Novel isoindoline compounds for potent and selective inhibition of prolyl dipeptidase DPP8

Article abstract of DOI:10.1016/j.bmcl.2004.11.023

DPP8 is a prolyl dipeptidase homologous to DPP-IV, which is a drug target for Type II diabetes. The biological function of DPP8 is not known. To identify potent and selective chemical compounds against DPP8, we have synthesized a series of isoquinoline an

Full text of DOI:10.1016/j.bmcl.2004.11.023

Bioorganic & Medicinal Chemistry Letters 15 (2005) 687–691
Novel isoindoline compounds for potent and selective inhibition
of prolyl dipeptidase DPP8
Weir-Torn Jiaang,a,* Yuan-Shou Chen,a Tsu Hsu,a Ssu-Hui Wu,a Chia-Hui Chien,a
Chung-Nien Chang,a Sheng-Ping Chang,b Shiow-Ju Leea and Xin Chena,*
aDivision of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 161,
Sec. 6, Minchiuan E. Rd., Neihu, Taipei 114, Taiwan, ROC
bDepartment of Obstetrics and Gynecology, Taipei Veterans General Hospital, No. 201,
Sec. 2, Shih-Pai Road, Taipei 112, Taiwan, ROC
Received 17 August 2004; revised 11 November 2004; accepted 11 November 2004
Available online 2 December 2004
Abstract—DPP8 is a prolyl dipeptidase homologous to DPP-IV, which is a drug target for Type II diabetes. The biological function
of DPP8 is not known. To identify potent and selective chemical compounds against DPP8, we have synthesized a series of isoquino-
line and isoindoline derivatives and have tested their inhibitory activity against DPP8, DPP-IV and DPP-II. Isoindoline derivatives
were found to be more potent DPP8 inhibitors than isoquinoline derivatives. Isoindoline with a 1-(4,40-difluor-benzhydryl)-piper-
azine group at the P2 site was observed to be a very potent DPP8 inhibitor, having an IC50 value of 14nM with at least a 2500-fold
selectivity over either DPP-IV or DPP-II. From SAR results, we speculate that the S1 site of DPP8 may be larger than that of DPP-
IV, which would allow the accommodation of larger C-terminal residues, such as isoquinoline or isoindoline.
Ó 2004 Elsevier Ltd. All rights reserved.
Dipeptidylpeptidase IV (DPP-IV, also known as CD26)
(EC 3.4.14.5) is a drug target for Type II diabetes. It is a
serine protease involved in the in vivo degradation of
two insulin-sensing hormones, glucagon-like peptide-1
(GLP-1) and glucose-dependent insulinotropic polypep-
tide (GIP).1,2 By extending the duration of the action by
GLP-1, animal studies preformed using DPP-IV inhibi-
tors have demonstrated stimulated insulin secretion,
inhibition of glucagon release, and slow gastric empty-
ing. Each of these effects has benefits in the control of
glucose homeostasis.3 DPP-IV knockout mice and rats
consistently display healthy phenotypes.4 When pre-
sented with a high concentration of glucose, these ani-
mals show an improved glucose tolerance, enhanced
insulin secretion, and an increased circulating GLP
and GIP activity.4 Therefore, selective and potent inhibi-
tors of the DPP-IV protein are effective in the treatment
of Type II diabetes.
DPP-IV belongs to the prolyl dipeptidase family, which
also includes DPP-II, DPP8 and DPP9.5 All these pro-
teases show prolyl dipeptidase activity by cleaving the
peptide bond at the penultimate proline residue.5 The
biological importance of these prolyl-cleaving pepti-
dases is supported by the fact that their known in vivo
substrates, that is, hormones, chemokines and neuro-
peptides, contain one or more proline residues, prevent-
ing their degradation by most proteases in vivo. As a
result, the processing and degradation of these peptides
requires the presence of these prolyl peptidases. Not sur-
prisingly, some prolyl peptidases have been found to
have important biological functions and are effective
drug targets, for example, DPP-IV and prolyl oligo-
peptidase.5,6
The in vivo functions of prolyl dipeptidases, other than
DPP-IV, are largely unknown. DPP-II is also known as
quiescent cell proline dipeptidase (QPP) or DPP-VII
(DPP7).5,7 The inhibition of DPP-II has been shown
to result in the apoptosis of quiescent T-cells.8 DPP8 is
a cytoplasmic protease with a 51% homology in amino
acid level with DPP-IV.9 Even though the in vivo func-
tion of DPP8 is not known, the administration of selec-
tive DPP8/DPP9 inhibitors in animals results in severe
Keywords: DPP8; Isoindoline; Isoquinoline; Prolyl dipeptidase; Type II
diabetes.
