Design, synthesis, and tripeptidyl peptidase II inhibitory activity of a novel series of (S)-2,3-dihydro-2-(4-alkyl-1H-imidazol-2-yl)-1H-indoles

Article abstract of DOI:10.1021/jm0202831

Butabindide, 1, was previously reported as a potent inhibitor (IC₅₀ = 7 nM) of the serine protease enzyme tripeptidyl peptidase II (TPPII), an endogenous protease that degrades cholecystokinin-8 (CCK-8). We found that 1 has some inherent chemical instability, yielding diketopiperazine 2 fairly readily under mimicked physiological conditions. We therefore prepared imidazoles 3, which are void of 1's inherent instability, and have found that our novel analogues maintained comparable TPPII inhibitory activity (e.g., for 3c, IC₅₀ = 4 nM) as 1.

Full text of DOI:10.1021/jm0202831

J . Med. Chem. 2002, 45, 5303-5310
5303
Design , Syn th esis, a n d Tr ip ep tid yl P ep tid a se II In h ibitor y Activity of a Novel
Ser ies of (S)-2,3-Dih yd r o-2-(4-a lk yl-1H-im id a zol-2-yl)-1H-in d oles
Henry J . Breslin,*,† Tamara A. Miskowski,† Michael J . Kukla,† William H. Leister,† Hans L. De Winter,‡
Diane A. Gauthier,† Maria V. F. Somers,‡ Danie¨lle C. G. Peeters,‡ and Peter W. M. Roevens‡
J ohnson & J ohnson Pharmaceutical Research & Development, L.L.C., Welsh and McKean Roads, P.O. Box 776,
Spring House, Pennsylvania 19477-0776, and Turnhoutseweg 30, B-2340 Beerse, Belgium
Received J uly 1, 2002
Butabindide, 1, was previously reported as a potent inhibitor (IC50 ) 7 nM) of the serine protease
enzyme tripeptidyl peptidase II (TPPII), an endogenous protease that degrades cholecystokinin-8
(CCK-8). We found that 1 has some inherent chemical instability, yielding diketopiperazine 2
fairly readily under mimicked physiological conditions. We therefore prepared imidazoles 3,
which are void of 1’s inherent instability, and have found that our novel analogues maintained
comparable TPPII inhibitory activity (e.g.,for 3c, IC50 ) 4 nM) as 1.
In tr od u ction
biochemically into two inactive metabolites, a penta-
peptide and a tripeptide, via a serine protease en-
zyme identified as tripeptidyl peptidase II (TPPII, EC
3.4.14.10).5 Butabindide (1) has been reported as a
potent inhibitor (7 nM) of TPPII and thus has been
viewed as a useful agent to increase endogenous levels
of CCK8. The biochemical inhibition of TPPII by 1 has
reportedly translated to the desired hypothesized in vivo
effects, i.e., mice dosed with 1 ate 50% less than saline-
control-dosed animals over a 1 h period.5
In 1998, 18% of the U.S. population could be classified
as obese compared to 12% in 1991.1 This is viewed as a
concern within our medical community because obesity
has been correlated with a number of diseases and
disorders, including diabetes, hypertension, gall bladder
disease, and osteoarthritis.2 More critically significant
is the determination that obesity is also attributable for
280000 deaths per year in the U.S.3 New therapeutic
antiobesity agents could help offset the morbidity and
economic drain caused by obesity-related diseases, while
concurrently yielding yearly revenues in the billion of
dollars for the marketers of such products.4
There are a number of new strategies currently being
explored to help attack the obesity problem, as described
recently in a comprehensive perspective of past and
present approaches.4 Among the many approaches being
explored is evaluation of various satiety-enhancing
agents. One reported endogenous satiety agent is the
octapeptide cholecystokinin-8 (CCK8), which has dem-
onstrated significant in vivo inhibitory feeding effects.
Some researchers have attempted to exploit this desired
effect of CCK8 by preparing non-peptidyl agonists of
CCK8, and these compounds are currently at various
stages of development. In lieu of CCK8 mimics, an
alternative approach recently reported involved efforts
toward enhancing CCK8’s natural response by inhibit-
ing CCK8’s normal degradation.5-8 CCK8 is degraded
From a chemistry perspective, we questioned the
inherent stability of 1. We were concerned that 1 might
spontaneously cyclize to generate the six-membered
diketopiperazine 2 with concurrent elimination of n-
butylamine (Scheme 1). We therefore ran a simple
stability study under mimicked physiological conditions,
i.e., 3 mg of 1 was dissolved in 1 mL of pH 7.0
phosphate-buffered aqueous solution and warmed to 38
°C. Under these conditions, we found 28% of 1 had
degraded to diketopiperazine 2 after 8 h. After 22 h, 57%
of 1 had been converted to 2.9 As a follow-up to these
findings, we confirmed the reported 7 nM inhibitory IC50
activity for 1 against the TPPII enzyme. Conversely, we
found a purified sample of 2 void of any TPPII enzy-
matic activity (IC50 > 10 µM).
On the basis of these results, we embarked to design
some bioisostere analogues of 1. We envisioned that our
analogues of 1 should possess greater inherent chemical
stability than 1. Concurrently we wanted our analogues
to maintain very similar chemical characteristics as 1,
since the biological activity of 1 had been honed via
combinatorial chemical variations. Following these guide-
* To whom correspondence should be addressed. Telephone: (215)
628-5610. Fax: (215) 628-4985. E-mail: HBreslin@prdus.jnj.com.
† J ohnson & J ohnson Pharmaceutical Research & Development.
‡ Turnhoutseweg 30.
Sch em e 1
10.1021/jm0202831 CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/26/2002

5304 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Breslin et al.
lines, we identified compounds of general structure 3
as potential preliminary targets.
designated active site. This docking revealed important
insights with respect to the key molecular interactions
between 1 and TPPII (Figures 1 and 2). Subsequent
overlapping of 3 with 1 in the active site of the TPPII
model confirmed that consistent binding interactions
were maintained for both molecules relative to key
TPPII protein residues (Figure 3). These molecular
modeling results supported our initial, intuitive notion
that minimal disruption to the character and conforma-
tion of 1 would occur by introduction of the imidazole
moiety of analogues 3.
On the basis of all the results, rationales, and ensuing
conclusions discussed above, synthetic efforts were
initiated toward imidazoles 3. Following are described
our initial experimental results related to the prepara-
tion and activities of 3.
