From TIBO to

Etravirine:20 Years of
Research on Non-
Nucleoside Inhibitors of
HIV-1 Reverse
Transcriptase
 Introduction to HIV
• The 20th century had faced two unexpected viral
outbreaks.

• During 1918-1919 emergence of an old virus causing

influenza A and killing over 25 million people
worldwide.

• In 1981 the world for the first time became aware of

another deadly disease outbreak, the ‘Acquired
Immunodeficiency Syndrome’ or AIDS caused by a
deadly virus ‘Human Immunodeficiency Virus’ or HIV
 Ancestry of HIV
• Scientist believed that ancestry of HIV virus is
SIVcpz (Simian Immunodeficiency Syndrome)
which are mainly found among the Chimpanzees
in west central Africa
• The transmission of the virus to humans
probably occurred while animals were
butchered for food and the virus transmitted
to human blood from infected animal blood
through human wounds.
• Huge migration of people from those areas to
other part of the world caused the virus to
spray out the world.
 HIV Cell Biology
•

http://www.avert.org/aidspicture.php?photo_id=504
 HIV Life Cycle

http://img.thebody.com/nmai/cycle.jpg
 Drug development-Making
Strategies
• One strategy is to destroy or repair the affected genes in human
T-cells.

• Gene Therapy is am important tool in this regard on

which researchers are working.
 Drug development-Making strategies
• And the second strategy is to inhibit HIV to infect genes into human T-cells.

This can be done in several ways,

 by stopping the virus from binding to CD4+ receptors
 inhibiting the fashioning of the capsid inside T-cell
 by inhibiting the replication of viral RNA
 by inhibiting replicated viral RNA from integrating into
human DNA
 by inhibiting the maturation of the new viral protein

__________________________________________________________________________________________________________________________________________________________________________

cartoon source-http://www.etoon.com
 Antiretroviral Drug
Classes
• Depending upon their way of action these
drugs can be classified in following groups,
• Nucleoside/Nucleotide Reverse
Transcriptase Inhibitors (NRTIs)
• Non-Nucleoside Reverse Transcriptase
Inhibitors (NNRTIs
• Protease Inhibitors (PIs)
• Fusion or Entry Inhibitors
• Integrase Inhibitors
• Maturation inhibitors
 History Begins
• Etrvirine a brand new drag which got the
FDA approval in January 2008
• The drug was marketed by Tibotec
Therapeutics, a subsidiary of
Johnson & Johnson
• Research work started in the year of 1987
at Janssen Pharmaceutica and under the
guidance of legendary Dr.Paul Janssen
 Discovery of TIBO and αAPA Series: The

first line of anti-HIV NNRTIs

• SAR was done on over 600 compounds from the Janssen compound
collection
• After selective screening in cell culture at virology laboratories at the
Rega Institute followed by a directed lead optimization, led to the
discovery of the first generation of NNRTIs aTIBO(R86183)*, an α-
APA(Loviride)* and a dipyridodiazepinone(Nevirapine) derivative; during the
period of 1995-1996.

O
Cl O Br H3C H
N
H
N
NH
N Br NH NH NH
S O NH2

TIBO(R86183) Loviride(R95845) Nevirapine

(1) (2) (3)

-----------------------------------------------------------------------------------
• *TIBO = tetrahydro-imidazo(4,5,1-jk) (1,4)-benzodiazepin-2(1H) thione
• α-APA = α-anilinophenylacetamides
 Information Obtained From First Line of
NNRTIs

• These derivatives were effective against wild-type HIV-1

virants
• But unfortunately had significantly lower potency when
tested against common NNRTI-resistant mutants
• But from the study of the crystal structure of the HIV-I
RT/TIBO, HIV-1RT/ α-APA and HIV-1 RT/nevirapine
complexes disclosed so many useful information regarding;
 mechanism of inhibitor binding on the enzyme
 conformational changes of that of inhibitors and the
binding sites on enzyme where the inhibitors bind

• All these information facilitated the further

research to design more potent NNRTIs.
 Information Obtained From First Line of
NNRTIs

