The Discovery of the CCR5 Receptor Antagonist, UK-427,857, A New Agent for the Treatment of HIV Infection and AIDS

Progress in Medicinal Chemistry – Vol. 43, Edited by F.D. King and G. Lawton q2005 Elsevier B.V. All rights reserved.

7 The Discovery of the CCR5 Receptor Antagonist, UK-427,857, A New Agent for the Treatment of HIV Infection and AIDS ANTHONY WOOD and DUNCAN ARMOUR Department of Chemistry, Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, UK

INTRODUCTION

239

HIGH-THROUGHPUT SCREENING AND ASSAYS

241

HIT-TO-LEAD STUDIES

244

BEYOND HIT-TO-LEAD

251

THE IDENTIFICATION OF UK-427,857

261

OTHER CCR5 ANTAGONISTS

266

CONCLUSION

268

REFERENCES

268

INTRODUCTION Since the identification of the human immunodeficiency virus (HIV) as the causative agent of AIDS in 1983 [1, 2], there have been intense efforts to develop effective treatment and control measures. Notwithstanding this, by 2003 the Joint United Nations Programme on HIV/AIDS estimated that 42 million people DOI: 1 0 . 1 0 1 6 / S 0 0 7 9 - 6 4 6 8 ( 0 5 ) 4 3 0 0 7 - 6

239

240

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

worldwide were infected with HIV, and that the virus had claimed the lives of more than 20 million people since the start of the epidemic [3, 4]. Furthermore, the rate of infection remains on the increase in the developed and developing world. Following the introduction of highly active antiretroviral therapy (HAART) regimes, using combinations of nucleoside/nucleotide reverse transcriptase and protease inhibitors, HIV has increasingly been considered to be a manageable, chronic disease [5]. However, there is still a need for new therapeutic agents with improved dosing regimes that are better tolerated with a reduced side effect burden [6]. Side effects and complicated dosing regimes have led to reduced patient compliance [7, 8] and contributed to the emergence of viral resistance. This is leading to a rise in numbers of those who have detectable viraemia during treatment with HAART, and those who are infected with HIV strains which are resistant to all three classes of drugs in the HAART regimes (estimated to be 5– 10% of patients undergoing therapy) [9]. It is also leading to a rise in the numbers of newly infected, treatment-naı¨ve patients who are already carrying virally resistant strains (estimated to be up to 20%) [9 – 11]. Consequently, therapies based on new mechanisms of action are particularly desirable. HIV gains entry into cells by fusing the lipid membrane of the virus with the host cell membrane, an event that is triggered by the interaction of proteins between the HIV envelope and cell surface receptors. The virus binds with its gp120 protein to the CD4 receptor forming a complex that undergoes a conformational change creating a co-receptor binding site for a chemokine receptor [12]. For M-tropic strains, which predominate during the initial phase of the disease and during transmission, this co-receptor is CCR5. T-tropic virus strains, which tend to predominate in the late stages of the disease, use the CXCR4 chemokine receptor (although approximately 50% of individuals remain infected with strains that maintain their dependence for CCR5 even in late stage disease). Chemokine receptor binding then triggers further conformational changes in the viral gp41 fusion protein unmasking the fusion peptide and facilitating its insertion into the host cell lipid bilayer and subsequent viral entry. Targeting the viral fusion process has become a new focus of research for the next generation of HIV antiretroviral therapies [13]. In contrast to existing antiretroviral agents that work after viral entry, these should have an advantage in that they will not need to access intracellular compartments. Other groups have focussed on blocking the interaction of the gp120 protein with CD4, e.g. BMS-806 [14], on developing CXCR4 receptor antagonists such as the bicyclam AMD3100 [15], or on blocking the formation of the necessary rearrangement of the gp41 protein [16]. This latter approach resulted in the development of the 36-amino-acid peptide Enfuvirtide (T-20), which was approved by the FDA in March 2003. Although this drug validates the viral

A. WOOD AND D. ARMOUR

241

fusion process as a viable target clinically for the treatment of HIV/AIDS, a complicated manufacturing process results in a high cost (approximately US$20,000 per patient year) and twice-daily subcutaneous injections cause a very high incidence (98% of patients) of local site irritation, both of which will probably limit its clinical utility [17]. When our project was initiated, we chose instead to target the CCR5 receptor, as genetic evidence had just become available to underpin the biological rationale for CCR5 blockade leading to an antiviral effect. A section of the human population has a 32 base pair deletion in the CCR5 coding region (D32). In homozygotes this results in a failure to express the receptor on the cell surface, and there is evidence that this group of people are resistant to infection with M-tropic HIV-1 [18]. More recently it has been shown that individuals who are heterozygous for the D32 gene show significantly longer progression times to the symptomatic stages of infection and appear to respond better to HAART treatment [19]. Moreover, both heterozygous and homozygous carriers of the D32 allele are apparently fully immunocompetent with no other obvious abnormalities, suggesting that the absence of CCR5 function may not be detrimental and that a CCR5 receptor antagonist should be well tolerated. This chapter describes the drug discovery programme that led to the identification of UK-427,857, a prototype CCR5 antagonist with excellent potency against lab-adapted and primary HIV-1 isolates, as a clinical candidate for the treatment of HIV. In particular, it deals with strategies for minimizing cardiac toxicity whilst maintaining ADME properties commensurate with low dose. HIGH-THROUGHPUT SCREENING AND ASSAYS The CCR5 receptor is a member of the family of G-protein coupled receptors (GPCRs) and is predicted to have a typical seven transmembrane structure [20]. This class of proteins is heavily represented in the ‘druggable genome’ [21], which gave us some encouragement that a drug-like ligand should be identifiable. The receptor binds the chemotactic chemokines, MIP-1a, MIP-1b and RANTES. Whilst these have been used to produce synthetic peptides such as AOP-RANTES [22], which have been useful for the validation of CCR5 as a target for HIV therapy, we did not believe that these offered a viable starting point for a low-molecular weight, orally bioavailable agent. Nor did we wish to pursue an antibody-based approach [23]. We, therefore, elected to use a high-throughput screen (HTS) to identify novel starting points for our programme. However, the selection of an appropriate screen was problematic. Initially, we did not have purified viral proteins available to us that would have been

