Structure-based optimization of potent PDK1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.05.070

In this Letter is described the structure-based design of potent dihydro-pyrazoloquinazolines as PDK1 inhibitors. Starting from low potency HTS hits with the aid of X-ray crystallography and modeling, a medicinal chemistry activity was carried out to improve potency versus PDK1 and selectivity versus CDK2 protein kinase.

Full text of DOI:10.1016/j.bmcl.2010.05.070

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4095–4099
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based optimization of potent PDK1 inhibitors
*
Mauro Angiolini , Patrizia Banfi, Elena Casale, Francesco Casuscelli, Claudio Fiorelli , Maria B. Saccardo,
Marco Silvagni, Fabio Zuccotto
Nerviano Medical Sciences Srl, Viale Pasteur 10, 20014 Nerviano, Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter is described the structure-based design of potent dihydro-pyrazoloquinazolines as PDK1
inhibitors. Starting from low potency HTS hits with the aid of X-ray crystallography and modeling, a
medicinal chemistry activity was carried out to improve potency versus PDK1 and selectivity versus
CDK2 protein kinase.
Received 26 April 2010
Revised 17 May 2010
Accepted 18 May 2010
Available online 8 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Kinase
Structure-based drug design
PDK1
CDK2
Scaffold
Signal transduction of many growth factors and oncogenes is
mediated by 3-phosphoinositide-dependent protein kinase-1
(PDK1), a master regulator of at least 23 related protein kinases
belonging to the AGC kinase family.1,2 PDK1 downstream transduc-
tion pathways involve a group of related serine-threonine protein
kinases like AKT, p70 ribosomal S6 kinase, and PKC that mediate vi-
tal cell processes such as cell survival and proliferation. Moreover,
about 50% of common human cancers including breast, lung, pros-
tate, and blood possess overstimulation of the PDK1 signaling,
making PDK1 an interesting therapeutic target in oncology.3
From the high throughput screening campaign of the corporate
chemical collection carried out on PDK1, dihydro-pyrazoloquinaz-
olines (general formula A—Fig. 1) emerged as one of the most inter-
esting chemical classes based on good cell permeability data and a
limited promiscuity profile compared to other active chemical
classes identified. Unfortunately such a chemotype was also char-
acterized by an intrinsic high affinity (nanomolar range) for CDK2
and PLK1 protein kinases.4,5 In order to investigate the possibility
of developing dihydro-pyrazoloquinazolines as PDK1 inhibitors, a
medicinal chemistry project was started with the primary aim of
improving potency against PDK1 and decreasing affinity versus
CDK2.
the kinase field it has been observed that, with a few exceptions,
the mode of binding of a particular scaffold is generally conserved
amongst different kinases. We hence assumed that the mode of
binding of scaffold A in PDK1 was similar to the one observed in
CDK2. An analysis of the ATP binding pocket was performed in or-
Structural data related to both CDK2 and PDK1 kinases with the
mode of binding of dihydro-pyrazoloquinazolines in CDK2 were
available at project inception.4b In several years of research in
* Corresponding author. Tel.: +39 0331581518.
E-mail address: mauro.angiolini@nervianoms.com (M. Angiolini).
Present address: Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova,
Italy.
Figure 1. General structure of PDK1 dihydro-pyrazoloquinazolines inhibitors A and
areas of potential SAR exploration. Chemical structures with biological activity of
compounds 1–2.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.070

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M. Angiolini et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4095–4099
Table 1
Different residues between CDK2 and PDK1 in the ATP binding region selected for
increasing potency and selectivity
Location
CDK2
PDK1
Glycine loop
Glycine loop
Glycine loop
Glycine loop
Gatekeeper
Hinge
Hinge
Hinge
Sugar region
Activation loop
Glu8
Lys86
Ser92
Phe93
Ser94
Leu159
Tyr161
Ala162
Asn164
Glu166
Thr222
Thr14
Tyr15
Gly16
Phe80
Phe82
Leu83
—
Asp86
Ala144
der to identify differences that could be exploited for selective drug
design. Interestingly the two binding sites are characterized by
several different but structurally equivalent residues some of
which could offer attractive opportunities in the development of
PDK1 selective compounds (Table 1).