We visualized that the imidazole of 3 would mimic
the amide of 1, which was supported by previous reports
of imidazole bioisostere equivalence for the amide
moiety.10 Consistent with our defined objectives, imid-
azoles 3 are devoid of 1’s instability liability but
maintain potentially desirable chemical features of 1,
even possessing both the H bonding donor and acceptor
character of 1’s amide group. These assumed chemical
characteristic similarities between structures 1 and 3
were further validated empirically through some mo-
lecular modeling work. We began our molecular model-
ing work by constructing a homology model for the
active site of TPPII, using the crystal structure of
subtilisin as a prototypical protease template.11 After
the TPPII receptor was designed, 1 was docked into its
Ch em istr y
To initiate our studies discussed above, we required
compounds 1 and 2 to evaluate their biological activities.
1 can be prepared as previously described7 (Scheme 2).
We altered from the reported preparation slightly,
which proved to be a bit more expedient for larger scale
preparation (Scheme 3). Ester 612 was prepared from
F igu r e 1. Putative binding mode of butabindide into a model of the active site of TPPII. The surface of the catalytic triad residues
is color-coded according to the underlying residue: Asp (red), His (blue), and Ser (orange).
F igu r e 3. Comparison of the docked structure of 1 (yellow
F igu r e 2. Analysis of the putative binding interactions
between butabindide and TPPII based on the docking of the
inhibitor into a model of the protein active site.
carbon atoms) with the imidazole mimic 3 (gray carbon atoms).
A number of important residues are shown. Putative hydrogen
bonds are indicated by thin green lines.

Indoles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5305
the commercially available S-indoline carboxylic acid 4,
which was then reacted neat with butylamine (n-
BuNH2) to yield amide 5 in good yield. Then 5 was
reacted with the activated ester of tert-butoxycarbonyl
(BOC) protected amino acid 7 to give 8, which was
subsequently deprotected with ethereal HCl. Pure 1
precipitated directly from this reaction mixture as its
HCl salt. Simply warming 1’s HCl salt neat at 165 °C
for 30 min yielded a pure sample of 2.
Synthesis of compounds 3 (Scheme 4) also began from
commercially available 4. Similar to the scheme em-
ployed for 1, the initial reaction was conversion of 4 to
6. Ester 6 was then treated with methanolic ammonia
at room temperature to yield amide 913 in respectable
yield as previously reported. 9 was acylated with acetyl
chloride (AcCl) in the presence of triethylamine (Et3N)
to give 10. Nitrile 11 was efficiently generated following
a literature dehydration procedure,14 simply by adding
trichloroacetyl chloride (Cl3CCOCl) to a 0 °C mixture
of primary amide 10 and Et3N in dichloromethane
(CH2Cl2). This procedure proved to be quite amenable
to larger scale preparations. To this point in the
synthetic scheme, no racemization had been observed,
as determined by chiral high-pressure liquid chroma-
tography (HPLC) analysis of crystallized 11.15 Imidate
12 was generated by the classical treatment of a nitrile
with HCl and alcohol to yield the crystalline product.
12 proved to be hygroscopic and was either quickly used
after filtering or maintained in an anhydrous environ-
ment. Generally 12 was immediately reacted with an
aminoketone 13 (two steps)16 to yield indoline-imid-
azoles 14. J udging by chiral HPLC, it was determined
that both crude 14a and crude 14b were 85:15 mixtures
of enantiomers, implying that some isomerization had
occurred in one of the two prior steps. Fortunately,
trituration of crude 14a with acetonitrile yielded opti-
cally pure material. Compound 14b proved to be slightly
less amenable to such purification but still yielded a
reasonable quantity of enantiomerically pure material.
The racemic material isolated from the synthesis of 14b
proved to be useful in pursuing product 3e, as will be
described below. 14 was deacetylated in refluxing 6 N
HCl to give 15. Generally, noramine 15 was used fairly
quickly after generation because air oxidation to the
indole proved to be somewhat problematic, as deter-
mined by HPLC/MS. Coupling of the appropriate
noramine 15 with preformed BOC-amino acid fluoride
16b17 generated compounds 17b, 17d , and 17e. Com-
Sch em e 2
Sch em e 3

5306 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Breslin et al.
pound 17c was prepared by coupling noramine 15a with
16c. Compounds 17b-d could be carried on in the
sequence without further purification because they were
prepared relatively cleanly from enantiomerically pure
samples of 15a or 15b (g99% ee). Compound 17e
required additional purification because it was prepared
from racemic predecessors 14b and 15b. Using the
racemic 15b as a starting material in the amide coupling
reaction with 16b yielded a mixture of diastereomers
17d and 17e as coproducts. Fortunately preparative
HPLC easily separated compound 17e as a single
compound from the previously identified 17d , which had
initially been generated from optically pure 15b. All
compounds 17 were subsequently deprotected easily
with trifluoroacetic acid (TFA) to yield related desired
targets 3b-e.
Resu lts a n d Discu ssion
Experimental details related to TPPII enzyme isola-
tion and subsequent determinations of TPPII inhibitory
activities are described in the Experimental Section. The
compiled biological results of TPPII inhibitory activities
are outlined in Table 1.
For our initial biological experiment, we reproduced
the reported TPPII inhibitory activity for 1 (7 nM). As
mentioned in the Introduction, for our initial chemical
experiments, we confirmed 1’s inherent instability, then
prepared a pure sample of 1’s major degradation prod-
uct, 2, for follow-up biological evaluation. Our plan was
to pursue one of two possible paths, dependent on the
relative activity of 2. If 2 had surprisingly proven to be
an extremely potent inhibitor of TPPII, we would have
prepared an array of diketopiperazines to explore the
related structure-activity relationship (SAR) of 2,
concluding that 1’s assumed activity was due to a small
percentage of 2 generated in situ during testing. How-
Racemic compound 3a was prepared in a similar
manner as described above, utilizing racemic indoline
15a as the starting material.
