Freezing the Bioactive Conformation to Boost Potency: The Identification of BAY 85-8501, a Selective and Potent Inhibitor of Human Neutrophil Elastase for Pulmonary Diseases

Human neutrophil elastase (HNE) is a key protease for matrix degradation. High HNE activity is observed in inflammatory diseases. Accordingly, HNE is a potential target for the treatment of pulmonary diseases such as chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchiectasis (BE), and pulmonary hypertension (PH). HNE inhibitors should reestablish the protease–anti-protease balance. By means of medicinal chemistry a novel dihydropyrimidinone lead-structure class was identified. Further chemical optimization yielded orally active compounds with favorable pharmacokinetics such as the chemical probe BAY-678. While maintaining outstanding target selectivity, picomolar potency was achieved by locking the bioactive conformation of these inhibitors with a strategically positioned methyl sulfone substituent. An induced-fit binding mode allowed tight interactions with the S2 and S1 pockets of HNE. BAY 85-8501 ((4S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-3,6-dimethyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile) was shown to be efficacious in a rodent animal model related to ALI. BAY 85-8501 is currently being tested in clinical studies for the treatment of pulmonary diseases.


Introduction
Humann eutrophil elastase (HNE;E C3.4.21.37) is am ember of the chymotrypsin-like family of serine proteases and is stored in the azurophil granules in the neutrophil cytoplasm. This highly active protease is able to break down mechanically important structures of the body's own cellular matrix (e.g.,p roteins such as elastin and collagen), as well as proteinsf oreign to the body (e.g.,o uter cell wall proteins of Gram-negative bacteria). Furthermore, the enzyme cleaves av ariety of endogenous ande xogenous proteins, tuning their biological activity such as activation of other bioactive proteases (e.g.,m atrix metalloproteinases, MMPs), liberation of growth factors, shedding of cell-surface-bound receptors, and degradation of endogenousp roteinase inhibitors (e.g.,t issuei nhibitors of metalloproteases,T IMPs) or exogenous virulence factors. [1] Thus, Humann eutrophil elastase (HNE) is ak ey protease for matrix degradation. High HNE activity is observed in inflammatory diseases. Accordingly,H NE is ap otential target for the treatment of pulmonary diseases such as chronic obstructive pulmonary disease (COPD), acutel ungi njury (ALI), acute respiratory distress syndrome (ARDS), bronchiectasis (BE), and pulmonary hypertension (PH). HNEi nhibitors should reestablisht he protease-anti-protease balance. By meanso fm edicinal chemistry an ovel dihydropyrimidinone lead-structure class was identified. Further chemical optimization yieldedo rally active compounds with favorable pharmacokinetics such as the chemical probe BAY-678. While maintaining outstanding target selectivity,p icomolar potencyw as achieved by locking the bioactive conformation of these inhibitors with as trategically positioned methyl sulfone substituent. An induced-fit binding mode allowed tight interactions with the S2 and S1 pockets of HNE. BAY85-8501 ((4S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-3,6-dimethyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile) was shown to be efficacious in ar odent animal model relatedt oA LI. BAY85-8501 is currently being tested in clinical studies fort he treatment of pulmonary diseases.
human neutrophil elastase plays ap ivotalr ole in tissue remodeling processes, as well as in the onset of inflammation and in host defense (innate immuner esponse).
