Fluoropyrrolidine amides as dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.12.040

Amides derived from fluorinated pyrrolidines and 4-substituted cyclohexylglycine analogues have been prepared and evaluated as inhibitors of dipeptidyl dipeptidase IV (DP-IV). Analogues which incorporated (S)-3-fluoropyrrolidine showed good selectivity for DP-IV over quiescent cell proline dipeptidase (QPP). Compound 48 had good pharmacokinetic properties and was orally active in an oral glucose tolerance test in lean mice.

Full text of DOI:10.1016/j.bmcl.2003.12.040

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1265–1268
Fluoropyrrolidine amides as dipeptidyl peptidase IV inhibitors
Charles G. Caldwell,a,* Ping Chen,a Jiafang He,a Emma R. Parmee,a
Barbara Leiting,b Frank Marsilio,b Reshma A. Patel,b Joseph K. Wu,b
George J. Eiermann,c Aleksandr Petrov,c Huaibing He,d Kathryn A. Lyons,d
Nancy A. Thornberryb and Ann E. Webera
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Metabolic Disorders-Diabetes, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
cDepartment of Animal Pharmacology, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
dDepartment of Basic Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 30 October 2003; revised 3 December 2003; accepted 9 December 2003
Abstract—Amides derived from fluorinated pyrrolidines and 4-substituted cyclohexylglycine analogues have been prepared and
evaluated as inhibitors of dipeptidyl dipeptidase IV (DP-IV). Analogues which incorporated (S)-3-fluoropyrrolidine showed good
selectivity for DP-IV over quiescent cell proline dipeptidase (QPP). Compound 48 had good pharmacokinetic properties and was
orally active in an oral glucose tolerance test in lean mice.
# 2004 Elsevier Ltd. All rights reserved.
Type-2 diabetes mellitus, also known as non-insulin-
dependent diabetes mellitus, accounts for >90% of the
cases of diabetes. This condition is characterized by
high levels of glucose resulting from progressive insulin
resistance and, at later stages of the disease, impairment
of insulin secretion.1 Glucagon-like peptide-1 (GLP-1) is
an incretin hormone secreted by intestinal L-cells in
response to food intake. The active form GLP-1[7-36]
amide is a 30-amino acid peptide which stimulates
insulin release while inhibiting hepatic glucose produc-
tion.2 Continuous subcutaneous infusion of active
GLP-1 in diabetic patients for 6 weeks has resulted in
reduction of blood glucose and hemoglobin A1c levels.3
GLP-1[7-36] amide is rapidly converted into the inactive
form GLP-1[9-36] amide by dipeptidyl peptidase IV
(DP-IV). This enzyme is a serine protease which cleaves
an N-terminal dipeptide from polypeptides, with a
preference for substrates having a proline or alanine
residue at the P1 site.1,2
derivatives 1 and 2 (Fig. 1) are potent inhibitors of DP-
IV, but related compounds lacking the electrophilic
nitrile group show a sharp decrease in activity.5 Addi-
tional selected examples of DP-IV inhibitors include
(S)-isoleucine thiazolidide (3) and cyclohexylglycine
pyrrolidide (4). Clinical trials of compound 3, which has
moderate potency versus DP-IV, resulted in improved
glucose tolerance in diabetic patients and healthy
The use of DP-IV inhibitors to increase levels of endo-
genous GLP-1[7-36] amide has potential as a therapeutic
treatment for diabetic patients.4 The cyanopyrrolidine
* Corresponding author. Tel.:+1-732-594-5439; fax:+1-732-594-5790;
e-mail: chuck_caldwell@merck.com
Figure 1. Inhibitors of DP-IV.
