The Discovery of CDP323, a Potent Alpha4 Integrin Antagonist Stuart Bailey, Roger Palframan, Gillian Watt and John Porter*

Current Immunology Reviews, 2012, 8, 135-140 135 The Discovery of CDP323, a Potent Alpha4 Integrin Antagonist Stuart Bailey, Roger Palframan, Gillian Watt and John Porter*

UCB Celltech, 216 Bath Road, Slough SL1 3WE, UK

Abstract: Blocking the action of alpha4 integrin would be expected to be of therapeutic benefit in the management of autoimmune diseases. Although this has been successfully demonstrated in the clinic with a monoclonal antibody for the treatment of multiple sclerosis, there are no small molecule alpha4 integrin antagonists on the market despite significant endeavour over the last 15-20 years. We review our efforts in this area, starting from a cyclic peptide based on an integrin recognition sequence and culminating in the low molecular weight clinical candidate, CDP323. We include a discussion on pre-clinical pharmacological data for CDP323. Keywords: Alpha4 integrin, CDP323, integrin antagonists, VLA-4 integrin.

INTRODUCTION known as Very Late Antigen 4, VLA-4) is formed by the interaction between alpha4 (CD49d) and beta1 (CD29) Alpha4 integrin is a clinically validated target for certain integrin subunits. Additionally, alpha4 can also associate autoimmune diseases. The anti-alpha4 integrin monoclonal ® with a beta7 subunit. The primary ligands for alpha4beta1 antibody (mAb) natalizumab (Tysabri , Biogen Idec/Elan) are the endothelial surface protein vascular cell adhesion has shown significant therapeutic efficacy in patients with molecule (VCAM) and the alternatively spliced connecting multiple sclerosis (MS) [1] and Crohn’s disease [2]. The segment 1 (CS-1) of the extracellular matrix protein alpha4 integrin subunit is expressed on the surface of fibronectin (FN). Alpha4beta7 (also known as lymphocyte leukocytes, including T-lymphocytes and monocytes, Peyer’s patch adhesion molecule 1, LPAM-1) binds to the forming non-covalent heterodimers with beta1 and beta7 mucosal vascular addressin cell adhesion molecule-1 integrin subunits to create functional cell adhesion molecules (MAdCAM-1) expressed on gut-associated lymphoid tissue [3]. Alpha4beta1 and alpha4beta7 are important for cell as well as FN and VCAM. Alpha4beta1 recognises key migration into tissues under both physiologic and pathologic amino acid sequences within the ligands, namely QIDS in conditions [4]. The pharmacologic mechanism of action of VCAM and LDV in the CS-1 of FN. natalizumab is thought to be antagonism of leukocyte alpha4beta1 and/or alpha4beta7 interaction with their The small-molecule alpha4 integrin antagonists that have counter receptors on endothelial cells, causing inhibition of so far been progressed into clinical trials can be classified pathogenic leukocyte migration into tissue [5]. into two structural types: (1) mimics of the LDV sequence, for example, the highly selective alpha4 integrin antagonist Monoclonal antibodies are currently delivered almost Bio-1211 [11, 12] compound 1 or (2) N-acylphenylalanine- exclusively by intravenous or subcutaneous routes of based compounds such as valategrast (R411) [13] compound injection, can be immunogenic [6] and relatively expensive 2, many of which exhibit dual inhibitory activity for integrin to manufacture. The successful clinical development of an alpha4beta1, as well as alpha4beta7. Our starting point, orally bioavailable, low molecular weight, potent and however, was the cyclic peptide R*CDThioPC* compound 3 selective alpha4 integrin antagonist would represent the [14] and we now summarize the medicinal chemistry that led next-generation of therapeutics for diseases where alpha4 to the clinical candidate CDP323 as well as some of the integrins have been shown to play a pivotal role [7]. Thus, associated pharmacology. over the past 15 years there have been intensive efforts to discover and develop new candidate low molecular weight R*CDThioPC* compound 3, Table 1, is active against alpha4 integrin antagonists [8]. However, in 2010 there are alpha4beta1 but also binds to the related integrin alpha5beta1 only a few candidates in clinical trials for the treatment of (VLA-5), presumably because of its derivation from the MS and Crohn’s disease, and none of these compounds has RGD sequence. RGD is another recognition sequence for a yet been approved in any indication. subset of integrins including alpha5beta1 but not alpha4beta1 or alpha4beta7. Replacing the arginine residue Structurally, integrins are formed by the non-covalent with more lipophilic groups led to more active and selective association of alpha and beta subunits [9]; currently, 18 analogues, for example, the cyclic tetrapeptide alpha units and 8 beta subunits are known and these can (adamantylcarbonyl)-C*DThioPC*-OH compound 4, Table assemble in a restricted manner, dependent on cell type, to 1. A substantial amount of work was done in exploring the generate 24 different heterodimeric human family members. SAR of these cyclic peptides but the key observation was to They can be grouped according to subunit composition or establish that the cysteine residue’s carboxylic acid was ligand binding properties [10]. Alpha4beta1 integrin (also important for activity. The aspartic acid residue, in contrast, could be replaced with an alanine, as for example in compound 5, Table 1, with only a small loss in potency [15]. *Address correspondence to this author at the UCB Celltech, 216 Bath Road, Slough SL1 3WE, UK; Tel: +44 1753 807429; Fax: +44 1753 The essential carboxylic acid presumably mimics the 536632; E-mail: [email protected]

