Synthesis of potential thrombin inhibitors. Incorporation of tartaric acid templates as P2 proline mimetics.

Article abstract of DOI:10.1016/S0968-0896(01)00426-6

With the objective to prepare novel non-peptidic thrombin inhibitors, bioisosteres of the inhibitory tripeptide D-Phe-Pro-Arg chain have been examined. Thus, the P1 Arg was replaced with p-amidinobenzylamine, an elongated homologue of the same and with 2,

Full text of DOI:10.1016/S0968-0896(01)00426-6

Bioorganic & Medicinal Chemistry 10 (2002) 1567–1580
Synthesis of Potential Thrombin Inhibitors. Incorporation of
Tartaric Acid Templates as P2 Proline Mimetics
Anders Dahlgren,a Jonas Branalt,b Ingemar Kvarnstrom,a Ingemar Nilsson,b
Djordje Musilc and Bertil Samuelssond,*,y
aDepartment of Chemistry, Linkoping University, S-581 83 Linkoping, Sweden
¨
¨
bAstraZeneca R&D, Medicinal Chemistry, S-431 83 Molndal, Sweden
¨
cAstraZeneca R&D, Structural Chemistry Laboratory, S-481 83 Molndal, Sweden
¨
dDepartment of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
Received 17 August 2001; accepted 29 November 2001
Abstract—With the objective to prepare novel non-peptidic thrombin inhibitors, bioisosteres of the inhibitory tripeptide d-Phe-Pro-
Arg chain have been examined. Thus, the P1 Arg was replaced with p-amidinobenzylamine, an elongated homologue of the same
and with 2,5-dichloro benzylamine. The P2-P3, d-Phe-Pro, was replaced with a novel tartaric acid template coupled to a series of
readily available, mainly lipophilic, amines. Some of these compounds exhibit promising thrombin inhibition activity in vitro, IC50
ꢀ5.9 mM. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
available thrombin inhibitors to regulate hemostasis
and thrombosis will provide new and efficacious drugs.2
It has been shown earlier that the tripeptide chain d-
Phe-Pro-Arg, which mimics parts of thrombin’s natural
substrate fibrinogen, exhibits thrombin-inhibiting prop-
erties.5 Considerable synthetic efforts have therefore been
directed towards mimicking this peptide. A number of
development candidates and clinical inhibitors like mela-
gatran6 (orally administered as the prodrug H 376/956)
have been developed based on this motif.2
There are four major classes of protease enzymes;
aspartic, serine, cysteine and metallo proteases, which
all catalyze hydrolysis of polypeptide bonds. This cata-
lytic activity regulates an immense number of biochem-
ical processes in both humans and microrganisms.1
Serine proteases (e.g., trypsin-like, a-chymotrypsin-like
or elastase-like) possess an active site catalytic triad
consisting of Ser 195, His 57 and Asp 102 residues.2 The
trypsin-like serine protease thrombin plays a central role
in hemostasis and thrombosis. Blood coagulation (or
fibrin formation) is achieved through a clotting cascade
involving enzymatic activation of 13 components —
factors I–XIII, where inactive precursors are sequen-
tially activated with amplification of the original signal
triggering the cascade. In the final event, thrombin con-
verts soluble fibrinogen to an insoluble meshwork of
fibrin in which blood cells are trapped, forming the
clot.3 Undesired blood clotting is the major culprit in a
number of cardiovascular diseases, that is, deep venous
thrombosis, pulmonary embolism, unstable angina,
restenosis following angioplasty and arterial thrombo-
sis.4 It is thus anticipated that small molecule orally
*Corresponding author. Tel.: +46-8-608-3104; fax: +46-8-608-3199;
e-mail: bertil.samuelsson@medivir.se
yAdditional address: Medivir AB, Lunastigen 7, S-141 44 Huddinge,
Sweden.
Figure 1. N-Acylproline (A) compared to some N-acylproline iso-
steres. B and C are taken from ref 8. D–F are isosteres used in this
paper.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00426-6

1568
A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
Scheme 2. Reagents: (a) and (b): see Scheme 1; (c) cyclohexylamine,
EDC, HOBt, Et3N, DMF; (d) NaOH, dioxane/water; (e) 4-(benzyl-
oxycarbonyl)amidinobenzylamine hydrochloride, EDC, HOBt, Et3N,
DMF; (f) H2/Pd-C, EtOH.
Scheme 1. Reagents: (a) diethoxymethane, BF3, i-PrOAc; (b) NaOH,
dioxane/water; (c) 4-(benzyloxycarbonyl)amidinobenzylamine hydro-
chloride, EDC, HOBt, Et3N, DMF.
The amino acid l-proline has frequently been explored
as a template and starting point for peptidomimetic
inhibitor design;7a,b,c compare inhibitors of human
immunodeficiency virus type 1 (HIV-1) protease7d and
angiotensin-converting enzyme (ACE).7e There are,
however, notably few reports of replacing proline with
five-membered carbocyclic ring isosteres in the tripep-
tide type motif d-Phe-Pro-Arg. Recently, we reported
on the synthesis of thrombin inhibitors containing two
novel N-acylproline isosteres, that is, trans-(1R,2R)-
cyclopentane-1,2-dicarboxylic acid and (2R)-cyclopent-
2-ene-1,2-dicarboxylic acid (Fig. 1).8
Scheme 3. Reagents: (a) AcCl, EtOH; (b) satd NH3 in MeOH; (c)
benzyl chloroformate, K2CO3, THF/water; (d) MsCl, pyridine; (e)
NaN3, DMF; (f) PPh3, Boc2O, MeOH; (g) TFA/CH2Cl2.
We now report on the synthesis of novel tartaric acid
based N-acylproline isosteres, that is, trans-(R,R)-1,3-
dioxolan-4,5-dicarboxamides9 (Fig. 1), and the incor-
poration of these P2 templates in a thrombin inhibitor
motif with p-amidinobenzylamine (Pab),10 an elongated
homologue of Pab and 2,5-dichloro benzylamine11 in
the P1 position and mainly lipophilic amines in the P3
position. Some of these compounds exhibit promising
thrombin inhibition activity in vitro, IC50 ꢀ5.9 mM.
lent yields. The overall yields for the three steps were
31–88% (Table 1). The primary amide 6t was synthe-
sized by stirring the monoester 4 in methanol saturated
with ammonia to convert the ester group to the primary
amide, followed by catalytic hydrogenation to remove
the protecting group, affording 6t in 96% yield over two
steps (Table 1).