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.040

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C. G. Caldwell et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1265–1268
volunteers.6 Introduction of the cyclohexylglycine sub-
unit of compound 4 produced an analogue which is
somewhat more potent than isoleucine pyrrolidide as a
DP-IV inhibitor.7
by Giardina and co-workers (see Scheme 1).13 Fluor-
ination of the (R)-3-hydroxypyrrolidine derivative 7
using DAST proceeded in good yield.14 Following
hydrogenolysis to remove the Cbz group and filtration
to separate the catalyst, addition of HCl and evapora-
tion of the solvent yielded the (S)-isomer 9. Inversion of
the stereochemistry of alcohol 7 was accomplished as
described by Giardina, and the enantiomer 10 was then
converted into (R)-3-fluoropyrrolidine hydrochloride
(11). Swern oxidation of the 3-hydroxypyrrolidine
intermediate 7 yielded ketone 12 which was fluorinated
in an overnight reaction using [bis(2-methoxy-
ethyl)amino]sulfur trifluoride (Deoxo-Fluor1) in
dichoromethane, or in 3 h using neat DAST. Deprotec-
tion yielded 3,3-difluoropyrrolidine hydrochloride 13.13
An earlier report from our laboratories described 4-
aminocyclohexylglycine derivatives (5) which are potent
DP-IV inhibitors having good pharmacokinetic proper-
ties.8 Augustyns has reported results for the (S)-3-fluoro-
pyrrolidine derivative 6 and the corresponding mixture
of epimeric 3-fluoropyrrolidides.9 The work which we
describe here is an examination of a series of DP-IV
inhibitors resulting from the combination of fluorinated
pyrrolidines with 4-substituted cyclohexylglycine sub-
units previously studied as their thiazolidine or pyrroli-
dine amides.8
l-Tartaric acid (14) reacted with benzylamine to give
the N-benzyl imide derivative which was reduced using
LAH to give (3S,4S)-1-benzyl-3,4-dihydroxypyrroli-
dine.15 After hydrogenolysis to remove the benzyl
group, the pyrrolidine nitrogen was protected to give
the Cbz derivative 15. The vicinal hydroxyl groups were
then activated by formation of the corresponding ditri-
flate, and displacement was accomplished by treatment
with tetrabutylammonium fluoride.16 Hydrogenolysis to
remove the Cbz group proceeded as for the previous
examples to give (3R,4R)-3,4-difluoropyrrolidine
hydrochloride (16). The enantiomeric (3S,4S)-isomer
was prepared from d-tartaric acid using the same syn-
thetic sequence.
Determinations of IC50 values for inhibition of DP-IV
and quiescent cell proline dipeptidase (QPP, also known
as DPP-II or DPP7) were carried out as described pre-
viously.10 Compounds were also tested against prolyl
endopeptidase, amino peptidase P, prolidase, and fibro-
blast activation protein a (FAP, also called seprase).11
Since significant inhibition (IC50 <10,000 nM) was not
observed for these other enzymes, data is presented for
DP-IV and QPP only. Measurements of binding to the
hERG potassium ion channel were obtained on com-
pounds of interest as a measure of general off-target
activity.12
The syntheses of (S)-3-fluoropyrrolidine hydrochloride
(9) and the corresponding (R)-isomer (11) were accom-
plished using modifications of the conditions described
The cyclohexylglycine amides 21–23 (Table 1) were pre-
pared by EDC-mediated coupling of the fluorinated
pyrrolidines with Boc-l-cyclohexylglycine, with sub-
sequent removal of the protecting group. The synthesis
of the 4-amino derivatives shown in Table 2 utilized the
4-azido intermediate 17 described earlier (see Scheme
2).9 Coupling of the pyrrolidines with 17 was followed
by hydrogenation to give amine 19. After acylation or
sulfonylation, removal of the Boc protecting group
provided the 4-substituted cyclohexylglycine derivatives
24–51.
The data presented in Table 1 compare the activities of
three fluorinated pyrrolidine derivatives of cyclohexyl-
Scheme 1. Reagents: (a) DAST, CH2Cl2, À78 ꢀC to rt; (b) H2, 10%
Pd/C, EtOH; (c) HCl; (d) AcOH, DEAD, Ph3P, PhCH3; (e) KOH,
,
EtOH, H2O; (f) (COCl)2, DMSO, Et3N, CH2Cl2; (g) Deoxo-Fluor1
CH2Cl2, rt, 15 h, or DAST (neat), rt, 3 h; (h) BnNH2, xylenes, reflux;
(i) LAH, THF; (j) H2, 20% Pd(OH)2/C, EtOH; (k) BnOC(O)Cl, (i-
Pr)2NEt, CH2Cl2; (l) Tf2O, pyridine, CH2Cl2; (m) (n-Bu)4NF, THF.