Sch em e 4

Indoles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5307
Ta ble 1. Inhibition of TPPII Enzyme
ates and final products are included in the Experimental
Section. Also, final products 3 and 2 were evaluated by high-
resolution mass spectrometry (HRMS) analyses (Autospec E
high-resolution magnetic sector mass spectrometer). These
HRMS measurements were carried out internally by our
spectrometry department in Spring House, PA. For an initial
key intermediate (14a ) and for a representative final product
(3d ), C, H, and N elemental analyses were carried out by our
internal analytical research department in Beerse, Belgium,
and were within the 0.4% limit values of the calculated percent
values for C, H, and N. All final products were also assayed
for homogeneity by thin-layer chromatography on Whatman
MK6F (1 in. × 3 in. × 250 µm) silica gel plates. Melting points
a
compd
IC50 (nM)
nb
1
2
7 ( 1
3
2
3
2
2
2
1
1
>10000
36 ( 4
6 ( 1
3a
3b
3c
3d
3e
14a
4 ( 1
23 ( 3
>10000
>10000
a
b
IC50’s from Sigma plot C/R curves. Number of total Sigma
plot C/R curves.
were determined on
a Thomas-Hoover Unimelt capillary
ever, finding 2 devoid of any desirable activity, we
pursued the alternative path of designing and preparing
bioisosteres of 1, with the requirement that the bio-
isosteres be inherently more stable to the cyclization
liability noted for 1. Pleasingly we found that our
imidazole bioisosteres 3 maintain good TPPII inhibitory
activities. More specifically, compound 3c (4 nM), the
direct analogue of 1, retains activity comparable to that
of 1 (7 nM).
melting point apparatus and are uncorrected. All reagents
were commercially available unless otherwise specified, and
all reactions were run under an inert atmosphere of Ar or N2
unless otherwise specified. Where required, preparative pu-
rifications were performed on a Gilson semipreparative HPLC
unit (column, YMC ODS-A 30 mm × 100 mm, 5 µm; temper-
ature, ambient; flow rate, 35 mL/min; mobile phase consisting
of (A) 10:90 acetonitrile/ water with 0.1% trifluoroacetic acid
or (B) 90:10 acetonitrile/ water with 0.1% TFA; gradient, linear
gradient from mobile phase A to mobile phase B over 9 min;
wavelength, 254 nm).
Our related SAR of other initial imidazoles prepared
(14a and 3a ,b,d ,e) is also reflective of the SAR previ-
ously described for 1.5 We found 14a to be devoid of any
desirable activity, supporting the conclusion of a need
for the related basic center. Our data also support the
SAR that some lipophilicity on the amino acid side chain
R correlates with improved activity. This is demon-
strated by comparing the single variable altered mol-
ecules 3a , 3b, and 3c [3c (R ) Et; 4 nM) g 3b (R ) Me;
6 nM) > 3a (R ) H; racemic, 36 nM]. A similar
conclusion that some lipophilicity is desirable at the R′
region of 3 for improved activity is supported by
comparing molecule 3b (R′ ) Pr; 6 nM) with singly
altered analogue 3d (R′ ) Et; 23 nM). Finally, compari-
son of compound 3d (S,S stereochemistry, 23 nM) with
its respective epimer 3e (R,S stereochemistry, >10000
nM) clearly demonstrates the enzymatic stereospecific-
ity requirement for good inhibitors.
Besides finding compounds at least as potent as 1,
our other initial criterion was that our compounds
should prove to be inherently more stable than 1. Since
imidazoles 3 are devoid of the cyclization potential noted
for 1, it was not surprising to find that under the same
mimicked physiological conditions where 1 degrades
57% over 22 h, compound 3c remains 100% pure after
the same time frame.
Molecu la r Mod elin g. A homology model of the active site
of TPPII was calculated using Modeller, version 4e (Accelrys
Inc., San Diego, CA), using the crystal structure of subtilisin
as the template.11 Docking of 1 was performed using the
program Gold, version 1.1, with the standard default settings.18
Visualization was done using the InsightII software of Accelrys
Inc. (San Diego, CA).
[S-(R*,R*)]-1-(2-Am in o-1-oxobu tyl]-N-bu tyl-2,3-dih ydr o-
1H -in d ole-2-ca r b oxa m id e H yd r och lor id e (1:1) (Bu t a -
bin d id e, 1). Ester 612 (21.62 g, 0.122 mol) was added neat to
n-BuNH2 (18 g, 0.245 mol) at 10 °C, and then the neat reaction
mixture was slowly warmed to room temperature. After 4 h,
an additional portion of n-BuNH2 (9 g, 0.122 mol) was added
to the reaction mixture. After another 2 h, the reaction mixture
was treated with hexane and the resulting solid was filtered,
rinsed liberally with hexane, and then dried under reduced
pressure to yield 24.13 g (90%) of 5 as a white solid.
Compound 7 (Sigma, 20.32 g, 0.1 mol) was dissolved in
CH2Cl2 (500 mL), then cooled to 0 °C. Then Et3N (14 mL, 0.1
mol) was added neat, followed by neat isobutyl chloroformate
(12.9 mL, 0.1 mol). This mixture was stirred for 1.5 h at 0 °C,
and then 5 was added neat. After 6 h, the reaction mixture
was extracted sequentially with H2O (200 mL), saturated
aqueous NaHCO3 (100 mL), 2 N citric acid (100 mL), saturated
aqueous NaHCO3 once again (100 mL), and then brine (100
mL). The organic phase was dried over MgSO4, filtered, and
concentrated under reduced pressure to yield 42.87 g of whitish
solid. This material was triturated in ice-cold Et2O, filtered,
and rinsed with ice-cold Et2O (150 mL). After it was air-dried,
there remained 26.35 g (65%) of 8 as a white solid (TLC, 40%
EtOAc/hexane, Rf ) 0.35, trace impurity at Rf ) 0.4; HPLC,
98.1%, one impurity).
Compound 8 was suspended in a 0 °C 1 N ethereal HCl
solution (500 mL), and then gaseous HCl was bubbled in for
20 min while stirring the mixture. The original heterogeneous
mix became homogeneous during the HCl addition. After the
HCl addition, the reaction mixture was warmed to room
temperature. Almost immediately a precipitate began to fall
out of solution. After the mixture was stirred for an additional
3 h, the reaction mixture was recooled to 0 °C and then filtered
and rinsed with ice-cold Et2O (200 mL). The resulting solid
was dried under high vacuum for 16 h to yield 19.51 g (93%)
of desired 1 as a white solid hydrochloride (1:1): mp 140-145
°C (TLC, 80:20:5 CHCl3/CH3OH/HCOOH, Rf ) 0.5, homoge-
neous; HPLC, 99%, one impurity); 1H NMR (DMSO-d6) δ 0.8-
0.9 (t, 3H), 0.95-1.05 (t, 3H), 1.2-1.35 (m, 2H), 1.35-1.5 (m,
2H), 1.75-2.05 (m, 2H), 3.05-3.2 (bd, 3H), 3.4-3.7 (m, 3H),
5.1-5.2 (d, 1H), 7.05-7.15 (t, 1H), 7.2-7.4 (m, 2H), 8.05-8.15
(d, 1H), 8.5-8.65 (bs, 3H), 8.75-8.85 (t, 1H).
In conclusion, we have identified novel imidazoles 3
as extremely potent inhibitors of TPPII, and they
possess greater inherent stability than the pharma-
cophore after which they were modeled, i.e., 1. We are
continuing further chemical and biological endeavors
around TPPII inhibitors, and these results will be
reported in subsequent publications.