242

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

of sufficient quality to construct a robust assay. We, therefore, chose to use a screen based on the inhibition of binding of radiolabelled MIP-1b to the CCR5 receptor stably expressed in HEK-293 cells [24] – a similar approach using MIP-1a has also been described by workers at Merck [25]. We could not be certain at the time that a compound that inhibited the binding of an endogenous chemokine would necessarily block the binding of the HIV gp120 protein. Indeed, more recent work using chimeric CCR5 receptors indicates that the binding domains of HIV gp-120 and MIP-1a are in fact distinct and separate [26, 27], although MIP-1a can act as an allosteric antagonist of gp120 binding. Furthermore, although the monoclonal antibody PRO140 blocks the binding of HIV-1 to the CCR5 receptor, it does not alter the binding of the natural chemokine ligands [23], again suggesting that the natural agonist and the viral proteins have different binding sites. However, since most small-molecule ligands of peptidergic GPCRs are not thought to function by disrupting the large surface area interactions of the protein –protein complex between the natural agonist and receptor, but rather to act allosterically to stabilize receptor conformations that bind the natural agonist less effectively, we believed that it should be possible to identify ligands that could prevent the binding of both the endogenous ligands and the HIV gp-120 protein. This turned out to be the case, although throughout our programme we did sometimes encounter discrepancies between the MIP-1b binding assay and our antiviral assays. Similar observations have been noted by others [28]. The HTS identified a number of hits which were then triaged [29]. One frequent criticism of the HTS approach is that it tends to produce large, lipophilic hits which prove difficult to optimise, particularly as medicinal chemistry programmes tend to increase the size of compound along the path from lead to drug [30, 31]. Indeed this has resulted in some questioning the value of the whole HTS approach to drug discovery. Apart from filtering out compounds which fell outside of Lipinski’s ‘Rule of Five’ [32] we also took into account target affinity and ligand efficiency (LE), as measured by heavy atom contributions to binding [33]. This latter criterion is particularly useful, as it allows the comparison of compounds with quite different affinities, and tends to favour smaller, low-molecular weight compounds. This reduces the risk of picking a starting point just based on its headline affinity, when, of all the properties that can be manipulated by the medicinal chemist, affinity is often the easiest to improve. A further compound was eliminated from consideration due to concerns over toxicity associated with a nitropyridine group, leaving us with four hits, two of which UK-107,543 (1) (MIP-1b IC50 0.4 mM, LE 0.29 kcal/mol/non-H atom), and UK-179,645 (2) (MIP-1b IC50 1.1 mM, LE 0.20 kcal/mol/non-H atom) formed the basis of the programme reviewed here.

A. WOOD AND D. ARMOUR

243

Both of these hits contain structural elements that have been termed ‘privileged structures’ [34 – 36] due to the frequency with which they are observed in drug discovery programmes. The origin of this effect has not been determined, although it has been hypothesized that it may reflect the existence of complementary binding sites within the proteins [37]. It may also be due to the effective display of functional groups that these templates inherently have due to conformational effects. Alternatively, it may be due to the composition of the compound screening collections that historically have been biased towards compounds from old discovery programmes. For the medicinal chemist, hits from privileged structures have the advantage of well-precedented chemistry, information on the potential liabilities of the template, but the potential disadvantage of polypharmacology and patentability concerns. Workers at Takeda [38], Merck [25] and Schering [39, 40] have reported isolating hits from their own independent HTS efforts which similarly also contained known GPCR pharmacophores. Neither of our hits, (1) and (2) could be considered to be ideal, having high molecular weight and lipophilicity, polypharmacology, weak binding affinity and no measurable antiviral activity. With considerations of the clinical need for a potent, durable and therefore, convenient and safe agent in mind, we established project goals aiming for a potent antiviral agent with an antiviral