To investigate the effect of changing stereochemistry at
the two stereogenic centers of the tartrate moiety, (À)-
diethyl-d-tartrate [(À)-1] was used as starting material.
The formation of the dioxolane ring provided (+)-2 in
72% yield. The diester (+)-2 was hydrolyzed to give the
monoacid (+)-3 in 75% yield. Since 6q (Tables 1 and 2)
proved to be the most potent compound in the series
described above we decided to use cyclohexylamine in
the P3-position of this particular target molecule. The
coupling of (+)-3 with cyclohexylamine gave the
monoamide 5 in 25% yield.18 Hydrolysis, coupling with
Pab(Z) and deprotection afforded the benzamidine
(ent)-6q in 51% yield over three steps (Scheme 2).
Results and Discussion
Compound (À)-2 was prepared from readily available
(+)-diethyl l-tartrate [(+)-1] (Scheme 1). Upon reacting
compound (+)-1 with diethoxy methane and boron tri-
fluoride in isopropyl acetate at reflux temperature,12 the
dioxolane derivative (À)-213 was formed in 82% yield.14
Treatment of the diester (À)-2 with aqueous sodium
hydroxide/dioxane gave the monoacid (À)-3 in 82%
yield. Coupling of (À)-3 with 4-(benzyloxycarbonyl)-
amidinobenzylamine [Pab(Z)]10 using N-ethyl-N0-(3-di-
methylaminopropyl)carbodiimide hydrochloride (EDC),
1-hydroxybenzotriazole (HOBt) and triethylamine in
dimethylformamide afforded the monoamide 4 (92%)
(Scheme 1), which was used for further couplings with
P3 amines.
To investigate the effect of a longer benzamidine side
chain in the P1 position,19 we synthesized compound 11
(Scheme 4). The required (4-amidinophenyl)-ethylamine
derivative 9 was synthesized starting from 2-(4-cyano-
phenyl)-ethanol (7) which was reacted with acetyl chlo-
ride in ethanol followed by stirring in methanol
saturated with ammonia. The resulting amidine moiety
was reacted with benzyl chloroformate to give the alco-
hol 8 in 74% yield over three steps. Compound 8 was
subsequently reacted with mesyl chloride in pyridine,
followed by sodium azide in dimethyl formamide. The
azide group was reduced using triphenylphosphine in
methanol in the presence of Boc anhydride20 to give 9 in
26% yield over three steps (Scheme 3).
For the synthesis of compounds 6a–6t, the monoamide
(monoester) 4 was hydrolyzed by treatment with sodium
hydroxide,17 followed by coupling of the monoacid with
the appropriate amine using either EDC/HOBt/Et3N/
DMF or diphenylphosphoryl azide (DPPA)/Et3N/
DMF or bis(2-oxo-3-oxazolidinyl)phosphinic chloride
(BOP-Cl)/Et3N/DMF, depending on the reactivity of
the amines. The final deprotection step, cleavage of the
benzyloxycarbonyl group by catalytic hydrogenation,
generally proceeded to give the final products in excel-

A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
1569
Removal of the Boc group with trifluoroacetic acid/
methylene chloride, followed by coupling with the
monoacid (À)-3 gave the monoamide 10 in 50% yield.
Hydrolysis, coupling with benzyl amine and deprotec-
tion afforded the benzamidine 11 in 86% yield over
three steps (Scheme 4).
Since the S2 pocket of thrombin seems to accommodate
more bulk than Pro in the P2-position, (cf. inogatran21
which contains a six-membered ring mimicking Pro in
d-Phe-Pro-Arg and showing substantial thrombin inhi-
biting properties), slightly bulkier P2-moieties were also
examined in compounds 17 and 21 (Scheme 5). Thus
(+)-diethyl-l-tartrate [(+)-1] was reacted with either
2-methoxypropene, or 1,1-diethoxyethane and cam-
phorsulfonic acid (CSA) in toluene22 to form the iso-
propylidene diethyl tartrate 1423 in 74% yield and the
ethylidine diethyl tartrate 1813b in 80% yield, respec-
tively. Hydrolysis afforded the monoacids 15 (47%) and
19 (69%, diastereomers 1:1), which were coupled with
Pab(Z) to give the monoamides 16 and 20 in 87 and
In order to investigate the effect of a different type of
side chain that has been reported to fit the S1-pocket of
thrombin, 2,5-dichlorobenzylamine11 was coupled to
the monoacid (À)-3, resulting in the monoamide 12 in
61% yield. Hydrolysis and coupling with cyclohexyl
amine afforded the diamide 31 in 76% yield over two
steps (Scheme 4).
Table 1. Structures, reaction conditions and yields for compounds 6a–6t
Compd
Amine R
Reagent b
A
Yield (%)
32
Compd
Amine R
Reagent b
A
Yield (%)
24
6a
6k
6b
6c
6d
A
B
31
52
53
6l
6m
6n
A
A
A
23
47
70
A
6e
6f
6g
6h
6i
Aa
Ab
A
60
50
42
42
32
55
6o
6p
6q
6r
6s
6t
C
C
A
C
C
D
49
64
88
71
61
96
Aa
Ac
A
6j
NH2
Reagent b: A: EDC, HOBt, Et3N, DMF; B: DPPA, Et3N, DMF; C: BOP–Cl, Et3N, DMF; D: Satd. NH3 in MeOH. The hydrolysis step a was not
included in the synthesis of 6t.
aBenzyloxycarbonyl-protected 6d and 6g were hydrolyzed by treatment with NaOH in dioxane/water followed by step c to yield 6e and 6h, respec-
tively.
bOH group protected by THP during coupling. The THP-group was removed quantitatively before step c by stirring in AcOH/THF/water 4:2:1.
cOH group protected by TBDMS during coupling. The TBDMS-group was removed quantitatively before step c by stirring in Bu4NF in THF.

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A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
Scheme 4. Reagents: (a) Boc-deprotected 9, EDC, HOBt, Et3N,
DMF; (b) NaOH, dioxane/water; (c) benzylamine, EDC, HOBt, Et3N,
DMF; (d) H2/Pd-C, EtOH; (e) 2,5-dichlorobenzylamine, EDC, HOBt,
Et3N, DMF; (f) NaOH, dioxane/water; (g) cyclohexylamine, EDC,
HOBt, Et3N, DMF.