CO H O 2 O CO H thioproline with homo-proline brought a reduction in H H 2 N N O N N activity, suggesting that the orientation of the two amide H O O carbonyl groups adjacent to the proline moiety was optimum N N H H in compound 8. We subsequently prepared a number of analogues in which the adamantyl carbonyl and thioproline 1 Bio1211 H2N NH groups were varied but none of these proved more active. Cl Cross screening against the integrins a5beta1 and HN CO2H O O alphaIIbbetaIIIa showed that these compounds were O HN Cl HN H selective for alpha4beta1 (IC50 >800 and 400 μM, N N O N O H respectively). O Cl O S HN O RO Table 1. In Vitro Activities for Compounds 3-8 N S H CO2H O Cl 3 Compound Alpha4 Beta1/VCAM IC50 (nM) Cell IC50 (nM)

2 R=CH2CH2N(C2H5)2 Valategrast, R411 3 7650 nd R

O 4 170 nd HN H N N 5 330 nd O O S HN O 6 7300 nd S CO2H 7 4100 nd

4 R=CO2H 8 280 nd 5 R=H nd: not done.

Having established the importance of the N-acetyl-l- H H N R N thioproline residue in conveying activity against the O O alpha4beta1 integrin, we derivatized this moiety with a S S S S O number of -amino acids, introducing a protein-based, O O ligand binding screen alongside the cell-based, adhesion N HO2C N HO C N H 2 H assay. This work [16] led to the O-benzylated tyrosine S analogue 9, Table 2, which afforded improved potency in 8

