PLANTEAMIENTO DEL PROBLEMA

Armed with the knowledge of both the classification of HIV-1 PR as an aspartic acid proteinase and of natural and synthetic substrates, the design of inhibitors was initiated by applying transition state mimicry. This concept, originally proposed by Pauling,1^3 is based on the premise that an enzyme has a higher affinity for the transition state than for either product(s) or reactant(s) and proved extremely effective when designing renin inhibitors.'^® In this approach, a Pi*Pi- pair are replaced with a proteolytically stable dipeptide surrogate. The task of developing the first inhibitors was considerably facilitated by the vast body of information accumulated from 2 0 years work into the design of inhibitors for renin.'^® Inhibitors of HIV-1 PR were first reported in 1989, designed by

"tailoring" renin inhibitors "to fit" HIV-1 These inhibitors were very potent with little residual renin-inhibitory properties.

Some successful examples of dipeptide mimetics (with the actual transition state analogue of the scissile bond boxed) are shown below (Fig. 1.8).

Reduced amide OH Pi Hydroxymethylene Hydroxymethylcarbonyl Pi O Hydroxyethylene Pi Pi' Hydroxyethylamine O OMe Pi O Phosphonamidate methyl ester Figure 1.8: Some dipeptide mimetics incorporated into HIV-1 PR inhibitors

Each mimetic incorporates some but not all of the features of the transition state. They all possess tetrahedral geometry at the position designed to mimic the carbonyl group of the scissile peptide bond. However, other required characteristics cannot be met on chemical grounds. Some mimetics {e.g. hydroxymethylcarbonyl and hydroxyethylamine derivatives) contain more backbone atoms and some contain fewer atoms {e.g.

hydroxymethylene derivatives). Intuitively, these mimetics might be expected to lead to poor inhibitors as their peptidic portions should be out of synchronisation with the pockets into which they are designed to fit. As will be described, however, some have made very good inhibitors.

It is useful to review some of the highlights of the work in this field, particularly as all four HIV-1 PR inhibitors currently on the market, resulted directly or indirectly from this approach. Furthermore, whilst describing the inhibitors, comments

will be made about the medicinal chemistry that led to their development. This should give some idea as to how much the state of the art has advanced over the last decade.

The earliest reported inhibitors incorporated the reduced amide surrogate. Many of the early compounds were of only modest potency (although inhibitors effective at subnanomolar concentrations have since been reported). However a co-crystal of an inhibitor containing this mimetic (11, MVT-101) and HIV-1 PR provided the first detailed three dimensional structure of HIV-1 PR in its inhibited form.^8 The modest inhibition of HIV-1 PR by most members of this series has been attributed to its comparative lack of functionality; this prevents effective hydrogen bonding to the catalytic aspartic acid residues.

HQ, Ac-Thr-llGs^^ ^ A^^GIn-Arg-NHz

i H n

11, MVT-101 12, SDZ-PRI-053 ^ OCH3

The archetypal aspartic acid PR inhibitor pepstatin A (3), contains the hydroxymethylene (statine) surrogate and is a moderate inhibitor of HIV-1 PR (IC5 0 ~ 1 |iM). Originally, however, use of this surrogate did not deliver potent inhibitors, probably because it failed to project a residue into Si*. This problem was solved through the development of a different synthetic route, which enabled the incorporation of substituents at the 2-position. This led to the development of a highly potent series of inhibitors culminating in 12 (SDZ-PRI-053; Ki = 9.5 nM).^05

Hydroxymethylcarbonyl (norstatine) derivatives had previously been used as renin inhibitors. In contrast to renin inhibitors, potency was only achieved with the amino acid proline in the Pi position {e.g. 13, KNI-1 0 2; IC5 0 = 89 nM).i®6 An extensive structure-activity study was undertaken, in which the pyrrolidine ring of proline was exchanged for heterocyclic 5-membered r i n g s . O n e exceptionally potent analogue

containing a thiazolidine ring (14, KNI-272; Kj = 5.5 pM) is active against a wide spectrum of HIV-1 strains and is currently undergoing phase II clinical trials. ^(^7,108

OH ^SCHa

Cbz-Asn-NF

Ph- - o |N ' o

13, KNI-102 14, KNI-272

The extremely high potency of 14 is attributed in part to conformational preorganisation of this compound caused by the norstatine-thioproline bond being locked in the trans configuration. This is supported both by quantum mechanical calculations and by the conformations seen in crystal structures: both the free and PR-bound forms of 14 are very similar. The binding of 14 in the active site of HIV-1 PR is therefore thought to be more favourable entropically than most other linear peptidomimetic inhibitors. Conformational preorganisation is an important factor behind the potency of the smaller non-peptidic compounds and will be discussed later (see section 1.6.5.2).

