Discovery of novel pyridazinylthioacetamides as potent HIV-1 NNRTIs using a structure-based bioisosterism approach

Article abstract of DOI:10.1039/c3md00028a

In continuation of our endeavors to develop new, potent, selective, and less toxic anti-HIV agents, we describe our structure-based bioisosterism design, synthetic strategy, and structure-activity relationship (SAR) studies that led to the identification of pyridazinylthioacetamides, a novel class of NNRTIs, isosteres of arylazolylthioacetanilide derivatives. Nearly all of the tested compounds inhibited HIV-1 strain IIIB replication in the lower micromolar concentration range (EC₅₀: 0.046-5.46 μM). Notably, the most promising compound 8k exhibited extremely potent inhibitory activity against HIV-1 replication with an EC₅₀ value of 0.046 μM, CC₅₀ of 99.9 μM and the viral selectivity index amounted to 2149. These values were much better than those of NVP (EC₅₀ = 0.09 μM) and DDC (EC₅₀ = 1.04 μM). Compound 8k also exhibited moderate inhibition of enzymatic activity with an IC₅₀ value of 4.06 μM, which was of the same order of magnitude as that of NVP (2.74 μM). Docking calculations were also performed to investigate the binding mode of compound 8k into the non-nucleoside binding site of HIV-1 RT and to rationalize some SARs. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3md00028a

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Discovery of Novel Pyridazinylthioacetamides As Potent HIV-
1 NNRTIs Using A Structure-Based Bioisosterism Approach
Yuning Song1, Peng Zhan 1*, Dongwei Kang 1, Xiao Li 1, Ye Tian1, Zhenyu Li1, Xuwang Chen 1,
Wenmin Chen1, Christophe Pannecouque 2, Erik De Clercq2 and Xinyong Liu1,*
1Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road,250012, Jinan, Shan-
dong,P.R.China
2Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
KEYWORDS: HIV-1, Reverse transcriptase,NNRTIs, Synthesis, SAR, Heterocycle, Pyridazinylthioacetamide, Structure-
based bioisosterism strategy
ABSTRACT:In continuation of our endeavors to develop new, potent, selective, and less toxic antiꢀ
HIV agents, we describe our structureꢀbased bioisosterism design, synthetic strategy, and structureꢀ
activity relationship (SAR) studies that led to the identification of pyridazinylthioacetamides, anovel
class of NNRTIs, isosteres of arylazolylthioacetanilide derivatives. Nearly all of the tested compounds
inhibited HIVꢀ1 strain IIIB replication at the lower micromolar concentration range (EC50: 0.046ꢀ
5.46ꢁM). Notably, the most promising compound8kexhibited extremely potent inhibitory activity
against HIVꢀ1 replication with EC50 value of 0.046ꢁM, CC50of 99.9ꢁM and the viral selectivity index
amounted up to 2149. These values were much better than those of NVP (EC50= 0.09 ꢁM) andDDC
(EC50= 1.04 ꢁM). Compound 8k also exhibited moderate inhibition of enzymatic activity with an IC50
value of 4.06 ꢂM, which was at the same order of magnitude as that of NVP (2.74 ꢂM). Docking calcuꢀ
lations were also performedto investigate the binding mode of compound 8k into the nonꢀnucleoside
binding site of HIVꢀ1 RT andto rationalize some SARs.