A chemical developability assessment of dihydro-pyrazoloqui-
nazoline hits led to the selection of compound 1 (Fig. 1) as starting
point for the optimization process despite its modest IC50 value
against PDK1 and the potent activity in the low nanomolar range
against CDK2.6
Dihydro-pyrazoloquinazoline 1 offered in fact several opportu-
nities for SAR studies allowing access to different areas of the ATP
binding site. With regard to the SAR exploration three regions of
scaffold A were identified as crucial: the amide in position 3, the
substitution on the nitrogen N1 of the pyrazole ring and the aro-
matic moiety installed on the amine in position 8 of the tricyclic
scaffold (Fig. 1).
Based on our initial assumption of a CDK2-like binding mode for
compound 1, the primary amide on pyrazole N1 would be placed in
proximity of Glu166 residue. The initial medicinal chemistry work
hence focused on the optimization of the interaction between the li-
gand and the negatively charged side chain. As a result compound 2
(Fig. 1), prepared following a Mitsunobu protocol, was obtained dis-
playing a sevenfold increase in potency versus PDK1 (IC50 = 374 nM)
and an almost unvaried high potency versus CDK2 (12 nM).5,7,8
The 3D structure of the complex between PDK1 and compound
2 was solved by X-ray crystallography at 2.0 Å resolution confirm-
ing our initial assumption on the mode of binding of the dihydro-
pyrazoloquinazoline scaffold in PDK1 (Fig. 2).9–11 The scaffold
indeed occupies the ATP pocket and binds to the hinge region
Figure 3. Chemical structure and biological activity for compounds 3–4–5.
through the amino-pyrimidine moiety. A first hydrogen bond is
established between the backbone NH of Ala162 and the nitrogen
of the pyrimidine ring in position 7 and an additional hydrogen
bond is observed between Ala162 carbonyl oxygen and the nitro-
gen of the anilino moiety in position 8 of the ligand. A further inter-
action between the conserved Lys111 and the carbonyl oxygen of
the amide in position 3 of the pyrazole is also observed.
The structural data also confirmed that, as expected, the in-
creased level of PDK1 related potency observed for compound 2
was the result of the favorable interaction between the positively
charged nitrogen of the piperidine ring and the Glu166 side chain.
The structurally equivalent residue in CDK2 is Asp86. Modeling
work suggested that, despite its shorter side chain, Asp86 is capa-
ble of establishing the same type of interaction as confirmed by the
good level of activity against CDK2 (12 nM). Clearly Glu166 could
not be targeted for selectivity but it proved to be a key element
to increase potency against PDK1. Further exploration of this inter-
action with linear chains containing basic residues like aminopro-
pyl (compound 3—Fig. 3) or aminoethyl (data not shown), allowed
a significant and effective step towards low nanomolar PDK1
inhibitors but also led to even more potent CDK2 compounds.
Compound 3 for instance had an IC50 value of 36nM against
PDK1 and 1 nM against CDK2.
Having proved that dihydro-pyrazoloquinazolines could pro-
vide potent PDK1 inhibitors, the second phase of the chemistry
project focused on targeting the CDK2 selectivity issue. In particu-
lar it was decided to follow two different strategies: one taking
advantage of specific differences in aminoacids between the two
kinases, the other one exploiting differences in the ATP binding site
morphology. The work focused on two sub-regions: (1) a small
cluster of residues (Ser92, Phe93, and Ser94) in the highly flexible
glycine-rich loop that are different between PDK1 and CDK2 and
(2) the hinge region, where the lack of a residue in CDK2 results
in a different binding site shape and size in that area.
Knowing the ligand mode of binding, we hypothesized that
secondary amide group in position 3 of the dihydro-pyrazoloqui-
nazoline scaffold might be in direct contact with the terminal
portion of the glycine-rich loop containing residues 92–94. An
Figure 2. X-ray structure of compound 2 (PDB 2xch) bound to PDK1 (blue ribbon)
at 2.0 Å resolution. Hydrogen bonds are represented as red dashed lines.

M. Angiolini et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4095–4099
4097
CDK2 activity. Similarly, ortho-substituted anilines were tolerated
by CDK2 but resulted detrimental for PDK1 inhibition. Thus, in or-
der to achieve potent PDK1 inhibitors having a selective profile
versus CDK2 it was decided to design new derivatives containing
both the amide moiety of compound 4 and the propylamino chain
of compound 3 on pyrazole N1 and, at the same time, explore the
meta-substitution on the aniline ring in position 8 to exploit the
difference between the two kinases at the hinge region. In fact,
in CDK2 the residue structurally equivalent to Asn164 in PDK1 is
missing. The lack of this residue leads to a change in backbone con-
formation with the Leu83 and His84 carbonyl groups encroaching
into the ATP binding site reducing the space available to the ligand.
