Organic &Biomolecular Chemistry

PAPER

Cite this: DOI: 10.1039/c3ob41165f

Received 6th June 2013,Accepted 16th July 2013

DOI: 10.1039/c3ob41165f

www.rsc.org/obc

Highly selective aza-nitrile inhibitors for cathepsin K,structural optimization and molecular modeling†

Xiao-Yu Yuan,a Ding-Yi Fu,a Xing-Feng Ren,a Xuexun Fang,b Lincong Wang,c

Shuxue Zouc and Yuqing Wu*a

As a new type of cathepsin K inhibitor, azadipeptide nitriles have the characteristics of proteolytic stability

and excellent inhibitory activity, but they exhibit barely any satisfactory selectivity. Great efforts have

focused on improving their selectivity toward cathepsin K. In this sequential study, we report the further

structural optimization, synthesis, molecular modeling, and in vitro enzymatic assays of a new series of

potent and selective inhibitors of cathepsin K without the P2–P3 amide linker. Significant selective

improvements were achieved for cathepsin K over L, S and B, and a triaryl meta-product 13’ possessed

the favorable balance between potency (Ki = 0.29 nM) and selectivity of cathepsin K over cathepsin

L (320-fold), S (1784-fold) and B (8566-fold). We undertook a covalent protein–ligand docking study to

explain the improved selectivity of several representative compounds. Such a selectivity improvement

would be useful to avoid harmful side effects in practical applications of these compounds.

1. Introduction

Emerging evidence has suggested that cathepsin K is theprimary enzyme involved in osteoclastic bone resorption,making it an important target for the treatment of osteoporo-sis.1 In addition, several studies have shown that cathepsin Kdeficiency leads to an increase in bone mineral density(BMD).2 Recent reports on the inhibition of cathepsin K havefocused on derivatives of aldehydes,3 ketoamides,4 semicarba-zones,5 and more recently nitriles.6 Among these cathepsin Kinhibitors, nitriles have attracted much interest due to thereversible formation of a thioimidate adduct resulting fromthe nucleophilic attack of the active-site cysteine at the nitrilecarbon.7 As a new type of cathepsin K inhibitor, azadipeptidenitriles have the characteristics of proteolytic stability andexcellent inhibitory activity, but they display barely any satis-factory selectivity.8 Therefore, great efforts to improve theirselectivity toward cathepsin K have been undertaken.8,9 Frizleret al. reported a series of selected azadipeptide nitriles, with

the best selective carba-analogue possessing a good selectivityprofile for cathepsin K against L (324-fold).8

A previous report that focused on improving potency indi-cated that the triaryl-P3 compound 13 with the P2–P3 amidelinker displayed good performance against cathepsin K withKi = 0.0031 nM and also acceptable selectivity over cathepsinsB and S (∼1000-fold).10 However, this compound achieved onlyan inferior selectivity toward cathepsins K and L (∼10-fold).In addition, the binding model based on covalent dockingbetween inhibitor 13 and cathepsin K indicated that the largeP3 moiety was located at the wide S1′ pocket, a location belowthe binding site, and at a site other than the S3 pocket whereit should be. Moreover, the binding model of the less potentpara-analogue 12 and cathepsin K indicated that although itsP3 moiety could reside at the S3 pocket, its third ring also pro-truded there with a torsion. All these results implied that thelinear para-P3 group together with the P2–P3 amide linker wastoo long to be accommodated well in the S3 pocket of cathep-sin K. Therefore, to improve binding, we designed a novelbackbone structure for the inhibitor that lacked the P2–P3amide linker (Table 1) to optimize its orientation and distancewith the S3 pocket. We performed the in vitro enzymatic assayson these new inhibitors toward cathepsins K, B, L and S, andwe employed covalent docking studies to better understandthe improved selectivity.

2. Results and discussion

In this study, a new series of azanitriles without the P2–P3amide linkage (n = 0, Table 1) were prepared and tested

†Electronic supplementary information (ESI) available: Synthetic details andcharacterizations of compounds 5′–7′ and 9′–13′, inhibition assays, representa-tive plots and IC50 values were available. See DOI: 10.1039/c3ob41165f

aState Key Laboratory of Supramolecular Structure and Materials, Jilin University,

No. 2699, Qianjin Street, Changchun, 130012 China. E-mail: yqwu@jlu.edu.cn;

Fax: +86-431-85193421; Tel: +86-431-85168730bKey Laboratory of Molecular Enzymology and Engineering of Ministry of Education,

College of Life Science, Jilin University, No. 2699 Qianjin Street, Changchun 130012,

ChinacCollege of Computer Science and Technology, Jilin University, No. 2699 Qianjin

Street, Changchun 130012, China

This journal is © The Royal Society of Chemistry 2013 Org. Biomol. Chem.