• The key design features learned from these structural studies

include the following
(1) All the inhibitors bind to the HIV-1 RT in a hydrophobic pocket (the non-
nucleoside inhibitor-binding pocket, or NNIBP) with a common binding mode which the
researcher termed as ‘butterfly-like’ mode. The wing I and wing II generally contain
aromatic rings.
 Information Obtained From First Line of
NNRTIs
(2) Binding of the inhibitors to the NNIBP is stabilized by the π-π stacking interactions
between the aromatic groups of inhibitors those of aromatic amino acids residues (especially
those of Tyr181, Tyr188, Tyr318, Trp229, and Phe227) in the binding pocket. Also the H-
bonding between the inhibitors and amino acid residues contribute towards the stability
(3) Binding of inhibitor in the NNIBP cause a significant change in the secondary
structure (orientation of the amino acid side chains and relative position of the secondary
structural elements) of the enzyme around NNIBP. These conformational changes in NNIBP
distort the precise geometry and\or mobility of the nearby polymerase catalytic site and
consequently DNA polymerization by HIV-1 RT is stopped as for that purpose mobility and
flexibility at polymerase active site is essential.
(4) In NNRTI resistant mutations the enzyme-RT impart conformational
rearrangements of the aromatic amino acid residues around NNIBP so that the π -π stacking
interactions between the aromatic groups of inhibitors those of aromatic amino acids
residues is reduced. Eventually the inhibitor becomes no longer effective. Since all these
first series of NNRTIs were conformationally rigid hence they were not able to adopt the
suitable conformation so that the wings can interact with the aromatic amino acid residues
in NNRTI resistant HIV mutants.
• New Ideas: All these useful information helped the researcher to design the more
NNRTIs and while doing so the kept the following ideas in mind.
 Aromaticity at the two wings should be restored and enhanced
 Flexibility need to be increased around the body/linker of the
two wings so that the inhibitor can adjust its own conformation
against NNR
 Discovery of Imidoyl Thioureas (ITUs) series: potent

2nd line of NNRTIs

• The researchers did an extensive SAR on α-APA series and came out
with some promising results (Table-1)
Table.1.Activity (EC50,µm) O Cl
Cl H
Compound E Y EC50(µM)
H Y N
4a CH2 o-NO2 >50 N
E
4b CO o-NO2 >50 Cl
4c CONH o-NO2 >50 Cl H2N O
O NH2
4d CSNH o-Cl >50
4e CSNH p-Cl 0.10 (4) Lov iride(5)
4f - o-Cl 0.4

• They noticed in general increasing the spacer length between two aryl
groups in loviride result in considerable decrease in activity
• But 4e which was surprisingly active compared to all other analogues
 Discovery of Imidoyl Thioureas (ITUs) series:
potent 2nd line of NNRTIs

• Further investigation of this anomaly along with SAR on a series of

ITU analogues with proper choice of the substitution pattern on
both aryl groups led to the discovery of the ITU derivative
R100943 as a potent NNRTI
• It exhibited good potency against HIV-1 and some Selected Single
Mutants (Table.2)
N
Table.2. Activity(EC50,µM)of R100943

Compound LAI(HIV-1) L100I K13N

Nevirapine (3) 0.032 0.32 6.3 Cl

Loviride(5) 0.013 0.05 1.3 Cl H
N NH
IR1009439(6) 0.003 0.51 0.59
NH S
ITU(R100943)

(6)
 Drawback of R100943
• The in vitro activity profile of ITU-R100943 vs HIV-1 (Table2.)
justified the consideration of it for clinical development
• But unfortunately the oxidative ring closure of the imidoyl thiourea
functionality, under metablolic condition proved to be a major liability
for this drug candidate as the cyclic thiadiazole derivative(7)
resulting from metabolic oxidation was inactive (EC50>1µM vs HIV-1)
N N

Oxidation
Cl Cl Cl Cl
N NH N NH

NH S N S
R100943 (7)
 Information obtained 2nd Series of
NNRTIs
• Crystal structure of RT\R100943 complex revealed that the
conformation of the inhibitor at NNIBP resembles a ‘U’ or
horseshoe shape (Fig.3) in contrast to butterfly shape as in the
case of TIBO or α-APA derivatives.
 Discovery of Diaryltrazine(DATA) 3rd Series
NNRTIs
• The key design features learned from the study of
ITU derivatives are,
(1)The horseshoe binding mode must be maintained in
the succeeding drug candidates

(2)Structural modification is needed to enhance

stability or the drug candidate against oxidative
ring closure

(3)The 4-cyanophenylgroup which bind at the eastern

section of the NNIBP must be kept constant in the
succeeding generations of the NNRTIs
 Discovery of Diaryltrazine(DATA) 3rd Series
NNRTIs
• To improve the metabolic stability of ITU-R100943 they
attempted to synthesize the cyanoguanidine isoster of ITU-
R100943 as outlined in the synthetic scheme below
N

Cl Cl

N N H
Cl Cl + H
Cl Cl
O N N N
N H H
2 N N H
N N H
N H C N C N 2
N H N
N C
(9)
(8)

(8) (9)

• But unexpectedly intended cyanoguanidine (8) was never isolated,

instead R106168 (9); the first representative of the DATA series
of NNRTIs was obtained
 Discovery of Diaryltrazine(DATA) 3rd
Series NNRTIs
• Surprisingly it is equally potent as the ITU-R100943 was
(EC50=0.006µ vs HIV-1)
• X-ray crystallography study of the RT bound R106168 complex
conformed the ‘U’ shape of bound inhibitor and also suggested that
the primary amino group is probably not interacting with the RT
enzyme
• Extensive SAR was done on anlogues of(9) through multiple
substitutions and\or additions of at various position on all the three
rings and on the two linkers
• After long in vitro analysis they came up with three clinically
important derivatives (Table-3) N

