Synthesis and Anti-HIV Profile of a Novel Tetrahydroindazolylbenzamide Derivative Obtained by Oxazolone Chemistry

Article abstract of DOI:10.1021/acsmedchemlett.8b00511

A new tetrahydroindazolylbenzamide derivative has been synthesized, characterized, and evaluated as HIV-inhibitor. The biological data revealed the ability to inhibit HIV proliferation with low cytotoxicity allowing for significant selectivity (EC_50<

Full text of DOI:10.1021/acsmedchemlett.8b00511

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Letter
Synthesis and anti-HIV profile of a novel tetrahydroindazolylbenzamide
derivative obtained by oxazolone chemistry
Angela Scala, Anna Piperno, Nicola Micale, Frauke Christ, and Zeger Debyser
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00511 • Publication Date (Web): 15 Dec 2018
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Synthesis and anti-HIV profile of a novel
tetrahydroindazolylbenzamide derivative obtained by oxazolone
chemistry
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Angela Scala,†,* Anna Piperno,† Nicola Micale,† Frauke Christ,‡ Zeger Debyser‡
† Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, V.le F. Stagno
D’Alcontres 31, Messina I-98166, Italy
‡ Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, UZ
St. Rafael, Kapucijnenvoer 33, BE-3000 Leuven, Belgium
KEYWORDS Tetrahydroindazole, azlactone, hydrazine, dimedone, reverse transcriptase.
ABSTRACT: A new tetrahydroindazolylbenzamide derivative has been synthesized, characterized and evaluated as HIV-inhibitor.
The biological data revealed the ability to inhibit HIV proliferation with low cytotoxicity allowing for significant selectivity (EC50
2.77 µM; CC50 118.7 µM; SI=68). The compound did not inhibit the viral integrase as demonstrated by in vitro studies. QPCR
experiments showed that the block of viral replication occurred at early replication steps, prior to integration, profiling it as a late
reverse transcription inhibitor. An efficient multi-step strategy was adopted for the synthesis of the scaffold, consisting of a sequential
ring-opening reaction of oxazol-5-(4H)-one with 1,3-diketone, followed by cyclocondensation with hydrazine and hydrolysis of the
nitrile to the desired carboxamide.
The infection caused by the human immunodeficiency virus
(HIV) induces a persistent and incurable disease with serious
health and socio-economical impact.1 HIV still causes
approximately three million deaths annually. Despite the
currently available drugs are unable to eradicate the virus, the
combined antiretroviral treatment (cART) has markedly
changed the evolution of the infection and transformed a deadly
Hsp90 is a molecular chaperone involved in a variety of
cellular processes that guides the folding, intracellular
disposition, and proteolytic turnover of many key regulators of
cell growth and differentiation.11 Hsp90 has been identified as
a therapeutic target in cancer and inhibitors of Hsp90 have
proven to be effective at driving cancer cells into apoptosis.12
Recently, Hsp90 inhibitors have also shown great promise in
the treatment of HIV infection.13 Tetrahydroindazolyl- and
tetrahydroindolyl-benzamide derivatives were recently
patented as Hsp90 inhibitors able to prevent integration of HIV
viral DNA into host cells.14 Additionally, nelfinavir and
ritonavir, lead inhibitors of the HIV protease, were found to
inhibit Hsp90 function in breast cancer cells, as an example of
“drug repurposing”.15-17
disease into
a
manageable chronic infection.2 The
pharmacological treatment of the HIV infection targets mostly
HIV proteins such as reverse transcriptase (RT), protease, and
integrase (IN) to specifically interfere with virus replication.3
Nevertheless, the identification of cellular factors involved in
the viral replication cycle has opened new avenues for the
development of HIV inhibitors.4 In the framework of our
studies, dealing with the design of biologically active
heterocycles,5-10 we have discovered a new compound
interfering in vitro with the HIV replication. It shares attractive
structural features with some inhibitors of the heat-shock
protein 90 (Hsp90), such as SNX-2112 and its prodrug SNX-
5422, the latter currently in phase I clinical trials as antitumoral
(Figure 1).
