(3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro- 1H -indol-2-one derivatives as dual inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.ejmech.2015.02.032

The HIV-1 Reverse Transcriptase (RT) is a validated and deeply explored biological target for the treatment of AIDS. However, only drugs targeting the RT-associated DNA polymerase (DP) function have been approved for clinical use.We designed and synthesised a new generation of HIV-1 RT inhibitors, based on the (3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one scaffold. These compounds are active towards both RT-associated functions, DNA polymerase and ribonuclease H. The structure, biological activity and mode of action of the new derivatives have been investigated. In particular, the nature of the aromatic group in the position 4 of the thiazole ring plays a key role in the modulation of the activity towards the two RT-associated functions.

Full text of DOI:10.1016/j.ejmech.2015.02.032

Accepted Manuscript
(3Z)-3-(2-[4-(Aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one
Derivatives as Dual Inhibitors of HIV-1 Reverse Transcriptase
Rita Meleddu, Simona Distinto, Angela Corona, Giulia Bianco, Valeria Cannas,
Francesca Esposito, Anna Artese, Stefano Alcaro, Peter Matyus, Dora Bogdand,
Filippo Cottiglia, Enzo Tramontano, Elias Maccioni
PII:
S0223-5234(15)00128-2
10.1016/j.ejmech.2015.02.032
EJMECH 7714
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 September 2014
Revised Date: 13 January 2015
Accepted Date: 19 February 2015
Please cite this article as: R. Meleddu, S. Distinto, A. Corona, G. Bianco, V. Cannas, F. Esposito,
A. Artese, S. Alcaro, P. Matyus, D. Bogdand, F. Cottiglia, E. Tramontano, E. Maccioni, (3Z)-3-(2-[4-
(Aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one Derivatives as Dual Inhibitors
of HIV-1 Reverse Transcriptase, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.02.032.
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ACCEPTED MANUSCRIPT
Graphical abstract
(3Z)-3-(2-[4-(Aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one Derivatives as
Dual Inhibitors of HIV-1 Reverse Transcriptase
Rita Meleddu, Simona Distinto, Angela Corona, Giulia Bianco, Valeria Cannas, Francesca Esposito, Anna
Artese, Stefano Alcaro, Peter Matyus, Dora Bogdand, Filippo Cottiglia, Enzo Tramontano, Elias Maccioni.
The synthesis of indolinone derivatives based on a previously identified analogue led to the
confirmation of this scaffold as a promising hit for the design of dual inhibitors of the two reverse
transcriptase associated functions, DNA polymerase and ribonuclease H (RNase H) activities.

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Title
(3Z)-3-(2-[4-(Aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one Derivatives as
Dual Inhibitors of HIV-1 Reverse Transcriptase
Authors
a
a
b
a
a
Rita Meleddu , Simona Distinto , Angela Corona , Giulia Bianco , Valeria Cannas , Francesca
b
c
c
d
d
a
Esposito , Anna Artese , Stefano Alcaro , Peter Matyus , Dora Bogdand , Filippo Cottiglia , Enzo
b
a*
Tramontano , Elias Maccioni .
Affiliations
a
Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124
Cagliari, Italy
b
Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria di
Monserrato, SS554, 09042 Monserrato, Cagliari, Italy
c
Dipartimento di Scienze della Salute, Università Magna Graecia di Catanzaro, Campus “S.
Venuta”, Viale Europa, 88100 Catanzaro, Italy
d
Department of Organic Chemistry, Hogyes Endre utca 7, 1092, Semmelweis University, Budapest,
Hungary
*
To whom correspondence should be addressed. S.D, Department of Life and Environmental
Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy; Tel: +39 0706758550; Fax
39 0706758553, E-mail: maccione@unica.it.
+

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Abstract
The HIV-1 Reverse Transcriptase (RT) is a validated and deeply explored biological target for the
treatment of AIDS. However, only drugs targeting the RT-associated DNA polymerase (DP)
function have been approved for clinical use. We designed and synthesised a new generation of
HIV-1 RT inhibitors, based on the (3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-
dihydro-1H-indol-2-one scaffold. These compounds are active towards both RT-associated
functions, DNA polymerase and ribonuclease H. The structure, biological activity and mode of
action of the new derivatives have been investigated. In particular, the nature of the aromatic group
in the position 4 of the thiazole ring plays a key role in the modulation of the activity towards the
two RT-associated functions.
Keywords: Antiviral agents, HIV-1 RT dual inhibitors, HIV-1, molecular modelling, RNase H,
(3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-ones
Abbreviations
HIV-1, Human Immunodeficiency Virus type 1; HAART, Highly Active Antiretroviral Therapy;
RT, Reverse Transcriptase; RTIs, RT inhibitors; NRTIs/NtRTIs, Nucleoside/Nucleotide RTIs;
NNRTI Non-Nucleoside RTIs; DP, DNA polymerase; RNase H, ribonuclease H; RHIs, RNase H
inhibitors; NNRTIBP, NNRTIs binding pocket; PLK4, polo-like kinase 4; BACE1, Beta-secretase
1; CRTH2 (DP2), chemoattractant receptor-homologous molecule expressed on Th2 cells (D2
prostanoid receptor); NOE, Nuclear Overhauser Effect; NMR, Nuclear Magnetic Resonance; R.T.,
room temperature; RNase H, Ribonuclease H; MM-GBSA, the Molecular Mechanics Generalized
Born/Surface Area; MMFFs, Merck Molecular Force Field; GB/SA, Generalized Born/Surface
Area; PRCG, Polak-Ribier Coniugate Gradient; QPLD, Quantum Mechanic-Polarized ligand
Docking.