244

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

IC90 similar to or better than, that of existing protease inhibitors, at least 100-fold selectivity over related targets, and pharmacokinetic properties commensurate with at worst b.i.d. dosing, with no CYP450 inhibition. We knew, therefore, that we would have to improve the quality of our starting point, to have any chance of delivering a high-quality compound with a good probability of surviving through preclinical and clinical studies. Therefore, the goal of our initial hit-to-lead studies was to optimise hits (1) and (2) by combining their most attractive features to produce novel, selective antagonists with enhanced ligand efficiency [33] and measurable antiviral activity. HIT-TO-LEAD STUDIES Most drug discovery groups define a hit-to-lead phase post-HTS when the viability of the hits is assessed through limited SAR studies, ideally performed with some elements of parallel/combinatorial chemistry [41]. This process uses less resource than a full medicinal chemistry programme, and aims to evaluate the potential of hits with a goal of reducing late-stage attrition. Our first goal was the replacement of the imidazopyridine in (1) as we believed that this was the cause of the profound type II cytochrome P450 2D6 inhibition [42] observed with this compound (CYP 2D6 IC50 40 nM). The inhibition of this enzyme can cause variable drug levels and serious safety concerns in combination therapy. Type II inhibitors generally contain a nitrogen heterocycle which can coordinate directly to the iron atom in the haeme unit of the enzyme, resulting in a large increase in the redox potential of the P450 and high occupation of the substrate binding site, leading to a dramatic reduction in turnover rates for the enzyme [43]. Modelling [44] indicated that the pyridine nitrogen in (1) was probably directly ligating to the haeme iron, (Figure 7.1), suggesting that the carbon analogue (3) would be preferable. The resultant benzimidazole (3), was a potent inhibitor of MIP-1b binding (MIP-1b IC50 4 nM), albeit still antivirally inactive, and was now a much weaker type I inhibitor of the 2D6 enzyme (CYP 2D6 IC50 710 nM). While still a concern, we felt that the resolution of this issue could wait while we searched for an antivirally active analogue. Another concern for us was the high lipophilicity of (3) – we introduced the amide which featured in (2), to increase the polarity of the template whilst keeping the molecular weight low. We reasoned that one of the phenyl rings of the benzhydryl group restricts the conformational space of the other, an effect known as hydrophobic collapse [45, 46], and that introduction of a more polar linker should not interfere with potency. Indeed, the benzamide (4) exhibited good chemokine receptor binding (MIP-1b IC50 45 nM), albeit slightly less potently than (3). Most encouragingly though, promising levels of antiviral activity could be measured, see Table 7.1. Subsequently structure (4) became the focus of our SAR investigations.

A. WOOD AND D. ARMOUR

Fig. 7.1 Modelling of the binding mode of (1) to cytochrome P450 2D6.

245

246

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

Table 7.1 MIP-1b INHIBITORY ACTIVITY AND ANTIVIRAL ACTIVITY OF SELECTED AMIDE ANALOGUES

R

MIP-1b a IC50 (nM)

AV b IC50 (nM)

(4)

45

210

(5)

100

740

(6)

820

9250

(7)

270

7110

(8)

50

700

(9)

430

.10000

40

75

(10)

a b

The concentration required to inhibit binding of [125I]MIP-1b by 50%. The concentration required to inhibit replication of HIVBaL into PM-1 cells by 50%.

A. WOOD AND D. ARMOUR

247

Initially, we investigated the SAR of different amide substituents, using parallel array chemistry. All such compounds were purified by either reversephase HPLC or semi-automated flash chromatography, and characterized by LC – MS [47]. Selected compounds were characterized further by NMR and microanalysis. Biological data for several representative compounds are summarized in Table 7.1. This set of analogues identified the benzamide (4), isopropylamide (8) and cyclobutyl amide (10) as the most potent analogues. Compounds with additional polarity, e.g. (6) and (9), showed a sharp decrease in potency suggesting that the amide substituent interacts with a predominantly lipophilic binding site on the CCR5 receptor. More bulky substituents such as the phenacetylamide (5) had decreased potency. The smallest ligand, acetamide (7), appeared to be less efficient for binding to CCR5. A homochiral synthesis of the two enantiomers unambiguously established that the activity resided with the ðSÞ enantiomer of the benzamide (11) (MIP-1b IC50 13 nM, AV IC50 190 nM), with the ðRÞ enantiomer (13) have much reduced affinity (MIP-1b IC50 580 nM), and no measurable antiviral activity (AV IC50 . 10 mM). Similarly, the homochiral synthesis of the ðSÞ enantiomer of the cyclobutyl amide (12), established that this was the active isomer (MIP-1b IC50 20 nM, AV IC50 73 nM).

248

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

The encouraging activity of (12) prompted us to continue our SAR investigations keeping the cyclobutyl substituent constant. However, (12) was still a Type I CYP2D6 inhibitor [42] when tested against a panel of cytochrome P450 enzymes. As CYP2D6 levels are polymorphic, being absent in 5 –9% of the Caucasian population [48], metabolic clearance can be variable for drugs with this interaction and drug – drug interactions are more common. ˚ away from a phenyl ring A pharmacophore that contains a basic amine 5 –7 A is common for both CYP2D6 inhibitors and substrates [49]. The basic centre is believed to interact with a key residue, Asp301, that is an important determinant for binding [50]. The lead (12) was readily docked into a model of the enzyme [44] (Figure 7.2). Believing that we could reduce the interaction of the key Asp301 residue in the 2D6 enzyme either by sterically encumbering the basic piperidine nitrogen or by reducing the basicity of the piperidine nitrogen, we designed a series of analogues which varied the central piperidine core (Table 7.2). Consistent with the steric encumbrance theory, both the tropanone-derived azacyclo-octyl derivatives (14) (the exo isomer) and (15) (the endo isomer), were devoid of 2D6 activity, and both proved to be highly potent inhibitors of viral replication. While we were initially surprised that both isomers were so similar in activity, 1H NMR analysis shows that the benzimidazole forces the endo substituted bridged piperidine ring in (15) into a boat conformation. The resulting structure overlaps well with that of the exo substituted analogue (14), as shown by molecular modelling (Figure 7.3). The bonus of enhanced binding affinity and improved antiviral activity for the tropanes (14) and (15), may be due to either an improved fit of the ligands

Fig. 7.2 Modelling of (12) bound to CYP2D6.

A. WOOD AND D. ARMOUR

249

Table 7.2 MIP-1b INHIBITORY ACTIVITY AND ANTIVIRAL ACTIVITY OF PIPERIDINE ANALOGUES

R

MIP-1b a IC50 (nM)

AV b IC90 (nM)

(14)

2

13

(15)

6

3

(16)

.1000

(17)

17

(18)

1.2

n.t.

