino group (compounds 31 and 35) possess quite interesting cytotoxic
activity, mainly the 5-chloro analogue 31, the IC50 value of which lies in the low micromolar
level. Overall, the most important finding concerns the activity of the 3-phenylpyrazolopyridines
43-46, which showed uniformly strong to good cytotoxicity. The most noticeable activity was
displayed by compound 46, which bears the 7-(3,4,5-trimethoxyphenyl)amino substituent. The
high antiproliferative activity of compound 46 was also confirmed, when it was tested against
DU145 prostate cancer cell line, where it was found to possess IC50 value 0.92 µM, against
A2058 melanoma cell line, where it exhibited IC50 value 0.89 µM, as well as against PC-3 cells
showing IC50 value 0.38 µM. It is of interest to notice that the precursor chloroderivative 44, was
found to be substantially less active when tested against PC-3 cell-line possessing an IC50 value
15 µM. Interestingly, all tested compounds (with the exception of 29 which was inactive against
SCOV3) were selectively more active against cancer cells compared to normal human ones (WI-
38). This is particularly evident regarding the most active compounds 46, 43, 31 and 44.
Cell-cycle perturbations induced after incubation of exponentially growing PC-3 cells with
compounds 44 and 46 for 48 h are given in Table 2 and in Figure 1. Both compounds inhibited
DNA synthesis of PC-3 cells by blocking cells at the G0/G1 phase. Compound 46 was much
more potent, since it led to the complete annulment of S phase, and, furthermore, it induced
apoptosis, as determined by the appearance of a sub-diploid peak in the DNA-content histogram.

In particular, compound 46 induced the appearance of 25.8% ( 0.3) apoptotic nuclei compared
to 0.7% ( 0.1) induced by the vehicle or 0.9% ( 0.2) by the chloroderivative 44 (Figure 1).
Table 2. Cell cycle phase distribution at 48 hours (%). a Exponentially growing PC-3 cells were
treated with the compounds 44 and 46, at concentration equal to their corresponding IC50 values,
or the corresponding DMSO concentration (vehicle).
Compound
44
G0/G1
S
G2/M
46.2 ( 3.0)
80.8 ( 4.4)
38.3 ( 0.7)
30.3 ( 0.4)
0.0 ( 0.0)
37.7 ( 2.2)
23.5 ( 3.4)
19.2 ( 4.4)
24.0 ( 2.9)
46
vehicle
a Mean ( standard deviation) of two independent experiments
Figure 1. Induction of apoptosis by the test compounds. Percentage of apoptotic nuclei in PC-3
cells treated for 48 hours with the indicated compounds at concentration equal to their
corresponding IC50 values, or with vehicle. Bars represent the mean of two independent
experiments (error bars: SD); ** indicates p<0.01, * indicates p<0.05 (Student's t-test).
In conclusion, we have synthesized a number of new 7-arylaminosubstituted pyrazolo[3,4-
c]pyridines and have determined their antiproliferative activity against pancreatic (MIA PaCa-2)
and ovarian (SCOV3) cancer cell lines. Following the structure-activity relationships extracted
by the target derivatives, we have further investigated the behavior of the most promising
derivative 46, against three additional cancer cell lines, where it exhibited potent antiproliferative

activity as well. This compound blocked the cell cycle at the G0/G1 phase and induced apoptosis
when studied against prostate PC-3 cancer cells.
Acknowledgements
Research reported in this publication included work performed in the Drug Discovery and
Structural Biology Core of City of Hope Comprehensive Cancer Center supported by the
National Cancer Institute of the National Institutes of Health under award number
P30CA033572. The content is solely the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.
Supplementary data concerning experimental procedures for the synthesis and biological
evaluation can be found as supplementary file.
References
1. Rosemeyer, H. Chem. Biodiversity 2004, 1, 361.
2. Ertl, P.; Jelfs, S.; Muhlbacher, J.; Schuffenhauer, A.; Selzer, P. J. Med. Chem. 2006, 49, 4568.
3. Legraverend, M.; Grierson, D. S. Bioorg. Med. Chem. 2006, 14, 3987.
4. Šála, M.; Kögler, M.; Plačková, P; Mejdrová, I.; Hřebabecký, H.; Procházková, E.; Strunin,
D.; Lee, G.; Birkus, G.; Weber, J.; Mertlíková-Kaiserová, H.; Nencka, R. Bioorg. Med. Chem.
