AntiAnti--viralsvirals::Inhibitors of HIV Protease

IntroductionAn estimated 58,300 adults were living with HIV in the UK at the end of 2004, of whom 19,700 (34%) were unaware of their infection. In 2005, there were at least 6,727 new diagnoses of HIV, contributing to a cumulative total of 78,883 reported by the end of March 2006. There have been 22,099 diagnoses of AIDS in the UK. It is known that at least 13,386 of these people have died.

Since 1999 there has been a steep increase in the number of HIV diagnoses. During 2005, reports show that at least 6,727 people were diagnosed with HIV in the UK. This number is expected to rise as further reports are received (there were 7,444 reported diagnoses in 2004). The major component of the rapid increase in recent years has been in heterosexually acquired infections. Although around 80% of these are contracted in countries with high HIV prevalence, infections acquired within the UK have also risen.

More information on UK HIV statistics: http://www.avert.org/uksummary.htm

Mode of ActionHIV is the causative agent of AIDS, which is defined as the development of certain opportunistic infections and with the catastrophic depletion of CD4+

lymphocytes. It is not yet understood how HIV causes AIDS!

Upon infection of the CD4+ cells, the reverse transcribed RNA genome of HIV is incorporated into the host genome. As the genome is integrated and HIV disease is chronic, and since there is no strategy for the excision of viral genomes from host DNA, treatment with anti-viral agents will be lifelong.

HIV carries its genetic information in the (+)-RNA strand, two copies of which are contained in the virus, along with the enzymes responsible for transcribing the information into the DNA of the host cell. Key to this replicative process is HIV reverse transcriptase enzyme (RT). Following formation of the (-)-DNA strand (from complimentary viral (+)-RNA strand), RT degrades the original viral RNA of the newly formed RNA-DNA duplex so that double stranded DNA can be prepared for integration into the host cell’s DNA.

Mode of ActionNucleoside analogues which are inhibitors of the HIV replication act, after phosphorylation by host cell kinases to their respective 5’-triphosphates, as inhibitors or chain terminators of HIV-RT. Typically chain terminators are devoid of the 3’-OH group required for chain extension. The first drug shown to have widespread utility against AIDS was zidovudine (AZT). Which was originally synthesised as a cancer treatment in 1964, but was shown to be ineffective. It was re-screened in the 1980’s as the AIDS epidemic swept across Western society.

As HIV is an RNA virus with no proofreading function it is inevitable that random genetic mutations will result in the production of drug resistant strains of the virus. Hence there is still much interest in the generation of new compounds effective against new or multi-drug resistant stains of the virus.

O

N3

HO

NH

N

O

OZidovudine aka AZT

Synthesis of Zidovudine

O

HO

HO

NH

N

O

O O

MsO

TrO

NH

N

O

O

OTrO

NH

N

O

O

N3

OHO

NH

N

O

O

N3

1) TrCl, py

2) MsCl, py

HCl, CHCl3, 4 oC

LiN3, DMF, 100 oC

zidovudine aka AZT

Starting material (2’-deoxy-lyxofuranosyl)thymine is expensive.

An Alternative Synthesis of Zidovudine

O

HO

HO

NH

N

O

O O

MsO

TrO

NH

N

O

O

OTrO N

N

O

O OTrO

NH

N

O

O

N3

OHO

NH

N

O

O

N3

1) TrCl, py

2) MsCl, py

80% AcOH, steam bath

K-phthalimide, DMF, H2O, 95 oC

zidovudine aka AZT

NaN3, DMF, H2O, reflux

mechanism?

Starting from thymidine a double inversion protocol was required to synthesise AZT.

Erectile Erectile DisfunctionDisfunction::Inhibitors of type-5 CGMP

Phosphodiesterase

Introduction

It is estimated that 10% of men suffer from erectile dysfunction, and that this figure is as much as 52% for men between 40 and 70 years old!

Sildenafil was discovered by a research team at Pfizer in Kent. The Pfizerresearch team started working on the programme in 1985 with the aim of discovering antihypertensive and antianginal compounds. They were studying compounds which inhibit phosphodiesterase (PDE). At that point, the team had no idea that their research would lead them to discover an anti-impotence drug. In 1989 sildenafil had been synthesised. Clinical trials of sildenafil were started in July 1991. In 1992, it was realised that the drug was not as effective as they had hoped. However, they were surprised because many of the patients that had tried it were reluctant to stop the trial, and wanted to continue taking the drug. The researchers finally learnt from some of the patients that one of the side effects of the drug was that it helped erections.

Robert F. Furchgott, Louis J. Ignarro and Ferid Murad

In 1998, Furcgott, Ignarro and Murad were awarded the Nobel Prize for Medicine for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system.

b. 1916

http://nobelprize.org/nobel_prizes/medicine/laureates/1998/

b. 1941 b. 1936

Mode of Action

Nitric Oxide (NO) is released with sexual stimulation from nerve endings and endothelial cells in the spongy erectile tissue, of the penis. The enzyme guanylate cyclase then converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). cGMP causes the smooth muscle to relax, which causes an inflow of blood which leads to an erection. cGMP is then hydrolysed back to the inactive GMP by phosphodiesteras type 5 (PDE5). The levels of cGMP are therefore controlled by the activation of cyclic nucleotide cyclase and the breakdown by PDE5. It is the latter that sildenafil acts upon.