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against human cathepsins K, B, L and S, aiming through theadjustment of the length of the P2–P3 linkage as well as theP3 group to develop inhibitors with improved selectivity forcathepsin K over the other enzymes, especially over cathepsin L.For that specificity, a series of phenyl derivatives with anextension at either the para- or meta-position of the phenylring (Scheme 1) were prepared.

We began this investigation by shortening the amide P2–P3linkage of the azadipeptide nitrile scaffold, as reported pre-viously.10 For this purpose, different synthesis protocols weredesigned for the synthesis of the parent compounds 1′ (para-)and 8′ (meta-phenyl derivative) according to a related report,11

and typical synthetic routes are illustrated in Scheme 1.Briefly, lithium di-isopropylamide (LDA), a non-nucleophilicstrong base, was used for the deprotonation of weakly acidic

compound a, which was then reacted with 1-iodo-2-methyl-propane to obtain the intermediate b. Subsequently, afterbeing activated via mixed anhydride, isobutyl chloroformateand 4-methylmorpholine, b was transformed into c after theaddition of 1,2-dimethylhydrazine. Finally, cyanogen bromidewas added to the produced c in MeOH to gain the respectiveparent compounds 1′ or 8′.

Further, a simple but high-yielding synthetic route10–12 wasused to extend the parent compounds 1′ and 8′, and the corres-ponding boronic acids or esters were employed to obtain thetarget products by Suzuki reaction (see Schemes S1 and S2,ESI†).

2.1. Biaryl P3 substituents

We synthesized the bi(hetero)aryl-derived nitriles 5′–7′(Table 1) from the parent compound 1′ and 4-(4,5-dimethyl-1,3-dioxolan-2-yl)pyridine, thiophen-2-ylmethanedio and phe-nylmethanediol, respectively (Scheme S2 in ESI†). After purifi-cation and characterization by 1H NMR, 13C NMR and massspectroscopy (see ESI†), the compounds were tested in theactive assays of human cathepsins K, L, S and B. They all dis-played a time-dependent binding behavior (see Fig. S1 in ESI†as representative). The Ki values of 5′–7′ on cathepsins K, L, Sand B were calculated and listed in Table 2, respectively. Allcompounds 5′–7′ exhibited sub- to nanomolar Ki values anddisplayed no significant differences in inhibition towardcathepsin K. The displayed moderate Ki values for cathepsin Kwere much less potent than the corresponding azadipeptidenitriles inhibitor 5–7, where inhibition in the low picomolarconcentration range toward cathepsin K was reported.10 Theloss of 2–3 orders of magnitude in inhibition potency maybe attributed to the loss of the amide P2–P3 linkage incompounds 5′–7′ which prevented formation of favorablehydrogen-bonds with cathepsin K; in previously reportedazadipeptide nitrile inhibitors, the amide linker formed ahydrogen bond with the carbonyl group of Gly66.10,13 A similarphenomenon was reported for the inhibitors of amino aceto-nitriles where a marked decrease in potency appeared whenthe amide bond of the P2–P3 linker was replaced by a phenylring.14

Despite the much reduced inhibition potency, compounds5′–7′ displayed obvious improvements in the selectivity profiles

Table 1 The structural scaffold of aza-nitrile (n = 0) (1’–13’) and azadipeptidenitrile (n = 1) (1–13) inhibitors

Nos.

Rn = 0 n = 1

1′ 1

5′ 5

6′ 6

7′ 7

8′ 8

9′ 9

10′ 10

11′ 11

12′ 12

13′ 13

Scheme 1 Synthesis routes for parent compounds 1’ (para-) and 8’ (meta-phenyl derivatives).

Paper Organic & Biomolecular Chemistry

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for cathepsin K over the other three enzymes, especially overcathepsin L. Both cathepsins K and L are endopeptidases, witha high sequence homology (60% identity, 76% similarity)between them which made it very difficult to be inhibitedselectively,15 and generally we considered a difference of 2–3orders of magnitude in Ki as a satisfactory result. Compound6′ showed the best selectivity profile (317-fold) for cathepsin Kover L, which was similar to the reported value.9 To a signifi-cant degree, the high selectivity might be due to the weakbinding affinity between 6′ and cathepsin L. This point will bediscussed below in the covalent docking studies.