steric pocket located 10 Å away from the DNA
polymerase active site, are a major component of
the current therapeutic regimens for AIDS, nameꢀ
ly as highly active antiretroviral therapy
(HAART). Nevertheless, the longꢀterm use of
NNRTIs is compounded by thee mergence of
drugꢀresistant viruses and potentially severe side
effects. Therefore, to alleviate these problems,
1. Introduction
The reverse transcriptase (RT) is one of the
main therapeutic targets for the design of antiꢀ
HIVꢀ1 drugs used in thetreatment of acquired
immunodeficiency syndrome (AIDS).1,2Among
RT inhibitors, the nonꢀnucleoside reverse tranꢀ
scriptase inhibitors (NNRTIs), binding to analloꢀ

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there is a substantial need for the identification of
novel NNRTIs with improved activity against
clinically relevant resistant mutants, and excelꢀ
lent pharmacokinetic and safety profiles.3,4
Encouraged by these promising results, and
with the aim tofurther explore thechemical feaꢀ
tures required for RT inhibition and identification
of the new compounds as potential NNRTIs
through application of the structureꢀbased biꢀ
oisosterismapproach, 23an excellent “followꢀon”ꢀ
basedlead optimization strategy 24 was carriedout
by modifying the centralcore structure of the lead
compounds to produce the desiredpotency, selecꢀ
tivity, and the required ADME profiles. Structurꢀ
al and structureꢀactivity relationship (SAR) studꢀ
ies of the known arylazolylthioacetanilides illusꢀ
tratedin the literature14ꢀ22 have pointed to the
presence ofkey chemical features that correlated
with the RT inhibitory ability, i.e., the aryl group
linked to the azole core fitted into the important
hydrophobic pocket and the carbonyl group of
the amide was able to establish a key hydrogenꢀ
bond through interaction with the backbone NꢀH
of K103. These functionalities are maintained in
a spatial arrangement within the NNRTIs binding
pocket bya positioning heterocyclic central core
inarylazolylthioacetanilides. There are differꢀ
ences in the electronic and conformational conꢀ
tribution of the fiveꢀmembered heterocyclic moiꢀ
ety to the binding of the inhibitors to
RT.15Additional branching moieties, such as the
Nꢀsubstitutedphenyl moiety located at the proꢀ
tein/solvent interface near a region of the protein
known to be flexible, can be present or extended
Among the structurally diverse HIVꢀ1
NNRTIs,
substituted
arylazolylthioacetaniꢀ
lidescaffolds have been of much interest for the
development of novel NNRTIs because oftheir
high potency and low toxicity against HIVꢀ1
wildꢀtype and resistant strains.5ꢀ11 Especialꢀ
12
ly,1,2,4ꢀtriazole derivatives VRXꢀ480733
and
13
RDEA806 , wereselected as candidates for furꢀ
ther studies. RDEA806, a promising newdrug
candidateundergoing phase IIa clinical trial (by
Ardea Biosciences Company), was highly effecꢀ
tive against mutant HIVꢀ1 strains and exhibited
reduced clinical adverse reactions and serum
halfꢀlife for onceꢀdaily dosing (Fig. 1).
Fig. 1.Azolylthioacetanilideꢀbased NNRTIs.
In our previous studies, we reported that a
series of new arylazolylthioacetanilide with strucꢀ
turally diverse azoles scaffolds possessed potent
antiꢀHIV activities in a cellꢀbased replicon sysꢀ
tem.14ꢀ22Among them, 1,2,3ꢀthiadiazole derivaꢀ
tive ZP7 (Fig. 1) exhibited the highestantiꢀHIVꢀ1
activity (EC50= 36.4 nM), inhibiting HIVꢀ
1replication in MTꢀ4 cells with sevenfold and
eightfold higher efficiency than nevirapine
(NVP)and delavirdine (DLV), respectively.15
2

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Scheme 1. Reagents and conditions: (i) NaH,
BrCH2CH(OEt)2, THF; (ii) KOH, EtOHꢀH2O; (iii) N2H4ꢀ
AcOHꢀWater; (iv) Br2, AcOH; (v) P2S5, Pyridine; (vi)
ClCH2CONHAr, triethylamine, THF.
to further optimize potency and/or physicochemiꢀ
cal properties.
Our structureꢀbased bioisosterism design of
new NNRTIs chemotypes was primarily focused
on the identification of additional heterocycle
core frame work with synthetic accessibility and
drugꢀlike properties, able to replace the central
chemical template of arylazolylthioacetanilides,
while keeping the above mentioned key interacꢀ
tion features in a spatial orientation appropriate to
fit into RT.
