Synthesis and evaluation of 2-pyridinylpyrimidines as inhibitors of HIV-1 structural protein assembly

Article abstract of DOI:10.1016/j.bmcl.2016.06.039

In an effort to identify an HIV-1 capsid assembly inhibitor with improved solubility and potency, we synthesized two series of pyrimidine analogues based on our earlier lead compound N-(4-(ethoxycarbonyl)phenyl)-2-(pyridine-4-yl)quinazoline-4-amine. In vitro binding experiments showed that our series of 2-pyridine-4-ylpyrimidines had IC₅₀values higher than 28 μM. Our series of 2-pyridine-3-ylpyrimidines exhibited IC₅₀values ranging from 3 to 60 μM. The congeners with a fluoro substituent introduced at the 4-N-phenyl moiety, along with a methyl at C-6, represent potent HIV capsid assembly inhibitors binding to the C-terminal domain of the capsid protein.

Full text of DOI:10.1016/j.bmcl.2016.06.039

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 2-pyridinylpyrimidines as inhibitors of
HIV-1 structural protein assembly
a
a
Milan Kozíšek , Ondrej Štepánek , Kamil Parkan a,y, Carlos Berenguer Albiñana b, Marcela Pávová a,c
,
ˇ
ˇ
ˇ
Jan Weber a, Hans-Georg Krӓusslich c, Jan Konvalinka a,d, Aleš Machara b,
⇑
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences and IOCB Research Center, Flemingovo n. 2, 166 10 Prague 6,
Czech Republic
b Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43 Prague 2, Czech Republic
c Department of Infectious Diseases, Virology, University Hospital Heidelberg, Im Neuenheimer Feld 324, 691 20 Heidelberg, Germany
d Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128 43 Prague 2, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to identify an HIV-1 capsid assembly inhibitor with improved solubility and potency, we syn-
thesized two series of pyrimidine analogues based on our earlier lead compound N-(4-(ethoxycarbonyl)
phenyl)-2-(pyridine-4-yl)quinazoline-4-amine. In vitro binding experiments showed that our series of
Received 24 May 2016
Revised 14 June 2016
Accepted 15 June 2016
Available online xxxx
2-pyridine-4-ylpyrimidines had IC50 values higher than 28
lM. Our series of 2-pyridine-3-ylpyrimidines
exhibited IC50 values ranging from 3 to 60 M. The congeners with a fluoro substituent introduced at the
l
4-N-phenyl moiety, along with a methyl at C-6, represent potent HIV capsid assembly inhibitors binding
to the C-terminal domain of the capsid protein.
Keywords:
Assay
Assembly
Capsid
Ó 2016 Elsevier Ltd. All rights reserved.
Inhibition
Pyrimidine
Human immunodeficiency virus type 1 (HIV-1) is the causative
agent of acquired immunodeficiency syndrome (AIDS). Since the
introduction of highly active antiretroviral therapy (HAART), the
mortality of AIDS patients has declined, and the life expectancy
of people with HIV infection has increased markedly. However,
treatment success can be compromised by the unwanted side-
effects of current medications and by selection of drug-resistant
viruses. Therefore, there remains a demand for novel approaches
and targets for therapeutic intervention in the viral life cycle.1,2
The HIV-1 virion is built up by assembly of approximately 2500
Gag polyprotein molecules that are organized inside the budding
virions.3 For the virus to mature, these polyproteins have to be
specifically processed by the viral protease into final viral proteins,
including matrix, nucleocapsid, small spacer peptides, and capsid
(CA).4–6 The latter possesses N- and C-terminal domains that are
tethered by a flexible linker. The conical shape of the mature HIV
capsid enclosing the viral genome stems from its architecture—
the CA subunits form hexamers and pentamers, contributing to
the overall fullerene-type structure.
At present, there is no HIV-1 assembly inhibitor on the market.
