New proline mimetics: Synthesis of thrombin inhibitors incorporating cyclopentane- and cyclopentenedicarboxylic acid templates in the P2 position. Binding conformation investigated by X-ray crystallography

Article abstract of DOI:10.1021/jm990557t

With the aim to prepare nonpeptidic thrombin inhibitors, the amino acids of the thrombin-inhibiting tripeptide chain D-Phe-Pro-Arg were replaced with isosteres. Arg was replaced with the more rigid P1 truncated p- amidinobenzylamine (Pab), Pro with either cyclopentane-1,2-dicarboxylic acid or cyclopentene-1,5-dicarboxylic acid, and D-Phe with a series of readily available lipophilic amines. One of the most potent compounds (25, pIC₅₀ = 6.01) in these series was cocrystallized with thrombin where the X-ray crystal structure provide insight to the structure-activity relationship (SAR).

Full text of DOI:10.1021/jm990557t

J . Med. Chem. 2000, 43, 1705-1713
1705
New P r olin e Mim etics: Syn th esis of Th r om bin In h ibitor s In cor p or a tin g
Cyclop en ta n e- a n d Cyclop en ten ed ica r boxylic Acid Tem p la tes in th e P 2
P osition . Bin d in g Con for m a tion In vestiga ted by X-r a y Cr ysta llogr a p h y
Daniel No¨teberg,†,| J onas Brånalt,† Ingemar Kvarnstro¨m,† Marcel Linschoten,‡ Djordje Musil,‡
J an-Erik Nystro¨m,‡ Guido Zuccarello,† and Bertil Samuelsson*,‡,§
Department of Chemistry, Linko¨ping University, S-581 83 Linko¨ping, Sweden, Department of Medicinal Chemistry,
AstraZeneca, S-431 83 Mo¨lndal, Sweden, and Department of Organic Chemistry, Stockholm University,
S-106 91 Stockholm, Sweden
Received November 8, 1999
With the aim to prepare nonpeptidic thrombin inhibitors, the amino acids of the thrombin-
inhibiting tripeptide chain D-Phe-Pro-Arg were replaced with isosteres. Arg was replaced with
the more rigid P1 truncated p-amidinobenzylamine (Pab), Pro with either cyclopentane-1,2-
dicarboxylic acid or cyclopentene-1,5-dicarboxylic acid, and D-Phe with a series of readily
available lipophilic amines. One of the most potent compounds (25, pIC50 ) 6.01) in these series
was cocrystallized with thrombin where the X-ray crystal structure provide insight to the
structure-activity relationship (SAR).
Ch a r t 1
In tr od u ction
Proteases represent a large and varied class of bio-
molecules that presently are under intense investigation
as targets for therapeutical intervention in disease.
However, the design and discovery of orally active and
selective protease inhibitors still remains a major chal-
lenge in drug discovery.1
Undesired blood clotting is the major culprit in a
number of cardiovascular diseases, i.e., deep venous
thrombosis, pulmonary embolism, unstable angina,
restenosis following angioplasty, and arterial thrombo-
sis.2 Thrombin, a member of the trypsin family of serine
proteases, plays a critical role in the blood coagulation
cascade. The procoagulant properties of thrombin are
exerted via the conversion of fibrinogen into a fibrin clot
and from activation of zymogens upstream in the
coagulation cascade.3 Moreover, thrombin is the most
potent stimulator of platelet aggregation known. Thus,
small molecule thrombin inhibitors to regulate hemo-
stasis and thrombosis are anticipated to provide power-
ful drugs, and indeed there has been a major focus on
low-molecular-weight thrombin inhibitors in recent
years.3b,4
Ch a r t 2. N-Acylproline in Comparison with the
N-Acylproline Isosteres Used in This Paper
The classical motif of thrombin inhibitors is the D-Phe-
Pro-Arg5 sequence mimicking thrombin’s natural sub-
strate, fibrinogen. A number of development candidates
and clinical inhibitors such as inogatran6 and melagat-
ran7 have been developed based on this motif (Chart
1).
The amino acid L-proline has frequently been explored
as a template and starting point for peptidomimetic
inhibitor design, cf. inhibitors of the human immuno-
deficiency virus type 1 (HIV-1) protease and angio-
tensin-converting enzyme (ACE). There are however
notably few reports of replacing proline with five-
membered carbocyclic ring isosteres in the tripeptide
type motif D-Phe-Pro-Arg. Cyclohexane-1,2-dicarboxylic
acid and 2-aminocyclohexanecarboxylic acid have been
reported as proline isosteres in thrombin inhibitors.8
Moreover, Turbanti et al.9 have incorporated cyclopen-
tane-1,2-dicarboxylic acids in ACE inhibitors. Recently
a (Z)-alkene isostere of a cis-Pro dipeptide was de-
scribed.10
We now report on the synthesis of two novel N-
acylproline isosteres, i.e., trans-(1S,2S)-cyclopentane-
1,2-dicarboxylic acid and (2R)-cyclopent-2-ene-1,2-di-
carboxylic acid (Chart 2), and furthermore the incor-
poration of these P2 templates in a thrombin inhibitor
* To whom correspondence should be addressed at: Medivir AB,
Lunastigen 7, SE-141 44 Huddinge, Sweden. Tel: +46 8 608 3100.
Fax: +46 8 608 3199. E-mail: bertil.samuelsson@medivir.se.
† Linko¨ping University.
‡ AstraZeneca.
§ Stockholm University.
| Current address: Organic Pharmaceutical Chemistry, Uppsala
University, Box 574, S-751 23 Uppsala, Sweden.
10.1021/jm990557t CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/14/2000

1706 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
No¨teberg et al.
Sch em e 1a
a
Reagents and conditions: (i) Raney nickel, methanol, reflux; (ii) NaOH, dioxane-water, 1:1; (iii) Pab(Z)‚HCl, EDC, HOBt, Et3N,
DMF.
Ta ble 1a
motif having p-amidinobenzylamine in the P1 position
and having lipophilic amines in the P3 position. Several
of these compounds showed promising thrombin inhibi-
tion activity in vitro, IC50 ∼ 1 µM. Moreover, the X-ray
crystal structure was determined for inhibitor 25 in
complex with thrombin, providing additional SAR for
this class of compounds.
Resu lts a n d Discu ssion
Ch em istr y. Numerous synthetic routes for the de-
sired enantiomerically pure trans-cyclopentane-1,2-di-
carboxylic acid derivatives have been reported, including
traditional resolution,11 enzymatic resolution,12 and
asymmetric synthesis.13 We chose to start with the
enantiomerically pure thioketal (-)-trans-(7R,8R)-1,4-
dithiaspiro[4.4]nonane-7,8-dicarboxylic acid dimethyl
ester [(-)-1], prepared earlier in our laboratory,14 which
was desulfurized using Raney nickel in refluxing metha-
nol to give the bisester (-)-2 in 85% yield (Scheme 1).15
Partial hydrolysis of (-)-2 with sodium hydroxide in
aqueous dioxane gave the corresponding monoester16
that was coupled with p-(benzyloxycarbonyl)amidinoben-
zylamine hydrochloride (Pab(Z)‚HCl)17 using EDC [N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide]-HOBt (N-
hydroxybenzotriazole) to give (-)-3 in 36% overall yield.
The corresponding (+)-3 was prepared from (+)-1 using
a similar procedure. The ester function of (-)-3 was
hydrolyzed using sodium hydroxide in water-dioxane,
and the resulting acid was directly coupled with various
amines (i.e., benzylamine, (R)-phenylglycine methyl
ester, cyclohexylamine, and N-ethylcyclohexylamine)
using EDC-HOBt or Bop-Cl [bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride] as coupling agents (Table 1). The
Z-group was removed by hydrogenolysis to give com-
pounds (-)-4, 5, 6,18 7, and 8 in overall yields ranging
from 41% to 67%. The corresponding (+)-4 was prepared
from (+)-3 using the same procedure in 67% yield.
a
Reagents and conditions: (i) NaOH, dioxane, water; (ii)
RNHR′, coupling agent, Et3N, DMF; (iii) (only for compound 6)
NaOH, dioxane, water; (iv) H2, Pd-C, ethanol.
Stevens et al.20 have reported on the synthesis of
racemic 1-formylcyclopentene-5-carboxylic acid (9)
(Scheme 2). This acid was thus converted to the methyl
ester using trimethoxymethane with acid catalysis to
give 10 in 97% yield. The aldehyde 10 was oxidized
using sodium chlorite with resorcinol as scavenger to
give the acid (()-11 in 81% yield. This acid was coupled
with benzylamine using EDC and HOBt as coupling
agents to give 12 in 75% yield (Table 2). Hydrolysis of
12 followed by coupling with Pab(Z)‚HCl and deprotec-
tion of the coupling product with triflic acid and anisole
in dichloromethane gave racemic 13 in 60% overall
yield. For the preparation of enantiomerically enriched
compounds, (()-11 was resolved with (-)-ephedrine to
give (-)-11 and (+)-11 in 17% and 21% yield, respec-
tively.21
For the synthesis of the chiral monoester of cyclopent-
2-ene-1,2-dicarboxylic acid, the corresponding enantio-
merically pure bismethyl esters19 were examined as a
potential precursor. Thus using the racemic compound
as a model, attempts were made to selectively hydrolyze
one of the esters using various metallo hydroxides or
using pig liver esterase (PLE). However, only modest
selectivity could be obtained. Moreover, the resulting
monoesters resisted chromatographic separation. In
view of these difficulties novel routes were investigated.
