Optimization of 1,4-diazepan-2-one containing dipeptidyl peptidase IV inhibitors for the treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmcl.2007.01.039

Following the discovery of N-acyl-1,4-diazepan-2-one as a novel pharmacophore for potent and selective DPP-4 inhibitors, optimization of this new lead with different substitution on the seven-membered ring resulted in several highly potent and selective,

Full text of DOI:10.1016/j.bmcl.2007.01.039

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1903–1907
Optimization of 1,4-diazepan-2-one containing dipeptidyl peptidase
IV inhibitors for the treatment of type 2 diabetes
Gui-Bai Liang,a,* Xiaoxia Qian,a Dennis Feng,a Tesfaye Biftu,a George Eiermann,b
Huaibing He,a Barbara Leiting,c Kathy Lyons,a Aleksandr Petrov,b Ranabir Sinha-Roy,c
Bei Zhang,c Joseph Wu,c Xiaoping Zhang,c Nancy A. Thornberryc and Ann E. Webera
aMerck Research Laboratories, Department of Medicinal Chemistry, Merck and Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
bMerck Research Laboratories, Department of Pharmacology, Merck and Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
cMerck Research Laboratories, Department of Metabolic Disorders, Merck and Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Received 15 December 2006; revised 9 January 2007; accepted 10 January 2007
Available online 24 January 2007
Abstract—Following the discovery of N-acyl-1,4-diazepan-2-one as a novel pharmacophore for potent and selective DPP-4 inhib-
itors, optimization of this new lead with different substitution on the seven-membered ring resulted in several highly potent and
selective, orally bioavailable, and efficacious DPP-4 inhibitors, such as 3R-methyl-1-cyclopropyl-1,4-diazepan-2-one derivative 9i
(DPP-4 IC50 = 8.0 nM) and 3R,6R-dimethyl-1,4-diazepan-2-one derivative 14a (DPP-4 IC50 = 9.7 nM).
Ó 2007 Elsevier Ltd. All rights reserved.
Glucose-dependent insulin secretion (GDIS) is of funda-
mental importance in blood sugar regulation and is the
object of intense research and development in both aca-
demia’s and pharmaceutical industry’s search for novel
treatments of type 2 diabetes. The incretin hormones
glucagon-like peptide 1 (GLP-1) and glucose-dependent
insulinotropic polypeptide (GIP) both play important
roles in GDIS,1,2 and therapeutic use of GLP-1 and
GIP as potential antidiabetic agents has been studied.3,4
Both hormones are deactivated rapidly in vivo through
the action of dipeptidyl peptidase IV (DPP-4), a serine
exopeptidase which cleaves a dipeptide from the N-ter-
minus.5,6 Thus, GLP-1 must be administered via contin-
uous infusion in order to have sustained efficacy.7,8
Development of DPP-4-resistant GLP-1 analogs, such
as exenatide,9 proved to be an effective way to circum-
vent this problem. Small-molecule inhibitors of DPP-4,
on the other hand, have been shown to prolong the
beneficial effects of endogenous GLP-1,10,11 as well as
to stabilize GIP.12 Human clinical trials of several
small-molecule DPP-4 inhibitors, including sitagliptin13
and vildagliptin,14 have shown improved glucose toler-
ance in diabetic patients.
Sitagliptin (1) is a b-alanine-based DPP-4 inhibitor con-
taining a triazolopiperazine moiety. Continued SAR
studies on 1 led to the discovery of 1,4-diazepan-2-one
derivatives (2a–d).15 In this paper, we report further
optimization of this new lead.
F
F
F
F
F
R
O
R'
NH2
NH2
O
O
N
N
N
N
NH
N
CF3
2a: R = H, R' = Me
2b: R = F, R' = Me
2c: R = F, R' = H
1: Sitagliptin
2d: R = F, R' = CH2CF3
First we extended the SAR to 1-alkylated derivatives,
probing the space that became vacant after removal of
the triazolo group in 1. Thus, intermediate 315 was alkyl-
ated under the typical NaH/R–X/DMF conditions
(Scheme 1). Partial epimerization at C2 was observed
and resulting diastereomers were separated after the
coupling step with 8.16 Alternatively, bulky alkyl
groups, such as tert-butyl, that do not undergo nucleo-
philic substitution under these conditions, were intro-
Keywords: Type 2 diabetes; DPP-IV inhibitors; 1,4-Diazepan-2-one.