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1. Introduction
The current approved treatment for Human Immunodeficiency Virus type 1 (HIV-1) infection is
based on the highly active antiretroviral therapy (HAART), that associates a combination of
antiviral agents, targeting different steps of the virus replication cycle [1, 2]. This multidrug
therapeutic regimen leads to the reduction of the amount of circulating virus, in some cases below
the current blood testing techniques detectable level, and allows high control of the infection.
Moreover, it leads to the reduction of drug resistance occurrence, decrease of mortality and
morbidity rates, and an overall improvement of patients quality of life [3]. However, due to the
chronic nature of HIV infection, a lifelong therapy is required and both adherence to treatment and
the management of drug-related toxicities are issues to deal with.
Although numerous strategies of simplified treatment have been explored in order to further
improve patient quality of life, while maintaining treatment success, RT inhibitors (RTIs), both
Nucleoside/Nucleotide RTIs (NRTIs/NtRTIs) and Non-Nucleoside RTIs (NNRTIs) are always
included in the HAART regimen [1, 2], due to the key role of RT in the early phase of the HIV-1
life cycle.
HIV-1 RT catalyses the reverse transcription process which consists of the conversion of a single
strand viral RNA into a double strand viral DNA via the formation of a RNA-DNA hybrid. To
perform this activity two main catalytic functions are associated in one enzyme, DNA polymerase
(DP) and ribonuclease H (RNase H).
However, despite the fact that both functions are validated drug targets, since also RNase H
function is essential for the reverse transcription process [4, 5], no inhibitor that target this
enzymatic activity has been introduced in therapy until now [6, 7].
2
+
Most RNase H inhibitors (RHIs), designed and studied so far, act by chelating the Mg ions
within the active site [8-15]. However other mechanisms of action have been reported for
hydrazone [16], naphthyridinone [17], anthraquinone [18] and propenone [19] derivatives.

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The latter compounds are capable of binding an allosteric pocket close to the NNRTIs binding
pocket (NNRTIBP) and of inhibiting the RNase H function by a long range allosteric effect.
We have recently reported the identification of a new scaffold for dual inhibition of both HIV-1
RT-associated RNase H and DP functions [20]. Among all the identified molecules, compound 1
(3Z)-3-(2-[4-(3,4-dihydroxyphenyl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-
one (Figure 1) resulted as the best performing derivative with IC value in the low micromolar
5
0
range [20].
Firstly, to gain insights into the drug novelty potential of compound 1, we wanted to investigate
2
+
its mode of action by measuring its capability to chelate Mg ions, since RNase H active site
2
+
inhibitors have been reported to chelate the Mg [21]. Secondly, we examined if cross resistance
with NNRTI resistant RTs could be observed for compound 1, since this behaviour has been
reported for those compounds that interact with the allosteric RNase H pocket, close to the
2
+
NNRTIBP [16, 18, 22]. Results showed that compound 1 did not chelate Mg (Figure 2), but its
activity was slightly affected when tested on the Lys103Asn and Tyr181Cys RTs, two NNRTI
resistant mutant enzymes [20]. These results suggested that compound 1 could have a mode of
action similar to previously reported hydrazone derivatives [16, 17]. Hence, we performed
biochemical and docking experiments that, in agreement with the biochemical data, indicated
compound 1 able to bind RT in the region indicated by Himmel et al in previous works [16, 17].
Therefore, in order to investigate the influence of structural modifications on the biological activity
of compound 1, we synthesised a small library of (3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-
ylidene)-2,3-dihydro-1H-indol-2-ones.
As in the original compound 1, we have conserved the indolinone and thiazole moieties that have
been reported as important fragments in a number of bioactive molecules, such as selective
chymase inhibitors [23], polo-like kinase 4 (PLK4) inhibitors [24], Beta-secretase 1 (BACE1)
inhibitors [25], chemoattractant receptor-homologous molecule expressed on Th2 cells
(
D2
prostanoid receptor) (CRTH2 (DP2)) antagonists [26], MAO inhibitors [27] anti-mycobacterial [28,

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29], anti-Candida [30], and antimicrobial [31]. In the present work, we introduced differently
substituted phenyl moieties at the position 4 of the thiazole ring in place of the 3,4-dihydroxyphenyl
group, and evaluated their ability to behave as dual RT-associated functions inhibitors (Figure 1).
Figure 1 here
2. Chemistry
(3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-ones
(EMAC2072-2082) were synthesized starting from 2,3-dihydro-1H-indole-2,3-dione. This was
reacted with thiosemicarbazide in 2-propanol to give 2-oxo-2,3-dihydro-1H-indol-3-
thiosemicarbazone. Equimolar amounts of semicarbazone synthesized and of the appropriate α-
halogeno-arylketone were then stirred in 2-propanol to give the desired final compounds, as
depicted in Scheme 1.
Scheme 1 here
Compounds EMAC2072-2082 were characterised by means of both analytical and spectroscopic
methods. Their analytical and spectroscopic properties are reported in Table S1 and S2.
The possible formation of both E and Z diastereoisomers along the C=N double bond was
investigated by Nuclear Overhauser Effect (NOE) experiments.
In each compound the irradiation of the indole CH-4 only changed the intensity of the neighbouring
CH protons, whereas upon irradiation of the hydrazone NH signal, no intensity change was
observed. This indicated that the two protons have no measurable NOEs since they are spatially too
far from each other. This distance is in agreement with the “Z” configuration. Therefore, Z-
configuration of each compound could be confirmed (Figure S1).
3. Results and discussion
Compounds EMAC2072-2082, were evaluated for their ability to inhibit both HIV-1 RT
associated RNase H and DP functions (Table 1) using as reference controls compound 1, Efavirenz

