Discovery of potent and selective phenylalanine based dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.03.055

anti-Substituted β-methylphenylalanine derived amides have been shown to be potent DPP-IV inhibitors exhibiting excellent selectivity over both DPP8 and DPP9. These are among the most potent compounds reported to date lacking an electrophilic trap. The mo

Full text of DOI:10.1016/j.bmcl.2005.03.055

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2533–2536
Discovery of potent and selective phenylalanine based
dipeptidyl peptidase IV inhibitors
Jinyou Xu,a,* Lan Wei,a Robert Mathvink,a Jiafang He,a You-Jung Park,a Huaibing He,a
Barbara Leiting,b Kathryn A. Lyons,a Frank Marsilio,b Reshma A. Patel,b 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
Received 7 February 2005; revised 10 March 2005; accepted 15 March 2005
Available online 9 April 2005
Abstract—anti-Substituted b-methylphenylalanine derived amides have been shown to be potent DPP-IV inhibitors exhibiting excel-
lent selectivity over both DPP8 and DPP9. These are among the most potent compounds reported to date lacking an electrophilic
trap. The most potent compound among these is 5-oxo-1,2,4-oxadiazole 44, which is a 3 nM DPP-IV inhibitor.
Ó 2005 Elsevier Ltd. All rights reserved.
F
Glucagon-like peptide-1 (GLP-1) is an incretin hormone
that is released from the gut during meals and serves as
an enhancer of glucose stimulated insulin release from
pancreatic b-cells. Chronic infusion of GLP-1 to pa-
tients with type 2 diabetes resulted in significant de-
creases in both blood glucose and hemoglobin A1c
levels;1 however, GLP-1 is rapidly degraded in plasma
by the serine protease dipeptidyl peptidase IV (DPP-
IV). Inhibition of DPP-IV increases the levels of endoge-
nous intact circulating GLP-1. Consequently, inhibition
of DPP-IV is rapidly emerging as a novel therapeutic
approach to the treatment of type 2 diabetes.2
O
H
N
S
O
N
O O
F
NH2
S
N
H
NH2
2
1
F
IC50(DPP-IV) = 420 nM
IC50(DPP8) = 2170 nM
IC50(DPP9) = 1600 nM
IC50(DPP-IV) = 48 nM
IC50(DPP8) = 993 nM
IC50(DPP9) = 2720 nM
O
O
N
N
NH2
S
NH2
S
4
3
Earlier reports from our laboratories described 4-amino
cyclohexylglycine and cyclopentylglycine analogs as po-
tent DPP-IV inhibitors lacking an electrophile.3 Among
the best compounds discovered from that series was 2,4-
difluorobenzenesulfonamide 1 (Fig. 1). Compound 1
was found to have good pharmacokinetic properties
and produced significant activity in an oral glucose tol-
erance test in lean mice. However, further development
of 1 was halted because it was subsequently found to ex-
hibit significant activity at both DPP8 (IC50 = 993 nM)
and DPP9 (IC50 = 2720 nM). Inhibition of these enz-
IC50(DPP-IV) = 968 nM
IC50(DPP8) > 100,000 nM
IC50(DPP9) > 100,000 nM
IC50(DPP-IV) = 460 nM
IC50(DPP8) = 220 nM
IC50(DPP9) = 320 nM
Figure 1. Lead DPP-IV inhibitors.
ymes has been correlated with toxicity in animals.4 De-
spite extensive SAR studies in this original a-
aminoacid series, little progress was made toward
improving the selectivity against DPP8 and DPP9.
The selectivity profile of thiazolidides 2 and 3 looked
very intriguing (Fig. 1). Although both compounds
exhibited similar potency toward DPP-IV, the threo-iso-
mer 25 is 10-fold more selective toward DPP8 and 5-fold
Keyword: DPP-IV.
*
Corresponding author. Tel.: +1 732 594 2217; fax: +1 732 594 5790;
e-mail: jinyou_xu@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.055

2534
J. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2533–2536
more selective toward DPP9 than allo-isomer 3. Appar-
ently, stereochemistry at the b-position in the a-amino
acid series has some effect on the selectivity over both
DPP8 and DPP9. We envisioned that we might be able
to improve the selectivity over DPP8 and DPP9 by
incorporating the ÔthreoÕ bias into the a-amino acid ser-
ies. To test this idea, we prepared b-methylphenylala-
nine thiazolidide 4. Although the potency of
compound 4 (DPP-IV, IC50 = 968 nM) was reduced
relative to compound 2, it did exhibit improved selectiv-
ity over both DPP8 and DPP9. Based on our previous
results in the a-amino series, we knew that P2-site could
be optimized to improve potency. The work described
here summarizes our initial efforts at optimizing the
potency and selectivity of this novel series of b-methyl-
phenylalanine based DPP-IV inhibitors.
diastereoselective bromination of the dibutylboryl eno-
late derived from 7 with N-bromosuccinimide (NBS)
followed by azide replacement with tetramethylguani-
dium azide (TMGA) afforded the desired compound 9
in excellent selectivity and isolated yield.