*
Corresponding author.Tel.: +1 732 594 3543; fax: +1 732 594 3007;
e-mail: gui-bai_liang@merck.com
duced via
a route outlined in Scheme 1. The
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.039

1904
G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1903–1907
O
O
and DPP-9 activities are similar, thus only DPP8 data
1) NaH, R"-X
are reported.20 A selectivity (DPP-4/DPP-8) ratio of
>1000 is targeted for the development candidates. Also
reported are inhibition data against quiescent cell pro-
line dipeptidase (QPP, also known as DPP-7).18,21 All
new compounds were tested inactive (IC50 > 50 lM)
against fibroblast activation protein.
R"
Boc
N
NH
HN
N
2) HCl, Dioxane
4
5
3
O
O
H2, Pt2O
iPr2NEt
CH2Cl2
O
Cl
+
O
NC
N
CHCl3, EtOH
HN
NC
As shown in Table 1, most 1-alkylated compounds (9a–
j) have very similar DPP-4 inhibition to their parent NH
compound 2a or 2b, even the one with a fairly large sub-
stituent (9g, IC50 = 10.5 nM), indicating a sizable hydro-
phobic pocket in that part of the DPP-4 active site. This
is consistent with the observation that polar groups,
such as a carboxylic acid group, reduce potency (9c,
IC50 = 69.9 nM). Since many of these compounds were
highly potent and select in our initial screening and
counter-screening, pharmacokinetic properties became
increasingly important in selecting candidates for fur-
ther evaluation.
O
N
O
O
HN
O
N
N
N
H2N
7
6a
6b
F
R
NH2
O
O
OH
R"
HN
N
1) Chiral Separation
2) HCl, Dioxane
8a-b
F
EDC, HOBt, CH3CN
F
4 or 7
R
O
N
NH2
O
9a-i
R"
N
The pharmacokinetic properties of several representa-
tive compounds in male Sprague–Dawley rats (1 mg/kg
iv, 2 mg/kg po) are summarized in Table 2. Consistent
with earlier observations, this class of compounds usual-
ly exhibits high plasma clearance and moderate oral bio-
availability in rats.15 In this case, trifluorophenyl
analogs have better PK parameters than the correspond-
ing difluorophenyl analogs, e.g., 9i versus 9d. Compared
to our early lead 2d,15 cyclopropyl analog 9i has im-
proved oral bioavailability, half-life, and exposure. Also
observed in these PK studies was the 1-dealkylated
metabolite (2a or2b), which itself is a potent DPP-4
inhibitor. In the case of 9e (1-methyl), the in vivo con-
version is as high as 17%. A cyclopropyl group (9d
and 9i) significantly reduced this conversion to about
3%.
F
Scheme 1.
commercially available adduct of tert-butylamine to
acrylonitrile was acylated with pyruvic acid via its acid
chloride to form precursor 5. In one pot under a hydro-
gen balloon in the presence of platinum oxide as cata-
lyst, the nitrile group of 5 was reduced to primary
amine 6a, which underwent intramolecular condensa-
tion with the keto group to form cyclic imine 6b. This
equilibrium was driven to completion by the further
reduction of 6b under the reaction conditions to the
desired 1-tert-butyl 1,4-diazepan-2-one 7. The carbodii-
mide-mediated amide bond formation with intermedi-
ates 8a and 8b (8a: R = H, 8b: R = F), followed
by chiral LC separation and subsequent deprotection,
yielded 1-alkyl 1,4-diazepan-2-one derivatives 9a–j.17
In an effort to further suppress this in vivo dealkylation
possibly through acyl iminium ion intermediates, a pro-
line derivative 12 was made and evaluated (Scheme 2).
Although compound 12 is a potent DPP-4 inhibitor
(IC50 = 23.9 nM), no overall improvement in drug expo-
sure was observed in PK studies on this compound in rat
(Table 2). The result indicated, however, that small sub-
stituents in the bottom half of the diazepanone ring may
be tolerated.