The idea we decided to follow was to introduce a group in this area
so that it could be tolerated by PDK1 but less by CDK2 due to unfa-
vorable steric clashes.
Modeling studies suggested that groups carrying hydrogen-
bonding atoms could also offer the opportunity to establish addi-
tional and selective interactions with the PDK1 protein backbone.
For instance, docking results suggested that the carbonyl group
of the dimethyl amide in meta-position of compound 5 (Fig. 3)
could give a hydrogen bond interaction with both positively
Figure 4. X-ray structure of compound
resolution). Hydrogen bonds are represented as red dashed lines.
4 (PDB 2xck) bound to PDK1 (2.3 Å
charged Ne of Lys86 and the hydroxyl group of Tyr161, placing
analysis of the HTS data related to the screened dihydro-pyrazolo-
quinazoline secondary amides, highlighted how substituents in
this position could indeed modulate CDK2 activity. More impor-
tant, bulky secondary amides showed a detrimental effect for
CDK2 inhibition but still retaining activity versus PDK1. Compound
4, for instance, one of the HTS hits belonging to this class, was char-
acterized by an IC50 of 713 nM for PDK1 and an IC50 of 319 nM for
CDK2. Moreover, docking studies of compound 4 in PDK1 sug-
gested the pyridinyl moiety could engage Ser94 in the glycine-rich
loop in an interaction with its sp2 nitrogen.12
Structural studies of compound 4 bound to PDK1 confirmed the
role of the 2-methyl-pyridin-4-ylmethyl amide in the kinase ATP
binding site (Fig. 4).11 As expected the pyridine ring sits under-
neath the glycine-rich loop with the 2-methyl group fitting nicely
in a small cavity made up by Gly91 backbone and Ser94 and Thr95
side chains. Also, the pyridine sp2 nitrogen establishes a hydrogen
bond with Ser94 determining a further interaction for PDK1, as al-
ready observed by Feldman et al.13
the dimethyl portion in the solvent exposed area at the same time
(Fig. 5). The corresponding residues in CDK2 are, respectively, Glu8
and Phe82.12
Compound 5 was subsequently prepared and when tested
showed an IC50 of 85 nM on PDK1. As expected the meta-substitu-
tion resulted in a further CDK2 activity loss with an IC50 of 500 nM,
a significant 50-fold lower than the value obtained for structure 1.
The data confirmed that the secondary amide in position 3 is a key
element to achieve the desired PDK1 selectivity profile versus
CDK2. When tested in a kinases panel, compound 5 showed com-
plete selectivity against Plk1, Gsk3-b, Lyn, Met, Mps1, Abl, Ack1,
Akt1, Alk, Bub1, Kdr, Jak1, Jak3, Zap70, Cdc7, Irk, Jak2, Eef2k, Fak,
p38a, Ret, Mst4, B-Raf, Ikk2, Igfr1, Egfr1, Fgfr1, Perk, Pak4, Brk,
Ck2, Nek6, Lck, Sulu1, Nim, Mapkapk2, Syk, Tyk, TrkA. The kinases
Aurora A (40 nM) and Aurora B (100 nM) were also inhibited by
compound 5.
The biological activity of the prepared compounds was also
evaluated in a cell based assay using both A2780 ovarian and
MCF7 breast cancer cell lines (Table 2).14 The dihydro-pyrazoloqui-
nazoline scaffold proved to have good cell permeability. Interest-
ingly, compound 3 was one of the most active compounds on
both cell lines despite the presence of the highly polar primary
amine residue. Unfortunately, compound 5 displayed only residual
activity in A2780 cell line. This is probably the result of different
factors like a relatively low potency against PDK1, a detrimental ef-
fect of the specific substituents on the physicochemical properties
of the molecule and a limited cross activity on other kinases (e.g.,
CDK2) which might be responsible for the cellular activity ob-
served for compounds 2, 3, and 4.
Activity data obtained during the HTS for other members of the
dihydro-pyrazoloquinazoline chemical class, suggested that typi-
cally an unsubstituted aniline in position 8 resulted in an enhanced
Scheme 1 reports the synthetic approach adopted to prepare
the PDK1 inhibitor 5.