Efficient preparations of the novel 2ꢀ(4ꢀ
(naphthalenꢀ1ꢀyl)pyridazinꢀ3ꢀylthio)ꢀNꢀ
acetamides (8) have been developed, which isoutꢀ
lined in Scheme 1. Using the establishedchemisꢀ
try, a fourꢀstage synthesis starting from commerꢀ
cially available 2ꢀ(naphthalenꢀ1ꢀyl) acetonitrile
(1) gave the 4ꢀ(naphthalenꢀ1ꢀyl)pyridazinꢀ3(2H)ꢀ
one (6) in high overall yield, which was the key
reaction in this synthetic route.25 Treatment of 6
with phosphorus pentasulfide in pyridine under
reflex afforded 4ꢀ(naphthalenꢀ1ꢀyl)pyridazineꢀ
3(2H)ꢀthione (7). The final pyridazinethioacetaniꢀ
lides (8) were synthesized by reaction of interꢀ
Fig. 2.The structureꢀbasedbioisosterism replacement of
azoles by pyridazine.
mediates
7
with suitable 2ꢀchloroꢀNꢀarylꢀ
To achieve this goal, a variety of scaffolds
easyto synthesize and suitable for appropriate
chemical functionalization was scrutinized by
athorough bibliographic research. Herein, a series
of novel 2ꢀ(4ꢀ(naphthalenꢀ1ꢀyl)pyridazinꢀ3ꢀ
ylthio)ꢀNꢀarylacetamide derivatives were syntheꢀ
sized(Fig.2), andtheir antiꢀHIV activities against
WT HIVꢀ1 and HIVꢀ2, as well as HIV RT inhibiꢀ
tory potency was evaluated.
substituted acetamides(or other alkyl halides) in
good yields. The 2ꢀchloroꢀNꢀphenyl acetamides
(or other alkyl halides) were synthesized accordꢀ
ing to the literature.26 To the best of our
knowledge, this is the first example of the synꢀ
thesis of pyridazinethioacetanilides.The syntheꢀ
sizedcompounds were characterized by MS, IR
1
and/or H NMR spectral data together with TLC
analysis.
2. Results and Discussion
2.1. Chemistry
2.2 Biological activities
2.2.1 Anti-HIV activities evaluation
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The test of the newly synthesized pyridaziꢀ
nylthioacetamide derivatives for activities against
HIVꢀ1 and HIVꢀ2 was performed inMTꢀ4 cell
cultures 27infected with wildꢀtype HIVꢀ1 (strain
IIIB) 28 and wildꢀtype HIVꢀ2 (strain ROD)29. The
methodology of the antiꢀHIV assay has been preꢀ
viously described.30,31 Meanwhile, nevirapine
(NVP), zidovudine (azidothymidine, AZT), dideꢀ
oxycytidine (DDC), delavirdine (DLV) and
efavirenz(EFV) were used as reference drugs.The
cytotoxicity of these compounds was determined
in parallel. Comparisons of inhibitory concentraꢀ
tion (EC50), cytotoxic concentration (CC50), and
SI (selectivity, given by the CC50/EC50 ratio) valꢀ
ues for different compounds are depicted in Taꢀ
ble 1.
which were much better than those of NVP
(EC50= 0.09 ꢁM) andDDC (EC50= 1.04 ꢁM).
Besides compound 8k, some other compounds,
8b, 8f,8g, 8h and 8j, were also endowed with the
high antiꢀHIVꢀ1 potencies (EC50= 0.19, 0.20,
0.13, 0.14 and 0.14ꢁM,respectively) and good
selectivity indices (SI = 177, 929,370, 1108 and
1438, respectively). Suchresults provide useful
information for further development of thisclass
of HIVꢀ1 NNRTIs.
