Isatin thiazoline hybrids as dual inhibitors of HIV-1 reverse transcriptase

Article abstract of DOI:10.1080/14756366.2016.1238366

A series of 3-3-{2-[2-3-methyl-4-phenyl-2,3-dihydro-1,3-thiazol-2-ylidene]hydrazin-1-ylidene-2,3-dihydro-1H-indol-2-one derivatives has been designed and synthesized to study their activity on both HIV-1 (Human Immunodeficiency Virus type 1) RT (Reverse Transcriptase) associated functions. These derivatives are analogs of previously reported series whose biological activity and mode of action have been investigated. In this work we investigated the influence of the introduction of a methyl group in the position 3 of the dihydrothiazole ring and of a chlorine atom in the position 5 of the isatin nucleus. The new synthesized compounds are active towards both DNA polymerase and ribonuclease H in the μM range. The nature of the aromatic group in the position 4 of the thiazole was relevant in determining the biological activity.

Full text of DOI:10.1080/14756366.2016.1238366

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Isatin thiazoline hybrids as dual inhibitors of HIV-1
reverse transcriptase
Rita Meleddu, Simona Distinto, Angela Corona, Enzo Tramontano, Giulia
Bianco, Claudia Melis, Filippo Cottiglia & Elias Maccioni
To cite this article: Rita Meleddu, Simona Distinto, Angela Corona, Enzo Tramontano, Giulia
Bianco, Claudia Melis, Filippo Cottiglia & Elias Maccioni (2016): Isatin thiazoline hybrids as dual
inhibitors of HIV-1 reverse transcriptase, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.1080/14756366.2016.1238366
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Date: 24 October 2016, At: 02:22

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2016
http://dx.doi.org/10.1080/14756366.2016.1238366
ORIGINAL ARTICLE
Isatin thiazoline hybrids as dual inhibitors of HIV-1 reverse transcriptase
Rita Meleddua, Simona Distintoa, Angela Coronab, Enzo Tramontanob, Giulia Biancoa, Claudia Melisa,
Filippo Cottigliaa and Elias Maccionia
b
aDepartment of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy; Department of Life and Environmental Sciences,
University of Cagliari, Cittadella Universitaria di Monserrato, Cagliari, Italy
ABSTRACT
ARTICLE HISTORY
Received 9 August 2016
Revised 5 September 2016
Accepted 5 September 2016
Published online 20 October
2016
A series of 3-3-{2-[2-3-methyl-4-phenyl-2,3-dihydro-1,3-thiazol-2-ylidene]hydrazin-1-ylidene-2,3-dihydro-1H-
indol-2-one derivatives has been designed and synthesized to study their activity on both HIV-1 (Human
Immunodeficiency Virus type 1) RT (Reverse Transcriptase) associated functions. These derivatives are ana-
logs of previously reported series whose biological activity and mode of action have been investigated. In
this work we investigated the influence of the introduction of a methyl group in the position 3 of the
dihydrothiazole ring and of a chlorine atom in the position 5 of the isatin nucleus. The new synthesized
compounds are active towards both DNA polymerase and ribonuclease H in the mM range. The nature of
the aromatic group in the position 4 of the thiazole was relevant in determining the biological activity.
KEYWORDS
HIV-1 therapeutic agents;
Isatin derivatives; RT dual
inhibitors
Introduction
simultaneously inhibit the two RT associated enzymatic functions,
namely DNA/RNA dependent polymerase (DDDP and RDDP) and
ribonuclease H (RNase H) functions, has been reported11–14. Isatin
based molecular hybrids have been reported as a valid scaffold for
the design of multi-target agents15–19, and in particular for the
dual inhibition of RT associated enzymatic functions12,14. Hence, in
continuation with our previous studies, we report on the synthesis
and the structure-activity relationships of a series of 3-[2-(3-
methyl-4-aryl-1,3-thiazol-2-ylidene)hydrazin-1-ylidene]-1H-indol-2-ones
whose activity has been evaluated towards both RT associated
enzymatic functions DDDP/RDDP and RNase H.
HIV-1 (Human Immunodeficiency Virus type 1) is one of the major
causes of death now days. There are different approaches to keep
under control this infection. The current approved treatment is
based on the highly active antiretroviral therapy (HAART), which
associates a combination of antiviral agents, targeting different
steps of the virus replication cycle1–3. This multidrug therapeutic
regimen leads to the reduction of the amount of circulating virus,
in some cases below the current blood testing techniques detect-
able 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 life4.
Methods
However, there is not a therapeutic regimen capable of com-
pletely eradicate the virus from the host and, therefore, due to the
chronic nature of HIV infection, a lifelong therapy is required.
Hence both adherence to treatment and the management of
drug-related toxicities are issues to deal with. In the light of the
above the design and synthesis of new and more effective anti-
viral agents is an attractive open field for medicinal chemists.