*
Corresponding authors. Tel.: +88 62 26534401x6113; fax: +88 62
27890264 (X.C.); e-mail addresses: wtjiaang@nhri.org.tw; xchen@
nhri.org.tw
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.023

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W.-T. Jiaang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 687–691
Table 1. Inhibition of DPP-IV, DPP8 and DPP-II by compounds 1–6a
toxic reactions, including alopecia, thrombocytopenia,
anemia, enlarged spleen, multiple histological patholo-
gies and increased mortality.10 In light of the physiolog-
ical importance of prolyl cleaving enzymes and the
toxicity associated with DPP8/DPP9, it is important to
have selective inhibitors targeting to each enzyme for
functional studies in vivo. In this work, we report on a
systematic search for novel, potent and selective DPP8
inhibition by isoindoline compounds.
Compd
DPP-IV
IC50 (nM)
DPP8
IC50 (nM)
DPP-II
IC50 (nM)
1
2
3
4
5
6
51
53
14,219
4573
27
>100,000
26,520
39,873
12
1660
63
364
19
68,985
31,259
>100,000
274
16
a IC50 determination was carried out as described in Ref. 12.
There are a number of DPP-IV inhibitors known
(Fig. 1). The cyanopyrrolidine derivatives 1 and 2 are
potent DPP-IV inhibitors developed by Novartis (Table
1).11,12 Compound 1 (also known as NVP-LAF237) has
progressed into mid-stage clinical trials and is showing
positive and encouraging results in the treatment of
Type II diabetes. Cyclohexylglycine-(2S)-cyanopyroli-
dine 3 is a potent DPP-IV inhibitor with an IC50 value
of 12nM.13 However, a related compound lacking a ni-
trile group, isoleucylthiazolidide 4 (Ile-Thia, or P32/98),
only shows modest DPP-IV inhibition potency, with an
IC50 value of 1.66lM.14 This compound has been shown
to be effective in improving glucose tolerance in both
healthy and NIDDM patients.15 During the course of
investigating inhibitors targeting DPP-IV, we have iden-
tified two novel and potent DPP-IV inhibitors: com-
pounds 5 and 6, which have IC50 values of 63 and
274nM, respectively. Structure–activity relationship
(SAR) studies have been carried out on these com-
pounds and the results will be published elsewhere.
There is a lack of structural information on the DPP8
protein. Due to the amino acid homology between
DPP-IV and DPP8, we speculate that they may have a
similar substrate-binding pocket. It is likely that some
DPP-IV inhibitors may have an inhibitory effect on
DPP8. Thus, the IC50 values for the inhibition of
DPP8 for compounds 1–6, were determined. As shown
in Table 1, N-substituted glycines at the P2 site and
the (2S)-cyanopyrrolidine at the P1 site (1 and 2) were
weak DPP8 inhibitors, with IC50 values of 14.2lM
and 4.5lM, respectively. However, a-carbon substituted
glycine at the P2 site (3–6) dramatically improved the
potency for DPP8 inhibition, showing IC50 values rang-
ing from 19 to 364nM. Thiazolidine (4) or (2S)-cyano-
pyrrolidine (5 and 6) at the P1 site yielded very potent
DPP8 inhibitors, though the selectivity shown between
DPP8 and DPP-IV was low (Table 1). Except for 1
and 6, compounds 2–5 showed weak inhibition against
DPP-II, with IC50 values ranging from 27 to 69lM
(Table 1).
Figure 1. Inhibitors of DPP-IV.
Table 2. Inhibitory activity of isoquinoline derivatives
Compd
P2
R
DPP8 IC50 (nM)
DPP-IV IC50 (nM)
DPP-II IC50 (nM)
7
Chga
H
3016
5962
>100,000
>100,000
>100,000
>100,000
>100,000
13,000
>100,000
>100,000
>100,000
>100,000
>100,000
>100,000
8
Chga
CN
H
9
PHgb
3,4-Difluoro-Phe
>100,000
>100,000
>100,000
>100,000
10
11
12
H
Pro
Trp
H
CN
a Cha = cyclohexylglycine.
b PHg = phenylglycine.