.1000

140

(Continued)

250

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857 Table 7.2 CONTINUED MIP-1b a IC50 (nM)

AV b IC90 (nM)

(19)

21.5

n.t.

(20)

39.7

n.t.

(21)

9

n.t.

(22)

8

R

53

n.t.: not tested. a The concentration required to inhibit binding of [125I]MIP-1b by 50%. b The concentration required to inhibit replication of HIVBaL into PM-1.

to the receptor, or possibly due to the enhanced rigidity of the 2-phenylpropylamine side chain as a consequence of gþ g2 (syn pentane) interactions [51]. The smaller azetidine (16) lost all binding affinity to CCR5. The thiagranatane (19) and endo-oxogranatane analogue (20) had reduced affinity when compared to the tropanes (14) and (15), and so appeared to offer little advantage. The piperidine derivatives (17) and (18), exo-oxogranatane (21) and 2,6 dimethyl piperidine (22) were potent inhibitors of chemokine binding. However, translation into antiviral activity was relatively poor, presumably for the same reasons that we have discussed earlier.

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251

Fig. 7.3 Overlap between exo and endo analogues (14) and (15).

Having established for (15), potent antiviral activity against a lab-adapted HIV strain (BaL) propagated using a CCR5 clone expressing cell line, we felt that it was important to confirm that the antiviral effect we observed was also translated to low passage primary origin viral isolates cultured in peripheral blood lymphocytes (PBLs). These assays are labour-intensive but may provide a more clinically relevant insight into the antiviral profile of the compound in light of the viral isolate and the host cell being of direct ex vivo origin, therefore, more closely mimicking the biological interactions that exist for pathogenesis in HIVinfected patients. Also, since the gp120 protein is a known variable epitope of HIV, it was also possible that sequence changes might result in a loss of antiviral activity. Encouragingly, (15) showed potent antiviral activity against a range of primary origin CCR5-tropic HIV isolates in PBLs, with IC50s ranging from 0.9 to 9.6 nM. Having established that we could achieve our primary pharmacological goals the hit-to-lead stage of the project was successfully concluded, resource was increased, and (15) was profiled more extensively. BEYOND HIT-TO-LEAD As compound (15) progressed through our safety screens, it became apparent that it was also a potent inhibitor of the human ether a-go-go-related gene (HERG) potassium channel [52], (99% inhibition at 1 mM). The function of HERG channels is to conduct the rapidly activating delayed rectifier potassium current (IKr), which has a key role in the control of cardiac rhythm [53]. HERG channel inhibition has risen to prominence recently in the drug discovery process as the predominant cause of acquired long QTc interval prolongation. A number of previously approved drugs which are associated with a prolongation of the QTc interval due to effects on IKr, have now been given black box warnings

252

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

or indeed have been withdrawn, because of a link to sudden cardiac death and ventricular arrythmia, a condition known as Torsades de Pointes [54] (see also chapter 1). Indeed, QTc prolongation has been reported to have caused issues during the clinical development of other CCR5 receptor antagonists such as SCH351,125 [55]. HERG channel inhibition has also been reported to be a problem for some analogues in the Merck series [56]. We were particularly concerned about this undesirable ion channel activity, as drugs to treat HIV are not given in isolation, but rather as cocktails of agents, to prevent the emergence of viral resistance. Many of the agents that are used in a clinical setting have interactions with cytochrome P450 enzymes that may profoundly affect the levels of drugs observed in the systemic circulation. This, combined with our desire to maintain a high plasma concentration of drug above the antiviral IC90 at all times, results in the demand for a large safety window. Thus, achieving good selectivity with respect to IKr blockade became a prime project objective. Unfortunately, all of the analogues in Table 7.2 proved to be unsuitable for further progression, whether due to a lack of antiviral efficacy or undesired HERG activity. Recent progress in the solution of several ion channel structures has greatly increased our understanding of the molecular determinants of binding of ligands to ion channels [57]. Although no crystal structures are available of the HERG channel itself, homology models based on the bacterial KcsA channel, coupled with site-directed mutagenesis studies [58], computational pharmacophore models [59], and a detailed analysis of the structure activity relationships of ligand series, have allowed us to build realistic homology models of the HERG channel, that can be used to predict how compounds might bind [60]. The HERG channel exists as a transmembrane spanning tetramer. By analogy with the crystal structure of the related KcsA channel, the protein consists of a large water filled pore that functions as a gateway from the intracellular side of the membrane to a set of helices that function as an ion selectivity filter by stripping the hydration shell from potassium ions and replacing these with backbone interactions, allowing the passage of ions to the extracellular side at diffusion limited rates. Basic compounds such as dofetilide are believed to bind in the channel, attracted by the negatively charged neck of the filter, making specific interactions with the aromatic amino acid residues lining the pore. Docking of both compounds into a model of the HERG ion channel [60] suggested that the introduction of polar groups into the amide substituent could increase selectivity against the HERG channel as more polar substituents would be less well accommodated by the hydrophobic amino acid residues that line the channel in the proximity of the aqueous pore, (Figure 7.4). The azetidine acetamide (23) (Table 7.3) had the desired effect substantially reducing channel affinity without impacting on antiviral potency. Further polar amide substituents are shown in Table 7.3. All analogues feature the endo substituted azabicyclo-octane as it was the most potent piperidine

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253

Fig. 7.4 Models of the HERG Channel. (a) HERG pharmacophore superimposed on the channel model, lipophilic binding areas shown as light grey rings, basic centre shown as a dark ball. Side chains are only shown for two of the four subunits of the channel protein for ease of viewing. (b) Dock of (15) into the HERG channel model.