Scheme 5. Reagents: (a) (for compound 14) 2-methoxypropene, CSA,
toluene; (a) (for 18) 1,1-diethoxyethane, CSA, toluene; (b) NaOH,
dioxane/water; (c) 4-(benzyloxycarbonyl)amidinobenzylamine hydro-
chloride, EDC, HOBt, Et3N, DMF; (d) NaOH, dioxane/water; (e)
benzylamine, EDC, HOBt, Et3N, DMF; (f) H2/Pd-C, EtOH.
61% (diastereomers 1:1) yield, respectively. Subsequent
hydrolysis of the monoesters, coupling with benzyl-
amine and catalytic hydrogenation provided 17 in 35%
yield over three steps and 21 as a diastereomeric mixture
(1:1) in 66% yield over three steps (Scheme 5).
Structure–activity relationship
The compounds in this series show low to modest affi-
nity for thrombin (Table 2), with compounds 6m, 6n, 6p
and 6q having IC50 values around or under 10 mM. In
order to provide further information on the lower affi-
nity of these compounds compared to PPACK24
(IC50=0.017 nM) compound 6m, which was the first of
the more active compounds in this series to be synthe-
sized, was cocrystallized in complex with a-thrombin
and subjected to X-ray analysis. Limited resources pre-
vented us from performing the adequate X-ray studies
on the slightly more active compounds 6p and 6q. A
direct comparison of affinity with that of PPACK may
not seem relevant since PPACK through its chloro-
methyl-keto group is covalently bond to His 57 and to
Ser 195, as shown in Figure 2, whereas the present
inhibitors do not have these interactions. However, it
has been shown that other non-covalent inhibitors such
as inogatran21 (IC50=22 nM) (Fig. 2), which also lacks
the chloromethyl-keto group, still has good affinity for
thrombin and interacts with thrombin in a way very
similar to PPACK.25
Some general remarks about the stability of the target
compounds
Since all of the final compounds contain either a five-
membered cyclic acetal or ketal, some instability could
be expected, especially under acidic conditions. How-
ever, the vast majority of the target compounds in this
report contains a methylene acetal, which requires
rather harsh conditions to be cleaved. In the case of the
ethylidene acetal found in 17 and the isopropylidene
ketal found in 21, reduced stability during prolonged
exposure to acidic conditions must of course be expec-
ted. It must be emphasized that the primary objective of
this report was to explore the in vitro thrombin inhibit-
ing properties of these novel acetal- and ketal-contain-
ing N-acylproline isostere derivatives.
The stability of the benzamidine part of the target
compounds is also worth some comments. We have
discovered that the final compounds should not be
stored as free bases, since degradation then occurs over
time. Storage of the corresponding salts (e.g., acetates,
hydrochlorides, etc.) is preferred.
The Connolly surface map of the X-ray crystal structure
of the a-thrombin–6m complex at 2.2 A resolution is
shown in Figure 3.
Thrombin inhibition data
The results of the biological testing of the final products
are listed in Table 2.
Figure 4 depicts the superimposition of the a-thrombin–
6m and a-thrombin–PPACK X-ray crystal structures.
Table 2. Thrombin IC50 values (mM)
Compd
IC50
Compd
IC50
Compd
IC50
Compd
IC50
Compd
IC50
6a
6b
6c
6d
6e
72
>13
14
43
42
6f
6g
6h
6i
>120
42
>120
110
22
6k
6l
6m
6n
6o
18
>120
8.5
11
>120
6p
6q
(ent)-6q
6r
6.0
5.9
48
6t
11
13
17
21
>120
>120
210
49
>130
83
6j
6s
46

A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
1571
The amidine group of 6m forms as expected a strong
salt bridge with Asp 189 in the S1-pocket, where the N–
O distances are 2.96 and 2.75 A, very similar to that of
the guanidine group of PPACK. The dioxolane ring of
6m occupies the hydrophobic S2-pocket of thrombin in
a way that resembles the interaction between the P2-
proline of PPACK and the S2-pocket. Additionally, the
N–H of the P1–P2 amide group of 6m makes a weak
hydrogen bond (N–O distance 3.45 A) with the car-
bonyl group of Ser 214, while the corresponding dis-
tance of PPACK is 2.87 A. However, 6m lacks the
hydrogen bond network to Gly 216, which is a promi-
nent feature of thrombin’s interaction with PPACK and
most non covalent bond inhibitors, including inogatran.
The N–O distance of the carbonyl group of the P2–P3
amide group in 6m to Gly 216 is 3.78 A and the N–H of
the amide group is pointing in the opposite direction
compared to that of the N–H of the d-Phe of PPACK.
To achieve hydrogen bonding with Gly 216 the P2-P3
amide bond in 6m would need to adopt an energetically
unfavorable cis geometry. Finally, the methyl group of
the P3 moiety of 6m, rather than the phenyl group,
occupies the S3-pocket. Thus, the interactions with the
hydrophobic P3-pocket are far from ideal and the
phenyl group interacts with the surrounding water
without any preferred orientation, which is verified by
the poorly defined electron density of this group in the
X-ray analysis. Modeling reveals that an orientation of
the phenyl group into the S3-pocket requires a move-
ment of the P2-residue of 6m, obviously resulting in an
overall less favorable interaction. Thus, the significantly
lower potency of 6m compared to that of PPACK and
inogatran can be explained by the absence of a hydro-
gen bond network between 6m and Gly 216, and also by
the decreased hydrophobic interaction between the
methyl group of 6m and the S3-pocket compared to the
interactions between the S3-pocket and the P3–d-Phe
group of PPACK and the corresponding P3–d-cyclo-
hexylalaninyl group of inogatran. Additionally,
PPACK binds covalently to His 57 and Ser 195,
whereas in our series of compounds these bonds, or
even non-covalent interactions, are lacking. Modeling
of 6l based on the X-ray analysis of the a-thrombin–6m
complex also explains, assuming similar conformation
of the P2–P3 moieties, why the (S)-1-phenylethyl amine
of 6l will interact less favorably with thrombin com-
pared to that of the (R)-1-phenylethyl amine of 6m. In
6l it is not possible to position the methyl group in the
S3-pocket, as this would force the phenyl group in steric
conflict with Trp 215. Thus, the phenyl group must be
oriented towards the S3-pocket, which results in unfa-
vorable steric interactions with the backbone of Asn 98.