A brief investigation of the structureꢀactivity
relationships (SARs) revealed that the nature of
the orthoand the para substitution at the phenyl
ring of the anilide moiety influenced the antiꢀHIV
activity remarkably. For instance, Table 1 also
revealed the potency order of the ortho substituꢀ
tion at the phenyl ring of the anilide moiety:NO2
(8k, EC50 = 0.046ꢁM) > halogen atoms｛Cl (8b,
EC50 = 0.19ꢁM), Br (8e, EC50 = 0.21ꢁM), F (8d,
EC50= 0.26ꢁM)｝> Me (8l, EC50 = 1.09ꢁM) >H
(8a, EC50= 1.93ꢁM). Introduction of small hyꢀ
drophobic substituents (methyl group or chlorine
atom) at the para substitution in the anilide moieꢀ
ty of 8k and 8b provided compounds 8j and
8cwith decreased the antiꢀHIV activities, respecꢀ
tively. Worthy of note, in the case of compound
8e, introduction of the methyl, 4ꢀacetyl and
methoxy carbonylgroups at the para position at
the phenyl ring of the anilide moiety led to 8f, 8g
and 8h with retained activity. Whereas introducꢀ
Table 1. AntiꢀHIV activity, cytotoxicity and selectivity
indices of 2ꢀ(4ꢀ(naphthalenꢀ1ꢀyl)pyridazinꢀ3ꢀylthio)ꢀNꢀ
arylacetamide derivatives (8aꢀ8p).
As shown in Table 1, nearly all of the tested
pyridazinylthioacetamide derivatives inhibitꢀ
edHIVꢀ1 strain IIIB replication in the lower miꢀ
cromolar concentrationrange (EC50: 0.046ꢀ
5.46ꢁM), exceptfor one compound 8o, which
showed an EC50 value >34.51ꢁM. Among them,
2ꢀ(4ꢀ(naphthalenꢀ1ꢀyl)pyridazinꢀ3ꢀylthio)ꢀNꢀ(2ꢀ
nitrophenyl)acetamide(8k) was the most promisꢀ
ing compound. It exhibited extremely potent inꢀ
hibitory activity against HIVꢀ1 replication with
an EC50 value of 0.046ꢁM, CC50value of 99.9ꢁM
and the viral selectivity amounted up to 2149,
4

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tion of ethoxy carbonyl in 8e led to 8i with deꢀ
None of the pyridazinylthioacetamides deꢀ
rivatives inhibited the replication of HIVꢀ2 ROD
in MTꢀ4 cells at a subtoxic concentration. Thereꢀ
fore, based on this fact and the above SAR conꢀ
clusions, it can be concluded that these comꢀ
pounds were specific for HIVꢀ1 and belonged to
typical NNRTIs.
creased activity.
Encouraged by these results, in order to find
more potent inhibitors and to furtherexplore the
salient features controlling the activity, further
modificationsat the protein/solvent interface were
performed. One Nꢀsubstituted pyridine acetamide
derivative 8m was also synthesized with acceptaꢀ
ble antiꢀHIVꢀ1 profile. Moreover, it is also worth
mentioning that the phenylethanone derivative 8n
2.2.2 HIV-1 RT inhibition assay
Compound 8k was evaluated in enzymatic
testsfor its ability to inhibit highly purified reꢀ
combinant HIVꢀ1 RT using poly(rC)ꢀoligo(dG)
as template primer32 to further confirm the drug
target of pyridazinꢀ3ꢀylthioacetamidederivatives.
The assay results of the compounds are summaꢀ
rized in Table 4. Compound8k exhibited moderꢀ
ate inhibition of enzymatic activitywith an
IC50value of 4.06 ꢂM, i.e. the same order of
magnitude as that of NVP (2.74 ꢂM).
and
3,4ꢀdihydroisoquinolinꢀ2(1H)ꢀylethanone
derivative 8p also demonstrated reasonable antiꢀ
viral activity, presumably due to the optimum
interactions with the NNRTIs binding pocket
(NNIBP).
The
3,4ꢀdihydroquinolinꢀ2(1H)ꢀ
ylethanone derivative 8o was essentially inactive,
which could be a result of the unfavorablesteric
interactions with the enzyme. We believe that
these compounds can be further optimized by
more appropriate substitutions in the proꢀ
tein/solvent interface, and work is currently onꢀ
going in this area.
Table 4. Inhibitory activity of compound8k against HIVꢀ1
RT.