replacement. Potassium channel activity could be avoided most effectively with strong hydrogen bonding groups, such as amides (23) and (24), and acid (25). However, the cell permeability as measured by apical to basal flux rate through a monolayer of Caco-2 cells [61] was compromised, predicting poor oral absorption in man. It is sometimes assumed that compounds which are fully ‘rule of five’ compliant [32], such as UK-395,859 (23) must have good permeability. However, for transcellular passive diffusion across membranes, although log P is an important consideration (and is incorporated in the rule of five), it can be more useful to treat this composite parameter in terms of its contributing factors – molecular weight and hydrogen bonding potential. As molecular weight increases one can achieve apparently acceptable lipophilicity by increasing hydrogen bonding potential. Unfortunately, this strategy has a major negative impact on permeability. For our series, hydrogen bonding potential, more than the overall log P; appears to determine the permeability [62, 63], and there is a clear relationship between cell permeability and the calculated polar surface area (PSA) of these molecules with the exception of compounds (26) and (27) (Table 7.3). ˚ 2 have Generally it is accepted that compounds with a PSA which exceeds 120 A 2 ˚ poor permeability, whereas compounds with PSA below 60 A have very good permeability [62, 63]. As this relationship is sigmoidal the transition from nonpermeable to permeable can be very sharp. It can be seen that, for this series of compounds, the transition from permeable to non-permeable is probably around ˚ 2, values in agreement with the work of others on other compound series 60 –80 A [64, 65]. Although the PSA of (26) and (27) are beyond this cut-off, cell

254

AVa IC90 (nM)

Kþ channel inhibitionb

CaCo-2 fluxc Papp (cm/s)

Polar surface area (A˚2)

(15)

3

80%, 300 nM

n.t.

45.1

(23)

1

0%, 300 nM

,1 £ 1026

76.9

(24)

1

0%, 300 nM

1.5 £ 1026

76.1

R

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

Table 7.3 ANTIVIRAL ACTIVITY, POTASSIUM CHANNEL ACTIVITY AND CACO-2 CELL ABSORPTION OF AMIDE SUBSTITUENTS WITH POLAR GROUPS

6

0%, 1 mM

,1 £ 1026

134.6

(26)

5

25%, 300 nM

10 £ 1026

122.8

(27)

3

10%, 300 nM

11 £ 1026

114.6

(28)

1

0%, 300 nM

4.9 £ 1026

66.0

(29)

3

16%, 1 nM

7 £ 1026

61.7

(30)

0.6

0%, 100 nM

23 £ 1026

58.9

A. WOOD AND D. ARMOUR

(25)

a

The concentration required to inhibit replication of HIVBaL into PM-1 cells by 90%. Percentage inhibition of tritium labelled dofetilide binding to HERG stably expressed on HEK-293 cells at different concentrations. c Apical to basal flux rate of compound through a monolayer of Caco-2 cells at 25 mM, pH 7.4. b

255

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THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

penetration does occur, presumably because of overestimation of the polarity of the tetrazole and nitrile groups (aided too perhaps, by the low molecular weight of (27)). We designed compounds with reduced hydrogen bonding potential in two ways. Firstly by relying on proximity effects for polar groups one can reduce the overall hydrogen bonding potential by isomerization, e.g. compounds (28) and (29), or alternatively, an aliphatic ether can be substituted for the amide function, thus exploiting an SAR element which sometimes improves the permeability of peptide isosteres [66 –68], e.g. compound (30). Both of these approaches improved permeability in line with our expectations and maintained good selectivity with respect to HERG binding. This compound (30) was profiled extensively. It is a potent inhibitor of binding of the chemokines MIP-1a, MIP-1b and RANTES, to CCR5. Interestingly, kinetic binding studies with radiolabelled (30) showed that the compound has a rapid onset rate onto the receptor, but has a slow offset from the receptor, with t1=2 ¼ 3:5 h [69]. It is highly selective for CCR5, is a functional antagonist and does not induce neutrophil chemotaxis to chemokines such as GRO, PAF, NAP-2, IL5 or C5A at concentrations up to 25 mM, highlighting its selectivity for CCR5 and the absence of the involvement of this receptor in these immune parameters. It is a potent inhibitor of HIVBaL in PM-1 cells and in peripheral blood mononuclear cells (PBMCs, IC90 ¼ 2 nM). This compound was devoid of cytotoxicity in parallel assays (up to 1 mM tested) and had no activity against the replication of the CXCR4-tropic isolate HIVIIIB in PBMCs, thereby supporting our conclusion that the antiviral activity of this compound was entirely attributable to blockade of CCR5. The compound was also clean in AMES. Unfortunately, when pharmacokinetic experiments on (30) were performed, the data was less favourable. Dog and human hepatocyte clearance was high, 27 and 14 ml/min/kg, respectively. In vivo, dog clearance was approaching liver blood flow and oral bioavailability was , 10% due to extensive first pass metabolism, which was predicted to be similar in man. Our attention turned to trying to define the lipophilicity window that would allow us to find compounds with adequate flux in combination with reasonable metabolic stability. Analysis of the data that we had on our series to date identified a narrow window of lipophilicity centred around a log D , 2:0; where compounds with both appropriate human liver microsomal stability and Caco-2 flux (apical to basolateral membrane) were found (Figure 7.5). It should be emphasized that focusing on this region does not guarantee success but merely increase the probability relative to other parts of the distribution where no solution is likely to be found. Our problem however, was even more multidimensional. We also needed to maintain antiviral potency and minimize affinity for the HERG channel. To address these issues, we decided to look back at some of our early SAR in search of compounds that were intrinsically more