Removal of auxiliary of 9 with hydrogen peroxide medi-
ated hydrolysis was followed by coupling with 3-(S)-flu-
oropyrrolidine, azide reduction and protection of the
primary amine to give the corresponding aryl bromide
11. Subsequent coupling of the aryl bromide 11 with aryl
boronic acid via Suzuki coupling followed by deprotec-
tion of the tert-butylcarbamate then gave the desired
compound 12q.
Inhibitors were tested for their selectivity profiles against
a variety of DPP-IV homolog and proline-specific en-
zymes including quiescent cell proline dipeptidase
(QPP/DPP-II), prolyl endopeptidase (PEP), amino pep-
tidase P (APP), prolidase, DPP8, and DPP9.7
The b-methylphenylalanine-based DPP-IV inhibitors
were prepared by EDC-mediated coupling of a-amino
acids with 3-(S)-fluoropyrrolidine. 3-(S)-Fluoropyrroli-
dine was chosen because it improved both the selectivity
and pharmacokinetic profile of these inhibitors as re-
ported before.3b A representative example of the synthe-
sis of these inhibitors is shown in Scheme 1.6 Activation
of the carboxylic acid 5 with trimethylacetyl chloride fol-
lowed by addition of lithiated oxazolidinone gave acyl-
oxazolidinone 6. The methylcopper reagent, generated
in situ by the treatment of methyl magnesium bromide
and CuBr–dimethyl sulfide complex, was added to the
acyloxazolidinone 6 to provide the methylated product
7 in more than 90% diastereoisomeric excess (de). The
With the goal of finding analogs of 3-(S)-fluoropyrroli-
dide 4a with increased potency,initially we looked at
substitution on the phenyl ring in 4a. Table 1 summa-
rizes the DPP-IV inhibitory properties of these a-amino-
acid pyrrolidides. Substitution at the 2- or 3-position on
the phenyl ring had little effect on or was detrimental to
activity.
Table 1. Inhibitory properties of selected DPP-IV inhibitors
O
N
R
+
Br
NH3
Br
Br
Ph
a, b
TFA-
F
c
O
N
COOH
Compd
R
IC50 (lM)
DPP-IV QPP DPP8 DPP9
O
6
O
5
7
Br
Ph
Ph
e
4a
H
2-F
1.4
1.1
0.48
4.2
59
19
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 >100
>100 78
Br
d
12a
12b
12c
12d
12e
12f
12g
12h
12i
N
N
O
O
2-F, 4-Cl
2-Cl, 4-F
2-CF3, 4-F
3-F
5.6
O
O
46
>100
14
O
O
54
8
3.7
1.1
Br
Br
Ph
3-Cl
5.7
7.8
11
5.2
23
N3
h, i
N3
f, g
3,5-DiF
3,4-DiF
3-Cl, 4-F
4-F
3.7
1.1
N
N
O
F
O
O
2.8
57
>100 95
34
O
12j
0.28
0.25
0.17
0.36
0.60
3.6
9
10
12k
12l
4-Br
4-Cl
3.6
5.9
5.9
5.2
>100 >100
>100 >100
TFA-
Br
NHBOC
j, k
+
12m
12n
12o
12p
12q
12r
12s
12t
12u
12v
12w
12x
12y
4-SCH3
4-CF3
61
67
NH3
N
O
>100 >100
>100 >100
>100 >100
F
N
4-SO2Me
4-OMe
4-Ph
36
14
3.0
F
0.77
0.12
2.0
O
11
12q
85
>100
3-F, 4-Ph
4-Piperidinyl
4-(2-Pyrazoloyl)
4-(2-Thiazolyl)
4-(2-Pyridyl)
4-(3-Pyridyl)
4-(4-Pyridyl)
4-(3,5-Pyrimidinyl)
48
28
>100 >100
>100 >100
Scheme 1. Reagents and conditions: (a) Et3N, t-BuCOCl, THF; (b) n-
BuLi, 4-(R)-4-phenyloxazolidinone, ꢀ78 °C to rt; (c) MeMgBr, CuBr–
Me2S, THF/Me2S (1/1), ꢀ60 °C to rt ; (d) (n-Bu)2BOTf, DIEA,
ꢀ78 °C, 1 h, then NBS, ꢀ78 °C to rt; (e) TMGA, CH3CN, rt; (f)
LiOH, H2O2, THF/H2O; (g) EDC, HOBT, DIEA, (S)-3-fluoropyrr-
olidine, DMF; (h) PPh3, THF/H2O, 60 °C; (i) BOC2O, NaHCO3, 1,4-
dioxane; (j) PhB(OH)2, toluene, EtOH, 2 N aq Na2CO3, Pd(dppf)2Cl2,
90 °C; (k) TFA/CH2Cl2, 1 h.