To avoid this the P2–P3 moiety has to move, thereby
penalizing other interactions, which evidently results in
the lower affinity of 6l. This will also explain the albeit
poor but reversed order of binding of the (S)-phe-
nylglycine analogue 6e (IC50=42 mM), which has the
same relative configuration as 6l and the (R)-phenylgly-
cine analogue 6h (IC50 >120 mM). Assuming a con-
formation of 6h with the P3-carboxyl group pointing
towards the S3-pocket in a similar fashion as the methyl
group of 6m seems less plausible, and most likely the
phenyl group of 6h is oriented towards the S3-pocket
even though this interaction is not optimal, resulting in
a significantly decreased potency. Regarding 6e, assum-
ing a binding conformation with the phenyl group
located in the S3-pocket as previously discussed for 6l,
the result will be an orientation of the carboxyl group
towards the surrounding water. Evidently, this gives a
somewhat improved potency of 6e compared to both 6h
and 6l.
Inhibitor 6q (IC50=5.9 mM), having a cyclohexylamine
as P3 substituent, is the most potent one in this series,
whereas the corresponding phenyl analogue 6n (IC50
=11.2 mM) is slightly less active. This can probably be
rationalized from a comparison of the directional vec-
tors imposed by the sp3 carbon of the N-connection of
the cyclohexyl group in 6q and the sp2 carbon of the
phenyl group in 6n, leading to the conclusion that the
cyclohexyl group seems to allow a more favorable fit in
the P3-pocket. The reversed stereochemistry of the two
P2-carboxy groups in (ent)-6q (Scheme 2) compared to
that of 6q resulted as predicted in a less potent although
not completely inactive compound.
Most phenyl analogues prepared exhibit low affinity for
thrombin. A noteworthy deviation from this is the
diphenylmethyl analogue 6p (IC50=6.0 mM), which in
contrast to 6m has to locate a phenyl group into the P3-
pocket assuming similar binding of the P1–P2 groups.
In both of the most likely conformations one of the
phenyl groups will be located in the surrounding water,
obviously not contributing to the affinity. However, we
have in several other projects observed that the diphe-
nylmethyl group often seems to increase potency. It
appears to be a general effect of increased hydro-
phobicity, which for reasons not clearly understood
favors the enzyme–inhibitor interaction compared to
that of the free inhibitor–water interactions. The intro-
duction of the 2,2-dimethyl and the 2-methyl groups in
17 and 21 respectively does not lower the potency com-
pared to that of the unsubstituted dioxolane analogue
6a. In view of the X-ray analysis of 6m this is somewhat
surprising, since the dioxolane ring very nicely fills the
lipophilic S2-pocket, even better than the proline of
PPACK, and an accommodation of additional groups
Figure 2. Structures of PPACK (IC50=0.017 nm), inogatran
(IC50=22 nM) and compound 6m (IC50=8.5 mM). The thrombin
nomenclature and the important interactions between PPACK and
thrombin are briefly outlined.

1572
A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
Figure 4. Connolly surface map of the X-ray structure of the a-
thrombin–6m complex (yellow) aligned with PPACK (magenta).
Figure 3. Connolly surface map of the X-ray structure of the a-
thrombin–6m complex at 2.2 A resolution.
thrombin inhibitors, having the proline templates in the
P2 position, p-amidinobenzylamine in the P1 position
and lipophilic amines in the P3 position, were 6q
(IC50=5.9 mM) and 6p (pIC50=6.0 mM). Further opti-
mization within this series of proline mimics should be
directed towards releasing the strain in the P2-P3 amide
linker by stabilization of the cis geometry around the
P2–P3 amido group.
Table 3. Statistics for X-ray crystallography data collection and
refinement
No of measurements
No of unique reflections
Data completeness
Rmerge
No of atoms in refined model
105,068
27,074
94.5 (91.6)a
0.083 (0.377)a
2618
b
protein
ligands
solvent
2329
29
260
Resolution range in refinement (A)
R.m.s. deviation for bond length (A)
angles (ꢁ)
Rcryst
Rfree
6.5–1.90
0.009
1.512
0.221
0.175
Experimental
c
Thrombin inhibition measurements
aThe numbers in parentheses are related to the data in the highest
resolution shell, that is from 2.02 to 1.90 A.
The thrombin inhibitor potency was measured with a
chromogenic substrate method in a Plato 3300 robotic
microplate processor (Rosys AG, CH-8634 Hom-
brechtikon, Switzerland), using 96-well, half volume
microtiter plates (Costar, Cambridge, MA, USA; Cat
No. 3690). Stock solutions of test substance in DMSO
(72 mL), 10 mmol/L, were diluted serially 1:3 (24+48
mL) 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 mL of test sample was
diluted with 124 mL assay buffer (0.05 mol/L Tris–HCl
pH 7.4, ionic strength 0.15 adjusted with NaCl, BSA 1
g/L), 12 mL of chromogenic substrate solution (S-2366,
Cromogenix, Molndal, Sweden) and finally 12 mL of a-
thrombin solution, (human a-thrombin, Sigma Chemi-
cal Co, St Louis, MO, USA; Cat No. T-6759), in buffer,
was added, and the samples were mixed. The final assay
concentrations were: Test substance 0.00068–13.3 mmol/
L, S-2366 0.30 mmol/L and a-thrombin 0.020 NIHU/
mL. The linear absorbance increase during a 40 min
incubation 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
bRmerge=ShSi(I(h,i)-<I(h)>)/ShSiI(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.
cRcryst=Shkl(|Fo-Fc|)/Shkl|Fo|. |Fo| and |Fc| are observed and calculated
structure factor amplitudes, respectively.
does not seem possible. Obviously the substituted ring
must move, but the introduction of favorable lipophili-
city compensates for that.
Finally, compound 13 was prepared to see if more lipo-
philic P1 groups in combination with the P2 tartaric
acid template would prove useful. The 2,5-dichloro
benzylamine group has been incorporated in new classes
of recently developed thrombin inhibitors.11 However,
this in our case results in an almost 100-fold decrease in
activity of 13 compared to that of the corresponding p-
amidinobenzylamine analogue 6q.