Starting from the intermediate 6 the synthetic key step was the
chemoselective introduction of the substituted anilino moiety at
Table 2
Cellular activities for compounds 1–5 in A2780 and MCF7 cell lines. Values are means
of at least two experiments
Compound
A2780 (
lM)
MCF7 (lM)
1
2
3
4
5
2.71
0.51
0.12
0.17
5.90
6.00
1.36
0.14
4.91
>10
Figure 5. Docking model of compound 5 (yellow) bound to PDK1. Hydrogen bonds
are represented as red dashed lines.

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M. Angiolini et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4095–4099
Scheme 1. Reagents and conditions: (a) KOH, EtOH, 50 °C, 90%; (b) DMF, 2-methyl-4-aminomethyl-pyridine, TBTU, DIPEA, 80%; (c) TFA, DCM, TES, rt, 75%; (d) Cs2CO3, DMF,
N-Boc-propylamino-bromide, rt, 70%; (e) dioxane, 110 °C, 2% Pd2(dba)3, 4% Xantphos, 2-(3-iodo-phenyl)-N,N-dimethyl-acetamide, Cs2CO3, 60%; (f) HCl 4 M, dioxane,
quantitative.
the position 8 of the tricyclic scaffold by the Hartwig–Buchwald
reaction with dihydro-pyrazoloquinazoline derivative 9 as nucleo-
philic reagent.6,15,16 This procedure avoided the low yield prepara-
tion of the 8-iodo-dihydro-pyrazoloquinazoline intermediate
normally used for the arylation step.4,5 Initial hydrolysis of the
ethyl-ester 6 with potassium hydroxide and coupling with 2-
methyl-4-aminomethyl-pyridine in presence of TBTU and DIPEA
afforded intermediate 7 in 80% yield. Removal of trityl protective
group from the pyrazole ring with trifluoroacetic acid furnished
the compound 8 and alkylation with Cs2CO3 with the propyl-ami-
no-bromide protected as Boc-derivative in DMF as solvent pro-
vided the compound 9 in good yield. The final product 5 was
achieved by the Hartwig–Buchwald arylation carried out in diox-
ane with Xantphos as ligand reagent, aryl-iodide and Pd2(dba)3
as the most efficient catalyst in 60% yield. Treatment with HCl
4 M in dioxane afforded the final compound 5.17
References and notes
1. Pearce, L. R.; Komander, D.; Alessi, D. R. Nat. Rev. Mol. Cell. Biol. 2010, 11, 9.
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2004, 15, 161.
3. (a) Atkinson, F. L.; Axten, J. M.; Cichy-Knight, M.; Moore, M. L.; Patei, V. K.; Tian,
X.; Wellaway, C. R.; Dunn, A. K. WO2010/019637, 2010.; (b) Nittoli, T.; Dushin,
R. G.; Ingalls, C.; Cheung, K.; Floyd, M. B.; Fraser, H.; Olland, A.; Hu, Y.; Grosu, G.;
Han, X.; Arndt, K.; Guo, B.; Wissner, A. Eur. J. Med. Chem. 2010, 45, 1379; (c)
Wuchrer, M.; Bruge, D.; Finsinger, D.; Graedler, U.; Dorsch, D.; Esdar, C.
WO2010/000364, 2010.; (d) Ramurthy, S.; Jain, R.; Lin, X.; NG, S. C.; Pfister, K.
B.; Rico, A.; Subramanian, S.; Wang, X. M. WO2009/153313, 2009.; (e) Kim, K.
H.; Wissner, A.; Floyd, M. B., Jr.; Fraser, H. L.; Wang, Y. D.; Dushin, R. G.; Hu, Y.;
Olland, A.; Guo, B.; Arndt, K. Bioorg. Med. Chem. Lett. 2009, 19, 5225; (f) Iorns, E.;
Lord, J. C.; Ashworth, A. Biochem. J. 2009, 417, 361; (g) Maira, S.-M.; Furet, P.;
Stauffer, F. Fut. Med. Chem. 2009, 1, 137; (h) Stauffer, F.; Maira, S. M.; Furet, P.;
Garcìa-Echeverrìa, C. Bioorg. Med. Chem. Lett. 2008, 18, 1027; (i) Peifer, C.;
Alessi, D. R. ChemMedChem 2008, 3, 1810.