Considering that the orientation of inhibitors in theenzyme active sites was crucially important for binding to theproteases,13 the corresponding meta-substituents, referred toas compounds 9′–11′ in Table 1, were also designed and syn-thesized. For the synthesis of the meta-products, the meta-scaffold 8′ was employed as the parent compound. Then theSuzuki reaction was used to obtain compounds 9′–11′ asdepicted in Scheme S2.† Bearing sub-nanomolar Ki values, themeta-products 9′–11′ showed a relatively better inhibitionpotency against cathepsin K in comparison to the para-derivednitriles. Such isomeric preference differed from other reportednitrile inhibitors against cathepsin K,10,16 but was consistentwith two previous observations14,17 where the para-linkedbiphenyl product lost considerable activity against cathepsin Kas compared to the corresponding meta-analogue, and theproper positioning of the second aryl group was suggested tobe critical in the inhibitory action. In addition, although thepotencies of inhibitors 5′–7′ were slightly less than 9′–11′, theselectivities of 5′–7′ for cathepsin K exceeded those of 9′–11′.Of note, para-derived aza-nitrile 6′ showed much better selecti-vity than the corresponding meta-product for cathepsin K over

L (317 vs. 38). In addition, 5′ displayed the largest improve-ment for the selectivity of cathepsin K over S (964 vs. 125) andB (1774 vs. 947) in comparison to its meta-isomer 9′. However,for further pharmaceutical development based on cathepsin K,such an improvement in selectivity was still not good enoughto avoid harmful side effects such as skin rashes and rarerincidences of morphea-like skin changes.18 Further structuraloptimization will be needed.

2.2. Triaryl P3 substituents

Recent results on azadipeptide nitrile cathepsin K inhibitorswith large triaryl P3 substitutions provided guidance for thedesign of more highly selective inhibitors.8,10 The P3 moietiesof compounds 7′ and 11′ were extended to obtain the triaryl-inhibitors 12′ and 13′, with the expectation of improving theselectivity for cathepsin K over L, S and B, as well as decreasingthe Ki values against cathepsin K. For the synthesis, thebromo-derivatives 1′ and 8′ were used as the parent com-pounds and biphenylboric acid was used for the extension toacquire the two triaryl-inhibitors 12′ and 13′, as illustrated inScheme S2.† The Ki values of 12′ and 13′ against cathepsins K,L, S, and B are also listed in Table 3. For comparison, theirselectivity profiles for cathepsin K over L, B and S, togetherwith the previously reported compounds 12 and 13, are shownin Table 3.

Compared to the corresponding biaryl inhibitor 7′, thepotency of the triaryl compound 12′ against cathepsin K actu-ally decreased and, in turn, its selectivity for cathepsin K overL also declined. However, in comparison to 7′, 12′ exhibited aremarkably improved selectivity profile for cathepsin K over B(>10 000-fold vs. 577-fold). Distinct from the improvement of12′ over 7′ which was limited to only cathepsin K over B, theoptimized meta-derivative 13′ displayed significant increases ofselectivity for cathepsin K over L, S and B in comparison to 11′.The large improvement in selectivity for cathepsin K over Bmight be related to the structural difference of the S2 pocketbetween the two enzymes,19 and the isobutyl P2 group wasmore suitable for that of cathepsin K, but not for B.

Having a structure similar to the bi(hetero)aryl-derivednitriles, the triaryl-P3 meta-derivative 13′ showed higher

Table 2 The inhibition potency and selectivity profiles of compounds 5’–7’ and9’–11’ toward cathepsins K, B, S and L, in comparison to those of 5–7 and 9–11

No. Kia (nM) Cat Kb

Selectivity

Cat Lb/K Cat Sc/K Cat Bb/K

5′ 0.79 156 964 17746′ 1.03 317 788 7147′ 1.93 154 328 5779′ 0.32 13 125 94710′ 0.79 38 96 44211′ 0.38 22 92 9665d 0.027 1.8 161 1116d 0.035 1.5 85 1337d 0.029 2.7 54 839d 0.104 0.4 32 3010d 0.054 0.3 59 9411d 0.137 0.1 10 30

a The Ki values were calculated from the corresponding IC50 and Kmvalues and the concentration of the substrate [S], by using theequation Ki = IC50/(1 + [S]/Km). IC50 were determined for at least fivedifferent inhibitor concentrations in triplicate. bHuman cathepsins K,L and B were assayed with the substrate of Z-Phe-Arg-AMC. cHumancathepsin S was assayed with the substrate of Z-Val-Val-Arg-AMC. d Thebioactivity values for these compounds originate from ref. 10, wherecompounds 5–7 and 9–11 were the analogues of 5′–7′ and 9′–11′,respectively.

