The Discovery of Potent, Selective, and Reversible Inhibitors of the House Dust Mite Peptidase Allergen Der p 1: An Innovative Approach to the Treatment of Allergic Asthma

Blocking the bioactivity of allergens is conceptually attractive as a small-molecule therapy for allergic diseases but has not been attempted previously. Group 1 allergens of house dust mites (HDM) are meaningful targets in this quest because they are globally prevalent and clinically important triggers of allergic asthma. Group 1 HDM allergens are cysteine peptidases whose proteolytic activity triggers essential steps in the allergy cascade. Using the HDM allergen Der p 1 as an archetype for structure-based drug discovery, we have identified a series of novel, reversible inhibitors. Potency and selectivity were manipulated by optimizing drug interactions with enzyme binding pockets, while variation of terminal groups conferred the physicochemical and pharmacokinetic attributes required for inhaled delivery. Studies in animals challenged with the gamut of HDM allergens showed an attenuation of allergic responses by targeting just a single component, namely, Der p 1. Our findings suggest that these inhibitors may be used as novel therapies for allergic asthma.


■ INTRODUCTION
At the heart of the clinical management of asthma lies a paradox: despite efficacious and safe therapies (e.g., β 2 agonist bronchodilators, inhaled corticosteroids, antileukotrienes, and an anti-IgE monoclonal antibody), the condition remains poorly controlled and its prevalence continues to increase. 1,2 The multiple factors underlying this paradox highlight a significant unmet need which might be better addressed by a completely different approach. Current standard of care in asthma is directed at downstream effector mechanisms, and the medicines involved act primarily by relieving symptoms. For novel therapies in development, target selection has been primarily driven by a focus on deeper understanding of the effector pathways of asthma, with the hope that new nonsteroidal interventions will reduce both the risk of disease exacerbations, which is the major goal of clinical management, and the potential for adverse events. However, experience shows that only limited success has been achieved by targeting individual downstream effectors in asthma, highlighting the need for new approaches.
For any condition, an alternative to symptom management is to target the major trigger or root cause. However, asthma is a complex spectrum of conditions rather than a homogeneous disease and on first inspection such an approach seems unfeasible. Asthma may be broadly divided into nonallergic and allergic types, with the latter, triggered by inhaled environmental allergens, predominating. Two pieces of epidemiological evidence suggest that design of an intervention directed toward a trigger of allergic asthma could be surprisingly tractable. First, a succession of studies highlight that, globally, the most important providers of allergen triggers are house dust mites (HDM). 3−12 Second, sensitization to HDM precedes the development of sensitization to allergens from unrelated sources. 13,14 Mechanistically, this longitudinal relationship exists because HDM facilitate sensitization to other agents by providing essential collateral priming events on which other allergens depend.
HDM are sources of more than 20 denominated allergen groups, 15 with those of group 1 being of particular interest because of their abundance, allergenicity, and their functional properties which promote sensitization to themselves and other allergens. 15−24 Sensitization to HDM allergens occurs through inhalation of this animal's fecal pellets, which, when they impact upon the airway mucosa, hydrate and release their contents. The group 1 allergens (e.g., Der p 1, Der f 1, Eur m 1) of the various HDM species form a distinct subfamily of C1 cysteine peptidases 25 whose sequences are sufficiently identical that targeting them with a single agent is a realistic possibility. 22 Two general peptidase-dependent mechanisms have been identified by which group 1 HDM allergens promote allergic sensitization and asthma. The first is their ability to cleave epithelial tight junctions by proteolytic attack on the transmembrane adhesion domains of occludin and claudin family proteins. 24,26 This cleavage results in the epithelial barrier becoming leaky, increasing the probability of contact of any allergen with dendritic antigen-presenting cells and permitting the migration of these cells, along with secondary effector cells, into the airway lumen. 6,27,28 Their second general mechanism as proteases is to activate signal transduction pathways of innate immunity which release chemokines and other mediators (e.g., IL-13, IL-33, TSLP, IL-25, CCL-20) 16,18,29 that are known to recruit the necessary effector cells and promote a TH 2 bias to immune responses. 6,18,23,29−31 Significantly, evidence suggests that some of these innate immune mechanisms are the focus of important genetic predispositions for allergic asthma. 32 Given the importance of HDM sensitization as a trigger for asthma and the increasing recognition that the peptidase activity of group 1 HDM allergens plays an important role in both its initiation and maintenance, the aim of our program was to develop small-molecule inhibitors of these pivotal allergens. We call these new drugs "allergen delivery inhibitors" (ADIs), and it is our hypothesis that an ADI compound would provide an effective inhaled treatment for patients suffering from allergic asthma. The compounds disclosed herein are the subject of a patent disclosure. 33 ■ RESULTS AND DISCUSSION Identification of Reversible Der p 1 Inhibitors. Prior to the commencement of our program, the only reported inhibitors of Der p 1 were irreversible acyloxymethyl ketone inhibitors ( Figure 1). 34 Given that asthma treatment will require chronic drug administration, we considered that compounds having an irreversible mechanism of action were inherently lacking in developability because of their potential to elicit adverse effects. We therefore sought to replace the irreversible binding motif with functional groups that could form a fully reversible covalent bond with the active site cysteine residue. An investigation of amino-ketones afforded compounds with initially encouraging potency against Der p 1, but we were unable to optimize inhibitory activity beyond that shown by compound 3. To enhance binding to the active site cysteine residue, we therefore examined alternative groups, including the corresponding pyruvamide analogues. This rapidly led to the identification of compound 5, which became the starting point for our discovery program ( Figure 1).
