From TIBO to From TIBO to Etravirine:20 Years of Etravirine:20 Years of

Research on Non-Research on Non-Nucleoside Inhibitors of Nucleoside Inhibitors of

HIV-1 Reverse HIV-1 Reverse TranscriptaseTranscriptase

From TIBO to From TIBO to Etravirine:20 Years of Etravirine:20 Years of

Research on Non-Research on Non-Nucleoside Inhibitors of Nucleoside Inhibitors of

HIV-1 Reverse HIV-1 Reverse TranscriptaseTranscriptase

Introduction to HIVIntroduction 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 HIVAncestry 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 BiologyHIV Cell Biology

•

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

HIV LifeHIV Life CycleCycle

http://img.thebody.com/nmai/cycle.jpg

Drug development-Making Drug development-Making StrategiesStrategies

• 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 Drug development-Making strategiesstrategies

• 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 RNAby inhibiting the replication of viral RNA by inhibiting replicated viral RNA from integrating into by inhibiting replicated viral RNA from integrating into human DNAhuman DNA by inhibiting the maturation of the new viral proteinby inhibiting the maturation of the new viral protein

__________________________________________________________________________________________________________________________________________________________________________

cartoon source-http://www.etoon.com

Antiretroviral Drug Antiretroviral Drug ClassesClasses

• Depending upon their way of action these drugs can be classified in following groups,

I.I. Nucleoside/Nucleotide Reverse Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs) Transcriptase Inhibitors (NRTIs)

II.II. Non-Nucleoside Reverse Transcriptase Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs Inhibitors (NNRTIs

III.III. Protease Inhibitors (PIs)Protease Inhibitors (PIs)

IV.IV. Fusion or Entry Inhibitors Fusion or Entry Inhibitors

V.V. Integrase Inhibitors Integrase Inhibitors

VI.VI. Maturation inhibitorsMaturation inhibitors

History BeginsHistory Begins

• Etrvirine a brand new drag which got the FDA approval in January 2008

• The drug was marketed by TibotecTibotec 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.

(1) (2) (3) -----------------------------------------------------------------------------------• *TIBO = tetrahydro-imidazo(4,5,1-jk) (1,4)-benzodiazepin-2(1H) thione

• α-APA = α-anilinophenylacetamides

O NH2

NH

Br

BrO

Loviride(R95845)

NH

NHNH NH

CH3

O

Nevirapine

Cl

N

NH

S

TIBO(R86183)

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.

InformationInformation Obtained From First Line of Obtained From First Line of NNRTIsNNRTIs

• 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: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 Aromaticity at the two wings should be restored and enhancedenhanced

Flexibility need to be increased around the body/linker of Flexibility need to be increased around the body/linker of the two wings so that the inhibitor can adjust its own the two wings so that the inhibitor can adjust its own

conformation against NNRconformation 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)

(4) Loviride(5)

• 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

Compound E Y EC50(µM)

4a CH2 o-NO2 >50

4b CO o-NO2 >50

4c CONH o-NO2 >50

4d CSNH o-Cl >50

4e CSNH p-Cl 0.10

4f - o-Cl 0.4

Cl

Cl

NH

E

O NH2

Y NH

O

NH2 O

Cl

Cl

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 R100943R100943 as a potent NNRTI

• It exhibited good potency against HIV-1 and some Selected Single Mutants (Table.2)

Table.2.Table.2. Activity(EC50,µM)of R100943

(6)

Compound LAI(HIV-1) L100I K13N

Nevirapine (3) 0.032 0.32 6.3

Loviride(5) 0.013 0.05 1.3

IR1009439(6) 0.003 0.51 0.59

Cl

Cl

NH

NH

S

NH

N

ITU(R100943)

Drawback of R100943Drawback 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 oxidative ring closure of the imidoyl thiourea functionality, under metablolic conditionthiourea 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)

Cl Cl

NH

N

S

NH

N

Cl Cl

N NH

N

N S R100943 (7)

Oxidation

Information obtained 2Information obtained 2ndnd Series of Series of NNRTIsNNRTIs

• Crystal structure of RT\R100943 complex revealed that the conformation of the inhibitor at NNIBP resembles a ‘U’ or ‘U’ or horseshoe shapehorseshoe shape (Fig.3) in contrast to butterfly shapebutterfly shape as in the case of TIBO or α-APA derivatives.

