Discovery of potent and selective orally bioavailable β-substituted phenylalanine derived dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.04.028

anti-Substituted biaryl β-methylphenylalanine derived amides have been shown to be potent DPP-IV inhibitors that suffer from suboptimal selectivity and pharmacokinetics. This letter describes the substitution of the β-methyl substituent with β-polar subst

Full text of DOI:10.1016/j.bmcl.2005.04.028

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3048–3052
Discovery of potent and selective orally
bioavailable b-substituted phenylalanine derived
dipeptidyl peptidase IV inhibitors
Scott D. Edmondson,a,* Anthony Mastracchio,a Joseph L. Duffy,a George J. Eiermann,c
Huaibing He,a Ida Ita,a Barbara Leiting,b Joseph F. Leone,a Kathryn A. Lyons,a
Amanda M. Makarewicz,a Reshma A. Patel,b Aleksandr Petrov,c Joseph K. Wu,b
Nancy A. Thornberryb and Ann E. Webera
aDepartment of Medicinal Chemistry, Merck & Co. Inc., PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Metabolic Disorders, Merck & Co. Inc., PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Pharmacology, Merck & Co. Inc., PO Box 2000, Rahway, NJ 07065, USA
Received 23 March 2005; revised 11 April 2005; accepted 15 April 2005
Abstract—anti-Substituted biaryl b-methylphenylalanine derived amides have been shown to be potent DPP-IV inhibitors that suffer
from suboptimal selectivity and pharmacokinetics. This letter describes the substitution of the b-methyl substituent with b-polar
substituents, culminating in the discovery of a b-dimethylamide substituted phenylalanine derivative with an excellent potency,
selectivity, and pharmacokinetic profile.
Ó 2005 Elsevier Ltd. All rights reserved.
Inhibition of dipeptidyl peptidase IV (DPP-IV) has re-
cently emerged as a promising new approach for the
treatment of type 2 diabetes mellitus.1 DPP-IV is the
enzyme responsible for inactivation of the incretin hor-
mones glucagon-like peptide 1 (GLP-1) and glucose-
dependent insulinotropic polypeptide (GIP). These two
hormones are secreted in response to nutrient ingestion,
and each enhances the glucose-dependent secretion of
insulin. Furthermore, GLP-1 has been shown in mam-
mals to stimulate insulin biosynthesis, inhibit glucagon
secretion, slow gastric emptying, reduce appetite, and
stimulate the regeneration and differentiation of islet
b-cells.1,2 More recently, the GLP-1 receptor has also
been implicated in learning and neuroprotection.3
and GIP levels, which leads to decreased blood glucose
levels, hemoglobin A1c levels, and glucagon levels.1,4
DPP-IV inhibitors offer a number of potential advanta-
ges over existing diabetes therapies including a lowered
risk of hypoglycemia, the potential for weight loss,
and the potential for the regeneration and differentiation
of pancreatic b-cells.
Previous work from these laboratories describes the con-
version of a moderately potent and poorly selective
dipeptidyl peptidase IV (DPP-IV) inhibitor (1) to potent
and selective b-methylphenylalanine derived DPP-IV
inhibitors with the general structure 2 (Fig. 1).5 The con-
version of the ethyl group of 1 to a biaryl group (2) in-
creased potency against DPP-IV as well as selectivity
over DPP8 and DPP9. Furthermore, the incorporation
of polar aryl groups into 2 further increased DPP-IV po-
tency and provided additional selectivity over off-target
enzymes (e.g., quiescent peptidyl peptidase—QPP) and
hERG.6 Unfortunately, the latter benefits were achieved
at the expense of inferior rat pharmacokinetic profiles.
Previous work in this series demonstrated that more ste-
rically demanding groups could be substituted for the b-
methyl group of 2 without sacrificing potency at
Due to rapid processing of GLP-1 and GIP by DPP-IV,
the half-lives of the active peptides in blood are extre-
mely short. Consequently, inhibition of DPP-IV in hu-
mans has been shown to increase circulating GLP-1
Keywords: Dipeptidyl peptidase IV; DPP-IV; DPP8; DPP9; QPP.
*
Corresponding author. Tel.: +1 732 594 0287; fax: +1 732 594
5350; e-mail: scott_edmondson@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.028

S. D. Edmondson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3048–3052
3049
Figure 1. Classic DPP-IV inhibitor isoleucyl thiazolidide (1) and
phenylalanine derived DPP-IV inhibitors.
Scheme 2. Synthesis of DPP-IV inhibitors 15–17. Reagents and
conditions: (a) NaBH4, MeOH; (b) NaCNBH3, (ClCH2)2, AcOH,
R2NH; (c) TFA, DCM.