Lett. 2016, 26, 2706.
5. Kang, D.; Fang, Z; Huang, B; Zhang, L.; Liu, H.; Pannecouque, C.; Naesens, L.; De Clercq,
E.; Zhan, P.; Liu, X. Chem. Biol. Drug Des. 2015, 86, 568.
6. Pace, S.; Brogin, G.; Stasi, M. A.; Riccioni, T.; Borsini, F.; Capocasa, F.; Manera, F.;
Tallarico, C.; Grossi, P.; Vacondio, F.; Bassi, M.; Bartoccini, F.; Lucarini, S.; Piersanti, G.;
Tarzia, G.; Cabri, W.; Minetti, P. ChemMedChem 2015, 10, 1149.
7. Sajith, A. M.; Abdul Khader, K. K.; Joshi, N.; Reddy, M. N.; Padusha, M. S. A.;
Nagaswarupa, H. P.; Joy, M. N.; Bodke, Y. D.; Karuvalam, R. P.; Banerjee, R.; Muralidharan,
A.; Rajendra, P. Eur. J. Med. Chem. 2015, 89, 21.
8. Demir, Z.; Guven, E. B.; Ozbey, S.; Kazak, C.; Atalay, R. C.; Tuncbilek, M. Eur. J. Med.
Chem. 2015, 89, 701.
9. Cai, Z. W.; Wei, D.; Schroeder, G. M.; Cornelius, L. A. M.; Kim, K.; Chen, X. T.; Schmidt, R.
J.; Williams, D. K.; Tokarski, J. S.; An, Y.; Sack, J. S.; Manne, V.; Kamath, A.; Zhang,Y.;
Marathe, P.; Hunt, J. T.; Lombardo, L. J.; Fargnoli, J.; Borzilleri, R. M. Bioorg. Med. Chem.
Lett. 2008, 18, 3224.
10. Patnaik, S.; Stevens, K. L.; Gerding, R.; Deanda, F.; Shotwell, J. B.; Tang, J.; Hamajima, T.;
Nakamura, H.; Leesnitzer, M. A.; Hassell, A. M.; Shewchuck, L. M.; Kumar, R.; Lei, H.;
Chamberlain S. D. Bioorg. Med. Chem. Lett. 2009, 19, 3136.
11. Bharate, S. B.; Mahajan, T. R.; Gole, Y. R.; Nambiar, M.; Matan, T. T.; Kulkarni-Almeida,
A.; Balachandran, S.; Junjappa, H.; Balakrishnan, A.; Vishwakarma, R. A. Bioorg. Med. Chem.
2008, 16, 7167.

12. Tucker, T. J.; Sisko, J. T.; Tynebor, R. M.; Williams, T. M.; Felock, P. J.; Flynn, J. A.; Lai,
M. T.; Liang, Y.; McGaughey, G.; Liu, M.; Miller, M.; Moyer, G.; Munshi, V.; Perlow-Poehnelt,
R.; Prasad, S.; Reid, J. C.; Sanchez, R.; Torrent, M.; Vacca, J. P.; Wan, B. L.; Yan, Y. J. Med.
Chem. 2008, 51, 6503.
13. Baviskar, A. T.; Banerjee, U. C.; Gupta, M.; Singh, R.; Kumar, S.; Gupta, M. K.; Kumar, S.;
Raut, S. K.; Khullar, M.; Singh, S.; Kumar R. Bioorg. Med. Chem. 2013, 21, 5782.
14. Chauhan, M.; Kumar, R. Bioorg. Med. Chem. 2013, 21, 5657.
15. Orlikova, B.; Chaouni, W.; Schumacher, M.; Aadil, M.; Diederich, M.; Kirsch G. Eur. J.
Med. Chem. 2014, 85, 450.
