Chinese Chemical Letters  2014, Vol.25 Issue (05):673-676   PDF    
Discovery of dipeptidyl peptidase IV (DPP4) inhibitors based on a novel indole scaffold
Peng-Fei Xiaoa,b, Rui Guoc, Shao-Qiang Huangb, Heng-Jun Cuic, Sheng Yec, Zhiyuan Zhangb     
a Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China;
b National Institute of Biological Sciences (NIBS), Beijing 102206, China;
c Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
Abstract : Dipeptidyl peptidase IV (DPP4) inhibitors are proven in the treatment of type 2 diabetes. We designed and synthesized a series of novel indole compounds that selectively inhibit the activity of DPP4 over dipeptidyl peptidase 9 (DPP9) (>200 fold). We further co-crystallized DPP4 with indole sulfonamide (compound 1 ) to confirm a proposed binding mode. Good metabolic stability of the indole compounds represents another positive attribute for further development.
Key words: DPP4 inhibitor     Type 2 diabetes     De novo design     Indole scaffold     Crystal structure    
1. Introduction

Incretin hormones,including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like-peptide-1 (GLP-1),stimulate insulin secretion,inhibit glucagon secretion,delay gastric empty- ing,and reduce food intake [1]. The rapid increasing plasma levels of both hormones in response to elevated serum glucose levels are negatively regulatedby dipeptidyl peptidase IV (DPP4) [2],a serine protease expressed in most tissues [3]. DPP4 regulates insulin secretion by the inactivation of GIP and GLP-1 through removing two amino acids from the N terminus of both hormones [4]. Enhancing the duration of endogenous incretin hormone by inhibiting DPP4 function is now a validated approach in treatment of type 2 diabetes [1]. Several DPP4 inhibitors have been approved by the FDA,such as sitagliptin [5],saxagliptin [6],linagliptin [7] and alogliptin [8a] while several others are in late stage clinical trials [9, 10].

Structure-based drug design (SBDD) has been extensively used in modern medicinal chemistry for the discovery of many drugs. In this paper we describe our effort in the de novo design and development of a series of potent and selective DPP4 inhibitors based on an indole scaffold utilizing this technology.

2. Experimental

The synthesis of compound 1 ,2 ,8a-8f and 11a-f (Scheme 1) began with 1-(1H-indol-3-yl)-N,N-dimethyl methylamine (3) [11]. Compound 3 was treated with MeI in THF,followed by heating with potassium phthalimide in anhydrous DMF at 150 ℃ to generate 4 [12]. Compound 5 were prepared from 4 using pyridinium tribromide at -10 ℃. The intermediate compound 6 was synthesized through Suzuki coupling reaction of compound 5 with 2,4-dichlorophenyl boronic acid in a mixture of toluene and EtOH at 105 ℃. Compounds 8a-8f and 1 were obtained by reacting 5 with R1-sulfonyl or acyl chloride,followed by the removal of the phthalimide group using NH2NH2in EtOH at room temperature. Similarly,11a-11f were readily synthesized in three steps including Suzuki coupling reaction between compound 5 and different phenyl boronic acids,sulfonamide formation using different alkyl sulfonyl chloride,and the removal of the phtha- limide group.

Scheme 1. Synthesisofcompounds1,2and8a-fand 11a-f .Reagentsandconditions:(a)MeI,THF,potassiumpthalimide,DMF,150 ℃for5 h,64%;(b)THF,CHCl3,pyridinium tribromide,-10 ℃ for 3 h,56%; (c) 2,4-dichlorophenylboronic acid,Pd(PPh3)4,LiCl,Na2CO3,PhMe,EtOH,105 ℃ for 4 h,30%; (d) R1-sulfonyl or acyl chloride,NaH,DMF,0 ℃ for 16 h,31%-50%; (e) NH2-NH2,EtOH r.t. for 1 h,84%-91%; (f) R2-phenyl boronic acid,Pd(PPh3)4,LiCl,Na2CO3,PhMe,EtOH,105 ℃ for 4 h,31%-57%; (g) R1-sulfonyl chloride, NaH,DMF,0 ℃ for 16 h,31%-50%.

Detailed synthesis procedure and characterization data of the synthesized compounds can be found in Supporting information.

3. Results and discussion

Before the initiation of the de novo design,we first studied the structure of several known DPP4-inhibitor complexes [8a, d] and identified several key interactions between the protein and the inhibitors. As shown in Fig. 1,alogliptin tightly binds in the active site of DPP4 through several important interactions. The cyano- benzyl group fits in the P1 hydrophobic pocket formed by Val656, Tyr631,Try662,Trp659,Tyr666 and Val711. The cyano group also formsahydrogenbondwithArg125.Anotherkeyinteractionisasalt bridge interaction between the aminopiperidine in alogliptin and residues Glu205/Glu206 in DPP4. The 4-carbonyl oxygen in the primidindioneinteractswith thebackboneNH ofTyr631througha hydrogen bond,while the pyrimidindione ring itself formsp-stack interactionwiththephenylringontheTyr547residue.FromDPP4- alogliptin and other co-crystal structures,we recapitulated the following important interactions: (a) hydrophobic filling in the P1 pocket,(b) salt bridge to Glu205/Glu206,(c) p-stacking and hydrogen bond with NH of Tyr631or OH of Ser630. Rather than starting from a screening hit,we decided to use the information described above to de novo design new chemical structures that could capture all or most important interactions with DPP4. An indole scaffold having certain functional groups was one of our designs.AsschematicallyshowninFig.2,amethylaminemotifatC- 3 provides a salt bridge to E205/E206,the phenyl group fills the P1 pocket,whileanacylgrouporalkylsulfonylgroupatN-1couldform oneortwopotentialhydrogenbondstoSer630,Tyr547andTyr361.