DPP-IV (data not shown). We hoped to introduce more
polar substituents at the b-position (3) in order to im-
prove the potency and selectivity profile of this promis-
ing new series of DPP-IV inhibitors.
tively, reductive amination of 12 followed by deprotec-
tion afforded diamines 16 and 17.
Clearly, the b-substituent of biaryl phenylalanines plays
a significant role in DPP-IV potency and selectivity (Ta-
ble 1). Unsubstituted biaryl phenylalanine 13, for exam-
ple, was more than an order of magnitude less potent
against DPP-IV than the b-methyl analog 14.5 Potency
could be further improved by changing the b-methyl
substituent from analog 14 to a carboxylic acid moiety.
Acid 18 (DPP-IV IC50 = 6.6 nM) was 10 times more po-
tent against DPP-IV and showed a superior overall
selectivity profile (Table 1).11 Similarly, dimethylamide
Inhibitors were synthesized using an effective combina-
tion of a chiral CBS reduction with a Kazmaier–Claisen
rearrangement (Scheme 1). The route begins with a Hor-
ner–Emmons reaction of aldehyde 4 to give the corre-
sponding methyl styrenone. A chiral reduction of this
enone with the (R)-2-methyl-CBS-oxazaborolidine re-
agent (R-CBS) then afforded allylic alcohol 5 in excellent
yield and enantioselectivity (typically 80–90% ee).7 The
optical activity could be further enriched (>98% ee) by
recrystallization from cyclohexane. Next, the allylic
alcohol was coupled with Boc-glycine to give 6. Expo-
sure of 6 to KazmaierÕs enolate-Claisen rearrangement
conditions8 followed by esterification of the resulting
acid with trimethylsilyl diazomethane then afforded N-
Boc amino ester 7 in excellent yield and stereoselectivity.
Hydrolysis of the ester was followed by coupling with
(S)-3-fluoropyrrolidine9 to give the corresponding
amide. Subsequent coupling of the aryl bromide with
aryl boronic acids via Suzuki coupling then gave 8.
Ozonolysis of 8 followed by oxidation of the resulting
aldehyde yielded acid 10, which could be deprotected
to afford analog 18.10 Alternatively, 10 could be coupled
with amines then deprotected to afford amides 20–22
and 24–53. The primary amide (11, R, R0 = H) was
dehydrated to the nitrile, which was then converted to
the corresponding tetrazole and deprotected to afford
23.
Table 1. Effects of polar substituents at the b-position of biaryl
phenylalanines
Compd
X =
IC50 (lM)
DPP-IV QPP DPP8
DPP9
13
14
15
16
17
18
19
20
21
22
23
H
Me
0.98
>100
2.7
15
2.5
0.7
>100
>100
87
>100
86
0.064
0.39
1.6
CH2OH
CH2NMe2
CH2N(CH2)4
CO2H
>100
16
49
>100
>100
>100
>100
>100
>100
>100
69
3.5
0.0066
0.22
0.11
>100
>100
>100
41
CO2Me
CONH2
CONHMe
CONMe2
Tetrazole
21
>100
>100
45
0.037
0.012
0.192
Additional analogs were synthesized from aldehyde 12
by reduction to the corresponding alcohol followed by
deprotection to give 15 (X = OH, Scheme 2). Alterna-
>100
>100
>100
>100
Scheme 1. Enantioselective synthesis of anti-substituted b-polar biaryl phenylalanine derived DPP-IV inhibitors. Reagents and conditions: (a)
(EtO)2POCH2COCH3, DBU, THF; (b) R-CBS, Catecholborane, tol, ꢀ78 ! ꢀ30 °C; (c) EDC, HOBt, Boc-Gly, DIEA, DCM; (d) LHMDS, ZnCl2,
THF, ꢀ78 °C; (e) TMSCHN2, Et2O, MeOH; (f) LiOH, H2O, THF, MeOH; (g) EDC, (S)-3-fluoropyrrolidine, HOBt, DIEA, DCM; (h) ArB(OH)2,
tol, EtOH, 2 N Na2CO3, Pd(dppf)2Cl2, D; (i) O3, MeOH, DCM then Me2S; (j) NaClO2, H2O, t-BuOH, isobutylene; (k) TFA, DCM; (l) EDC, HOBt,
RRÕNH, DIEA, DCM; (m) cyanuric chloride, DMF; (n) NaN3, tol, Et3N–HCl, D.