A general point worth raising about the KNI series of norstatine inhibitors is that they all incorporate an unnatural amino acid, phenylnorstatine [(2R,3S)-3-amino-2~ hydroxy-4-phenylbutyric acid]. Unnatural amino acids have been widely employed to stabilise peptidomimetic HIV-PR inhibitors towards proteolysis. This is because they are not recognised by cellular proteinases. A similar strategy employed to evade metabolic degradation has been the use of D-amino acids in place of the natural L-amino acids. Compounds that contain such surrogates have been shown to retain inhibitory activity, although modifications to side chains need to be engineered on account of the reversal of the stereochemistry.

Five different transition state mimetics were directly compared in a study conducted by Dreyer et al. in which the surrogates were incorporated into the substrate Ser-Ala-Ala-Phe*Pro/Gly-Val-Val-OCH3.ii^ The reduced amide analogue gave the

poorest inhibition (Ki = 19 |iM). The hydroxyethylene-containing compound gave the greatest inhibition (Ki = 62 nM).m This outcome guaranteed an extensive investigation of the hydroxyethylene suiTOgate and has ultimately yielded an inhibitor (15, indinavir; Ki = 0.34 nM),ii^ which is now in clinical use.^®'^

Modifications to the hydroxyethylene surrogate have led to other novel inhibitors. Through computer modelling s t u d i e s , i t was calculated that an analogue of 15 bound to HIV-1 PR such that the aminoindan NH and the a-CH were nearly coplanar (the comparable atoms and bonds are shown in red in 15). Incorporation of these atoms into a 5-membered lactam ring (shown in red in 16) combined with additional stmctural features has delivered a series of potent inhibitors including 16 (IC5 0 = 5 nM).i^3 These y-lactams have shown significant oral bioavailability in dogs,^^^ no doubt as a result of the elimination of the indan amide bond.

15, indinavir Ph OH OH Pb 16

Compound 16 is another example of the constraining of an inhibitor into its bioactive conformation similar to that described previously for 14. Such confonnationally constrained peptidomimetic inhibitors have been extensively reported. There are many publications which describe the advantages of macrocycles over the acyclic peptidic inhibitors from which they are derived. Benefits include the protection of amide bonds from proteolysis, increased water solubility (and lipid solubility),!!^ and the entropie benefits of conformational preorganisation akeady described.

In a highly novel approach to the construction of rigid molecular stitictures, the Amos B. Smith III group have developed |3-strand peptidomimetic inhibitors, analogues of which have recently been shown to have good bioavailabilities, These compounds were

designed with the knowledge that HIV-1 PR binds its substrates and inhibitors by generating p-pleated sheets.!!"^ Flanking the hydroxyethylene surrogate with linked pyrrolinones as |3-strand mimetics enabled a reduction in the number of amide bonds. Compound 17 (IC5 0 =1.3 nM) was more active in a cc//-based anti-HIV assay than corresponding peptidic inhibitor 18 (IC5 0 = 0.4 nM), despite the in vitro IC50 values indicating the reverse. This suggested that the premise that lowering the number of peptide bonds aids the transport of these inhibitors into cells is co r re c t . ! !^418

H H

°P h '

18

A second type of compound, developed by the Gani group at St Andrews, has also been shown to aid in cell-uptake properties. By incorporating the phosphonamidate methyl ester transition state surrogate in a series of compounds derived from one of the HIV-1 cleavage sequences, moderate inhibitors of PR were derived. All the analogues prepared had a marked ability to enter cells, as demonstrated by the approximate equivalence of IC50 values found for both solution- and cell-based assays.!!^

Finally, the hydroxyethylamine surrogate has arguably proved to be the most successful of the transition state mimetics in generating potent HIV-1 PR inhibitors. Extensive structure-activity studies of inhibitors containing the hydroxyethylamine analogue of the Phe-Pro scissile bond have shown that inhibitors could be truncated to occupy only 8 3-8 2'.^^°

Further modification of the Py proline to the decahydroisoquinoline group led to the discovery of 19 (saquinavir; Ki = 0.1 nM),!20 the first HIV-1 PR inhibitor to be approved for clinical use.!®^ Further manipulation enabled truncation of the N-terminal

end to just Pi and ? 2 and led to the identification of 2 0 (nelfinavir; Ki = 2 nM),i^^ which has improved bioavailability compared to 19 and is itself now approved for clinical use. 1^4

OH OH N HO

J<