Discovery of ((4R,5S)-5-amino-4-(2,4,5-trifluorophenyl)cyclohex-1-enyl)-3- (trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H-yl)methanone (ABT-341), a highly potent, selective, orally efficacious, and safe dipeptidyl peptidase IV inhibitor

Article abstract of DOI:10.1021/jm060955d

Dipeptidyl peptidase IV (DPP4) deactivates glucose-regulating hormones such as GLP-1 and GIP, thus, DPP4 inhibition has become a useful therapy for type 2 diabetes. Optimization of the high-throughput screening lead 6 led to the discovery of 25 (ABT-341),

Full text of DOI:10.1021/jm060955d

J. Med. Chem. 2006, 49, 6439-6442
6439
Chart 1. Structures of Selected DPP4 Inhibitors in Clinical
Trials
Discovery of ((4R,5S)-5-Amino-4-(2,4,5-
trifluorophenyl)cyclohex-1-enyl)-(3-
(trifluoromethyl)-5,6-dihydro-
[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone
(ABT-341), a Highly Potent, Selective, Orally
Efficacious, and Safe Dipeptidyl Peptidase IV
Inhibitor for the Treatment of Type 2 Diabetes
Zhonghua Pei,* Xiaofeng Li, Thomas W. von Geldern,
David J. Madar, Kenton Longenecker, Hong Yong,
Thomas H. Lubben, Kent D. Stewart, Bradley A. Zinker,
Bradley J. Backes, Andrew S. Judd, Mathew Mulhern,
Stephen J. Ballaron, Michael A. Stashko, Amanda K. Mika,
David W. A. Beno, Glenn A. Reinhart, Ryan M. Fryer,
Lee C. Preusser, Anita J. Kempf-Grote, Hing L. Sham, and
James M. Trevillyan
Chart 2
Metabolic Disease Research, Global Pharmaceutical Research and
DeVelopment, Abbott Laboratories, Abbott Park, Illinois 60064-6098
ReceiVed August 7, 2006
Intensive research efforts have resulted in the discovery of a
number of potent DPP4 inhibitors.7 Pioneering work led to the
identification of first-generation DPP4 inhibitors such as 18 and
29 (Chart 1). They are potent but may lack optimal selectivity10
against other related peptidases such as DPP811 and DPP9.12
Because inhibition of DPP8/9 was linked to toxicity in animal
studies,13 efforts were made to identify second-generation
inhibitors that are selectiVe. Another study demonstrated that
high levels of GLP-1 should be maintained for 24 h for optimal
glycemic control.14 These results provided impetus for us and
others to identify potential third-generation DPP4 inhibitors that
are not only potent and selective, but also possess pharmaco-
kinetic profiles that provide g90% DPP4 inhibition for g24 h
for maximal efficacy, particularly in more severe diabetic
patients (e.g., HbA1c > 9%).15 Preclinical studies will give
indications as to whether this goal is achieved by known DPP4
inhibitors 3,16 4,17 5,18 or our new compound, although a
definitive answer will await clinical trials in humans.
In this work, cyclohexene-constrained phenethylamine 6
(Chart 2) was identified by high-throughput screening of the
Abbott compound collection and subsequently confirmed as a
weak DPP4 inhibitor with a Ki of 0.82 µM, as assayed with
human DPP4 using Gly-Pro-7-amido-methylcoumarin (Gly-Pro-
AMC) as the substrate.19 After initial SAR exploration revealed
that (1) 2,4-dichlorophenyl increases potency over 2-chlorophen-
yl; (2) the most fruitful position on the cyclohexene ring to
modify is C5, as evidenced by compound 7 (Ki < 0.14 µM),
acid 8 was identified as a lead with a Ki of 0.045 µM.