3050
S. D. Edmondson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3048–3052
Table 3. Effect of changing the right-hand side amide
22 (DPP-IV IC50 = 12 nM) showed improved potency
and overall selectivity compared to 14. With the excep-
tion of these two analogs and monomethylamide 21,
the remainder of the b-polar groups gave analogs with
reduced potency and/or selectivity against DPP-IV.
Consequently, an investigation of various amide groups
at the b-position was initiated.
Compd
X =
IC50 (lM)
DPP-IV
0.10
QPP
DPP8
>100
DPP9
>100
With the exception of aminotetetrazole 28, tertiary
amides at the b-position were more potent against
DPP-IV than secondary amides (Table 2). Tetrazole 28
was equipotent to 22, but suffered from a drop in selec-
tivity against DPP8. Pyrrolidide amide 32 proved to be
the most potent of the tertiary amides (DPP-IV
IC50 = 9.2 nM), but this analog also showed reduced
selectivity against all of the counterscreens. Attention
was next focused on replacing the fluoropyrrolidide moi-
ety, which is an effective P1-site proline mimic for DPP-
IV.9,12
35
36
–N(CH2)3
>100
58
0.017
>100
>100
37
38
0.029
9.4
>100
22
>100
66
–N(CH2)4
0.0075
26
94
39
0.030
37
>100
40
41
42
0.027
0.0091
0.230
7.1
8.5
75
8.0
46
26
In general, only small changes were tolerated at the P1-
site (Table 3). While four- and five-membered rings each
showed good activity against DPP-IV, a substantial
drop in potency was observed with six-membered rings
(DPP-IV IC50 >500 nM for 44 and 45). Pyrrolidide 38,
thiazolidide 41, and cyanopyrrolidide 43 had compara-
ble or increased potency relative to 22, but each of these
analogs exhibited increased inhibition of DPP8 and
DPP9. Especially noteworthy was the very potent
cyanopyrrolidide 43 (DPP-IV IC50 = 3.3 nM), which
shows significant activity at both DPP8 (IC50 = 2.1 lM)
and DPP9 (IC50 = 2.2 lM). Since activity in these
homologs has been correlated with toxicity in animals,13
further pursuit of this analog was halted. Also notewor-
thy was fluoroazetidide 36, which was similar to 22 with
respect to DPP-IV potency and selectivity.
13
>100
>100
43
0.0033
40
2.1
2.6
44
45
–N(CH2)5
0.55
0.52
38
77
>100
>100
>100
>100
Table 4. Influence of various biaryl groups
Because the introduction of polar biaryl groups afforded
increased potency and selectivity in the b-methyl series,5
we decided to incorporate some of the improved aryl
Compd Ar =
IC50 (lM)
DPP-IV QPP DPP8
DPP9
46
47
48
49
50
51
52
53
2,4-F2Ph
3,4-F2Ph
2,5-F2Ph
3-CNPh
0.013
0.021
0.056
0.043
15
44
11
57
29
81
57
89
40
14
>100
81
Table 2. Effects of amide groups at the b-position of biaryl phenyl-
alanines
6.9
0.46
>100
>100
57
3-(Tetrazole)Ph 0.0022
>100
>100
>100
100
3-(MeSO2)Ph
3-pyr
4-pyr
0.021
0.019
0.020
>100
12
>100
Compd –NRR0 =
IC50 (lM)
DPP-IV QPP DPP8 DPP9
groups into the b-dimethylamide scaffold (Table 4). As
expected, polar aryl groups afforded compounds with
comparable or improved selectivity profiles. Most
noteworthy among these analogs were tetrazole 50,
methylsulfone 51, and 4-pyridyl analog 53
(IC50Õs = 2.2, 21, and 20 nM, respectively). Each of these
analogs shows acceptable selectivity profiles in the
counterscreens.
24
25
26
27
28
29
30
31
32
33
34
–NHEt
0.11
90
76
>100
26
26
>100
>100
>100
>100
>100
>100
>100
>100
62
–NHi-Pr
–NHc-Pr
–NH(CH2)2OH 0.094
0.099
0.071
55
>100
>100
43
32
19
–NHtetrazole
–NMeEt
–NEt2
0.011
0.020
0.059
0.044
0.0092
0.045
0.067
18
52
48
–N(CH2)3
–N(CH2)4
–NMe(OMe)
–NHNMe2
5.3
5.6
>100
>100
47
15
27
22
>100
>100
Inhibitors that possessed superior potency and selectivity
profiles were evaluated for their rat pharmacokinetic

S. D. Edmondson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3048–3052
3051
Table 5. Pharmacokinetic properties of selected DPP-IV inhibitors in
the rat (1/2 mpk iv/po) and hERG binding
plasma concentrations in this assay. The observed levels
of DPP-IV inhibition in the PD assay did not, however,
correspond to the expected efficacy of 22 in light of the
mouse DPP-IV potency (murine IC50 = 50 nM).