Using the same procedure, vide supra, compound (+)-
11 was coupled with piperidine, cyclohexylamine, and
cyclohexylmethylamine to give compounds 15, 17, and

New Proline Mimetics
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1707
Sch em e 2a
a
Reagents and conditions: (i) Cl2CHCOCl, Et3N, hexane; (ii) 1
M NaHCO3, 80 °C; (iii) HC(OCH3)3, TsOH; (iv) NaClO2, resorcinol,
NaH2PO4, t-BuOH-water, 2:1; (v) (-)-ephedrine, EtOAc, recrys-
tallization.
F igu r e 1. Lipophilic Connolly suface from the X-ray crystal
structure of human R-thrombin in complex with inhibitor 25.
Ta ble 2a
Boc-deprotected prior to the coupling reaction using a
1:1 mixture of trifluoroacetic acid and dichloromethane.
Str u ctu r e-Activity Relation sh ip an d X-r ay Cr ys-
ta llogr a p h ic Da ta . The compounds synthesized show
less affinity for thrombin compared to melagatran or
inogatran (Chart 1). In the following, possible explana-
tions will be given as well as some qualitative SAR
considerations for the relative affinities within each of
the two series.
1. tr a n s-(1S,2S)-Cyclop en ta n e-1,2-d ica r boxylic
Acid Der iva tives. With respect to the analogues based
on the trans-(1S,2S)-cyclopentanedicarboxylic acid scaf-
fold (Table 1), their low affinities as compared to
classical inhibitors can be explained at least partly by
the different geometry as compared to proline. The
planar proline P3 amido bridge is fundamentally altered
by an sp3 carbon in cyclopentane, thereby changing the
angle under which P2 is connected to P3. This obviously
influences the possibility of the carbonyl group to act
as a hydrogen bond acceptor for the NH of Gly216 in
thrombin. On the basis of some preliminary docking
studies using the Sybyl molecular modeling software,
a hydrogen bond with Gly216 should be possible de-
pending on the rest of the P3 moiety. The best compound
(7, IC50 ) 1.32 µM) in this series has a cyclohexyl in
the P3 position. All of the phenyl analogues are less
active. A possible explanation for this might be that the
positioning of a phenyl group in P3 is rather critical due
to its interaction with the π-electron system of Trp215.
An optimal interaction between these two moieties
results from either a perpendicular or a parallel relative
orientation of the ring systems, whereas optimal inter-
actions of a cyclohexane with Trp215 are based on
lipophilicity only, thereby making the geometry of the
interaction less critical.
a
Reagents and conditions: (i) RNHR′, coupling agent, Et3N,
DMF; (ii) NaOH, dioxane, water; (iii) Pab(Z)‚HCl, coupling agent,
Et3N, DMF; (iv) TFMSA, anisole, CH2Cl2.
19. In view of the promising results, vide infra, obtained
from 17 with cyclohexylamine in the P3 position ad-
ditional amines were also investigated, i.e., aniline,
N-ethylcyclohexylamine, N-ethylaniline, and N-ethoxy-
cyclohexylamine resulting in compounds 21, 23, 25, and
31. N-Ethoxycyclohexylamine was prepared from N-
cyclohexylhydroxylamine (26) in two steps. Compound
26 was Boc-protected using Boc-anhydride and Na2CO3
in a 1:1 mixture of water and tetrahydrofuran to give
the N-protected N-cyclohexylhydroxylamine 27 in 41%
yield along with the O-Boc-protected derivative 28 in
39% yield. O-Alkylation of 27 using EtI and NaOH in
refluxing methanol gave 29 in 84% yield, which was
2. (2R)-Cyclop en t-2-en e-1,2-d ica r boxylic Acid s.
These derivatives are closer mimics of proline than the
above-discussed cyclopentane derivatives (Table 2). Next
to the active cyclohexyl analogues 17, 23, and 31, also
the phenyl derivative 25 was found to have a reasonable
affinity. To get a better understanding of the interaction
of this compound, 25 was cocrystallized with thrombin.
The structure of this complex is shown in Figure 1.
With respect to the hydrogen-onding interactions of
25, the following observations can be made: The benz-

1708 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
No¨teberg et al.
Ta ble 3. Parameters and Statistics from the Data Collection
and Refinement of the Thrombin-(5R)-Cyclopentene-1-
carbonyl-N-ethylphenylamide-5-carbonyl-p-amidinobenzylamide
Complex
amidino group forms a salt bridge with the carboxylate
of Asp189 (N-O distances being 2.74 and 2.78 Å). The
positioning of the benzamidino moiety as a whole is
identical with other X-ray structures of complexes of
highly active analogues (unpublished results). The NH
in the P1-P2 linker forms a neat hydrogen bond with
the main chain carbonyl of the catalytic Ser256 (N-O
distance is 3.07 Å). In contrast to this the hydrogen bond
of the carbonyl group in the P2-P3 linker of 25 is far
from optimal, the O-N distance being 3.35 Å.
no. of measurements
no. of unique reflections
data completeness
257369
45814
89.7 (75.5)*
0.039 (0.299)*
2618
a
Rmerge
no. of atoms in refined model
protein
2329
ligands
29
solvent
260
The binding conformation of 25 is not optimal in the
P2-P3 linker. The torsional angle of the cyclopentene
double bond relative to the R-carbonyl group is not
planar. The deviation from planarity is 30.1° thereby
giving rise to a considerable loss in binding affinity.
Besides this imperfection in the binding conformation,
the P2-P3 amido group also deviates from planarity by
an amount of 20°. Moreover, the P2-P3 amido group
adopts a cis-geometry around the N-carbonyl-carbon
plane, directing the phenyl group into the S3 pocket.
This would favor secondary amines (tertiary amides) in
the P3 position as primary amines, which preferentially
adopt a trans-geometry, giving rise to unfavorable
binding to the enzyme. Notably however, the angle
between the planes of the ligand’s phenyl group in P3
and the Trp215 indole plane is a near optimal 93.4°.
resolution range in refinement (Å)
rms deviation
18.5-1.55
bond length (Å)
angles (deg)
0.015
1.853
0.247
0.285
b
Rcryst
Rfree
a
Rmerge ) ShSi(I(h,i) - I(h) )/ShSi I(h,i), where I(h,i) is the
intensity value of the ith measurement of h and I(h) is the
corresponding mean value of h for all i measurements of h. Rcryst
) Shkl(|Fo - Fc|)/Shkl |Fo|, where |Fo| and |Fc| are observed and
calculated structure factor amplitudes, respectively. *The numbers
in parentheses are related to the data in the highest resolution
shell, i.e., from 1.63 to 1.55 Å.
b
[amino(imino)methyl]benzyl}-N2-phenyl-N2-ethyl-2-cyclopen-
tene-1,2-dicarboxamide (25)-thrombin structure. The refine-
ment was performed using REFMAC (CCP4 package) with
subsequent runs of X-PLOR.25 Statistics for X-ray data col-
lection and refinement are presented in Table 3.
P la sm a Coa gu la tion Mea su r em en ts. The thrombin in-
hibitor potency was measured with a chromogenic substrate
method, in a Plato 3300 robotic microplate processor (Rosys
AG, CH-8634 Hombrechtikon, Switzerland), using 96-well,
half-volume microtiter plates (Costar, Cambridge, MA, cat. no.
3690). Stock solutions of test substance in DMSO (72 µL), 10
mM, were diluted serially 1:3 (24 + 48 µL) with DMSO to
obtain 10 different concentrations, which were analyzed as
samples in the assay, together with controls and blanks. The
dilutions of each test substance were analyzed consecutively,
row-wise on the microtiter plate, with wash cycles between
substances to avoid cross-contamination. 2 µL of test sample
was diluted with 124 µL of assay buffer (0.05 M Tris-HCl, pH
7.4, ionic strength 0.15 adjusted with NaCl, BSA 1 g/L); 12
µL of chromogenic substrate solution (S-2366, Cromogenix,
Mo¨lndal, Sweden) and finally 12 µL of R-thrombin solution
(human R-thrombin, Sigma Chemical Co., St. Louis, MO, cat.
no. T-6759), both in buffer, were added, and the samples were
mixed. The final assay concentrations were test substance
0.00068-13.3 µM, S-2366 0.30 mM, R-thrombin 0.020 NIHU/
mL. The linear absorbance increase during a 40-min incuba-
tion at 37 °C was used for calculation of percent inhibition for
the test samples, as compared to blanks without inhibitor. The
IC50 value, corresponding to the inhibitor concentration which
caused 50% inhibition of the thrombin activity, was calculated
from a log dose vs inhibition curve.