Conclusion
A novel proline mimetic, that is, trans-(R,R)-1,3-diox-
olane-4,5-dicarboxamides embedded in a thrombin
inhibitor motif, has been prepared. The best of these

A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
1573
inhibitor concentration which caused 50% inhibition of
the thrombin activity, was calculated from a log dose
versus inhibition curve.
(1 M, 20.5 mL, 20.5 mmol) was added dropwise during
30 min at room temperature. The reaction mixture was
then stirred for an additional hour at room temperature.
After acidification with 1 M aqueous hydrochloric acid
to pH 2–3 the solution was extracted several times with
dichloromethane. The combined organic layers were
dried, filtered and concentrated. Finally flash column
chromatography (toluene/ethyl acetate/acetic acid
20:20:1) provided the monoacid.
X-ray crystallography
Human a-thrombin was purchased from Enzyme
Research Laboratories, Inc., South Bend, IN, USA, and
hirugen from American Diagnostica, Inc., Greenwich,
CT, USA. Hirugen–thrombin complex was prepared
according to the method of Skrzypczak-Jankun et al.26
The crystallization was done as described previously.8
The X-ray diffraction data were collected on a MAR-II
imaging plate system, MARResearch, Hamburg, Ger-
many, using Cu Ka radiation from a rotating anode.
The data was reduced and scaled using DENZO and
SCALEPACK27 programs. The hirugen–a-thrombin
structure previously examined in our laboratory was
used in the refinement of the 6m–thrombin complex
structure. The refinement was performed using
REFMAC (CCP4 package)28 with subsequent runs of
CNX.29 Statistics for X-ray data collection and refine-
ment are presented in Table 3.
Procedure B: Peptide coupling (typical procedure). The
monoacid (8.13 mmol) was dissolved in dimethyl for-
mamide (50 mL). The amine (12.20 mmol) and 1-
hydroxy benzotriazole (HOBt) (12.05 mmol) were
added and the pH was adjusted to 7–8 with triethyl-
amine. The mixture was cooled to 0 ꢁC and N-ethyl-N0-
(3-dimethylaminopropyl)carbodiimide
hydrochloride
(EDC) (12.31 mmol) was added. The reaction mixture
was stirred at 0 ꢁC for 1 h and at room temperature
overnight. The solvent was evaporated and the crude
product was purified by flash column chromatography.
Procedure C: Ester hydrolysis+peptide coupling+depro-
tection of benzyloxycarbonyl protecting group (typical
procedure). To a solution of the monoester (mono-
amide) (0.21 mmol) in dioxane/water 1:1 (5 mL) was
added aqueous sodium hydroxide (1 M, 0.50 mL, 0.50
mmol) and the solution was stirred for 20 min at room
temperature. After neutralization using 1 M aqueous
hydrochloric acid the solvents were evaporated and
coevaporated with toluene 3 times. The resulting
monoacid was coupled with the appropriate amine
using Procedure B. The afforded diamide was dissolved
in ethanol (5 mL) and palladium on carbon (10%, 10
mg) was added and the mixture was hydrogenated
(atmospheric pressure) at room temperature for 45 min.
The suspension was filtered through a fine filter paper.
Acetic acid (10 equivalents) was added and the solution
was stirred at room temperature for 20 min. After eva-
poration the remainder was freeze dried in water.
General methods
NMRspectra were recorded on a Bruker AF 250 instru-
ment using CDCl3, methanol-d4 or D2O with TMS as an
internal standard. The NMRmeasurements of the ben-
zamidine final products were performed on the acetates
unless otherwise noted. Mass spectral data was obtained
in positive ion mode using a double focusing Finnigan
MAT900S equipped with electrospray interface. Reso-
lution: 5000 (10% valley definition). Two PEG refer-
ences were used, one on either side (on the mass scale)
of the mass of the compounds that were analyzed. Spray
voltage: 1.2 kV. The infusion rate was 1.2 microliters/
min. Temperature of the capillary heater: 230 ꢁC. Opti-
cal rotations were measured in CHCl3, H2O or metha-
nol solutions on a Perkin–Elmer 141 polarimeter. The
optical rotations of the benzamidine final products were
measured on their respective acetate salts. TLC was
carried out on Merck precoated 60 F254 plates using
UV-light and charring with ethanol/sulfuric acid/acetic
acid/p-anisaldehyde 90:3:1:2 or ethanol/acetic acid/colli-
dine/ninhydrine 500:150:20:1 for visualisation. Column
chromatography was performed using silica gel 60 (0.040–
0.063 mm, Merck). Organic phases were dried over anhy-
drous magnesium sulfate. Concentrations were per-
formed under diminished pressure (1–2 kPa) at a bath
temperature of 40 ꢁC. The benzamidine final products
were sent as acetates for elemental analysis, and some-
times some or all of the acetic acid was lost during drying/
heating before the actual analysis. Some of the benzami-
dine final products gave very poor results in the elemental
analyses, perhaps due to instability of the compounds.
Those compounds were instead analyzed using HRMS.
Procedure D. As Procedure C except for the coupling
step where diphenylphosphoryl azide (DPPA) was used
instead of EDC and HOBt.
Procedure E. As Procedure C except for the coupling
step where bis(2-oxo-3-oxazolidinyl)phosphinic chloride
(BOP-Cl) was used instead of EDC and HOBt.