The general synthesis of cyclohexene-constrained phenethyl-
amines is outlined in Scheme 1. Thermal Diels-Alder reaction
between nitrostyrenes 9 and 2-trimethylsiloxy-1,3-butadiene
gave racemic ketones 10 after acidic workup. Vilsmeier bro-
moformylation of ketones 10 furnished bromoaldehydes 11 in
modest yields.20 After the aldehydes were reduced to alcohols
12, the bromine atom was removed to afford alcohols 13. The
alcohol group was transformed to a mesylate group, which was
replaced with amines. Reduction of the nitro group and
subsequent reverse-phase HPLC purification afforded amines
8 or 14 as TFA salts. Alternatively, the nitro group of 12b was
reduced with zinc, and the resulting amino group was protected
with a Boc group to afford 15, which was then debrominated
Abstract: Dipeptidyl peptidase IV (DPP4) deactivates glucose-
regulating hormones such as GLP-1 and GIP, thus, DPP4 inhibition
has become a useful therapy for type 2 diabetes. Optimization of the
high-throughput screening lead 6 led to the discovery of 25 (ABT-
341), a highly potent, selective, and orally bioavailable DPP4 inhibitor.
When dosed orally, 25 dose-dependently reduced glucose excursion
in ZDF rats. Amide 25 is safe in a battery of in vitro and in vivo tests
and may represent a new therapeutic agent for the treatment of type 2
diabetes.
Dipeptidyl peptidase IV (DPP4,a also known as CD26) is a
110-kDa serine protease that is ubiquitously distributed in the
body. It modulates biological activities of a number of peptides
by cleaving two amino acid residues from the N-terminus.1 Two
of the most prominent endogenous substrates of DPP4 are
glucagon-like peptide-1 (GLP-1) and glucose-dependent insuli-
notropic polypeptide (GIP, also known as gastric inhibitory
polypeptide), both of which play important roles in regulating
blood glucose levels.2 DPP4 knock-out mice not only showed
reduced degradation of both GLP-1 and GIP, but were also
healthy without any major immune phenotype (despite the
putative importance of DPP4 for immune function), showed
improved glucose tolerance, and were resistant to body weight
gain on a high-fat diet.3 DPP4-deficient Fisher rats also showed
similar phenotypes.4 Furthermore, chronic treatment with DPP4
inhibitors preserved islet function in diabetic mice and improved
â-cell survival and islet cell neogenesis in streptozotocin-induced
diabetic rats,5 which suggest that DPP4 inhibitors might be able
to induce production of new â-cells in type 2 diabetics and thus
prevent the progression of the disease. More importantly, several
clinical trials in humans show that small-molecule DPP4
inhibitors are well-tolerated, lower blood glucose and/or HbA1c
levels, and increase glucose tolerance.6 All the above evidence
suggests that DPP4 inhibitors have the potential to be novel,
safe, and useful antidiabetic agents.7
* To whom correspondance should be addressed. Phone: 847-935-6254.
Fax: 847-938-1674. E-mail: zhonghua.pei@abbott.com.
a Abbreviations: DPP4, dipeptidyl peptidase IV; GLP-1, glucagon-like
peptide-1; GIP, glucose-dependent insulinotropic polypeptide; OGTT, oral
glucose tolerance test; HbA1c, glycosylated (or glycated) hemoglobin A.
10.1021/jm060955d CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/07/2006

6440 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Letters
Table 1. SAR Summary of DPP4 Inhibitorsa
Scheme 1a
a Values reported are the mean of at least two runs using human DPP4.
Potencies were unchanged when rat DPP4 was used. b Racemic. c Chiral.
d Ki in 10% of human serum.
a Reagents and conditions: (a) trimethylsiloxy-1,3-butadiene, toluene,
120 °C, then TFA; (b) PBr3, DMF/CH2Cl2, 0 °C to rt; (c) NaBH4 or
NaBH(OAc)3, EtOH/CH2Cl2; (d) HCO2H, n-Bu3N, cat. Pd(PPh3)2Cl2, DMF,
80 °C; (e) MsCl, CH2Cl2, TEA, 0 °C; (f) R2R3NH, CH2Cl2, TEA; (g) Zn,
HOAc/MeOH, reflux; then HPLC; (h) (Boc)2O, THF;. (i) Ar′OH, DBAD,
PPh3, toluene, 80 °C; (j) TFA, CH2Cl2, rt; (k) Dess-Martin periodinane,
CH2Cl2; (l) NaBH3CN, Ar′NH2; (m) NaClO2, isoprene, NaH2PO4 buffer,
DMSO; (n) R4R5NH, TBTU, TEA, DMF, rt; (o) TMSCHN2, MeOH; (p)
chiral prep. HPLC; (q) NaOH, THF/H2O, rt.
to alcohol 16. Mitsunobu reaction between 16 and a phenol,
then subsequent removal of the Boc group, yielded ether 17.