Con clu sion
Two new proline mimetics, i.e., trans-(1S,2S)-cyclo-
pentane-1,2-dicarboxylic acid and (2R)-cyclopent-2-ene-
1,2-dicarboxylic acid, incorporated in a thrombin inhibi-
tor motif have been prepared. The best of these thrombin
inhibitors, having the proline templates in the P2
position, p-amidinobenzylamine in the P1 position, and
lipophilic amines in the P3 position, were 23 (IC50
)
0.89 µM), 25 (IC50 ) 0.98 µM), and 31 (IC50 ) 0.87 µM)
all having a secondary amine in the P3 position. The
X-ray crystal structure of compound 25 complexed with
thrombin shows that further optimization within this
series of proline mimics should be directed toward
releasing the strain in the P2-P3 linker and improving
the hydrogen bonding with Gly216. Moreover, further
stabilization of the cis-geometry around the P2-P3
amido group should lead to more potent thrombin
inhibitors.
Exp er im en ta l Section
X-r a y Cr ysta llogr a p h y. Human R-thrombin was pur-
chased from Enzyme Research Laboratories, Inc., South Bend,
IN, and hirugen from American Diagnostica, Inc., Greenwich,
CT. Hirugen-thrombin complex was prepared according to the
method of Skrzypczak-J ankun et al.22 and the sample was
concentrated to 5.5 mg/mL. The crystallization was carried out
at 20 °C by vapor diffusion method using 24-30% PEG 4000
and 0.1 M sodium phosphate buffer (SPB) at pH 7.3. A single
crystal was used for soaking overnight in 30% PEG 4000, 0.1
M SPB, pH 7.3, containing 0.5 mg/mL of the inhibitor. The
crystal belongs to monoclinic space group C2 with cell con-
stants of a ) 69.96 Å, b ) 71.80 Å, c ) 71.49 Å, a ) g ) 90°,
b ) 100.2°, with one protein molecule per asymmetric unit.
The X-ray data to 1.55 Å resolution were collected on a
MAR-II imaging plate system, MAR Research, Hamburg,
Germany, using Cu KR radiation from a rotating anode. The
data were reduced and scaled using DENZO23 and SCALA
from the CCP424 package.
Gen er a l Meth od s. 1H and 13C NMR spectra were recorded
on a Bruker 250 instrument using CDCl3, methanol-d4 or D2O.
The shifts are reported in ppm (δ scale) and all J values are
in Hz. TLC was performed on Merck precoated 60 F254 plates.
The spots were visualized with UV light (254 nm) and ethanol/
sulfuric acid/acetic acid/p-anisaldehyde (90:3:1:2). Column
chromatography was performed using silica gel 60 (0.040-
0.063 mm, Merck). Optical rotations were measured in CHCl3
or methanol solutions at room temperature using a Perkin-
Elmer 141 instrument. Organic phases were dried over
anhydrous MgSO4.
tr a n s-(1R,2R)-Cyclop en ta n e-1,2-d ica r boxylic Acid Di-
m eth yl Ester [(-)-2]. A suspension of Raney nickel was
filtered, washed with water until pH ∼ 7 and added to a
solution of (-)-trans-(7R,8R)-1,4-dithiaspiro[4.4]nonane-7,8-
dicarboxylic acid dimethyl ester [(-)-1]14 (1.94 g, 6.83 mmol)
in methanol (80 mL). After refluxing the mixture for 4 h, it
was cooled, filtered, concentrated, and purified by column
The hirugen-R-thrombin structure previously solved in our
laboratory was used in the refinement of the (1R)-N1-{4-

New Proline Mimetics
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1709
chromatography (toluene-ethyl acetate, 7:1) to give (-)-2 (1.06
g, 85%) as a colorless liquid. (-)-2: [R]D22 -73.7 (c 1.7, CHCl3)
50.3, 50.5, 128.1, 128.3, 128.4, 128.9, 129.1, 129.5, 140.1, 147.2,
168.2, 176.8, 176.9. Anal. (C22H26N4O2‚4.3MeOH‚0.6H2O) C,
H, N.
[lit.13b (+)-2: [R]D +71.9 (c 1.6, CCl4)]; H NMR (250 MHz,
CDCl3) identical with ref 13b; 13C NMR (62.9 MHz, CDCl3) δ
25.4, 30.4, 47.1, 51.7, 175.2.
22
1
tr a n s-(1S,2S)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-
ben zyl-1,2-cyclop en ta n ed ica r boxa m id e [(+)-4]. Compound
(+)-4 (44 mg, 67%) was prepared from (+)-3 (65 mg, 0.15 mmol)
according to the method for the preparation of (-)-4. (+)-4:
tr a n s-(1S,2S)-Cyclop en ta n e-1,2-d ica r boxylic Acid Di-
m eth yl Ester [(+)-2]. Compound (+)-2 (0.10 g, 78%) was
prepared from (+)-1 (0.20 g, 0.70 mmol)14 according to the
method for the preparation of (-)-2. (+)-2: [R]D22 +70.9 (c 0.7,
22
[R]D +21.9 (c 0.7, MeOH). Anal. (C22H26N4O2‚4.1MeOH‚
1.4H2O) C, H, N.
CHCl3) [lit.13b [R]D +71.9 (c 1.6, CCl4)].
22
tr a n s-(1R,2R,2′R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-
N2-[2′-(m et h yl)ca r b oxylb en zyl]-1,2-cyclop en t a n ed ica r -
boxa m id e (5). Compound 5 (28 mg, 66%) was prepared from
(-)-3 (48 mg, 0.110 mmol) according to the method for the
preparation of (-)-4 using phenylglycine methyl ester instead
tr a n s-Meth yl (1R,2R)-2-{4-[Am in o(ben zyloxyca r bon yl-
im in o)m et h yl]b en zyla m in oca r b on yl}cyclop en t a n eca r -
boxyla te [(-)-3]. To a solution of [(-)-2] (0.86 g, 4.6 mmol) in
dioxane-water (1:1) (30 mL) was added aqueous NaOH (1.0
M, 4.6 mL) dropwise during 30 min at room temperature. After
2 h, the reaction mixture was acidified with concentrated
hydrochloric acid to pH ∼ 2-3 and extracted five times with
dichloromethane (10 mL). The combined organic phases were
dried, filtered, concentrated and purified by column chroma-
tography (toluene-ethyl acetate-acetic acid, 50:50:1) to give
trans-(1R,2R)-cyclopentane-1-carboxylic acid methyl ester 2-car-
boxylic acid (0.38 g, 48%) as an oil.13c All of the monoacid was
then dissolved in DMF (10 mL) containing p-(benzyloxycar-
bonyl)amidinobenzylamine hydrochloride (Pab(Z)‚HCl) (0.76
g, 2.4 mmol) and N-hydroxybenzotriazole (HOBt) (0.475 g, 3.5
mmol). The mixture was cooled to 0 °C, N-(3-dimethylamino-
propyl)-N′-ethylcarbodiimide hydrochloride (EDC) (0.665 g, 3.5
mmol) was added and triethylamine was added dropwise until
pH ∼ 8. After 1 h, the ice bath was removed and the solution
was stirred at room temperature overnight. The solvent was
evaporated and the residue was purified by column chroma-
tography (toluene-ethyl acetate, 1:1) to give (-)-3 (0.85 g, 88%,
42% overall) as white needles (ethyl acetate, hexane). (-)-3:
22
1
of benzylamine. 5: [R]D -37.4 (c 0.6, MeOH); H NMR (250
MHz, methanol-d4) δ 1.68-1.88 (4 H, m), 1.95-2.12 (2 H, m),
2.98-3.18 (2 H, m), 3.68 (3 H, s), 4.36-4.57 (4 H, m), 5.46 (1
H, s), 7.30-7.43 (5 H, m), 7.50-7.59 (2 H, d, J ) 8.6 Hz), 7.72-
7.82 (2 H, d, J ) 8.6 Hz); 13C NMR (62.9 MHz, methanol-d4)
δ 26.4, 32.0, 32.1, 43.7, 49.7, 50.3, 53.0, 58.4, 128.5, 128.8,
129.0, 129.1, 129.6, 129.9, 137.3, 147.1, 168.3, 172.7, 177.0,
180.9. Anal. (C24H28N4O4‚1.5MeOH‚0.7H2O) C, H, N.
tr a n s-(1R,2R,2′R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-
N2-(2′-ca r boxylben zyl)-1,2-cyclop en ta n ed ica r boxa m id e
(6). Compound 6 (15 mg, 56%) was prepared from (-)-3 (28
mg, 0.064 mmol) according to the method for the preparation
of 5, with the exception that the peptide coupling product was
treated with 3 equiv of NaOH (1 M) in water-dioxane (1:1, 3
mL) at 0 °C. The mixture was stirred for 30 min, neutralized
with dilute hydrochloric acid and evaporated. This product was
then used in the hydrogenation step. The mobile phase in the
final purification was methanol-water (25:75). 6: [R]D22 -8.6
(c 1.1, MeOH); 1H NMR (250 MHz, methanol-d4) δ 1.67-1.91
(4 H, m),1.93-2.18 (2 H, m), 2.94-3.17 (2 H, m), 4.36-4.61 (4
H, m), 5.48 (1 H, s), 7.32-7.45 (5 H, m), 7.46-7.57 (2 H, d, J
) 8.6 Hz), 7.72-7.82 (2 H, d, J ) 8.6 Hz); 13C NMR (62.9 MHz,
methanol-d4) δ 26.3, 31.8, 31.9, 43.6, 43.7, 49.9, 50.4, 59.0,
128.0, 128.6, 128.6, 129.0, 129.1, 129.7, 136.8, 147.3, 168.1,
174.0, 176.5, 177.3. Anal. (C23H26N4O4‚1.5MeOH‚0.8H2O) C,
H, N.
