Optimization of the anti-cancer activity of the phosphatidylinositol-3 kinase pathway inhibitor PITENIN-1: Switching thiourea with 1,2,3-triazole

Article abstract of DOI:10.1039/c4md00109e

We previously reported encouraging in vitro and in vivo anti-cancer activity of N-((3-chloro-2-hydroxy-5-nitrophenyl)carbamothioyl)benzamide (termed PITENIN-1). In the current work, we describe the structure-activity relationship study of the PIT-1 series, based on the replacement of a central thiourea unit with 1,2,3-triazole, which leads to increased liver microsomal stability, drug likeness and toxicity towards cancer cells.

Full text of DOI:10.1039/c4md00109e

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Optimization of the anti-cancer activity of the
phosphatidylinositol-3 kinase pathway inhibitor
PITENIN-1: switching thiourea with 1,2,3-triazole†
Cite this: Med. Chem. Commun., 2014,
5, 1359
Yadagiri Kommagalla,a Sinziana Cornea,b Robert Riehle,c Vladimir Torchilin,c
b
a
*
*
Alexei Degterev and Chepuri V. Ramana
Received 8th March 2014
Accepted 2nd May 2014
We previously reported encouraging in vitro and in vivo anti-cancer activity of N-((3-chloro-2-hydroxy-5-
nitrophenyl)carbamothioyl)benzamide (termed PITENIN-1). In the current work, we describe the structure–
activity relationship study of the PIT-1 series, based on the replacement of a central thiourea unit with 1,2,3-
triazole, which leads to increased liver microsomal stability, drug likeness and toxicity towards cancer cells.
DOI: 10.1039/c4md00109e
www.rsc.org/medchemcomm
Overactivation of PI3K signaling is a common feature of many F10 syngeneic xenogra models by the dimethyl analog of
types of human cancer.1 Increased activation of a variety of PI3K PIT-1.3,4
effectors, through their binding to the PI3K product, lipid PIP3,
Despite the promising initial results, PITs displayed obvious
provides multiple growth, survival, migration and metabolic limitations, including high micromolar activity as well as
advantages to cancer cells. Not surprisingly, small molecule multiple non-drug-like features. In particular, nitrophenyl and
targeting of the PI3K pathway, including direct inhibition of thiourea moieties of PIT-1 represent potential toxicity concerns
PI3K isoforms as well as its multiple effectors (Akt, mTOR, and metabolic liabilities. Initial analysis of the PIT-1 series
PDK1, etc.) has attracted major attention. While many revealed surprisingly specic SAR for a micromolar compound
approaches focused on targeting enzymatic activities in the suggesting several changes to the molecule, leading to some
PI3K network, we and others have recently described a new increase in activity and changes in targeting different PH
approach aimed at targeting a universal central step in PI3K domains.3,4 In particular, the addition of two methyl groups to
signal transduction, i.e. binding of PIP3 to PH domains of the phenyl ring in PIT-1 (DM-PIT-1, Fig. 1) resulted in some
effector proteins.2 In particular, we have developed two new increase in activity and improved incorporation into long-
classes of small molecule antagonists of PIP3, termed PIT-1 [N- circulating PEG–PE micelles for in vivo delivery.3,5 Furthermore,
((3-chloro-2-hydroxy-5-nitrophenyl)carbamothioyl)benzamide] changes to the nitrophenyl ring were identied. However, these
and PIT-2 [(Z)-5-(2-benzyl-5-hydroxy-4-nitrobenzylidene)-2-thio- results did not address the main limitations of the PIT-1 series,
xothiazolidin-4-one] (Fig. 1).3,4 These two structurally dissimilar which was the target of our current work.
molecules displayed very similar activities in cancer cells,
In the processes of rening the initially identied structural
including induction of apoptosis and metabolic stress and scaffold of the PIT-1, our rst concern was to replace the
inhibition of cell migration and invasion. Furthermore, both susceptible thiourea unit with a stable bioisostere.6 The 1,2,3-
PIT-1 and PIT-2 displayed synergistic toxicity with TRAIL in triazole structural motif has attracted our attention in this
human glioblastoma U87MG cells. These activities have been regard considering the fact that the triazole is
linked to the inhibition of Akt signaling and actin remodeling
by ARF6, two pathways regulated by PI3K.3,4 These in vitro
a safe
activities of PITs translated into the signicant inhibition of
tumor growth and lung metastasis formation in 4T1 and B16-
aDivision of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, 411008,
India. E-mail: vr.chepuri@ncl.res.in; Fax: +91–20–25902629
bDepartment of Developmental, Molecular and Chemical Biology, Tus University, 136
Harrison Avenue, Boston, MA 02111, USA. E-mail: alexei.degterev@tus.edu
cDepartment of Pharmaceutical Sciences, Bouve College of Health Sciences,
Northeastern University, 140 The Fenway, Boston, MA 02115, USA
Fig. 1 Structures of the two classes (PIT-1 and PIT-2) of inhibitors of
the phosphatidylinositol-3 kinase (PI3K) signaling pathway, termed
PITENINs (PITs) and the newly designed 2nd generation triazole-PITs.