4. 4a Traquandi, G.; Ciomei, M.; Ballinari, D.; Casale, E.; Colombo, N.; Croci, V.;
Fiorentini, F.; Isacchi, A.; Longo, A.; Mercurio, C.; Panzeri, A.; Pastori, W.;
Pevarello, P.; Volpi, D.; Roussel, P.; Vulpetti, A.; Brasca, M. G. J. Med. Chem. 2010,
53, 2171; (b) Brasca, M. G.; Traquandi, G.; Vianello, P.; Quartieri, F.; Panzeri, A.;
Pevarello, P.; Vulpetti, A.; Roletto, F.; D’Alessio, R.; Amboldi, N.; Ballinari, D.;
Cameron, A.; Casale, E.; Cervi, G.; Colombo, M.; Colotta, F.; Croci, V.; Fiorentini,
F.; Isacchi, A.; Mercurio, C.; Moretti, W.; Pastori, W.; Ciomei, M. J. Med. Chem.
2009, 52, 5152.
In summary, HTS dihydro-pyrazoloquinazolines hits were opti-
mized to increase PDK1 potency and to falter activity on CDK2.
Potency was increased about 75-fold (compound 1—PDK1 IC50
=
2740 nM, compound 3—PDK1 IC50 = 36 nM) establishing charge
interactions with Glu166 whereas selectivity towards CDK2 was
obtained by developing secondary amide analogues (PDK1/CDK2
activity ratio was improved from 274 for compound 1 to 0.17 for
compound 5). The strategy was supported by crystallographic con-
firmation of the mode of binding of the analyzed compounds. Fur-
ther optimization is required to achieve a therapeutic level of
cellular activity.
5. Beria, I.; Brasca, M. G.; Caruso, M.; Ballinari, D.; Borghi, D.; Bossi, R. T.;
Cappella, P.; Ceccarelli, W.; Ciavolella, A.; Cristiani, C.; Croci, V.; De Ponti,
A.; Fachin, G.; Ferguson, R. D.; Lansen, J.; Moll, J.; Pesenti, E.; Posteri, H.;
Perego, P.; Rocchetti, M.; Volpi, D.; Valsasina, B.; Bertrand, J. A.; Storici, P. J.
Med. Chem. 2010, 53, 3532.
6. Traquandi, G.; Brasca, M. G.; Polucci, P.; Vianello, P.; Ferguson, R.; Quartieri, F.;
Panzeri, A.; Pevarello, P.; Vulpetti, A.; Roletto, F.; D’Alessio, R.; Fancelli, D.
WO2004/104007, 2004.
7. Caruso, M.; Beria, I.; Brasca, M. G.; Ferguson, R. D.; Posteri, H.; Valsasina, B.
WO2008/074788, 2008.
8. In vitro kinase activity assays: IC50 values were determined as reported in Ref. 6.
9. Biondi, R. M.; Komander, D.; Thomas, C. C.; Lizcano, J. M.; Deak, M.; Alessi, D. R.;
van Aalten, D. M. EMBO J. 2002, 21, 4219.
Acknowledgments
We would like to thank Daniele Donati and Gabriella Brasca for
valuable discussions; Francesca Quartieri and Roberto Marcucci for
supporting the chemistry activity.
10. (a) Otwinowski, Z.; Minor, W.. In Methods in Enzymology; Carter, C. W., Sweet,
R. M., Eds.; Academic Press: San Diego, 1997; Vol. 276, pp 307–326; (b) Emsley,
P.; Cowtan, K. Acta Crystallogr., Sect. D 2004, 2126.

M. Angiolini et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4095–4099
4099
11. Crystals for soaking were transferred to
a
solution of 2.2 M ammonium
(CellTiter-Glo) from Promega. IC50 values were calculated with the rate of
percent of growth versus the untreated control.
15. Yin, J.; Zhao, M. M.; Huffman, M. A.; McNamara, J. M. Org. Lett. 2002, 20, 3481.
16. Loones, K. T. J.; Maes, B. U. W.; Dommisse, M. A.; Lemière, G. L. F. Chem.
Commun. 2004, 21, 2466.