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257

Fig. 7.5 A graph of human liver microsome stability and Caco-2 flux against log D for the benzimidazole series indicating our key target window for log D:

active than our benzimidazoles, in the hope that these would allow us to reduce molecular weight and hence reduce lipophilicity, without having to increase the hydrogen bonding potential. In the course of our early programme we had looked at alternative heterocycles as replacements for the benzimidazole moiety on the original piperidine ring system (Table 7.4). We were particularly intrigued by the 1,2,4triazole (36), a compound that achieved good CCR5 affinity despite its low molecular weight (, 400). Initially, lack of antiviral activity for this compound had limited our interest. However, we now felt that the dramatically reduced lipophilicity of the triazole gave us greater flexibility to modify and optimize potential hydrophobic interactions with the receptor to improve antiviral activity. We felt that this reduced lipophilicity also gave the triazole (36) an advantage over the essentially equipotent oxadiazoles (31) and (32). Transfering our learning from the benzimidazole series, we synthesized the isomeric exo and endo tropane isomers of the triazoles for direct comparison. These compounds were now also prepared as single ðSÞ enantiomers, and to ensure that we explored our target lipophilicity window properly, we prepared both 3,5-dimethyl and 3-isopropyl-5-methyl substituted triazole analogues (Table 7.5). The 3,5-dimethyltriazoles (37) and (38) display similar potencies. However, increasing the steric demands of the substituents of the triazole with the 3-isopropyl-5-methyltriazoles (39) and (40), results in a clear separation of activity between the exo and endo series. From NMR studies it is again apparent

258

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

Table 7.4 MIP-1b INHIBITORY ACTIVITY AND ANTIVIRAL ACTIVITY OF PIPERIDINE ANALOGUES

R

MIP-1b a IC50 (nM)

AV b IC50 (nM)

cLog P

(13)

40

75

3.2

(31)

90

2920

1.4

(32)

70

(33)

n.t.

1.4

730

.10000

0.5

(34)

540

.10000

0.8

(35)

900

.10000

1.1

(Continued)

A. WOOD AND D. ARMOUR

259

Table 7.4 CONTINUED R (36)

MIP-1b a IC50 (nM)

AV b IC50 (nM)

cLog P

49

.10000

0.8

n.t.: not tested. a The concentration required to inhibit binding of [125I]MIP-1b by 50%. b The concentration required to inhibit replication of HIVBaL into PM-1.

that to minimize 1,3-diaxial strain between the triazole and the carbon bridge of the tropane, in the endo series the tropane adopts a pseudo-boat conformation, rather than the normally preferred chair conformation, as adopted by exo series. As a consequence of these conformational effects the triazole lies in approximately the same geometric position for both the exo and endo series. As such, it is difficult to rationalize the dramatically deleterious effect observed for isopropyl substitution in the endo series, other than to postulate that the greater steric crowding for (40) results in a subtle conformational difference that is less well tolerated by the receptor. From the antiviral data alone there was little to discriminate between compounds (38) and (39), so both were progressed further (Table 7.6). Table 7.5 ANTIVIRAL ACTIVITY OF TRIAZOLE TROPANE ANALOGUES Exo

Endo

(37) R ¼ Me AV IC90 ¼ 13 nMa (38) R ¼ i-Pr AV IC90 ¼ 8 nM

(39) R ¼ Me AV IC90 ¼ 6 nM (40) R ¼ i-Pr AV IC90 ¼ 101 nM

a

The concentration required to inhibit replication of HIVBaL into PM-1.

260

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857 Table 7.6 IN VITRO DATA FOR COMPOUNDS (38) AND (39)

(38) (39) a

HLM (min)

Kþ channel inhibitiona

Log D

Caco-2 ( £ 1026 cm/s)

Polar surface area (A˚2)

55 45

30%@ 300 nM 33%@ 300 nM

1.6 1.3

4.5 3.9

75.5 87.8

Percentage inhibition of tritium labelled dofetilide binding to HERG stably expressed on HEK-293 cells at different concentrations.

Calculated polar surface areas indicated that both compounds lie in the range that we had previously observed as being in the transition zone from permeable to non-permeable, and this was confirmed by the moderate Caco-2 flux values that were obtained for both. Although compound (38) showed some inhibition of the HERG potassium channel in vitro, it was assessed further as a potential candidate for clinical

Fig. 7.6 Graph of percentage change in inhibition of dofetilide binding or purkinje fibre APD prolongation for (38) versus concentration of drug, expressed as a multiple of the predicted clinical concentration (based on a AV IC90 8 nM for (38)), with comparison curves for terfenadine, terfenadine and ketoconazole, and an ‘ideal’ candidate profile.

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development. The compound was potent and stable and showed acceptable absorption and dose predictions [70] based on dog (t1=2 ; 7 h; Clou 172 ml/min/kg, F 43%, absorption . 80%, estimated human dose 100 mg b.i.d.) and rat pharmacokinetics (absorption 20%, estimated human dose 350 mg b.i.d.). The dofetilide binding assay that we use routinely as a high-throughput screen to measure inhibition of the HERG channel, while effective as a screening tool, is limited by being based on a single channel type, which has been cloned and expressed to a high level in a cell line. Therefore, (38) was tested in dog Purkinje fibre to better assess the QTc risk potential. This has the advantage of giving a view of the functional relevance of any and all ion channel inhibition for a compound. Unfortunately, (38) showed a terfenadine-like selectivity window between our predicted free plasma levels needed to maintain antiviral coverage and QTc threshold effects (Figure 7.6). This meant that drug –drug interactions with CYP450 inhibitors could lead to clinical QTc prolongation, allowing for substantial variation in total exposure and peak to trough concentrations when co-administered with other agents such as Ritonavir, frequently used as a component of a HAART regime. Despite this setback we were encouraged that a modest increase in the antiviral potency of (38) to , 1 nM levels whilst maintaining the other properties at a similar level, would deliver a candidate with an acceptable therapeutic window. This optimism, however, had to be tempered with the knowledge that antiviral potency appeared to be strongly linked to lipophilicity. For example, homologation of the cyclobutyl amide (38) to the cyclopentyl amide (41) (AV IC90 ¼ 1.6 nM, Log D 2.1, HLM 21 min, Caco-2 5.6 £ 1026 cm/s) gave both a predictable increase in potency and a predictable reduction in stability.

