at the heart of the 
 clinical management of asthma lies a paradox : 
 despite efficacious and safe therapies ( e.g. , 2 agonist 
 bronchodilators , inhaled corticosteroids , antileukotrienes , and an 
 anti - ige monoclonal antibody ) , the condition remains poorly controlled 
 and its prevalence continues to increase . 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 ) . second , sensitization to hdm precedes the development of sensitization 
 to allergens from unrelated sources . 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 , 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 . 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 whose sequences are sufficiently identical that targeting 
 them with a single agent is a realistic possibility . 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 . 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 . 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 ) that are known to recruit the 
 necessary effector cells and promote a th2 bias to immune 
 responses . significantly , evidence suggests 
 that some of these innate immune mechanisms are the focus of important 
 genetic predispositions for allergic asthma . 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 . prior 
 to the commencement of our program , the only reported inhibitors of 
 der p 1 were irreversible acyloxymethyl ketone inhibitors ( figure 1 ) . 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 ) . reversible der p 1 inhibitors 
 were derived from a series of irreversible 
 inhibitors by modifying the cysteine binding motif . 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 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 . pyruvamide - motif inhibitors were 
 synthesized from the corresponding -hydroxy amides ( 7 ) by oxidation with the dess the -hydroxy amide intermediates were 
 constructed by two synthetic approaches : either a modified passerini 
 reaction ( 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 . in - solution chemistry 
 used a stepwise process which involved , as a typical first step , the 
 coupling of a p2 amino acid ester ( 11 ) with 
 a boc - protected p3 amino acid ( 10 ) under low - temperature 
 mixed - anhydride coupling conditions to avoid epimerization of the 
 p3 chiral center ( scheme 2 ) . ( i ) ibuococl , n - methylmorpholine , 40 c , thf ; ( ii ) tfa , dcm 
 or 4 n 
 hcl / dioxane ; ( iii ) p4-co2h , tbtu , dipea , dcm ; 
 ( iv ) lioh , thf / h2o . 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 -hydroxy 
 amide ( scheme 3 ) . for route b , a variety of -hydroxy amides ( 7 ) were synthesized by coupling -amino--hydroxy amides 
 ( 15 ) with dipeptide acids ( 6 ) under similar 
 conditions to those previously described ( scheme 3 ) . the -amino--hydroxy amides ( 15 ) were made in seven steps from cbz - protected valine 16 ( scheme 4 ) . ( i ) ibuococl , n - methylmorpholine , 
 40 c , thf ; ( ii ) dess martin 
 periodinane , dcm ; ( iii ) pnc , pyridine , tfa , dcm . ( i ) n - methyl - o - methyl - hydroxyl amine , edc , dipea , dcm ; ( ii ) lah , thf ; 
 ( iii ) nahso3 , nacn , h2o / thf ; ( iv ) 6 n hcl , dioxane , 
 100 c ; ( v ) boc2o , et3n , meoh ; ( vi ) diphosgene , 
 h2np , dcm ; ( vii ) tfa , dcm or 6 n hcl dioxane , thf . 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 ) . 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 p1 subunit 
 is able to form an interaction with the carbonyl of tyr 169 , and the 
 p2 subunit forms a donor acceptor pair with the 
 backbone carbonyl and nh of asp 74 ( figure 2a ) . model of compound 5 bound to der p 1 ( pdb : 2as8 ) and cat b ( pdb : 1gmy ) . ( a ) proposed binding 
 mode of compound 5 ( green ) to der p 1 active site . ( b ) 
 proposed binding mode of compound 5 to cat b active site . ( c , d ) proposed incorporation of dimethyl group ( pink ) as part of the 
 p3 substituent bound to der p 1 and cat b respectively . the bulky p3 residue is anticipated to clash with tyr 75 
 in cat b. 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 
 p1 substituent would hinder nonspecific interactions . increasing 
 the branching at p1 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 p1 substituent resulted in 
 a 1020-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 s3 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 p3 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 potency against 
 cat b with only a modest impact on der p 1 activity . compound 22 also showed resistance to airway macrophages , with no significant 
 degradation observed over 2 h. further modeling suggested that 
 the s2 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 . consistent with these observations , the methyl substituent at p2 generally provided the best balance of potency and selectivity 
 for der p 1 ( 19 vs 24 ) . preliminary investigations 
 into the p1 , p2 , 
 and p3 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 s4 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 . 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 