Low molecular weight peptides are often characterized by poor oral bioavailability. This has been attributed to their propensity for proteolytic cleavage, and the presence of a high proportion of hydrogen bond donors and acceptors, in conjunction with a relatively flexible scaffold; factors likely to hinder passive absorption in the gut. Conversely, these  properties may be beneficial for an inhaled drug as they would minimize absorption of any inadvertently swallowed portion of a dose, thereby limiting adverse systemic effects. In our view, these considerations made a peptidic scaffold an ideal template for the design of an inhaled Der p 1 inhibitor.
To obtain in vivo efficacy that was compatible with delivery from a range of inhaler devices, we required a compound with high inhibitory potency against Der p 1. Upon the basis of empirical estimates of target exposure, we therefore set a template IC 50 ≤ 20 nM for the target. While compound 5 fulfilled this criterion, a developable candidate requires other features that impact on in vivo efficacy, notably those properties that would affect the retention of the compound at the site of action: permeability, lipophilicity, and stability in the lungs. Additionally, increasing the intrinsic selectivity of our initial lead over closely related human cysteine peptidases, notably cathepsin B (Cat B) (Figure 1), was desirable because intrinsic selectivity combined with low systemic exposure would reduce the risk of off-target events. This risk would be subject to further mitigation because of the entirely extracellular interaction between target and inhibitor, whereas the potential off-target enzymes have largely intracellular dispositions which would require compounds to be highly membrane permeant for enzymes to be at risk of inhibition. Pharmaceutical properties were also taken into account early in the program so as to generate compounds that would be suitable for use in a dry-powder inhaler (DPI), our preferred device. The requirement to be able to consistently produce particles of optimal respirable size places stringent constraints on the solid-phase behavior of compounds that are to be delivered by DPI. For example, the need to be compatible with micronization requires a stable, nonhygroscopic crystalline form with a melting point >100°C. Aqueous solubility was required to balance adequate drug dissolution for efficacy with an enduring effect while avoiding the potential for irritancy associated with low solubility compounds. Compound 5 lacked selectivity and was extensively (>50%) degraded by airway macrophages over a 2 h period, suggesting that its duration of action in vivo would be insufficient. However, its potency and the scope for structural variation suggested it was a promising lead for optimization.
Synthetic Chemistry. Pyruvamide-motif inhibitors were synthesized from the corresponding α-hydroxy amides (7) by oxidation with the Dess−Martin periodinane reagent (Scheme 1). The α-hydroxy amide intermediates were constructed by two synthetic approaches: either a modified Passerini reaction 35 (route A) or the use of cyanohydrin chemistry (route B). Both routes required capped dipeptide acids (6), produced either by solid-or solution-phase synthesis. Solid-phase synthesis was carried out using standard Fmoc chemistry on Wang resin. 36 Insolution chemistry used a stepwise process which involved, as a typical first step, the coupling of a P 2 amino acid ester (11) with a Boc-protected P 3 amino acid (10) under low-temperature  For route A, the dipeptide acids (6) were typically coupled with the appropriate amino alcohol (13) followed by oxidation to give the corresponding aldehyde (14). A modified Passerini reaction was then used to produce the α-hydroxy amide (Scheme 3). 35 For route B, a variety of α-hydroxy amides (7) were synthesized by coupling β-amino-α-hydroxy amides (15) with dipeptide acids (6) under similar conditions to those previously described (Scheme 3). The β-amino-α-hydroxy amides (15) were made in seven steps from Cbz-protected valine 16 (Scheme 4).
Finally, some compounds were made by routes C and D, each being a variation on route B where the order of events was changed to aid the synthesis of particular analogues (Scheme 1).