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(EFV), and RDS 1643 [13], a diketoacid inhibitor of the RNase H function that binds the catalytic
site.
Table1 here
All tested compounds, with the exception of compound EMAC2081, exhibited dual inhibitory
activity towards both HIV-1 RT functions, confirming that the (3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-
yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one scaffold could be considered a promising
candidate for the design of HIV-1 RT dual functions inhibitor [20]. Interestingly, while compound 1
showed a specificity index (SpI), expressed as the ratio of the RNase H IC value versus the DP
50
IC value, favourable to the DP function (SpI = 3,5), all new derivatives exhibited a SpI favourable
5
0
to the RNase H function. These results suggest that the compounds activity and specificity on the
two functions is strongly influenced by the presence and the nature of the substituents in the 4-
phenylthiazole system.
In fact, in the case of compound EMAC2081, bearing the un-substituted 4-phenylthiazole
moiety, almost no activity towards both functions was observed. Conversely, the introduction of a
4-(4-biphenyl)thiazole moiety (EMAC2076) led to one of the most potent compounds, with a SpI
of 0.11. However, the introduction of a 4-(3-nitrophenyl)thiazole (EMAC2078) appears as the most
performing substitution with respect to DP and RNase H inhibition. Instead, the introduction of the
2,4-difluorophenyl thiazole (EMAC2077) or the 4-(4-biphenyl)thiazole (EMAC2076) is effective
towards the RNase H inhibition. Therefore, a mixture of steric and electronic effects should be
blended in order to achieve the best activity towards the two RT associated functions. Moreover, the
double substitution with halogen atoms in the positions 2 and 4 of the 4-phenyl thiazole is also
effective with respect to the mono-substitution. Thus, compounds EMAC2072 and EMAC2073 are
less potent than their di-substituted analogues EMAC2082 and EMAC2077.
In this respect, fluorine demonstrated to be more efficient than chlorine, probably due to the
higher capability to act as hydrogen bond acceptor. Indeed the size of fluorine is intermediate
between that of hydrogen and oxygen and its van der Waals radius (1.47 Å) could be compared to

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those of hydrogen (1.2 Å) and oxygen (1.57 Å). As a matter of fact, we have observed that small
changes in structure have dramatic effects on activity.
Dual activity might originate from a set of ligand-enzyme interactions, such as the formation of
complexes between the ligand and the NNRTIBP, the RNase H allosteric site, and the RNase H
catalytic site. Therefore, a mode of action investigation by means of biochemical and computational
methods was performed, in order to identify the mechanism of action and the most probable binding
modes of these compounds.
Firstly, we verified the possibility that, differently from compound 1, EMAC derivatives could
2
+
bind the RNase H catalytic site through an interaction with the Mg ions, measuring their spectrum
of absorbance in the absence and in the presence of MgCl , observing no differences (Figure 2).
2
Hence, it is possible to assume that EMAC derivatives do not inhibit the RNase H function by
2
+
binding the Mg ions in the catalytic site.
Figure 2 here
Next, we investigated the possible binding of EMAC compounds either in the NNRTIBP or in
the RNase H allosteric site close to the NNRTIBP by studying the effect of single point mutation. In
particular the activity of EMAC2076 on Tyr181Cys and Lys103Asn mutated RT was investigated,
since these mutations are commonly selected in NNRTI resistance [32]. The same mutated enzymes
were evaluated using the NNRTI EFV and RDS1643, as controls (Table 2) [13]. Results showed
that EMAC2076 retains its potency of inhibition on both RT-associated functions when tested on
Lys103Asn and Tyr181Cys RTs. Its behaviour is analogue to that previously reported for
compound 1: mutated residues were not essential for the interaction of inhibitor with the HIV-1 RT.
Table 2 here
Subsequently, we further investigated the NNRTIBP by checking if EMAC2076 and the NNRTI
EFV were able to simultaneously bind to RT. Hence, we performed a revised Yonetani-Theorell

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model [33] that allows to discriminate between inhibitors that compete for a single binding site (and
are kinetically mutually exclusive) and inhibitors that bind to two non-overlapping binding sites
(and are kinetically not mutually exclusive). In this revised model, the plot of the reaction velocity
reverse (1/v) observed in the presence of different concentrations of the first inhibitor, in the
absence or in the contemporaneous presence of the second inhibitor, leads to a series of lines which
are parallel if the two inhibitors compete for the same binding site, whereas they intersect if the
inhibitors bind to different enzyme sites. Results showed that the dual inhibitor EMAC2076 is not
kinetically mutually exclusive with EFV for the binding to the NNRTIBP, suggesting that both
compounds can bind to RT at the same time (Figure 3). In addition, since the calculation of the
interaction constant α, allows to estimate the degree of binding interference of the two inhibitors
[
33],we calculated this constant for all three obtained curves in the presence of EFV with respect to
the one without EFV. The obtained α value was 2.6 ± 0.17, thus indicating a negative influence of
the binding of one inhibitor with respect to the other. Overall, these results fully support the
hypothesis that EMAC2076 binds an allosteric site close-by and not overlapping with the NNRTI
binding pocket. In the light of these data and considering that EMAC derivatives activity is not
affected when assayed on common resistant mutants we can consider these compounds as
promising hit candidates.
Figure 3 here
The binding mode of compound EMAC2076 with RT HIV-1 has been further investigated by
docking experiments, according to the same protocol described in our previous work [20]. The
allosteric RNase H binding pocket of RT is mainly lipophilic with aromatic residues. Therefore,
hydrophobic interactions play crucial role on determining anti-HIV activity. Moreover, this pocket
is characterized by an L shape expanding from the region next to catalytic DP site to the NNRTIBP
[
34].