There are few reports of non-peptidic compounds targeting CTD of
CA.7–11 The other reported compounds bind to the NTD of CA and
some of them affect capsid assembly. Three independent binding
sites have been defined in the NTD. Acylhydrazones, thioureas,
benzodiazepines, benzimidazoles and the urea-based derivative
CAP-1, bind to the hydrophobic pocket at the base of the helical
bundle.12–16 A second binding site interacts with the ligand PF-
3450074.17 A third ligand-binding site lies near the flexible cyclo-
philin A binding loop.18 In general, the reported CA NTD-binding
compounds exhibit mostly micromolar affinity towards CA
in vitro and weak antiviral activity. However, several effective
nanomolar inhibitors have been reported.15,17
Abbreviations: AIDS, acquired immunodeficiency syndrome; CA, capsid; CAI,
capsid assembly inhibitor; CTD, C-terminal domain; HAART, highly active antiretro-
viral therapy; HIV, human immunodeficiency virus; ITC, isothermal titration
calorimetry; NMR, nuclear magnetic resonance; TLC, thin layer chromatography;
TBTU, N,N,N0,N0-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate.
The discovery by Sticht et al.19 of a 12-mer peptide (CAI) that is
capable of binding to the CA C-terminal domain (CTD) and inhibits
assembly of immature and mature-like particles in vitro enabled
the development of a competitive binding assay designed to select
CTD ligands. This new screening assay for assembly inhibitors
⇑
Corresponding author. Tel.: +420 221 95 1325.
E-mail address: macharaa@natur.cuni.cz (A. Machara).
Department of Chemistry of Natural Compounds, University of Chemistry and
y
Technology, Technická 5, Prague 166 28, Czech Republic.
http://dx.doi.org/10.1016/j.bmcl.2016.06.039
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
ˇ
Please cite this article in press as: Kozíšek, M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.039

ˇ
M. Kozíšek et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
2
Table 1
O
Structure–activity relationship of the pyridine-4-yl derivatives
COOEt
R2
HN
N
HN
N
NH
N
N
N
R1
N
N
N
N
Figure 1. 4-Aminophenylquinazoline derivatives that bind to the C-terminal
Compound
R2
IC50
28
(lM)
domain of the HIV-1 capsid protein.7
11*
3
COOEt
COOEt
O
Cl
12*
>100
(i)
(ii)
NH2.HCl
NH
N
R1
N
HN
R1
N
13*
14*
>100
>100
C(=O)NH(CH2)2F
Ac
Y
Y
Y
X
X
X
1
3-6
7-10
: X=CH, Y=N
2: X=N, Y=CH
F
R2
15*
81
36
8
NH
COOH
F
HN
N
(iii-iv)
(vi,iv)
(v)
N
16**
17**
18**
3
COOEt
N
R1
N
R1
Y
X
>100
C(=O)NH(CH2)2F
Ac
Y
11-21
: X=CH, Y=N
22-45: X=N, Y=CH
X
54
5
Scheme 1. Synthesis of pyrimidines 11–45. R1 = H, CH3. The following reagents and
conditions were used: (i) ethyl formylacetate sodium salt, ethanol, reflux or methyl
acetoacetate, NaHCO3, ethanol, reflux; (ii) neat POCl3, 100 °C; (iii) aniline, HCl,
dioxane, reflux or HCl, ethanol, water, reflux; (iv) HCl in dioxane; (v) aminobenzoic
acid, HCl, ethanol, water, reflux; (vi) amine, TBTU, Et3N, DMF.
Ac
F
19**
20**
21**
>100
>100
C(=O)NH(CH2)2F
F
Ac
utilizes an amplified luminescent proximity assay system
(AlphaScreen). Briefly, interaction of CAI linked to donor beads
with CA CTD on acceptor beads results in a strong luminescent sig-
nal originating from energy transfer from excited donor to acceptor
beads. The presence of an effective assembly inhibitor causes the
beads to separate, resulting in decreased luminescence.
We previously identified a set of unique capsid assembly inhibi-
tors binding to CA CTD from a high throughput screen.7 Because
the inhibitors compete with CAI, we proposed that they bind into
a conserved hydrophobic groove in CA CTD.20 We further opti-
mized the identified hits to improve the inhibitory activity, result-
ing in the compounds shown in Figure 1. These compounds exhibit
moderate affinity to the binding site in vitro, as determined by
AlphaScreen assay and ITC.7
To improve the pharmacokinetics and binding of those quinazo-
line compounds, we searched for derivatives with superior solubil-
ity. From previous work,7 it was obvious that the 4-N-phenyl and
2-pyridinyl moieties are crucial for binding. Therefore, we decided
to replace the parent quinazoline scaffold with a smaller, and less
hydrophobic, heterocyclic pyrimidine.