Modeling the Binding Mode to Der p 1. To assist the design of analogues of compound 5, a computational model was constructed based on the crystal structure of Der p 1 (PDB code 2AS8) and on the structures of a number of peptidic inhibitors bound to the C1 family of clan CA peptidases (PDB codes 1TU6 and 2BDL). Compound 5 was built and minimized within the active site of the Der p 1 crystal structure. The electrophilic carbonyl of the pyruvamide was positioned to form a covalent interaction with the catalytic cysteine residue (Cys 34), while the peptide backbone of 5 was oriented to follow a similar trajectory to that of the other peptidic inhibitors. This minimized structure revealed a number of putative hydrogen-bonding interactions which anchored compound 5, thereby allowing the side chains to interact with the specificity pockets of the enzyme. On the basis of this binding model, we propose that the amide carbonyl of the pyruvamide unit interacts with the backbone NH of Cys 34, the NH of the P 1 subunit is able to form an interaction with the carbonyl of Tyr 169, and the P 2 subunit forms a donor− acceptor pair with the backbone carbonyl and NH of Asp 74 ( Figure 2a).
Improving Selectivity. Significant off-target activity of compound 5 against Cat B was revealed by counter-screening against human cysteine peptidases, notably certain members of the C1 family. We therefore investigated SAR around compound 5 with the aim of improving the selectivity profile (Table 1). We established that the P′ cyclohexyl group could be replaced with a benzyl group without a significant effect on Der p 1 potency or selectivity (for comparison see 19 and 20; similar effects seen for other analogues, data not shown), so these two groups were used interchangeably for SAR comparisons. As the pyruvamide motif has the potential to interact indiscriminately with nucleophilic cysteine or serine residues of other peptidases, we hoped that increasing the steric bulk of the P 1 substituent would hinder nonspecific interactions. Increasing the branching at P 1 to iso-propyl (19) retained inhibitory potency on Der p 1 but did not significantly improve selectivity over Cat B. It did however improve resistance to processing by airway macrophages, with ∼70% remaining unchanged after 2 h. The iso-propyl group was retained for further investigations because further branching to tert-butyl (21) reduced the potency against Der p 1. Of further note, removal of the P 1 substituent resulted in a 10−20-fold drop in Der p 1 potency (see Supporting Information, Appendix 5, compound 76).
Comparison of the reported crystal structures of Der p 1 and Cat B revealed that the S 3 pocket is more capacious in Der p 1 than in Cat B, mainly due to the presence of a Thr in Der p 1 (Thr 74) instead of Tyr in Cat B (Tyr 75). Cathepsins S (Cat S) and K (Cat K) also possess similar large groups at this position (Phe 70 in the case of Cat S and Tyr 67 in the case of Cat K). We hypothesized that increasing the steric bulk at P 3 would increase selectivity with respect to these enzymes (Figure 2c,d), and it was therefore pleasing to find that switching from benzyl (5) to tert-butyl (22) increased selectivity over Cat B while maintaining good inhibitory potency on Der p 1. Similarly, introducing geminal-dimethyl substitution onto the benzyl group (23) reduced inhibitory   Further modeling suggested that the S 2 pocket was shallow in Der p 1 compared to cathepsins B, K, or S; effective inhibitors of these enzymes tend to have groups larger than methyl at this position. 37−39 Consistent with these observations, the methyl substituent at P 2 generally provided the best balance of potency and selectivity for Der p 1 (19 vs 24).
Modification of Molecular and Physicochemical Properties. Preliminary investigations into the P 1 , P 2 , and P 3 positions had shown that we could obtain good inhibitory potency against Der p 1 and significantly improve the selectivity over Cat B. We next turned to the N and C terminal groups with the aim of modifying physicochemical properties to optimize the endurance and pharmaceutical properties of inhibitors. Simultaneously, we hoped to take advantage of any preferences shown in the S 4 and S′ pockets to further enhance Der p 1 potency and selectivity with respect to Cat B.
A number of chemical design philosophies have been adopted to enhance the duration of action of small-molecule drugs aimed at other respiratory targets. 40,41 Approaches considered potentially applicable to ADIs involved the incorporation of features to confer either low permeability or increased binding to lung tissue. Permeability can be reduced by increasing molecular weight and/or PSA, whereas lung tissue retention may be enhanced by combining increasing lipophilicity with a basic or quaternary ammonium group. However, because it was uncertain how effective or welltolerated any of these tactics would be in the case of Der p 1 inhibitors, we decided to modify their molecular and physical properties as widely as possible, thereby maximizing the scope to manipulate their in vivo and pharmaceutical behavior.