Herein, we report the synthesis and the HIV profile of a new
tetrahydroindazolylbenzamide derivative prepared by ring
opening reaction of 4-methyl-2-phenyl oxazol-5-(4H)-one 1
with dimedone 2, followed by cyclocondensation of the resulted
1,3,3’-tricarbonyl derivative 3 with 4-cyanophenylhydrazine 4
and the subsequent hydration of the benzonitrile 5 into
benzamide 6. The synthetic procedure is depicted in Scheme 1.
OR
Triketone 3 was conveniently prepared by an efficient
chemo- and regioselective method, which entails the reaction of
5,5-dimethylcyclohexane-1,3-dione 2 with oxazolone 1 in
acetonitrile under microwave irradiation.18 The asymmetrical
oxazolone 1 was obtained in turn by cyclodehydration of
commercial N-benzoyl-D,L-alanine in acetic anhydride.19 The
cyclocondensation20 of 3 with hydrazine 4 in refluxing ethanol
efficiently yielded the tetrahydroindazolone 5. The reaction
proceeded regioselectively yielding only the 1,3-disubstituted
regioisomer 5, due to the higher nucleophilicity of the external
A)
B)
O
R=H
SNX2112
O
NH
H2N
R=glycine
H2N
ester mesylate
SNX5422
N
N
H
N
N
N
Ph
CF3
O
O
O
Figure 1. A) Structures of SNX2112 and SNX5422; B) the
novel tetrahydroindazolylbenzamide derivative reported herein.
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nitrogen of arylhydrazine 4. Finally, the chemoselective
corresponding carboxylic acid group by acid hydrolysis, with
the simultaneous acid-promoted N-deprotection of the
functionalized side-chain that led to the interesting amino acid
derivative 7 as hydrochloride (Scheme 1).
1
2
3
4
conversion of the cyano group of 5 into amide derivative 6 was
performed in a mixture ethanol/dimethylsulfoxide, by using
sodium hydroxide and hydrogen peroxide as a catalyst.
Alternatively, the nitrile moiety of 5 was converted into the
5
6
Scheme 1. Synthetic strategy for the preparation of compounds 3-7
7
8
O
O
O
9
N
O
+
H2N
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O
Ph
2
1
( )-
N
N
a)
H
N
c)
NC
b)
Ph
O
CN
O
OH
O
H
6
( )-
N
N
N
H
Ph
+
N
O
Ph
O
O
HO
d)
NH NH2
O
( )-
O
(
)-
3
4
5
N
N
NH3Cl
7
( )-
O
Reagents and conditions: (a) anhydrous CH3CN, DBU, MW, reflux 85°C, 1 h; (b) abs. EtOH, reflux, 5 h; (c) EtOH, NaOH, DMSO, H2O2,
rt, 24 h; (d) 6N HCl, glacial CH3COOH, reflux, 2 days.
The structures of new compounds 5, 6 and 7 were determined
on the basis of analytical and spectroscopic data (See
Supporting Information).
inhibitors (Figure 2, panel B). It rather profiled as a RT inhibitor
with a stark reduction in formation of the reverse transcribed
DNA, yet, to less extend than observed for AZT (Figure 2, panel
A). Next, both the formation of 2-LTRs and integrated provirus
were reduced to background levels (Figure 2, panel B and C,
To the best of our knowledge, the introduction of a
functionalized side-chain at the C3-pyrazole carbon of
tetrahydroindazolylbenzamide scaffold is unprecedented, as
only alkyl or aryl substituents (e.g. Me, Et, i-Pr, CF3,
cyclohexyl, Ph, thienyl, etc.) have been introduced so far.14,21,22
In principle, using different amino acids as starting materials for
the oxazolone formation, the proposed synthetic methodology
might give efficient entry into facile diversification of the side-
chain, allowing to maximize the molecular diversity of the final
tetrahydroindazolylbenzamide. Moreover, the amino group
could be further derivatized to improve the pharmaceutical
properties including pharmacokinetic and druggability, i.e. by
conjugation with biopolymers.
respectively),
demonstrating
once
more
that
tetrahydroindazolylbenzamide 6 acts at early replication steps
prior to integration. In time of addition experiments a similar
profile was observed confirming that 6 profiles as a late reverse
transcription inhibitor (data not shown).