THE IDENTIFICATION OF UK-427,857 We decided to focus firstly on making further small changes to the triazole heterocycle (Table 7.7). As can be seen for analogues (42 –46), simply varying the triazole substituent was generally deleterious for antiviral activity. Furthermore, inhibition of the HERG channel was moderately increased for

262

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857 Table 7.7 ANTIVIRAL ACTIVITY OF TRIAZOLE ANALOGUES

R

AVa IC90 (nM)

HLM (min)

Log D

HERG Channel (%I @ 300 nM)

(38)

8

55

1.6

30%

(42)

9

n.t.

1.1

34%

(43)

60

19

2.2

53%

(44)

.100

22

2.3

46%

(45)

n.t.

32

2.0

48%

98

1.0

42%

(46)

77

(Continued)

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Table 7.7 CONTINUED AVa IC90 (nM)

HLM (min)

Log D

HERG Channel (%I @ 300 nM)

(47)

29

56

2.1

65%

(48)

1

13

2.3

85%

R

n.t. not tested. a The concentration required to inhibit replication of HIVBaL into PM-1.

all of these analogues, generally tracking with increased lipophilicity (but not always, see, for example, compound (46). Replacement of the 1,2,4-triazole with a 1,3,4-triazole gave compound (47), which possessed dramatically improved cell permeability (Caco-2 30 £ 1026 cm/s, apical –basal) for a compound with an identical molecular weight. This is presumably due to the smaller dipole moment of the 1,3,4-triazole (3.0 Debye) compared to the 1,2,4-triazole (6.1 Debye) resulting in overall weaker hydration for compound (47). However, HERG channel inhibition was also increased and antiviral activity reduced. The polarity of the triazole may be important for reducing the level of interaction with the lipophilic aromatic residues that line the channel mouth. Certainly, the triazole moiety lies in a region in our HERG pharmacophore model where lipophilicity is preferred, and so may have a similar effect to the polar amide groups described earlier. The imidazole analogue (48) possessed improved antiviral potency. However, both reduced microsomal stability and ion channel effects limited our interest in this series. We adjusted our focus to modifying the amide moiety within the series and our synthesis was such that the amide coupling could be undertaken as the final step. This again enabled us to use parallel chemistry techniques to assess the potential chemical space of this region as rapidly as possible. Within Table 7.8 we have described a small number of analogues that demonstrate our key SAR learning from these modifications. As we noted before, replacement of the cyclobutyl amide in (38) with the cyclopentyl amide (41), gave an increase in antiviral potency and cell permeability; however, microsomal stability decreased. This can all be attributed to an increase in lipophilicity. Excitingly though, when we tested (41)

264

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857 Table 7.8 ANTIVIRAL ACTIVITY OF AMIDE ANALOGUES

R

MIP1b (nM)

AVa IC90 (nM)

HLM (min)

Log D

HERG Channel (%I @ 300 nM)

(49)

5

n.t.

43

1.9

n.t.

(50)

3

n.t.

95

1.5

n.t.

(51)

15

n.t.

.120

n.t.

n.t.

(52)

5

14

.120

1.8

14%

(53)

26.5

125

n.t.

n.t.

0%

(54)

2

2.1

0%

1.0

51

n.t. not tested. a The concentration required to inhibit replication of HIVBaL into PM-1.

in the dofetilide binding assay it appeared that the ion channel effects were decreasing, presumably due to a steric clash with the channel. Mass spectrometry data on the products of microsomal metabolism of (41) suggested that metabolism was primarily occurring on the left-hand side amide, so the trifluoropropionoyl amide (51) and trifluorobutanoyl amide (52) were prepared as blocking groups. Although showing relatively poor antiviral potency, (52) displayed excellent microsomal stability and again improved ion channel effects compared to (38). It was apparent that modification of the amide could

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simultaneously limit oxidative metabolism and increase steric demands to generate selectivity over the HERG potassium channel. Using this knowledge gave the impetus to prepare the 4,40 -difluorocyclohexylamide, UK-427,857 (54) which possessed excellent antiviral potency and reasonable microsomal stability combined with good selectivity over potential ion channel effects. Subsequently (54) was tested at 1,000 nM and showed no significant binding to the HERG potassium channel. The compound also possessed the required levels of aqueous solubility and could be crystallized as the free base (Figure 7.7). With these promising biological and physiochemical properties, the compound was progressed to in vivo pharmacokinetic profiling. Predictions to man suggested that an oral 100 mg dose twice daily would provide a free drug concentration of greater than the antiviral IC90 throughout the dosing regime. UK-427,857 (54) was also found to show no significant inhibition (IC50 . 100 mM) of any of the major P450 isoforms tested (1A2, 2C9, 3A4) and , 50 mM against CYP2D6. This is . 30,000-fold the target free plasma concentration, suggesting that the drug will not affect metabolism of coadministered agents. This would be a key benefit when compared to currently marketed therapies [71]. The potency of UK-427,857 (54) was encouraging when tested against HIVBaL in peripheral blood monocytes (PBMCs) where the receptor is expressed under its native conformations (IC90 ¼ 6 nM), but was even more exciting when extensively tested against primary origin HIV isolates. In a PBMC assay, UK-427,857 showed potent cross-clade antiviral activity against all CCR5-tropic isolates with a geometric mean IC90 of 2.03 nM. UK-427,857 (54) possesses high selectivity over a range of other chemokine receptors, particularly CCR2b which is the most closely related chemokine receptor genetically [72], as well as a range of pharmacologically relevant targets [73]. As with previous compounds in this series, no cytotoxicity or activity against CXCR4-tropic HIV