Discovery of Diaryltrazine(DATA) 3Discovery of Diaryltrazine(DATA) 3rdrd Series Series

NNRTIsNNRTIs • The key design featuresdesign 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) 3Discovery of Diaryltrazine(DATA) 3rdrd Series NNRTIsSeries NNRTIs

• To improve the metabolic stability of ITU-R100943 they attempted to synthesize the cyanoguanidine isostercyanoguanidine isoster of ITU-R100943 as outlined in the synthetic scheme below

(8) (9) • But unexpectedly intended cyanoguanidineintended cyanoguanidine (8) was never

isolated, instead R106168 (9);R106168 (9); the first representative of the DATA series of NNRTIs was obtained

Cl Cl

N H

N H2

NH

NC NC N

OCl Cl

N H

NH

N

N H

N

N C

Cl Cl

N N H

N

N N

N H2

+

(8)(9)

Discovery of Diaryltrazine(DATA) Discovery of Diaryltrazine(DATA) 33rdrd Series NNRTIs 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‘U’ shape of bound inhibitor and also suggested that the primary amino groupprimary 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)

Table.3Table.3.Activity of DATAs

(10)

Comp. R Y LAI(HIV-1) L100I K103N

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

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

10c 2,6-di-Me-4-CN NH 0.001 0.25 0.008

Y

N N

N

N

R

10

Drawbacks DATA Series of Drawbacks DATA Series of DerivativesDerivatives

• Despite of satisfactory potency against HIV-1 and other single mutants DATAs were completely inactiveinactive against HIV double double mutantsmutants in clinical settings

• Hence more structural modifications were required

Development of Development of Diarylpyrimidines (DAPYs): Diarylpyrimidines (DAPYs):

Discovery of EtravivirineDiscovery 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

• All these derivatives exhibited good anti-viral activity vs HIV single mutants

Cl Cl

N

N NH

NH2

N

Cl Cl

N

N NH

NH2

N

Cl Cl

N N

NH

NH2

N

11c11b11a

Development of Development of Diarylpyrimidines (DAPYs): Diarylpyrimidines (DAPYs):

Discovery of EtravivirineDiscovery of Etravivirine

• Researchers then decided to monitor the effect of replacing the primary ammine group

• The activity profile of these derivatives we satisfactory against HIV single mutants (Table.4.), but insignificant against HIV double mutants

Table.4.Table.4.Activity(Activity(EC50,µMEC50,µM) ) of 12a-dof 12a-d

O

N

N NH

N

NH

N

N NH

N

O

N

N NH

N

N

N

N

NH NH

N

12a12b 12d12c

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 Development of Diarylpyrimidines (DAPYs): Diarylpyrimidines (DAPYs):

Discovery of EtravivirineDiscovery 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)

Table.5.Table.5.Activity(EC50,µM) of 13a-cActivity(EC50,µM) of 13a-c

(13)(13)

• The activity of The activity of 13b&13c against against very recalcitrant L100I+K130NL100I+K130N double mutant was reason for excitement

Comp R Y LAI(HIV-1) L100I K103N Y181C L100I+K103N K103N+Y181C

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

13c CN NH 0.0004 0.007 0.0004 0.010 0.037 0.032

Y

N

N NH

R

N

Br

Finally Discovery of Finally Discovery of EtravirineEtravirine

• 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)

• 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

O

N

N NH

N

Br

N

NH2

Etravirine

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

Color codes: orange, EC50 > 0.1 Color codes: orange, EC50 > 0.1 µµM; yellow, 0.1 > EC50 > 0.01 M; yellow, 0.1 > EC50 > 0.01 µµM; and light yellow, EC50 < M; and light yellow, EC50 < 0.01 0.01 µµM.M.

Activity of EtravirineActivity 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.

Table.7.Table.7.

• Reason Behind the Activity of etravirineReason 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.

Comp. L100I+K103N K103N+Y181CComp. L100I+K103N K103N+Y181C

Nevirapine nb >10Nevirapine nb >10

Delaviridine nb >10Delaviridine nb >10

Efavirezine >10 0.040Efavirezine >10 0.040

Etravirine 0.019 0.004Etravirine 0.019 0.004

O

N

N NH

N

Br

N

NH2

Etravirine

Beyond Etravirine-The Discovery of Beyond Etravirine-The Discovery of

Rilpivirine (TMC278)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

Ralpivirine

NH

N

N NH

N

N

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