The anti-HIV activity of compounds 5, 6 and 7 was evaluated
in vitro in a classic MTT-MT4 assay23 (see Supporting
Information). The benzamide 6 showed a remarkable ability to
inhibit HIV proliferation and low cytotoxicity (EC50 2.77 µM;
CC50 118.7 µM) allowing for significant selectivity (SI=68);
conversely, both the precursor benzonitrile 5 and the carboxylic
derivative 7 did not show any antiviral activity, pointing out the
essential role of the primary benzamide moiety at the N1-
position of the pyrazole ring.
To further elucidate the mechanism of action, the effects of
compound 6 on HIV integrase activity have been analyzed in an
in vitro strand transfer assay. No activity was observed (data not
shown) excluding HIV-IN as a drug target for this class of
molecules. This finding was confirmed in cell culture by QPCR
analysis (see Supporting Information). Indeed compound 6 did
not show the increase in 2-LTRs typically seen for integration
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(3) Soriano, V.; Fernandez-Montero, J. V.; Benitez-Gutierrez, L.; de
Figure 2. Analysis of the mechanism of action of 6 by QPCR
analysis of HIV DNA species. Compounds were added at 50-fold
EC50 value: control (DMSO, brown), compound 6 (light blue), and
AZT (green). Panel A: Analysis of the RT activity by measurement
of late RT transcripts. While AZT blocks reverse transcription
completely, compound 6 shows a partial inhibition of reverse
transcription. Panel B: Analysis of 2-LTRs circle formation as a
measure of the block of integration. Alike AZT, compound 6 does
not induce formation of 2-LTRs circles demonstrating that the
antiviral activity is blocked at an earlier step (RT). Panel C:
Analysis of the integration event. No provirus formation resulting
from integration is observed.
Mendoza, C.; Arias, A.; Barreiro, P.; Pena, J. M.; Labarga, P. Dual
antiretroviral therapy for HIV infection. Expert Opinion on Drug
Safety, 2017, 16, 923-932.
(4) Duerr, R.; Keppler, O.; Christ, F.; Crespan, E.; Garbelli, A.;
Maga, G.; Dietrich, U. Targeting Cellular Cofactors in HIV Therapy.
Topics in Medicinal Chemistry 2015, 15, 183-222.
(5) Scala, A.; Micale, N.; Piperno, A.; Rescifina, A.; Schirmeister,
T.; Kesselring, J.; Grassi, G. Targeting of the leishmania mexicana
cysteine protease CPB2.8DCTE by decorated fused benzo[b]
thiophene scaffold. RSC Advances, 2016, 6, 30628-30635.
(6) Rescifina, A.; Scala, A.; Sciortino, M.T., Colao, I.; Siracusano,
G.; Mazzaglia, A.; Chiacchio, U.; Grassi, G. Decorated 6,60,7,70-
tetrahydro-1H,10H-2,30-biindole scaffold as promising candidate for
recognition of the CDK2 allosteric site. Med Chem Commun, 2015, 6,
311-318.
(7) Scala, A.; Cordaro, M.; Grassi, G.; Piperno, A.; Barberi, G.;
Cascio, A.; Risitano, F. Direct synthesis of C3-mono-functionalized
oxindoles from N-unprotected 2-oxindole and their antileishmanial
activity. Bioorg. Med. Chem 2014, 22, 1063-1069.
(8) Scala, A.; Cordaro, M.; Mazzaglia, A.; Risitano, F.; Venuti, A.;
Sciortino, M. T.; Grassi, G. Aldol-type compounds from water-soluble
indole-3,4-diones. Synthesis, kinetics and antiviral properties. Mol.
Diversity 2013, 17, 479-488.