Alcohol 16 was oxidized to aldehyde 18, which underwent
reductive amination and subsequent removal of the Boc group
to give 19. Aldehyde 18 was further oxidized to acid 20, which
was coupled with an amine, followed by removal of the Boc
group to provide amide 22.
To make optically pure inhibitors, acid 20 was transformed
to racemic ester 23, which was resolved by chiral HPLC to give
optically pure ester 24. Ester 24 was hydrolyzed under basic
conditions to give chiral acid 21, which was converted to
optically pure final product 25 in a similar manner to that
described for amide 22.
Table 1 summarizes the SAR of the DPP4 inhibition and
selectivity over DPP8 and DPP9. After dichlorophenyl was
switched to a less hydrophobic, equipotent trifluorophenyl at
the P1 position, bicyclic amine 14 (Ki ) 12 nM) was identified
and showed significantly improved potency over acid 8. Moving
the phenyl ring closer to the cyclohexene ring and introducing
an amidophenyl group resulted in 19 with a single-digit Ki of 6
nM. Analog 17, with an ether linker, was also very potent, with
a Ki of 3.6 nM. However, all of these analogs had limited
selectivity over DPP8 and DPP9 (e.g., amine 14 was only 6-fold
selective over DPP9). Replacement of the flexible linkers with
a more rigid amide significantly improved the selectivity. Thus,
amide 22 had a Ki of 4.7 nM against DPP4 and was 1800-fold
and 3000-fold selective over DPP8 and DPP9, respectively.
Figure 1. X-ray crystal structure of 25/huDPP4 complex. Color-
coding: all nitrogen atoms are in blue, all oxygen atoms in red and
fluorine atoms in cyan. Carbon atoms of 25 are in green, while the
carbon atoms of the protein are in gray. The protein surface is shown
with atom coloring.
Finally, introduction of a known bicyclic amine16 provided 25
(A-916165), which showed excellent potency (Ki ) 1.3 nM)
and selectivity, and the potency was maintained even in the
presence of 10% human serum (Ki ) 1.6 nM). Neither was there
a change in potency when rat DPP4 was used.
The structure of the amide 25/huDPP4 complex was solved
by X-ray crystallography. As shown in Figure 1, the trifluo-
rophenyl occupies the hydrophobic S1 pocket.21 The amino
group on the cyclohexene ring is in close proximity to the side
chains of Glu205 and Glu206 for an electrostatic interaction.
The carbonyl oxygen of 25 is oriented toward a water mole-
cule positioned for a bridging hydrogen-bonding interaction
with the side chain of Arg669. A favorable hydrophobic
interaction of the heterocycle with the side chain of Phe357 is
observed.
Amide 25 showed superior pharmacokinetic (PK) profiles in
all three species tested (Table 2). Amide 25 is characterized by
having good exposure across the species, good oral half-lives
(5.4 to 6.7 h), large volumes of distribution (7.19 to 3.20 L/kg),
and excellent oral bioavailabilities (54 to 104%). Based on the
PK in these three species, it is predicted that a 150 mg dose of

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6441
Table 2. Selected PK Parameters of Amide 25 in Rat, Dog, and
a benign cardiovascular profile at plasma concentrations vastly
higher than those required to achieve efficacy in vivo. As such,
amide 25 produced no physiologically relevant effects on mean,
systolic, or diastolic arterial pressure, heart rate, cardiac output,
pulmonary arterial pressure, pulmonary vascular resistance, or
systemic vascular resistance at the highest concentration tested
(23.9 ( 1.3 µg/mL) and produced only a small reduction in
ventricular contractility at the same concentration. Also, con-
sistent with the absence of hERG binding, amide 25 produced
no increase in the QT-interval corrected for changes in heart
rate.22 Amide 25 caused no toxicological effects in a 5-day study
in rats when dosed up to 1000 mg/kg/day. Taken together, the
combination of superb potency and pharmacokinetic profiles
offers the potential for amide 25 to reach maximal efficacy
achievable by the mechanism of DPP4 inhibition (as limited
by the GLP-1 and GIP secretory capacity). Based on its
combined profile of excellent potency, selectivity, efficacy, and
safety, amide 25 was selected as a drug development candidate
(ABT-341).