[R]D -22.1 (c 1.1, CHCl3); mp 131-132 °C; 1H NMR (250
22
MHz, CDCl3) δ 1.60-2.19 (6 H, m), 2.81-3.13 (2 H, m), 3.62
(3 H, s), 4.22-4.45 (2 H, m), 5.20 (2 H, s), 6.82-6.91 (1 H, m),
7.10-7.46 (7 H, m), 7.70-7.78 (2 H, d, J ) 8.5 Hz); 13C NMR
(62.9 MHz, CDCl3) δ 25.4, 30.2, 30.4, 43.0, 47.9, 48.4, 52.0,
67.2, 127.3, 127.7, 128.2, 128.4, 129.0, 133.4, 136.7, 143.0,
164.6, 168.1, 174.6, 176.0. Anal. (C24H27N3O5‚0.4EtOAc) C, H,
N.
tr a n s-(1R,2R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-
cycloh exyl-1,2-cyclop en t a n ed ica r b oxa m id e (7). Com-
pound 7 (51 mg, 67%) was prepared from (-)-3 (90 mg, 0.210
mmol) according to the method for the preparation of (-)-4
using cyclohexylamine instead of benzylamine and bis(2-oxo-
3-oxazolidinyl)phosphinic chloride (Bop-Cl) (107 mg, 0.420
tr a n s-Meth yl (1S,2S)-2-{4-[Am in o(ben zyloxyca r bon yl-
im in o)m et h yl]b en zyla m in oca r b on yl}cyclop en t a n eca r -
boxyla te [(+)-3]. Compound (+)-3 (0.30 g, 21%) was prepared
from (+)-2 (0.60 g, 3.26 mmol) according to the method for the
22
preparation of (-)-3. (+)-3: [R]D +20.1 (c 0.5, CHCl3); mp
22
mmol) in DMF (3 mL) instead of EDC and HOBt. 7: [R]D
130-131 °C. Anal. (C24H27N3O5) C, H, N.
-37.4 (c 0.5, MeOH); 1H NMR (250 MHz, methanol-d4) δ 1.05-
2.15 (16 H, m), 2.85-3.08 (2 H, m), 3.52-3.71 (1 H, m), 4.35-
4.57 (2 H, m), 7.48-7.54 (2 H, d, J ) 8.3 Hz), 7.72-7.80 (2 H,
d, J ) 8.5 Hz); 13C NMR (62.9 MHz, methanol-d4) δ 26.1, 26.2,
26.3, 26.6, 32.2, 33.8, 43.6, 45.9, 50.3, 50.5, 51.7, 65.4, 128.3,
129.0, 129.1, 147.2, 168.2, 176.0, 178.0. Anal. (C24H28N4O4‚
2.1MeOH‚1.8H2O) C, H, N.
tr a n s-(1R,2R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-
ben zyl-1,2-cyclop en ta n ed ica r boxa m id e [(-)-4]. To a solu-
tion of compound (-)-3 (84 mg, 0.19 mmol) in an ice-cold
mixture of dioxane-water (1:1) (5 mL) was added NaOH (1.0
M, 0.6 mL) dropwise during 30 min. The mixture was stirred
for an additional hour, neutralized with dilute aqueous
hydrochloric acid, evaporated and evaporated twice with
toluene. The crude carboxylic acid was dissolved in DMF (1
mL) containing benzylamine (0.10 mL, 0.86 mmol) and HOBt
(40 mg, 0.29 mmol). The mixture was cooled to 0 °C and EDC
(57 mg, 0.29 mmol) was added. The pH of the mixture was
adjusted to ∼8 with triethylamine. After 1 h, the ice bath was
removed and the solution was stirred at room temperature
overnight. After evaporation of the solvent, the residue was
purified by column chromatography (ethyl acetate) and dis-
solved in ethanol (5 mL). Palladium on carbon (3 mg) was
added and the mixture was stirred under hydrogen atmo-
sphere for 1 h. After filtration, acetic acid (0.1 mL) was added
and the mixture was stirred for an additional hour. After
evaporation of the solvents, the residue was purified by
preparative HPLC (Kromasil C-18; water-methanol-triethyl-
amine, 24:75:1) to give (-)-4 (40 mg, 41%) as a white powder.
tr a n s-(1R,2R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-
cycloh exyl-N2-eth yl-1,2-cyclop en ta n ed ica r boxa m id e (8).
Compound 8 (28 mg, 66%) was prepared from (-)-3 (48 mg,
0.110 mmol) according to the method for the preparation of
(-)-4 using N-ethylcyclohexylamine instead of benzylamine
22
and Bop-Cl instead of EDC and HOBt. 8: [R]D -18.8 (c 0.6,
CHCl3); 1H NMR (250 MHz, CDCl3) δ 1.03-2.18 (19 H, m),
3.02-3.45 (2 H, m), 3.65-3.81 (1 H, m), 4.32-4.53 (2 H, m),
7.44-7.52 (2 H, d, J ) 8.6 Hz), 7.69-7.86 (2 H, d, J ) 8.6 Hz);
13C NMR (62.9 MHz, methanol-d4) δ 15.2, 26.3, 26.9, 27.1, 31.7,
32.2, 32.9, 37.9, 43.5, 48.6, 50.6, 51.2, 55.8, 58.5, 128.3, 128.9,
129.1, 147.1, 168.1, 176.7, 177.3. Anal. (C24H28N4O4‚2.4MeOH‚
2.5H2O) C, H, N.
(()-2-F or m ylcyclop en t-2-en eca r boxylic Acid (9).16 To
an ice-cold, vigorously stirred solution of dichloroacetyl chloride
(35.4 g, 0.24 mol) and freshly distilled cyclopentadiene (15.4
g, 0.24 mol) in hexane (250 mL) was added triethylamine (24.3
g, 0.24 mol) in hexane (200 mL) over a period of 2 h. When all
the triethylamine was added, the ice bath was removed and
the mixture was stirred vigorously overnight. The reaction
22
(-)-4: [R]D -22.1 (c 0.5, MeOH); 1H NMR (250 MHz,
methanol-d4) δ 1.72-1.90 (4 H, m), 2.00-2.14 (2 H, m), 2.98-
3.07 (2 H, m), 4.30-4.49 (4 H, m), 7.11-7.38 (5 H, m), 7.42-
7.51 (2 H, d, J ) 8.5 Hz), 7.67-7.72 (2 H, d, J ) 8.5 Hz); 13C
NMR (62.9 MHz, methanol-d4) δ 26.3, 32.0, 32.2, 43.6, 44.1,

1710 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
No¨teberg et al.
mixture was filtered, the filter cake was washed with hexane,
the solvent was removed, and the obtained orange liquid (7,7-
dichlorobicyclo[3.2.0]hept-2-en-6-one) was, without further
purification, suspended in an aqueous solution of NaHCO3 (1
M, 1 L). The mixture was heated to 75 °C with stirring for 5
h. After cooling, the mixture was filtered, washed three times
with diethyl ether (50 mL), acidified with concentrated hy-
drochloric acid to pH ∼ 2, saturated with ammonium chloride
and extracted several times with ethyl acetate-tetrahydro-
furan (1:1). The combined organic phases were dried, filtered
and evaporated. Column chromatography (toluene-ethyl ace-
tate-acetic acid, 50:50:1) and crystallization from cyclohexane
gave 9 (7.5 g, 16% overall) as white needles. 9: mp 90-91 °C;
1H NMR (250 MHz, CDCl3) δ 2.22-2.48 (2 H, m), 2.55-2.90
(2 H, m), 3.71-3.86 (1 H, m), 7.05-7.11 (1 H, dd, J ) 2.5, 4.0
Hz), 9.78 (1 H, s); 13C NMR (62.9 MHz, CDCl3) δ 28.1, 33.1,
47.1, 144.6, 155.8, 177.9, 189.1. Anal. (C7H8O3) C, H.
(()-2-F or m ylcyclop en t -2-en eca r b oxylic Acid Met h yl
Ester (10). To a solution of 9 (34 mg, 0.24 mmol) in tri-
methoxymethane (1.5 mL) was added p-toluenesulfonic acid
monohydrate (20 mg, 0.11 mmol) and the solution was stirred
overnight. Water (1 mL) was added and the mixture was
stirred for an additional 30 min. The mixture was extracted
twice with dichloromethane and the combined organic phases
were dried, filtered and evaporated to give 10 (36 mg, 97%)
as a colorless oil. 10: 1H NMR (250 MHz, CDCl3) δ 2.09-2.22
(1 H, m), 2.28-2.45 (1 H, m), 2.53-2.90 (2 H, m), 3.70 (3 H,
s), 3.71-3.82 (1 H, m), 7.00-7.10 (1 H, m), 9.75 (1 H, s); 13C
NMR (62.9 MHz, CDCl3) δ 28.6, 33.1, 46.9, 52.2, 145.6, 154.4,
174.4, 188.4. Anal. (C8H10O3‚0.35CHCl3) C, H.