W.-T. Jiaang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 687–691
Table 3. Structure–activity relationship at C-terminal P1 site
689
Compd
R
DPP8 IC50 (nM)
27
DPP-IV IC50 (nM)
12
DPP-II IC50 (nM)
3
39,873
>100,000
27,357
7
3016
1448
78
>100,000
31,000
1034
13
14
15
>100,000
16,520
24
15,000
To improve on the selectivity and to maintain the po-
tency for DPP8, we replaced the P1 site of compound
3 with isoquinoline, generating (S)-cyclohexylglycine
isoquinoline 7. Compound 7 inhibited DPP8, showing
an IC50 value of 3lM, and no detectable DPP-IV or
DPP-II inhibitory activity (Table 2). The addition of
a nitrile group on isoquinoline, forming the 3-cyano
derivative 8, did not improve potency, nor enhance the
selectivity towards DPP8. Therefore, isoquinoline com-
pounds conferred moderate selectivity towards DPP8,
with no DPP-IV or DPP-II inhibitory activity detected.
Due to the weak activity of compounds 7 and 8, we
focused further optimization on the N-terminal P2 site.
Phenylglycine at the P2 site with isoquinoline 9 resulted
in a complete loss of inhibitory activity against DPP8,
DPP-IV, and DPP-II. Similarly, compounds with 3,4-
difluoro-Phe 10 or Pro 11 groups at the P2 site did not
inhibit any of these proteases. A Trp group at the P2 site
with 3-cyanoisoquinoline 12 did not exhibit DPP8 or
DPP-II inhibition, although it did exhibit a weak inhib-
itory activity towards DPP-IV, showing an IC50 value of
13lM.
the SI value increased to 625- and 688-fold over DPP-
IV and DPP-II, respectively. Thus, compound 15 is a
potent DPP8 inhibitor, with a good selectivity against
either DPP-IV or DPP-II .
Next, we aimed to improve further the selectivity of the
isoindoline compound 15 over DPP-IV and DPP-II,
preferably by eliminating any detectable inhibitory
activity. Since compounds 5 and 6 exhibited potent
DPP8 inhibition with an aspartic acid derivative at the
P2 site (Table 1), a series of isoindolines with aspartic
acid derivatives at the P2 site, compounds 16–18, were
synthesized (Scheme 1). The synthetic strategy was as
follows. Compounds 16–18 were synthesized from
Isoquinoline at P1 site may not be the optimum site for
DPP8 inhibition, and further modification on the P1 site
was therefore carried out. First, we replaced the isoquino-
line by other ring structures, piperidine 13, pyrrolidine 14
and isoindoline 15 (Table 3). Compared to isoquinoline
7, piperidine 13 was twice as active towards DPP8 inhi-
bition, but retained a weak DPP-IV and DPP-II inhibi-
tory activity. Upon decreasing the ring size to a
five-member ring, the pyrrolidine 14 gave an IC50 value
of 78nM and 1 lM against DPP8 and DPP-IV, respec-
tively, with no inhibitory activity observed against
DPP-II.14 The selectivity index (SI) of 14 for DPP8 ver-
sus DPP-IV inhibition was about 13-fold. Introduction
of isoindoline at the P1 site 15 greatly improved DPP8
inhibition, showing an IC50 value of 24nM. Moreover,
Scheme 1. Synthesis of aspartic acid derivatives: (a) DCC, HOSu, l,4-
dioxane/CH2Cl2; (b) H2, 5% Pd–C, MeOH; (c) DCC, dichlorome-
thane; (d) TFA, CH2Cl2.

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W.-T. Jiaang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 687–691
Table 4. Inhibitory activity and selectivity of isoindoline derivatives
Compd
R
DPP8 IC50 (nM)
555
DPP-IV IC50 (nM)
>100,000
DPP-II IC50 (nM)
20,891
16
17
18
150
>100,000
>100,000
>100,000
14
38,377
Boc-L-aspartic acid 4-benzyl ester 19; the DCC coupling
of 19 with isoindoline was followed by hydrogenation to
give acid 20. Compound 20 was then coupled with the
primary or secondary amines, 1,2,3,4-tetrahydroiso-
against DPP8, DPP-IV and DPP-II. Isoquinoline deri-
vatives are weak DPP8 inhibitors, with IC50 values of
3–6lM, though they generally lack any activity against
DPP-IV or DPP-II. Excitingly, isoindoline derivatives
are very potent DPP8 inhibitors, with IC50 values below
20nM. Isoindoline 18 with 1-(4,40-difluor-benzhydryl)-
piperazine at the P2 site is a very potent DPP8 inhibitor,
with an IC50 value at 14nM: a 2500-fold selectivity over
either DPP-IV or DPP-II. From the SAR studies pre-
sented in this paper, we speculate that the S1 site of
DPP8 may be larger than that of DPP-IV, allowing
the accommodation of larger C-terminal residues, such
as isoquinoline or isoindoline, at the P1 site.