Here, we report evaluation of two series of N-4-phenyl-2-
pyridinylpyrimidines that are substituted at the N-phenyl moiety.
Pyridine-4-ylpyrimidines (compounds 11–21) and pyridine-3-
ylpyrimidines (compounds 22–45), shown in Scheme 1, are
bioisosteres that act as new classes of capsid assembly inhibitors
binding to CA CTD.
83
7
The IC50 value was determined as the compound concentration sufficient for
decreasing the AlphaScreen signal by 50%. For experimental details, see Supple-
mentary materials.
2,4-Disubstituted pyrimidine derivative (R1 = H).
2,4,6-Trisubstituted pyrimidine derivative (R1 = Me).
*
**
The compounds reported here were synthesized according to
the general route outlined in Scheme 1. The commercially available
3-/4-cyanopyridines were first converted to corresponding amidi-
nes 1 and 2 by treatment with sodium methoxide and later with
ammonium chloride.21 The resulting amidines were either com-
bined with the sodium salt of ethyl formylacetate, which was pre-
pared by aldol condensation of ethyl acetate with ethyl formate, or
with methyl acetoacetate to yield pyrimidinones 3–6.21,22
Subsequent treatment23 with phosphorus oxychloride yielded
4-chloropyrimidines 7–10, which were subjected to nucleophilic
displacement of the chlorine atom by variously substituted ani-
lines.7,24 To prepare the carboxamide series (13, 17, 20, 28–31,
39–44), a different synthetic approach was used. An appropriate
chloropyrimidine was first treated with aminobenzoic acid, and
when TLC indicated its disappearance, the entire reaction mixture
was evaporated to dryness and subjected to standard amidation
using TBTU as a coupling agent. We found that this stepwise
approach was superior to the nucleophilic aromatic substitution
carried out with corresponding N-alkyl aminobenzamides in terms
of yield and versatility. Finally, all prepared compounds were con-
verted into the corresponding hydrochloride salts.
We explored the structure–activity relationship (SAR) of these
compounds with regard to substitution of the N-phenyl moieties.
From previous work,7 we concluded that the attachment of
electron-withdrawing groups lead to the decrease of in vitro IC50
values determined by AlphaScreen assay. In particular, carbonyl
and carboxyl functionalities were superior to sulfonamides and
carboxamides, except for N-2-fluoroethylcarboxamide.
We started with preparation of pyridine-4-ylpyrimidines,
which are congeners of the compounds shown in Figure 1. The first
round of SAR studies revealed that ablation of the annulated ben-
zene ring from the parent quinazoline led to a significant decrease
ˇ
Please cite this article in press as: Kozíšek, M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.039

ˇ
M. Kozíšek et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 2
Table 3
Structure–activity relationship of the 2-(pyridine-3-yl) pyrimidine derivatives
Structure–activity relationship of the 6-methyl-2-(pyridine-3-yl) pyrimidine
derivatives
R2
NH
R2
NH
N
N
N
N
N
N
Compound
R2
IC50
19
(lM)
Compound R2
IC50
M)
EC50
M)
CC50
M)
(
l
(
l
(
l
22
1
COOEt
COOEt
33
34
21
12
3
2
n.d.
n.d.
COOEt
Ac
23
24
21
2
50 12
77 39
EtOOC
F
Ac
F
>100
35
36
37
10
25
9
4
3
23
1
64 10
n.d.
25
26
27
28
29
30
53
39
34
2
COOEt
Ac
n.d.
Ac
F
8
5
4
Ac
Ac
2
>100
n.d.
>100
Ac
F
38
>100
n.d.
C(=O)NH(CH2)2F
C(=O)NH(CH2)2F
C(=O)NH(CH2)2F
F
F
43 15
73 12
39
40
8
3
1
1
25
20
1
44
35
7
3
C(=O)NH(CH2)2F
C(=O)NH(CH2)2F
C(=O)NH(CH2)2F
F
F
1
30
31
32
C(=O)NH(CH2)2F
41
42
15
52
4
6
>100
n.d.