The activity of the versatile proteaseH NE is tightly controlled by:1 )channeling the potentially dangerous protease to specialized compartments (e.g.,s torage granula and phagolysosomes), and 2) the presence of extracellular neutralizing endogenouss erine protease inhibitors (SERPINs), for example, a-1 antitrypsin (AAT, also known as a-PI) and elafin, which maintain the crucial balance of the protease and itsa nti-proteases. [2][3][4] An imbalance in elastase activity might contribute to the onset and progression of many inflammatory diseases ( Figure 1) with an impact on organ tissue integrity,e specially in cardiopulmonary diseases, such as chronic obstructive pulmonaryd isease (COPD), bronchiectasis (BE), pulmonary arterial hypertension (PAH), and pulmonary fibrosis. Elastase knock-out in rodents or anti-protease transgenic animals reveal as ignificant protection or resistance against experimental lung emphysema, [5] pulmonary hypertension (PH), [6] pulmonary fibrosis, [7] and myocarditis. [8] Individualsw ith antitrypsin deficiency (AATD) reveal dramatically lower levelso fA AT and have an increasedr isk of suffering from lung emphysema. [9] Notably,e lastase knock-out mice are vulnerable to infection with Gramnegative bacteria. [10] So far,various HNE inhibitors have been described; [12,13] however,o nly af ew chemical entitiesh ave hada no verall profile suitable for clinical testing. The first potent elastase inhibitors to reach the clinic were biologicals such as elafin [11] (Figure 2). The first small-molecule inhibitors were electrophilicc ompounds including serine acylators such as sivelestat (1) [14][15][16] or transition-state mimetics such as freselestat (2). [17] Recently,A s-traZeneca reported phase II studies with the reversible HNE in-hibitor AZD 9668 (3)i np atients with pulmonary diseases:H ere as mall four-week treatment study with 56 cystic fibrosis patients revealed modulation of some secondary endpoint biomarkers( e.g.,u rinary desmosine concentration), but did not show as ignificant improvement in primary endpoints (clinical outcomes, e.g.,l ung function FEV 1 or quality of life). [18] Af urther small four-week treatment signal-searching study with 38 BE patients showedapromising improvement in lung function (FEV 1 )a nd trends for decreases in sputum inflammatorym arkers. [19] For both indications, longera nd larger studies would be neededt oc onfirmt he initial findings. In two larger 12-week treatment studies with nearly 1500 COPD patients in total, no clinicalb enefit and no effecto nb iomarkers of inflammation or tissue degradation could be demonstrated. This might have been due to the rather short treatment period and the heterogeneityo ft his disease (a post-hoc analysisr evealed an improvement in lung functioni nasubgroup of patients with ac hronic bronchitis phenotype). [20,21] In general, combining potency with selectivity is al arge hurdle fors mall-molecule serine protease inhibitors. Herein we report the discovery of an ew,h ighly potent and selective neutrophil elastase inhibitor,B AY 85-8501,w ith an unprecedented  a) Biologicals such as elafin [11] werethe first class of HNE-inhibiting drugs. These compoundsare only suitable for i.v.application. b) As asecond prominent class, covalent drugs thatacylateserine in the active site of the enzyme,s uch as sivelestat(1), or transition-state mimetics, such as freselestat (2), were described. At remendouse ffort was required to find the first reversible and selective small-molecule drugs as at hird class. Ap rominent example is alvelestat (3).

Results and Discussion
In the search for better HNE inhibitors, we performed ah ighthroughput screen (HTS) of our small-molecule compound library.F rom the hit list, we identified hexahydroquinoline 4 as the most promising and structurally unique starting point for exploratory chemistry (Table 1). The racemic HTS hit 4 displayed moderate in vitro potency (IC 50 :0 .9 mm). Upon separation of the enantiomers, stereospecific activity became evident as the R enantiomer 5 proved to be fivefold more potent (IC 50 : 0.2 mm)t han the nearly inactive S enantiomer 6 (7 mm). Due to high lipophilicity and am olecular weight > 500 Da, the lipophilic binding efficiency [24] (LipE = 2.5) of the screening hit was poor (Table 1). Unfortunately,b othh exahydroquinolines 5 and 6 showedu ndesirablei nhibition of human CYP2C9 and/or CYP 3A4.
To decrease the molecular weight and ring count, the corresponding ring-opened dihydropyridine analogues 7-9 were prepared (Table 2). Fortunately,t he ring-opened analogue 7 was fivefold more potent than our screening hit 4.T he poten- [b] Clog D (pH 7.5) was calculated by using ah ighlypredictive method developed at Bayer,b ased on data points of experimentally determined log D values of internal pharmaceutical compounds and the Simulations Plus pK a predictor. [23] [c]Calculated as LipE = pIC 50 Àlog D. [24] [d] The capacity of test compounds to inhibit human CYP 2C9 and CYP 3A4 was investigated with pooled human liver microsomes as enzyme source and selective standard substrates (SupportingI nformation); IC 50 valuesw ere derived from enzyme activity data (pH 7.4) in the presence/absence of various compound concentrations and diclofenac/midazolam as CYP 2C9/CYP 3A4 substrate.  www.chemmedchem.org tially unfavorable nitro group of 7 was subsequently converted into at rifluoromethyl group (in 8)w ithoutl oss in potency.A s as econd step towarda ne ven more drug-like molecule, we exchanged the northern bromo substituent for other,p otentially more stable, electron-withdrawing groups. The linear cyano substituent of 9 enabled a1 0-foldp otencyi ncrease coupled with as ignificant improvement in the lipophilic binding efficiency (LipE = 4.0, Clog D = 3.7). Unfortunately,a ll 2-aminodihydropyridine-type compounds 7-9 revealed inhibitory potencyt owardC YP 2C9 and/or CYP 3A4.