(()-Cyclop en t -2-en e-1,2-d ica r b oxylic Acid 1-Met h yl
Ester [(()-11]. To a solution of 10 (5.6 g, 0.036 mol) in tert-
butyl alcohol (100 mL) and water (50 mL) were added
resorcinol (6.0 g, 0.063 mol), NaH2PO4‚H2O (7.6 g, 0.055 mol)
and Na2ClO2 (80%, 6.3 g, 0.044 mol). The mixture was stirred
for 5 h, quenched with saturated Na2SO3 (10 mL), washed
three times with ethyl acetate, acidified with concentrated
hydrochloric acid to pH ∼ 2 and extracted three times with
dichloromethane. The combined organic phases were dried,
filtered and evaporated and the residue was recrystallized from
ether-hexane to give (()-11 (5.0 g, 81%) as white crystals.
(()-11: mp 75-76 °C; 1H NMR (250 MHz, CDCl3) δ 2.02-
2.80 (4 H, m), 3.70 (3 H, s), 3.71-3.92 (1 H, m), 7.01-7.11 (1
H, m), 10.50 (1 H, broad s); 13C NMR (62.9 MHz, CDCl3) δ
28.8, 32.8, 49.0, 52.2, 134.6, 149.6, 169.4, 174.9. Anal. (C8H10O4)
C, H.
overnight. Hydrochloric acid (1 M) was added until pH ∼ 2
and the mixture was extracted twice with dichloromethane.
The combined organic phases were dried, filtered, evaporated
and purified by column chromatography (toluene-ethyl ace-
tate, 3:1) to give 12 (0.130 mg, 75%) as white crystals (from
diethyl ether-hexane). 12: mp 93-94 °C; 1H NMR (250 MHz,
CDCl3) δ 2.15-2.32 (2 H, m), 2.41-2.74 Hz (2 H, m), 3.65 (3
H, s), 3.78-3.87 (1 H, m), 4.41-4.52 (1 H, dd, J ) 5.5, 14.8
Hz), 4.49-4.61 (1 H, dd, J ) 5.9, 15.0 Hz), 6.54 (1 H, broad s),
6.62-6.70 (1 H, m), 7.10-7.39 (5 H, m); 13C NMR (62.9 MHz,
CDCl3) δ 28.5, 32.3, 43.5, 49.7, 52.2, 127.5, 127.8, 128.2, 128.7,
129.0, 141.0, 164.4, 174.9. Anal. (C15H17NO3) C, H, N.
(()-N1-{4-[Am in o(im in o)m et h yl]b en zyl}-N2-b en zyl-2-
cyclop en ten e-1,2-d ica r boxa m id e (13). To a solution of
compound 12 (41 mg, 0.16 mmol) in dioxane-water (1:1, 2 mL)
was added NaOH (1.0 M, 0.5 mL) dropwise during 30 min at
0 °C. The mixture was stirred for an additional hour, acidified
to pH ∼ 2 with concentrated hydrochloric acid, and extracted
several times with dichloromethane. The combined organic
phases were dried, filtered and concentrated. The crude
carboxylic acid was redissolved in DMF (1 mL) containing Pab-
(Z)‚HCl (0.077 g, 0.24 mmol) and HOBt (0.033 g, 0.24 mmol).
The mixture was cooled to 0 °C and EDC (46 mg, 0.24 mmol)
was added. The pH of the reaction mixture was adjusted to
∼8 with triethylamine. After 1 h, the ice bath was removed
and the solution was stirred at room temperature overnight.
Hydrochloric acid (1 M) was added until pH ∼ 2 and the
mixture was extracted twice with dichloromethane. The
combined organic phases were dried, filtered, concentrated,
purified by column chromatography (ethyl acetate) and dis-
solved in dichloromethane (30 mL). A mixture of trifluo-
romethanesulfonic acid, anisole and dichloromethane (10:3:
100) was added under stirring until the solution was red (∼1
mL). After 15 min the solution was applied to a silica column
and eluted with methanol/ethyl acetate/acetic acid (40:10:1).
The crude product was purified by preparative HPLC (Kro-
masil C-18; water-methanol-triethylamine, 24:75:1) to give
13 (36 mg, 60%) as a white powder. 13: 1H NMR (250 MHz,
D2O) δ 2.02-2.17 (1 H, m), 2.41-2.59 (1 H, m), 2.65-2.57 (2
H, m), 3.91-4.03 (1 H, m), 4.42-4.63 (4 H, m), 6.83-6.92 (1
H, m), 7.27-7.43 (5 H, m), 7.46-7.55 (2 H, d, J ) 8.3 Hz),
7.62-7.71 (2 H, d, J ) 8.3 Hz); 13C NMR (62.9 MHz, D2O) δ
31.1, 35.1, 45.3, 54.0, 129.0, 129.4, 130.1, 130.5, 131.3, 139.4,
140.7, 145.8, 147.3, 167.0, 169.9, 184.4. Anal. (C22H24N4O2‚
0.8MeOH‚1.3H2O) C, H, N
(1R)-2-P ip er id in ylca r bon yl-2-cyclop en ten e-1-ca r boxy-
lic Acid Meth yl Ester (14). To an ice-cold solution of (+)-11
(36 mg, 0.21 mmol) in DMF (1 mL) was added Bop-Cl (0.108
g, 0.42 mmol). The pH of the reaction mixture was adjusted
to ∼8 with triethylamine. After stirring the mixture at 0 °C
for 30 min, piperidine (36 mg, 0.42 mmol) was added and the
solution was stirred at room temperature overnight. Hydro-
chloric acid (1 M) was added until pH ∼ 2 and the mixture
was extracted twice with dichloromethane. The combined
organic phases were dried, filtered, evaporated and purified
by column chromatography (toluene-ethyl acetate, 3:1) to give
Op t ica l R esolu t ion of (()-11. Compound (()-11 (5.0 g,
0.029 mol) and (-)-ephedrine (2.5 g, 0.015 mol) were dissolved
in ethyl acetate and the solution was allowed to stand at 0 °C
overnight. The white precipitate was collected by filtration and
recrystallized from ethyl acetate until the optical rotation of
22
the precipitate became constant ([R]D +19.8 (c 1.0, CHCl3)).
The precipitate was then dissolved in 40 mL of dichlo-
romethane and 1 M NaHSO4 was added until the pH of the
water layer became ∼3. The organic layer was dried, filtered
and concentrated to give (+)-11 (1.07 g, 21%) as white crystals.
22
22
14 (44 mg, 88%) as a colorless syrup. 14: [R]D +22.9 (c 2.0,
(+)-11: [R]D +56.0 (c 1.0, CHCl3). The combined mother
CHCl3); 1H NMR (250 MHz, CDCl3) δ 1.50-1.71 (6 H, m),
2.01-2.19 (1 H, m), 2.22-2.70 (3 H, m), 3.50-3.62 (4 H, m),
3.67 (3 H, s), 3.95-4.09 (1 H, m), 5.90-5.95 (1 H, m); 13C NMR
(62.9 MHz, CDCl3) δ 24.7, 26.4, 27.2, 32.5, 48.1, 51.7, 51.9,
133.0, 136.4, 166.6, 174.7. Anal. (C13H19NO3‚1.05CHCl3) C, H,
N.
liquors were evaporated, (-)-ephedrine (1.0 g) was added and
the above procedure was repeated. The optical rotation became
22
constant at [R]D -63.4 (c 1.0, CHCl3) and the corresponding
acid was shown to be (-)-11 (0.86 g, 17%). (-)-11: [R]D22 -47.2
(c 1.0, CHCl3). The above procedures were continued until all
of the starting material was resolved. Chiral HPLC (Chiralcel
OD; isoheptane-2-propanol-acetic acid, 80:20:1; 0.5 mL/min)
gave the following retention times: (+)-isomer ) 9 min, (-)-
isomer ) 17 min. The following purities were recorded: (+)-
isomer ) 90% ee, (-)-isomer ) 96% ee.
(1R)-N-{4-[Am in o(im in o)m eth yl]ben zyl}-2-(1-p ip er id i-
n ylca r bon yl)-2-cyclop en ten e-1-ca r boxa m id e (15). To an
ice-cold solution mixture of compound 14 (40 mg, 0.17 mmol)
in dioxane-water (1:1) (2 mL) was added NaOH (1.0 M, 0.5
mL) dropwise during 30 min. The mixture was stirred for an
additional hour, acidified to pH ∼ 2 with dilute hydrochloric
acid, and extracted several times with dichloromethane. The
combined organic phases were dried, filtered and concentrated.
The crude carboxylic acid was redissolved in ice-cold DMF (1
mL) containing Bop-Cl (86 mg, 0.34 mmol). The pH of the
mixture was adjusted to ∼8 with triethylamine. After stirring
(()-2-Ben zyla m in oca r bon yl-2-cyclop en ten e-1-ca r box-
ylic Acid Meth yl Ester (12). To an ice-cold solution of (()-
11 (0.113 g, 0.66 mmol) in DMF (1 mL) containing benzylamine
(0.107 g, 1.0 mmol) and HOBt (0.135 g, 1.0 mmol) was added
EDC (0.192 g, 1.0 mmol). The pH of the reaction mixture was
adjusted to ∼8 with triethylamine. After 1 h, the ice bath was
removed and the solution was stirred at room temperature

New Proline Mimetics
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1711
22
1
the mixture at 0 °C for 30 min, Pab(Z)‚HCl (0.108 g, 0.34 mmol)
was added and the solution was stirred at room temperature
overnight. Hydrochloric acid (1 M) was added until pH ∼ 2
and the mixture was extracted twice with dichloromethane.