Exp er im en ta l Section
Compounds listed in Table 1 (1, 2, 3a -e, and 14a ) were all
characterized by 360 MHz 1H NMR (Bruker AM 360 WB),
mass spectrometry (Finnegan 3300), and HPLC (>98% purity
at 214 and 254 nm; Hewlett-Packard series 1050 HPLC with
a 3 µm, 3.3 mm × 50 mm Supelco AZB+ C18 column, using a
gradient mobile phase of 4:96:0.1 acetonitrile/water/TFA to
100:0:0.1 acetonitrile/water/TFA over 8.5 min at a flow rate
1
of 1.20 mL/min and a total run time of 9.5 min). H NMR of
key intermediates was also run on the 360 MHz NMR
1
spectrometer. The related H NMR results for both intermedi-

5308 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Breslin et al.
(3S,10a S)-Eth yl-2,3,10,10a -tetr a h yd r op yr a zin o[1,2-a ]-
in d ole-1,4-d ion e (2). Compound 1‚HCl (0.076 g, 0.000 22 mol)
was placed neat in an oil bath prewarmed to 140 °C and was
stirred as the temperature of the bath was elevated slowly.
After 15 min, the temperature of the oil bath had risen to 152
°C and bubbling of the starting material was noted. After an
additional 15 min, the oil bath had reached a temperature of
168 °C and the reaction had become a homogeneous, stirring
oil. After the mixture was stirred for an additional 15 min at
168 °C, heating was discontinued. After the mixture was
cooled, the brown residue was triturated with Et2O (10 mL)
and filtered through Dicalite. The filtrate was treated with
hexane (15 mL) and cooled to 0 °C. The resulting solid was
filtered and rinsed three times with hexane (1 mL each). After
it was air-dried, there remained 12 mg (23%) of 2 as a white
solid: mp 169-170 °C (TLC, 80:20:5 CHCl3/CH3OH/HCOOH,
Rf ) 0.75; HPLC, 100%); 1H NMR (CDCl3) δ 1.05-1.15 (t, 3H),
1.90-2.05 (m, 1H), 2.10-2.20 (m, 1H), 3.35-3.45 (dd, 1H),
3.6-3.7 (dd, 1H), 4.1-4.2 (t, 1H), 4.75-4.85 (t, 1H), 5.8-5.9
(bs, 1H), 7.05-7.15 (t, 1H), 7.25-7.3 (m, 2H), 8.1-8.15 (d, 1H).
HRMS calcd for C13H14N2O2: 230.1056. Found: 230.1056.
(S)-1-Acetyl-2,3-d ih ydr o-1H-in d ole-2-ca r boxa m ide (10).
(S)-2,3-Dihydro-1H-indole-2-carboxamide13 (9) (4.86 g, 0.030
mol) was suspended in trichloromethane (CHCl3) (400 mL).
The mixture was cooled to 0 °C. Et3N (6.26 mL, 0.045 mol)
was added neat followed by neat AcCl (3.21 mL, 0.045 mol),
which was added dropwise over 2 min. After 30 min, TLC (80:
20:5 CHCl3/MeOH/HCOOH) showed that the reaction was
incomplete. Maintaining the reaction mixture at 0 °C, more
Et3N (6.26 mL) was added, followed 15 min later with dropwise
addition of AcCl (3.21 mL). TLC showed the reaction was still
incomplete (∼80%). Therefore, third portions of Et3N (6.26 mL)
and AcCl (3.21 mL) were added. After an additional 15 min,
ice-cold water (200 mL) was added. The mixture was stirred
for 10 min, and the solid was filtered and rinsed with water
(3 × 100 mL) and CHCl3 (2 × 75 mL). The sample was allowed
to dry overnight, yielding 4.71 g (77%) of 10 as a white solid:
(18.45 g, 0.188 mol). The mixture was heated to reflux under
argon. To this was slowly added a solution of 13a 16 (12.93 g,
0.094 mol) in MeOH (95 mL) over 45 min. After the addition
was complete, the mixture was allowed to stir overnight at
reflux, then concentrated. The concentrate was taken up in
CH2Cl2 and washed with saturated aqueous NaHCO3. The
aqueous layer was extracted with a second portion of CH2Cl2.
The combined organic extracts were dried over MgSO4, filtered,
and concentrated to an oily residue. The residue was triturated
with Et2O, and the resulting solid was filtered. This filtered
solid (1.13 g, 9%) proved to be a racemic mixture of desired
14a , as determined by chiral column analysis.15 The resulting
filtrate from this Et2O trituration was concentrated under
reduced pressure, and the resulting residue was triturated
with ice-cold CH3CN. The resulting solid was filtered to yield
4.97 g of product as a white solid. This material was further
triturated in Et2O to yield 4.67 g of pure product 14a as a white
solid. The combined filtrates from the latter two triturations
were combined, concentrated under reduced pressure, and
purified by flash silica gel column chromatography to yield an
additional 0.83 of 14a . The combined return was 5.53 g (44%)
of desired 14a as a white solid: mp 174-175°C (TLC, 10%
CH3OH in CH2Cl2, Rf ) 0.42, homogeneous); chiral LC
analysis,15 99% ee; 1H NMR (DMSO-d6) δ 0.8-0.9 (t, 3H),
1.45-1.55 (m, 2H), 1.95-2.1 (bs, 3H), 2.35-2.45 (t, 3H), 3.05-
3.2 (d, 2H), 3.55-3.7 (t, 3H), 5.5-5.6 (d, 2H), 6.55-6.65 (bs,
1H), 6.95-7.05 (t, 1H), 7.1-7.25 (m, 2H), 7.95-8.1 (bs, 1H),
11.6-11.8 (bs, 1H). Anal. Calcd for C16H19N3O′: C, 71.35; H,
7.11; N, 15.60. Found: C, 71.38; H, 6.92; N, 15.61.
(S)-1-Acet yl-2,3-d ih yd r o-2-(4-et h yl-1H -im id a zol-2-yl)-
1H-in d ole (14b). 14b was prepared in a similar manner as
14a , with the exception that 13b was used in place of 13a .
Workup varied somewhat from that for 14a . The following
details are based on a 0.015 mol scale of 12. After the original
reaction mixture was concentrated, the residue was partitioned
between CH2Cl2 and saturated aqueous NaHCO3. The aqueous
phase was washed with a second portion of CH2Cl2 and the
organic phases were combined, dried over Na2SO4, filtered, and
concentrated under reduced pressure, yielding a yellow oil.