To further explore the central ring system, dihydro-2-pyridones 10-13 were pursued next ( Table 3). As hift from nitrogen to oxygen at C2 was indeed possible, leading to 10 with an additional stereocenter at C3, which was unfortunately inclinedt oe pimerize. Omitting the ester functionality at C3 streamlined the system to only one stereocenter at C4 (compounds [11][12][13]. In this series, nearly unchanged target potency and overall less inhibitory potencytowardC YP 3A4 were observed. A5 -cyano substituent yieldedt he most potent compound 13.H owever,s ynthetic accessibility and stereochemical integrity were potential limitations of this pyridone class. To tackle these issues, we capitalized on the highS AR flexibility at C3 that allowed another major structural transformation. Introduction of ar ing nitrogen at position 3l ed to 1,4-dihydropyrimidinones, which were especiallyp otent against HNE (Table 4). Enantiopurec ompounds of this type were readily availableb yB iginelli chemistry [25] coupled with chiral chromatography (Supporting Information). Impressive flexibility was observed with respectt ol igand polarity in the equatorial sphere at N3. Either potentially anionic or cationic compounds, such as carboxylic acids 16 and 19,oramines 21 and 24,inhibited HNE with good to excellent potency. Furthermore, an overall decreased inhibitory potency of the 1,4-dihydropyrimidinones against CYP isoforms turned out to be as ignificant improvement. For example, up to ac oncentration of 50 mm, amide 23 elicited no observable inhibition toward CYP 2C9 and CYP 3A4. Due to its overall balanced profile with respect to polarity and potency, BAY-678 (20)w as selected for in-depth in vitro and in vivo characterization as ac hemical probe candidate for HNE (see below).
Apparently,a ll compounds from the 1,4-dihydropyrimidinones series 14-24 showeds ignificantly lower potency against rat neutrophil elastase (RNE,T able 4). Obtaining compounds with decent potencyn ot only against HNE but against neutrophil elastase in rodents as well seemed more desirable foru pcomingi nv ivo studies with rats and mice. For this reasonw e continued our efforts to further optimizep otency versusH NE, hoping that this would ultimately lead to compounds with im-provedRNE inhibition as well.

X-ray crystallographic investigationofb ound ligands
Ligand-protein co-crystallization experiments and modeling were appliedt od esign better HNEb inders within our 1,4-dihydropyrimidinone series:X -ray crystallography showedt he anticipated folding structure forH NE, typical for chymotrypsinlike serine proteases.U pon binding, the equatorial C2 carbonyl moiety of ligand 19 formed as trong hydrogen bond to Val216 [26] (Figure 3a). More significantly,b inding of the inhibitor 19 was driven by its shape complementarity with the binding subsites of HNE. Thec lamp-like ligand 19 fits perfectly into the S1 and S2 subsites of HNE, with both phenyl moieties perpendicularly directed away from the central core ( Figure 3b). In particular,t he hydrophobic S1 subsite was fully occupied by the southern meta-(trifluoromethyl)phenylm oiety of ligand 19.
In contrast, the S2 subsite found in the apo structure of HNE was at best as hallow groove, clearly not providing sufficient room for binding of the northern para-cyanophenyl moiety; however,i nt he ligand-bound structure, Leu99B had been moved toward the bulk solvent, thus expandingt he lipophilic S2 to al arger subsite, ideally suited to accommodate the large northern cyanophenyl residue (Figure 3c). In our complete series of co-crystallization structures we observed an inducedfit binding mode. [27] All bound inhibitors from the dihydropyrimidinone series were characterized by rotational dihedral angles of the northern cyanophenyl moiety of~908-1358 ( Figure 4) with ap reference of dihedral angles of~1108 (mean: 111.68).