Compd Clp (mL/min/kg) t1/2 (h) F (%) hERG IC50 (lM)
18
22
28
32
38
50
0.25
4.8
3.1
3.5
16
67
76
4.6
41
1.5
1.2
>100
In order to resolve this apparent disconnect between effi-
cacy and in vitro potency, the potency of 22 against
mouse and human DPP-IV was measured in the pres-
ence of increasing amounts of mouse and human serum,
respectively (Table 7). Potency against mouse DPP-IV
steadily dropped with increasing serum concentrations,
displaying an IC50 = 230 nM at 50% mouse serum. This
serum shift was attributed to high levels of non-covalent
plasma protein binding. Protein binding was even more
pronounced in human serum, with an IC50 = 387 nM at
50% human serum. The measured potency of 22 in
mouse in the presence of 50% serum (IC50 = 230 nM)
agrees well with the pharmacodynamic study described
above. Thus, the high levels of protein binding observed
with 22 explain the apparent disconnect between efficacy
and plasma drug levels observed in the mouse pharma-
codynamic model.
55
0.20
1.7
<1
21
18
16
18
2.0
0.74
100
<1
properties (Table 5). Activity at hERG was also mea-
sured as an indicator of general off-target activity.6,14
Although 22 showed moderate activity against hERG,
this compound displayed excellent oral bioavailability
(67%) and half-life (3.5 h) in the rat. More potent com-
pounds such as 18, 28, and 50 all possess low bioavail-
abilities compared to 22. Analogs 32 and 38 are roughly
equipotent to 22 and display acceptable pharmacokinetic
profiles, but each of these compounds was inferior in
terms of selectivity against DPP8, DPP9, and hERG.
Consequently, 22 was chosen for further evaluation (Ta-
ble 6). We were pleased to find that this compound dis-
played excellent oral bioavailabilities and half-lives in
dogs and rhesus monkeys (Table 7).
In conclusion, optimization of anti-substituted b-polar
substituents of biarylphenylalanine derived DPP-IV
inhibitors led to the discovery of the potency and selec-
tivity enhancing dimethylamide group at the b-position.
Further investigation into pyrrolidine right-hand side
replacements and biaryl left-hand side substituents led
to the discovery of a series of highly potent and selective
phenylalanine derived DPP-IV inhibitors. Since com-
pound 22 exhibits the best balance of potency, selectiv-
ity, and rat pharmacokinetics of this series, this
compound was profiled further. Compound 22 possesses
an excellent pharmacokinetic profile in three species and
excellent in vivo efficacy in a lean mouse OGTT. Never-
theless, the off-target ion channel activity at hERG com-
bined with the observed serum shift of 22 has prompted
further investigation into optimization of this series of
inhibitors.
An oral glucose tolerance test (OGTT) was used to as-
sess the ability of 22 to improve glucose tolerance in
mice. In lean animals, 22 was orally administered 1 h
prior to dextrose challenge and significantly reduced
blood glucose excursion in a dosage-dependent manner
from 0.1 mg/kg (21% reduction) to 3.0 mg/kg (64%
reduction). In the corresponding pharmacodynamic
(PD) assay, compound-mediated DPP-IV inhibition
and plasma compound concentrations were dosage-
dependent 10 min following dextrose challenge. At the
1.0 mg/kg dosage, the plasma concentration of 22 was
940 nM and plasma DPP-IV activity was inhibited by
>75%.15 The administration of 22 at dosages from 1.0
to 3 mg/kg also significantly increased active GLP-1
Acknowledgments
Table 6. Pharmacokinetic properties of 22 in the dog and rhesus
monkey (1/2 mpk iv/po)
The authors thank W. P. Feeney, J. E. Fenyk-Melody, J.
C. Hausamann, S. A. Iliff, C. N. Nunes, A. S. Parlapi-
ano, G. M. Seeburger, and K. G. Vakerich for dosing
the animals used in pharmacokinetic experiments.
References and notes
Species
Clp (mL/min/kg)
t1/2 (h)
F (%)
Dog
Rhesus
1.5
2.4
6.1
4.7
90
56
1. For lead DPP-IV references, see: (a) Mentlein, R. Expert
Opin. Invest. Drugs 2005, 14, 57; (b) Weber, A. E. J. Med.