In this respect, the identification of a multiple-acting molecule,
able to inhibit different steps of the virus replication cycle, appears
as a promising approach for the design of new and likely more
efficient antiviral agents. Such agent should combine a dual func-
tion targeting capability in only one molecule5–8. Albeit HIV-1
Reverse Transcriptase (RT) has been the first and most investigated
target for the therapy of HIV-1 infected patients, RT inhibitors
(RTIs) are within the most represented components of HAART3. RT
plays a key role in the HIV-1 replication cycle and is therefore a
strategic target for the design of new therapeutic agents to com-
bat the virus infection9. The molecular aspects of the RT/drug
interaction have been reviewed by some of us and indication for
Materials and apparatus
Starting materials and reagents were obtained from commercial
suppliers and were used without purification. Chemical reagents
were purchased form Sigma-Aldrich (St. Louis, MO). RNA–DNA
labeled sequences were purchased from Metabion international AG.
All melting points were determined on a Stuart SMP11 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 dir-
ectly introduced into the ion source. Found mass values are in
agreement with theoretical ones. Melting points, yield of reactions
and descriptive data of derivatives EMAC 3039–3064 are reported
in Table 1.
1H-NMR spectra (Table 2) were registered on a Varian 500 MHz
spectrometer (Palo Alto, CA). All samples were measured in
DMSO-d6 and CDCl3. Chemical shifts are reported referenced to
the solvent in which they were measured. Coupling constants J
are expressed in hertz (Hz). Elemental analyzes were obtained on
the design of dual-acting RT inhibitors outlined10. Moreover the a Perkin–Elmer 240 B microanalyser (Waltham, MA). Analytical data
identification, design and synthesis of small molecules, able to of the synthesized compounds are in agreement within 0.4% of
CONTACT Elias Maccioni
maccione@unica.it; Simona Distinto
s.distinto@unica.it
Department of Life and Environmental Sciences, University of Cagliari,
Cagliari, Italy
ß 2016 Informa UK Limited, trading as Taylor & Francis Group

2
R. MELEDDU ET AL.
Table 1. Analytical data of derivatives EMAC 3039–3064.
C–H–N
Compound
M.w.
Yield %
M.p. ꢀC.
Crystals color
Cryst. solvent
Calc.
Found
EMAC 3039
EMAC 3040
EMAC 3041
EMAC 3042
EMAC 3043
EMAC 3044
EMAC 3045
EMAC 3046
EMAC 3047
EMAC 3048
EMAC 3049
EMAC 3050
EMAC 3051
EMAC 3052
EMAC 3053
EMAC 3054
EMAC 3055
EMAC 3056
EMAC 3057
EMAC 3058
EMAC 3059
EMAC 3060
EMAC 3061
EMAC 3062
EMAC 3063
449.75
433.29
494.20
460.30
491.40
440.31
406.84
460.30
484.19
429.33
445.33
415.30
439.75
484.19
467.74
528.65
494.75
525.85
474.76
441.28
494.65
518.54
463.68
479.68
449.65
71.15
84.25
91.70
76.05
82.40
81.75
73.75
79.30
84.65
59.40
83.00
61.40
69.35
59.56
57.70
70.00
66.70
58.95
53.70
27.20
57.60
29.89
59.10
51.00
55.60
>250d
210–212
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250d
>250ꢀ
>250ꢀ
>250ꢀ
>250ꢀ
>250ꢀ
Orange
Orange
Orange
Orange
Orange
Orange
Orange
Orange
Yellow
Light orange
Orange
Ocher yellow
Yellow
Orange
Orange-yellow
Orange
Orange
Orange
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
Water/ethanol
C, 58.61; H, 3.55; N, 15.19
C, 61.35; H, 3.72; N, 15.90
C, 52.31; H, 3.17; N, 13.56
C, 56.98; H, 3.45; N, 18.46
C, 70.22; H, 4.42; N, 13.65
C, 63.49; H, 3.65; N, 19.49
C, 58.37; H, 3.27; N, 15.13
C, 56.98; H, 3.45; N, 18.46
C, 53.61; H, 3.00; N, 13.89
C, 65.50; H, 4.63; N, 16.08
C, 62.62; H, 4.43; N, 15.37
C, 64.65; H, 4.22; N, 16.75
C, 53.61; H, 3.00; N, 13.89
C, 53.61; H, 3.00; N, 13.89
C, 55.89; H, 3.13; N, 14.48
C, 48.29; H, 2.70; N, 12.51
C, 52.24; H, 2.92; N, 16.92
C, 64.79; H, 3.85; N, 12.59
C, 57.94; H, 3.07; N, 17.78
C, 53.40; H, 2.74; N, 13.84
C, 52.24; H, 2.92; N, 16.92
C, 49.39; H, 2.53; N, 12.80
C, 59.60; H, 3.95; N, 14.63
C, 57.21; H, 3.79; N, 14.05
C, 58.61; H, 3.55; N, 15.19
C, 58.57; H, 3.53; N, 15.17
C, 61.45; H, 3.70; N, 15.87
C, 52.33; H, 3.15; N, 13.53
C, 56.95; H, 3.41; N, 18.43
C, 70.27; H, 4.43; N, 13.62
C, 63.51; H, 3.62; N, 19.44
C, 58.38; H, 3.23; N, 15.11
C, 57.02; H, 3.44; N, 18.44
C, 53.60; H, 2.98; N, 13.87
C, 65.48; H, 4.61; N, 16.06
C, 62.61; H, 4.45; N, 15.33
C, 64.63; H, 4.23; N, 16.68
C, 53.58; H, 3.01; N, 13.85
C, 53.61; H, 2.99; N, 13.88
C, 55.88; H, 3.14; N, 14.44
C, 48.31; H, 2.67; N, 12.48
C, 52.20; H, 2.94; N, 16.89
C, 64.76; H, 3.84; N, 12.57
C, 58.02; H, 3.05; N, 17.77
C, 53.38; H, 2.76; N, 13.80
C, 52.21; H, 2.94; N, 16.88
C, 49.37; H, 2.50; N, 12.74
C, 59.58; H, 3.97; N, 14.60
C, 57.18; H, 3.81; N, 14.00
C, 58.56; H, 3.54; N, 15.16
Orange-yellow
Yellow
Orange
Red
Orange-red
Red
Orange
the theoretical values. TLC chromatography was performed using described in literature23. The lowest energy conformers were con-
sidered for the following studies. These were obtained from the
starting conformations through a conformational search by means
silica gel plates (Merck F 254, Kenilworth, NJ), spots were visual-
ized by UV light.