A variety of groups were investigated in the P 4 position. Additional small lipophilic substituents on the phenyl ring were tolerated (data not shown), as were fused rings which could be used to increase molecular weight and log D. Furthermore, some of these groups gave improved selectivity over Cat B (compounds 25, 26, and 27, Table 2). Replacement of the Table 2. Impact of Modifying the P 4 Substituent on log D, Der p 1 Potency, and Selectivity over Cat B a Contains ∼30% of P 1 R epimer. b ND = not determined. c Single determination measured as described in Supporting Information.  phenyl group with 5-or 6-membered heterocycles reduced log D but, with the exception of the 4-pyridyl group (29), these decreased the potency against Der p 1. It was also possible to incorporate a basic center in the form of an N-methylpiperidine group (31) which could be quaternized to give 32, which as expected showed excellent aqueous solubility (∼1.6 mmol when shaken in PBS 7.4 for 2 h). The S 4 pocket in Der p 1 is surrounded by a number of Tyr residues (Tyr 169, Tyr 216, Tyr 218), and we speculate that the positively polarized Nmethyl hydrogen atoms of 32 are able to make favorable interactions with these residues, thereby resulting in a potent Der p 1 inhibitor.

Journal of Medicinal Chemistry
Having shown that cyclohexyl and benzyl gave good activity in the P′ position, we next turned to exploration of the SAR in this region. Because the parent compounds, such as 22, were lipophilic, we initially focused on reducing log D because this would increase aqueous solubility and mitigate any potential risk of irritancy arising from the accumulation of poorly soluble compounds in the lung. Replacement of the C-4 cyclohexyl carbon with a heteroatom lowered both Der p 1 and Cat B inhibitory activity and was not pursued further (Table 3, 33 and 34). It transpired that a more successful means of lowering log D was to reduce the ring size. Replacement of the cyclohexyl group (22) with a cyclopropyl group (35) reduced the measured log D by ∼1.5 log units while maintaining high potency against Der p 1 and selectivity over Cat B, whereas removal of the P′ group caused attrition of selectivity even though potency was acceptable (36). However, simple substitution with a methyl group was sufficient to restore selectivity (37). Regardless of these findings, we discovered that the synthesis of P′ benzyl analogues was a more fruitful approach to the modification of physiochemical properties and the optimization of both potency and selectivity. In general, lipophilic groups in the para position were less active than more polar groups, especially those containing a hydrogen bond donor. In particular the introduction of a p-SONH 2 group, as in compound 39, gave high Der p 1 potency and excellent selectivity over Cat B. Introduction of an α-methyl group showed a preference for the R enantiomer (42). Basic groups could also be appended to the phenyl ring as shown by compound 41.
To further probe the SAR of the P′ pocket, we elected to explore a series of glycinamide analogues, reasoning that the glycinamide moiety could mimic the peptidic backbone of a substrate molecule and that the amide substituent would allow us to modify the molecular and physical properties. Furthermore, this subseries would have an increased PSA, thereby reducing compound permeability in both the lung and the gut. Serendipitously, this was an effective way of removing the undesired Cat B activity. Compound 45, containing a basic motif, showed excellent Der p 1 inhibitory activity and good selectivity over Cat B. Moreover, combining this modification with a tert-butyl group in P 3 resulted in compound 48 that was both potent against Der p 1 and which showed no significant inhibition of Cat B at 2.5 μM. Alternatively, replacing the Nmethyl group with a bulkier iso-propyl group (49) or introducing a substituent on the α position of the glycinamide (50), reduced inhibition of Cat B without the need to introduce the tert-butyl group at P 3 . As can be seen from Table 3, glycinamides that were potent against Der p 1 spanned a range of lipophilicity. Additionally, it was possible to convert the piperazine group to a quaternary ammonium compound (52) with good Der p 1 activity and excellent selectivity over Cat B.
To further examine the selectivity of inhibitors produced by P′ variation, compounds were counter-screened against a wider panel of proteases and it was pleasing to see that good selectivity was attained over a diverse range of targets. As general exemplification of a compound which displays encouraging potency against the Group 1 HDM peptidase allergens, data for compound 38 are presented in Appendix 7 of the Supporting Information.
Pharmaceutical Properties. A key goal of the program was to produce potential candidate drugs with stable crystalline forms compatible with delivery by DPI and with confidence that they would be compatible with other devices. Several approaches were taken to increase the likelihood of obtaining crystalline compounds. However, it was difficult to predict with confidence which would crystallize; after establishing a viable approach, approximately half of the compounds examined crystallized readily, and it is probable that others could be crystallized with further effort. Tactics used to favor crystallinity are described in the Supporting Information. The fact that satisfactory crystalline properties were common and associated with molecules whose profiles could be manipulated by structural alterations at multiple locations meant that we were able to prosecute the program with confidence that all of the required characteristics for a candidate drug were achievable within this series.
Efficacy in Vivo. Having demonstrated that it was possible to obtain potent, selective inhibitors of Der p 1 spanning a range of physicochemical properties and that no overriding issues with crystallinity existed, experiments were carried out to elucidate which features were most strongly correlated with in vivo efficacy. All of the compounds chosen for this work showed no degradation when incubated with rat airway epithelial cells or airway macrophages for 2 h, thereby minimizing metabolic instability as a variable in these studies.