>100
n.d.
68
38
9
5
F
F
C(=O)NH(CH2)2F
F
F
O
The IC50 value was determined as the compound concentration sufficient for
decreasing the AlphaScreen signal by 50%. For experimental details, see
Supplementary materials.
N
N
F
43
44
20
54
7
8
n.d.
n.d.
n.d.
n.d.
C6H4
O
O
N
N
in inhibition potency, as evident from the IC50 values of com-
pounds 11–14 (Table 1). We hypothesized that the lack of benzene
ring in these compounds affected their lipophilicity, and therefore
we attempted to compensate for the absence of a benzene ring by
introduction of a methyl group at C-6 (16–19). However, this alter-
ation did not lead to improved inhibition potency.
Next, we introduced an additional fluorine atom on the N-phe-
nyl moiety of compounds 17 and 19, because derivative 15 with a
3,4-difluorophenyl moiety revealed some, albeit weak, inhibition.
The resulting compounds 20 and 21 did not benefit from the pres-
ence of a fluorine atom.
The second SAR round was performed with an isomeric scaffold
possessing a pyridine-3-yl moiety (Table 2). Based on the inhibi-
tory activities of carboxyl and carbonyl derivatives 22–23 and
26–27, it is evident that this series has superior binding capabili-
ties than that presented in Table 1. Furthermore, the solubility of
all prepared pyridine-3-yl derivatives in aqueous systems was bet-
ter than that of pyridine-4-yl congeners. The screening revealed
that an ethyl ester moiety is more suitable than an acetyl moiety.
Furthermore, the SAR data indicate that meta and para substituted
compounds have similar potency, whereas ortho substituted
ethoxycarbonyl derivative 24 was inactive. As seen with the first
C6H4
O
F
F
45
22
4
n.d.
n.d.
F
The IC50 value was determined as the compound concentration sufficient for
decreasing the AlphaScreen signal by 50%. Anti-HIV-1 activity (EC50) was calculated
as the concentration of compound required to inhibit virus replication in MT-4 cells
by 50%. The CC50 value represents the compound concentration reducing the cell
viability by 50%. For experimental details, see Supplementary information. n.d.
= not determined.
series, derivatives with an additional fluorine atom appeared to
be less active (compare 22 and 28 with 25, 29, 30 and 31).
In the third SAR round Table 3, we intended to explore the effect
of a methyl group introduced at C-6 of the pyrimidine core, similar
to what was done with the first series (see 16–21). The inhibition
activity of ethyl ester derivative 33 was unaffected by the addition
of a methyl substituent. Surprisingly, acetyl derivatives 34 and 35
appeared to be much more sensitive to the methyl introduction.
ˇ
Please cite this article in press as: Kozíšek, M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.039

ˇ
M. Kozíšek et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
4
Their binding was significantly improved by the presence of a
methyl moiety (compare 26 and 27 with 34 and 35). Fluorinated
acetyls 36–38 were of comparable potency or were weaker, which
is in good agreement with the data shown in Table 2.
for his valuable comments and Hillary E. Hoffman for manuscript
editing.
Supplementary data
Introduction of 2-fluoroethylcarboxamide led to the discovery
of compound 39, which has one of the highest inhibition potencies
found in this SAR cycle. To further explore the potential of the
2-fluoroethylcarboxamide derivative, we prepared three monoflu-
orinated congeners. Introduction of the fluorine atom at the meta
position (beside the carboxamide, see 40) furnished the tightest
ligand binding to CA CTD. In addition to primary amides, we also
prepared two secondary amides containing a piperazine scaffold
with a fluorophenyl (43) or carbonyl (44) moiety at N-4. Both com-
pounds exhibited moderate micromolar binding to CA in the com-
petitive assay. Trifluorophenyl derivative 45 had similar inhibitory
activity as its congener 32 and displayed a one-order-of-magnitude
weaker affinity to CA compared to 40, the tightest binding com-
pound reported here (Table 3).