The combined organic phases were dried, filtered, concen-
trated, purified by column chromatography (ethyl acetate) and
dissolved in dichloromethane (40 mL). A mixture of trifluo-
romethanesulfonic acid, anisole and dichloromethane (10:3:
100) was added under stirring until the solution was red (∼1
mL). After 15 min the solution was applied to a silica column
and eluted with methanol/ethyl acetate/acetic acid (40:10:1).
The crude product was purified by preparative HPLC (Kro-
masil C-18; water-methanol-triethylamine, 24:75:1) to give
piperidine. 20: colorless syrup; [R]D +4.9 (c 1.5, CHCl3); H
NMR (250 MHz, CDCl3) δ 2.15-2.39 (2 H, m), 2.41-2.72 (2
H, m), 3.70 (3 H, s), 3.81-3.93 (1 H, m), 6.74-6.82 (1 H, m),
7.0-7.12 (1 H, t, J ) 7.4 Hz), 7.22-7.32 (2 H, t, J ) 7.8 Hz),
7.52-7.61 (2 H, d, J ) 8.1 Hz), 8.43 (1 H, broad s); 13C NMR
(62.9 MHz, CDCl3) δ 28.4, 32.6, 49.8, 52.4, 120.0, 124.1, 128.9,
129.0, 138.0, 138.5, 141.8, 162.7, 175.2. Anal. (C14H15NO3) C,
H, N.
(1R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-p h en yl-2-
cyclop en ten e-1,2-d ica r boxa m id e (21). Compound 21 (13
mg, 19%) was prepared from 20 (27 mg, 0.102 mmol) according
22
to the method for the preparation of 15. 21: [R]D +13.5 (c
0.7, MeOH); 1H NMR (250 MHz, methanol-d4) δ 2.00-2.16 (1
H, m), 2.25-2.43 (1 H, m), 2.58-2.75 (2 H, m), 3.85-3.98 (1
H, m), 4.35-4.68 (2 H, m), 6.82-6.89 (1 H, m), 7.05-7.13 (1
H, t, J ) 7.5 Hz), 7.25-7.35 (2 H, t, J ) 7.7 Hz), 7.51-7.62 (4
H, m), 7.63-7.72 (2 H, d, J ) 8.3 Hz); 13C NMR (62.9 MHz,
methanol-d4) δ 29.5, 34.0, 43.6, 52.8, 121.9, 125.3 129.0, 129.1,
129.8, 139.2, 140.5, 141.7, 165.8, 168.3, 177.3. Anal. (C21H22N4O2)
C, H, N.
22
15 (33 mg, 47%) as a white powder. 15: [R]D +87.8 (c 0.7,
1
MeOH); H NMR (250 MHz, methanol-d4) δ 1.42-1.75 (6 H,
m), 1.92-2.11 (1 H, m), 2.21-2.72 (3 H, m), 2.46-3.71 (4 H,
m), 3.82-3.97 (1 H, m), 4.32-4.60 (2 H, m), 6.05-6.11 (1 H,
m), 7.41-7.57 (2 H, d, J ) 8.3 Hz), 7.70-7.82 (2 H, d, J ) 8.3
Hz); 13C NMR (62.9 MHz, methanol-d4) δ 25.5, 27.0, 27.7, 29.2,
33.7, 43.6, 54.7, 128.5, 129.0, 136.8, 138.1, 146.7, 149.9, 167.8,
168.7, 175.3. Anal. (C20H26N4O2‚0.8MeOH‚1.8H2O) C, H, N.
(1R)-2-Cycloh exyl(eth yl)am in ocar bon yl-2-cyclopen ten e-
1-ca r boxylic Acid Meth yl Ester (22). Compound 22 (88 mg,
100%) was prepared from (+)-11 (51 mg, 0.30 mmol) according
to the method for the preparation of 14 using N-ethylcyclo-
(1R)-2-Cycloh exylam in ocar bon yl-2-cyclopen ten e-1-car -
boxylic Acid Meth yl Ester (16). Compound 16 (39 mg, 85%)
was prepared from (+)-11 (31 mg, 0.182 mmol) according to
the method for the preparation of 14 using cyclohexylamine
instead of piperidine. 16: white crystals from ether-hexane;
22
hexylamine instead of piperidine. 22: colorless syrup; [R]D
+74.1 (c 1.1, CHCl3); 1H NMR (250 MHz, CDCl3) δ 0.92-1.82
(13 H, m), 1.95-2.32 (2 H, m), 2.33-2.65 (2 H, m), 3.15-3.41
(2 H, m), 3.58 (3 H, s), 3.75-4.13 (2 H, m), 5.77-5.95 (1 H,
m); 13C NMR (62.9 MHz, CDCl3) δ 14.7, 21.4, 25.4, 25.8, 27.0,
31.8, 32,6, 36.3, 51.8, 52.2, 58.2, 132.5, 137.4, 167.6, 174.5.
Anal. (C16H25NO3‚0.25EtOAc) C, H, N.
[R]D +12.8 (c 1.2, CHCl3); mp 108-109 °C; 1H NMR (250
22
MHz, CDCl3) δ 1.05-1.48 (4 H, m), 1.50-1.79 (4 H, m), 1.84-
2.00 (2 H, m), 2.17-2.30 (2 H, m), 2.39-2.72 (2 H, m), 3.70 (3
H, s), 3.70-3.90 (2 H, m), 5.97-6.13 (1 H, m), 6.60-6.68 (1 H,
m); 13C NMR (62.9 MHz, CDCl3) δ 24.7, 25.8, 28.6, 32.2, 33.0,
48.0, 49.8, 52.2, 138.4, 140.4, 163.5, 175.0. Anal. (C14H21NO3)
C, H, N.
(1R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-cycloh exyl-
N2-et h yl-2-cyclop en t en e-1,2-d ica r b oxa m id e (23). Com-
pound 23 (26 mg, 23%) was prepared from 22 (70 mg, 0.251
mmol) according to the method for the preparation of 15. 23:
(1R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-cycloh exyl-
2-cyclop en ten e-1,2-d ica r boxa m id e (17). Compound 17 (21
mg, 39%) was prepared from 16 (32 mg, 0.127 mmol) according
[R]D +3.4 (c 0.5, MeOH); 1H NMR (250 MHz, methanol-d4) δ
22
22
to the method for the preparation of 15. 17: [R]D +33.4 (c
1.02-1.85 (13 H, m), 1.96-2.13 (1 H, m), 2.22-2.40 (1 H, m),
2.41-2.75 (2 H, m), 3.50-3.72 (1 H, m), 3.83-3.97 (2 H, m),
3.95-4.12 (1 H, m), 4.32-4.58 (2 H, m), 5.95-6.15 (1 H, m),
7.44-7.57 (2 H, d, J ) 7.7 Hz), 7.65-7.83 (2 H, d, J ) 7.8 Hz);
13C NMR (62.9 MHz, methanol-d4) δ 15.1, 26.8, 29.1, 32.7, 33.8,
37.6, 43.6, 55.1, 59.9, 128.1, 129.1, 135.1, 139.0, 147.2, 168.1,
170.1, 176.4. Anal. (C22H30N4O2‚0.4MeOH‚1.8H2O) C, H, N.