7.9
1.7
1.5
0.72 >100 >100
63
>100
1.5
0.24
0.11
0.11
0.58
4.6
4.8
>100 >100
>100 >100
>100 >100
3.1
10.0
57
>100

J. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2533–2536
2535
Table 3. Inhibitory properties of selected DPP-IV inhibitors
Lipophilic groups were well tolerated at the 4-position.
Introduction of a fluorine at the 4-position of the phenyl
ring increased potency by 5-fold (12j). An 8-fold in-
crease in potency was achieved when a chlorine was
introduced at the 4-position (12l). Incorporating a phe-
nyl group at the 4-position of the phenyl ring increased
the potency by 10-fold (12q). Replacement of the pen-
dant phenyl group in 12q with a heterocycle usually re-
sulted in a decrease in potency. However, the potency of
3-pyridyl analog 12w and 4-pyridyl analog 12x was
comparable to 12q.
O
N
+
NH3
TFA-
F
Het
Compd
R
IC50 (lM)
QPP DPP8
DPP-IV
0.14
DPP9
N
N
N
N
37
38
39
0.87
0.96
1.1
58
>100
>100
56
N
In order to further increase the potency and selectivity
over QPP, we looked at substitution on the pendant
phenyl group in the biphenyl lead 12q. The results are
summarized in Table 2. Substitution at the 20-position
on the pendant phenyl ring had little effect on the po-
tency. Polar groups were well tolerated at the 30-posi-
tion. Incorporating a carboxylic acid group at this
position (20) increased potency as well as selectivity
(>170-fold window over inhibition of QPP). Analogs
incorporating a polar group at the 40-position were all
less potent than 12q. Introduction of a fluorine at the
40-position of the phenyl ring increased potency by 2-
fold (25).
N
0.069
0.095
>100
47
O
H
O
N
N
HN
HN
40
41
42
0.11
0.098
0.14
0.83
0.35
0.096
17
57
N
H
O
N
HN
>100
55
>100
>100
N
O
N
N
Since polar groups were well tolerated at 30-position, we
decided to incorporate a heterocycle at that position to
N
N
N
43
44
0.006
0.003
2.9
1.1
>100
>100
52
N
H
Table 2. Inhibitory properties of selected DPP-IV inhibitors
N
O
>100
O
N
H
O
N
+
NH3
TFA-
R
F
see if it could improve the potency and selectivity of this
series. The results are summarized in Table 3. Neutral or
basic heterocycles had little effect on potency. However,
analogs incorporating an acidic heterocycle such as
tetrazole or 5-oxo-1,2,4-oxadiazole exhibited improved
potency and selectivity (43 and 44).
Compd
R
IC50 (lM)
DPP-IV
QPP
DPP8
DPP9
12q
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
H
0.13
0.10
0.086
0.11
0.13
0.15
0.075
0.15
0.056
0.11
0.14
0.027
0.10
0.064
0.51
0.13
0.39
0.71
0.44
0.37
0.42
0.24
0.15
0.06
0.037
3.0
1.1
85
>100
>100
>100
>100
>100
>100
>100
>100
>100
45
2-F
2-Cl
>100
>100
>100
>100
>100
>100
>100
>100
23
0.38
3.8
2-OMe
2-CF3
2-Me
Representative analogs were selected for evaluation of
pharmacokinetic properties in the rat and possible ion
channel activity as a measure of general off-target acti-
vity (Table 4). The latter is illustrated here with binding
to the hERG potassium channel.8 Compound 25 exhibi-
ted excellent pharmacokinetic properties in rats (F =
85%). However, it had demonstrable ion channel bind-
ing (hERG binding Ki = 1100 nM). Analogs incorporat-
ing a polar group at the 30-position generally exhibited
enhanced selectivity over QPP and ion channel binding.