This material was triturated with Et2O (40 mL) to yield a solid
precipitate. The solid was filtered and rinsed with ice-cold Et2O
to yield 0.46 g of white solid. This filtered solid proved to be
the racemic mixture of desired 14b (12%), as determined by
chiral column analysis.15 The resulting Et2O filtrates were
combined and concentrated under reduced pressure to give
2.66 g of yellow tinted solid. This material was triturated in
Et2O (20 mL) to give an additional portion of solid. This solid
was filtered and rinsed with Et2O twice to yield 0.89 g of
desired 14b as a white solid: mp 136-139 °C (TLC, 80:20:5
CHCl3/CH3OH/HCOOH, Rf ) 0.7, homogeneous; chiral LC
analysis,15 99% ee). The resulting filtrate was again concen-
trated to yield 1.56 g of residue, which was purified by
preparative HPLC to yield an additional 0.34 g of 14b. The
mp >260 °C (TLC, 80:20:5 CHCl3/CH3OH/HCOOH, Rf
)
1
0.60); H NMR (DMSO-d6) δ 2.05 (s, 3H), 2.95-3.05 (d, 1H),
3.55-3.65 (dd, 1H), 4.85-4.95 (dd, 1H), 6.9-7.0 (t, 1H), 7.1-
7.25 (m, 2H), 7.25-7.35 (bs, 1H), 7.7-7.8 (bs, 1H), 8.0-8.1 (d,
1H).
(S)-1-Acetyl-2,3-d ih yd r o-1H-in d ole-2-ca r bon itr ile (11).
10 (4.13 g, 0.020 mol) was suspended in CH2Cl2 (175 mL). The
mixture was cooled to 0 °C, and Et3N (8.44 mL, 0.061 mol)
was added neat. Cl3CCOCl (3.39 mL, 0.030 mol) in CH2Cl2 (20
mL) was added dropwise over 20 min. After 2 h, ice/water (200
mL) was added, the phases were separated, and the organic
phase was reextracted with 3 N HCl and then with a saturated
aqueous NaHCO3 solution. The organic phase was dried over
MgSO4, filtered, and concentrated under reduced pressure to
yield 4.61 g of brown solid. The solid was triturated with ice-
cold Et2O (30 mL) and filtered and the resulting solid was
rinsed with ice-cold Et2O (twice), yielding 3.12 g (83%) of 11
as a tan solid: mp 134-135 °C (TLC, 80:20:5 CHCl3/CH3OH/
HCOOH, Rf ) 0.85); 1H NMR (CDCl3) δ 2.35-2.5 (bs, 3H),
3.35-3.75 (bm, 2H), 5.05-5.2 (bd, 0.5H), 5.35-5.5 (bd, 0.5H),
7.05-7.15 (t, 1H), 7.15-7.35 (m, 2.5H), 8.1-8.2 (bd, 0.5H).
1
total return of optically pure 14b was 1.23 g (32%): H NMR
(DMSO-d6) δ 1.05-1.15 (bs, 3H), 1.95-2.1 (bs, 3H), 2.3-2.5
(bs, 2H), 3.1-3.2 (d, 1H), 3.55-3.65 (t, 1H), 5.55-5.65 (d, 1H),
6.4-6.5 (bs, 0.5H), 6.65-6.75 (bs, 0.5H), 6.95-7.05 (t, 1H),
7.15-7.3 (m, 2H), 7.95-8.05 (d, 1H).
(S)-Eth yl 1-a cetyl-2,3-d ih yd r o-1H-in d ole-2-ca r boxim i-
d a te Mon oh yd r och lor id e (12). Compound 11 (2.81 g, 0.0151
mol) was suspended in Et2O (200 mL). Ethanol (0.97 g, 0.0214
mol) was added, and the mixture was cooled to 0 °C. HCl (gas)
was bubbled in for 45 min. The mixture was removed from
the ice bath and stirred. After 20 min, a residue began to collect
on the sides of the flask. The residue was scratched with a
spatula, and a white solid precipitated out. After 1 h, the
sample was filtered, rinsed with Et2O, and air-dried quickly,
yielding 3.99 g (99%) of 12. Compound 12 was immediately
placed under Ar and was generally used for subsequent
reactions within the same working day (TLC, 80:20:5 CHCl3/
CH3OH/HCOOH, Rf ) 0.80).
(S)-2,3-Dih yd r o-2-(4-p r op yl-1H -im id a zol-2-yl)-1H -in -
d ole (15a ). Compound 14a (0.46 g, 0.001 70 mol) was com-
bined with aqueous HCl (6 N aqueous, 20 mL) and immedi-
ately warmed in a 100 °C oil bath under a nitrogen atmosphere.
After 3.5 h, the heat was removed and the sample was cooled
to 0 °C. Then 3 N NaOH (35 mL) was slowly added. Basifica-
tion to neutral pH was completed with saturated aqueous
NaHCO3. The aqueous solution was then extracted with CHCl3
(2 × 40 mL). The organic extracts were combined, dried over
Na2SO4, and filtered, and the resulting solution of 15a was
used for subsequent reactions without further purification
(TLC, 80:20:5 CHCl3/CH3OH/HCOOH, Rf ) 0.55).
(S )-2,3-Dih yd r o-2-(4-e t h yl-1H -im id a zol-2-yl)-1H -in -
d ole (15b). Compound 15b was prepared in a similar manner
as 15a and was also used for subsequent reactions without
(S)-1-Acetyl-2,3-d ih yd r o-2-(4-p r op yl-1H-im id a zol-2-yl)-
1H-in d ole (14a ). Intermediate 12 (0.047 mol) in methanol
(MeOH) (200 mL) was treated with potassium acetate (KOAc)

Indoles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24 5309
further purification (TLC, 80:20:5 CHCl3/CH3OH/HCOOH, Rf
) 0.65).
1H), 7.15-7.3 (m, 2H), 8.0-8.2 (bs, 1H). HRMS calcd for
C16H20N4O (MH+): 285.1715. Found: 285.1722.
(S)-1,1-Dim eth yleth yl [1-(flu or oca r bon yl)p r op yl]ca r -
ba m a t e (16c). (S)-2-(tert-butoxycarbonylamino)butyric acid
(2.03 g, 0.010 mol) dissolved in CH2Cl2 (25 mL) was placed in
a cooling bath at -10 °C. While the reaction mixture was being
stirred, pyridine (0.77 g, 0.010 mol) was added neat, followed
by 2,4,6-trifluoro-1,3,5-triazine (4.66 g, 0.0345 mol) neat. After
the mixture was stirred for 1 h, ice-cold water (75 mL) was
added. More CH2Cl2 (45 mL) was added, and the mixture was
shaken. The organic phase was separated and washed with
ice-cold water again (100 mL), and then the organic phase was
dried over MgSO4, filtered, and concentrated to yield 2.29 g
(total weight (TW) ) 2.05 g) of crude 16c, which was used
without further purification.