Table.3.Activity
Table.3 of DATAs
Comp. R Y LAI(HIV-1) L100I K103N R

10a 2,4,6-tri-Me NH 0.0003 0.013 0.003 Y N

10b 2,4,6-tri-Me O 0.0006 0.020 0.003 N N

10
10c 2,6-di-Me-4-CN NH 0.001 0.25 0.008
(10)
 Drawbacks DATA Series of
Derivatives

• Despite of satisfactory potency

against HIV-1 and other single
mutants DATAs were completely
inactive against HIV double mutants
in clinical settings
• Hence more structural modifications
were required
 Development of Diarylpyrimidines
(DAPYs): Discovery of Etravivirine
• With the help of molecular modeling researcher
decided to replace the triazine ring in DATA series of
NNRTIs with pyrimidine ring which led to the discovery
of DAPY series of NNRTIs

N N

Cl Cl
Cl Cl Cl Cl
N NH
N NH NH
N
N N N

NH 2 NH2
NH 2

11a 11b 11c

• All these derivatives exhibited good anti-viral activity

vs HIV single mutants
 Development of Diarylpyrimidines
(DAPYs): Discovery of Etravivirine
• Researchers then decided to monitor the effect of replacing the primary
ammine group
N N N
N N

O N NH HN N NH O N NH
HN N NH
N N N
N
12b 12c 12d
12a

• The activity profile of these derivatives we satisfactory against HIV single

mutants (Table.4.), but insignificant against HIV double mutants

Table.4.Activity(EC50,µM) of 12a-d
Comp. LAI(HIV-1) L100I K103N L100I+K103N K103N+Y181C

12a 0.001 0.018 0.004 >10 0.044

12b 0.003 0.022 0.003 8.2 1.1

12c 0.0004 0.034 0.002 1.1 0.037

12d 0.001 0.073 0.003 0.80 0.094

 Development of Diarylpyrimidines
(DAPYs): Discovery of Etravivirine
• Researchers then decided to explore the impact of substitution
at the 5-position of the pyrimidine ring
• They were able to synthesize 13a-c and measured their in-vitro
activity which were very promising (Table-5) R
N

Table.5.Activity(EC50,µM) of 13a-c
Y N NH
Comp R Y LAI(HIV-1) L100I K103N Y181C L100I+K103N K103N+Y181C
N
Br
13a Me NH 0.006 0.011 0.007 0.036 0.35 0.28

13b CN O 0.001 0.007 0.001 0.022 0.049 0.025

(13)

13c CN NH 0.0004 0.007 0.0004 0.010 0.037 0.032

• The activity of 13b&13c against very recalcitrant

L100I+K130N double mutant was reason for excitement
 Finally Discovery of Etravirine
• Finally, introduction of an amino group at the 6-position of the
pyrimidine ring in 13b, afforded the long awaited NNRTI, the
‘Etravirine’ ( or TMC125 or R165335)
N N

O N NH

N
Br
NH2
Etravirine

• It is a compound with low-Nanomolar potency against wild

type HIV-1 and a large spectrum of clinically relevant
HIV single and double mutants
 Activity of Etravirine
• The overall in vitro potency file of etravirine compared to other first and
second generation NNRTIs against HIV-1 and other selected single
mutants is shown in Table.6

Table.6.Activity(EC50,µM) of Etravirine

Color codes: orange, EC50 > 0.1 µM; yellow, 0.1 > EC50 > 0.01 µM; and light yellow, EC50 < 0.01 µM.
 Activity of Etravirine
• But the potency of etravirine against the double mutants
L100I+K103N and K103N+Y181C (Table.7) makes it as an extraordinary
drug candidate.
N N
Table.7.
Comp. L100I+K103N K103N+Y181C
Nevirapine nb >10
Delaviridine nb >10

Efavirezine >10 0.040 O N NH

Etravirine 0.019 0.004 N

Br
NH2
Etravirine

• Reason Behind the Activity of etravirine

The torsional flexibility around the bonds connecting the two wings
with the pyrimidine rings permits etravirine to access more than one
conformationally distinct mode.
This enable the drug bind NNRTI resistant single and double HIV
mutants enzyme in more than one conformationally distinct mode.
 Beyond Etravirine-The Discovery of
Rilpivirine (TMC278)

• Research work is on to improve the pharmacokinetics properties of etravirine

while maintaining its activity profile against wild-type HIV-1 and clinically
relevant HIV-1 mutants
• This effort has led to the discovery of Rilpivirine which exhibited an
exceptional activity profile
• It entered phase-III clinical trials on April 2008,with an estimated study
completion date on August 2010
N

HN N NH

Ralpivirine
 Conclusion
• Though so many effective HIV anti-
retrovirals are available most of them
are very expensive and only rich people
can afford those medication
• Research should be continued to find
out effective drugs which will be
cheaper and accessible to all.
 References
1. Bart L De Corte,J.Med. Chem. 2005,48,1689-1696

2. J.Med. Chem. 2004, 47, 2550-2560

3. Nature Structural Biology, volume 2,Number 5, May 1995, 407-415

4. Web Sources-www.wikipedia.org
Thank you and have a
nice day