24
€
of MacroModel version 7.2 (Schrodinger LLC, New York, NY) , con-
sidering MMFFs25 as force field and solvent effects by adopting
the implicit solvation model Generalized Born/Surface Area (GB/
SA) water26. The simulations were performed allowing 1000 steps
Monte Carlo analysis with Polak–Ribier Coniugate Gradient (PRCG)
method and a convergence criterion of 0.05 kcal/(molÅ).
Biological assay
HIV-1 RT-associated DNA polymerase-independent RNase H
activity determination
The HIV RT-associated RNase
H activity was measured as
described20. Briefly, 20 ng of HIV-1 wt RT was incubated for 1 h at
37 ꢀC in 100 mL reaction volume containing 50 mM Tris HCl pH 7.8,
6 mM MgCl2, 1 mM dithiothreitol (DTT), 80 mM KCl, 250 nM hybrid
RNA/DNA (50-GTTTTCTTTTCCCCCCTGAC-30-Fluorescein, 50-CAAAAG
AAAAGGGGGGACUG-30-Dabcyl). Reactions were stopped by add-
ition of EDTA and products were measured with a with a
Perkin–Elmer Victor 3 multilabel counter plate reader at excitatio-
n–emission wavelength of 490/528 nm (Table 3).
Protein preparation
The coordinates for RT enzymes were taken from the RCSB Protein
Data Bank27 (PDB codes 1vrt28, 2zd129, 1ep430, 3qo931, 1rti28,
1tv632, 3lp233). The proteins were prepared by using the Maestro
Protein Preparation Wizard protocol22.
Docking experiments
QMPL default settings were applied34. The docking grids were
defined by centering on W229 and Q500. The grid boxes of the same
size (46ꢁ 46 ꢁ 46 Å) covered overall the whole p66 subunit. Best sol-
utions were subjected to post-docking procedure and analysed.
5000 steps of the Polak–Ribier conjugate gradient (PRCG) mini-
mization method were conducted on the top ranked theoretical
complexes using AMBER force field35. The optimization process
was performed up to the derivative convergence criterion equal to
HIV-1 RT-associated RNA dependent DNA polymerase activity
determination
The HIV-1 RT-associated RNA-Dependent DP activity was measured
as previously described21. Briefly, 20 ng of HIV-1 wt RT was incubated
for 30 min at 37 ꢀC in 25 mL volume containing 60 mM Tris–HCl pH
8.1, 8 mM MgCl2, 60 mM KCl, 13 mM DTT, 2.5 mM poly(A)-oligo(dT),
100 mM dTTP. Enzymatic reaction was stopped by addition of EDTA.
Reaction products were detected by picogreen addition and meas-
ured with a Perkin–Elmer Victor 3 multilabel counter plate reader at
excitation–emission wavelength of 502/523 nm.
0.1 kJ/(mol Å)ꢃ1. The binding free energies were computed apply-
ꢂ
ing molecular mechanics and continuum solvation models with
the molecular mechanics generalized Born/surface area (MM-GBSA)
method36.
Molecular modelling studies
Figures
Ligand preparation
The resulting best complexes were considered for the binding
Theoretical 3D models of the compounds were built by means of modes graphical analysis with LigandScout (inte:Ligand, Vienna,
38
Austria)37 and Pymol (Schrodinger LLC, New York, NY) .
Maestro GUI22. Considering the configuration of double bonds
€

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
Table 2. 1H NMR data of derivatives EMAC 3039–3064.