3. Molecular modeling
Figure 3.(A) Predicted binding mode and molecular dockꢀ
ing of compound 8k into theallosteric site of HIVꢀ1 RT
(PDB code: 3DLG); (B) Superimposition of the docked
conformations of 8k (white) and ZP7 (purple) in the HIVꢀ1
RT (PDB code: 3DLG); (C) Superimposition of the docked
conformations of 8k (white) and RDEA806(red) in the
HIVꢀ1 RT (PDB code: 3DLG). The docking resultsareꢀ
shown by PyMOL. Hydrogen bonds are indicated by
dashed lines.
On the whole, we found that the SARfeaꢀ
tures of the pyridazinylthioacetamides were
grossly consistent with the previously observed
arylazolylthioacetanilide NNRTIs.14ꢀ21 These
results confirm the important role of heterocyclic
core and the pyridazinylthioacetamide motifas a
valuable lead series for the design of the next
generation NNRTIs.
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In order to gain a better understanding of how 8k
interacts with RT NNIBP, a hypothetical molecuꢀ
lar modelof the complex was constructed using
AutodockVina[http://vina.scripps.edu] based on
our previously reported methodology (Fig.3).The
structure of HIV RT (ID: 3DLG) was acquired
from the Protein Data Bank (www.rcsb.org). The
resulting models showed that 8k adopted the typꢀ
ical “butterfly” conformation that is characteristic
ofmany NNRTIs (Fig. 3).The binding features
were similar to those of the previous hypothetical
molecularmodels obtained with arylazolylthioꢀ
acetanilide derivatives (RDEA806 and ZP7).
The left hand side naphthalene ring of 8k laid in
a hydrophobic cleft formed by Lue 234, Tyr188,
combination of hydrophobic interactions and πꢀ
stacking appears to be what governs the binding
of the pyridazinꢀ3ꢀylthioacetamide to RT. Conseꢀ
quently, the design of new arylazinylthioacetaniꢀ
lide derivatives will be further guided by the
model of interaction between the lead compound
8k and the NNIBP of RT.
4. Conclusion
In our onꢀgoing efforts to identify structuralꢀ
ly diverse compounds with HIV inhibitory activiꢀ
ty,we have identified a novel series of 2ꢀ(4ꢀ
(naphthalenꢀ1ꢀyl)pyridazinꢀ3ꢀylthio)ꢀNꢀ
arylacetamide by the structureꢀbased bioisosterꢀ
ism approach and also exploredthe SAR in the
protein/solvent interface near a flexible region of
the RT. Most active compounds showed activity
in the lowmicromolecular range with EC50 values
(varying from 0.046 to 5.46ꢁM).The most acꢀ
tivecompound, 8k showed activity against wildꢀ
type HIVꢀ1 in the low micromolar range (EC50 =
0.046ꢂM). As inhibitor of the WT RT of HIVꢀ1,
compound 8k showed inhibitory concentrations
comparable to the reference drug NVP. Because
oftheir excellent potency, these pyridazinꢀ3ꢀ
ylthioacetamidederivatives may have potenꢀ
tialand should be further pursued as nextꢀ
generation NNRTIs.Inaddition, molecular modelꢀ
ing studies are being carried out todetermine the
effect of structural features on antiꢀHIV activity.
These results have prompted further investigation
Trp229,
and
had
an
apparent
πꢀ
stackinginteraction with Tyr188. The amide carꢀ
bonyl of the ligand established a hydrogen bond
withthe backbone NH of Lys103. In addition,
potential interactions were probably present beꢀ
tweenthe pyridazin linker and residue Lys103.
Indeed the conformational and electronic contriꢀ
butionof the heterocyclic linkers have a signifiꢀ
cant effect on the binding of the inhibitors in the
NNIBP, which indicates the importance of cenꢀ
tral heterocycles for antiꢀHIV activity. Moreover,
the para substitution in the anilide moiety (carꢀ
boxyl in RDEA806) extends out of the hydroꢀ
phobic pocket and points tothe solventꢀexposed
regions, suggesting that there is a very large
space for substitution at this position. In all, the
6

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(3) Jayaweera, D.; Dilanchian, P. New therapeutic landscape of
NNRTIs for treatment of HIV: a look at recent data. Expert. Opin.