Monkeya
AUC
Cmax
Tmax
t1/2
Vss
F
(%)
(ng‚hr/g)
(ng/mL)
(hr)
(hr)
(L/kg)
rat
iv
po
iv
po
iv
po
2920
1948
6019
6285
3557
1905
5.3
5.4
7.0
6.7
4.3
6.3
7.19
4.03
3.20
195
718
345
4.7
67
104
54
dog
0.58
2.7
monkey
a Dosed at 5 mg/kg in rats (n ) 6) and 2.5 mg/kg in dogs and monkeys
(n ) 6). AUC, area under curve; Cmax, max. concentration; Tmax, time when
max. concentration was achieved; t1/2, terminal half-life; Vss, steady-state
volume of distribution; F, oral bioavaibility.
In summary, aided with structural information, we were able
to quickly optimize the high-throughput screening lead 5, leading
to the discovery of amide 25, a highly potent, selective, orally
efficacious, and potentially third-generation DPP4 inhibitor.
When dosed orally, amide 25 dose-dependently reduced glucose
excursion, increased active GLP-1 levels, and decreased glu-
cagon levels in ZDF rats. Amide 25 is safe in a battery of in
vitro and in vivo safety tests and may represent a new
therapeutic agent for the treatment of type 2 diabetes.
Figure 2. Effects of amide 25 on glucose excursion in female ZDF
rats (n ) 10/group).
amide 25 once a day will provide g90% DPP4 inhibition for
24 h in humans.22
Acknowledgment. The authors thank Larry R. Solomon,
Marc R. Lake, Rohinton Edalji, and Elizabeth H. Fry for DPP4
preparation, M. S. Diehl and P. A. Sonders for mini-Ames and
micronucleus tests, James Limberis and Zhi Su for hERG
binding, Chong Chen for preparative chiral HPLC, Zhengping
Tian and Daniel J. Plata for providing 8b, and Michael Duvall
for a two-week rat safety study.
Next, we profiled amide 25 in our efficacy model. Briefly,
10-week old, female Zucker diabetic fatty (ZDF) rats were dosed
with either vehicle or amide 25 orally after an overnight fast.
Four hours later (t ) 0), the rats were allowed free access to a
highly palatable, macronutrient balanced food source during the
next 4 h. This model is analogous to oral glucose tolerance tests
(OGTT) used clinically to routinely evaluate glycemic control,
except that the “challenge” is a liquid mixed meal composed
of fats, proteins, and carbohydrates, thus a more realistic
representation of the nutritional makeup of normal food. Plasma
glucose levels were measured at six time points over the course
of 4 h, and the change of plasma glucose levels from the baseline
is shown in Figure 2. Amide 25 caused a dose-dependent
reduction in glucose excursion. The reduction as measured by
area under curve (AUC) was 23, 37, and 51% at 0.3, 1.0, and
3.0 mpk, respectively. Consistent with the mechanism of action,
active GLP-1 levels (measured at t ) 10 min) increased by
151%, 163% and 291% at 0.3, 1.0 and 3.0 mpk, respectively.
Glucagon levels (measured at t ) 30 min) decreased by 36, 46,
and 61% at 0.3, 1.0, and 3.0 mpk, respectively. The plasma
concentration of amide 25 reached 120 ng/mL (t ) 0), pro-
viding 98% (t ) 0) and 96% (t ) 240 min) inhibition of DPP4
at 3 mpk.
Besides being efficacious in vivo, amide 25 also possesses
an excellent safety profile. It showed no inhibition of major
liver metabolic enzymes such as CYP3A4, CYP2D6, and
CYP2C9 (IC50 > 30 µM). It was negative in both mini-Ames
and clastogenicity tests23 (up to 2000 µg/well). It exhibited no
hERG binding (dofetilide Ki > 50 µM, IC50 > 300 µM in patch
clamp using HEK 293 cells). When amide 25 was tested against
a panel of 74 receptor-binding/ion channel assays (Cerep
screening) at a concentration of 10 µM,24 it did not display
control-specific binding by 50% or greater in any of the 74
assays. When administered intravenously using an anesthetized,
comprehensively instrumented dog model,25 amide 25 exhibited
Supporting Information Available: Experimental procedures
including characterization data for compounds and dog cardiovas-
cular safety data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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JM060955D