Compound
1H NMR
EMAC 3039
1H NMR (CDCl3) d(ppm): 8.29 (d, 1H) (dm; J ¼ 7.48 Hz; 1H); 8.10 (s; 1H); 7.49 (d, 1H) (dm, J ¼ 7.95 Hz; 1H); 7.36 (d, 1H); (dm; J ¼ 8.03 Hz; 1H);
7.24 (s, 1H); 7.04 (t, 1H) (dm; J ¼ 7.60 Hz; 1H); 6.89 (d, 1H) (dm; J ¼ 7.64 Hz; 1H); 6.31 (s, 1H); 3.58 (s, 3H).
1H NMR (DMSO-d6) d(ppm): 10.48 (s, 1H); 8.22 (d; 1H) (dm, J ¼ 7.39 Hz; 1H); 7.64 (dt, 1H) (dm, J ¼ 5.35, 8.25 Hz; 1H); 7.49 (d, 1H) (dm,
J ¼ 7.45 Hz; 1H); 7.38 (td, 1H) (dm; J ¼ 1.63,8.74 Hz; 1H); 7.25 (td, 1H) (dm, J ¼ 1.48, 7.69 Hz; 1H); 6.99 (q, 1H) (dm, J ¼ 7.20 Hz; 1H); 6.84
(m, 1H); 3.50 (s, 3H).
EMAC 3040
EMAC 3041
EMAC 3042
EMAC 3043
1H NMR (DMSO-d6) d(ppm): 10.48 (s, 1H); 8.22 (d, 1H) (dm, J ¼ 7.54 Hz; 1H); 7.74 (d, 1H) (dm, J ¼ 8.43 Hz; 1H); 7.54 (d, 1H) (dm; J ¼ 8.12 Hz;
1H); 7.47 (d, 1H) (dm, J ¼ 7.01 Hz; 1H); 7.24 (m, 1H); 6.98(dt, 1H) (dm, J ¼ 7.61, 15.13 Hz; 1H); 6.83(m, 1H); 3.53 (s, 3H).
1H NMR (CDCl3) d(ppm): 8.38 (d, 1H) (dm, J ¼ 8.20 Hz; 1H); 8.29 (d, 1H) (dm, J ¼ 7.54 Hz; 1H); 7.63 (d, 1H) (dm, J ¼ 8.23 Hz; 1H); 7.50 (s, 1H);
7.26 (m, 1H); 7.06 (t, 1H) (dm; J ¼ 7.36 Hz; 1H); 6.86 (d, 1H) (dm; J ¼ 7.84 Hz; 1H); 6.46 (s, 1H); 3.63 (s, 3H).
1H NMR (DMSO-d6) d(ppm): 10.56 (s, 1H); 8.24 (d, 1H) (dm, J ¼ 7.52 Hz; 1H); 7.83 (dd, 1H) (dm, J ¼ 4.30,8.21 Hz; 1H); 7.75 (m, 1H); 7.68 (dd,
1H) (dm, J ¼ 5.55,7.98 Hz; 1H); 7.50 (m, 1H); 7.42 (t, 1H) (dm, J ¼ 7.37 Hz; 1H); 7.25 (m, 1H); 7.00 (dt, 1H); (dm, J ¼ 7.51, 12.06 Hz; 1H); 6.89
(t, 1H) (dm, J ¼ 9.92 Hz; 1H); 6.84 (t, 1H) (dm; J ¼ 7.75, 18.98 Hz; 1H); 3.57 (s, 3H).
EMAC 3044
EMAC 3045
EMAC 3046
EMAC 3047
1H NMR (DMSO-d6) d(ppm): 10.51 (s, 1H); 8.22 (d, 1H); (dm, J ¼ 7.46 Hz; 1H); 8.01 (dd, 1H) (dm, J ¼ 6.34,8.30 Hz; 1H); 7.81 (dd, 1H) (dm,
J ¼ 5.59, 8.10 Hz; 1H); 7.47 (d, 1H); (dm, J ¼ 7.29 Hz; 1H); 7.25(m, 1H); 7.00(m, 1H); 6.85(m, 1H); 3.42 (s, 3H).
1H NMR (CDCl3) d(ppm): 8.47(s, 1H); 8.29 (d, 1H) (dm; J ¼ 7.51 Hz; 1H); 7.40 (q, 1H) (dm, J ¼ 7.86 Hz; 1H); 7.25 (m, 1H); 7.04 (m, 1H); 6.99 (m,
1H); 6.91(d, 1H); (dm, J ¼ 7.68 Hz; 1H); 6.37(s, 1H); 3.52 (s, 3H).
1H NMR (DMSO-d6) d(ppm): 1052 (s, 1H); 8.39 (s, 1H); 8.36 (dm, 1H); 8.05 (d, J ¼ 8 Hz; 1H); 7.83 (t, J ¼ 8; 1H); 7.47 (d, J ¼ 7.5 Hz; 1H); 7.23 (t,
J ¼ 7.5 Hz; 1H); 7.01 (s, 1H); 6.97 (t, J ¼ 7.5 Hz; 1H); 6.80 (d, J ¼ 8 Hz; 1H); 3.52 (s, 3H).