† Electronic supplementary information (ESI) available: Synthesis and
characterization data and spectra of all the new compounds. See DOI:
10.1039/c4md00109e
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Scheme 1 Synthesis of alkynones 3a–e (eqn (1)) and their Cu-catalyzed [3 + 2]-cycloaddition (eqn (2)) with selected 2-azidophenols 4a–e.
Table 1 (Contd.)
Table 1 The synthesis of and screening results with 1,4-disubstituted
1,2,3-triazolesa
EC50 (mmol)
EC50 (mmol)
S. No
10
Triazole (yield, code)
Alone
With TRIALb
98.4
S. No
1
Triazole (yield, code)
Alone
With TRIALb
51.6
104.0
103.6
11
12
13
64.1
>100
>100
41.6
>100
>100
2
3
4
5
>100
48.9
8.1
>100
42.3
8.0
14
15
16
>100
>100
85.2
>100
>100
74.9
>100
>100
6
57.8
30.6
7
8
9
>100
>100
89.1
>100
64.4
70.5
17
11.99
16.2
3.15
14.0
18
a
Experiments were performed in A2780 cells as described in the ESI.
TRIAL – tumor necrosis factor-related apoptosis-inducing ligand.
b
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bioequivalent surrogate for the amide bond and that this potential second-generation PITenins (PITs), in anticipation of
concept has found its application in the area of anticancer better antitumor activity.
agents as well as in developing non-nucleoside reverse tran-
Scheme 1 reveals the salient features of the synthesis of the
scriptase inhibitors.7,8 As shown in Fig. 1, triazole-PITs have newly designed triazole PITENINS. The alkynone precursors 3a–
been designed as the structural mimics of DM-PIT-1 and as e were prepared by following a two-step sequence consisting of
the addition of ethynylmagnesium chloride to corresponding
aldehydes 2a–e and subsequent IBX oxidation. The azidophe-
nols 4a–f were synthesized from the corresponding amino-
phenols by following the standard azidation procedures and are
used immediately. The copper catalyzed [3 + 2] cycloaddition
reaction of alkynones 3 with azidophenols 4 was carried out
under established click reaction conditions (20 mol% CuSO4,
20 mol% Na-ascorbate in tert-BuOH–water, at rt) to obtain the
requisite 1,2,3-triazole PITENINS.9 Table 1 summarizes the
details of the compounds synthesized. All the new compounds
have been characterized completely with the help of spectral
and analytical data.
Scheme 2 Synthesis of triazole 1fa.
Fig. 2 Increased activity of 1ea. (A) Increased inhibition of PI(3,4)P2/Akt-PH domain binding by 1ea. Lipid overlay experiment was performed as
described in ref. 3. (B) Inhibition of S6 phosphorylation by 1ea. U87MG or A2780 cells were treated with indicated concentrations of inhibitors for
7 h, followed by Western blotting using phospho-S6 and total S6 antibodies. (C) Increased cytotoxicity of 1ea compared to DM-PIT-1 in multiple
cell types. Cells were treated with indicated concentrations of inhibitors alone or in combination with 1 mg mLꢀ1 TRAIL for 24 h. Cell viability was
determined using the CellTiter-Glo assay. (D) 1ea efficiently blocks the migration of A2780 cells. Wound healing assay was performed as
previously described.4 Cell monolayers were treated with indicated concentrations of inhibitors, followed by a scratch wound. The size of the cell
free area was photographed at indicated periods of time. Quantification of the open wound area is shown on the right.