(9) Scala, A.; Cordaro, M.; Risitano, F.; Colao, I.; Venuti, A.;
Sciortino, M.T.; Primerano, P.; Grassi, G. Diastereoselective
multicomponent synthesis and anti-HSV-1 evaluation of dihydrofuran-
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In summary, the synthesis and the anti-HIV profile of a novel
tetrahydroindazolylbenzamide
derivative
obtained
by
oxazolone chemistry have been reported. Compound 6 showed
low cytotoxicity (CC50 118.7), a remarkable anti-HIV activity
(EC50 2.77 µM) and significant selectivity (SI=68). The pivotal
role of the primary benzamide moiety at the N1-position of the
pyrazole ring emerged by the absence of antiviral activity for
benzonitrile 5 and carboxylic derivative 7. Preliminary studies
carried out to elucidate the mechanism of action pointed out that
it profiles as a late reverse transcription inhibitor.
(10) Scala, A.; Cordaro, M.; Mazzaglia, A.; Risitano, F.; Venuti, A.;
Sciortino, M.T.; Grassi, G. Synthesis and anti HSV-1 evaluation of
novel indole-3,4-diones. Med. Chem. Comm. 2011, 2, 172-175.
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M. M.; Workman, P. Maximizing the Therapeutic Potential of Hsp90
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Materials and Methods; Synthetic procedures; Drug susceptibility
assay; Quantification of different HIV-1 DNA species during HIV
infection by real-time PCR; 1H and 13C NMR spectra (PDF file).
(13) Smith, A. P.; Haystead, T. A. J. Hsp90: a key target in HIV
infection. Future Virol. 2016, 12, 55-59.
(14) Orlemans, E. O. M.; Haynes, B. F.; Ferrari, G.; Haystead, T.;
Kwiek, J. J. Use of tetrahydroindazolylbenzamide and
tetrahydroindolylbenzamide derivatives for the treatment of Human
Immunodeficiency Virus (HIV) and Acquired Immune Deficiency
Syndrome (AIDS). From U.S. Pat. Appl. Publ. (2016), US
20160143884 A1 20160526.
(15) Shim, J. S.; Rao, R.; Beebe, K.; Neckers, L.; Han, I.; Nahta, R.;
Liu, J. O. Selective Inhibition of HER2-Positive Breast Cancer Cells
by the HIV Protease Inhibitor Nelfinavir. J. Natl. Cancer Inst. 2012,
104, 1576-1590.
(16) Bernstein, W. B.; Dennis, P. A. Repositioning HIV protease
inhibitors as cancer therapeutics. Current Opinion in HIV and AIDS,
2008, 3, 666-675.
(17) Blumenthal, G. M.; Gills, J. J.; Ballas, M. S.; Bernstein, W. B.;
Komiya, T.; Dechowdhury, R.; Morrow, B.; Root, H.; Chun, G.;
Helsabeck, C.; Steinberg, S. M.; LoPiccolo, J.; Kawabata, S.; Gardner,
E. R.; Figg, W. D.; Dennis, P. A. A phase I trial of the HIV protease
inhibitor nelfinavir in adults with solid tumors. Oncotarget 2014, 5,
8161-8172.
AUTHOR INFORMATION
Corresponding Author
* Tel: +39 (0)90 6765515. E-mail: ascala@unime.it
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
HIV, human immunodeficiency virus; cART, combined
antiretroviral treatment; RT, reverse transcriptase; IN, integrase;
Hsp90, heat-shock protein 90; EC50, half maximal effective
concentration; CC50 half maximal citotoxic concentration; SI,
selectivity index; QPCR, quantitative polymerase chain reaction;
2-LTR, two-long long terminal repeats; DNA, deoxyribonucleic
acid; AZT, azidothymidine.
(18) Cordaro, M.; Grassi, G.; Risitano, F.; Scala, A. A new
construction of diversely funzionalized oxazoles from enolizable cyclic
1,3-dicarbonyls and 5(4H)-oxazolones. Synlett 2009, 1, 103-105.
(19) Piperno, A.; Scala, A.; Risitano, F.; Grassi, G. Oxazol-5-(4H)-
Ones. Part 1. Synthesis and Reactivity as 1,3-dipoles. Curr. Org.
Chem., 2014, 18, 2691-2710.
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