Pharmacother.2012,13, 2601ꢀ2612.
into alternative heterocyclic systems which will
be disclosed shortly.
(4) (a) Zhan, P.; Liu, X.; Li, Z. Recent advances in the discovery
and development of novel HIVꢀ1 NNRTI platforms: 2006ꢀ2008
update. Curr. Med. Chem.2009, 16, 2876ꢀ2889.(b) Zhan, P.; Liu,
X.; Li, Z.; Pannecouque, C.; De Clercq, E.Design strategies of
AUTHOR INFORMATION
Corresponding Authors
*
Peng Zhan. Tel: +86531 88382005; fax: +86531
88382731; Eꢀmail address:zhanpeng@sdu.edu.cn
Xinyong Liu. Tel: +86531 88380270; fax: +86531
*
novelNNRTIs
to
overcomedrugresistance.
Curr.
Med.
88382731; Eꢀmail address:xinyongl@sdu.edu.cn
Chem.2009, 16, 3903ꢀ3917.(c) Zhan, P.; Chen, X.; Li, D.;Fang,
Z.; De Clercq, E.; Liu, X.Structural diversity, pharmacophore
similarity, and implications for drug design. Med. Res. Rev. 2011,
Apr 26. doi: 10.1002/med.20241.(d) Zhan, P.; Liu, X.Novel HIVꢀ
1 nonꢀnucleoside reverse transcriptase inhibitors: a patent review
Present Addresses
44, West Culture Road,250012, Jinan, Shandong, P.R.
China
Funding Sources
The financial support from the National Natural Science
(2005 ꢀ 2010) Expert Opin Ther Pat. 2011, 5, 717ꢀ796.
.
Foundation of China (No.81273354, NSFC No.81102320,
No.30873133, No.30772629, No.30371686), Key Project
of NSFC for International Cooperation (No.30910103908),
Research Fund for the Doctoral Program of Higher Educaꢀ
tion of China (No.20110131130005, 20110131120037),
Independent Innovation Foundation of Shandong Universiꢀ
ty (IIFSDU, No.2010GN044), Shandong Postdoctoral Inꢀ
(5)
Zhan, P.; Li, Z.; Liu, X.; De Clercq,
promising class of HIVꢀ1
E.Sulfanyltriazole/tetrazoles:
a
NNRTIs. Mini. Rev. Med. Chem. 2009, 9, 1014ꢀ1023.
(6) Wang, Z.; Wu, B.; Kuhen, K. L.; Bursulaya, B.; Nguyen, T.
N.; Nguyen, D. G.; He, Y. Synthesis and biological evaluations
of sulfanyltriazoles as novel HIVꢀ1 nonꢀnucleoside reverse
transcriptase inhibitors. Bioorg. Med. Chem. Lett.2006,16,
4174ꢀ4177.
novation
Science
Research
Special
Program
(No.201002023), China Postdoctoral Science Foundation
funded project (No.20100481282, 2012T50584) and KU
Leuven(GOA 10/014)is gratefully acknowledged.
(7) De La Rosa, M.; Kim, H. W.; Gunic, E.; Jenket, C.; Boyle, U.;
Koh, Y. H.; Korboukh, I.; Allan, M.; Zhang, W.; Chen, H.; Xu,
W.; Nilar, S.; Yao, N.; Hamatake, R.; Lang, S. A.; Hong, Z.;
Zhang ,Z.; Girardet, J. L.Triꢀsubstituted triazoles as potent nonꢀ
nucleoside inhibitors of the HIVꢀ1 reverse transcriptase.
Bioorg. Med. Chem. Lett. 2006,16, 4444ꢀ4449.
ACKNOWLEDGMENT
We thank K. Erven, K. Uyttersprot and C. Heens fortechnical
assistance with the HIV assays.
ABBREVIATIONS
(8) Muraglia, E.; Kinzel, O. D.; Laufer, R.; Miller, M. D.; Moyer,
G.; Munshi, V.; Orvieto, F.; Palumbi, M. C.; Pescatore, G.;
Rowley, M.; Williams, P. D.; Summa, V.Tetrazole
thioacetanilides: potent nonꢀnucleoside inhibitors of WT HIV
reverse transcriptase and its K103N mutant. Bioorg. Med.