 well - tolerated 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 . 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 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 pbs7.4 for 2 h ) . the s4 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 n - methyl 
 hydrogen atoms of 32 are able to make favorable interactions 
 with these residues , thereby resulting in a potent der p 1 inhibitor . contains 
 30% of p1 r epimer . single determination measured as 
 described in supporting information . 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 - sonh2 group , as in compound 39 , gave high 
 der p 1 potency and excellent selectivity over cat b. introduction 
 of an -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 . single determination measured as 
 described in supporting information . 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 p3 resulted in compound 48 that was both potent against der p 1 and which showed no 
 significant inhibition of cat b at 2.5 m . alternatively , replacing 
 the n - methyl group with a bulkier iso - propyl group ( 49 ) or introducing a substituent on the 
  position of the glycinamide ( 50 ) , reduced inhibition 
 of cat b without the need to introduce the tert - butyl 
 group at p3 . 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 . 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 . 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 . 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 . 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 d7.4 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 peptidase - dependent 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 . ( a ) plot of psa (  ) versus measured log d7.4 for 
 compounds used to explore the effects 
 of physiochemical properties on in vivo efficacy . symbols denote the 
 anticipated ionization state at ph 7.4 : circle ( neutral ) , square ( positively 
 charged basic center ) , and diamond ( positively charged , quaternary 
 ammonium ) . ( b ) percentage reduction in the number of eosinophils recovered 
 by bal 48 h after challenge of nonsensitized rats with a natural mixture 
 of hdm allergens following a single intratracheal dose of test compound 
 2 h prior to challenge . compounds were dosed at a drug : target molar 
 ratio of 15:1 ( compound 38 ) or 50:1 ( other compounds ) . ( c ) compound 52 exemplifies the endurance of protection 
 ( > 6 h ) by quaternary amines . data are shown as the percentage reduction 
 in bal eosinophil numbers following hdm allergen challenge after administration 
 of a single intratracheal dose of 52 ( 67 nmol / kg ) administered 
 at the stated times prior to hdm allergen challenge . in ( b ) and ( c ) , 
 data are mean values  se , with 10 animals per treatment group . 
 * 0.001 ( 1-way anova ) compared to controls 
 which were not treated with 52 . 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 ) . effect of single dose pretreatment with compound 32 ( 12 
 g / kg ; 18 nmol / kg ) on the recruitment of cells to the 
 airways 48 h following challenge with mixed hdm allergens in sensitized 
 bn rats . ( a ) data for total nucleated cells recovered by bal ; ( b ) corresponding 
 data for eosinophils . p < 0.01 versus 
 vehicle ( veh)-challenged animals sensitized to hdm . * * p < 0.001 versus animals pretreated with vehicle and then challenged 
 with mixed hdm allergens ( 1-way anova ) . 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 , 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 off - targets 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 cmax < 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 . bioavailability 
 based upon comparison 
 with a 1 mg / kg ( 12 mol / kg according to compound ) iv 
 dose . the p4 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 . in vitro enzymatic assays were performed 
 as described in experimental section . compounds were dosed as intratracheal 
 aerosols 6 h prior to challenge of groups of 10 animals with the hdm 
 allergen mixture . dose delivered to each animal was 40 g / kg 
 at a drug : target molar ratio of 50:1 . 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 . 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 . 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 p3 and p. 
 in the case of the p3 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 incorporating a quaternary ammonium group , display superior 
 in vivo efficacy in a rodent hdm allergen challenge model . a number 
 of the compounds have a high - melting crystalline form and show low 
 levels of exposure when dosed orally , two further properties which 
 are desirable in an inhaled drug . epidemiological studies highlight 
 the importance of hdm allergy 
 as both an asthma trigger and a facilitator of allergic sensitization 
 generally . 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 th2-polarized adaptive immunity . 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 . this is supported by 
 our in vivo experiments 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 . the syntheses of key compounds are described below . 