Fig. 1. Cocrystal structure of Alogliptin in the DPP4 active site.

Fig. 2. A model of compound 1 in the DPP4 active site showing structure based design.

To evaluate the design,we first synthesized methylsulfonyl derivative 1 and acetyl derivative 2,which showed inhibitory activity against DPP4 at an IC50 concentration of 232 nmol/L and 38% inhibition at 10 umol/L,respectively (Table 1). Furthermore, compound 1 also showed more than 200 fold of selectivity over DPP9. According to our modeling results,the sulfonyl group in compound 1 could form more favorable hydrogen bonds with Ser630 and Tyr547 residues of DPP4,thus explained the better activity of compound 1. To verify our modeling results and our hypotheses,we co-crystallized compound 1 and DPP4 (Table 2). The detail structural information (Fig. 3) revealed the mode of binding ofcompound 1 withDPP4in theactive site. The indolering forms a cation-p interaction with the guanidinium moiety of Arg125,the methylamine motif forms salt bridges to Glu205/ Glu206,and the 2,4-dichlorobenzene group effectively fits in the P1 pocket. Comparing the proposed binding mode and the real crystal structure,the inhibitor conformation was as predicted except the amine orientation. The proposed hydrogen bond between the sulfonyl group and Tyr 547 and the orientation of the methylsulfonyl group are in good agreement with the crystal structure. However,the residue Tyr547 of DPP4 rotates 90 degrees intheco-crystalstructurethusdifferedfromthestructureofDPP4- Alogliptin and other DPP4 complexes,which causes the loss of the hydrophobic interactions.

Table 1
Selected data for indole scaffold analogs (R1substitution).

Table 2
X-ray data and refinement statistics.

Fig. 3. Cocrystal structure of 1 in the DPP4 active site.

The encouraging results motivated us to develop structure activityrelationship(SAR)bothonthealkylsulfonylandArgroups. Basedontheanalysisofourcrystalstructure,wedecidedtomodify the R1 group on the sulfonyl group to generate more interactions with the phenyl ring of Tyr547 in order to improve the potency. According to the SAR shown in Table 1,similar to compound 1 ethylsulfonyl(8a),isopropylsulfonyl (℃) and isobutylsulfonyl(8d) analogs demonstrate good potency against DPP4,while the propylsulfonyl (℃) and cyclopropylsulfonyl (8d) analogs are relativelyweak. Our interpretationfor the SARis that the hydrogen bond between the indole compound and the Tyr547 is not as strong comparing with the hydrogen bond between Alogliptin and Tyr631,while the phenol group in Tyr547 is more flexible compared with the backbone NH in the Tyr631. Therefore, increasing the p-stack interactions with the Tyr547 might not be beneficial.

The P1 pocket of DPP4 is the most important area utilized by others in their inhibitor design; therefore our second optimization study was focused on modifying the Ar group on the indole core. With knowledge learned from others [8a, d],the 2,4-di-Cl phenyl group was used in our first inhibitor design (compound 1). To understand SAR at this position,compounds with mono or di- substituted phenyl group (11a-11f) were synthesized. The inhibition testing results (Table 3) showed that only 2,4- disubstituted phenyl compounds (℃ and 11d) were tolerated and all other modifications caused significant activity loss,which indicated that the P1 pocket might be rigid and sensitive to the substituents on the phenyl ring.

Table 3
Selected data for indole scaffold analogs (R2substitution).

In general,the indole sulfonamide analogs are stable in the liver microsome metabolic stability test (see Table 1),indicating that indole sulfonamide is a suitable scaffold for further development. DPP9 belongs to the same family of DPP4 and blocking its function could cause some undesired side effects [13]. Therefore the most potent compounds (8a-8e and 1) were also tested against DPP9 enzymatic activity,and more than 200 fold selectivity has been achieved for DPP4 over DPP9.

4. Conclusion

In conclusion,we have de novo designed and developed a series of indole DPP4 inhibitors using computer modeling and known DPP4-inhibitor complex structures. The proposed binding mode of the indole compound in the active pocket overlapped well with that observed from the co-crystal structureof DPP4 and compound 1. Preliminary SAR around indole core has been developed and a series of potent and selective DPP4 inhibitors with favorable physical properties were obtained. Further optimization around the core is in progress.


We thank Yongfen Ma and Xiao Liu for experimental assistances; and Sheng Huang and Jianhua He at the Shanghai Synchrotron Radiation Facility (SSRF) for on-site assistance. This work was supported in part by funds from the Ministry of Science and Technology (No. 2014CB910300),the Natural Science Foundation of Zhejiang Province (No. R2100439) and the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20110101110122).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at

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