Chem.Lett. 2006, 16, 2748ꢀ2752.
SAR,structureꢀactivityrelationship; RT, reverse transcriptase;
NNRTIs, nonꢀnucleoside reverse transcriptase inhibitors;
HAART, highly active antiretroviral therapy; AIDS, acquired
immunodeficiency syndrome; NNIBP, NNRTIs binding pocket.
REFERENCES
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and biologicalevaluation of
a new series of 2ꢀ{[4ꢀ(3,4ꢀ
dichlorophenyl)ꢀ1,2,3ꢀ thiadiazolꢀ5ꢀyl]sulfanyl}acetanilides as
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randomized controlled trial of shortꢀterm activity, safety, and
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evaluation of novel N'ꢀarylideneꢀ2ꢀ[1ꢀ(naphthalenꢀ1ꢀyl)ꢀ1Hꢀ
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(13) Li, D.; Zhan, P.; De Clercq, E.; Liu, X.Strategies for the
design of HIVꢀ1 nonꢀnucleoside reverse transcriptase inhibitors:
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De
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Liu,
X.
Arylazolylthioacetanilide. part 8 ☆ : Design, Synthesis and
Biological Evaluation of Novel 2ꢀ(2ꢀ(2,4ꢀDichlorophenyl)ꢀ2Hꢀ
1,2,4ꢀtriazolꢀ3ꢀylthio)ꢀNꢀarylacetamides As Potent HIVꢀ1
Inhibitors. Eur. J. Med. Chem.2011,46, 5039ꢀ5045.
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Clercq, E.1,2,3ꢀThiadiazole thioacetanilides as a novel class of
potent HIVꢀ1 nonꢀnucleoside reverse transcriptase inhibitors.
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(15) Zhan, P.; Liu, X.; Li, Z.; Fang, Z.; Li, Z.; Wang, D.;
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synthesis and preliminary SAR studies.Bioorg. Med. Chem.
2009,17, 5920ꢀ5927.
Synthesis
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thiazolylthioacetamides derivatives as a novel class of potential
antiviral agents. Arch. Pharm. Res.2012, 35, 975ꢀ86.
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tion.Curr. Opin. Drug Discov. Devel.2001, 4, 471ꢀ478. (c) Liꢀ
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9

Medicinal Chemistry Communications
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Cl
Cl
Cl
N
N
N
N
S
N
H
H
H
N
N
N
N
S
S
N
S
Br
Br
N
N
O
O
O
Br
SO2NH2
COOK
VRXꢀ480773
EC50 = 0.14 ꢁM (WT)
EC50 = 0.23 ꢁM (K103N/Y181C)
RDEAꢀ806
ZP7
Br
EC50 =36.4 nM, SI>6460
Fig. 1.Azolylthioacetanilideꢀbased NNRTIs.
N
R2
N
R2
N
N
H
H
N
N
R1
S
S
X1
N
O
O
X2
R4
R4
R
Structure-Based
Bioisosterism Design
Triazole thioacetanilide
N
N
R2
S
N
H
Het
N
N
S
S
X3
O
O
R4
R
X3 = CH, N
Pyridazinꢀ3ꢀylthioacetanilide
1,2,3ꢀThiadiazole thioacetanilide
Lead structures
Target compounds
Fig. 2.The structureꢀbased bioisosterismreplacement of azoles by pyridazine.
EtO
EtO
OEt
COOH
EtO
OEt
N
O
NH
CN
CN
O
O
iii
i
ii
iv
1
2
3
4
5
N
N
N
N
NH
NH
N
R2
N
H
Het
N
S
O
S
S
X1
O
O
X2
vi
v
R4
6
7
8
Scheme 1. Reagents and conditions: (i) NaH, BrCH2CH(OEt)2, THF; (ii) KOH, EtOHꢀH2O; (iii) N2H4ꢀ
AcOHꢀWater; (iv) Br2, AcOH; (v) P2S5, Pyridine; (vi) ClCH2CONHAr, triethylamine, THF.