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The obtained [EMAC2076•RT] complexes were subjected to a post-docking procedure based on
energy minimization and subsequent binding free energies calculation. The free energies of binding
were obtained applying molecular mechanics and continuum solvation models using the molecular
mechanics Generalized Born/Surface Area (MM-GBSA) method [35].
To further validate the docking pose, the experiment was carried out also on Tyr181Cys mutated
enzyme. The best docking poses (Figure 4) show that Tyr181Cys mutation did not change the
ligand binding mode (Table S3 of SD).
Moreover, the evaluated interaction energies are comparable (-43.87 vs -42.89 kcal/mol). This is
in agreement with the biological data (Table S3 of SD).
The best docking pose for EMAC2076 shows the molecule involved in Van-der-Waals contacts
with Leu228, Val108, Pro226 side-chains. The biphenyl substituent is oriented towards NNRTIBP
and stabilized by an array of aromatic π−π interactions involving Tyr188, Phe227 and Trp229.
However, the rigidity of this group does not allow the compound to enter more deeply in the L
pocket. Stacking contacts are also possible with Tyr181Cys mutation, which was shown to confer
no resistance to this inhibitor (Table 2). Interestingly, the biphenyl hydrophobic substituent orients
the compound with the indolinone portion outside the pocket, while the previously reported
compound 1 seems to prefer a 180°rotated orientation. This is probably due to the presence, in the
case of compound 1, of the more polar di-hydroxyphenyl moiety with respect to the lipophilic
biphenyl substituent of compound EMAC2076. Such polar substituent confers a higher water
affinity and flips the ligand with the thiazole portion oriented to the outer, solvent exposed, part of
the enzyme cavity. Furthermore, this orientation is stabilised through a favourable π−π stacking
between the indolinone portion and Trp229 (Figure 4 and Table S3). However, in our opinion, the
binding mode of EMAC2076 offers more space for chemical optimization, since the activity of
these derivatives could be further improved with a wide variety of hydrophobic and heterocyclic
substituents in position 4 of thiazole ring and by keeping the indolinone portion oriented towards

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the solvent with appropriate substitutions. Indeed, the presence of an aromatic ring seems important
to allow stacking interactions with the highly conserved Phe227 and Trp229 residues [36].
On the whole, the good agreement between the drug resistance mutation profile, Yonetani plot
and the docking poses represents a further validation of the obtained results and corroborates the
hypothesis that EMAC2076 binds the RNAse H allosteric site close-by but not overlapping with
the NNRTI binding pocket.
Figure 4 here
4
. Conclusion
The present study describes a simple method for the synthesis of indolinone derivatives. These
compounds show interesting dual inhibitory activity on both RNase H and DP HIV-1 RT-associated
functions, leading to inhibitors that may completely block RT activities. Biochemical and molecular
modelling studies confirm that, as the hit compound 1, also the new derivatives are able to bind an
allosteric pocket that modulates both RT activities. Furthermore, mutagenesis studies helped to
elucidate the mechanism of action and showed that the most active compound EMAC2076 displays
a favourable drug resistance profile since its activity is conserved on most common NNRTI resistant
mutants. Molecular modelling experiments evidenced the orientation of compound EMAC2076 in
the allosteric RNase H binding pocket with the biphenyl moiety pointed towards the NNRTIBP.
Since the system seems really susceptible to the nature of the aromatic substituents in the position 4
of thiazole ring, the activity could be further improved with a wide variety of substituents, and
heterocyclic systems and through an accurate modulation of electronic, steric and solvation effects.
Hence these new derivatives could be considered as a key step towards the design of dual
DP/RNase H HIV 1 RT inhibitors.
5
. Experimental Section
5.1 Material and General methods

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Starting materials and reagents were obtained from commercial suppliers and were used without
purification. All melting points were determined by the capillary method on a Büchi-540 capillary
melting points apparatus and are uncorrected. Electron ionization mass spectra were obtained by a
Fisons QMD 1000 mass spectrometer (70 eV, 200 mA, ion source temperature 200°C). Samples
were directly introduced into the ion source. Found mass values are in agreement with theoretical
ones. Melting points, yield of reactions and the analytical data of derivatives are reported in Tables
S1, S2, and S3 of SD.
All samples were measured in DMF-d or DMSO-d solvent at 278.1 K temperature on a Bruker
7
6
AVANCE III 500 MHz spectrometer. Chemical shifts are reported relative to TMS (δ = 0) and/or
1
5
referenced to the solvent in which they were measured. (In the N chemical shift assignments we
applied the spectrometer’s digital reference which is calibrated to liq. NH3 δ = 0 ppm.) Coupling
constants J are expressed in hertz (Hz).
Elemental analyses were obtained on a Perkin–Elmer 240 B microanalyser. Analytical data of the
synthesised compounds are in agreement within ± 0.4 % of the theoretical values. TLC
chromatography was performed using silica gel plates (Merck F 254), spots were visualised by UV
light.
5
.2
yl)hydrazono)indolin-2-one (general procedure)
The synthesis is accomplished by a two step procedure
a) Synthesis of 2-(2-oxoindolin-3-ylidene)-hydrazinecarbothioamide
,5g (3.8 mmol) of indolinone-2,3-dione and 0,35g (3.8 mmol) of thiosemicarbazide were
Synthesis
of
compound
EMAC
2072
(Z)-3-(2-(4-(4-chlorophenyl)thiazol-2-
0
introduced in a three necked flask and dissolved with 25 mL of 2-propanol at 50°C. 5 drops of
CH3COOH were added to the mixture reaction as catalyst. After a few minutes the formation of an
abundant yellow precipitate is observe. The reaction was monitored with TLC (eluent