A series of similarly potent compounds with a range of measured log D 7.4 values and PSA values (Figure 3a) were chosen for study in rats challenged with a natural mixture of allergens from Dermatophagoides pteronyssinus, i.e., a diverse mixture containing the full spectrum of HDM allergens including our drug target, Der p 1. We had previously discovered that when delivered by intratracheal aerosol to rats, the proteolytic activity of Der p 1 in this mixture recruited inflammatory cells to the airways in the absence of prior sensitization because the proteolytic activity triggered innate immune responses. The nature and time-course of this cell recruitment was similar to that which occurs in animals sensitized to HDM but was of smaller magnitude. As a first step, we therefore exploited this finding of a peptidasedependent innate response to compare the activity of selected Der p 1 inhibitors. In brief, this model involved intratracheal aerosolization of a solution of the test compound into rats using a Penn-Century device, followed 2 h later by challenge with a mixture of HDM allergens delivered by the same method.  Bronchoalveolar lavage (BAL) was performed 48 h after allergen challenge as the optimal time to evaluate any effects on the recruitment of eosinophils (data not shown). The compounds under investigation contained a range of groups that would be expected to be neutral or positively charged at physiological pH. The percentage inhibition of eosinophil recruitment when challenged with the HDM allergens was recorded (Figure 3b). There was a clear trend toward more lipophilic compounds showing better efficacy (compare compounds 27, 29, and 38 to compounds 45, 47, and 53). Good efficacy was also achieved for the quaternary ammonium compounds 32 and 52 with good endurance of action (>6 h protection from a single dose) evident for both (Figure 3c). Further details of the duration of protection achieved with a selection of inhibitors are presented in Appendix 8 of the Supporting Information.

Journal of Medicinal Chemistry
Demonstration of potency and endurance of action against the HDM allergen target in the innate response model led us to investigate efficacy in an IgE-dependent context in animals actively sensitized to a mixture of HDM allergens. Figure 4a shows that a single dose of compound 32 administered 2 h prior to allergen challenge significantly blunted the increase in total nucleated cells recoverable from the airways by BAL. At the 48 h sampling point, the majority of this reduction was accounted for by the marked inhibition of eosinophil recruitment (Figure 4b).
Systemic Exposure. The PK behavior of a range of compounds was measured in rats to assess the extent of any systemic exposure which might lead to side effects. Systemic exposure following drug delivery by inhalation can arise from the portion of the dose that is inadvertently swallowed, creating the potential for both on-and off-target systemic effects, so part of our design strategy was to minimize oral absorption. Further studies are planned to assess the extent of systemic exposure directly from the lung.
For existing antiasthma medicines, the systemic effects being avoided are primarily on-target actions. One approach to avoid systemic effects is to introduce a metabolic "soft-spot" so that biotransformation occurs predictably to an inactive metabolite. In this program, the therapeutic target is nonhuman and it is found only in the airways after inhalation, so systemic exposure to ADIs is concerned solely with off-target effects. Although offtargets can be predicted by homology with the therapeutic target, unexpected effects of both the parent drug and its metabolites remain a possibility. Our preferred path was therefore more general, relying on the minimization of free drug levels in plasma by varying PPB and oral bioavailability, alone or in combination. Selected compounds were therefore profiled using a 5 mg/kg oral dose in fed rats with the aim of generating compounds with a free concentration C max < 100 nM. The oral dose was chosen primarily to allow accurate quantitative analysis because in practice the actual dose of an inhaled compound would be >300-fold less. In these studies, a number of compounds showed encouragingly low levels of free drug (Table 4). For the smaller lipophilic analogue 35, high oral bioavailability was observed, but for higher MW analogues such as 38 and 28, the combination of reduced oral bioavailability and high PPB resulted in negligible levels of free drug even at doses more than 2 orders of magnitude higher than those that would be given therapeutically. As anticipated, the quaternization of amines, as demonstrated by compounds 32 and 52, was a particularly effective means of reducing oral absorption.
Further Optimization. The P 4 and P′ substituents were subsequently varied to generate a wider range of compounds with the desired potency/selectivity profile ( Table 5). The most promising compounds from the entire compound set were subsequently evaluated in depth using in vivo models.
With low systemic exposure, excellent Der p 1 potency and selectivity, high aqueous solubility, and efficacy beyond 6 h, compounds 32 and 52 exemplify one approach to the design of Der p 1 inhibitors (Table 6). Furthermore, Table 6 demonstrates that by using Der p 1 as the chemical design template and the screening target, it was possible to obtain potent inhibitors of an orthologous group 1 HDM allergen from another HDM species, D. farinae, confirming their behavior as a single drug target.