quinoline,
6,7-dimethoxy-1,2,3,4-tetrahydroisoquino-
line, or 1-(4,40-difluor-benzhydryl)-piperazine to yield
21. Removal of the Boc protecting group of 21 with tri-
fluoroacetic acid yielded the desired 4-substituted aspar-
tic acid derivatives 16, 17 and 18, respectively.
As shown in Table 4, by changing 2-cyanopyrrolidine to
isoindoline in compounds 16–18, the selectivity for
DPP8 increased dramatically. All these isoindoline com-
pounds showed varying degrees of DPP8 inhibition with
no detectable DPP-IV inhibitory activity. Compounds
16 and 18 showed very weak DPP-II inhibitory activity,
while 17 did not inhibit DPP-II at all. Among these
compounds, compound 18 was a very potent inhibitor
for DPP8, with an IC50 value of 14nM: a 2500-fold
selectivity over DPP-IV and DPP-II.
Acknowledgements
We would like to thank Dr. Yu-Sheng Chao for his sup-
port and encouragement, and Dr. Peter Nell for criti-
cally reading the manuscript and making suggestions.
The study was financially supported by National Health
Research Institutes, Taiwan.
The crystal structure of the DPP8 protein has yet to be
elucidated. Our results suggest that the S1 site of DPP8
is larger than that of DPP-IV, and can thus allow larger
moieties, such as isoquinoline 7 and 8 and isoindoline
15–18, to be accommodated. As a result, larger P1
groups with either isoindoline 15–18 or isoquinoline
derivatives 7 and 8 were more potent inhibitors towards
DPP8 than DPP-IV. Consistently, smaller sized moie-
ties, such as the five-member ring pyrrolidine derivatives
3–6 and 14, inhibited DPP8 as well as DPP-IV, while the
six-membered ring piperidine 13 inhibited DPP8 by a
magnitude of twenty times better than it did DPP-IV.
Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/
j.bmcl.2004.11.023.
References and notes
1. Mentlein, R. Regul. Pept. 1999, 85, 9.
In summary, to find potent and selective chemical com-
pounds against prolyl dipeptidase DPP8, we have syn-
thesized a series of isoquinoline and isoindoline
derivatives, and have tested their inhibitory activity
2. Zhu, L.; Tamvakopoulos, C.; Xie, D.; Dragovic, J.; Shen,
X.; Fenyk-Melody, J. E.; Schmidt, K.; Bagchi, A.; Griffin,
P. R.; Thornberry, N. A.; Roy, R. S. J. Biol. Chem. 2003,
278, 22418.

W.-T. Jiaang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 687–691
691
3. (a) Pospisilik, J. A.; Stafford, S. G.; Demuth, H. U.;
McIntosh, C. H.; Pederson, R. A. Diabetes 2002, 51, 2677;
(b) Ahren, B.; Holst, J. J.; Martensson, H.; Balkan, B. Eur.
J. Pharmacol. 2000, 404, 239; (c) Villhauer, E. B.;
Brinkman, J. A.; Naderi, G. B.; Dunning, B. E.; Mangold,
B. L.; Mone, M. D.; Russell, M. E.; Weldon, S. C.;
Hughes, T. E. J. Med. Chem. 2002, 45, 2362; (d) Deacon,
C. F.; Danielsen, P.; Klarskov, L.; Olesen, M.; Holst, J. J.
Diabetes 2001, 50, 1588; (e) Sudre, B.; Broqua, P.; White,
R. B.; Ashworth, D.; Evans, D. M.; Haigh, R.; Junien, J.
L.; Aubert, M. L. Diabetes 2002, 51, 1461.
4. (a) Marguet, D.; Baggio, L.; Kobayashi, T.; Bernard, A.