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ethylacetate:exane 7:2). After 24 hours the reaction is completed and the solid filtered. The desired
compound is a yellow solid. R.f.: 0.33 (eluent ethylacetate:exane 7:2); M.P.: >250°C; Yield: 97%
1
H NMR (DMF) δ(ppm): 12.72 (s; 1H); 11.34 (s; 1H); 9.21 (s; 1H); 9.05 (s; 1H); 7.66-7.64 (m;
1H); 7.42-7.34 (m; 1H); 7.17-7.10 (m; 1H); 7.06-7.02 (m; 1H)
1
3
C NMR (DMF) δ(ppm): 180.1; 163.3; 143.1; 132.4; 131.6; 122.8; 121.1; 120.7; 111.5
1
5
N NMR (DMF) δ(ppm): 170.0; 135;6; 110.4
Synthesis of (Z)-3-(2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazono)indolin-2-one EMAC 2072
.5g (2.3 mmol) of (Z)-1-(2-oxoindolin-3-ylidene)thiosemicarbazide and 0.54g (2.3 mol) of 2-
b)
0
bromo-4’-chloroacetophenone were stirred at r.t. in 30 ml of 2-propanol. After few minutes the
formation of an abundant light yellow-orange precipitate was observed. The reaction was monitored
by TLC (eluent exane:ethylacetate 7:2) and after 24 hours it was completed. The desired compound
is a yellow-orange solid. R.f.:0.85 (eluent exane:ethylacetate 7:2); M.P.: >250°C; Yield: 73%
5.3. NMR Final Compounds characterisation:
5.3.1.1 (Z)-3-(2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazono)indolin-2-one (EMAC2072)
1
H NMR (DMF-d7) δ(ppm): 13.55 (s, 1H); 11.43 (s; 1H); 8.07-8.02 (m; 2H); 7.82 (s; 1H); 7.63
(dm; J=7.6 Hz; 1H); 7.58-7.53 (m; 2H); 7.41 (td; J=7.6 Hz; 1.1 Hz; 1H); 7.16 (td; J=7.6 Hz; 1.0
Hz; 1H); 7.10 (dm; J=7.6 Hz; 1H)
1
3
C NMR (DMF-d7) δ(ppm): 166.9; 164.0; 150.6; 142.1; 133.6; 133.1; 132.7; 130.9; 129.2; 127.9;
122.8; 120.5; 120.3; 111.5; 107.7
1
5
N NMR (DMF-d7) δ(ppm): 156.2; 135.7
5.3.2 (Z)-3-(2-(4-(4-fluorophenyl)thiazol-2-yl)hydrazono)indolin-2-one (EMAC2073)
1
H NMR (DMSO-d6) δ(ppm): 13.33 (s; 1H); 11.25 (s; 1H); 7.97-7.90 (m; 2H); 7.60 (s; 1H); 7.53
dm; J= 7.5 Hz; 1H); 7.34 (tm; J= 7.5 Hz; 1H); 7.29-7.17 (m; 2H); 7.09 (tm; J= 7.5 Hz; 1H); 6.99-
(
6.92 (m; 1H)

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1
3
C NMR (DMSO-d6) δ(ppm): 166.4; 163.2; 161.9 (d; J= 244.6 Hz); 150.0; 141.3; 132.2; 130.6;
130.5; 127.8 (d; J= 8.3 Hz) 122.4; 119.9; 119.8; 115.6 (d; J= 21.6 Hz); 111.1; 106.6
1
5
N NMR (DMSO-d6) δ(ppm): 137.1
5.3.3 (Z)-3-(2-(4-(4-bromophenyl)thiazol-2-yl)hydrazono)indolin-2-one (EMAC2074)
1
H NMR (DMF-d7) δ(ppm): 13.53 (s; 1H); 11.42 (s; 1H); 7.99-7.93 (m; 2H); 7.82 (s; 1H); 7.71-
7.65 (m; 2H); 7.61 (dm; J= 7.6 Hz; 1H); 7.39 (tm; J= 7.6 Hz; 1H); 7.14 (tm; J= 7.6 Hz; 1H); 7.08
(dm; J= 7.6 Hz; 1H)
1
3
C NMR (DMF-d7) δ(ppm): 167.0; 164.0; 150.7; 142.1; 133.9; 132.7; 132.1; 130.9; 128.1; 122.8;
121.6; 120.5; 120.3; 111.5; 107.8
1
5
N NMR (DMF-d7) δ(ppm): 156.1; 135.7
5.3.4 (Z)-3-(2-(4-(4-nitrophenyl)thiazol-2-yl)hydrazono)indolin-2-one (EMAC2075)
1
H NMR (DMF-d7) δ(ppm): 13.55 (s; 1H); 11.42 (s;1H); 8.38-8.32 (m; 2H); 8.30-8.24 (m; 2H);
8.12 (s; 1H); 7.62 (dm; J= 7.7 Hz; 1H); 7.40 (td; J= 7.7 Hz; 1.2 Hz; 1H); 7.15 (td; J= 7.7 Hz; 0.7
Hz; 1H); 7.08 (dm; J= 7.7 Hz; 1H)
1
3
C NMR (DMF-d7) δ(ppm): 167.3; 163.9; 149.7; 147.2; 142.2; 140.7; 133.1; 131.1; 127.0; 124.5;
122.9; 120.4; 120.3; 111.6; 111.6
1
5
N NMR (DMF-d7) δ(ppm): 155.8; 135.7
5.3.5 (Z)-3-{2-[4-(4-biphenyl)- thiazol-2-yl)hydrazono)indolin-2-one (EMAC2076)
1
H NMR (DMF-d7) δ(ppm): 13.56 (s; 1H); 11.41 (s; 1H); 8.13-8.08 (m; 2H); 7.85-7.75 (m; 5H);
7.63 (d; J= 7.6 Hz; 1H); 7.55-7.49 (m; 2H); 7.44-7.36 (m; 2H); 7.15 (tm; J= 7.6 Hz; 1H); 7.08 (dm;
J= 7.6 Hz; 1H)
1
3
C NMR (DMF-d7) δ(ppm): 166.8; 164.0; 151.6; 142.1; 140.4; 140.3; 133.8; 132.6; 130.9; 129.4;
127.9; 127.4; 127.0; 126.7; 122.8; 120.5; 120.2; 111.5; 107.1

