Lead optimization of [(S)-γ-(arylamino)prolyl]thiazolidine focused on γ-substituent: Indoline compounds as potent DPP-IV inhibitors

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Abstract

Dipeptidyl peptidase IV (DPP-IV) inhibitors are looked to as a potential new antidiabetic agent class. A series of [(S)-γ-(arylamino)prolyl]thiazolidine compounds in which the electrophilic nitrile is removed are chemically stable DPP-IV inhibitors. To discover a structure for the γ-substituent of the proline moiety more suitable for interacting with the S2 pocket of DPP-IV, optimization focused on the γ-substituent was carried out. The indoline compound 22e showed a DPP-IV-inhibitory activity 100-fold more potent than that of the prolylthiazolidine 10 and comparable to that of NVP-DPP728. It also displayed improved inhibitory selectivity for DPP-IV over DPP8 and DPP9 compared to compound 10. Indoline compounds such as 22e have a rigid conformation with double restriction of the aromatic moiety by proline and indoline structures to promote interaction with the binding site in the S2 pocket of DPP-IV. The double restriction effect provides a potent inhibitory activity which compensates for the decrease in activity caused by removing the electrophilic nitrile.

Graphical abstract

Lead optimization of the DPP-IV inhibitor [(S)-γ-(arylamino)prolyl]thiazolidine was conducted with focus on the γ-substituent. Compounds with an indoline structure at the γ-position showed potent activity, with the representative compound 22e 100-fold more potent than the prolylthiazolidine 10 and comparable to NVP-DPP728 despite its lack of an electrophilic group.

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Introduction

Dipeptidyl peptidase IV (EC 3.4.14.5, DPP-IV) is a serine protease which recognizes an amino acid sequence having proline or alanine at the second position from the N-terminal and produces dipeptide.1 DPP-IV is widely distributed in mammalian tissues and plays several physiological roles; in particular its role as a peptidase that rapidly inactivates glucagon-like peptide 1 (GLP-1) has drawn interest.2 GLP-1 is secreted in response to meal ingestion and stimulates insulin secretion.3 It has been suggested that potentiation and extension of the action of GLP-1 by DPP-IV inhibition would stimulate insulin secretion after meal ingestion only,4 and DPP-IV inhibitors have therefore come to be seen as a potential new type of antidiabetic agent free of side effects such as hypoglycemia and exhaustion of pancreatic beta-cells. In particular a potent and long-acting inhibitor might offer advantages in exploiting DPP-IV inhibition. Recent in vivo studies indicate that inhibitory selectivity for DPP-IV over other related prolyl dipeptidases, such as DPP8 and DPP9, is one of the key issues for clinical use since inhibition of DPP8 and/or DPP9 has produced profound toxicity in animal studies.5

Several DPP-IV inhibitors have been reported (Fig. 1),6 a number of which are substrate analogs of the P2-P1 fragment. As proline mimics at the P1 part, (S)-2-cyanopyrrolidine and thiazolidine structures are used. (S)-2-Cyanopyrrolidine inhibitors, for example, NVP-DPP728 (1),7, 8, 9 LAF237 (2),9, 10 and BMS-477118 (3),11 which contain a nitrile group as an electrophilic trap for the Ser630 of the catalytic triad, have been reported as potent inhibitors. Thiazolidine inhibitors, which lack an electrophilic nitrile group, are generally of only modest potency. In contrast, P32/98 (4) improved glucose tolerance in patients with diabetes and in healthy volunteers despite exhibiting intrinsic moderate inhibitory properties.12, 13

We previously reported that a series of [(S)-γ-(arylamino)prolyl]-(S)-2-cyanopyrrolidine compounds had a potent inhibitory activity (Fig. 2b).14 The representative compound 5 is an analog conformationally restricted using a proline structure and which mimics the folded conformation of the conformationally flexible compound NVP-DPP728 to allow interaction of the (5-cyano-2-pyridyl)amino moiety with the S2 pocket as shown in Figure 2a. As a result, compound 5 has 5-fold more potent inhibitory activity than NVP-DPP728. In addition, compound 5 with an arylamino group introduced at the γ-position has over 10-fold more potent inhibitory activity than the prolyl-(S)-2-cyanopyrrolidine 616 and the (R)-isomer 7.14