1H NMR (DMSO-d6) d(ppm): 10.49 (s, 1H); 8.22 (d, 1H) (dm, J ¼ 7.56 Hz; 1H); 7.91(dd, 1H) (dm, J ¼ 2.06, 6.23 Hz; 1H); 7.81 (dd, 1H) (dm,
J ¼ 7.19,8.31 Hz; 1H); 7.60 (ddd, 1H) (dm, J ¼ 2.10, 5.88, 8.30 Hz; 1H); 7.47 (d, 1H) (dm, J ¼ 7.39 Hz; 1H); 7.23(m, 1H); 6.97(m, 1H); 6.80(d,
1H) (dm; J ¼ 7.79 Hz; 1H); 3.51 (s, 3H).
EMAC 3048
EMAC 3049
EMAC 3050
EMAC 3051
1H NMR (CDCl3) d(ppm): 8.30 (m, 1H); 8.11 (s, 1H); 7.86(s, 1H); 7.30 (q, 1H); 7.24 (dd, 3H); (dm, J ¼ 1.35, 7.69 Hz; 1H); 7.04 (m, 1H); 6.88(d,
1H); 6.28(s, 1H); 6.37(s, 1H); 3.52 (s, 3H).
1H NMR (CDCl3) d(ppm): 8.30 (d, 1H); (dm, J ¼ 7.54 Hz; 1H); 7.94 (s, 1H); 7.34(s, 1H) (dm, J ¼ 8.77 Hz; 1H); 7.24 (m, 1H); 7.05 (d, 1H) (dm,
J ¼ 7.49 Hz; 1H); 7.01 (m, 1H) (dm, J ¼ 8.75 Hz; 1H); 6.86 (d, 1H) (dm, J ¼ 7.66 Hz; 1H); 6.25(s, 1H); 3.88 (s, 3H); 3.58 (s, 3H).
1H NMR (CDCl3) d(ppm): 8.30(d, 1H); (dm, J ¼ 7.42 Hz; 1H); 7.98(s, 1H); 7.50 (q, 1H) (dm, J ¼ 3.69 Hz; 1H); 7.42 (dd, 1H) (dm, J ¼ 2.97, 6.43 Hz;
1H); 7.25 (t, 1H); (dm, J ¼ 7.83 Hz; 1H); 7.05 (t, 1H) (dm, J ¼ 7.42 Hz; 1H); 6.88 (d, 1H) (dm, J ¼ 7.64 Hz; 1H); 6.31 (s, 1H); 3.60 (s, 3H).
1H NMR (CDCl3) d(ppm): 8.29 (d, 1H) (dm, J ¼ 7.45 Hz; 1H); 7.75 (s, 1H); 7.57(d, 1H) (dm, J ¼ 10.05 Hz; 1H); 7.41 (d, 1H) (dm, J ¼ 8.48 Hz; 1H);
7.35 (d, 1H) (dm, J ¼ 8.18 Hz; 1H); 7.24 (d, 1H) (dm, J ¼ 6.97 Hz; 1H); 7.04 (d, 1H) (dm, J ¼ 7.55 Hz; 1H); 6.86 (d, 1H) (dm, J ¼ 7.75 Hz; 1H);
6.35 (s, 1H); 3.46 (s, 3H).
EMAC 3052
EMAC 3053
EMAC 3054
EMAC 3055
EMAC 3056
1H NMR (CDCl3) d(ppm): 8.27(d, 1H) (dm, J ¼ 2.16 Hz; 1H); 7.99 (s, 1H); 7.50 (d, 1H) (dm, J ¼ 8.55 Hz; 1H); 7.37 (d, 1H) (dm, J ¼ 8.49 Hz; 1H);
7.22 (dd, 1H) (dm, J ¼ 2.19, 8.30 Hz; 1H); 6.81 (d, 1H) (dm, J ¼ 8.33 Hz; 1H); 6.36 (s, 1H); 3.61 (s, 3H).
1H NMR (CDCl3) d(ppm): 8.28(s, 1H); 7.63 (d, 1H) (dm, J ¼ 17.38 Hz; 1H); 7.49 (s, 1H); 7.41 (m, 1H); 7.20 (q, 1H) (dm, J ¼ 8.33 Hz; 1H); 6.79
(d, 1H) (dm, J ¼ 8.32 Hz; 1H); 6.35 (s, 1H); 3.58 (s, 3H); NH not detected
1H NMR (CDCl3) d(ppm): 8.28 (d, 1H) (dm, J ¼ 2.22 Hz; 1H); 7.65 (t; 1H) (dm; J ¼ 9.12 Hz; 1H); 7.30 (dd, 1H) (dm, J ¼ 6.98, 8.73 Hz; 1H); 7.22
(dd, 1H) (dm, J ¼ 2.20, 8.26 Hz; 1H); 6.80 (d, 1H) (dm, J ¼ 8.26 Hz; 1H); 6.37 (s, 1H); 3.61 (s, 3H); NH not detected.