[2S-[1(R*),R*]]-r-Meth yl-2,3-d ih yd r o-â-oxo-2-(4-p r op yl-
1H -im id a zol-2-yl)-1H -in d ole-1-et h a n a m in e (3b ). TFA (4
mL) was cooled to 0 °C and added to 17b (0.68 g, 0.0017 mol)
with stirring. After 15 min, the excess TFA was removed under
reduced pressure. The residue was dissolved in water (12 mL)
and extracted with Et2O (2 × 15 mL). The aqueous phase was
basified with saturated aqueous NaHCO3 and then reextracted
with Et2O (5 mL), then with CHCl3 (2 × 40 mL). The CHCl3
organic phases were combined, dried (Na2SO4), filtered, and
concentrated under reduced pressure to leave 0.51 g of clear
oil. Hexane (35 mL) was added, and the mixture was held
overnight. The material was then scratched, and the resulting
solid was filtered and rinsed (ice-cold hexane, 3 × 2 mL). After
it was air-dried, there remained 0.237 g (45%) of 3b as a white
solid: mp 68-69 °C (TLC, 80:20:5 CHCl3/CH3OH/HCOOH, Rf
) 0.3, homogeneous; HPLC, 100%); 1H NMR (CDCl3) δ 0.85-
0.9 (t, 3H), 1.15-1.2 (d, 3H), 1.45-1.55 (m, 2H), 2.35-2.45 (t,
2H), 3.3-3.4 (m, 3H; 2H exchangeable on treatment with D2O;
post D2O treatment, d, 1H), 3.55-3.7 (m, 2H), 5.65-5.75 (d,
1H), 6.55-6.65 (bs, 1H), 6.95-7.05 (t, 1H), 7.1-7.2 (t, 1H),
7.2-7.3 (d, 1H), 8.05-8.2 (bs, 1H). HRMS calcd for C17H22N4O
(MH+): 299.1872. Found: 299.1881.
[2S-[1(R*),R*]]-R-Eth yl-2,3-d ih yd r o-â-oxo-2-(4-p r op yl-
1H -im id a zol-2-yl)-1H -in d ole-1-et h a n a m in e Hyd r och lo-
r id e (3c). Compound 17c (1.24 g, 0.003 mol) and TFA (4 mL),
both precooled in an ice bath, were combined. After 10 min,
the mixture was concentrated. The concentrate was dissolved
in water and extracted twice with Et2O. The aqueous layer
was separated and basified with saturated NaHCO3. The
resulting basic aqueous solution was then extracted twice with
CHCl3. The combined CHCl3 organic extracts were dried over
MgSO4, filtered, and concentrated. The residue was flash-
chromatographed on a silica gel column, eluting with 25:1
CHCl3/MeOH. The desired fractions were combined and
concentrated under reduced pressure to yield 0.33 g of clear
oil. This material was dissolved in Et2O (10 mL) and treated
with 3 mL of 1 M HCl in Et2O. The precipitate was filtered,
rinsed with Et2O, and dried under vacuum to yield 0.24 g (19%)
of desired product 3c as a white solid: mp >200°C (TLC, 80:
20:5 CHCl3/CH3OH/HCOOH, Rf ) 0.5, homogeneous; HPLC,
99.4%, one impurity). NMR of 3c’s free base, as the 3c salt
itself, yielded a noninterpretable spectra containing multiple
rotamers with poor resolution. 1H NMR of free base 3c (DMSO-
d6): δ 0.85-0.95 (m, 6H), 1.4-1.55 (m, 3H), 1.65-1.75 (m, 1H),
2.35-2.45 (t, 2H), 3.35-3.45 (m, 3H; 2H exchangeable on
treatment with D2O; post D2O treatment, d, 1H), 3.45-3.55
(bt, 1H), 3.55-3.7 (m, 1H), 5.65-5.75 (d, 1H), 6.5-6.6 (bs, 1H),
7-7.1 (t, 1H), 7.15-7.25 (t, 1H), 7.25-7.3 (d, 1H), 8.1-8.2 (bd,
1H). HRMS calcd for C18H24N4O (MH+): 313.2028. Found:
313.2014.
{2-Oxo-2-[2-(4-p r op yl-1H -im id a zol-2-yl)-2,3-d ih yd r o-
in d ol-1-yl]eth yl}ca r ba m ic Acid ter t-Bu tyl Ester (17a ).
Fluorocarbonylmethylcarbamic acid tert-butyl ester17 (16a ) was
reacted with racemic 15a in the typical manner as described
for intermediate 17b. The crude 17a (51% over two steps) was
used in the subsequent step without further purification (TLC,
90:9:1 CHCl3/CH3OH/NH4OH, Rf ) 0.22).
[2S-[1(R*),R*]]-{1-Meth yl-2-oxo-2-[2-(4-p r op yl-1H-im id -
azol-2-yl)-2,3-dih ydr oin dol-1-yl]eth yl}car bam ic Acid ter t-
Bu tyl Ester (17b). N-Methylmorpholine (NMM, 0.17 g, 0.0017
mol) was added to a 0 °C solution of intermediate 15a (0.39 g,
0.0017 mol) in CHCl3 (75 mL). Then intermediate 16b17 (0.33
g, 0.0017 mol) was added neat. After 0.5 h, the reaction
mixture was washed with water (50 mL), saturated aqueous
NaHCO3 (50 mL), and then brine (50 mL), dried over Na2SO4,
filtered, and concentrated, yielding 0.77 g (TW ) 0.68 g over
two steps) of 17b as an oil, which was used without further
purification (TLC, 80:20:5 CHCl3/MeOH/HCOOH, Rf ) 0.75).
[2S-[1(R*),R*]]-{1-[2-(4-P r op yl-1H -im id a zol-2-yl)-2,3-
d ih yd r oin d ole-1-ca r b on yl]p r op yl}ca r b a m ic Acid ter t-
Bu tyl Ester (17c). Starting from intermediate 16c, compound
17c was prepared in a similar manner as 17b and was also
used for the subsequent reaction without further purification,
with the crude yield proving to be ∼100% over two steps (TLC,
80:20:5 CHCl3:MeOH:HCOOH, Rf ) 0.8).
[2S-[1(R*),R*]]-{1-Meth yl-2-oxo-2-[2-(4-eth yl-1H-im id -
azol-2-yl)-2,3-dih ydr oin dol-1-yl]eth yl}car bam ic Acid ter t-
Bu tyl Ester (17d ). Starting from intermediate 15b, compound
17d was prepared in a similar manner as 17b and was also
used for the subsequent reaction without further purification,
with the crude yield proving to be ∼100% over two steps (TLC,
80:20:5 CHCl3/MeOH/HCOOH, Rf ) 0.7).
