4-Amino cyclohexylglycine analogues as potent dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.10.016

Substituted 4-amino cyclohexylglycine analogues were evaluated for DP-IV inhibitory properties. Bis-sulfonamide 15e was an extremely potent 2.6 nM inhibitor of the enzyme with excellent selectivity over all counterscreens. 2,4-Difluorobenzenesulfonamide 1

Full text of DOI:10.1016/j.bmcl.2003.10.016

Bioorganic & Medicinal Chemistry Letters 14 (2004) 43–46
4-Amino cyclohexylglycine analogues as potent dipeptidyl
peptidase IV inhibitors
Emma R. Parmee,a,* Jiafang He,a Anthony Mastracchio,a Scott D. Edmondson,a
Larry Colwell,a George Eiermann,c William P. Feeney,e Bahanu Habulihaz,a
Huaibing He,a Ruth Kilburn,d Barbara Leiting,b Kathryn Lyons,a Frank Marsilio,b
Reshma A. Patel,b Aleksandr Petrov,c Jerry Di Salvo,d 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 Animal Pharmacology, Merck & Co. Inc., PO Box 2000, Rahway, NJ 07065, USA
dDepartment of Immunology and Rheumatology, Merck & Co. Inc., PO Box 2000, Rahway, NJ 07065, USA
eDepartment of Comparative Medicine, Merck & Co. Inc., PO Box 2000, Rahway, NJ 07065, USA
Received 6 August 2003; revised 6 October 2003; accepted 14 October 2003
Abstract—Substituted 4-amino cyclohexylglycine analogues were evaluated for DP-IV inhibitory properties. Bis-sulfonamide 15e
was an extremely potent 2.6 nM inhibitor of the enzyme with excellent selectivity over all counterscreens. 2,4-Difluoro-
benzenesulfonamide 15b and 1-naphthyl amide 16b, however, combined an acceptable in vitro profile with good pharmacokinetic
properties in the rat, and 15b was orally efficacious at 3 mpk in an OGTT in lean mice.
# 2003 Elsevier Ltd. All rights reserved.
Inhibition of dipeptidyl peptidase IV (DP-IV), a proline
specific serine dipeptidase, is a novel therapeutic
approach to the treatment of type 2 diabetes.1 DP-IV
rapidly cleaves the active form of the incretin hormone
glucagon-like peptide 1 (GLP-1) (GLP-1[7-36] amide) to
its inactive form (GLP-1[9-36] amide) with a half-life of
approximately one minute and is thought to be the pri-
mary enzyme responsible for this hydrolysis.1c GLP-1
plays an important role in glucose-dependent insulin
biosynthesis and secretion in humans. Infusion of GLP-
1 has been shown to normalize both postprandial and
fasting glucose in diabetics.2 Additionally, chronic
administration of GLP-1 has resulted in significant
decreases in both fasting plasma glucose and HbA1c.3
Inhibition of DP-IV, therefore, is expected to sig-
nificantly reduce inactivation of GLP-1[7-36] and
should lead to an increase in circulating levels of the
active form of the hormone. Supporting evidence for
this comes from DP-IV deficient mice, which have ele-
vated levels of GLP-1[7-36] amide.4 Thus, DP-IV inhi-
bitors act as indirect stimulators of insulin secretion by
stabilization of GLP-1.
Importantly, in contrast to many current therapies for
type 2 diabetes, this mechanism potentially carries with
it only a low risk of hypoglycemia since GLP-1 stimu-
lates insulin release solely in the presence of elevated
plasma glucose levels. Other possible advantages of DP-
IV inhibitors as a treatment for diabetes include the fact
that no weight gain is anticipated and there are poten-
tial long-term beneficial effects on beta cell function.5
Several small molecule inhibitors of DP-IV have been
reported in the literature and progressed into clinical trials
with positive results.6,7 Of these, compounds 1 and 2 are
the most potent inhibitors of DP-IV (IC50=22 and 34 nM,
respectively),8 but both contain an electrophilic nitrile (Fig.