 all commercially 
 available solvents and reagents 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 lc - ms using an analytical 
 c18 column ( phenomenex luna c18 ( 2 ) 150 mm  4.6 mm , 
 5 m ) , using a diode array detector and an a : b gradient starting 
 from 95% a:5%b at a flow rate of 1.5 ml / min , where eluent a was 0.1% 
 formic acid / h2o and eluent b was 0.1% formic acid / mecn 
 or 0.1% formic acid / meoh . mass spectra were obtained using a waters 
 zq4000 single quadrupole or a micromass ultima triple quadrupole mass 
 spectrometer . silica gel ( 60  , 4063 m , fisher ) 
 or cartridges ( biotage snap cartridges kp - sil or isolute si - ii cartridges ) 
 were used for flash chromatography . routine analytical thin layer 
 chromatography was performed on precoated plates ( alugram , silg / uv254 ) . reverse - phase preparative hplc was carried out on a waters zq instrument 
 using mass - directed purification on a preparative c18 column 
 ( phenomenex luna c18 ( 2 ) , 100 mm  21.2 mm , 5 m ) . depending upon the retention time and the degree of separation of 
 the desired product from any impurities an a : b gradient was employed 
 starting from high % a low % b at a flow rate of 20 ml / min . the following 
 combinations of a and b were typically used : a = h2o + 
 0.1% formic acid , b = meoh + 0.1% formic acid , or a = h2o + 0.1% tfa , b = mecn + 0.1% tfa , or a = 10 mm nh4hco3 ( aq ) , b = meoh . all compounds tested were at least 95% pure 
 by lc - ms unless otherwise stated . in all cases pyruvamide inhibitors 
 of der p 1 were synthesized 
 by oxidation of the corresponding -hydroxyamide ( 7 ) using the same general procedure . to a stirred solution of 7 ( 1 equiv ) in anhydrous dcm 
 ( 1 ml/25250 mg of alcohol ) 
 and anhydrous dmf ( 1035% v / v depending upon solubility ) at 
 ambient temperature was added dess 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 nahco3 ( aq ) ( 1 volume ) and na2s2o3 ( aq , 10% w / v ) . the mixture was stirred for 30 min , diluted 
 with ethyl acetate ( 10 volumes ) , and washed with saturated nahco3 ( aq ) ( 2  5 volumes ) , deionized water ( 5 volumes ) , and 
 brine ( 5 volumes ) . the organic layer was subsequently dried over mgso4 , 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 . compound 5 was made using route 
 a : 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 12 h. the resulting mixture 
 was diluted with etoac ( 150 ml ) and washed with saturated nahco3 ( aq ) ( 2  100 ml ) and brine ( 1  100 ml ) . the organic 
 layer was dried over mgso4 , 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 nahco3 ( aq ) ( 3 g in 30 ml ) was added and the 
 mixture stirred for 30 min . the organic layer was separated 
 and washed with saturated nahco3 ( aq ) ( 2  50 ml ) 
 and brine ( 50 ml ) then dried over mgso4 , filtered , and 
 evaporated to yield a pale - yellow oil . trituration with et2o gave 14a as white solid ( 683 mg , 26% ) ; [ m + h ] 438 . h nmr ( ( cd3)2so , 400 
 mhz ) :  9.40 ( 1h , s , c(o)h ) , 8.58 ( 1h , d , j = 8.3 hz , nh ) , 8.33 ( 1h , d , j = 7.3 hz , nh ) , 8.27 
 ( 1h , d , j = 7.1 hz , nh ) , 7.807.75 ( 2h , m , 
 arh ) , 7.547.49 ( 1h , m , arh ) , 7.477.41 ( 2h , m , arh ) , 
 7.407.35 ( 2h , m , arh ) , 7.297.23 ( 2h , m , arh ) , 7.197.13 
 ( 1h , m , arh ) , 4.754.68 ( 1h , m , chc(o ) ) , 4.414.33 ( 1h , 
 m , chc(o ) ) , 4.104.03 ( 1h , m , chc(o ) ) , 3.13 ( 1h , dd , j = 13.9 , 4.0 hz , 1 of ch2ph ) , 2.98 ( 1h , dd , j = 13.9 , 11.1 hz , 1 of ch2ph ) , 1.801.61 ( 1h , m ) , 1.521.44 