0.8, MeOH); 1H NMR (250 MHz, methanol-d4) δ 1.11-1.48 (6
H, m), 1.55-1.88 (6 H, m), 1.99-2.12 (1 H, m), 2.19-2.37 (1
H, m), 2.42-2.82 (2 H, m), 3.54-3.75 (1 H, m), 3.73-3.83 (1
H, m), 4.33-4.62 (2 H, m), 6.60-6.68 (1 H, m), 7.49-7.58 (2
H, d, J ) 8.3 Hz), 7.70-7.80 (2 H, d, J ) 8.3 Hz); 13C NMR
(62.9 MHz, methanol-d4) δ 26.7, 29.5, 33.7, 33.7, 33.8, 43.6,
49.5, 52.7, 128.4, 129.0, 129.1, 140.1, 140.5, 147.1, 166.6, 168.1,
177.2. Anal. (C20H26N4O2‚0.6MeOH‚1.1H2O) C, H, N.
(1R)-2-Eth yl(p h en yl)a m in oca r bon yl-2-cyclop en ten e-1-
ca r boxylic Acid Meth yl Ester (24). Compound 24 (60 mg,
73%) was prepared from (+)-11 (51 mg, 0.30 mmol) according
to the method for the preparation of 14 using N-ethylaniline
instead of piperidine. 24: colorless syrup; [R]D22 +213.6 (c 0.7,
CHCl3); 1H NMR (250 MHz, CDCl3) δ 1.05-1.20 (3 H, m),
1.78-1.96 (1 H, m), 2.01-2.40 (3 H, m), 3.56-3.76 (2 H, m),
3.70 (3 H, s), 3.91-4.10 (1 H, m), 5.48-5.57 (1 H, m), 7.12-
7.50 (5 H, m); 13C NMR (62.9 MHz, CDCl3) δ 12.8, 27.2, 32.7,
45.0, 51.3, 51.7, 127.3, 128.1, 129.2, 137.4, 140.1, 143.1, 165.8,
175.0. Anal. (C16H19NO3‚0.5EtOAc) C, H, N.
(1R)-2-Cycloh exylm et h yla m in oca r b on yl-2-cyclop en -
ten e-1-ca r boxylic Acid Meth yl Ester (18). Compound 18
(30 mg, 51%) was prepared from (+)-11 (38 mg, 0.224 mmol)
according to the method for the preparation of 14 using
cyclohexylmethylamine instead of piperidine. 18: colorless
syrup; [R]D +43.2 (c 1.1, CHCl3); 1H NMR (250 MHz, CDCl3)
22
δ 0.82-1.03 (2 H, m), 1.10-1.32 (4 H, m), 1.40-1.58 (1 H, m),
1.59-1.80 (4 H, m), 2.19-2.30 (2 H, m), 2.40-2.72 (2 H, m),
3.01-3.27 (2 H, m), 3.70 (3 H, s), 3.71-3.82 (1 H, m), 6.20-
6.40 (1 H, m), 6.63-6.71 (1 H, m); 13C NMR (62.9 MHz, CDCl3)
δ 25.9, 26.4, 28.6, 30.8, 32.2, 38.0, 45.7, 49.8, 52.3, 138.2, 140.6,
164.4, 175.0. Anal. (C15H23NO3) C, H, N.
(1R)-N1-{4-[Am in o(im in o)m et h yl]b en zyl}-N2-p h en yl-
N2-et h yl-2-cyclop en t en e-1,2-d ica r b oxa m id e (25). Com-
pound 25 (17 mg, 19%) was prepared from 24 (54 mg, 0.198
mmol) according to the method for the preparation of 15. 25:
(1R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-cycloh ex-
ylm eth yl-2-cyclop en ten e-1,2-d ica r boxa m id e (19). Com-
pound 19 (23 mg, 51%) was prepared from 18 (27 mg, 0.102
mmol) according to the method for the preparation of 15. 19:
[R]D +65.6 (c 0.4, CHCl3); 1H NMR (250 MHz, D2O) δ 0.95-
22
1.13 (3 H, t, J ) 7.2 Hz), 1.62-1.82 (1 H, m), 2.04-2.43 (3 H,
m), 3.50-3.73 (2 H, m), 3.81-4.00 (1 H, m), 4.32-4.53 (2 H,
m), 6.10-6.25 (1 H, m), 7.05-7.25 (2 H, m), 7.33-7.49 (3 H,
m), 7.49-7.59 (2 H, d, J ) 8.2 Hz), 7.72-7.85 (2 H, d, J ) 8.3
Hz); 13C NMR (62.9 MHz, D2O) δ 14.6, 31.2, 34.9, 45.3, 48.3,
55.3, 129.5, 130.4, 130.7, 131.4, 132.2, 132.5, 139.4, 143.7,
147.3, 169.1, 172.3, 184.1. Anal. (C23H26N4O2‚2.2MeOH‚
0.5H2O) C, H, N.
22
[R]D +26.1 (c 0.9, MeOH); 1H NMR (250 MHz, methanol-d4)
δ 0.81-1.02 (2 H, m), 1.12-1.35 (4 H, m), 1.40-1.59 (1 H, m),
1.60-1.80 (4 H, m), 1.94-2.13 (1 H, m), 2.20-2.39 (1 H, m),
2.42-2.74 (2 H, m), 2.96-3.13 (2 H, m), 3.75-3.90 (1 H, m),
4.35-4.60 (2 H, m), 6.61-6.68 (1 H, m), 7.50-7.59 (2 H, d, J
) 8.3 Hz), 7.70-7.80 (2 H, d, J ) 8.3 Hz); 13C NMR (62.9 MHz,
methanol-d4) δ 27.0, 27.6, 29.6, 32.0, 33.6, 39.3, 43.6, 46.7, 52.6,
128.0, 128.7, 129.0, 140.1, 140.4, 147.3, 167.5, 168.2, 176.2.
Anal. (C21H28N4O2‚1.2MeOH‚1.1H2O) C, H, N.
N -C y c lo h e x y l-N -t er t -b u t y lo x y c a r b o n y lh y d r o x y l-
a m in e (27). Na2CO3 (1.4 g, 13.2 mmol) was added slowly to a
solution of N-cyclohexylhydroxylamine hydrochloride (26; 1.0
g, 6.6 mmol) and di-tert-butyl dicarbonate (1.6 g, 7.3 mmol) in
water (20 mL) and tetrahydrofuran (10 mL). After 30 min at
room temperature ethyl acetate (20 mL) was added and the
organic phase was dried, filtered, concentrated and purified
(1R)-2-P h en ylam in ocar bon yl-2-cyclopen ten e-1-car box-
ylic Acid Meth yl Ester (20). Compound 20 (57 mg, 44%) was
prepared from (+)-11 (90 mg, 0.53 mmol) according to the
method for the preparation of 14 using aniline instead of

1712 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
No¨teberg et al.
(4) (a) Sanderson, P. E. J .; Naylor-Olsen, A. M. Thrombin Inhibitor
Design. Curr. Med. Chem. 1998, 289. (b) Brundish, D. E.
Thrombin Inhibitors. Curr. Opin. Ther. Patents 1992, 1457. (c)
Exp. Opin. Ther. Patents 1997, 7, 651. (d) Stone, S. R. Thrombin
inhibitors. A New Generation of Antithrombotics. Trends Car-
diovasc. Med. 1995, 5, 134. (e) Ripka, W. C.; Vlasuk, G. P.
Antithrombotics/Serine Proteases. In Annual Reports in Medici-
nal Chemistry; Bristol, J . A., Ed.; Academic: San Diego, 1997;
Vol. 32, pp 71-89. (f) Wiley, M. R.; Fisher, M. J . Small-molecule
Direct Thrombin Inhibitors. Exp. Opin. Ther. Patents 1997, 7,
1265.
by column chromatography. Elution with toluene-ethyl ace-
tate (39:1) afforded N-cyclohexyl-O-tert-butyloxycarbonylhy-
droxylamine (28; 0.55 g, 39%) as a colorless syrup. Further
elution with toluene-ethyl acetate (9:1) afforded 27 (0.59 g,
1
41%) as white crystals (from hexane). 27: mp. 88-90 °C; H
NMR (250 MHz, CDCl3) δ 1.02-1.40 (3 H, m), 1.47 (9 H, s),
1.52-1.89 (7 H, m), 3.62-3.81 (1 H, m), 7.82 (1 H, s); 13C NMR
(62.9 MHz, CDCl3) δ 25.4, 25.7, 28.4, 29.3, 58.3, 81.2, 157.0.
Anal. (C11H21NO3) C, H, N.
N-Eth oxy-N-ter t-bu tyloxycar bon ylcycloh exylam in e (29).
To an ice-cold solution of 27 (0.57 g, 2.65 mmol) and potassium
hydroxide (0.32 g, 5.7 mmol) was added ethyl iodide (0.42 mL,
5.25 mmol). The mixture was stirred at 0 °C for 1 h and then
refluxed overnight. Pentane (30 mL) was added and the
precipitated potassium iodide was removed by filtration. The
mixture was then concentrated and purified by column chro-
matography (pentane-diethyl ether, 9:1) to afford 29 (0.54 g,
84%) as a colorless liquid. 29: 1H NMR (250 MHz, CDCl3) δ
1.00-1.47 (4 H, m), 1.17-1.25 (3 H, t, J ) 7.1 Hz), 1.48 (9 H,
s), 1.52-1.87 (6 H, m), 3.68-3.82 (1 H, tt, J ) 3.5, 11.6 Hz),
3.82-3.93 (2 H, q, J ) 7.1 Hz); 13C NMR (62.9 MHz, CDCl3) δ
(5) Bajusz, S. The Story of D-Phe-Pro-Arg-H, the Likely Anticoagu-
lant of the Future. Bioke´mia 1990, 14, 127.
(6) (a) Gustafsson, D.; Elg, M.; Lenfors, S.; Borjesson, I.; TegerNils-
son, A. C. Effects of Inogatran, a new Low-Molecular Weight
Thombin Inhibitor, in Rat Models of Venous and Arterial
Thrombosis, Thrombolysis and Bleeding Time. Blood Coag.
Fibrinolysis 1996, 7, 69. (b) TegerNilsson, A. C.; Bylund, R.;
Gustafsson, D.; Gyzander, E.; Eriksson, U. In vitro Effects of
Inogatran, a Selective Low Molecular Weight Thrombin Inhibi-
tor. Thromb. Res. 1997, 85, 133.