1).9 Inhibitors lacking an electrophile, while more stable,
are generally of only modest intrinsic potency. One such
example is (S)-isoleucine thiazolidide 3, which showed
good effects in limited clinical trials despite moderate inhi-
bitory properties (DP-IV IC50=420 nM).7,8
* Corresponding author. Tel.: +1-732-594-6626; fax: +1-732-594-
9473; e-mail: emma_parmee@merck.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.016

44
E. R. Parmee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 43–46
The cis isomer 6 was obtained in a 36% yield from 5
accompanied by the epimeric alcohol and fully reduced
cyclohexyl compound. Alcohol 6 was converted to its
methanesulfonate, followed by installation of the amine
masked as its azide, which was carried through the
subsequent saponification and amide coupling to afford
amide 8. The amino functionality was then released
from the azide by treatment with triphenylphosphine in
aqueous tetrahydrofuran, followed by acylation and
deprotection to give compounds 11–17.13 Deprotection
of the tert-butylcarbamate group in 9 gives the parent
amine 10.
Figure 1. Reported DP-IV inhibitors.
Table 1 summarizes the inhibitory properties of amino
containing thiazolidides. As can be seen from analogue
10, a primary amino moiety at the 4-position was not
well tolerated. Attenuation of the basicity by acylation
of the nitrogen, however, provided a series of potent
thiazolidide containing DP-IV inhibitors (IC50<60
nM). Sulfonamide derivatives 11a–e were equipotent 20
nM inhibitors of the enzyme. Differences in selectivity
over QPP were however evident, as the window for DP-
IV inhibition ranged from 50-fold for 4-tri-
fluoromethoxyphenyl compound 11b to 9-fold for 2-
Related to compound 3, (S)-cyclohexylglycine pyrroli-
dide 4 (R=H, X=CH2, DP-IV IC50=320 nM)8,10 has
been reported as an inhibitor of DP-IV and we selected
this as a scaffold for further modification in an effort to
prepare DP-IV inhibitors of higher intrinsic potency but
lacking an electrophile. We decided to install an amino
substituent (R=NH2) in the 4-position of the cyclo-
hexyl ring of compound 4 and investigate the effect of a
variety of aromatic acyl and sulfonyl capping groups on
DP-IV inhibitory properties. Initially a thiazolidine ring
was employed as the proline mimic (X=S). Inhibitors
were also assessed for their selectivity profiles against a
variety of DP-IV homologues and proline-specific
enzymes [including prolyl endopeptidase, amino pepti-
dase P, prolidase, quiescent prolyl peptidase (QPP/DPP-
II), and seprase (FAP)].11 Significant off-target activity
(<10,000 nM), however, only presented with QPP and
hence only these data will be discussed. QPP is an
enzyme reported to have DP-IV-like activity, although
its exact physiological function is not currently well
understood.12 The QPP inhibitory properties of com-
pounds 1–4 showed that nitrile containing analogues
exhibited excellent selectivity for DP-IV (1 and 2 at least
300-fold), while analogues 3 and 4 were 5–10-fold less
selective.
naphthyl derivative 11e. Incorporating
a
urea
substituent onto the benzenesulfonamide as in analogue
11f14 increased potency (DP-IV IC50=7.5 nM) as well
as selectivity (>250-fold window over inhibition of
QPP). Amide derivatives 12 showed a similar range of
activities against DP-IV, with 3-quinolyl analogue 12g
being the most potent (DP-IV IC50=9 nM), while the 1-
naphthyl derivative 12b exhibited both excellent potency
and a 60-fold window over inhibition of QPP. While the
carbamate and urea analogues 13 and 14 were also
potent inhibitors of DP-IV, benzyl carbamate 13a was
the only analogue to exhibit significant selectivity over
QPP (36-fold). The epimeric cis analogues of 11a,b,e,
12c, and 13a were prepared from the trans isomer of
alcohol 6 in an identical manner to that described
above. They were all found to be significantly less
potent inhibitors of DP-IV (IC50’s 100–500 nM) and no
selectivity over QPP was observed (data not shown).