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1
5
N NMR (DMF-d7) δ(ppm): 156.5; 135.7
5.3.6 (Z)-3-(2-(4-(2,4-difluorophenyl)thiazol-2-yl)hydrazono)indolin-2-one (EMAC2077)
1
H NMR (DMF-d7) δ(ppm): 13.52 (s; 1H); 11.39 (s; 1H); 8.23-8.14 (m; 1H); 7.62 (d; J= 7.6 Hz;
1H); 7.58(d; J= 2.4 Hz; 1H); 7.45-7.36 (m; 2H); 7.25 (td; J= 8.5 Hz; 2.5 Hz; 1H); 7.15 (td; J= 7.6
Hz; 0.7 Hz; 1H); 7.07 (dm; J= 7.6 Hz; 1H)
1
3
C NMR (DMF-d7) δ(ppm): 166.4; 164.0; 162.3 (dd; J= 248.0 Hz; 12.4 Hz); 160.4 (dd; J= 251.9
Hz; 12.3 Hz); 144.7 (dd; J= 2.6 Hz; 0.9 Hz); 142.1; 132.8; 131.2 (dd; J= 9.6Hz; 4.8 Hz); 131.0;
22.9; 120.5; 120.3; 119.1 (dd; J= 11.3Hz; 3.7 Hz); 112.3 (dd; J= 21.4Hz; 3.4 Hz); 111.5; 111.2 (d;
J= 15.0 Hz); 104.9 (t; J= 26.5 Hz)
1
1
5
N NMR (DMF-d7) δ(ppm): 155.9; 135.5
5.3.7 (Z)-3-(2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazono)indolin-2-one (EMAC2078)
1
H NMR (DMF-d7) δ(ppm): 13.57 (s; 1H); 11.41 (s; 1H); 8.84-8.76 (m; 1H); 8.46 (dm; J= 8.1 Hz;
1H); 8.26 (dm; J= 8.1 Hz; 1H); 8.09 (s; 1H); 7.80 (t; J= 8.1 Hz; 1H); 7.63 (d; J= 7.6 Hz; 1H); 7.41
(tm; J= 7.6 Hz; 1H); 7.15 (tm; J= 7.6 Hz; 1H); 7.08 (dm; J= 7.6 Hz; 1H)
1
3
C NMR (DMF-d7) δ(ppm): 167.3; 164.0; 149.5; 149.0; 142.2; 136.3; 133.0; 132.2; 131.0; 130.7;
122.9; 122.9; 120.6; 120.4; 120.3; 111.5; 109.6
1
5
N NMR (DMF-d7) δ(ppm): 155.9; 135.5
5.3.8 (Z)-3-(2-(4-p-tolylthiazol-2-yl)hydrazono)indolin-2-one (EMAC2079)
1
H NMR (DMF-d7) δ(ppm): 13.53 (s; 1H); 11.38 (s; 1H); 7.91-7.86 (m; 2H); 7.66 (s; 1H); 7.62 (d;
J= 7.6 Hz; 1H); 7.39 (td; J= 7.6 Hz; 1.2 Hz; 1H); 7.30-7.25 (m; 2H); 7.15 (td; J= 7.6 Hz; 0.8 Hz;
1H); 7.07 (dm; J= 7.6 Hz; 1H); 2.35 (s; 3H)
1
3
C NMR (DMF-d7) δ(ppm): 166.6; 164.0; 152.0; 142.0; 138.0; 132.4; 132.1; 130.8; 129.7; 126.1;
22.8; 120.5; 120.2; 111.5; 106.0; 20.8
1

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1
5
N NMR (DMF-d7) δ(ppm): 156.4; 135.5
5.3.9 (Z)-3-(2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazono)indolin-2-one (EMAC2080)
1
H NMR (DMF-d7) δ(ppm): 13.53 (s; 1H); 11.40 (s; 1H); 7.95-7.91 (m; 2H); 7.63-7.59 (dm; J= 7.7
Hz; 1H); 7.56 (s; 1H); 7.39 (td; J= 7.7 Hz; 1.1 Hz; 1H); 7.15 (td; J= 7.7 Hz; 1.0 Hz; 1H); 7.08 (dm;
J= 7.7 Hz; 1H); 7.06-7.02 (m, 2H); 3.85 (s; 3H)
1
3
C NMR (DMF-d7) δ(ppm): 166.6; 164.0; 159.9; 151.8; 142.0; 132.4; 130.8; 127.5; 127.5; 122.8;
120.5; 120.2; 114.3; 111.5; 104.7; 55.3
1
5
N NMR (DMF-d7) δ(ppm): 156.6; 135.6
5.3.10 (Z)-3-(2-(4-phenylthiazol-2-yl)hydrazono)indolin-2-one (EMAC2081)
1
H NMR (DMF-d7) δ(ppm): 13.54 (s; 1H); 11.41 (s; 1H); 8.01-7.97 (m; 2H); 7.74 (s; 1H); 7.62
dm; J= 7.7 Hz; 1H); 7.49-7.44 (m; 2H); 7.42-7.34 (m; 2H); 7.17-7.10 (m; 1H); 7.08 (dm; J= 7.7
(
Hz; 1H)
1
3
C NMR (DMF-d7) δ(ppm): 166.8; 164.0; 151.9; 142.1; 134.7; 132.6; 130.9; 129.1; 128.3; 126.1;
22.8; 120.5; 120.2; 111.5; 106.9
1
1
5
N NMR (DMF-d7) δ(ppm): 156.2; 135.6
5.3.11 (Z)-3-(2-(4-(2,4-dichlorophenyl)thiazol-2-yl)hydrazono)indolin-2-one (EMAC2082)
1
H NMR (DMF-d7) δ(ppm): 13.51 (s; 1H); 11.39 (s; 1H); 8.05 (d; J= 8.5 Hz; 1H); 7.85 (s; 1H);
7
.75 (d; J= 2.2 Hz; 1H); 7.63 (dm; J= 7.6 Hz; 1H); 7.58 (dd; J= 8.5 Hz; 2.2 Hz; 1H); 7.40 (td; J=
.6 Hz; 1.2 Hz; 1H); 7.15 (td; J= 7.6 Hz; 1.0 Hz; 1H); 7.07 (dm; J= 7.6 Hz; 1H)
C NMR (DMF-d7) δ(ppm): 166.1; 164.0; 147.1; 142.1; 133.6; 132.6; 132.9; 132.2; 132.1; 131.0;
30.4; 128.1; 122.9; 120.5; 120.3; 112.9; 111.5
7
1
3
1
1
5
N NMR (DMF-d7) δ(ppm): 155.6; 135.5