Synthetic experimentals
trans-(4R,5R)-1,3-Dioxolane-4,5-dicarboxylic acid di-
ethyl ester [(À)-2]. To a solution of (+)-diethyl l-tar-
trate [(+)-1)] (16.61 g, 80.5 mmol) in isopropyl acetate
(100 mL) were added diethoxymethane (10.1 g, 96.6
mmol) and boron trifluoride–diethyl ether complex
(27.4 g, 193 mmol), and the mixture was refluxed for 7
h. The reaction mixture was quenched with saturated
aqueous sodium hydrogen carbonate and the layers
were separated. The water phase was extracted several
times with isopropyl acetate, and the combined organic
layers were dried, filtered and concentrated. The crude
product was purified by silica gel flash chromatography
General synthetic procedures
Procedure A: Monohydrolysis of diesters (typical proce-
dure). The diester (19.7 mmol) was dissolved in water/
dioxane (1:1, 100 mL), and aqueous sodium hydroxide

1574
A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
(toluene/ethyl acetate 9:1) to give compound (À)-2
trans-(4R,5R)-N1 -{4-[Amino(imino)methyl]benzyl}-N2 -
benzyl - 1,3 - dioxolane - 4,5 - dicarboxamide (6a). Com-
pound 6a (30 mg as the acetate salt, 32%) was prepared
from 4 (95 mg, 0.21 mmol) and benzylamine according
to Procedure C. Chromatography mobile phase after
the coupling step: ethyl acetate. 6a: a2D2À8.4 (c 0.2,
H2O); 1H NMR(methanol- d4, 250 MHz) d 1.91 (s, 3H),
4.44 (s, 2H), 4.54 (s, 2H), 4.68 (d, J=4.0 Hz, 1H), 4.71
(d, J=4.0 Hz, 1H), 5.21 (s, 1H), 5.23 (s, 1H), 7.21–7.38
(m, 5H), 7.53 (d, J=8.4 Hz, 2H), 7.77 (d, J=8.4 Hz,
2H); 13C NMR(methanol- d4, 62.9 MHz) d 24.0, 43.4,
43.8, 79.4, 79.5, 97.9, 128.3, 128.4, 128.5, 129.1, 129.2,
129.6, 139.6, 146.6, 168.3, 171.7, 172.1, 181.2. HRMS
m/z calcd for C20H23N4O4 (MH+): 383.1719. Found:
383.1727.
(14.33 g, 82%) as a slightly yellow oil. (À)-2: a2D2À75.1 (c
1
1.5, CHCl3) (lit:13cÀ77.9); H NMR(CDCl , 250 MHz)
3
in agreement with ref 13c; 13C NMR(CDCl
62.9 MHz) in agreement with ref 13c.
,
3
trans-(4S,5S)-1,3-Dioxolane-4,5-dicarboxylic acid diethyl
ester [(+)-2]. Compound (+)-2 (3.00 g, 72%) was pre-
pared from (À)-diethyl d-tartrate [(À)-1] (4.00 g, 19.4
mmol) according to the method for the preparation of
(À)-2. (+)-2: a2D2: +75.7 (c 3.4, CHCl3). Anal. calcd for
C9H14O6: C, 49.54; H, 6.47. Found: C, 49.81; H, 6.60.
trans-(4R,5R)-1,3-Dioxolane-4,5-dicarboxylic acid mono-
ethyl ester [(À)-3)]. Compound (À)-3 (3.18 g, 82%, a
slightly orange liquid) was synthesized from (À)-2 (4.29
g, 19.7 mmol) according to Procedure A. (À)-3: aD22
trans-(4R,5R)-N1 -{4-[Amino(imino)methyl]benzyl}-N2 -
benzyl-N2-methyl-1,3-dioxolane-4,5-dicarboxamide (6b).
Compound 6b (21 mg as the acetate salt, 31%) was
prepared from 4 (68 mg, 0.15 mmol) and N-methyl
benzylamine according to Procedure C. 6b (rotational
isomers a and b; a/b=1.62:1): a2D2À31.0 (c 0.8, MeOH);
1H NMR(methanol- d4, 250 MHz) d 2.00 (s, 3H), 2.90
(s, 3H, isomer b), 3.11 (s, 3H, isomer a), 4.50–4.73 (m,
4H, a and b), 4.96 (d, J=3.6 Hz, 1H, isomer a), 5.00 (d,
J=3.8 Hz, 1H, isomer b), 5.07 (d, J=3.8 Hz, 1H, iso-
mer b), 5.14 (d, J=3.6 Hz, 1H, isomer a), 5.22 (s, 1H,
isomer a), 5.23 (s, 1H, isomer b), 5.24 (s, 1H, isomer b),
5.27 (s, 1H, isomer a), 7.21–7.41 (m, 5H, a and b), 7.48–
7.59 (m, 2H, a and b), 7.71–7.81 (m, 2H, a and b); 13C
NMR(methanol- d4, 62.9 MHz) d 20.9, 34.4, 35.2, 43.3,
52.4, 53.7, 77.9, 78.0, 78.6, 78.7, 128.0, 128.3, 128.6,
128.8, 128.9, 129.2, 129.8, 130.0, 137.6, 137.9, 146.8,
168.2, 170.3, 170.5, 172.8, 172.9, 181.3. Anal. calcd for
C21H24N4O41.5HOAc: C, 59.25; H, 6.22; N, 11.52.
Found: C, 59.24; H, 6.27; N, 11.65.
1
À70.7 (c 1.7, CHCl3); H NMR(CDCl , 250 MHz) d
3
1.35 (t, J=7.1 Hz, 3H), 4.31 (q, J=7.1 Hz, 2H), 4.82 (s,
2H), 5.27 (s, 1H), 5.30 (s, 1H), 8.73 (br s, 1H); 13C
NMR(CDCl , 62.9 MHz) d 14.1, 62.3, 76.5, 76.9, 97.6,
3
169.1, 174.1. Anal. calcd for C7H10O6: C, 44.21; H, 5.30.
Found: C, 44.01; H, 5.40.
trans-(4S,5S)-1,3-Dioxolane-4,5-dicarboxylic acid mono-
ethyl ester [(+)-3]. Compound (+)-3 (1.70 g, 75%) was
prepared from (+)-2 (2.50 g, 11.5 mmol) according to
the method for the preparation of (À)-3. (À)-3: aD22
+68.0 (c 0.9, CHCl3). Anal. calcd for C7H10O6: C,
44.21; H, 5.30. Found: C, 43.87; H, 5.05.