6 R=CO2H both the protein and cell based assays. An important 7 R=H breakthrough was the discovery that an `L to D' switch in the stereochemistry of the thioproline residue afforded a carboxylic acid residue in the recognition sequence of magnitude improvement in activity of compound 10, Table alpha4beta1’s ligands. Although cyclic peptides have been 2. When the analogous stereochemical switch was performed developed as therapeutic drugs, they generally suffer from on the tyrosine residue in a related analogue, a decrease in poor pharmacokinetics and rapid metabolism. Indeed, activity of two orders of magnitude was observed. plasma stability studies on compound 3 revealed rapid Incorporation of the 2, 6-dichlorobenzyl compound 11, degradation (t 3 min). Although depeptidisation and Table 2 (initially introduced to improve plasma stability of stabilising modifications were considered, this approach, in the benzoyl derivative) improved activity, particularly in the our opinion, would be both demanding and without cell-based assay. Cross-screening against a panel of 7 other sufficient guarantee of success. Instead, we attempted to integrins showed that compound 11 was over 6000 fold more determine which were the key pharmacophoric groups in a selective for alpha4beta1, with the exception of the related cyclic peptide such as 5 and use this information to design integrin alpha4beta7, where it displayed a 5-fold selectivity. simpler, acyclic structures more amenable to SAR studies. Compound 11 was active in a sheep model of antigen During the initial experiments, we isolated N,N- induced early and late phase bronchoconstriction followed diadamantylcarbonyl-l-cystine 6, Table 1, as a by-product. by an airway hyperresponsiveness to inhaled methacholine. This compound displayed significant activity prompting When administered orally, 2 h prior to antigen challenge, further investigation. More interestingly, the analogue 7, compound 11 (30 mg/kg) caused a marked inhibition of both Table 1, in which one of the carboxylic acid groups was early and late phase bronchoconstriction and blocked the removed completely, was also active and represented a expression of airway hyperresponsiveness. Replacement of significantly simplified structure. The inhibitors 6 and 7 had the benzyl group with a dichloropyridylamide, compound a relationship to the potent cyclic peptides, for example 12, Table 2, gave even greater potency and selectivity compound 4, with the essential cysteine-derived carboxylic (though retaining alpha4beta7 activity). acid common to both structures and the proximal Disappointingly, these compounds and their analogues adamantylcarbonyl group of the acyclic analogues displayed rapid clearance in a number of species thereby mimicking the (thio)proline residues of the cyclic peptides. hindering further development. That this rapid clearance was Support for this idea was delivered by making the a result of an active transport mechanism was shown by the appropriate proline acyclic analogues, for example presence in the bile of only trace amounts of metabolites compound 8, Table 1. Interestingly, replacement of the compared to the parent compounds. We postulated that the The Discovery of CDP323, a Potent Alpha4 Integrin Antagonist Current Immunology Reviews, 2012, Vol. 8, No. 2 137

-amido group, although apparently essential for potency, was responsible for the rapid clearance in this series. In an attempt to move away from the lead acyl derivatives but O O retain the features believed necessary for inhibitory activity, O 9 L Thioproline we envisaged incorporating the carbonyl of the a- N HO C N 10 D Thioproline aminocarbonyl group into a heterocycle, to obtain N-(triazin- 2 H S 1,3,5-yl)-phenylalanine derivatives such as compound 13

[16]. In this way, we retained a degree of structural and Cl Cl N electrostatic similarity but also ensured that the -amino O group remained non-basic and also possessed a hydrogen Cl HN atom, features felt to be necessary for potent alpha4 integrin Cl O O O antagonists. In order to gain some understanding of the SAR O O N N for clearance it was considered essential to have access to a HO C N HO2C N 2 H H relatively high throughput screen. The method chosen was S S the isolated perfused rat liver (IPRL) [17] whereby five 11 12 compounds (including a reference compound) could be dosed as a cassette. The elimination of each compound from Cl the perfusate was expressed in terms of a rate constant, k, N and normalised to the reference compound. The higher the O value of k the more rapidly the compound was cleared, for Cl HN R -1 13 R=OMe example compound 12 having an IPRL k of 3.7h . 14 R=NH(CH ) OH N N 2 2 Table 2. In Vitro Activities for Compounds 9-12 HO2C N N OMe H Cl Cl N Compound Alpha4 beta1/VCAM IC50 (nM) Cell IC50 (nM) N O O O Cl HN 9 220 19250 Cl HN O S O O 10 25 1480 N N

HO2C N HO C N H N 11 5 34 2 H H 12 0.7 0.7 16 15 Table 3. In Vitro Activities and Clearance for Compounds 13- Triazine compound 13, Table 3, displayed a clearance of 16