Table 3 The potency and selectivity profiles of compounds 12’–13’ in compari-son to those of 12–13

Nos Kia (nM) Cat K

Selectivity

Cat L/K Cat S/K Cat B/K

12′ 6.54 31 324 >10 00013′ 0.29 320 1784 856612b 0.124 5.6 2.1 1213b 0.0031 10 1061 1016

a The Ki values were calculated from the corresponding IC50 and Kmvalues and the concentration of the substrate [S], by using theequation Ki = IC50/(1 + [S]/Km). IC50 were determined for at least fivedifferent inhibitor concentrations in triplicate. b The bioactivity valuesfor these compounds originate from ref. 10, where the compounds 12and 13 were the analogues of 12′ and 13′, respectively.

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potency than the corresponding para-derivative 12′ for cathep-sin K. The selectivity of 13′ against cathepsin K over L and Swas significantly improved with respect to that of 12′, anoutcome that was consistent with the reports for the corres-ponding azadipeptidic nitrile inhibitors 12 and 13.10

Although the selectivity profile of 13′ for cathepsin K over Lremained at almost the same level as the best azadipeptideinhibitor previously reported (324-fold),8 a 10-fold (0.29 vs.2.9 nM) enhancement in potency for 13′ against cathepsin Kwas achieved here. In addition, it was particularly gratifying tonote that 13′ exhibited a significantly enhanced selectivityprofile for cathepsin K over S (∼1700-fold), much higherthan the previously reported selectivity (∼140-fold).8 Thus, theobtained triaryl meta-derivative 13′ displayed a favorablebalance between cathepsin K potency and selectivity over cath-epsins L, S and B, among all the reported inhibitors.

2.3. Molecular modelling

To provide insight into the inhibition mechanism and to esti-mate the binding affinities of inhibitors, especially theirlocation and orientation with respect to the active site of theenzymes, we applied an advanced molecular docking programGOLD 5.1 (The Cambridge Crystallographic Data Center, Cam-bridge, U.K.) for covalent docking. The selective inhibitorswere manually modified to the corresponding thioimidates,20

and then the resulting addition products were drawn andenergy minimized in ChemBioOffice (Cambridgesoft,Cambridge, MA). They were then exported to Discovery Studioand subjected to diverse conformation generation and themaximum conformation was set to 1000. The aim of this stepwas to generate a conformer library for each ligand which con-sisted of prospective conformers instead of exhaustive con-formational sampling for every rotary bond. At the end, GOLD5.1 was used to covalently dock each conformer of the in-hibitors to the corresponding enzyme. Finally, each pose wasranked according to its GoldScore fitness function, and thehighest scoring pose was chosen as the binding conformationand used for further analysis.21

As part of our ongoing efforts in the area of cathepsin Kinhibitors, the removal of the P2–P3 amide linkage wasachieved, and this series of inhibitors displayed an improvedselectivity profile. Further SAR around these selective cathep-sin K inhibitors may be discovered through the molecularmodeling studies.

Based on the largely improved selectivity of compound 6′toward cathepsins K and L, it was modeled into these twoendopeptidases (Fig. 1A and B) to explore its location andorientation. Evaluation of the resulting poses showed that forcompound 6′ in cathepsin K, the isobutyl group resided well inthe S2 pocket, and the P3 group in the S3 pocket which wasformed by Cys36, Gly23, Ser24, Gly64 and Gly65.22 In addition,the parental phenyl ring formed an edge-to-face interactionwith Tyr75, and the N-atom of the thioimidate was locatedwithin hydrogen-bonding distance (2.69 Å) to the NH group ofGln19. As one of the papain-like cysteine proteases, cathepsinL has a structure analogous to cathepsin K, where the residues

Cys25 and His163 were in the middle of a “V”-shaped activesite cleft, forming the catalytic site of the enzyme.23 Asobserved in Fig. 1B, for inhibitor 6′ and cathepsin L, the iso-butyl group of the inhibitor was not packed into the S2 pocketformed by Leu69, Met70, Ala135, Met161, Asp162, Gly164 andAla214,24 but extended into the solvent. This binding orien-tation might be attributed to the fact that the S2 pocket ofcathepsin L generally preferred large aromatic groups as thebi(hetero)aryl group.25 However, the P3 group showed a littlecrowding in the S2 pocket and there was no residue to corre-spond with the S3 pocket. Thus, compound 6′ could hardlyinhibit cathepsin L, which greatly improved its selectivity forcathepsin K over L, explaining well the experimental data.

With the triaryl groups in P3 position, inhibitor 13′ wasmodeled into both cathepsins K and L by using an identicalprocess as 6′.