However, the series of [(S)-γ-(arylamino)prolyl]-(S)-2-cyanopyrrolidine compounds seems to be unstable in neutral aqueous solution (e.g., for compound 5, T1/2decomp = 1.3 h at pH 6.8 aqueous solution15). The instability may result from the intra-molecular cyclization of the nucleophilic amine in the proline moiety and the electrophilic nitrile in the (S)-2-cyanopyrrolidine moiety. We previously reported that compound 8, in which the (S)-2-cyanopyrrolidine moiety is converted to a thiazolidine structure, has improved chemical stability and a long half-life in plasma (t1/2 = 5.27 h); but compound 8 showed 100-fold decreased inhibitory activity compared to the (S)-2-cyanopyrrolidine compound 5 (Fig. 2b and c). This effect demonstrates that the nitrile group of (S)-2-cyanopyrrolidine compounds plays a dominant part in the exhibition of inhibitory activity by realizing interaction with the Ser630 of the catalytic triad. On the other hand, the thiazolidine analogs have displayed similar features to the (S)-2-cyanopyrrolidine compounds; the restricted compound 8 is 10-fold more potent than the flexible compound 917 and 20-fold more potent than the prolylthiazolidine 10. These results indicate that introducing a substituent at the γ-position of the proline moiety of the prolylthiazolidine core structure provides potent DPP-IV inhibition with improved chemical stability and a good pharmacokinetic profile.15

In the present study, to compensate for the decrease in activity caused by the conversion of the (S)-2-cyanopyrrolidine moiety to a thiazolidine structure, we optimized the arylamino structure at the γ-position of the proline moiety. The resulting compound 22e (IC50 = 4.8 nmol/L), with the aromatic moiety at the γ-position restricted using an indoline structure, is 100-fold more potent than the prolylthiazolidine 10 (IC50 = 538 nmol/L) and has comparable activity to NVP-DPP728 (IC50 = 1.4 nmol/L) despite its lack of an electrophilic nitrile. Its inhibitory selectivity for DPP-IV over DPP8 and DPP9 is ca. 100-fold. The rigid conformation realized by the double restriction with proline and indoline structures is concluded to be effective for improving DPP-IV-inhibitory activity.

Section snippets

Chemistry

Compounds bearing a non-cyclized nitrogen at the γ-position of the proline moiety of the ((S)-γ-substituted prolyl)thiazolidine structure (15ah, 17ai) were prepared from ((S)-γ-aminoprolyl)thiazolidine 1315 (Scheme 1). Introduction of an alkyl, acyl, sulfonyl or carbamoyl group at the γ-amino group of 13, followed by removal of the Boc group with HCl/AcOEt, afforded the N-mono-introduced compounds 15ah. The further modified compounds 17ai were prepared by alkylation or acylation of the

Results and discussion

The ((S)-γ-substituted prolyl)thiazolidine and related compounds were evaluated for DPP-IV-inhibitory activity in human and rat plasma by fluorescence assay using Gly-Pro-MCA (Table 1, Table 2, Table 3, Table 4).

In a previous study of ((S)-γ-substituted prolyl)-(S)-2-cyanopyrrolidine, we noted the importance of the aromatic moiety at the γ-position of the proline moiety for DPP-IV inhibition.14 First, we examined various spacers between this aromatic moiety and the proline moiety. In [(S

Conclusion

A series of [(S)-γ-(arylamino)prolyl]thiazolidine compounds with the electrophilic nitrile removed are chemically stable DPP-IV inhibitors.15 In the present study, we carried out optimization focused on the γ-substituent of these compounds. The resulting compounds bearing an indoline structure at the γ-position showed potent DPP-IV inhibitory activity. The representative compound 22e (IC50 = 4.8 nmol/L) had 50-fold more potent activity than the flexible compound 9 (IC50 =  270 nmol/L) and 100-fold

Chemistry

1H NMR spectra were measured on a Bruker DPX-300 instrument or on a Bruker AMX-500 with tetramethylsilane as the internal standard; chemical shifts are reported in parts per million (ppm, δ units). Splitting patterns are designated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; br s, broad singlet. Mass spectra (MS) were recorded on a JEOL JMS-700 instrument operating in the chemical ionization (CI) mode. Electron analysis for carbon, hydrogen, and

Acknowledgments

We thank Kumiko Yoshida and Tadashi Hatano for their technical support. We also thank Dr. Tohru Nakajima and Dr. Takao Kondo for their insight and guidance during the course of this work.

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