trans-(4R,5R)-5-{4-[Amino(benzyloxycarbonylimino)me-
thyl]benzylaminocarbonyl} - 1,3 - dioxolane - 4 - carboxylic
acid ethyl ester (4). Compound 4 (3.0 g, 92%, a color-
less solid) was prepared from (À)-3 (1.54 g, 8.13 mmol)
and 4-(benzyloxycarbonyl)amidinobenzylamine hydro-
chloride [Pab(Z)HCl] according to Procedure B. Chro-
matography mobile phase: ethyl acetate. 4: a2D2À21.3 (c
0.6, CHCl3); 1H NMR(CDCl 3, 250 MHz) d 1.31 (t,
J=7.1 Hz, 3H), 4.27 (q, J=7.1 Hz, 2H), 4.46 (m, 2H),
4.67 (d, J=3.4 Hz, 1H), 4.80 (2, J=3.4 Hz, 1H), 5.14 (s,
1H), 5.20 (s, 2H), 5.23 (s, 1H), 6.97 (bs, 1H), 7.13 (bs,
1H), 7.22–7.45 (m, 7H), 7.80 (d, J=8.3 Hz, 2H), 9.47
trans-(4R,5R)-N1-{4-[Amino(imino)methyl]benzyl}-N2,N2-
dibenzyl - 1,3 - dioxolane - 4,5 - dicarboxamide (6c). Com-
pound 6c (25 mg as the acetate salt, 52%) was prepared
from 4 (46 mg, 0.10 mmol) and dibenzylamine accord-
ing to Procedure D. 6c: a2D2À25.0 (c 0.2, H2O); 1H NMR
(D2O, 250 MHz) d 1.90 (s, 3H), 4.43 (s, 2H), 4.47–4.70
(m, 4H), 4.84 (d, J=4.3 Hz, 1H), 5.04 (d, J=4.3 Hz,
1H), 5.21 (s, 1H), 5.27 (s, 1H), 7.10–7.21 (m, 5H), 7.31–
(bs, 1H); 13C NMR(CDCl , 62.9 MHz) d 14.1, 42.7,
3
62.1, 67.2, 77.2, 77.6, 96.9, 127.6, 127.8, 128.0, 128.2,
128.4, 133.8, 136.6, 142.0, 165.0, 167.8, 169.2, 169.5.
Anal. calcd for C23H25N3O7: C, 60.65; H, 5.53; N, 9.23.
Found: C, 60.85; H, 5.60; N, 9.13.
7.41 (m, 7H), 7.64 (d, J=8.3 Hz, 2H); 13C NMR(D O,
2
62.9 MHz) d 25.0, 44.0, 51.1, 51.9, 77.5, 79.4, 99.0,
128.5, 129.4, 129.6, 129.8, 130.6, 130.7, 137.1, 137.6,
146.0, 167.9, 172.0, 172.9, 183.3. HRMS m/z calcd for
C27H29N4O4 (MH+): 473.2189. Found: 473.2204.
trans-(4S,5S)-5-Cyclohexylaminocarbonyl-1,3-dioxolane-
4-carboxylic acid ethyl ester (5). Compound 5 (185 mg,
25%, a colorless syrup) was prepared from (+)-3 (515
mg, 2.71 mmol) and cyclohexylamine according to Pro-
cedure B. Chromatography mobile phase: ethyl acetate.
trans-(4R,5R,20S)-N1-{4-[Amino(imino)methyl]benzyl}-N2-
[20-(methyl)carboxylbenzyl] - 1,3 - dioxolane - 4,5 - dicarbox-
amide (6d). Compound 6d (71 mg as the acetate salt,
53%) was prepared from 4 (125 mg, 0.27 mmol) and
(S)-phenylglycine methyl ester according to Procedure
5: a2D2 +56.0 (c 0.3, MeOH); 1H NMR(CDCl
,
3
250 MHz) d 1.10–1.50 (m, 5H), 1.33 (t, J=7.1 Hz, 3H),
1.58–1.80 (m, 3H), 1.83–2.00 (m, 2H), 3.80 (m, 1H), 4.28
(q, J=7.1 Hz, 2H), 4.61 (d, J=3.6 Hz, 1H), 4.80 (d,
J=3.6 Hz, 1H), 5.18 (s, 1H), 5.24 (s, 1H), 6.42 (br s,
22
1
C. 6d: aD À27.8 (c 0.2, H2O); H NMR(methanol- d4,
250 MHz) d 1.93 (s, 3H), 3.72 (s, 3H), 4.52 (bs, 2H), 4.70
(m, 2H), 5.25 (bs, 2H), 5.55 (s, 1H), 7.32–7.45 (m, 5H),
7.52 (d, J=8.3 Hz, 2H), 7.78 (d, J=8.3 Hz, 2H); 13C
NMR(methanol- d4, 62.9 MHz) d 24.1, 43.4, 53.2, 57.9,
79.1, 79.3, 98.0, 128.7, 128.8, 129.1, 129.7, 130.0, 136.5,
136.8, 146.6, 168.2, 171.0, 171.1, 172.1, 181.7. Anal.
1H); 13C NMR(CDCl , 62.9 MHz) d 14.1, 24.7, 25.4,
3
32.9, 32.0, 48.1, 61.9, 77.0, 77.7, 96.8, 167.7, 169.7.
Anal. calcd for C13H21NO5: C, 57.55; H, 7.80; N, 5.16.
Found: C, 57.40; H, 7.93; N, 5.03.

A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
1575
calcd for C22H24N4O61.5H2CO3: C, 52.90; H, 5.10; N,
10.50. Found: C, 53.02; H, 5.30; N, 10.61.
preparation of 6e. 6h: a2D2À2.6 (c 0.2, MeOH); 1H NMR
(methanol-d4, 250 MHz) d 1.92 (s, 3H), 4.53 (s, 2H),
4.60–4.66 (m, 2H), 5.20–5.27 (m, 3H), 7.20–7.45 (m,
5H), 7.51 (d, J=8.3 Hz, 2H), 7.76 (d, J=8.3 Hz, 2H);
13C NMR(methanol- d4, 62.9 MHz) d 24.0, 43.4, 60.1,
79.1, 79.2, 97.9, 128.3, 128.4, 128.5, 129.2, 129.4, 140.7,
146.4, 146.5, 168.1, 170.7, 171, 172.2, 181.9. HRMS m/z
calcd for C21H23N4O6 (MH+): 427.1618. Found:
427.1625.