 ( 1h , m ) , 1.341.22 ( 7h , m ) , 0.870.82 ( 3h , m ) . to a stirred solution of 14a ( 218 
 mg , 0.5 mmol ) in anhydrous dcm ( 2 ml ) at 0 c 
 was added cyclohexylisonitrile ( 74 l , 0.6 mmol ) and pyridine 
 ( 161 l , 2.0 mmol ) followed by dropwise addition of tfa ( 74 
 l , 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 l , 0.5 mmol ) and tfa ( 37 l , 0.5 mmol ) was added 
 and the reaction mixture stirred for 18 h. the mixture was diluted 
 with dcm ( 10 ml ) , washed with saturated nahco3 ( aq ) ( 2 
  15 ml ) and brine ( 15 ml ) , and the organic layer then dried 
 over mgso4 , filtered and evaporated . the crude material 
 was oxidized following general procedure i to give compound 5 ( 76 mg , 27% ) ; [ m + h ] 563 . h nmr 
 ( ( cd3)2so , 400 mhz ) :  8.58 ( 1h , d j = 8.6 hz , nh ) , 8.55 ( 1h , d , j = 8.4 hz , 
 nh ) , 8.28 ( 1h , d , j = 7.6 hz , nh ) , 8.22 ( 1h , d , j = 7.1 hz , nh ) , 7.807.74 ( 2h , m , arh ) , 7.557.48 
 ( 1h , m , arh ) , 7.477.41 ( 2h , m , arh ) , 7.407.35 ( 2h , 
 m , arh ) , 7.307.23 ( 2h , m , arh ) , 7.197.14 ( 1h , m , arh ) , 
 5.004.94 ( 1h , m , chco ) , 4.764.66 ( 1h , m , chco ) , 4.424.34 
 ( 1h , m , chco ) , 3.603.50 ( 1h , m , nhch(cyclohexyl ) ) , 3.12 ( 1h , dd , j = 13.8 and 3.4 hz , 
 1 of ch2ph ) , 2.96 ( 1h , dd , j = 13.8 and 11.2 hz , 1 of ch2ph ) , 1.801.40 ( 7h , m ) , 1.391.00 ( 12h , m ) , 
 0.82 ( 3h , t , j = 6.9 hz , ch3 ) . to cbz - val - oh ( 16 ) ( 50.0 g , 199 mmol ) , n , o - dimethylhydroxylamine hydrochloride ( 38.8 g , 398 mmol ) and edchcl 
 ( 47.7 g , 249 mmol ) in dcm ( 500 ml ) was added dipea ( 87 ml , 497 mmol ) , 
 and the reaction mixture stirred at ambient temperature for 20 h , 
 after which time it was diluted with dcm ( 200 ml ) , washed with 1 m 
 hcl ( aq ) ( 3  200 ml ) , 1 m naoh ( aq ) ( 200 ml ) , saturated nahco3 ( aq ) ( 200 ml ) , and brine ( 300 ml ) . the organic layer was 
 dried over mgso4 , filtered , and the solvent removed under 
 vacuum to give the desired compound as a colorless oil ( 51.2 g ; ms 
 [ m + h ] 295 ) . a portion of this ( 33 g , 112 mmol ) 
 was dissolved in anhydrous thf ( 300 ml ) at 30 to 40 
 c , and lialh4 ( 4.3 g , 113 mmol ) was added portionwise 
 over a period of 45 min . the mixture was warmed to 0 c and stirred 
 for 2 h. the reaction mixture was quenched at 0 c with 1 m khso4 ( 330 ml ) then 10% w / v rochelle s salt ( aq ) ( 330 ml ) 
 was added and the mixture stirred for 20 min then extracted with etoac 
 ( 2  700 ml ) . the combined organic phases were washed with 10% 
 w / v rochelle s salt ( aq ) ( 330 ml ) and brine ( 450 ml ) , dried 
 over mgso4 , filtered , and concentrated under vacuum to 
 obtain a clear oil ( 26.3 g , major peak ; [ m + h ] 236 ) . 
 the oil was dissolved in meoh ( 150 ml ) and cooled to 0 c . a 
 solution of nahso3 ( 11.9 g , 114 mmol ) in h2o 
 ( 230 ml ) was added and the mixture stirred at 0 c for 2.5 h. 
 the resulting mixture was added to a solution of nacn ( 8.5 g , 174 
 mmol ) in h2o ( 150 ml ) and etoac ( 450 ml ) also at 0 c . 
 after 1 h , this was allowed to warm to ambient temperature and was 
 stirred for 20 h. the etoac layer was separated and the aqueous layer 
 extracted with etoac ( 2  500 ml ) . the combined organic extracts 
 were washed with brine ( 400 ml ) , dried over mgso4 , filtered , 
 and concentrated to give compound 17 ( 29.7 g , crude , 