Strategy to tune conformational space
Locking the dihedral angle of the northern phenyls ubstituent into the preferred bioactive conformation (Figure 4) wase nvisaged as ad esign strategy to improveb inding efficiency.A s ab ase case, we assessed conformational freedom of the N3and C2'-unsubstituted system 22 with relaxeds can,d ensityfunctional calculations. [30] Rotation appeared to be nearly unhindered, with ab arrier of~10 kJ mol À1 and al owest-energy conformation at a( suboptimal) dihedral angle of~1408 Figure 3. Induced-fit binding mode. Protease (HNE) residues are showninstick representation (white) with transparent Connolly-like surface. [28,29] Ligand 19 (purple) is shown in ball-and-stickmodel (oxygen:r ed, nitrogen:b lue, fluorine:c yan);h ydrogen bonds are depicted as broken yellow lines. a) Structure of HNE in complex with 19.L igand 19 interactswith HNE by ahydrogen bond(3.1 ) formed between the C2 carbonyl oxygen atom of the centralpyrimidine ring and the Val216 backboneamide of HNE. b) Binding to the S1 and S2 subsites is governed by exact protein-ligand shape complementarity of the northern and southern phenyl spheres of 19.c )Binding conformation of 19 overlaid on the binding site of apo-HNE. In the apo structure the S2 subsite next to Leu99 is not large enough to accommodate the northern para-cyanophenyl ring. Binding is only possible through an induced-fitmechanism, in which Leu99B is rotatedtoward the bulk solvent, thus expanding the lipophilic S2 pocket.  [27] for the X-ray data of 17.c )The dihedral angle was measured at the northern cyanophenyl moiety along N3ÀC4ÀC1'ÀC2' (highlighted in red), as indicated for compound 19. Apparently,t his system in its free state wasn ot pre-oriented for HNE binding.
Next, the more hindered N3methylated and C2'-trifluoromethylateds ystem 27 was analyzed. According to our protein X-ray structures, substitution at C2' of the northern cyanophenyl sphere ('C2'-north')w as considered to be feasible. This position projected into the solventa nd accordingly was not likely to compromise the binding mode; instead, it was well suited to introducealarge rotational barrier at the C4ÀC1' axis. Additional substituents at N3 (and C5)o f the dihydropyrimidinone core would reinforce that effect by furtherl ocking the system at the equatorial-northerni nterphase. According to our calculations, ligand 27 wase xpected to have significantly less rotational freedom along the C4ÀC1' axis, pre-orienting the system with ar otational barrier of > 40 kJ mol À1 at ad ihedral angle of~1208,v ery close to the 'ideal' bioactive dihedrala ngle of~1108.

Synthesis and assessment of C2'-north substituted systems
The synthesis of C2'-north-substituted systems turned out to be challenging (Table 5). Benzaldehydes with + Ms ubstituents at the ortho position provedt ob el ess reactive startingm aterials for the Biginelli reaction. Accordingly,e lectron-donating substituents had to be avoided at the pyrimidineforming stage of the synthesis forc ompounds 25-30 (Supporting Information).
Whereas N3 alkylation (22!25)o nly improved potencyt wofold, trifluoromethylation at C2'-north (22!26)a dvanced the IC 50 by af actor of eight. Yet, the combination of both substituents at N3 and C2' (22!27)b oosted potencyb ym ore than two orders of magnitude in as ynergistic fashion,v alidating our design hypothesis. The double conformational lock resultedi nh igh lipophilic binding efficiency (LipE = 7.0). Still, compound 27 was not an ideal candidate, with log D > 3( at pH 7.5). Therefore, we decided to replacet he lipophilic trifluoromethyl group by am ore polar,l ess lipophilic alternative whiler etaining the double conformational lock. Figure 5. Lockingthe bioactive conformation with substituents at N3 and C2'.C onformational analysisoffree ligands based on modeling. Relaxed coordinate scan of the rotation of the cyanophenyl moiety of 22 and 27 from 08 to 1808 in steps of 28.D epictedi s the dihedral angle along N3ÀC4ÀC1'ÀC2'.W hereasthe unsubstituteds ystem 22 has very low rotationbarriers around the 'non-ideal'd ihedral angle of 1408,t he substituted system 27 is locked with significantly higher rotationbarriers at 1208,w hich is closeto the 'ideal'd ihedral binding angleof1 108.E nergies were calculated using the B3LYP/6-31G* density functional technique. [30]  [b] log D (pH 7.5) was determined by reversed-phase HPLC at physiologicalp H7.5. A series of standards were injectedf or which log D has alreadyb een determined using definitive analyticalm ethods (a homologous series of n-alkanones). Plotting of the retention times against their log D generated ac alibration curve. The retention time of the testc ompound was then compared with the calibration curve leading to its log D.