(7) (a) Eriksson, H.; Eriksson, U.; Frison, L.; Thorsen, M. Intrave-
nous treatment of Acute Deep Venous Thrombosis (DVT) with
Melagatran,
a Synthetic Low Molecular Weight Thrombin
Inhibitor. Thromb. Haemostasis 1997, (Suppl.) OC-2411. (b)
Eriksson, B. I.; Carlsson, S.; Halvarsson, M.; Risberg, B.;
Mattsson, C. Antithrombotic Effect of Two Low Molecular
Weight Thrombin Inhibitors and a Low-Molecular Weight He-
parin in a Caval Vein Thrombosis Model in the Rat. Thromb.
Haemostasis 1997, 78, 1404. (c) Gustafsson, D.; Antonsson, T.;
Bylund, R.; Eriksson, U.; Gyzander, E.; Nilsson, I.; Elg, M.;
Mattsson, C.; Deinyu´m, J .; Pehrsson, S.; Karlsson, A.; So¨rensen,
H. Effects of Melagatran, a New Low-molecular-weight Throm-
bin Inhibitor, on Thrombin and Fibrinolytic Enzymes. Thromb.
Haemostasis 1998, 79, 110.
13.4, 25.4, 25.7, 28.3, 29.5, 59.0, 72.0, 80.8, 156.9. Anal. (C13H25
NO3) C, H, N.
-
(1R)-2-Cycloh exyl(eth oxy)a m in oca r bon yl-2-cyclop en -
ten e-1-ca r boxylic Acid Meth yl Ester (30). Compound 30
(36 mg, 76%) was prepared from (+)-11 (30 mg, 0.18 mmol)
according to the method for the preparation of 14 using
N-ethoxycyclohexylamine instead of piperidine. N-Ethoxycy-
clohexylamine was prepared from 29: To a solution of 29 in
dichloromethane was added trifluoroacetic acid and the mix-
ture was stirred for 5 min. After evaporation the crude organic
salt was dissolved in dichloromethane and washed with
NaHCO3 (1 M), dried, filtered and evaporated. The resulting
N-ethoxycyclohexylamine was used directly without further
(8) (a) Semple, J . E.; Rowley, D. C.; Brunck, T. K.; Ripka, W. C.
Synthesis and Biological Activity of P2-P4 Azapeptidometic P1-
arginal and P1-ketoarginineamide Derivatives: a Novel Class
of Serine Protease Inhibitors. Bioorg. Med. Chem. Lett. 1997, 7,
315. (b) Harmat, N. J . S.; Di Bugno, C.; Criscuoli, M.; Giorgi,
R.; Lippi, A.; Martinelli, A.; Monti, S.; Subissi, A. 1,2-Disubsti-
tuted Cyclohexane Derived Tripeptide Aldehydes as Novel
Selective Thrombin Inhibitors. Bioorg. Med. Chem. Lett. 1998,
8, 1249.
(9) Turbanti, L.; Cerbai, G.; Di Bugno, C.; Giorgi, R.; Garzelli, G.;
Criscuoli, M.; Renzetti, A. R.; Subissi, A.; Bramati, G.; DePriest,
S. A. 1,2-Cyclomethylenecarboxylic Monoamide Hyddroxamic
Derivatives. A Novel Class of Non-Amino Acid Angiotensin
Converting Enzyme Inhibitors. J . Med. Chem. 1993, 36, 699.
(10) Hart, S. A.; Sabat, M.; Etzkorn, F. A. Enantio- and Regioselective
Synthesis of a (Z)-Alkene cis-Proline Mimic. J . Org. Chem. 1998,
63, 7580-7581.
22
purification. 30: colorless syrup; [R]D +25.9 (c 0.5, CHCl3);
1H NMR (250 MHz, CDCl3) δ 1.00-1.42 (4 H, m), 1.12-1.21,
(3 H, t, J ) 7.1 Hz), 1.47-1.93 (6 H, m), 1.95-2.12 (1 H, m),
2.18-2.33 (1 H, m), 2.37-2.72 (2 H, m), 3.65 (3 H, s), 3.75-
4.07 (3 H, m, CH), 4.05-4.27 (1 H, m), 6.50-6.57 (1 H, m);
13C NMR (62.9 MHz, CDCl3) δ 13.4, 25.4, 25.7, 27.5, 29.9, 30.4,
32,8, 51.4, 51.9, 58.1, 72.6, 136.8, 139.4, 166.6, 174.9. Anal.
(C16H25NO4) C, H, N.
(1R)-N1-{4-[Am in o(im in o)m eth yl]ben zyl}-N2-cycloh exyl-
N2-eth oxy-2-cyclop en ten e-1,2-d ica r boxa m id e (31). Com-
pound 31 (30 mg, 47%) was prepared from 30 (36 mg, 0.13
mmol) according to the method for the preparation of 15. 31:
(11) Goldsworthy, L. J .; Perkin, W. H. Resolution of trans-Cyclopen-
tane-1:2-Dicarboxylic Acid. J . Chem. Soc. 1914, 2639.
(12) Morimoto, Y.; Terao, Y.; Achiwa, K. Enzymes and Catalysts. I.
Pig Liver Esterase-Catalyzed Hydrolysis of Heterocyclic Di-
esters. Chem. Pharm. Bull. 1987, 35, 2266.
22
[R]D +9.6 (c 0.4, MeOH); 1H NMR (250 MHz, methanol-d4) δ
1.02-1.85 (13 H, m), 1.96-2.13 (1 H, m), 2.22-2.40 (1 H, m),
2.41-2.75 (2 H, m), 3.50-3.72 (1 H, m), 3.83-3.97 (2 H, m),
3.95-4.12 (1 H, m), 4.32-4.58 (2 H, m), 5.95-6.15 (1 H, m),
7.44-7.57 (2 H, d, J ) 7.7 Hz), 7.65-7.83 (2 H, d, J ) 7.8 Hz);
13C NMR (62.9 MHz, methanol-d4) δ 15.1, 26.8, 29.1, 32.7, 33.8,
37.6, 43.6, 55.1, 59.9, 128.1, 129.1, 135.1, 139.0, 147.2, 168.1,
170.1, 175.2. Anal. (C22H30N4O3‚1.3MeOH‚0.8H2O) C, H, N.
(13) (a) Studer, A.; Hintermann, T.; Seebach, D. Synthesis and First
Applications of a New Chiral Auxiliary (tert-Butyl 2-(tert-Butyl)-
5,5-dimethyl-4-oxoimidazolidine-1-carboxylate). Helv. Chim. Acta
1995, 78, 1185. (b) Roush, W. R.; Gillis, H. R.; Ko, A. I.
Stereochemical Aspects of the Intramolecular Diels-Alder Reac-
tions of Deca-2,7,9-trienoate Esters. 3. Thermal Lewis Acid
Catalyzed, and Asymmetric Cyclizations. J . Am. Chem. Soc.
1982, 104, 2269. (c) Horton, D.; Usui, T. Synthesis of Optically
Pure di- to tetra-Substituted Cyclopentanes from Sugar-Derived
Norbornene Derivatives. Carbohydr. Res. 1991, 51. (d) Langer,
T.; Illich, M.; Helmchen, G. Diastereoselective Oxidative Cou-
pling of Chiral Carboxylic Acid Derivatives. Tetrahedron Lett.
1995, 36, 4409. (d) Misumi, A.; Iwanaga, K.; Furuta, K. Yama-
moto, H. Simple Asymmetric Construction of Carbocyclic Frame-
work. Direct Coupling of Dimenthyl Succinate with 1,ω-
Dihalides. J . Am. Chem. Soc. 1985, 107, 3343. (e) Furuta, K.;
Iwanaga, K.; Yamamoto, H. Condensation of (-)-Dimenthyl
Succinate Dianion with 1,ω-Dihalides: (+)-(1S,2S)-Cyclopro-
pane-1,2-Dicarboxylic Acid. Org. Synth. 1988, 67, 76.
(14) (a) No¨teberg, D.; Brånalt, J .; Kvarnstro¨m, I.; Classon, B.;
Samuelsson, B.; Nillroth, U.; Danielson, U. H.; Karle´n, A.;
Hallberg, A. Synthesis of Enantiomerically Pure cis and trans
2-Aminocyclopentanecarboxylic Acids. Use of Proline Replace-
ments in Potential HIV-Protease Inhibitors. Tetrahedron 1997,
53, 7975. (b) Rosenquist, Å.; Kvarnstro¨m, I.; Svensson, S. C. T.;
Classon, B.; Samuelsson, B. Synthesis of Enantiomerically Pure
trans-3,4-Substituted Cyclopentanols by enzymatic resolution.
Acta Chem. Scand. 1992, 46, 1127.
Ack n ow led gm en t. We gratefully acknowledge As-
traZeneca Mo¨lndal for financial support and for provid-
ing biological screening and the X-ray crystal structure.
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(15) NMR and optical rotation of (-)-2 were in agreement with the
literature values.13b

New Proline Mimetics
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1713
-47.3 (c 1.3, CHCl3) [lit.19 (5S)-cyclopentene-1,5-dicarboxylic acid
(16) NMR and optical rotation of this monoester were in agreement
with the literature values.13c
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22
methyl ester: [R]D -56.5 (c 0.7, CHCl3)]. Comparing with
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an ee > 90%.
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(21) The resolved (-)-11 was methylated using trimethoxymethane
22
and pTsOH to give the corresponding dimethyl ester: [R]D
J M990557T