Inhibitors were synthesized from alcohol 6, prepared
from (S)-4-hydroxyphenylglycine by esterification and
protection of the amine as its tert-butylcarbamate fol-
lowed by hydrogenation of the aromatic ring using cat-
alytic platinum oxide in acetic acid at 45 psi (Scheme 1).
Selected derivatives were also prepared in the pyrrolidide
series, and their inhibitory properties are summarized in
Scheme 1. Synthesis of 4-amino cyclohexylglycine analogues: (a) HClg, MeOH, 45 ꢀC; (b) BOC2O, DIEA, EtOAc; (c) PtO2, AcOH, H2, 45 psi, 16 h;
(d) MsCl, DIEA, CH2Cl2; (e) NaN3, DMF, 60 ꢀC; (f) aq LiOH, THF; (g) thiazolidine or pyrrolidine, EDC, HOBt, DIEA, DMF; (h) PPh3, aq THF;
(i) acylating agent, Et3N, CH2Cl2; (j) TFA, CH2Cl2.

E. R. Parmee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 43–46
Table 1. Inhibitory properties of selected DP-IV inhibitors8
45
Table 2. Inhibitory properties of selected DP-IV inhibitors8
Entry
Ar
DP-IV IC50, nM
QPP IC50, nM
Entry
Ar
DP-IV IC50, nM
QPP IC50, nM
1
2
3
4
10
—
—
—
22
34
420
320
2700
23
22
24
24
18
7.5
33
33
53
38
15
21
9
25
29
17
20
10
55
17
6600
>100,000
15,000
19,000
—
15a
15b
15c
15d
15e
16a
16b
16c
17
4-CF3OPh
2,4-diFPh
2,3,4-triFPh
4-Me2NCONHPh
4-CF3CH2SO2NHPh
3,4-diFPh
89
88
98
26
2.6
190
46
64
94
6400
8800
8200
12,000
15,000
5000
8600
4500
(R=H, X=CH2)
—
4-IPh
4-CF3OPh
3-CNPh
2,3,4-triFPh
2-Naphthyl
4-Me2NCONHPh
4-IPh
4-CF3OPh
3,4-diFPh
3,4-diClPh
1-Naphthyl
2-Naphthyl
3-Quinolinyl
Ph
3,4-diClPh
1-Naphthyl
2-Naphthyl
4-IPh
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
12g
13a
13b
13c
13d
14a
14b
14c
14d
280
1100
820
960
170
2000
340
360
690
230
900
230
230
900
170
1-Naphthyl
3-Quinolinyl
Ph
4100
Table 3. hERGbinding and rat pharmacokinetic properties (1/2 mpk
iv/po) of selected DP-IV inhibitors
Entry hERGbinding Ki (nM) Clp (mL/min/kg) t1/2 (h) F (%)
11b
12c
12e
14a
15a
15b
15d
15e
16b
17
2800
5900
22,000
1900
5700
21
30
22
74
31
53
21
95
13
28
4.7
2
85
81
51
12
38
36
1
1.2
2.3
3.6
1.8
2.4
0.1
2.3
4.0
170
200
210
350
170
380
35,000
>100,000
>100,000
17,000
40,000
4-CF3OPh
3,4-diClPh
3,4-diFPh
0
30
52
35
Table 2. These analogues were at least 3-fold less potent
than their thiazolidide congeners, but exhibited
improved selectivity over QPP. Thus, sulfonamide 15a
was 4-fold less potent than 11b, but was more than 70-
fold selective over inhibition of QPP. Further improve-
ments in selectivity were achieved. For example, 2,4-
difluorobenzenesulfonamide 15b (DP-IV IC50=88 nM)
displayed a 100-fold window over QPP. Increasing the
polarity of the phenyl substituents again resulted in
dramatic improvements in the in vitro profile of these
compounds, exemplifed with N,N-dimethylurea 15d14
(DP-IV IC50=26 nM, >450-fold selective over QPP). A
more profound improvement in intrinsic activity was
obtained, however, with bis-sulfonamide 15e14 which is
a highly potent 2.6 nM inhibitor of DP-IV with >5000-
fold selectivity over inhibition of QPP. This analogue
represents the most potent DP-IV inhibitor reported to
date lacking an electrophile on the pyrrolidide ring. In
the amide series, the 1-naphthyl derivative 16b was
again the most potent and selective analogue (DP-IV
IC50=46 nM, 180-fold selective over inhibition of
QPP).