  1:1 mixture of diastereomers ) as a clear gummy liquid ; [ m 
 + h2o ] 280 . the mixture was used without further 
 purification in the synthesis of 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 ) . the residue was washed with et2o ( 2  50 ml ) to give a gummy solid . the crude mixture was dissolved 
 in methanol ( 100 ml ) , and et3n ( 9.0 ml , 64 mmol ) was added . di - tert - butyl 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 
 mgso4 , filtered , and concentrated under vacuum to give 
 a white , waxy solid . this was further purified on a biotage isolute 
 ( ist)-nh2 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 ( 1.5 g , 27% from compound 16 ) . 18a : [ m  h ] 246 ; h nmr ( ( cd3)2so , 400 mhz ) :  12.4 
 ( 1h , br s ) , 6.46 ( 1h , d , j = 10.0 hz ) , 5.38 ( 1h , 
 br s ) , 3.83 ( 1h , d , j = 6.8 hz ) , 3.653.59 
 ( 1h , m ) , 1.991.91 ( 1h , m ) , 1.36 ( 9h , s ) , 0.810.76 
 ( 6h , m ) . 18b : ms [ m  h ] 246 ; h nmr ( ( cd3)2so , 400 mhz ) :  12.44 
 ( 1h , br s ) , 6.21 ( 1h , d , j = 10.0 hz ) , 4.95 ( 1h , 
 br s ) , 4.11 ( 1h , d , j = 1.6 hz ) , 3.533.47 
 ( 1h , m ) , 1.74 ( 1h , m ) , 1.35 ( 9h , s ) , 0.910.83 ( 6h , m ) . the 
 single diastereomer 18a could be isolated by dissolving 
 the mixture of diastereomers in chcl3 and adding n - pentane to afford isomer 18a as a white precipitate 
 that could be collected by filtration . compound 22 was prepared as 
 a white solid from compound 6b ( 17 mg , 10% ) using a similar 
 procedure to that of compound 5 , with the exception that 
 the aldehyde intermediate was purified by reverse - phase preparative 
 hplc using a h2o + 0.1% tfa / mecn + 0.1% tfa gradient at 
 50 c followed by lyophilization to remove the solvent . h nmr ( ( cd3)2so , 400 mhz ) :  8.53 ( 1h , d , j = 8.1 hz , nh ) , 
 8.19 ( 1h , d , j = 7.1 hz , nh ) , 7.98 ( 1h , d , j = 7.8 hz , nh ) , 7.88 ( 1h , d , j = 9.2 hz , 
 nh ) , 7.867.82 ( 2h , m , arh ) , 7.587.52 ( 1h , m , arh ) , 
 7.507.44 ( 2h , m , arh ) , 5.04 ( 1h , dd , j = 
 7.8 , 5.3 hz , chco ) , 4.50 ( 1h , d , j = 9.2 hz , chco ) , 
 4.494.41 ( 1h , m , chco ) , 3.613.50 ( 1h , m , nhch(cyclohexyl ) ) , 2.222.12 ( 1h , m , chme2 ) , 1.731.63 ( 4h , m ) , 1.611.52 
 ( 1h , m ) , 1.341.19 ( 7h , m ) , 1.141.02 ( 1h , m ) , 1.00 
 ( 9h , s , t - bu ) 0.89 ( 3h , d , j = 6.8 
 hz , ch3 ) , 0.81 ( 3h , d , j = 6.8 hz , ch3 ) . compound 27 was prepared as 
 a white solid from compound 6c ( 32 mg , 62% ) using a similar 
 procedure to that of 22 ; [ m + h ] 574 . h nmr ( ( cd3)2so , 400 mhz ) :  9.30 
 ( 1h , t , j = 6.3 hz , nh ) , 8.96 ( 1h , d , j = 4.3 hz , arh ) , 8.72 ( 1h , d , j = 9.0 hz , nh ) , 8.21 
 ( 1h , d , j = 6.8 hz , nh ) , 8.12 ( 1h , d , j = 7.8 hz , nh ) , 8.08 ( 1h , d , j = 7.8 hz , arh ) , 8.05 
 ( 1h , dd , j = 8.3 hz , 0.8 hz , arh ) , 7.847.78 
 ( 1h , m , arh ) , 7.687.62 ( 1h , m , arh ) , 7.48 ( 1h , d , j = 4.3 hz , arh ) , 7.357.29 ( 2h , m , arh ) , 7.287.22 
 ( 3h , m , arh ) , 5.06 ( 1h , dd , j = 7.8 , 5.4 hz , chco ) , 
 4.47 ( 1h , d , j = 9.0 hz , chco ) , 4.544.45 
 ( 1h , m , chco ) , 4.36 ( 1h , dd , j = 14.7 , 6.3 hz , 1 
 of ch2ph ) , 4.30 ( 1h , dd , j = 14.7 , 6.3 hz , 1 of ch2ph ) , 2.252.16 ( 1h , m , chme2 ) , 1.24 ( 3h , d , j = 7.1 hz , ch3 ) , 1.03 
 ( 9h , s , t - bu ) , 0.91 ( 3h , d , j = 
 6.8 hz , ch3 ) , 0.84 ( 3h , d , j = 6.8 hz , 
 ch3 ) . to a solution of 6d ( 350 mg , 1.1 mmol ) in anhydrous thf ( 10 ml ) at 40 