[2R-[1(S*),R*]]-{1-Meth yl-2-oxo-2-[2-(4-eth yl-1H-im id -
azol-2-yl)-2,3-dih ydr oin dol-1-yl]eth yl}car bam ic Acid ter t-
Bu tyl Ester (17e). Compound 17e was prepared in a similar
manner as 17b, except the starting material 15b used for this
reaction was racemic; i.e., it contained 50% of the R enanti-
omer. Also, after the typical workup, the crude product was
purified by preparative HPLC to separate 17e from 17d .
Compound 17e (27%) eluted in the front run, with an ad-
ditional sample of 17d (29%) eluting in the latter fractions.
The resulting 17e, after lyophylization, was used for the
subsequent reaction without further purification (TLC for 17e
same as for 17d , 80:20:5 CHCl3/MeOH/HCOOH, Rf ) 0.7).
2-Am in o-1-[2-(4-p r op yl-1H-im id a zol-2-yl)-2,3-d ih yd r o-
in d ol-1-yl]eth a n on e (3a ). TFA (10 mL) was cooled to 0°C
and added to 17a (0.40 g, 0.0010 mol) with stirring. After 15
min, the excess TFA was removed under reduced pressure.
The residue was partitioned between aqueous NaHCO3 and
CHCl3. The CHCl3 organic phase was dried (MgSO4), filtered,
and concentrated under reduced pressure. The residue was
triturated in CH3CN to yield a white solid, which was filtered
and rinsed with a small amount of ice-cold CH3CN to yield
0.137 g (48%) of 3a as a white solid: mp 168-170 °C (TLC,
90:9:1 CHCl3/CH3OH/NH4OH, Rf ) 0.37, homogeneous; HPLC,
98.6%, one impurity); 1H NMR (DMSO-d6) δ 0.85-0.9 (t, 3H),
1.45-1.6 (m, 2H), 2.35-2.45 (t, 2H), 3.1-3.2 (bd, 2H), 3.5-
3.7 (m, 2H), 5.55-5.65 (d, 1H), 6.55-6.65 (bs, 1H), 7.0-7.1 (t,
[2S-[1(R*),R*]]-r-Meth yl-2,3-d ih yd r o-â-oxo-2-(4-eth yl-
1H-im id a zol-2-yl)-1H-in d ole-1-eth a n a m in e (3d ). Starting
from 17d , 3d was prepared and worked up in a similar manner
as was 3b to yield 60% of 3d as a white solid: mp 116-118
°C (TLC, 80:20:5 CHCl3/CH3OH.HCOOH, Rf ) 0.25, homoge-
neous; HPLC, 99.3%, one impurity); 1H NMR (DMSO-d6) δ
1.1-1.15 (t, 3H), 1.2-1.25 (d, 3H), 1.45-1.55 (m, 2H), 2.4-
2.5 (t, 2H), 3.35-3.45 (m, 3H; 2H exchangeable on treatment
with D2O; post D2O treatment, d, 1H), 3.6-3.75 (m, 2H), 5.7-
5.75 (d, 1H), 6.55-6.65 (bs, 1H), 7.0-7.1 (t, 1H), 7.2-7.25 (t,
1H), 7.25-7.3 (d, 1H), 8.1-8.25 (bs, 1H). Anal. Calcd for
C
16H20N4O)′: C, 67.58; H, 7.09; N, 19.70. Found: C, 67.31; H,
6.90; N, 19.54.
[2R-[1(S*),R*]]-r-Meth yl-2,3-d ih yd r o-â-oxo-2-(4-eth yl-
1H-im id a zol-2-yl)-1H-in d ole-1-eth a n a m in e (3e). Starting
from 17e, 3e was prepared and worked up in a similar manner
as was 3b to yield 32% of 3e as a white solid: mp 73-74 °C
(TLC, 80:20:5 CHCl3/CH3OH/HCOOH, Rf ) 0.3, homogeneous;
HPLC, 99.8%, one impurity); 1H NMR (DMSO-d6) δ 0.7-0.85
(bs, 3H), 1.05-1.15 (t, 3H), 1.85-2.0 (bs, 2H, both exchange-
able on treatment with D2O), 2.35-2.45 (m, 1H), 2.45-2.55
(m, 1H), 3.05-3.2 (d, 1H), 3.5-3.7 (m, 2H), 5.8-5.9 (bd, 1H),

5310 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 24
Breslin et al.
(9) We ran the stability study in a Fisher versa-bath at a constant
temperature of 38 °C at pH 7.0 (0.05 M potassium phosphate
mono-basic-sodium hydroxide buffer solution; Fisher SB108-
500). The percentage of degradation was measured by HPLC
(monitoring area percent at 214 and 254 nm on a HP series 1050
HPLC instrument).
6.5 (s, 0.5H), 6.7 (s, 0.5H), 7.0-7.1 (t, 1H), 7.2-7.3 (m, 2H),
8.05-8.2 (bs, 1H), 11.65-11.9 (bd, 1H, exchangeable on
treatment with D2O). HRMS calcd for C16H20N4O (MH+):
285.1715. Found: 285.1723.
Tr ip ep tid yl P ep tid a se II (TP P II) En zym e Isola tion
a n d Rela ted Deter m in a tion of In h ibitor y Activity. TPPII
enzyme was purified from a rat liver postlysosomal fraction
using DEAE ion exchange chromatography followed by an
S-200 size exlusion column and hydroxyl apatite chromatog-
raphy.19 To the protein that eluted at 50 mM potassium
phosphate, glycerol was added to a final concentration of 30%
and the protein was stored at -70 °C.
TPPII activity was evaluated at room temperature using
100 µM H-Ala-Ala-Phe-AMC as the substrate in an assay
buffer of pH 7.5 consisting of 50 mM KH2PO4, 1 mM EGTA, 1
mM DTT, 100 µM Bestatin, and 100 nM Thiorphan. Inhibitors
were added at a final DMSO concentration of 1%. Assay
incubations were measured in the fluoroscan (Labsystems) at
405 nm (excitation at 355 nm) in the linear part of the reaction
(less than 15% of hydrolysis).5 IC50 values were determined
using Sigma plot C/R curves.