These compounds were evaluated in vitro for their inhi-
bition of DPP-4 activity18 and selectivity against DASH
family members.19 Among them, selectivity against
DPP-8 and DPP-9 was of particular concern since safety
studies using a selective DPP-8/9 dual inhibitor suggest
that inhibition of DPP-8 and/or DPP-9 is associated
with profound toxicity in preclinical species.20 DPP-8
Table 1. Activities of 1-substituted diazepanone (9) DPP-4 inhibitors
R00
Compound
R
DPP-4 IC50 (nM)
QPP IC50 (nM)
DPP-8 IC50 (nM)
2a
9a
9b
9c
9d
2b
9e
9f
H
H
H
H
H
F
F
F
F
F
F
F
H
Et
11.5
20.7
11.6
69.9
9.0
>100,000
>100,000
>100,000
>100,000
>100,000
59,000
58,000
38,000
29,000
>100,000
45,000
46,000
21,000
53,000
15,000
42,000
22,000
55,000
CH2CO2Me
CH2CO2H
cPr
H
Me
6.6
6.6
>100,000
78,000
17,000
CH2CH2OH
CH2CH2OBn
CH2CF3
cPr
15.1
10.5
19.2
8.0
9g
9h
9i
56,000
46,000
35,000
9j
tBu
9.6

G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1903–1907
Table 2. Mean pharmacokinetic parameters of selected 1,4-diazepanone analogs in rats
1905
Compound
Pharmacokinetic parameter
Clp (mL/min/kg)
t1/2 (h)
Foral (%)
Cmax (lM)
Conversion to 2a or 2b
2d
9a
9d
9e
9h
9i
94
152
132
113
82
2
36
7.4
19
29
23
45
21
8.1
0.09
0.03
0.05
0.13
0.08
0.57
0.04
0.01
n/a
n/a
0.8
1.6
1.3
1.7
4.3
1.4
2.0
3.4%
17%
n/a
51
3%
n/a
12
15f
152
97
n/a
Clp, plasma clearance; Foral, oral bioavailability; Cmax, peak plasma concentration after indicated oral dosing. Pharmacokinetic parameters were
obtained following a iv (1 mg/kg) or po (2 mg/kg) dose (amorphous trifluoroacetic acid salt) in water. n/a, data not available.
O
O
As shown in Table 3, the chirality at C5 (13a vs 13b) has
little effect on DPP-4 inhibition (IC50 ratio of 1:1.4). The
ratio increases to moderate at C6 (14a:14b = 1:3.4) in
favor of the (S) configuration and to quite large at C7
(15a:15b = 1:26) in favor of the (R) configuration. The
change in preferred configuration is rather a result of
nomenclature than the actual spatial orientation of the
methyl group. It is clear that substitution at C5 (13a
and 13b vs 2c) is not readily tolerated by the enzyme
resulting in a 5-fold decrease in potency. On the other
hand, C6 (14a and 14b, IC50 = 9.7 and 33 nM, respec-
tively) and C7 (15a, R-diastereomer, IC50 = 15.1 nM)
substitutions appeared to be well-tolerated, and both
potencies and selectivities remain excellent, comparable
to 2b.
H2, Pt2O
SOCl2
O
OH
O
NC
N
CHCl3, EtOH
+
iPr2NEt, CH2Cl2
HN
10
NC
F
F
F
O
N
O
NH2
O
1) 8, EDC, HOBt
HN
11
N
N
2) Chiral Separation
3) HCl, Dioxane
12
Scheme 2.
Based on the route outlined in Scheme 3, we were able to
synthesize the 5-, 6-, and 7-methyl analogs 13, 14, and
15, respectively. Reductive animation of D-alanine meth-
yl ester with desired aldehydes, readily obtained from
corresponding Weinreb amides, forms the first nitrogen
link between the top and bottom pieces. After protecting
group manipulation, cyclization to diazepanone was
accomplished by treatment with tert-butylmagnesium
bromide.15 From previous studies, we have learned that
the (R) configuration at C2 is required for DPP-4 inhib-
itory activity.15 As a result of the second methylation,
there is a pair of diastereomers to be evaluated. In the
case of the 5-methyl analogs, the diastereomers were
separated, but their absolute configurations have yet to
be determined. For 6- and 7-methyl analogs, enantio-
merically pure starting materials were used, and there-
fore, the configurations of the final products are known.
Next, we modified 14a–14b by replacing the 2-methyl
group with other alkyl groups identified in our previous
studies, such as ethyl and trifluoroethyl groups.15 In this
case, however, such replacements do not result in
improvement in potency as we had observed previously
(Table 4). Although both diastereomeric methyl groups
are well-tolerated at the C6 position, the 6,6-dimethyl
analog (14g, IC50 = 142 nM) is not. It is much less active
than either one of the monomethyl analog 14a or 14b.