As observed in the docked complexes (Fig. 2A and B), theisobutyl group of 13′ was settled in the S1′ pocket and theP3 group was located in a broad pocket below the binding site,oriented perpendicular to the S3 pocket. A similar bindingmode had been reported for a ketone inhibitor in cathepsin K,where the terminal phenyl of the 4-phenoxyphenyl sulfona-mide formed an interaction with Trp184.26 Inhibitor 13′formed hydrogen-bonds with cathepsin K, between the CvOof the P1–P2 linker and the imidazole NH of His162 (2.65 Å)with the N-atom in the thioimidate and the NH2 in Gln19(2.26 Å) and that of the imidazole NH in His162 (2.26 Å). Here,the second phenyl ring in the P3 group of 13′ formed a face-to-face aromatic interaction with the indole ring in Trp184. Dis-tinct from cathepsin K, the isobutyl group of compound 13′was located in the S2 pocket in cathepsin L while the extendedP3 group was bound in the S3 pocket. The hydrogen bondswere observed only between the thioimidate N-atom with the

Fig. 1 The docked modes of 6’ in the active site of human cathepsin K (panelA, left, pdb id: 1VSN) and cathepsin L (panel B, right, pdb id: 2XU5). The inhibi-tor is rendered as a stick representation.

Fig. 2 The docked modes of 13’ in the active site of human cathepsin K (panelA, left, pdb id: 1VSN) and cathepsin L (panel B, right, pdb id: 2XU5). The inhibi-tor is rendered as a stick representation.

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imidazole NH of His163 (2.22 Å) and that of the NH2 in Gln19(2.49 Å) when 13′ was modeled in cathepsin L. That is, theexcellent binding mode of 13′ in cathepsin K not onlyenhanced the potency for its inhibition toward cathepsin Kbut also improved the selectivity against L, further explainingthe experimental results of a better selectivity profile for thisinhibitor with respect to cathepsin K against L.

From the literature,27 it is known that the nitrile inhibitorbinds through formation of a thioimidate ester with Cys29 ofcathepsin B at the active site, with the backbone NH of Cys29and the side-chain amide of Gln23 playing a role in stabiliz-ation of the intermediate by hydrogen bonding. In addition,the tether length of the phenyl moiety was reduced with theuse of a diphenylacetyl group to occupy the hydrophobicpocket S3, leaving the S2 pocket with enough room for anadditional hydrophobic moiety attached to the phenyl group,especially for 3-methyl phenylalanine.27 Moreover, it wasreported that the S′ pocket of cathepsin B was unique amongpapain superfamily proteases, where His110 and His111 inthis pocket had previously been shown to bind carboxylategroups, and the histidine region of S′ had been accessed via P1substitution. However, the cathepsin K inhibitors of our serieswere synthesized neglecting the S′ pocket of cathepsin B, andthe isobutyl group did not match well with the S2 pocket,while tri-phenyl rings of the P3 group did not reside as wellwith the S3 pocket as the diphenylacetyl group. In Fig. 3, thephenyl tether length of the P3 group was very long andinclined toward the S′ pocket, and the P2 group did not formany lipophilic interactions with the cathepsin B enzyme, butrather was directed away from the active site into the solventwater. In this way, the S3 portion of the cathepsin B enzymewas completely vacant. It is not difficult to imagine that thesecompounds were not good inhibitors of cathepsin B. Thus,compounds 12′ and 13′ showed swinging selectivity profilesfor cathepsin K against B.

3. Conclusions

In this study, a new approach was used to remove the hydrogenbond donated by the P2–P3 amide linkage of inhibitors to thebackbone oxygen of Gly66 in the cathepsin enzymes, whichresulted in highly cathepsin K-selective aza-nitrile inhibitorsby further structural optimization of the P3 group. With a sub-nanomolar Ki value for cathepsin K, the selectivity profiles

against cathepsins S and B were all improved by one or severalthousand folds, especially against cathepsin L, the enzymewith the highest degree of homology to cathepsin K. Thepresent inhibitors were better than the previously reportedselective compounds. Such a study would be helpful in devel-oping the treatment of postmenopausal osteoporosis and mayserve as a model for planned clinical trials in human subjects.Under the premise of further exceeding the values of potencyand selectivity given here, we shall present the design of novelcathepsin K inhibitors with even higher selectivity profiles in asubsequent article.

Acknowledgements

The present work was supported by the projects of NSFC(no. 20973073, 20934002 and 91027027) and the State KeyLaboratory of Supramolecular Structure and Materials, JilinUniversity.

Notes and references

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Fig. 3 The docked modes of 12’ (panel A, left) and 13’ (panel B, right) in theactive site of human cathepsin B (pdb id: 1GMY). The inhibitor is rendered as astick representation.

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