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5.4 Biochemistry studies
5.4.1 HIV-1 RT purification.
Heterodimeric RT was expressed essentially as described [37].
.4.2 HIV-1 RT-associated DNA polymerase-independent RNase H activity determination.
5
The HIV RT-associated RNase H activity was measured as described [38]. Briefly, 20 ng of HIV-1
wt Reverese Transcriptase was incubated for 1 h at 37 °C in 100 µL reaction volume containing 50
mM Tris HCl pH 7.8, 6 mM MgCl2, 1 mM dithiothreitol (DTT), 80 mM KCl, 250nM hybrid
RNA/DNA
(5’-GTTTTCTTTTCCCCCCTGAC-3’-Fluorescein,
5’-
CAAAAGAAAAGGGGGGACUG-3’-Dabcyl). Reactions were stopped by addition of EDTA and
products were measured with a with a PerkinElmer Victor 3 multilabel counter plate reader at
excitation- emission wavelength of 490/528 nm.
5
.4.3 HIV-1 RT-associated RNA dependent DNA polymerase activity determination.
The HIV-1 RT-associated RNA-Dependent DP activity was measured as previously described [39].
Briefly,20 ng of HIV-1 wt Reverese Transcriptase was incubated for 30 min at 37 °C in 25 µL
volume containing 60 mM Tris-HCl pH 8.1, 8 mM MgCl2, 60 mM KCl, 13 mM DTT, 2.5 µM
poly(A)-oligo(dT), 100 µM dTTP. Enzymatic reaction was stopped by addition of EDTA. Reaction
products were detected by picogreen addition and measured with a PerkinElmer Victor 3 multilabel
counter plate reader at excitation- emission wavelength of 502/523 nm.
Chemical reagents were purchased form Sigma-Aldrich srl. RNA- DNA labeled sequences were
purchased from Metabion international AG.
5.4.4 The Yonetani-Theorell analysis.
The Yonetani-Theorell analysis was perfomed according to the official protocol [33]
The calculation of the interaction constant α, allows to estimate the degree of interference of the two
inhibitors for the binding, was performed according to the following equation, assuming that Kꢀꢁꢂꢃ

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=
IC for non-competitive inhibitors in accordance with Prusoff–Cheng equation [40] and equal to
5
0
12 nM in our system.
slopeꢄwithꢄEFV
[nMꢄEFV]
=
1 +
slopeꢄwithoutꢄEFV
αꢄKꢀꢁꢂꢃ
5.5 Molecular Modeling
5.3.1 Ligand preparation.
The ligand was built within Maestro GUI and its geometry was optimized. The lowest energy
conformer was considered for the following studies. This was obtained with MacroModel version
7.2 [41], considering MMFFs [42] as force field and solvent effects by adopting the implicit
solvation model Generalized Born/Surface Area (GB/SA) water [43]. The simulation was
performed allowing 1000 steps Monte Carlo analysis with Polak-Ribier Coniugate Gradient
(PRCG) method and a convergence criterion of 0.05 kcal/(molÅ).
5.5.2. Protein Preparation.
The atomic coordinates of the protein with pdb code 3LP2 [17] were imported into the Maestro
module available in the Schrödinger package[44]. The protein was further optimized using the
Protein Preparation Wizard[44].
5.5.3 Docking and post-docking procedure.
The docking experiments were performed as described in our previous work [20] applying
Quantum Mechanic-polarized ligand docking (QPLD) [45]. In order to better take into account the
induced fit phenomena, the most energy favoured generated complexes were fully optimized with
the AMBER* united atoms force field [46] in GB/SA implicit water [43], setting 10000 steps
iteractions analysis with Polak-Ribier Coniugate Gradient (PRCG) method and a convergence
criterion of 0.1 kcal/(molÅ). Analysis of the results of the complex minimization was carried out
taking into account the state equations (free energy of complex formation) computed at 300 K,
applying molecular mechanics and continuum solvation models with the molecular mechanics