Table 3. Activities of 5-, 6-, 7-methyl diazepanone DPP-4 inhibitors
F
R'
R'
O
O
F
R'
O
O
NH2
O
O
N
N
N
NH
NH
Boc
HN
NH
Boc
HN
F
H
R"
1) LAH, -40C
2) D-Ala-OMe
1) Cbz-X
2) HCl
N
CO2Me
N
R"
14a-b
15a-b
13a-b
R"
O
O
Compound R0 R00
DPP-4
IC50 (nM) IC50 (nM) IC50 (nM)
QPP
DPP-8
16
17
O
Cbz
N
H2
tBuMgBr
CH2Cl2
H2N
CO2Me
Cbz
2c
H
H
142
765
1050
6.6
>100,000
44,000
42,000
>100,000
>100,000
46,000
N
NH
Mea
Mea
H
Pd-C
13a
13b
2b
H
18
19
H
Me
>100,000
59,000
38,000
F
F
O
Boc
14a
14b
15a
15b
Me (S)-Me 9.7
Me (R)-Me 33.0
Me (R)-Me 15.1
Me (S)-Me 397
21,000
93,000
F
1) 8, EDC, HOBt
O
NH
O
71,000
72,000
73,000
HN
NH
2) Chiral Separation
3) HCl, Dioxane
38,000
70,000
N
NH
14a
20
a Diastereomeric methyl groups. Absolute configurations have yet to
be determined.
Scheme 3.

1906
G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1903–1907
Table 4. Activities of 6-methyl diazepanone 14 DPP-4 inhibitors
Table 6. Mean pharmacokinetic parameters of 14a and 15a in rat, dog,
and monkey
F
F
Pharmacokinetic
parameter
Rat
Dog
15a
Monkey
R'
NH2
O
O
14a
15a
14a
14a
15a
N
NH
Clp (mL/min/kg)
T1/2 (h)
Foral (%)
92.8
1.5
87.0
1.5
8.1
8.1
77
8.5
7.2
88
39.4
4.4
42.3
4.7
F
14
R"
49
0.14
64
0.17
59
0.29
71
0.43
Cmax (lM)
1.21
1.29
Compound R0
R00
DPP-4
IC50 (nM) IC50 (nM) IC50 (nM)
QPP
DPP-8
Clp, plasma clearance; Foral, oral bioavailability; Cmax, peak plasma
concentration after indicated oral dosing. Pharmacokinetic parameters
were obtained following a iv (1 mg/kg) or po (2 mg/kg) dose in water.
14a
14b
14c
14d
14e
14f
14g
Me
(S)-Me 9.7
(R)-Me 33.1
(S)-Me 37.3
(R)-Me 60.3
38,000
71,000
61,000
90,000
31,000
42,000
17,000
21,000
93,000
41,000
Me
Et
Et
>100,000
16,000
89,000
inhibitors, and addition of a methyl group at C6 or
C7 does provide compounds with improved oral
bioavailability and drug exposure.15 In dogs, plasma
clearance is much reduced, resulting in excellent phar-
macokinetic parameters including longer half-lives and
good drug exposure. Plasma clearance in monkeys is rel-
atively high for both compounds 14a and 15a (39, and
42 mL/min/mg, respectively), but oral bioavailability
(59% and 71%, respectively) remains very good.
Although predicting pharmacokinetic parameters in
human is very difficult, based on clinical studies with
sitagliptin,22 good drug exposure and long half-lives
suitable for once-a-day dosing can be expected for both
compounds.
CH2CF3 (S)-Me 24.6
CH2CF3 (R)-Me 143
Me
di-Me 142
26,000
Similar modifications were done on C7 methylated dia-
zepanone 15a (Table 5, only active diastereomers are
shown). Benzyl analogs in this series (15d and 15f,
IC50 = 1.49 and 2.77 nM, respectively) have superior
potencies compared to the parent 2-methyl analog 15a,
consistent with our observation from previous SAR
studies on 2a–c.15 However, they suffer from reduced
selectivity. Although pyridomethyl analog 15e has com-
parable potency and selectivity, it has an unacceptable
pharmacokinetic profile (Table 2). Replacement of the
7-methyl group with other substituents (15g–i) resulted
in no improvement in terms of potency and selectivity,
indicating limited space in this region of the enzyme
active site.