3 mL/min/kg, a t of 1.6 h and 9% bioavailability in the rat

(10 mg/kg p.o. and i.v.). Further derivatisation with a range Alpha4 Beta1/ -1 Compound Cell IC50 (nM) IPRL k h of alkoxy and amino groups led to a moderate improvement VCAM IC50 (nM) in potency (with respect to activity in the cell-based assay) as in, for example, compound 14, Table 3. In general the more 13 8 900 1.1 lipophilic the substituents, the better the potency and the 14 7 320 1.8 lower the rate of clearance though this is probably a reflection of the enhanced plasma protein binding of these 15 1 40 4.4 compounds. Modification of the phenylalanine substituent 16 1.5 120 0.11 led to a loss in potency, although the rates of clearance remained similar, suggesting that this feature is not key for recognition by the active transporter. N-(pyrimidin-4-yl) and which has been recognised as an isostere for -amino acids N-(pyridin-2-yl) replacements for the triazine were also as well as for other functional groups, such as studied [18] where introduction of sulphonyl functionality cyanoguanidines [19]. Derivatising the cyclobutenedione, improved potency. However, despite varying the initially with primary unbranched amines [20], gave a physiochemical properties all compounds displayed rapid relatively flat SAR in the protein-based assay yet noticeable clearance, for example, compound 15, Table 3 had a trends in the cell-based assay where, as the length of the clearance of 27 mL/min/kg, a t of 0.5h and oral carbon chain increased, an increase potency was observed bioavailability of 4% after dosing in the rat (10 mg/kg po that reached a maximum with the n-propyl analogue 16. The and iv). This work failed to identify any analogues with IPRL clearance rate constant of these compounds was potencies less than 100 nM in the cell-based assay, potencies reasonably low but reached a minimum of 0.1 h-1 with of this order were considered inappropriate to show efficacy compound 16, translating to a clearance of 0.1 mL/min/kg in in animal models and consequently this series was not the rat. Branched primary and secondary amines gave further developed. products that had good potency in the protein and cell based assays but much higher rates of clearance than their primary Continuing our search for suitable replacements for the amine counterparts. Obviously, the squarate amino acid amide believed responsible for the rapid clearance, we substituent [20] had a profound effect on clearance, and investigated the 3,4-diamino-3-cyclobutene-1,2-dione group although we were unable to rule out plasma protein binding,

138 Current Immunology Reviews, 2012, Vol. 8, No. 2 Bailey et al. we speculated that conformational differences may have relationship with log concentration of compound 19 was some influence. However, concerns about the stability of the obtained (Fig. 1c) suggesting that there was no evidence of cyclobutenedione group led to a continued search for hysteresis or proteresis. Individual data points were fitted to suitable alternatives. a direct link, sigmoid Imax model with maximal inhibition of To this end, a series of 3-aminocyclobut-2-en-1-ones 100% as described in the methods. The following model parameters were estimated; an IC value of 57ng.ml-1 were synthesized as suitable replacements for the 50 (109nM) and a Hill slope coefficient of 0.75 (Fig. 1d). cyclobutanedione functionality [21]. Initially, this work Cl retained the dichloropyridyl amide ‘top’ group and gave N structures such as compound 17 which were potent inhibitors O in both enzyme and cell assays. However, a number of compounds in this series that were potent in the alpha4 Cl HN integrin cell assay failed to demonstrate in vivo activity in a O rat lymphocyte trafficking model, despite retaining the required level of exposure. We have subsequently been able RO2C N H Br to correlate the lack of in vivo activity of some of these compounds with a significant reduction in potency when 17 R=H a4b1/VCAM protein IC 0.37nM; cell IC 1.5nM bovine serum albumin (BSA) was changed to rat serum 50 50 18 R=HO(CH2)2 Mouse bioavailabilty = 2.2% (15mpk) albumin (RSA) in the cell binding assays. The IC50 values of several key compounds were therefore evaluated in the presence of rat, mouse and human serum albumin (HSA) to ensure they possessed sufficient inhibition in the plasma of the corresponding target animal species and thus, have more N N chance to demonstrate activity in vivo. We suspected that the HN general shifts in potencies seen in our and other laboratories could well be due to differentiated non-specific binding to O non-target plasma proteins (matrix effect). RO2C N Compound 17 showed only 1-2% bioavailability in either H Br the rat or mouse what, so an ester prodrug [22] strategy was 19 R=H a4b1/VCAM protein IC50 1.1nM; cell IC50 13nM initiated. However, from more than 15 prodrug esters 20 R=Et Mouse bioavailabilty = 27% (15mpk) synthesized, the best mouse bioavailability seen was still only 2% for the 2-hydroxyethyl analogue 18. In an attempt Experimental autoimmune encephalomyelitis (EAE) is to rid the compounds of all amide bonds and effectively de- the animal model of Multiple Sclerosis. Here we used myelin peptidising the structures, we embarked on a heterocyclic oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) replacement strategy of the ‘top’ dichloropyridylamide induced EAE in C57BL/6 mice, which produces a chronic moiety. 2,7-naphthyridines were found to show best effects disease profile [24]. Compound 20 administered once daily and loss of the ‘top ring’ amide resulted in a lowering of by the subcutaneous route significantly reduced EAE disease PSA and an expected improvement in PK properties and symptoms when administered either prophylactically at bioavailability. Consequently, compound 19 showed good 100mpk or therapeutically (10-100mpk), prior to immunisat- potency in both enzyme and cell assays but mouse ion, or therapeutically, at the onset of disease symptoms. bioavailability was still low. The same ester prodrug strategy Compound 20 reduced both maximal and cumulative disease yielded the ethyl ester compound 20, which showed score by both dosing regimens and also reduced disease reasonable bioavailability in the mouse (27% at 15mpk). incidence following prophylactic dosing [24, 25]. The pre-clinical pharmacology of Compound 19 or 20 was Compound 20, designated CDP323, entered clinical trials evaluated across a range of in vivo rodent models from cell in November 2003 for the treatment of relapsing Multiple trafficking to allergic lung inflammation and animal models Sclerosis. of multiple sclerosis. Here we report the pharmacokinetic Alpha4 antagonist antibodies, as exemplified by and pharmacodynamic (PK/PD) relationship and the effect natalizumab, are currently enjoying success in the clinic, of compound 19. The pharmacokinetic/pharmacodynamic though a number of issues such as cost, potential (PK/PD) relationship was investigated by following oral immunogenicity and mode of administration suggest that administration of compound 19 (15mpk) and inhibition of there is scope for small molecule, orally bioavailable VLA4/VCAM-1 binding was used as an indicator of antagonists. Starting from a cyclic peptide and using a blockade of target [23]. The pharmacokinetic profile and classical medicinal chemistry approach we obtained a corresponding inhibition of VLA4/VCAM-1 binding phenylalanine core structure. Extensive SAR studies on this determined at fixed time-points are shown in Fig. (1a) and structure allowed us to gain insights into the pharmacophore (1b), respectively. and to identify at an early stage potential DMPK issues such A graphical display of the relationship between the as rapid biliary clearance. Our efforts to solve these issues concentration of compound 19 and inhibition of led us through a number of different series, ultimately VLA4/VCAM-1 ligand binding with the data points leading to the clinical candidate CDP323 and have outlined connected in time sequence was made to define the pattern its pharmacological profile. of the pharmacodynamic relationship. A sigmoidal The Discovery of CDP323, a Potent Alpha4 Integrin Antagonist Current Immunology Reviews, 2012, Vol. 8, No. 2 139