Data contain procedures used for preparation of intermediates;
synthesis of pyrimidines; general procedure for the synthesis of
pyrimidine-4-amines; AlphaScreen methods; competition of sol-
uble free CAI, scrambled control peptide CAIctrl, compounds 40
and 20 with CAI-biotin for binding to GST-CA measured by
AlphaScreen; anti-HIV-1 and cytotoxicity testing; copies of 1H
and 13C NMR spectra.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.06.
039. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
The anti-HIV-1 activities (EC50) of the compounds were deter-
mined by measuring inhibition of viral infectivity in MT-4 cells,
1. De Clercq, E. Adv. Pharmacol. 2013, 67, 317.
2. Tintori, C.; Brai, A.; Fallacara, A. L.; Fazi, R.; Schenone, S. Curr. Opin. Pharmacol.
2014, 18, 1.
in parallel with
a cytotoxicity assay (for details, see the
Supplementary material). Briefly, two-fold serial dilutions of each
compound were added to MT-4 cells infected with the HIV-1 labo-
ratory strain NL4-3 expressing human Renilla luciferase. Virus
replication was assessed after 3 days by measuring luciferase activ-
ity. Cell toxicity was evaluated by XTT assay.
We observed weak but significant and reproducible antiviral
activity for compounds 35, 39 and 40. These compounds also
scored well in the competitive CA binding assay. As expected, bind-
ing to HIV CA per se is not sufficient to ensure antiviral activity.
Many factors, including cell entry through the intact plasma mem-
brane, serum protein binding and compound stability, can compro-
mise the overall antiviral activity of the compounds. In addition,
we detected slight cytotoxicity for some of the active compounds
(see comparison of EC50 and CC50 values).
In summary, we have explored the SAR of pyridinylpyrimidines
as in vitro HIV capsid ligands that bind to the CTD of the CA pro-
tein. We have described three series of pyridinylpyrimidines that
exhibited improved activities upon substitution with methyl at
C-6 along with introduction of a fluoro functionality at the N-4-
phenyl moiety. Thus, our studies indicate that quinazoline, which
was the central core in our previously reported inhibitors, can be
replaced with a pyrimidine ring. Compounds containing the pyri-
dine-3-yl moiety tended to have better solubility and proved to
be superior CA binders compared to pyridine-4-ylpyrimidines.
Compound 40, with a 2-fluoroethyl carbamide motif, exhibited
the tightest binding with a moderate activity against HIV-1 in tis-
sue culture. Further studies focused on fluorinated pyridinylpyrim-
idines are needed to fully analyze the potential of this class of
compounds as HIV assembly inhibitors.
3. Konvalinka, J.; Krausslich, H. G.; Muller, B. Virology 2015, 479.
4. Sundquist, W. I.; Krausslich, H. G. Cold Spring Harbor Perspect. Med. 2012, 2,
a006924.
5. Briggs, J. A.; Krausslich, H. G. J. Med. Biol. 2011, 410, 491.
6. Ganser-Pornillos, B. K.; Yeager, M.; Pornillos, O. Adv. Exp. Med. Biol. 2012, 726,
441.
ˇ
ˇ
7. Machara, A.; Lux, V.; Kozíšek, M.; Grantz Šašková, K.; Štepánek, O.; Kotora, M.;
Parkan, K.; Pávová, M.; Glass, B.; Sehr, P.; Lewis, J.; Müller, B.; Krausslich, H. G.;
Konvalinka, J. J. Med. Chem. 2016, 59, 545.
8. Machara, A.; Konvalinka, J.; Kotora, M. ChemistrySelect 2016, 1, 101.
9. Lampel, A.; Bram, Y.; Ezer, A.; Shaltiel-Kario, R.; Saad, J. S.; Bacharach, E.; Gazit,
E. ACS Chem. Biol. 2015, 10, 1785.
10. Curreli, F.; Zhang, H.; Zhang, X.; Pyatkin, I.; Zagorodnikov, V.; Altieri, A.;
Debnath, A. K. Bioorg. Med. Chem. 2011, 19, 77.