■ CONCLUSION
This program has identified compounds which create an innovative approach to the treatment of allergic asthma. Unusually, the therapeutic target is nonhuman, it is contacted by inhalation, and its engagement with an inhibitor is extracellular, features which are attractive for chemical design. Starting from leads which act irreversibly, we designed potent, reversible inhibitors of Der p 1. Clinically, Der p 1 is widely considered to be archetypal of group 1 allergens from all HDM species and is used as a surrogate measure of environmental exposure to HDM generally. Immunological reactivity to group 1 HDM allergens is globally prevalent and is found in >90% of patients who are allergic to HDM. 9,15,42 Collectively, these strategic factors make HDM the major domestic trigger of asthma attacks. Our work validates Der p 1 as an archetype for drug design because we were able to demonstrate the principle that leading compounds were equally effective as inhibitors of the orthologous group 1 allergen from another clinically significant HDM species. The cysteine peptidase activity of Der p 1 and, by inference, other group 1 HDM allergens, is of functional significance to the development of allergic disease through general mechanisms which are targeted by these new inhibitors. 22 We call the new compounds "allergen delivery inhibitors" to reflect the sentinel events which are blocked by this intervention.
Candidate ADIs were generated by optimizing interactions with the binding pockets and improvements in selectivity were largely achieved by modifying the substituents interacting at P 3 and P′. In the case of the P 3 position, this can be rationalized by the presence of a more open substrate-binding pocket in Der p 1. Additionally, by modifying their terminal groups, we have demonstrated that Der p 1 inhibitors with a wide range of physiochemical properties can be identified, allowing the impact of such properties on in vivo efficacy to be explored. These studies show that lipophilic compounds, and those Epidemiological studies highlight the importance of HDM allergy as both an asthma trigger and a facilitator of allergic sensitization generally. 42 This central importance of HDM and their allergens accords with the sentinel roles that innate mechanisms play in the development of allergic asthma through the activation of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) which are linked to the expression of TH 2 -polarized adaptive immunity. 15 HDM, with their environmental pervasiveness and extensive repertoire of allergens of diverse biological activity, are understandably decisive regulators of innate responses operating through such mechanisms. While the exact nature of the collateral priming mechanisms activated by HDM exposure is incompletely understood, compelling evidence implicates cysteine peptidase group 1 allergens as key components. 6,16,18,[22][23][24]37 This is supported by our in vivo experi-ments where we show, for the first time, that the targeted inhibition of a group 1 allergen by suitably optimized compounds substantially reduces cellular inflammation following challenge with a mixture comprising more than 20 different allergens. This suggests that ADIs have the potential to influence inter alia a broad spectrum of innate pathways which form the general mechanism which underpins the development, maintenance, and, ultimately, escalation of allergic asthma. The emergence of promising candidates from this program, including one nominated for development, will enable evaluation of a new therapeutic approach to allergic asthma based upon treating its root cause rather than by amelioration of its symptoms.

■ EXPERIMENTAL SECTION
The syntheses of key compounds are described below. All commercially available solvents and reagents were used without further purification unless otherwise noted. NMR spectra were measured with a Bruker DRZ 400 MHz spectrometer; chemical shifts are expressed in ppm and are aligned relative to the residual solvent peak, e.g., 2.5 ppm for DMSO. Coupling constants (J) are recorded in Hz. The purity of test compounds was determined by reverse-phase Table 5. Further Optimization of the P 4 and P′ Groups with Respect to Der p 1 Potency and Selectivity over Cat B In all cases pyruvamide inhibitors of Der p 1 were synthesized by oxidation of the corresponding α-hydroxyamide (7) using the same general procedure.
General Procedure I: Oxidation of α-Hydroxyamides (7) to Pyruvamides. To a stirred solution of 7 (1 equiv) in anhydrous DCM (1 mL/25−250 mg of alcohol) and anhydrous DMF (10−35% v/v depending upon solubility) at ambient temperature was added Dess−Martin periodinane (1.6 equiv) in portions. The reaction mixture was stirred at ambient temperature and monitored by LC-MS until full conversion to product pyruvamide had occurred (typically 1 h to 1 day). Where necessary, additional Dess−Martin periodinane was added to complete the oxidation. The reaction mixture was quenched by addition of saturated NaHCO 3 (aq) (1 volume) and Na 2 S 2 O 3 (aq, 10% w/v). The mixture was stirred for ∼30 min, diluted with ethyl acetate (10 volumes), and washed with saturated NaHCO 3 (aq) (2 × 5 volumes), deionized water (5 volumes), and brine (5 volumes). The organic layer was subsequently dried over MgSO 4 , filtered, and evaporated. Purification by reverse-phase preparative HPLC was generally followed by lyophilization to give the desired pyruvamide.