[ c] Clog D (pH 7.5) was calculated by using ah ighly predictive methodd eveloped at Bayer, based on data points of experimentally determined log D values of internal pharmaceutical compounds andthe Simulations Plus pK a predictor. [23] [d] Calculated as LipE = pIC 50 Àlog D. [24] [e] The potency of testcompounds to inhibit human CYP 2C9 and CYP 3A4 was investigated with pooled human liver microsomes as enzyme source and selective standard substrates (SupportingI nformation); IC 50 values were derived from enzyme activity data (pH 7.4) in the presence/absence of various compoundc oncentrations and diclofenac/midazolam as selective CYP 2C9/CYP 3A4 substrate. Indeed, with as ulfone group, potencyc ould again be advancedb yafactor of ten (22!28). Combination of the C2'sulfone with am ethyl group at N3 enhanced potency by nearly two orders of magnitude relative to 22,y ielding BAY85-8501 (29,H NE IC 50 :6 5pm)w ithaformidable lipophilic binding efficiency (LipE 7.2). The C2'-north position also tolerated the slightly basic sulfoximine [31] residue, yieldingc ompound 30 with improved solubility (Table 5). Due to its overall balanced technical profile, BAY85-8501 (29)w as selected for in-depth in vitro and in vivo testing (see below).
BAY85-8501 was synthesized in an ine-step sequence, with deliberate introduction of the electron-withdrawings ulfone substituent prior to the Biginelli reaction in order to increase electrophilicity and reactivity of the corresponding benzaldehyde 34 (Scheme 1). Separation of enantiomers 35 was subse-quently achieved by HPLC on chiral phase. The cyano group at the dihydropyrimidinone was installed from carboxylic acid 37 via amide 38 by dehydration with the Burgess reagent.
For ab etter understanding of the binding mode with our novel conformationally locked systems, 28 was co-crystallized with HNE ( Figure 6), which revealed ab inding mode nearly identicalt ot hat of ligand 19 (Figure 3). The N3ÀC4ÀC1'ÀC2' dihedral angle of 109.58 was very close to the assumed optimum of 1108 (Supporting Information). The sulfone moiety pointed outward from the active site while one of itso xygen atoms was hydrogen bondedt o awater molecule, gaining further binding energy.

In-depth in vitro testing of BAY-678 and BAY8 5-8501
We ran the biochemical inhibition assayf or BAY85-8501 also in the presence of 1mm hydrogen peroxide to mimic in vivo conditions of oxidative stress in an inflammatory environment. Under these harsh circumstances, the IC 50 only shiftedb yafactor of two toward 140 pm,indicatinggood oxidative stability.
To confirm the exceptionally high potencyo fB AY-678 (20)a nd BAY85-8501 (29), we measured enzyme reaction velocities with different substrate concentrations at various inhibitor concentrationsa nd extrapolated the inhibition constants( K i )f rom Dixon plots (Supporting Information). Both compounds revealed (substrate) competitive inhibition, further confirming their binding into the active site of the enzyme. However,t he K i values toward rodento rthologous enzymes were about two orders of magnitude higher than toward HNE (Table 6). Beneficially,B AY-678 andB AY 85-8501 revealed no inhibition against 21 relateds erine proteases, up to an inhibitor concentration of 30 mm (Table 6).
Next we investigated the binding kinetics of BAY85-8501 (Table 7). BAY85-8501 showedalong residence time of~17 min. According to published data, [33] 29 binds to HNE as rapidlya st he endogenous a-proteinase inhibitor (aPI). The latter,h owever,b eing ap rotein, shows am uch longer residence time, leading to pseudo-irreversible binding characteristics.

Pharmacokinetic studies
Various dihydropyrimidinones showed overall promising pharmacokinetic data in rodents (Table 8). While both early dihydropyrimidinones 17 and 20 revealed medium clearance in rodents as ar esult of oxidation at C4, the additional electronwithdrawing effect of the sulfone C2'-north substituent in BAY85-8501 resulted in metabolic stabilization of the drug. BAY85-8501 (29)d isplayed low clearancea nd improved halflife in rats, and no inhibitory potency toward CYP isoforms (Tables5and8 ). Am ain role of elastase inhibitors in lung diseases could be the prevention of lung injury driven by chronic inflammation (Figure 1). We set up ar apid, preventive in vivom odel that was designed to reflect basic aspects of lung diseases, such as ALI, by combining an exogenous 'noxa' [triggered by HNE or porcinep ancreatic elastase (PPE)] with an endogenousi nflammation that developed over time and was driven by murine neutrophil elastase (MNE).