1H NMR (CDCl3) d(ppm): 8.39 (d, 1H) (d, J ¼ 8.67 Hz; 1H); 8.28 (s, 1H); 7.64 (m, 1H); 7.49 (s, 1H); 7.24 (d, 1H) (dm; J ¼ 12.22 Hz; 1H); 6.79
(m, 1H); 6.49 (d, 1H) (dm; J ¼ 16.75 Hz; 1H); 3.65 (s, 3H).
1H NMR (CDCl3) d(ppm): 8.30 (d, 1H); (d, J ¼ 2.21 Hz; 1H); 8.18 (s, 1H); 7.73(d, 1H) (dm, J ¼ 8.17 Hz; 1H); 7.65 (d, 1H) (dm, J ¼ 7.55 Hz; 1H);
7.50 (d, 1H) (dm, J ¼ 7.86 Hz; 1H); 7.41 (t, 1H) (dm; J ¼ 7.29Hz; 1H); 7.21 (m; 1H); 6.83 (d, 1H) (dm, J ¼ 8.21 Hz; 1H); 6.76 (d, 1H) (dm,
J ¼ 8.22 Hz; 1H); 6.41 (s, 1H); 3.68 (s, 3H).
EMAC 3057
EMAC 3058
1H NMR (DMSO) d(ppm): 10.61 (s, 1H); 8.18 (d. 1H) (dm, J ¼ 2.25 Hz; 1H); 8.02(dd, 1H) (dm, J ¼ 6.72, 8.38 Hz; 1H); 7.82 (dd, 1H) (dm, J ¼ 6.17,
8.23 Hz; 1H); 7.28 (ddd, 1H) (dm, J ¼ 2.24, 8.22, 26.39 Hz; 1H); 7.04 (s, 1H); 6.84 (dd, 1H) (dm, J ¼ 8.26, 29.93 Hz; 1H); 3.54 (s, 3H).
1H NMR (DMSO) d(ppm): 10.63 (s, 1H); 8.17 (d, 1H) (dm, J ¼ 2.21 Hz; 1H); 7.68(qd, 1H) (dm, J ¼ 6.35, 8.53 Hz; 1H); 7.53 (tdd, 1H), (dm,
J ¼ 2.46, 7.91, 10.16 Hz; 1H); 7.31 (ddt, 1H) (dm, J ¼ 2.76, 5.70, 11.48 Hz; 1H); 7.02 (s, 1H); 6.87 (d, 1H) (dm, J ¼ 8.28 Hz; 1H); 6.81(d, 1H)
(dm, J ¼ 8.29 Hz; 1H); 3.47 (s, 3H).
EMAC 3059
EMAC 3060
EMAC 3061
EMAC 3062
EMAC 3063
1H NMR (CDCl3) d(ppm): 8.38 (d, 1H); (d, J ¼ 8.38 Hz; 1H); 7.79 (d; 1H) (dm, J ¼ 7.58 Hz; 1H); 7.74 (t, 1H) (dm, J ¼ 8.02 Hz; 1H); 7.67 (s, 1H);
7.24 (dd, 1H) (dm, J ¼ 2.07, 8.08 Hz; 1H); 6.80 (d, 1H) (dm, J ¼ 8.29 Hz; 1H); 6.75 (d, 1H) (dm, J ¼ 8.27 Hz; 1H); 6.49 (s; 1H); 3.65 (s, 1H).
1H NMR (CDCl3) d(ppm): 8.27 (d, 1H); (d, J ¼ 2.11 Hz; 1H); 7.92 (s, 1H); 7.60 (d, 1H) (dm, J ¼ 8.37 Hz; 1H); 7.55 (s, 1H); 7.27 (m, 1H); 6.81
(d, 1H) (dm, J ¼ 8.29 Hz; 1H); 6.40 (s, 1H); 3.62 (s, 1H).
1H NMR (DMSO) d(ppm): 10.60 (s, 1H); 8.17 (s, 1H); 7.47 (d, J ¼ 8 Hz; 2H); 7.34 (d, J ¼ 8 Hz; 2H); 7.29 (d, J ¼ 8 Hz; 1H); 6.85 (d, J ¼ 8 Hz; 1H);
3.56 (s, 3H); 3.29 (s, 3H).
1H NMR (DMSO) d(ppm): 10.60 (s, 1H); 8.18 (d, 1H) (dm, J ¼ 2.15 Hz; 1H); 7.52(d, 1H) (dm, J ¼ 8.54 Hz; 1H); 7.29 (dd, 1H); (dm, J ¼ 2.22,
8.28 Hz; 1H); 7.09 (d, 1H) (dm, J ¼ 8.53 Hz; 1H); 6.86 (d, 1H) (dm, J ¼ 8.29 Hz; 1H); 6.81 (s, 1H); 3.83 (s, 3H); 3.57 (s, 3H).
1H NMR (CDCl3) d(ppm): 8.29 (d, 1H) (d; J ¼ 2.14 Hz; 1H); 7.69 (s; 1H); 7.51 (m, 1H); 7.43 (m, 1H); 7.21(m, 1H); 6.79 (d, 1H); (dm, J ¼ 8.25 Hz;
1H); 6.37 (s, 1H); 3.63 (s, 1H); NH not detected.