Mode of Action

enzyme guanylate cyclase activated

increased level of cGMP

phospodiesterase type 5

GTP GMP

smooth muscle

relaxation

inflow of blood

erection

sildenafil (Viagra)

cell membrane

NO release during sexual stimulation

NH

N

N

O

NH2N

O

OHOO

POO

cGMP

Mode of Action

Men who suffer from erectile dysfunction often produce too little amounts of NO. This means that the small amount of cGMP they produce is broken down at the same rate and therefore doesn't have the time to accumulateand cause a prolonged vasodilation effect. Sildenafil works by inhibiting the enzyme PDE5 by occupying its active site. This means that cGMP is not hydrolysed as fast and this allows the smooth muscle to relax. Sildenafil is a potent and highly selective inhibitor of PDE5.

N

HNN

N

O

O

SN

N

O O

sildenafil aka Viagra

C6H8O7

CO2Et

OO

N

HNN

N

O

Pr

EtO

NN

Pr

H2NOC

H2N

OEtCOCl

N

HN

Pr

EtO2C

N

HNN

N

O

Pr

EtO

SN

N

O

O

NN

Pr

H2NOC

NH

EtOO

NN

Pr

HO2C

Synthesis of sildenafil

N2H4, H2O 1) Me2SO4

2) NaOH, H2O

1) HNO3, H2SO42) SOCl2

3) NH4OH4) SnCl2

Et3N, DMAP, CH2Cl2

mechanism?

intermediates?

NaOH, EtOH/H2O

mechanism?

1) ClSO3H

2) N-methylpiperazine, EtOH sildenafil aka Viagra

OEtCO2H

NN

Pr

H2NOC

H2N NN

Pr

H2NOC

NH

EtOO

S N NOO

OEtCO2H

SO2Cl

N

HNN

N

O

Pr

EtO

SN

N

O

O

CO2HOEt

SN

N

O

O

Alternative Synthesis of sildenafil

NaOH, EtOH/H2O

sildenafil aka Viagra

SOCl2, ClSO3H N-methylpiperazine

carbonyldiimidazole

The overall yield of sildenafil via this second route can be as high as 52%,which compares favourably with the first route in which the overall yield is 28%. The actual commercial route used by Pfizer has not been published.

The Future:The Future:A Change of Strategy?

A recent review has highlighted the success and limitations of the current approach to the discovery of new drug molecules. The data was taken from the process groups of 3 of the major pharmaceutical companies in the UK. It was shown that:

1) The average number of synthetic steps across all 3 companies was 8.

2) 20% of all reactions were heteroatom arylations or alkylations.15% of all reactions were deprotection reactions.12% of all reactions were acylations (e.g. amide formation).Only 11% were actual C-C bond forming reactions.

As all 3 companies use the same types of reactions and adhere to the dogma of Lipinski’s Rule of Five, it is unsurprising that they generate the same types of molecular structures as potential drugs and hence explore the same area of chemical space.

Diversity Oriented SynthesisA strategy designed to explore greater areas of chemical space than those accessed by traditional med chem approaches is diversity oriented synthesis (DOS). A comparison of (DOS) with the more conventional target oriented synthesis (TOS) is given below.

Target-Oriented Synthesis:Convergent

SingleTarget

Simple ComplexRetrosynthetic

Analysis

DiverseTarget

Structures

Diversity-Oriented Synthesis:Divergent

Simple &Similar

Complex &Diverse

ForwardSynthetic

Analysis

Reproduced with the kind permission of Dr. David Spring, University of Cambridge

Library

FocusedDOS 1DOS 2MDDR

Average 'Chemical Space' occupied per compound

1.0810.0928.1028.09

STDDEV(PC1)

4.9013.1811.7817.31

STDDEV(PC2)

0.461.031.601.44

STDDEV(PC3)

0.500.741.491.15

Reproduced with the kind permission of Dr. David Spring, University of Cambridge

Natural Products

As well as DOS, the manipulation of chemical entities found in nature (natural products) can also provide diverse lead structures for investigation and evaluation as potential drug candidates.

For example drugs such as Taxol (anti cancer), cyclosporin A (immuno-suppressant), digoxin (heart failure), ivermectin (river blindness) and lovastatin (cholesterol lowering) are all natural products or derived fromnatural products. It is hard to see how these drugs would have been discovered via a Lipinski Rule of Five driven medicinal chemistry research project.

O

O

H

O

HO O

O O

OH

OH

H

H

HO

O

OHOH

O O

O O

O

O

OO

OMeHO

H

OMe

H

OH

HOH

H

O

Lovastatin

cyclosporin A

3

digoxin

ivermectin

O

O

AcO OOH

AcOHBzO

HO

BzHN

O

OH

Ph

taxol

Natural Products

You should have an appreciation of the types of molecules targeted by medicinal chemists and be able to discuss the pros and cons in the syntheses of a number

of drug molecules. You should aim to familiarise yourselves with each chemical transformation discussed

in the course and with the mechanisms of those highlighted. Some rudimentary understanding of the

importance of each drug and its mode of action should have also been acquired.

End of the Course