The synthesis of pyruvamide compounds via routes A, B, C, or D required the synthesis of intermediates 6a−l, 8a−c, 9a, and 15a−i. The syntheses of these compounds are described in the Supporting Information.
N To a solution of acid 6a (2.0 g, 5.9 mmol) in anhydrous THF (50 mL) at −40°C was added N-methylmorpholine (2.0 mL, 18.2 mmol) and i-BuOCOCl (0.8 mL, 6.4 mmol), and the reaction mixture was stirred for 1 h at −40°C. A solution of (S)-2-amino-1-hexanol (757 mg, 6.4 mmol) in dry THF (5 mL) was then added dropwise and the reaction mixture stirred at −40°C for 6 h before warming to room temperature slowly over 1−2 h. The resulting mixture was diluted with EtOAc (150 mL) and washed with saturated NaHCO 3 (aq) (2 × 100 mL) and brine (1 × 100 mL). The organic layer was dried over MgSO 4 , filtered, and evaporated to give a crude oil. The oil was purified by flash chromatography on silica, eluting with EtOAc to 30% MeOH/EtOAc to give a white solid (1.6 g; MS [M + H] + 440). The solid was dissolved in anhydrous DCM (40 mL) and anhydrous DMF (8 mL) and Dess−Martin periodinane (1.5 g, 3.4 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h and monitored by LC-MS; if the reaction had not gone to completion, a further amount of Dess−Martin periodinane was added and the reaction mixture stirred until completion (in this instance 450 mg, 1.1 mmol was added and the reaction mixture stirred for a further 2 h). The solvent was removed by evaporation and the crude mixture redissolved in EtOAc (50 mL). A solution of sodium thiousulfate in saturated NaHCO 3 (aq) (3 g in 30 mL) was added and the mixture stirred for ∼30 min. The organic layer was separated and washed with saturated NaHCO 3 (aq) (2 × 50 mL) and brine (50 mL) then dried over MgSO 4 , filtered, and evaporated to yield a pale-yellow oil. Trituration with Et 2 O gave 14a as white solid (683 mg, 26%); [M + H] + 438. 1  Compound 5. To a stirred solution of 14a (218 mg, 0.5 mmol) in anhydrous DCM (2 mL) at 0°C was added cyclohexylisonitrile (74 μL, 0.6 mmol) and pyridine (161 μL, 2.0 mmol) followed by dropwise addition of TFA (74 μL, 1.0 mmol). The reaction mixture was stirred at 0°C for 10 min and then allowed to warm to ambient temperature. After 4 h at room temperature, analytical LC-MS suggested that considerable starting material remained. Therefore, further cyclohexylisonitrile (61 μL, 0.5 mmol) and TFA (37 μL, 0.5 mmol) was added and the reaction mixture stirred for 18 h. The mixture was diluted with DCM (10 mL), washed with saturated NaHCO 3 (aq) (2 × 15 mL) and brine (15 mL (S)-3-tert-Butoxycarbonylamino-2-hydroxy-4-methyl-pentanoic Acid (18). To a solution of 18 (5.1 g, 19.5 mmol) in 1,4-dioxane (90 mL) was added concentrated HCl (aq) (90 mL) and anisole (1.5 equiv), and the mixture was heated to 110°C for 18 h. The reaction mixture was cooled to ambient temperature and concentrated under vacuum to remove the dioxane. The mixture was then washed with EtOAc, and the residue was further concentrated under vacuum at 40°C to remove the HCl (aq). Residual water was removed by azeotroping with toluene. The residue was washed with Et 2 O (2 × 50 mL) to give a gummy solid. The crude mixture was dissolved in methanol (100 mL), and Et 3 N (9.0 mL, 64 mmol) was added. Di-tertbutyl dicarbonate (4.7 g, 22 mmol, ∼1.1 equiv based upon crude solid) was added portionwise, and the reaction mixture was stirred at ambient temperature for 20 h. The reaction mixture was concentrated in vacuo, and the residue was dissolved in EtOAc (100 mL) and 1N NaOH (aq) (75 mL). The organic phase was separated, and the aqueous phase was washed further with EtOAc (2 × 100 mL) to remove any nonpolar/nonacidic impurities. The aqueous layer was then acidified (pH ∼2) with 2 N HCl and extracted with EtOAc (3 × 100 mL). The combined organic phases were dried over MgSO 4 , filtered, and concentrated under vacuum to give a white, waxy solid. This was further purified on a Biotage Isolute (IST)-NH 2 cartridge (25 g/150 mL). The cartridge was first equilibrated with MeOH (75 mL), MeCN (75 mL) and EtOAc (75 mL). The crude mixture was then loaded in 5% MeOH/EtOAc (50 mL) and washed with EtOAc (2 × 75 mL) and MeCN (75 mL). The desired mixture of diastereomeric acids was then eluted by washing with MeCN containing 1% formic acid (350 mL). A 1:1 diastereomeric mixture of the desired compounds was obtained, as a white solid, following evaporation of the solvent under vacuum (1. 16 Automated screening of inhibitory activity toward group 1 HDM peptidase allergens was performed as described elsewhere 25,43 with the minor modification that Der p 1 or Der f 1 were incubated with inhibitors for 20 min prior to initiation of reactions by addition of substrate. In initial screening, the activity of each compound was measured using a series of 10 doubling dilutions from a starting concentration of 50 μM. Compounds showing optimizable activity were cherry-picked for multiple (up to six) rounds of rescreening to confirm activity and stability and to determine IC 50 values over a range of concentrations that were compound-directed by initial screening. Assays were performed in triplicate for calculation of mean ± SE IC 50 values.