(10) (a) Chen, J . J .; Zhang, Y.; Hammond, S.; Dewdney, N.; Ho, T.;
Lin, X.; Browner, M. F.; Castelhano, A. L. Design, synthesis,
activity, and structure of a novel class of matrix metalloprotein-
ase inhibitors containing a heterocyclic P2′-P3′ amide bond
isostere. Bioorg. Med. Chem. Lett. 1996, 6 (13), 1601-1606. (b)
Thompson, S. K.; Murthy, K. H. M.; Zhao, B.; Winborne, E.;
Green, D. W.; Fisher, S. M.; DesJ arlais, R. L.; Tomaszek, T. A.,
J r.; Meek, T. D.; Gleason, J . G.; Abdel-Meguid, S. S. Rational
Design, Synthesis, and Crystallographic Analysis of a Hydroxy-
ethylene-Based HIV-1 Protease Inhibitor Containing a Hetero-
cyclic P1′-P2′ Amide Bond Isostere. J . Med. Chem. 1994, 37 (19),
3100-3107. (c) Gordon, T.; Hansen, P.; Morgan, B.; Singh, J .;
Baizman, E.; Ward, S. Peptide Azoles: A New Class of Biologi-
cally-Active Dipeptide Mimetics. Biomed. Chem. Lett. 1993, 3
(5), 915-920.
(11) (a) Fitzpatrick, P. A.; Steinmetz, A. C.; Ringe, D.; Klibanov, A.
M. Enzyme crystal structure in a neat organic solvent. Proc.
Natl. Acad Sci U.S.A. 1993, 90, 8653. (b) Bode, W.; Papamokos,
E.; Musil, D. The high-resolution X-ray crystal structure of the
complex formed between subtilisin Carlsberg and eglin c, an
elastase inhibitor from the leech Hirudo medicinalis. Structural
analysis, subtilisin structure and interface geometry. Eur. J .
Biochem. 1987, 166, 673.
(12) Martens, J .; Dauelsberg, C.; Behnen, W.; Wallbaum, S. Enantio-
selective catalytic borane reductions of achiral ketones: syn-
thesis and application of two chiral â-amino alcohols from (S)-
2-indolinecarboxylic acid. Tetrahedron: Asymmetry 1992, 3 (3),
347-50.
(13) Stanton, J . L.; Ksander, G. M. Preparation and formulation of
glutamylindoline carboxylates and related compounds as anti-
hypertensives and for treatment of congestive heart failure. U.S.
Patent 4,678,800, J uly 7, 1987.
(14) Saednya, A. Conversion of Carboxamides to Nitriles Using
Trichloroacetyl Chloride/Triethylamine as a Mild Dehydrating
Agent. Synthesis 1985, 184-185.
(15) Enantiomeric purity was determined using a Chiralpak AD (4.6
mm × 250 mm, 10 µm) HPLC column from Chiral Technologies
(temperature, ambient; flow rate, 0.8 mL/min; mobile phase,
(80% hexane)/(20% 90:10:0.05 hexane/ethanol/trifluoroacetic
acid); wavelength, 254 nm).
Ack n ow led gm en t. We appreciate the assistance of
William J ones for obtaining high-resolution mass spec-
tral data.
Refer en ces
(1) Mokdad, A. H.; Serdula, M. K.; Dietz, W. H.; Bowman, B. A.;
Marks, J . S.; Koplan, J . P. The Spread of the Obesity Epidemic
in the United States, 1991-1998. J AMA, J . Am. Med. Assoc.
1999, 282, 1519-1522.
(2) Must, A.; Spadano, J .; Coakley, E. H.; Field, A. E.; Colditz, G.;
Dietz, W. H. The Disease Burden Associated With Overweight
and Obesity. J AMA, J . Am. Med. Assoc. 1999, 282, 1523-1529.
(3) Allison, D. B.; Fontaine, K. R.; Manson, J . E.; Stevens, J .;
VanItallie, T. B. Annual Deaths Attributable to Obesity in the
United States. J AMA, J . Am. Med. Assoc. 1999, 282, 1530-1538.
(4) Kordik, C. P.; Reitz, A. B. Pharmacological Treatment of
Obesity: Therapeutic Strategies. J . Med. Chem. 1999, 42, 181-
201.
(5) Rose, C.; Vargas, F.; Facchinetti, P.; Bourgeat, P.; Bambal, R.
B.; Bishop, P. B.; Chan, S. M. T.; Moore, A. N. J .; Ganellin, C.
R.; Schwartz, J .-C. Characterization and Inhibition of a Chole-
cystokinin-Inactivating Serine Peptidase. Nature 1996, 380,
403-409.
(6) Ganellin, C. R.; Bishop, P. B.; Bambal, R. B.; Chan, S. M. T.;
Law, J . K.; Marabout, B.; Luthra, P. M.; Moore, A. N. J .;
Peschard, O.; Bourgeat, P.; Rose, C.; Vargas, F.; Schwartz, J .-
C. Inhibitors of Tripeptidyl Peptidase II. 2. Generation of the
First Novel Lead Inhibitor of Cholecystokinin-8-Inactivating
Peptidase: A Strategy for the Design of Peptidase Inhibitors.
J . Med. Chem. 2000, 43, 664-674.
(16) Suzuki, M.; Iwasaki, T.; Miyoshi, M.; Okumura, K.; Matsumoto,
K. Synthesis of amino acids and related compounds. 6. New
convenient synthesis of R-C-acylamino acids and R-amino ke-
tones. J . Org. Chem. 1973, 38 (20), 3571-3575.
(17) Carpino, L. A.; Mansour, E.-S. M. E.; Sadat-Aalaee, D. tert-
Butyloxycarbonyl and benzyloxycarbonyl amino acid fluorides.
New, stable rapid-acting acylating agents for peptide synthesis.
J . Org. Chem. 1991, 56 (8), 2611-2614.
(18) J ones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and Validation of a Genetic Algorithm for Flexible
Docking. J . Mol. Biol. 1997, 267, 727-748.
(19) Balow, R. M.; Ragnarsson, U.; Zetterqvist, O. Purification,
substrate specificity, and classification of tripeptidyl peptidase
II. J . Biol. Chem. 1986, 261 (5), 2409-2417.
(7) Rose, C.; Vargas, F.; Bourgeat, P.; Schwartz, J .-C.; Bishop, P.
B.; Bambal, R. B.; Ganellin, C. R.; Leblond, B.; Moore, A. N. J .;
Chan, S.; Zhao, L. Tripeptidylpeptidase inhibitors, methods of
synthesis, and use of inhibitors in treatment of gastrointestinal
and mental disorders. Patent WO 9635805 A2, November 14,
1996.
(8) Schwartz, J .-C.; Rose, C.; Vargas, F.; Ganellin, C. R.; Zhao, L.;
Samad, S.; Chen, Y. Preparation of new 1-(2-aminobutyryl)-
indoline-2-carboxylic acid amides as tripeptidyl peptidase inhibi-
tors. Patent WO 9933801 A1, J uly 8, 1999.
J M0202831