Chem. 2004, 47, 4135; (c) Deacon, C. F. Diabetes 2004, 53,
2181; (d) Deacon, C. F.; Ahren, B.; Holst, J. J. Expert
Opin. Invest. Drugs 2004, 13, 1091; (e) Holst, J. J.; Deacon,
C. F. Curr. Opin. Pharmacol. 2004, 4, 589.
2. For lead GLP-1 references, see: (a) Holst, J. J. Curr. Opin.
Endocrin. Diabetes 2005, 12, 56; (b) Knudsen, L. B. J.
Med. Chem. 2004, 47, 4128; (c) Vahl, T. P.; DÕAlessio, D.
A. Expert Opin. Invest. Drugs 2004, 13, 177.
Table 7. Potency of 22 in the presence of increasing amounts of mouse
and human serum (mouse and human DPP-IV IC50Õs are given in nM)
Species
0%
Serum
3%
Serum
15%
Serum
50%
Serum
80%
Serum
Mouse
Human
—
12
50
18
71
39
230
387
—
376
3. During, M. J.; Cao, L.; Zuzga, D. S.; Francis, J. S.;
Fitzsimons, H. L.; Jiao, X.; Bland, R. J.; Klugmann, M.;

3052
S. D. Edmondson et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3048–3052
1
Banks, W. A.; Drucker, D. J.; Haile, C. N. Nat. Med.
2003, 9, 1173.
10. All final compounds were characterized by H NMR and
LC–MS.
4. Ahren, B.; Landin-Olsson, M.; Jansson, P.-A.; Svensson,
M.; Holmes, D.; Schweizer, A. J. Clin. Endocrin. Metab.
2004, 89, 2078.
5. Xu, J; Wei, L.; Mathvink, R.; He, J.; Park, Y.-J.; He, H.;
Leiting, B.; Lyons, K. A.; Marsilio, F.; Patel, R. A.; Wu, J.
K.; Thornberry, N. A.; Weber, A. E.; Parmee, E. R.
Bioorg. Med. Chem. Lett. 2005, 15, 2533.
6. IC50 determinations for hERG was carried out as
described in Friesen, R. W.; Ducharme, Y.; Ball, R. G.;
Blouin, M.; Boulet, L.; Cote, B.; Frenette, R.; Girard, M.;
Guay, D.; Huang, Z.; Jones, T. R.; Laliberte, F.; Lynch, J.
J.; Mancini, J.; Martins, E.; Masson, P.; Muise, E.; Pon,
D. J.; Siegel, P. K. S.; Styhler, A.; Tsou, N. N.; Turner, M.
J.; Young, R. N.; Girard, Y. J. Med. Chem. 2003, 46,
2017.
7. (a) Corey, E. J.; Helal, C. J. Tetrahedron Lett. 1995, 36,
9153; (b) Corey, E. J.; Bakshi, C. J. Tetrahedron Lett.
1990, 31, 611.
8. (a) Kazmaier, U. Liebigs Ann. Recl. 1997, 285; (b)
Kazmaier, U. Angew. Chem., Int. Ed. Engl. 1994, 33, 998.
9. Caldwell, C. G.; Chen, P.; He, J.; Parmee, E. R.; Leiting,
B.; Marsilio, F.; Patel, R. A.; Wu, J. K.; Eiermann, G. J.;
Petrov, A.; He, H.; Lyons, K. A.; Thornberry, N. A.;
Weber, A. E. Bioorg. Med. Chem. Lett. 2004, 14, 1265.
11. IC50 determinations for DPP-IV and other proline pepti-
dases were carried out as described in 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.
12. SAR at the P1 position of dipeptide a-amino amide
inhibitors has been investigated with DPP-IV and QPP
(DPP-II). For a lead reference, see: Senten, K.; Veken, P.
V.; De Meeser, I.; Lambeir, A.-M.; Scharpe, S.; Haemers,
A.; Augustyns, K. J. Med. Chem. 2003, 46, 5005.
13. Lankas, G.; Leiting, B.; Sinha Roy, R.; Eiermann, G.;
Biftu, T.; Kim, D.; Ok, H.; Weber, A. E.; Thornberry, N.
A. Diabetes 2004, 53, A2.
14. IC50Õs against human calcium and sodium channels were
also measured, and activity at hERG is indicative of
activity at these other ion channels.
15. It should be noted that the % inhibition as determined
using the in vitro assay underestimates the % inhibition
achieved in vivo, since compound 22 is a competitive,
reversible inhibitor and the assay of plasma
DPP-IV activity requires dilution of plasma (resulting in
dilution of the total inhibitor) and the presence of
substrate that competes with inhibitor for binding to the
enzyme.