Results indicates that most of the EMAC isatin derivatives are able
to inhibit both RDDP and RNase H functions at mM concentrations,
indicating that these derivatives could represent a good starting
point for the design of new and more efficient dual HIV-1 RT
inhibitors. With the aim of obtaining more information on the
structure activity relationships and to achieve insights into the
binding mode of compounds EMAC 3039–3063, we performed
blind docking studies on the wt-HIV-1 RT heterodimer by applying
the QM-polarized ligand docking protocol (QMPLD)34,39 consider-
ing two grid box: one centered in W229 (NNRTIBP) and one in
Q500 (RNAse domain), in order to include all the p66 subunit39. In
particular, we carried out ensemble docking experiments40 using
Results and discussion
The synthesis of the isatin derivatives EMAC 3039–3063 is illus-
trated in Figure 1. Firstly, the 1-amino-3-methylisothiourea (1) was
obtained by direct reaction between methylisothiocyanate and
hydrazine hydrate (ratio 1:1), at rt, using ethanol as solvent.
Secondly, the condensation between substituted isatin and com-
pound 1, at reflux condition, using ethanol, gave the desired
thiosemicarbazones.
Finally, EMAC 3039–3063 derivatives were obtained in good
yields by reaction of compound 2 with differently substituted
bromo or chloro acetophenones in isopropanol. All the synthe-
sized compounds were submitted to biological assays to evaluate
their ability to inhibit both RT-associated enzymatic functions. seven different crystal structures to take into account the

4
R. MELEDDU ET AL.
Table 3. Activity of compounds EMAC 3039–3063 on HIV-1 RT-associated enzymatic functions RDDP and RNase H.
Compound
R
RNase H – IC50 (lM)
RDDP – IC50 (lM)
Compound
EMAC 3052
RNase H – IC50 (lM)
RDDP – IC50 (lM)
EMAC 3039
EMAC 3040
EMAC 3041
EMAC 3042
EMAC 3043
EMAC 3044
EMAC 3045
EMAC 3046
EMAC 3047
EMAC 3048
EMAC 3049
EMAC 3050
EMAC 3051
4 Cl
4 F
4 Br
17.5 1.3
28 1.0
29 1.0
17.2 1.1
25 1.3
21 2.1
12.5 0.6
24 0.5
24 0.4
15.9 1.2
27 2.0
19.9 5.4
15.0 0.3
24 3.0
30 5.0
100 2.0
15.6 1.6
35 9.0
27 0.7
30 2.0
69 4.0
50 3.0
29 1.0
17.9 3.0
15.0 6.0
19.6 0.6
24 0.8
18.9 1.1
23 1.0
21 0.5
10.0 0.5
27 3.0
21 0.9
17.5 2.2
16.7 1.0
19.0 1.0
15.1 5.1
25 5.0
//
39 3.0
31 2.6
34 2.8
26 1.5
9.5 1.5
28 2.3
54 5.0
60 3.2
98 2.0
85 7.0
28 0.5
100 10
//
EMAC 3053
EMAC 3054
EMAC 3055
EMAC 3056
EMAC 3057
EMAC 3058
EMAC 3059
EMAC 3060
EMAC 3061
EMAC 3062
EMAC 3063
Not synthesized
4 NO2
4C6H5
4 CN
2-4 F
3 NO2
3-4 Cl
4 CH3
4 OCH3
H
2-4 Cl
Figure 1. Synthetic pathway to compounds EMAC 3039–3063; reagents and conditions: (i) methylisothiocyanate, hydrazine hydrate, ethanol, rt; (ii) 1-amino-3-methyliso-
thiourea, substituted isatin, ethanol, reflux; (iii) 2, substituted acetophenones, isopropanol, rt.
We focused our attention on the most active compounds of
target flexibility. The same protocol was successfully applied in
the two series: EMAC 2045 and EMAC 2056.
The best RT complexes obtained by the docking experiments
were subjected to a post-docking procedure based on energy
our previous studies12,13. In fact, the high NNRTIBP plasticity allows
different orientations of Y181, Y188, Y183 and primer grip
b12–b13 hairpins41. The overall volume of this pocket is 620–720
minimization and successive binding free energy calculation.
Å3 and is characterized by a L shape. Its volume is approximately
The binding free energies were obtained applying molecular
more than twice the one occupied by most of the known NNRTIs.
mechanics and continuum solvation models using the
Thus it offers many binding possibilities to small molecules3. This
explains the large variety of chemical scaffolds of this class of
molecular mechanics generalized Born/surface area (MM-GBSA)
method36.
inhibitors, whose shapes was often described in a creative way:
e.g. “butterfly”42, “horseshoe”43 and “dragon”31. These inhibitors
are able to lock the enzyme into an inactive conformation.