To test inhibitory potential against Cat S, reaction mixtures comprised 70 μL of reaction buffer (0.1 M sodium phosphate/2 mM EDTA, pH 7.4), 10 μL of recombinant human Cat S (25 nM in reaction buffer and 10 μL DTT (2 mM final concentration). Reactions were initiated by addition of 10 μL of substrate (Z-Phe-Arg-AMC, 20 μM final concentration in assay). Reactions were performed at 30°C and progress followed by excitation/emission at 320/420 nm.
In all cases, for reactions involving inhibitors, the amount of reaction buffer was adjusted to 60 μL, the inhibitor added as a 10 μL aliquot, and incubated for 20 min with the enzyme prior to reaction start.
Counter-screening against a broader range of proteases, with exemplification for compound 38, is described in Appendix 7 of the Supporting Information.
Allergen Challenge. Brown Norway strain rats (male, 300−350 g at time of allergen challenge, 12−16 weeks old, Harlan UK Ltd.) were used to explore the pharmacological properties of ADIs. To examine the relationship between the physicochemical properties of compounds and their duration of protection through the intended mechanism of action, animals were used without prior sensitization to HDM so that allergen challenge activated only IgE-independent innate mechanisms.
For other studies, rats were actively sensitized to a natural mixture of HDM allergens prepared from laboratory cultures of Dermatophagoides pteronyssinus. Animals were sensitized by the ip route in the absence of additional adjuvant on day 0, 7, and 14. This protocol is known to result in the development of allergic sensitization as judged by the appearance of allergen-specific IgE. For standardization purposes, sensitization mixtures were normalized to contain 10 μg Der p 1 of known catalytic activity, as previous studies indicated that this yielded satisfactory adjuvantless responses. Negative control groups comprised naïve, unsensitized animals or those sham-sensitized with vehicle solution.
Test ADIs were delivered as intratracheal aerosols of 25−30 μm mass median diameter from a Penn-Century IA-1B microsprayer at various times prior to allergen challenge. Allergen or vehicle challenge was similarly delivered by Penn-Century microsprayer on day 21 of the protocol. These procedures were conducted under anesthesia with isoflurane in oxygen. Test ADIs were generally dosed in a constant 50:1 molar ratio to the Der p 1 content of the allergen challenge mixture. In practice, this resulted in doses of 35−45 μg/kg being delivered to animals, except in the case of compound 38, where the maximum dose used was 12 μg/kg (20 nmol/kg).
All in vivo studies were conducted within the jurisdiction of, and in accordance with, the UK Animals (Scientific Procedures) Act, 1986, in an AAALAC-accredited facility. Groups typically comprised 10 animals randomly assigned to treatment. Treatments were coded prior to formulation so that staff responsible for dosing were unaware of test substance identity.
Pulmonary Leukocyte Accumulation. Animals were killed by sodium pentobarbital overdose 48 h after allergen challenge because this is the optimal sampling point to assess the recruitment of eosinophils (our unpublished observations). The airways were lavaged with 3 × 4 mL aliquots of Hanks' Balanced Salt Solution and the recovered cells pooled and counted automatically (ADVIA; Bayer Healthcare, Diagnostics Division, UK). Differential counts were obtained after the preparation of smears by cytocentrifugation and the staining of methanol-fixed cells with buffered eosin and methylene blue/azur 2 (Speedy-Diff; ClinTech Ltd., Guildford, Surrey UK). Cells were counted by an independent observer using light microscopy under oil immersion (×1000). Statistical analyses of cell data were made by one-way ANOVA and Holm−Sidak test. ■ ASSOCIATED CONTENT * S Supporting Information Synthesis details for intermediates 6a−l, 8a−c, 9a, and 15a−i; synthesis details for leading subsidiary compounds; sequence overlays of Der p1, Cat B, Cat S, and Cat K; methods for PK studies, PPB measurement, stability assays with rat lung cells, duration of protection profiles for 12 selected compounds in rats, protease-counter screening, XRPD, DSC, and log D 7.4 determination. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.