Fig. 7.7 X-ray crystal structure of UK-427,857 (54).

266

THE DISCOVERY OF THE CCR5 RECEPTOR ANTAGONIST, UK-427,857

isolates was observed, supporting the conclusion that antiviral activity was entirely attributable to CCR5 blockade [74, 75]. Interestingly, UK-427,857 activity seemed insensitive to changes in multiplicity of infection (MOI) in our antiviral assays and showed no variation in potency against MIP-1b binding to CCR5 at varying chemokine concentrations. Receptor offset studies using tritiated UK-427,857 indicated that the non-competitive behaviour is probably a consequence of slow receptor offset kinetics ðt1=2 . 8 hÞ [75]. This profile was achieved with some trade-off in our permeability goals leading to a predicted bioavailability of 10% driven by 20% permeability and 50% first pass loss. We rationalized this parameter as the most appropriate one to compromise on for a number of reasons. Firstly, absorption could be readily evaluated early in the clinical development programme. In addition to this, whilst UK-427,857-like Caco-2 flux is likely to give incomplete absorption, it does not guarantee it! This is especially true for low dose, soluble, stable compounds, that are not significant Pgp substrates, as under these conditions slow absorption can be achieved without significant gut wall metabolismmediated first pass loss [32, 76]. Since, UK-427,857 has good metabolic stability, is only a moderate Pgp substrate (as shown by knock-out mouse exposure studies) and is readily soluble we were confident that the molecule possessed at least the right profile to mitigate against low permeability. UK-427,857 (54) was, therefore, progressed as a clinical candidate for the treatment of HIV infected individuals. We were delighted when Phase I human data confirmed that at a clinical exposure of 100 mg we achieved our desired objective of maintaining plasma levels above the IC90 at trough within the window provided by a b.i.d. regime. Not too surprisingly, given the slow absorption profile the Tmax is relatively late and we do see food effects with respect to the pharmacokinetic profile [77]. Phase II monotherapy studies with UK-427,857 resulted in impressive efficacy (, 1.42 log reduction in circulatory HIV RNA copy number) following 10 days monotherapy at 100 mg b.i.d. [78]. Further details of the clinical evaluation of this compound will be reported in due course. OTHER CCR5 ANTAGONISTS A number of other approaches to CCR5 antagonists have been reported in the literature. It is not our intention to review all of these here as such reviews already exist [79, 80], but rather to briefly highlight a few key compounds of interest which have undergone or are undergoing clinical investigation. Workers at Takeda have described TAK-779 (55), a quaternary ammonium compound intended for subcutaneous delivery [38, 81]. Although potent in vitro, clinical development appears to have been curtailed, at least partly due to adverse reactions associated with the route of administration.

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Several development compounds have been reported by workers at ScheringPlough including Sch-351125/Sch-C (56) [39, 40] including some preliminary clinical data [82], and Sch-417690/Sch-D (57) [83]. Sch-D has been reported to be more potent in vitro, have reduced HERG affinity and similarly, also to be under clinical investigation.

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Workers at Ono have discovered the spirodiketopiperazine AK602/ONO4128/GW873140 (58), a compound which is now in Phase II clinical trials [84]. Although very little data has been reported to date on this compound, it is a development from a series that this group has described previously [85]. CONCLUSION The path from the HTS hits (1) and (2) to the development candidate UK427,857 (54) proved to be challenging, taking two and a half years and 965 analogues. At times it appeared impossible to achieve the delicate balance of antiviral activity, metabolic stability, absorption and ion channel activity that UK-427,857 represents. A large number of colleagues too numerous to list here from an array of disciplines were instrumental in the identification of UK427,857 through their contributions to the HTS, chemical synthesis, biological, pharmacokinetic and safety screening, and many more colleagues have contributed in the subsequent development and clinical phases of the programme. We acknowledge all of their efforts, and hope that UK-427,857 or another CCR5 receptor antagonist eventually finds its way into the armoury of therapeutic agents for the treatment of HIV and AIDS. REFERENCES [1] Barre-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. (1983) Science 220, 868 –871. [2] Popovic, M., Sarin, P.S., Robert-Gurroff, M., Kalyanaraman, V.S., Mann, D., Minowada, J. and Gallo, R.C. (1983) Science 219, 856 –859. [3] http://www.unaids.org. [4] Fauci, A.S. (2003) Nat. Med. 9, 839 –843. [5] Pomerantz, R.J. and Horn, D.L. (2003) Nat. Med. 9, 867–873. [6] Carr, A. (2003) Nat. Rev. Drug. Disc. 2, 624–634. [7] Duran, S., Save`s, M., Spire, B., Cailleton, V., Sobel, A., Carrieri, P., Salmon, D., Moatti, J.-P. and Leport, C. (2001) AIDS 15, 2441–2444.

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