However, incorporation of the polar functionality was
2.5
2.5
3-CO2Et
3-NHSO2Me
3-COOH
3-SO2Me
3-SO2NH2
3-NHSO2CF3
3-CONH2
4-F
0.49
0.25
9.2
2.9
3.6
>100
>100
39
>100
59
37
1.3
0.82
2.7
88
64
86
4-CF3
4-OMe
4-Me
0.67
0.33
1.1
>100
>100
>100
>100
>100
>100
>100
79
74
77
4-NHSO2Me
4-CO2Et
4-CO2H
4-SO2Me
3,5-DiF
2,5-DiF
3,4-DiF
2,4-DiF
1.8
>100
>100
>100
>100
9.8
Table 4. Pharmacokinetic properties of selected DPP-IV inhibitors in
the rat (1/2 mpk iv/po) and hERG binding
0.12
8.6
Compd Clp (mL/min/kg) t1/2 (h) F (%) hERG IC50 (lM)
1.2
2.0
20
25
43
44
50.8
8.6
0.4
2.2
0.3
1.1
2.3
85
>100
1.1
1.3
2.0
>100
42
>100
>100
48
>100
132.4
23.9
3.5
4.2
59
28
2.5

2536
J. Xu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2533–2536
Patel, R. A.; Petrov, A.; Di Salvo, J.; Wu, J. K.;
Thornberry, N. A.; Weber, A. E. Bioorg. Med. Chem. Lett.
2004, 14, 43; (b) 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.; Thorn-
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Thornberry, N. A.; Weber, A. E. Bioorg. Med. Chem. Lett.
2004, 14, 859.
detrimental to pharmacokinetic properties as evidenced
by the extremely poor oral bioavailability of acid 20 and
heterocycles 43 and 44.
In summary, we have discovered a novel series of potent
and selective DPP-IV inhibitors. These are among the
most potent compounds reported to date lacking an
electrophilic trap. The most potent compound among
these is heterocycle 44, which is a 3 nM DPP-IV inhibi-
tor with good selectivity over related proline peptidases
and ion channel activity. However, this compound lacks
oral bioavailability in rats. More lipophilic compounds
such as 25 exhibited excellent oral bioavailability in rats,
but suffered from poor selectivity over QPP and ion
channel activity. These preliminary results suggest that
there is a fine balance among lipophilicity, selectivity,
and oral bioavailability. Incorporation of a polar group
at the b-position led to further optimization of these
properties. These results will be described in the due
course.
4. Lankas, G.; Leiting, B.; Sinha Roy, R.; Eiermann, G.;
Biftu, T.; Kim, D.; Ok, H.; Weber, A. E.; Thornberry, N.
A. Diabetes 2004, 53(S2), A2.
5. (a) Scho¨n, E.; Born, I.; Demuth, H. U.; Faust, J.; Neubert,
K.; Steinmetzer, T.; Barth, A.; Ansorge, S. Biol. Chem.
Hoppe-Seyler 1991, 372, 305; (b) Sto¨ckel-Maschek, A.;
Mrestani-Klaus, C.; Stiebitz, B.; Demuth, H. U.; Neubert,
K. Biochim. Biophys. Acta 2000, 1479, 15.
6. Yuan, W.; Hruby, V. J. Tetrahedron Lett. 1997, 38, 3853.
7. (a) None of the compounds in this report showed any
significant inhibition against APP, PEP, and prolidase
(IC50s > 10,000 nM). For assay conditions for DPP-IV and
QPP inhibition, see: 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; (b) All multiple determinations of the IC50 values were
with 1.4-fold of the reported average; (c) IC50 determina-
tions for hERG were carried as described in Lankas, G.;
Leiting, B.; Sinha Roy, R.; Eiermann, G.; Biftu, T.; Kim,
D.; Ok, H.; Weber, A. E.; Thornberry, N. A. Inhibition of
DPP8/9 Results in Toxicity in Preclinical Species: Potential
Importance of Selective Dipeptidyl Peptidase IV Inhibition
for Treatment of Type 2 DM. Presented at the American
Diabetes Association 64th Scientific Session, Orlando, FL,
June, 2004, Abstract 7-OR.
8. IC50 determinations for hERG were carried 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.
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