 c was added n - methylmorpholine ( 240 l , 
 2.1 mmol ) and i - buococl ( 180 l , 1.2 mmol ) 
 and the reaction mixture stirred for 1 h. a solution of compound 15a ( 420 mg , 1.2 mmol ) in dry thf ( 5 ml ) and n - methylmorpholine ( 180 l , 1.6 mmol ) was then added dropwise 
 and the reaction mixture stirred at 40 c for 6 h before 
 warming to room temperature over 12 h. the solvent was evaporated 
 in vacuo and the crude residue dissolved in etoac ( 50 ml ) then washed 
 with saturated nahco3 ( aq ) ( 2  50 ml ) and brine ( 50 
 ml ) . the organic layer was dried over mgso4 , filtered , 
 and evaporated to give a white solid . oxidation using general procedure 
 i gave compound 28 as a white solid ( 277 mg , 47% ) ; [ m 
 + h ] 535 . h nmr ( ( cd3)2so , 400 mhz ) :  9.29 ( 1h , t , j = 6.5 hz , nh ) , 
 8.52 ( 1h , d , j = 6.8 hz , nh ) , 7.99 ( 1h , d , j = 7.7 hz , nh ) , 7.71 ( 1h , d , j = 7.6 hz , 
 arh ) , 7.647.56 ( 2h , m , arh ) , 7.527.46 ( 1h , m , arh ) , 
 7.357.29 ( 2h , m , arh ) , 7.287.22 ( 3h , m , arh ) , 5.05 
 ( 1h , dd , j = 7.7 , 5.5 hz , chco ) , 5.00 ( 1h , d , j = 18.4 hz , 1 from dihydro - isoindolone ring ) , 4.84 ( 1h , 
 s , chco ) , 4.76 ( 1h , d , j = 18.4 hz , 1 from dihydro - isoindolone 
 ring ) , 4.404.27 ( 3h , m , ch2ph and chco ) , 2.262.14 ( 1h , m , chme2 ) , 1.16 ( 3h , d , j = 7.1 hz , ch3 ) , 1.05 ( 9h , s , t - bu ) , 0.92 ( 3h , d , j = 6.8 hz , ch3 ) , 0.83 ( 3h , d , j = 6.8 
 hz , ch3 ) . compound 29 was prepared as 
 a white solid from compound 6e ( 59 mg , 5% ) using a similar 
 procedure to that of 22 ; [ m + h ] 516 . h nmr ( ( cd3)2so , 400 mhz ) :  8.758.72 
 ( 2h , m , arh ) , 8.57 ( 1h , d , j = 8.3 hz , nh ) , 8.38 
 ( 1h , d , j = 9.4 hz , nh ) , 8.24 ( 1h , d , j = 7.1 hz ) , 8.02 ( 1h , d , j = 7.8 hz , nh ) , 7.817.78 
 ( 2h , m , arh ) , 5.04 ( 1h , dd , j = 7.8 and 5.3 hz , chco ) , 
 4.52 ( 1h , d , j = 9.4 hz , chco ) , 4.474.40 
 ( 1h , m , chco ) , 3.603.45 ( 1h , m , nhch(cyclohexyl ) , 2.222.12 ( 1h , m , chme2 ) , 1.721.64 ( 4h , m ) , 1.611.54 ( 1h , m ) , 1.341.17 
 ( 7h , m ) , 1.121.04 ( 1h , m ) , 1.00 ( 9h , s , t - bu ) , 0.89 ( 3h , d , j = 6.8 hz , ch3 ) , 
 0.80 ( 3h , d , j = 6.8 hz , ch3 ) . compound 32 was prepared 
 in its formate salt form from compounds 6f and 15a ( 19 mg , 20% ) using a similar procedure to that of compound 28 , yielding a white solid ; [ m ] 558 . h nmr ( ( cd3)2so , 400 mhz ) :  9.31 ( 1h , 
 t , j = 6.3 hz , nh ) , 8.42 ( 1h , s hc(o)o from formate ) , 8.25 ( 1h , d , j = 6.8 hz , nh ) , 8.13 ( 1h , d , j = 9.8 hz ) , 8.09 ( 1h , j = 7.8 hz , nh ) , 7.357.28 ( 2h , m , arh ) , 7.287.21 
 ( 3h , m , arh ) , 5.05 ( 1h , dd , j = 7.8 , 5.3 hz , chco ) , 
 4.414.23 ( 4h , m , ch2ph and 
 2  chco ) , 3.403.20 ( 4h , m , me2n(ch2)2 ) , 3.12 
 ( 3h , s , nch3 ) , 3.02 ( 3h , s , nch3 ) , 2.602.55 ( 1h , m , piperidine ring adjacent to c(o)nh ) , 
 2.242.14 ( 4h , m , piperidine ring ) , 1.18 ( 3h , d , j = 7.0 hz , ch3 ) , 0.930.87 ( 12h , m , t - bu and ch3 ) , 0.82 ( 3h , d , j = 6.8 hz , 
 ch3 ) . compound 38 was prepared from 
 compounds 6b and 15a , as a white solid ( 170 
 mg , 50% ) using a similar procedure to that of 28 ; [ m 
 + h ] 523 . h nmr ( ( cd3)2so , 400 mhz ) :  9.24 ( 1h , t , j = 6.3 hz , nh ) , 
 8.18 ( 1h , d , j = 7.1 hz , nh ) , 8.13 ( 1h , d , j = 7.8 hz , nh ) , 7.88 ( 1h , d , j = 9.5 hz , 
 nh ) , 7.867.82 ( 2h , m , arh ) , 7.577.52 ( 1h , m , arh ) , 
 7.507.45 ( 2h , m , arh ) , 7.357.29 ( 2h , m , arh ) , 7.277.22 