16. Wenglowsky, S.; Moreno, D.; Rudolph, J.; Ran, Y.; Ahrendt, K. A.; Arrigo, A.; Colson, B.;
Gloor, S. L.; Hastings G. Bioorg. Med. Chem. Lett. 2012, 22, 912.
17. Ducray, R.; Ballard, P.; Barlaam, B. C.; Hickinson, M. D.; Kettle, J. G.; Ogilvie, D. J.;
Trigwell, C. B. Bioorg. Med. Chem. Lett. 2008, 18, 959.
18. Negi, A.; Ramarao, P.; Kumar, R. Mini-Rev. Med. Chem. 2013, 13, 653.
19. Radi, M.; Dreassi, E.; Brullo, C.; Crespan, E.; Tintori, C.; Bernardo, V.; Valoti, M.;
Zamperini, C.; Daigl, H.; Musumeci, F.; Carraro, F.; Naldini, A.; Filippi, I.; Maga, G.; Schenone,
S., Botta, M. J. Med. Chem. 2011, 58, 2610.
20. Kourafalos, V. N.; Marakos, P.; Pouli, N.; Townsend, L. B. J. Org. Chem. 2003, 68, 6466.
21. Tite, T.; Lougiakis, N.; Marakos, P.; Pouli, N. Synlett 2009, 2927.
22. Tite, T.; Lougiakis, N.; Myrianthopoulos, V.; Marakos, P.; Mikros, E.; Pouli, N.; Tenta, R.;
Fragopoulou, E.; Nomikos, T. Tetrahedron 2010, 66, 9620.
23. Pino, L. N.; Zehrung W. S. III. J. Am. Chem. Soc. 1955, 77, 3154.
24. Roe, A.; Seligman R. B. J. Org. Chem. 1955, 20, 1729.
25. Marakos, P.; Pouli, N.; Wise, D. S.; Townsend, L. B. Synlett 1997, 561.
26. Chapman, D.; Hurst, J. J. Chem. Soc., Perkin Trans. 1, 1980, 2398.
27. Michailidou, M.; Giannouli, V.; Kotsikoris, V.; Papadodima, O.; Kontogianni, G.; Kostakis,
I. K.; Lougiakis, N.; Chatziioannou, A.; Kolisis, F. N.; Marakos, P.; Pouli, N.; Loutrari, H. Eur.
J. Med. Chem. 2016, 121, 143.
28. Hanan, E. J.; Eigenbrot, C.; Bryan, M. C.; Burdick, D. J.; Chan, B. K.; Chen, Y.; Dotson, J.;
Heald, R. A.; Jackson, P. S.; La, H.; Lainchbury, M. D.; Malek, S.; Purkey, H. E.; Schaefer, G.;
Schmidt, S.; Seward, E. M.; Sideris, S.; Tam, C.; Wang, S.; Yeap, S. K.; Yen, I.; Yin, J.; Yu, C.;
Zilberleyb, I.; Heffron, T. P. J. Med. Chem. 2014, 57, 10176.
29. Nam, S.; Wen, W.; Schroeder, A.; Herrmann, A.; Yu, H.; Cheng, X.; Merz, K. H.;
Eisenbrand, G.; Li, H.; Yuan, Y. C.; Jove, R. Mol. Oncol. 2013, 7, 369.
30. Kostakis, I. K.; Tenta, R.; Pouli, N.; Marakos, P.; Skaltsounis, A.-L.; Pratsinis, H.; Kletsas,
D. Bioorg. Med. Chem. Lett. 2005, 15, 5057.
31. Gong, J. P.; Traganos, F.; Darzynkiewicz, Z. Anal. Biochem. 1994, 218, 314.

Graphical abstract
NH2
NHAr
OMe
H
H
N
N
MeO
MeO
N
N
N
N
N
CH2R1
Cl
NH
H
N
R2
R2
NO2
R1= H, CH(CH3)2
N
R2= H, CH(CH3)2, Ph
N
46
IC50 (µM): MIA PaCa-2: 3.8; SCOV3: 0.73; DU145:
0.92; A2058: 0.89; PC-3: 0.38