In pharmacodynamic studies, oral glucose tolerance
tests (OGTT) in lean male mice were conducted to deter-
mine the efficacy of these DPP-4 inhibitors. Compounds
9i, 14a, and 15a or water (vehicle) were administered
60 min prior to an oral dextrose challenge (5 g/kg). Con-
trol animals received water only. The glucose AUC was
determined from 0 to 120 min. Percent inhibition values
for each treatment were generated from the AUC data
normalized to the water-challenged controls, and per-
cent inhibition of DPP-4 in plasma was measured at
each dose level.
Thus, the two most promising compounds 14a and 15a
were chosen for extensive pharmacokinetic screening,
and the data are summarized in Table 6. Across the spe-
cies in rat, dog, and monkey, 14a and 15a exhibited
good to excellent pharmacokinetic profiles. Similar to
the 1-alkylated analogs 9 in Table 2, these two com-
pounds also showed high plasma clearance in rats. This
appears to be common among b-alanine-based DPP-4
OGTT data on 9i, 14a, and 15a are summarized in Table 7.
For 14a, significantly reduced blood glucose excursions
were observed in a dose-dependent manner from
0.03 mg/kg (27% inhibition), 0.1 mg/kg (46% inhibition),
to 0.3 mg/kg (49% inhibition). Because of the highly po-
Table 5. Activities of 7-substituted diazepanone 15 DPP-4 inhibitors
tent inhibition of 14a against mouse DPP-4 (IC50
=
F
F
1.0 nM), the fully efficacious level was reached at a
low dose of 0.1 mg/kg. In a separate OGTT of 14a, plas-
ma DPP-4 inhibition, compound concentration, and
active GLP-1 level were measured 20 min after dextrose
challenge. At a dose of 0.1 mg/kg, the corresponding
R'
NH2
O
O
N
NH
F
15
R"
Compound R0
R00
DPP-4
QPP
DPP-8
IC50 (nM) IC50 (nM) IC50 (nM)
Table 7. Oral glucose tolerance test of 9i, 14a, and 15a in lean mice
15a
15c
15d
15e
15f
15g
15h
15i
Me
(R)-Me 15.1
CH2CF3 (R)-Me 34.3
2-MeBn (R)-Me 1.49
2-F-Bn (R)-Me 2.77
38,000 21,000
42,000 12,000
6,800 19,000
Compound
Glucose AUC % vehicle at indicated
dose (mg/kg)
0.03
0.1
0.3
1.0
3.0
18,000 5,300
27,000 34,000
>100,000 76,000
49,000 20,000
8,700 19,000
2-PyMe (R)-Me 11.0
a
(R)-Me CF3
9i
n/a
27
n/a
46
28
49
34
40
52
52
43
50
55
139
14a
15a
(R)-Me (R)-Et 29.6
(R)-Me 2-F–Bna 126
n/a
19
C57BL/6N male lean mice were used for the test. n/a, data not
available.
a Presumed (R) configuration based on DPP-4 activies.

G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1903–1907
1907
7. Zander, M.; Madsbad, S.; Madsen, J. L.; Holst, J. J. The
Lancet 2002, 359, 824.
8. Ahren, B. E. Drug Discovery Today: Therapeutic Strate-
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9. Heine, R. J.; van Gaal, L. F.; Johns, D.; Mihm, M. J.;
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plasma concentration of 14a reached 4.4 nM, and an
uncorrected 72% inhibition of plasma DPP-4 activity
was observed,23 resulting in about a 3-fold increase in
active GLP-1 levels, and full efficacy in glucose reduc-
tion. Because 9i (mouse DPP-4 IC50 = 18 nM) and 15a
(mouse DPP-4 IC50 = 26 nM) are much less potent than
14a against mouse DPP-4, higher doses (3 mg/kg for 9i
and 1 mg/kg for 15a, respectively) have to be used to
reach maximum efficacy based on at least 80% inhibition
of plasma DPP-4. Nonetheless, all three compounds
showed good efficacy in glucose reduction, comparable
to that of sitagliptin.16 Good correlations among plasma
concentration, DPP-4 inhibition, GLP-1 elevation (data
not shown), and glucose AUC reduction strongly sug-
gest that the observed enhancement in glucose tolerance
is indeed mechanism based and mediated at least in part
by the GLP-1.