M.; Pierres, M.; Nielsen, P. F.; Ribel, U.; Watanabe, T.;
Drucker, D. J.; Wagtmann, N. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 6874; (b) Conarello, S. L.; Li, Z.; Ronan,
J.; Roy, R. S.; Zhu, L.; Jiang, G.; Liu, F.; Woods, J.;
Zycband, E.; Moller, D. E.; Thornberry, N. A.; Zhang, B.
B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6825; (c)
Nagakura, T.; Yasuda, N.; Yamazaki, K.; Ikuta, H.;
Yoshikawa, S.; Asano, O.; Tanaka, I. Biochem. Biophys.
Res. Commun. 2001, 284, 501.
L.; Russell, M. E.; Hughes, T. E. J. Med. Chem. 2003, 46,
2774; (b) Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.;
Dunning, B. E.; Mangold, B. L.; Mone, M. D.; Russell,
M. E.; Weldon, S. C.; Hughes, T. E. J. Med. Chem. 2002,
45, 2362.
12. The IC50 determinations were performed as described in
Ref. 8 and were measured at least twice with independent
triplicate samples. The data obtained were reproducible
with less than 15% difference in the independent experi-
ments. The standard deviation in each data point was less
than 15% of the IC50 value presented. Sigma plots were
used to obtain the IC50 values. DPP-IV and DPP8 were
purified as described by Chen et al. in ÔProtein Expression
and PurificationÕ, 2004; Vol. 35, pp 142–146. The Purifi-
cation of DPP-II was carried out as described in Ref. 8
with the modification. The detailed expression and puri-
fication of DPP-II will be described elsewhere. The buffers
used in the DPP-IV, DPP8 and DPP-II assays were 2mM
Tris–HCl at pH8.0, PBS buffer and 125mM of sodium
phosphate buffer at pH5.5, respectively. The substrates
used for DPP-IV and DPP8 were Gly-Pro-pNA at
concentrations of 0.5mM and 2.5mM, respectively, while
the substrate used for DPP-II was Lys-Ala-pNA at
concentration of 1mM.
5. Rosenblum, J. S.; Kozarich, J. W. Curr. Opin. Chem. Biol.
2003, 7, 496.
6. Polgar, L. Cell. Mol. Life Sci. 2002, 59, 349.
7. Chiravuri, M.; Huber, B. T. Apoptosis 2000, 5, 319.
8. Leiting, B.; Pryor, K. D.; Wu, J. K.; Marsilio, F.; Patel, R.
A.; Craik, C. S.; Ellman, J. A.; Cummings, R. T.;
Thornberry, N. A. Biochem. J. 2003, 371, 525.
9. Abbott, C. A.; Yu, D. M.; Woollatt, E.; Sutherland, G.
R.; McCaughan, G. W.; Gorrell, M. D. Eur. J. Biochem.
2000, 267, 6140.
10. Lankas, G.; Leiting, B.; Roy, R. S.; Eiermann, G.; Biftu,
T.; Kim, D.; Ok, H.; Weber, A.; Thornberry, N. A.
Abstracts, 7-OR, 63rd Annual American Diabetes Asso-
ciation meeting, Orlando, FL, June 2004; American
Diabetes Association.
13. Ashworth, D. M.; Atrash, B.; Baker, G. R.; Baxter, A. J.;
Jenkins, P. D.; Jones, D. M.; Szelke, M. Bioorg. Med.
Chem. Lett. 1996, 6, 1163.
14. Parmee, E. R.; He, J.; Mastracchio, A.; Edmondson, S.
D.; Colwell, L. E.; Eiermann, G.; Feeney, W. P.; Habu-
lihaz, B.; He, H.; Kilburn, R.; Leiting, B.; Lyons, K.;
Marsilio, F.; Patel, R.; Petrov, A.; di Salvo, J.; Wu, J. K.;
Thornberry, N.; Weber, A. E. Bioorg. Med. Chem. Lett.
2004, 14, 43.
15. (a) Pospisilik, J. A.; Sta.ord, S. G.; Demuth, H.-U.;
Brownsey, R.; Parkhouse, W.; Finegood, D. T.; McIn-
tosh, C. H. S.; Pederson, R. A. Diabetes 2002, 51, 943; (b)
Sorbera, L. A.; Revel, L.; Castaner, J. Drugs Future 2001,
26, 859.
11. (a) Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.;
Burkey, B. F.; Dunning, B. E.; Prasad, K.; Mangold, B.