sulfate, 0.1 Tris pH 8.5, 20% glycerol in which compounds 2 and 4 were
dissolved up to saturation. Diffraction data were collected at the ESRF
(Grenoble, France). Data were processed using the HKL package (Otwinoski
and Minor, 1997). Model correction and structure refinement were
iteratively carried out with COOT (Emsley, 2004) and Refmac (CCP4,
1994). The final R-factor achieved for each complex was of 20.2% (Rfree
17. Procedure for the synthesis of compound 5 by the Hartwig–Buchwald reaction
(Step (e) of Scheme 1). To a suspension of (3-{8-amino-3-[2-methyl-pyridin-4-
ylmethyl)-carbamoyl]-4,5-dihydro-1,2,7,9-tetraaza-cyclopenta[a]naphthalen-
1-yl}-propyl)-carbamic acid-tert-butyl ester (0.142 mmol; 1 equiv) 9 in dry
dioxane (1.5 ml) under argon atmosphere, Cs2CO3 (0.2 mmol; 1.4 equiv),
Xantphos (0.0056 mmol; 0.04 equiv), Pd2(dba)3 (0.0028 mmol; 0.02 equiv)
and 2-(3-iodo-phenyl)-N,N-dimethyl-acetamide (0.2 mmol; 1.4 equiv) were
added. The suspension was heated 8 h at 100 °C and the solvent removed in
vacuum. The residue extracted in DCM-water was purified by flash column
chromatography (AcOEt/MeOH 98:2) affording the corresponding product as
Boc-protected compound in 60% yield. Final treatment with 10 equiv of HCl
4 M in dioxane gave compound 5 as a white hydrochloride salt. 1H NMR
(400 MHz, DMSO): d 2.15 (q, 2H, NCH2CH2CH2NH2, J = 6.58 Hz), 2.73 (s, 3H,
CH3Pyr), 2.80–2.85 (m, 4H, NCH2CH2CH2NH2, –CH2CH2–), 2.86 (s, 3H,
CH3NCH3CO), 3.00 (t, 2H, –CH2CH2–, J = 7.56 Hz), 3.03 (s, 3H, CH3NCH3CO),
3.69 (s, 2H, ArCH2CONMe2), 4.65 (d, 2H, CONHCH2Pyr, J = 5.97 Hz), 4.90 (t, 2H,
NCH2CH2CH2NH2, J = 6.58 Hz), 6.86 (d, 1H, HAr, J = 7.68 Hz), 7.27 (t, 1H, HAr,
J = 7.68 Hz), 7.50 (br s, 1H, HAr), 7.62 (d, 1H, HAr, J = 7.68 Hz), 7.77 (d, 1H, HPyr,
J = 6.34 Hz), 7.82 (br s, 1H, HPyr), 7.97 (br s, 3H, NCH2CH2CH2NH2ÁHCl), 8.43 (s,
1H, HPyrim), 8.71 (d, 1H, HPyr, J = 6.34 Hz), 9.10 (t, 1H, CONHCH2Pyr,
23.3%) for compound
2 and 20.5% (Rfree 24.4%) for compound 4.
Crystallographic data with the corresponding structure factors files for
compounds 2 and 4 have been deposited with the Protein Data Bank (PDB)
under the accession code 2xch and 2xck.
12. Docking simulations were carried out using the mcdock algorithm
implemented in the FLO_QXP package: McMartin, C.; Bohacek, R. S. J.
Comput. Aided Mol. Des. 1997, 11, 333.
13. Feldman, R. I.; Wu, J. M.; Polokoff, M. A.; Kochanny, M. J.; Dinter, H.; Zhu, D.;
Biroc, S. L.; Alicke, B.; Bryant, J.; Yuan, S.; Buckman, B. O.; Lentz, D.; Ferrer, M.;
Withlow, M.; Adler, M.; Finster, S.; Chang, Z.; Arnaiz, O. D. J. Biol. Chem. 2005,
280, 19867.
14. Cellular assays: A2780 (ECACC) were grown in an RPMI-1640 medium
supplemented with heat-inactivated 10% fetal calf serum. MCF7 (ECACC)
were grown in MEM medium supplemented with heat-inactivated 10% fetal
calf serum. Cells were seeded at different densities ranging from 2500 to 5000
cells/well in black 96-well plates with the appropriate complete medium. After
24 h the plates were treated with the compound to be tested and incubated for
72 h at 37 °C under a 5% CO2 atmosphere. At the end of the incubation, cells
were lysed and the ATP content in the well used as a measure of viable cells.
J = 5.97 Hz), 9.59 (s, 1H, NHPyrim). HRMS calcd for
C
30H36N9O2 (MH+):
This was determined with
a
thermostable firefly luciferase based assay
554.2987. Found: 554.2982.