10

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N
N
NO2
H
N
S
O
8k
EC50 = 46 nM
(A)
(B)
(C)
Figure 3.(A) Predicted binding mode and molecular docking of compound 8k into theallosteric site of
HIVꢀ1 RT (PDB code: 3DLG); (B) Superimposition of the docked conformations of 8k (white) and
ZP7 (purple) in the HIVꢀ1 RT (PDB code: 3DLG); (C) Superimposition of the docked conformations of
8k (white) and RDEA806 (red) in the HIVꢀ1 RT (PDB code: 3DLG). The docking results are shown by
PyMOL. Hydrogen bonds are indicated by dashed lines.
Table 1. AntiꢀHIV activity, cytotoxicity and selectivity indices of 2ꢀ(4ꢀ(naphthalenꢀ1ꢀyl)pyridazinꢀ3ꢀ
ylthio)ꢀNꢀarylacetamide derivatives (8aꢀ8p).
11

Medicinal Chemistry Communications
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N
N
R2
H
N
S
X1
O
X2
R4
CC50
(ꢁM)
EC50(ꢁM)
SI
Code
X1
X2
R2
R4
HIVꢀ1
IIIB
HIVꢀ2
ROD
HIVꢀ1
IIIB
HIVꢀ2
ROD
HIVꢀ1
IIIB
105
HIVꢀ2
ROD
< 1
<1
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
H
Cl
H
H
>173.91
>34.98
203.0
173.91
8a
8b
8c
8d
8e
8f
1.93
34.25
>283.87
204.39
31.53
34.98
231.63
240.60
31.31
177
0.19
0.42
0.26
0.21
0.20
0.13
0.14
0.56
0.14
0.046
1.09
0.27
Cl
Cl
>231.63
>240.60
>31.31
>684
799
<1
F
H
<1
Br
H
151
<1
Br
Me
>215.34
>48.54
182.40
46.31
215.34
48.54
929
<1
Br
COMe
COOMe
COOEt
Me
370
<1
8g
8h
8i
Br
>245.87
>239.28
>243.91
>134.71
>150.46
>164.42
158.74
237.36
204.42
99.90
>245.87
>239.28
243.91
134.71
150.46
164.42
1106
420
×1
Br
×1
NO2
NO2
Me
Cl
1438
2149
130
<1
8j
H
<1
8k
8l
H
141.90
156.06
<1
H
573
<1
8m
N
Cl
Cl
N
S
>55.02
54.55
55.02
10
<1
8n
5.46
O
12

Page 13 of 13
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N
N
N
S
>34.51
>34.99
34.51
34.99
<1
<1
8o
O
N
N
N
S
>36.45
34.02
36.45
29
<1
8p
1.15
O
NVPe
AZTe
DDCe
EFVe
DLVe
0.09
0.021
>15.02
249.5
>15.02
249.5
>168
0.0045
1.28
12221
>93
56907
>74
1.04
>94.69
>6.34
>94.69
>6.34
0.0054
0.03257
>1187
>1096
>36.19
>36.19
aEC50: concentration of compound required to achieve 50% protection of MTꢀ4 cells against HIVꢀ1ꢀinduced cytotoxicity, as determined by
the MTT method. In bold are the values of active compounds.
bCC50: concentration required to reduce the viability of mockꢀinfected cells by 50%, as determined by the MTT method.
cSI: selectivity index (CC50/EC50). The SI values: x 1 stand for ≥1or<1.
eThe data were obtained from the Rega Institute for Medical Research, KU Leuven, Belgium.
Table 2. Inhibitory activity of compound 8k against HIVꢀ1 RT.
NVP
2.74
Compd.
8k
IC50(ꢂM)a
4.06
a50% of the inhibitory concentration of tested compounds required to inhibit biotin deoxyuridine triphosphate (biotinꢀdUTP) incorporaꢀ
tioninto the HIVꢀ1 RT by 50%.
13