 ( 3h , m , arh ) , 5.03 ( 1h , dd , j = 7.8 , 5.6 hz , chco ) , 
 4.50 ( 1h , d , j = 9.5 hz , chco ) , 4.494.40 
 ( 1h , m , chco ) , 4.384.26 ( 2h , m , ch2ph ) , 2.252.15 ( 1h , m , chme2 ) , 1.20 ( 3h , d , j = 7.1 hz , ch3 ) , 1.01 ( 9h , s , t - bu ) , 0.89 ( 3h , d , j = 6.8 hz , ch3 ) , 0.82 ( 3h , d , j = 6.8 
 hz , ch3 ) . compound 45 was prepared as 
 its tfa salt and was isolated as a white solid from compound 6a ( 16 mg , 14% ) using a similar procedure to that of 22 ; [ m + h ] 607 . h nmr ( ( cd3)2so , 400 mhz ) :  9.80 ( 1h , s , menh ) , 8.71 ( 1h , t , j = 5.7 hz , nh ) , 8.58 
 ( 1h , d , j = 8.3 hz , nh ) , 8.32 ( 1h , d , j = 7.1 hz , nh ) , 8.09 ( 1h , d , j = 7.6 hz , nh ) , 7.797.75 
 ( 2h , m , arh ) , 7.547.49 ( 1h , m , arh ) , 7.477.41 ( 2h , 
 m , arh ) , 7.407.36 ( 2h , m , arh ) , 7.297.23 ( 2h , m , arh ) , 
 7.197.13 ( 1h , m , arh ) , 5.08 ( 1h , dd , j = 
 7.6 , 5.1 hz , chco ) , 4.754.68 ( 1h , m , chco ) , 4.504.30 
 ( 2h , m , chco + 1 other ) , 3.152.90 ( 3h , m ) , 3.503.30 
 ( 4h , m partially obscured by hod residual ) , 3.152.90 ( 4h , 
 m , ch2ph + 2 others ) , 2.82 ( 3h , 
 s , nch3 ) , 2.262.16 ( 1h , m , chme2 ) , 1.26 ( 3h , d , j = 7.1 hz , ch3 ) , 0.91 ( 3h , d , j = 6.8 hz , ch3 ) , 0.83 ( 3h , d , j = 6.8 hz , ch3 ) . compound 47 was prepared as 
 a white solid from compound 6a ( 8 mg , 5% ) using a similar 
 procedure to that of 22 ; [ m + h ] 594 . h nmr ( ( cd3)2so , 400 mhz ) :  8.62 
 ( 1h , t , j = 5.8 hz , nh ) , 8.58 ( 1h , d , j = 8.3 hz , nh ) , 8.32 ( 1h , d , 7.3 hz , nh ) , 8.08 ( 1h , d , j = 7.8 hz , nh ) , 7.797.75 ( 2h , m , arh ) , 7.547.48 ( 1h , 
 m , arh ) , 7.467.41 ( 2h , m , arh ) , 7.407.36 ( 2h , m , arh ) , 
 7.297.23 ( 2h , m , arh ) , 7.187.13 ( 1h , m , arh ) , 5.06 
 ( 1h , dd , j = 7.8 , 5.3 hz , chco ) , 4.754.68 
 ( 1h , m , chco ) , 4.504.40 ( 1h , m , chco ) , 4.04 ( 1h , dd , j = 16.7 , 5.8 , 1 of ch2co - morpholine ) , 3.98 ( 1h , dd , j = 16.7 , 5.8 , 1 of 
 ch2co - morpholine ) , 3.603.52 
 ( 4h , m , morpholine ) , 3.453.39 ( 4h , m , morpholine ) , 3.12 ( 1h , 
 dd , j = 13.6 , 3.4 hz , 1 of ch2ph ) , 2.97 ( 1h , dd , j = 13.6 , 11.3 hz , 
 1 of ch2ph ) , 2.272.17 ( 1h , 
 m , chme2 ) , 1.26 ( 3h , d , j = 7.1 hz , ch3 ) , 0.90 ( 3h , d , j = 6.8 hz , ch3 ) , 0.83 ( 3h , d , j = 7.1 
 hz , ch3 ) . compound 52 was prepared 
 as its formate salt and isolated as a white solid ( 40 mg , 37% ) from 
 compounds 6a and 15b using a similar procedure 
 to that of 28 ; [ m ] 621 . h nmr 
 ( ( cd3)2so , 400 mhz ) :  8.78 ( 1h , t , j = 5.7 hz , nh ) , 8.69 ( 1h , d , j = 8.6 hz , 
 nh ) , 8.49 ( 1h , d , j = 7.3 hz , nh ) , 8.47 ( 1h , s , hc(o)o ) , 8.15 ( 1h , d , j = 7.6 hz , nh ) , 7.827.76 
 ( 2h , m , arh ) , 7.5347.48 ( 1h , m , arh ) , 7.467.41 ( 2h , 
 m , arh ) , 7.407.35 ( 2h , m , arh ) , 7.297.23 ( 2h , m , arh ) , 
 7.187.13 ( 1h , m , arh ) , 5.07 ( 1h , dd , j = 
 7.6 , 5.2 hz , chco ) , 4.764.68 ( 1h , m , chco ) , 4.494.39 
 ( 1h , m , chco ) , 4.12 ( 1h , dd , j = 16.9 , 5.7 hz , 1 
 of ch2co ) , 4.07 ( 1h , dd , j = 16.9 , 5.7 
 hz , 1 of ch2co ) , 3.863.74 ( 4h , m , piperazine ring ) , 
 3.443.34 ( 2h , m , piperazine ring ) , 3.203.09 ( 8h , m , 
 including n(ch3)2 ) , 3.042.92 ( 2h , m ) , 2.262.16 ( 1h , m , chme2 ) , 1.27 ( 3h , d , j = 7.1 
 hz , ch3 ) , 0.91 ( 3h , d , j = 6.8 hz , ch3 ) , 0.83 ( 3h , d , j = 6.8 hz , ch3 ) . compound 53 was prepared as 