Intratracheali nstillation of HNEi nto the lungs of mice caused severe injury,l eading to lung hemorrhage and inflammation that were quantified 1h after the HNE challenge by measuring hemoglobin concentrations and neutrophil count in the bronchoalveolar lavage fluid (BALF;F igure 7a,1 st ). In this model the exogenous HNE noxaw as the primary cause of injury and lung hemorrhage.A ccordingly,t he degree of primary injury was directly dependento nt he amount of HNE given. However,t he subsequenti nflammation that was primarily driven by the endogenous MNE also contributed to secondary injury effects, developing over time. Based on picomolar potencya gainst HNE as well as single digit potencyv ersus MNE, BAY85-8501 (29)c ompletely prevented the development of lung injury and subsequenti nflammation when administered 1h prior to the HNE noxa. In the 0.01 mg kg À1 dose group, hemoglobinc oncentration was already significantly decreased ( Figure 7b). At ad ose of 0.1mgkg À1 ,asignificant effect on neutrophil count was observed (Figure 7c). In this setup, efficacy was predominantly driven by potency against HNE (K i = 0.08 nm).
Next we changedt he experimental setup to an exogenous PPE noxa (Figure 7a,2 nd ): Intratracheali nstillation of PPE caused severe lung hemorrhage and inflammation which were quantified by measuring hemoglobin concentrations and neutrophil count in BALF 1hafter the noxa. As the highly HNE-selective inhibitor BAY85-8501 had no effect on PPE, BAY85-8501 could not prevent the primary lung injury in this setup. Nevertheless, BAY85-8501 could inhibit MNE, the endogenous driver of inflammation ands econdary injury,a lthough with decreasedp otency.C onsequently,t he effects of BAY85-8501 on inflammation and secondary injury were weaker at this point, and only observed at 30-foldh igher doses (Figure 7d,e). Efficacy was predominantly driven by potencya gainst MNE (K i = 6nm)i nt his second setup. Ultimately,i nv itro potency against Figure 6. Co-crystallization of 28 with HNE. Protease residues are shown in stick representation with transparent Connolly-like surface;l igand 28 is shown in ball-and-stick representation (oxygen:r ed, nitrogen:b lue, fluorine: cyan, sulfur:y ellow). Ligand 28 has ad ihedral angle of 109.58 at the northern cyanophenyl moiety along N3ÀC4ÀC1'ÀC2'.T he image was generated with PyMOL. [29]  [a] K i valueswere extrapolated from Dixon plots (SupportingI nformation). As expected, K i values showed good correlation to the IC 50 values. [24] [b] Murine neutrophil elastase.
[c] IC 50 values for 21 related serine proteases, including porcine pancreatic elastase (PPE), were determined by applying functional biochemical assays with the respective isolated enzyme and the appropriate fluorogenic peptide substrate (Supporting Information). [a] The on-rates at which elastase inhibitors bind to the target wered etermined by applying af unctional biochemical assay using as ubstrate with am odifiedf luorescent label,M eOSuc-AAPV-umbelliferyl;t hisa llows very sensitive detection of substrate hydrolysis on the millisecond timescale, in the presence or absence of elastase inhibitor. Usingn onlinear regression of the reaction progress curves, the observed rate constant of the onset of inhibition (k obs )w as obtained and plotteda gainst the inhibitor concentration. The slope of the linear regression revealed the estimated k on value (Supporting Information).
[b] Calculated from k on and K i according to the equation k off = k on K i .[ c] Data from Sinden et al. [33] [d] Calculated with K i~1 10 À14 m,t aken from Beattye tal. [32] [e] Calculated according to target residence time, 1/k off .

Conclusions
In summary,w eh ave evolved as ub-micromolar lipophilic screening hit into ah ighlys elective picomolar HNE inhibitor with lower molecular weight (Scheme 2). Boosting of the lipophilic binding efficiency by nearly five was possible by locking the bioactive conformation of our pyrimidinonel ead series on the basis of at horough conformational understanding of the induced-fit binding mode. Electronic modulation of the northern hemispherei mproved in vitro and in vivo pharmacokinetics data significantly.BAY 85-8501 (29)has shown in vivoefficacy in various preclinical animal models, resultsthat will be published in due course.B AY 85-8501 is currently in clinical testing for the treatment of pulmonary diseases.