Blind docking calculations indicated the presence of two
energetically favored sites that we named as Pocket 1 and
Pocket 2. Pocket 1 is located close to the polymerase triad and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
Figure 2. Putative binding modes of EMAC2045 and EMAC2056 and critical residues individuated for their binding in the pocket 1: (a, d) EMAC2045-HIV-1 RT complex and
EMAC2056-HIV-1 RT complex; (b and e) close-up into the EMAC2045 and EMAC2056 binding site; (c and f) 2D depiction of EMAC2045 and its respective interactions with RT
residues. pale yellow sphere indicates hydrophobic interactions with lipophilic residues. Red arrow indicates a hydrogen bond (HB) acceptor interaction, while the violet
sphere represents the aromatic p ꢃ p stacking interaction.
Figure 3. Putative binding modes of EMAC2045 and EMAC2056 and critical residues individuated for their binding in the pocket 2: (a, d) EMAC2045-HIV-1 RT complex and
EMAC2056-HIV-1 RT complex; (b and e) close-up into the EMAC2045 and EMAC2056 binding site; (c and f) 2D depiction of EMAC2045 and its respective interactions with RT
residues. pale yellow sphere indicates hydrophobic interactions with lipophilic residues. Red arrow indicates a hydrogen bond (HB) acceptor interaction, green HB
donor, while the violet sphere represents the aromatic p ꢃ p stacking interaction.
comprehend the whole
L shaped NNRTIBP. Conversely, the 3. The two functions inhibition seems explicable by the binding
second putative binding pocket is located in the RNase H to at least two sites: one (pocket 1) responsible for the polymer-
ase activity, and the second (pocket 2), responsible for the
RNase H inhibitory activity. In particular, EMAC 2056 is better
accommodated compared to EMAC 2045 into the pocket 1
since is able, with its di-phenyl substituent, to take contact with
Tyr188, Tyr181, Tyr183 Trp229 by p ꢃ p interactions. Overall the
complexes are mainly stabilized by several hydrophobic
interactions.
domain, below the RNase H active site. Some new RNase H
inhibitors which are able to bind this pocket, have been already
described by Felts44 and by our previous studies12–14. Most
likely, these compounds are able to prevent the correct anchor-
ing of the nucleic acid in the RNase H domain and, therefore,
inhibit the RNase H hydrolytic function. Putative binding modes
of EMAC 2045 and EMAC 2056 are depicted in Figures 2 and

6
R. MELEDDU ET AL.
In pocket
2
the compounds are sandwiched between
inhibitors of HIV-1 reverse transcriptase. Chem Med Chem
2014;9:1869–79.
14. Meleddu R, Distinto S, Corona A, et al. (3Z)-3-(2-[4-(aryl)-1,3-
thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one deriv-
atives as dual inhibitors of HIV-1 reverse transcriptase. Eur J
Med Chem 2015;93:452–60.
15. Agamennone M, Belov DS, Laghezza A, et al. Fragment-
based discovery of 5-arylisatin-based inhibitors of matrix
metalloproteinases 2 and 13. Chem Med Chem. 2016;11:
1892–8.
16. Chen G, Ning Y, Zhao W, et al. Synthesis, neuro-protection
and anti-cancer activities of simple isatin Mannich and Schiff
bases. Lett Drug Des Discov 2016;13:395–400.
the two subunits p66 and p51. In fact, the complexes are stabi-
lized by interactions with both A (p66) and B (p51) chains
(Figure 3).
Conclusions
In this research we have designed and synthesized a library of isa-
tin based derivatives EMAC 3039–3063 to evaluate their activity
towards both RT-associated enzymatic functions RDDP and RNase
H. Our data indicate that the isatin derivatives are generally able
to inhibit both RDDP and RNase H functions al mM concentrations
and that these molecules are a good starting point for the design
of new dual HIV-1 RT inhibitors. Nevertheless, considering the high
potential of the isatin scaffold we will pursue in our efforts to
identify new isatin based anti-viral agents.
17. Tavari M, Malan SF, Joubert J. Design, synthesis, biological
evaluation and docking studies of sulfonyl isatin derivatives
as monoamine oxidase and caspase-3 inhibitors. Med Chem
Commun 2016;7:1628–39.
18. Rane RA, Karunanidhi S, Jain K, et al. A recent perspec-
tive on discovery and development of diverse thera-
peutic agents inspired from isatin alkaloids. Curr Top
Med Chem (Sharjah, United Arab Emirates) 2016;16:
1262–89.
Disclosure statement
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
19. Ozgun DO, Yamali C, Gul HI, et al. Inhibitory effects of
isatin Mannich bases on carbonic anhydrases, acetylcholin-
esterase, and butyrylcholinesterase. J Enzyme Inhib Med
Chem. 2016;31:1498–1501.
20. Corona A, Di Leva FS, Thierry S, et al. Identification of highly
conserved residues involved in inhibition of Hiv-1 Rnase H
function by diketo acid derivatives. Antimicrob Agents
Chemother 2014;58:6101–10, 11.
21. Suchaud V, Bailly F, Lion C, et al. Development of a series of
3-hydroxyquinolin-2(1H)-ones as selective inhibitors of HIV-1
reverse transcriptase associated RNase H activity. Bioorg
Med Chem Lett 2012;22:3988–92.
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