Scheme 2. Reagents: (a) EDC, HOBt, Et3N, DMF; (b) H2, 10% Pd/C,
EtOH; (c) RCl, (i-Pr)2NEt, CH2Cl2; (d) HCl, CH3OH.

C. G. Caldwell et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1265–1268
1267
Table 1. Cyclohexylglycine amides
In the 3,4-difluorobenzoyl series of compounds (24–28)
shown in Table 2, the (S)-3-fluoro analogue 26 and the
3,3-difluoro analogue 28 are equipotent with the thia-
zolidide 24 as DP-IV inhibitors, while the (R)-3-fluoro
derivative 26 shows a slight drop in potency. Both 26
and 27 have improved selectivity versus QPP.
Compd
X–Y
DP-IV
IC50
(nM)
QPP
IC50
(nM)
hERG
Ki
(nM)
For the 2-(trifluoromethyl)benzoyl series of derivatives,
the (S)- and (R)-3-fluoropyrrolidides 30 and 31 showed
approximately 100-fold selectivity for DP-IV versus
QPP, although their IC50 values for DP-IV had
increased to >200 nM. The 3,3-difluoropyrrolidine
derivative 32 was more potent as a DP-IV inhibitor,
although selectivity over QPP was relatively low. A
similar trend was observed for the Cbz derivatives 33–
37, with 3,3-difluoropyrrolidide 37 showing the greatest
potency versus DP-IV, while having only 15-fold selec-
tivity over QPP. Both the (3R,4R)- and (3S,4S)-3,4-
difluoropyrrolidine derivatives 38 and 39 had decreased
activity as DP-IV inhibitors.
20
4
21
22
23
S-CH2
CH2-CH2
CHF-CH2 (S)–
CHF-CH2 (R)–
CF2-CH2
89
320
170
340
73
2800
19,000
20,000
38,000
1900
28,000
—
>90,000
>90,000
>90,000
glycine to the parent compound as well as to the thi-
azolidide. Pyrrolidide 4 is approximately 4-fold less
potent than thiazolidide 20 in the DP-IV assay. The (R)-
3-fluoro analogue 22 shows no improvement relative to
pyrrolidide 4 in DP-IV potency, although it does show
some increase in selectivity versus QPP. The (S)-3-fluoro
derivative 21 shows improved DP-IV potency, while
retaining 100-fold selectivity versus QPP. The 3,3-
difluoro analogue 23 is approximately equipotent to the
thiazolidide 20 as a DP-IV inhibitor, although it has lost
the increased selectivity versus QPP seen for the mono-
fluoropyrrolidides 21 and 22.
Among the 4-(trifluoromethoxy)benzenesulfonamides in
Table 2, the (S)-3-fluoro analogue 42 and the 3,3-
difluoro analogue 44 had potency versus DP-IV similar
to the thiazolidide 40. The 3,3-difluoropyrrolidine deri-
vative once again showed relatively low selectivity for
inhibition of DP-IV over QPP. The (R)-3-fluoro com-
pound 43 had a moderate decrease in DP-IV potency
relative to thiazolidide 40, while the (3R,4R)- and
(3S,4S)-3,4-difluoropyrrolidine derivatives (45 and 46)
both had ꢁ10-fold reductions in activity versus DP-IV.
Table 2. 4-Aminocyclohexylglycine derivatives
In the 2,4-difluorobenzenesulfonamide series of com-
pounds (47–51), the (3R,4R)-3,4-difluoropyrrolidine
derivative 51 was the least active DP-IV inhibitor. The
3,3-difluoropyrrolidine analogue 50 showed the greatest
potency versus DP-IV, but the selectivity of this com-
pound over QPP was relatively low. The (S)- and (R)-3-
fluoropyrrolidides 48 and 49 both showed good potency
with IC50 values of 48 and 58 nM, respectively, for
DP-IV inhibition. Potassium channel activity as mea-
sured by hERG binding was weak, with Ki values
approximately 1000-fold higher than the IC50 for DP-IV
inhibition. In addition, compound 48 showed 250-fold
selectivity for inhibition of DP-IV over QPP.