11. Thenin-Houssier, S.; de Vera, I. M. S.; Pedro-Rosa, L.; Brady, A.; Richard, A.;
Konnick, B.; Opp, S.; Buffone, C.; Fuhrmann, J.; Kota, S.; Billack, B.; Pietka-Ottlik,
M.; Tellinghuisen, T.; Choe, H.; Spicer, T.; Scampavia, L.; Diaz-Griffero, F.;
Kojetin, D. J.; Valente, S. T. Antimicrob. Agents Chemother. 2016, 60, 2195.
12. Kelly, B. N.; Kyere, S.; Kinde, I.; Tang, C.; Howard, B. R.; Robinson, H.; Sundquist,
W. I.; Summers, M. F.; Hill, C. P. J. Mol. Biol. 2007, 373, 355.
13. Tian, B.; He, M.; Tang, S.; Hewlett, I.; Tan, Z.; Li, J.; Jin, Y.; Yang, M. Bioorg. Med.
Chem. Lett. 2009, 19, 2162.
14. Li, J.; Tan, Z.; Tang, S.; Hewlett, I.; Pang, R.; He, M.; He, S.; Tian, B.; Chen, K.;
Yang, M. Bioorg. Med. Chem. 2009, 17, 3177.
15. Lemke, Ch. T.; Titolo, S.; von Schwedler, U.; Goudreau, N.; Mercier, J. F.;
Wardrop, E.; Faucher, A. M.; Coulombe, R.; Banik, S. S. R.; Fader, L.; Gagnon, A.;
Kawai, S. H.; Rancourt, J.; Tremblay, M.; Yoakim, Ch.; Simoneau, B.;
Archambault, J.; Sundquist, W. I.; Mason, S. W. J. Virol. 2012, 86, 6643.
16. Jin, Y.; Tan, Z.; He, M.; Tian, B.; Tang, S.; Hewlett, I.; Yang, M. Bioorg. Med. Chem.
2010, 18, 2135.
17. Blair, W. S.; Pickford, C.; Irving, S. L.; Brown, D. G.; Anderson, M.; Bazin, R.; Cao,
J.; Ciaramella, G.; Isaacson, J.; Jackson, L.; Hunt, R.; Kjerrstrom, A.; Nieman, J. A.;
Patrick, A. K.; Perros, M.; Scott, A. D.; Whitby, K.; Wu, H.; Butler, S. L. PLoS
Pathog. 2010, 6, e1001220.
18. Goudreau, N.; Lemke, Ch. T.; Faucher, A. M.; Grand-Maitre, Ch.; Goulet, S.;
Lacoste, J. E.; Rancourt, J.; Malenfant, E.; Mercier, J. F.; Titolo, S.; Mason, S. W.
ACS Chem. Biol. 2013, 8, 1074.
19. Sticht, J.; Humbert, M.; Findlow, S.; Bodem, J.; Muller, B.; Dietrich, U.; Werner,
J.; Krausslich, H. G. Nat. Struct. Mol. Biol. 2005, 12, 671.
20. Ternois, F.; Sticht, J.; Duquerroy, S.; Krausslich, H. G.; Rey, F. A. Nat. Struct. Mol.
Biol. 2005, 12, 678.
Acknowledgments
21. Medwid, J. B.; Paul, R.; Baker, J. S.; Brockman, J. A.; Du, M. T.; Hallett, W. A.;
Hanifin, J. W.; Hardy, R. A.; Tarrant, M. E.; Torley, L. W.; Wrenn, S. J. Med. Chem.
1990, 33, 1230.
22. Lesher, G. Y.; Singh, B.; Mielens, Z. E. J. Med. Chem. 1982, 25, 837.
23. Maurya, H. K.; Gupta, A. RSC Adv. 2014, 4, 22106.
This work was supported by the Grant Agency of the Czech
Republic (grant 13-19561S) and by a program of the University
Excellence Center of Charles University (UNCE). M.P. and J.W. were
supported by a grant from the Ministry of Education, Youth and
Sports of the Czech Republic (LK11207). We thank Martin Kotora
24. Sirisoma, N.; Pervin, A.; Nguyen, B.; Crogan-Grundy, C.; Kasibhatla, S.; Tseng,
B.; Drewe, J.; Cai, S. X. Bioorg. Med. Chem. Lett. 2009, 19, 2305.
ˇ
Please cite this article in press as: Kozíšek, M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.039