hERGpotassium channel was significantly improved by
replacement of the trifluoromethoxyphenyl group with a
2,4-difluorophenyl moiety in analogue 15b which was
now devoid of hERGbinding up to concentrations of
35000 nM whilst maintaining good pharmacokinetic
properties in the rat. Analogues 15d and 15e, which had
displayed enhanced selectivity over QPP (vide supra),
displayed negligible ion channel binding (hERGbinding
Ki >100,000 nM). Incorporation of the polar function-
ality was, however, detrimental to pharmacokinetic
properties as evidenced by the extremely poor oral
bioavailability of both these analogues.
Mimicking the simple sulfonamides, amides 12c,e, and
16b also possessed acceptable to good pharmacokinetic
properites in the rat, with the naphthyl derivatives show-
ing the best selectivity profiles over hERGbinding
(Ki>17,000 nM). Urea 14a exhibited one of the poorest
overall profiles, with significant affinity for the hERG
channel, a high clearance and low oral bioavailability.
Carbamate 17 had the best pharmacokinetic profile of the
pyrrolidide analogues (F=52%) and was devoid of ion
channel activity up to concentrations of 40,000 nM.
Representative analogues were selected for evaluation
of pharmacokinetic properties in the rat and possible
ion channel activity as a measure of general off-target
activity. The latter is illustrated here with binding to the
hERGpotassium channel ( Table 3).15 In the sulfon-
amide series, trifluoromethoxyphenyl analogues (11b
and 15a) had demonstrable ion channel binding (hERG
binding Ki=2800 and 5700 nM, respectively). These
compounds, however, had excellent pharmacokinetic
properties with a moderate clearance and long half-life
(4–5 h). The thiazolidide ring appeared to impart
superior oral bioavailability in the rat [F=85% (entry
1) vs 38% (entry 5)]. Selectivity over binding to the
In summary, we have discovered a novel series of potent
and selective DP-IV inhibitors. These are among the
most potent compounds reported to date lacking an
electrophilic trap. Notable among these is bis-sulfona-
mide 15e, which is a 2.6 nM DP-IV inhibitor with an
exceptional selectivity profile over both QPP and ion
channel binding. This compound, however, lacks oral
bioavailability in the rat. Sulfonamide 15b and amide
16b both exhibit an acceptable 100-fold window over
QPP, with excellent ion channel selectivity and good
pharmacokinetic properties in the rat. Sulfonamide 15b
was selected for assessment in the dog where it had an

46
E. R. Parmee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 43–46
excellent pharmacokinetic profile with a moderate clear-
ance (14 mL/min/kg), 6 h half-life, and oral bioavail-
ability equal to 100%. This compound was evaluated in
vivo for glucose lowering potential. Thus, a dose of 3 mpk
given 1 h prior to an oral glucose tolerance test (OGTT)
in lean C57BL6/N mice resulted in a 36% reduction in
glucose excursion when compared to vehicle.
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This important result confirmed the possible utility of
substituted 4-aminocyclohexylglycine DP-IV inhibitors
as orally active hypoglycemic agents for the treatment
of type-2 diabetes. It was subsequently found that
modifications to the pyrrolidide ring resulted in a fur-
ther enhancement in both the selectivity and
pharmacokinetic profile of these inhibitors. These
alterations will be the subject of a future publication.
Acknowledgements
We would like to thank Mr Don Hora, Mr Paul Cun-
ningham, and Ms Gina Seeburger for the dog
pharmacokinetic study.
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