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The initial screening of 1aa against growth of A2780 human 930 mL hꢀ1 kgꢀ1 and bioavailability of 85.1% following
ovarian cancer cells revealed that 1aa has comparable activity i.p. administration. Furthermore, 1ea, unlike DM-PIT-1,
with the corresponding PIT-1 analogue.10 In addition, the cor- complied with 4 out of 5 Lipinski rules, with the exception of
responding methyl ether 1af was completely inactive consistent liphophilicity (calculated log P ¼ 5.3). Overall, our SAR analysis
with the importance of the free –OH group in the original describes a new analog of PIT-1 with signicantly improved
thiourea series.3 In this course of SAR studies, we have installed anti-cancer activity and pharmacological properties, which may
the dimethyl groups and isopropyl groups on the ring A along present a promising molecule for further analysis in mouse
with variable functional groups on the ring C. However, this xenogra models.
approach was not effective. Replacing the aryl ring A with a
naphthyl unit has been also examined, albeit with no
improvement in the activity. In contrast, we found that the
Conclusions
addition of the –CF3 group at the C-3 and C-5 positions on ring A In summary, a structure–activity relationship (SAR) study of the
in combination with a chloro group at the C-4 position of the N-((3-chloro-2-hydroxy-5-nitrophenyl)carbamothioyl)benza-
ring C (1ea) showed excellent improvement (20 times higher mide (PIT-1) series revealed that increased liver microsomal
than that of the DM-PIT-1) in anti-tumor activity.
stability and toxicity towards cancer cells could be achieved by
In order to examine whether the addition of an extra replacing the central thiourea unit with the 1,2,3-triazole
hydrogen bond donor in between the triazole and ring A will heterocycle unit. Secondly, the nitro group of the nitrophenyl
have any effect, the compound 1fa was synthesized (Scheme 2). moiety can be replaced with chlorine, removing another
The compound 1fa has shown good activity, which, however is potential liability. Finally, the addition of two triuoromethyl
lower than that of 1ea.
moieties to the second phenyl ring of the molecule further
Having examined a wide range of analogs, compound 1ea increased the activity, but also resulted in increased lip-
was selected for further progress in the direction of examining ophilicity. Overall, a derivative incorporating all of the modi-
its pharmacological properties. The mode of activity of 1ea has cations (i.e. 1ea) displayed good stability in vitro and in vivo and
been studied by the lipid overlay assay, measuring the binding signicantly increased the toxicity against a number of cancer
of the AktPH domain to PI(3,4)P2 spotted on the nitrocellulose cell lines alone and also in combination with TRAIL. Interest-
membrane, as previously described.3 Compound 1ea displayed ingly, compound 1ea displayed particularly robust activity in
substantially higher activity compared to PIT-1 (Fig. 2A). We inhibiting the mammalian Target Of Rapamycin Complex 1
further examined the inhibition of PI3K/Akt signaling in human (TORC1) signaling downstream from Akt. The mechanism of
glioblastoma U87MG and ovarian carcinoma A-2780 cells. this inhibition is currently under investigation.
Interestingly, we observed very robust inhibition of the TORC1/
p70S6K/S6 pathway downstream from Akt, which correlated
with the cytotoxicity of the compounds and suggested that the
Acknowledgements
increased activity of 1ea translated into more specic inhibition We would like to thank Dr Jinbo Lee for helpful discussions.
of a particular pathway downstream from PI3K/Akt (Fig. 2B). CVR and YK thank CSIR (India) for funding this project (12 FYP
Other targets of the compound 1ea in the PI3K/Akt signaling ORIGIN program, CSC0108) and for a fellowship to YK. This
pathway remain to be fully elucidated in the future.
work was supported in part by the NIH/NCI U54CA151881 grant
Subsequently, the cytotoxicity of new PIT analogs in three to V.T. and A.D.
different cancer cell lines (U87MG, A2780 and T47D) has
revealed that 1ea displayed the highest activity in all cases
Notes and references
(Fig. 2C). In addition to activation of cell death, PITs displayed
several additional important properties. First, we found that
PIT-1 analogs reverse the resistance of cancer cells to anti-
cancer cytokine TRAIL. This led to synergistic cytotoxicity of
PIT-1 and TRAIL when applied in combination.3 This useful
property was retained with 1ea (Fig. 2C and Table 1). Second,
PIT-1 analogs not only increased the cell death, but also sup-
pressed the cell migration and invasion through the attenuation
of actin cytoskeleton remodeling.4 Consistently, 1ea displayed
signicantly increased activity in the cell migration assay
(Fig. 2D).
Finally, one of the major goals of our study was to improve
the pharmacological properties of PIT-1/DM-PIT-1. DM-PIT-1
displayed T1/2 ¼ 1.8 min (CL ¼ 1262 mL minꢀ1 mgꢀ1) in mouse
liver microsomal stability assay in vitro. Compound 1ea dis-
played T1/2 ¼ 119 min (CL ¼ 19.4 mL minꢀ1 mgꢀ1) in the same
assay. Pharmacokinetic analysis following i.v. injection of 1 mg
kgꢀ1 of the drug showed reasonable T1/2 ¼ 3.22 h with CL of
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