Compd
R
X–Y
DP-IV QPP hERG
IC50 IC50 Ki
(nM) (nM) (nM)
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
CO(3,4-F2C6H3)
CO(3,4-F2C6H3)
CO(3,4-F2C6H3)
CO(3,4-F2C6H3)
CO(3,4-F2C6H3)
CO(2-(CF3)C6H4)
CO(2-(CF3)C6H4)
CO(2-(CF3)C6H4) CHF-CH2 (R)–
CO(2-(CF3)C6H4)
Cbz
S-CH2
CH2-CH2
CHF-CH2 (S)–
CHF-CH2 (R)–
CF2-CH2
53
190 5000 29,000
54 5000 19,000
110 4500 73,000
690 5900
55
67
660 16,000
2000 39,000
S-CH2
CHF-CH2 (S)–
210 25,000
250 21,000
—
—
—
—
CF2-CH2
S-CH2
CH2-CH2
CHF-CH2 (S)–
CHF-CH2 (R)–
CF2-CH2
70
25
94
56
88
27
1100
900
4100 40,000
5200 37,000
3500 43,000
410 42,000
Studies of the pharmacokinetic properties of (S)-3-
fluoropyrrolidide 48 revealed that this compound was
53% orally bioavailable in rats (see Table 3), while
studies in dogs showed 75% oral bioavailability. The
compound was found to have a moderate clearance in
dogs of 9.1 mL/min/kg and a half-life of 5.9 h. Peak
Cbz
Cbz
Cbz
Cbz
Cbz
CHF-CHF (R,R)– 360 3200
CHF-CHF (S,S)– 830 3000
—
—
Cbz
SO2(4-(CF3O)C6H4)
SO2(4-(CF3O)C6H4)
SO2(4-(CF3O)C6H4) CHF-CH2 (S)–
SO2(4-(CF3O)C6H4) CHF-CH2 (R)–
SO2(4-(CF3O)C6H4)
SO2(4-(CF3O)C6H4) CHF-CHF (R,R)– 270 6100
SO2(4-(CF3O)C6H4) CHF-CHF (S,S)– 870 7500
SO2(2,4-F2C6H3)
SO2(2,4-F2C6H3)
SO2(2,4-F2C6H3)
SO2(2,4-F2C6H3)
S-CH2
CH2-CH2
22
89
35
1100 2800
6400 5700
8300 5500
Table 3. Pharmacokinetic properties of compound 48
75 12,000 6000
23 600 5300
Rat
Dog
CF2-CH2
—
—
Dosage:
iv
po
1 mg/kg
2 mg/kg
24 mL/min/kg
2.5 L/kg
1.6 h
1 mg/kg
2 mg/kg
9.1 mL/min/kg
4.4 L/kg
5.9 h
CH2-CH2
CHF-CH2 (S)–
CHF-CH2 (R)–
CF2-CH2
88 8800 35,000
48 12,000 49,000
Clp
Vdss
58
21
9400 52,000
t1/2
Cmax (po)
Oral bioavailability
810
—
—
0.40 mM
53%
1.0 mM
75%
SO2(2,4-F2C6H3) CHF-CHF (R,R)– 250 8100

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C. G. Caldwell et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1265–1268
concentrations following oral dosing at 2 mg/kg were
1.0 mM. Compound 48 was also examined in an oral
glucose tolerance test. Dosing of the compound in lean
C57BL6/N mice at 3 mg/kg resulted in a 42% reduction
in the glucose excursion relative to vehicle-treated
controls.
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In summary, modification of the substituted 4-amino-
cyclohexylglycine pyrrolidide lead structure by intro-
duction of fluorine substituents on the pyrrolidine ring
has been examined. The (3S,4S)- and (3R,4R)-3,4-
difluoropyrrolidine derivatives were less active inhibi-
tors of DP-IV, but the 3,3-difluoropyrrolidine analogues
showed good potency against the enzyme. Selectivity
versus QPP for the 3,3-difluoropyrrolidine analogues
was similar to that observed for the thiazolidine deriv-
atives. Compounds derived from (S)-3-fluoropyrrolidine
showed good potency versus DP-IV, with these com-
pounds generally having slightly greater activity than
the (R)-3-fluoropyrrolidides. The (S)-3-fluoro derivative
48 was found to have good pharmacokinetic properties
and produced significant activity in an oral glucose tol-
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Acknowledgements
The authors would like to thank Dr. Phil Eskola, Mr.
Robert Frankshun, and Mr. Joseph Leone for large-
scale preparations of several intermediates used in this
work.
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