 its tfa salt and was isolated as a white solid ( 30 mg , 18% ) from compounds 6 g and 15c using a similar procedure to that 
 of 28 ; [ m + h ] 657 . h nmr ( ( cd3)2so , 400 mhz ) :  9.80 ( 1h , br s , menh ) , 8.768.69 ( 2h , m ) , 8.29 
 ( 1h , d , j = 7.1 hz ) , 8.17 ( 1h , d , j = 7.6 hz ) , 7.97 ( 1h , d , j = 8.3 hz ) , 7.93 ( 1h , 
 d , j = 8.1 hz ) , 7.74 ( 1h , d , j = 
 8.1 hz ) , 7.547.47 ( 2h , m , arh ) , 7.447.36 ( 4h , m , arh ) , 
 7.357.29 ( 2h , m , arh ) , 7.297.22 ( 1h , m , arh ) , 5.09 
 ( 1h , dd , j = 7.6 , 5.0 hz , chco ) , 4.904.82 
 ( 1h , m , chco ) , 4.574.47 ( 1h , m , chco ) , 4.464.30 ( 1h , 
 m ) , 4.223.92 ( 3h , m ) , 3.302.78 ( 7h , m ) , 2.79 ( 3h , 
 s , nch3 ) , 2.272.17 ( 1h , m , chme2 ) , 1.30 ( 3h , d , j = 7.1 hz , ch3 ) , 0.91 ( 3h , d , j = 6.8 hz , ch3 ) , 0.84 ( 3h , d , j = 6.8 hz , ch3 ) also 
 1  h under hod 3.403.20 ppm . automated 
 screening of inhibitory activity toward group 1 hdm peptidase allergens 
 was performed as described elsewhere 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 m . compounds showing optimizable activity were cherry - picked 
 for multiple ( up to six ) rounds of rescreening to confirm activity 
 and stability and to determine ic50 values over a range 
 of concentrations that were compound - directed by initial screening . reaction mixtures comprised 10 l of human liver 
 cat b ( 0.5 nm final concentration , merck chemicals ltd . uk ) preactivated 
 by 2.5 mm 1,4-dithioerythritol ( dte , sigma - aldrich ) at 37 c 
 for 10 min , 70 l of reaction buffer ( 0.1 m naac - hac , ph 4.5 , 
 0.2 m nacl ) , and 10 l of 22.5 mm dte ( 2.5 mm final concentrations ) . reactions were initiated by adding 10 l of 59 m substrate 
 abz - gly - ile - val - arg - ala - lys - dnp - oh ( merck chemicals ltd . , uk ) dissolved 
 in reaction buffer . progress curves were followed at 30 c by 
 detection of fluorescence at 320/420 nm ( excitation / emission ) . to test inhibitory potential against cat s , reaction mixtures comprised 
 70 l of reaction buffer ( 0.1 m sodium phosphate/2 mm edta , 
 ph 7.4 ) , 10 l of recombinant human cat s ( 25 nm in reaction 
 buffer and 10 l dtt ( 2 mm final concentration ) . reactions were 
 initiated by addition of 10 l of substrate ( z - phe - arg - amc , 
 20 m 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 l , the inhibitor added as 
 a 10 l 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 . brown norway strain rats ( male , 
 300350 g at time of allergen challenge , 1216 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 g der p 1 of known catalytic 
 activity , as previous studies indicated that this yielded satisfactory 
 adjuvantless responses . negative control groups comprised nave , 
 unsensitized animals or those sham - sensitized with vehicle solution . test adis were delivered as intratracheal aerosols of 2530 
 m 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 . 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 3545 g / kg 
 being delivered to animals , except in the case of compound 38 , where the maximum dose used was 12 g / 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 . treatments were coded prior 
 to formulation so that staff responsible for dosing were unaware of 
 test substance identity . 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 . , cells were counted by an independent 
 observer using light microscopy under oil immersion ( 1000 ) .
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 .