14. Ahren, B.; Landin-Olsson, M.; Jansson, P. A.; Svensson,
M.; Holmes, D.; Schweizer, A. J. Clin. Endocrinol. Metab.
2004, 89, 2078.
In summary, following the discovery of the novel b-ala-
nine-derived 1,4-diazepanone-containing DPP-4 inhibi-
tors, we have explored different substitution patterns
of the diazepanone ring through continued SAR studies.
Representative compounds, such as 1-substituted 9i,
6-substituted 14a, and 7-substituted 15a, all have good
potency against DPP-4, and good selectivity against
QPP, DPP-8, and other closely related peptidases. They
have good to excellent pharmacokinetic properties
across species from rat and dog to monkey. And they
have shown good efficacy after oral dosing in rodents
at dose levels correlated well to their intrinsic potency.
Optimization of this promising lead continues as we
try to explore specific interactions in the enzyme active
site identified by structural biology, and with input from
computer modeling.
15. Biftu, T.; Feng, D.; Qian, X.; Liang, G.-B.; Kieczykowski,
G.; Eiermann, G.; He, H.; Leiting, B.; Lyons, K.; Petrov,
A.; Sinha-Roy, R.; Zhang, B.; Scapin, G.; Patel, S.; Gao,
Y.-D.; Singh, S.; Wu, J.; Zhang, X.; Thornberry, N. A.;
Weber, A. E. Bioorg. Med. Chem. Lett. 2007, 17, 49.
16. Kim, D.; Wang, L.; Beconi, M.; Eiermann, G. J.; Fisher,
M. H.; He, K.; Hickey, G. J.; Kowalchick, J. E.; Leiting,
B.; Lyons, K.; Marsilio, F.; McCann, M. E.; Patel, R. A.;
Petrov, A.; Scapin, G.; Patel, S. B.; Roy, R.; Wu, J.;
Wyvratt, M. J.; Zhang, B. B.; Zhu, L.; Thornberry, N. A.;
Weber, A. E. J. Med. Chem. 2005, 48, 141.
1
17. (a) All new compounds were characterized by H NMR
and LC-MS prior to submission for biological evaluation;
(b) All multiple determinations of the IC50 values were
within 1.5-fold of the reported average.
18. 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.
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Acknowledgments
20. Lankas, G. R.; Leiting, B.; Sinha Roy, R.; Eiermann, G.
J.; Beconi, M. G.; Biftu, T.; Chan, C.-C.; Edmondson, S.;
Feeney, W. P.; He, H.; Ippolito, D. E.; Kim, D.; Lyons, K.
A.; Ok, H. O.; Patel, R. A.; Petrov, A. N.; Pryor, K. A.;
Qian, X.; Reigle, L.; Woods, A.; Wu, J.; Zaller, D.; Zhang,
X.; Zhu, L.; Weber, A. E.; Thornberry, N. A. Diabetes
2005, 54, 2988.
21. Maes, M.-B.; Lambier, A.-M.; Gilany, K.; Senten, K.;
Van der veken, P.; Leiting, B.; Augustyns, K.; Scharpe, S.;
De Meester, I. Biochem. J. 2005, 386, 315.
We thank Dr. Phil Eskola, Regina Black, Mark Levorse,
Joe Leone, Bob Frankshun, Amanda Makarewicz,
Daniel Kim, and Dr. Derek Von Langen of Synthetic
Services Group for large scale synthetic support, and
Dr. Bernard Choi and Eric Streckfuss of Analytical Sup-
port Group for open access LC-MS service.
22. Herman, G. A.; Stevens, C.; van Dyck, K.; Bergman, A.;
Yi, B.; De Smet, M.; Snyder, K.; Hilliard, D.; Tanen, M.;
Tanaka, W.; Wang, A. Q.; Zeng, W.; Musson, D.;
Winchel, G.; Davies, M. J.; Ramael, S.; Gottesdiener, K.
M.; Wagner, J. A. Clin. Pharmacol. Ther. 2005, 78, 675.
23. It should be noted that % inhibition as determined using
the in vitro assay under estimates the % inhibition
achieved in vivo, as compound 14a is a competitive,
rapidly reversible inhibitor and assay of plasma DPP-4
activity requires (1) dilution of plasma which results in a
dilution of the total inhibitor, and (2) presence of substrate
that competes with inhibitor for binding to the enzyme.
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