(a) (b)

1000 100

75 100

10 25

19] (ng/ml) [compound (n=2-5) (n=2-5) 1 0 0.0 2.5 5.0 7.5 10.0 0.0 2.5 5.0 7.5 10.0

Time (hr)

Time (hr) binding VLA4/VCAM-1 of % inhibition

ex vivo Compound 19 (15mg/kg p.o.) 100

100

t5 75 t4 75 t6 t2 t3 t7 50 50 t1 25 25 t9 t8 binding % ligand 0 0 1 10 100 1000 -91 10-8 100-7 1000-6 10000-5 [compound 19] ng/ml inhibition% of VLA4/VCAM-1binding [compound 20]: ng/ml

Fig. (1). The pharmacokinetic profile of compound 20 following oral dosing at 15mg/kg (a) and corresponding ligand binding (b) timecourse in adult male Balb/c mice (c) the relationship between inhibition of ligand binding and [compound 19] linked in time sequence, and (d) relationship between % ligand binding and [compound 19] concentration described by an IMAX model.

(a) (b) Compound 20 (s.c. u.i.d.)

100mg/kg 3 3 30mg/kg Vehicle 10mg/kg Compound 20 (100mg/kg sc uid) Vehicle only 2 2

1 1

0 0

Mean Disease Score (+/- sem)

Mean Disease Score(+/- sem) Mean Disease 0 10 20 30 0 10 20 30 PSD PSD

Fig. (2). The effect of compound 20 on disease score in Mog35-55-induced EAE in C57BL/6 mice following (a) prophylactic and (b) therapeutic dosing regimen. Data is shown as mean disease score +/- sem, n=10-15/group. PSD: post-sensitisation day.

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Received: July 22, 2010 Revised: July 13, 2011 Accepted: July 15, 2011