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generalized Born [35]. The resulting complexes were considered for the binding modes graphical
analysis with Pymol [47] and Maestro [44].
Acknowledgements
This work was supported by RAS grant LR 7/2007 CRP2_450, CRP-24915, and Istituto Superiore
di Sanità grant RF-PAD-2009-1304305 n. 40H98. Rita Meleddu PhD grant was founded by
Fondazione Banco di Sardegna.
Appendix. Supplementary material
Supplementary material associated with this article: Chemical-physical, spectroscopic and
analytical data of derivatives EMAC2072-2082, NOE effect for E and Z configuration, docking
scores, post-docking energies and main active site residues interactions, NMR spectra, MS
fragmentation. Supplementary material associated with this article can be found, in the online
version, at…
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[
Captions
Figure 1. Schematic representation of compound 1 and new derivatives.
Scheme 1. Synthesis of (Z)-3-(2-(4-arylthiazol-2-yl)hydrazono)indolin-2-one derivatives EMAC
2
072-2082. Reagents and solvents: (i) 2-propanol, AcOH; (ii) 2-propanol, room temperature (r.t).
2
+
Figure 2. Effect of MgCl on the spectrum of absorbance of RHIs. Chelation of Mg by RDS1643
2
(panel A), Compound 1 (panel B) and EMAC2076 (panel C). UV–vis spectrum was measured with
compound alone (unbroken line) or in the presence of 6 mM MgCl (dotted line).
2
Figure 3. Yonetani–Theorell plot of the combination of EMAC7026 and EFV on the HIV-1 RT
RDDP activity. HIV-1 RT was incubated in the presence of EMAC7026 alone (●) or in presence
different concentrations of EFV:2 nM (▼), 8 nM (■), 16 nM ( ).
Figure 4. Putative binding mode of a) compound 1 –wtRT HIV-1, and d) EMAC2076-wt RT
HIV-1; g) EMAC2076- Tyr181Cys-RT HIV-1; b) e) h) 2D representation of compound and
binding pocket interacting residues; c) f) i) Residues involved in the complex stabilization sorted by
number of contacts between ligand and receptor.

ACCEPTED MANUSCRIPT
Table 1. EMAC derivatives effects on the HIV-1 RT-associated functions
H
O
N
N
H
N
S
N
Ar
RNase H
DP
c
Compound
1
Ar
a
b
SpI
IC (µM)
IC (µM)
50
50
OH
OH
3.2 ± 1.2
0.9 ± 0.3
3.5
EMAC2072
10.6 ± 0.8
6.4 ± 1.5
20.0 ± 6.0
23.9 ± 5.7
0.53
0.26
Cl
F
EMAC2073
EMAC2074
14.9 ± 2.8
2.9 ± 0.8
81.0 ± 15
34.0 ± 5
0.18
0.08
Br
EMAC2075
EMAC2076
NO2
2.5 ± 0.4
22.0 ± 1
0.11
EMAC2077
EMAC2078
EMAC2079
2.6 ± 0.5
2.8 ± 0.8
9.0 ± 0.9
18.5 ± 2.5
13.0 ± 5
0.14
0.21
0.20
F
F
NO
2
45.0 ± 2.5
CH
3
EMAC2080
4.7 ± 0.5
100
26.0 ± 5
71.6 ± 16
19.5 ± 2.5
0.18
1.3
OCH
3
EMAC2081
EMAC2082
Efavirenz
RDS1643
3.9 ± 1.5
0.20
Cl
Cl
d
ND
0.012 ± 0.003
ND
10.1 ± 2.2
a
Compound concentration required to reduce the HIV-1 RT-associated RNase H activity by 50%;
Compound concentration required to reduce the HIV-1 RT-associated RNA-dependent DNA polymerase activity by
0%;
b
5
c
Specificity Index: ratio between compound concentration required to reduce the HIV-1 RT-associated RNase H
activity by 50% and compound concentration required to reduce the HIV-1 RT-associated DP activity by 50% (IC50
RNase H/IC DP);
5
0
d
ND, not done.

ACCEPTED MANUSCRIPT
Table 2: Inhibition of wild-type HIV-1 and the mutants HIV-1 RT associated activities by
EMAC2076 expressed as IC (µM)
5
0
HIV-1 wt RT
HIV-1 Tyr181Cys RT
HIV-1 Lys103Asn RT
a
b
a
b
a
b
Compound
EMAC2076
RDS1643
RNase H
2.5 ± 0.4
10.1 ± 2.2
DP
RNase H
DP
RNase H
DP
22.0 ± 1
2.7 ± 0.8
9.6 ± 1.8
15.4 ± 2.3
3.42 ± 0.9
9.2 ± 0.9
18.8 ± 2.7
ND
c
c
ND
ND
0
.280 ±
.070
c
c
Efavirenz
ND
0.012 ± 0.003
ND
0.680 ± 0.200
ND
0
a
Compound concentration required to reduce the HIV-1 RT-associated RNase H activity by 50%
b
Compound concentration required to reduce the HIV-1 RT-associated RNA-dependent DNA polymerase activity by
c
5
0%. ND, not done.

ACCEPTED MANUSCRIPT
Figure 1
S
S
O
O
HN
N
HN
N
OH
N
N
HN
HN
Ar
OH
HIT compound 1
EMAC 2072-2082
Scheme 1
S
NH2
C
S
NH
N
H
O
H N
2
N
C
NH2
O
O
N
i
N
H
H
R
O
ii
X
S
N
O
R
N
N
H
N
H
EMAC 2072-2082
R: 4-Cl, 4-F, 4-Br, 4-NO2, 4-C H 2,4-F, 3-NO2, 4-CH3, 4-OCH3, H, 2,4-Cl
6
5,
X: Cl or Br

ACCEPTED MANUSCRIPT
Figure 2
3
3
2
2
1
1
0
0
,5
,0
,5
,0
,5
,0
,5
,0
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
3
3
2
2
1
1
0
0
,5
,0
,5
,0
,5
,0
,5
,0
A
B
C
2
00
300
400
500
600
200
300
400
500
600
2
00
300
400
500
600
wavelenght (nm)
wavelenght (nm)
wavelenght (nm)