trans-(4R,5R,20S)-N1 -{4-[Amino(imino)methyl]benzyl}-
N2-(20-carboxylbenzyl)-1,3-dioxolane-4,5-dicarboxamide
(6e). Compound 6e (10 mg as the acetate salt, 100%)
was prepared by dissolving benzyloxycarbonyl pro-
tected 6d (13 mg, 0.023 mmol) in dioxane/water 1:1 fol-
lowed by the addition of aqueous NaOH (1 M, 0.10 mL,
0.10 mmol). The solution was stirred at room tempera-
ture for 30 min, after which the mixture was neutralized
with 1 M aqueous hydrochloric acid followed by eva-
poration of the solvents. The remainder was then sub-
jected to catalytic hydrogenation as described in
trans-(4R,5R,20R)-N1 -{4-[Amino(imino)methyl]benzyl}-
N2 -[20 -(hydroxymethyl)benzyl]-1,3-dioxolane-4,5-dicar-
boxamide (6i). Compound 6i (66 mg as the acetate salt,
32%) was prepared from 4 (228 mg, 0.50 mmol) and
TBDMS protected (R)-phenyl glycinol according to
Procedure C. The TBDMS group was removed quanti-
tatively before the benzyloxycarbonyl group deprotec-
tion by stirring in tetrahydrofuran (10 mL) and
tetrabutylammonium fluoride3 H2O (64 mg, 0.20 mmol),
followed by evaporation of the solvent and filtering
through a short silica plug with ethyl acetate/methanol
22
1
Procedure C. 6e: aD À29.4 (c 0.5, MeOH); H NMR
(methanol-d4, 250 MHz) d 1.92 (s, 3H), 4.55 (s, 2H),
4.63 (m, 2H), 5.21–5.25 (m, 3H), 7.19–7.48 (m, 5H), 7.52
(d, J=8.2 Hz, 2H), 7.78 (d, J=8.2 Hz, 2H); 13C NMR
(methanol-d4, 62.9 MHz) d 23.9, 43.4, 60.1, 79.1, 79.2,
97.9, 128.4, 128.5, 129.2, 129.4, 140.7, 140.8, 146.6,
168.1, 170.2, 170.4, 172.2, 182.1. HRMS m/z calcd for
C21H23N4O6 (MH+): 427.1618. Found: 427.1623.
22
1
5:1 as eluent. 6i: aD À22.5 (c 0.4, MeOH); H NMR
(D2O, 250 MHz) d 1.83 (s, 3H), 3.78 (d, J=6.3 Hz, 2H),
4.46 (s, 2H), 4.63 (d, J=4.2 Hz, 1H), 4.71 (d, J=4.2 Hz,
1H), 4.95 (t, J=6.3 Hz, 1H), 5.15 (s, 1H), 5.16 (s, 1H),
7.22–7.37 (m, 5H), 7.42 (d, J=8.3 Hz, 2H), 7.67 (d,
J=8.3 Hz, 2H); 13C NMR(D 2O, 62.9 MHz) d 25.9,
45.1, 58.2, 66.5, 80.2, 80.3, 99.3, 129.3, 129.5, 129.6,
130.4, 130.7, 131.6, 141.0, 146.7, 169.1, 173.6, 174.0,
184.3. HRMS m/z calcd for C21H25N4O5 (MH+):
413.1825. Found: 413.1822.
trans-(4R,5R,20S)-N1 -{4-[Amino(imino)methyl]benzyl}-
N2 -[20 -(hydroxymethyl)benzyl]-1,3-dioxolane-4,5-dicar-
boxamide (6f). Compound 6f (41 mg as the acetate salt,
50%) was prepared from 4 (79 mg, 0.17 mmol) and
THP-protected (S)-phenylglycinol according to Proce-
dure C. The THP group was removed in quantitative
yield before the benzyloxycarbonyl group deprotection
step, by stirring in acetic acid/tetrahydrofuran/water
4:2:1 (10 mL) for 3 h followed by evaporation of the
solvents. 6f: a2D2 +11.9 (c 1.0, MeOH); H NMR(D O,
trans-(4R,5R,20S)-N1 -{4-[Amino(imino)methyl]benzyl}-
N2-[20-(methyl)carboxylphenylethyl]-1,3-dioxolane-4,5-di-
carboxamide (6j). Compound 6j (74 mg as the acetate
salt, 55%) was prepared from 4 (118 mg, 0.26 mmol)
and (S)-phenylalanine methyl ester according to Proce-
1
2
250 MHz) d 1.93 (s, 3H), 3.90 (d, J=6.1 Hz, 2H), 4.57
(s, 2H), 4.75 (bs, 2H), 5.04 (t, J=6.1 Hz, 1H), 5.28 (s,
1H), 5.30 (s, 1H), 7.35–7.48 (bs, 5H), 7.52 (d, J=7.8 Hz,
2H), 7.78 (d, J=7.8 Hz, 2H); 13C NMR(D 2O,
62.9 MHz) d 26.0, 45.1, 58.3, 66.6, 80.3, 80.4, 99.4,
129.4, 129.6, 129.8, 130.4, 130.8, 131.6, 140.9, 146.8,
169.1, 173.5, 174.1, 184.3. HRMS m/z calcd for
C21H25N4O5 (MH+): 413.1825. Found: 413.1823.
22
1
dure C. 6j: aD À22.7 (c 0.4, MeOH); H NMR(metha-
nol-d4, 250 MHz) d 1.97 (s, 3H), 3.07 (dd, J=13.9, 9.1
Hz, 1H), 3.23 (dd, J=13.9, 5.4 Hz, 1H), 3.71 (s, 3H),
4.52 (s, 2H), 4.55 (d, J=3.9 Hz, 1H), 4.57 (d, J=3.9 Hz,
1H), 4.73 (dd, J=9.1, 5.4 Hz, 1H), 5.10 (s, 1H), 5.18 (s,
1H), 7.22 (m, 5H), 7.51 (d, J=8.4 Hz, 2H), 7.77 (d,
J=8.4 Hz, 2H); 13C NMR(methanol- d4, 62.9 MHz) d
20.9, 37.9, 43.3, 52.9, 54.9, 79.1, 79.1, 97.9, 127.9, 128.2,
128.6, 129.2, 129.5, 130.3, 138.0, 146.7, 168.0, 171.4,
172.0, 172.9, 175.3. Anal. calcd for C23H26N4O60.60-
MeOH2.00H2O: C, 55.60; H, 6.41; N, 10.99. Found: C,
55.41; H, 6.56: N, 10.78.
trans-(4R,5R,20R)-N1 -{4-[Amino(imino)methyl]benzyl}-
N2-[20 -(methyl)carboxylbenzyl]-1,3-dioxolane-4,5-dicar-
boxamide (6g). Compound 6g (18 mg, 42%) was pre-
pared from
4 (45 mg, 0.099 mmol) and (R)-
phenylglycine methyl ester according to Procedure C.
6g: a2D2À23.3 (c 0.2, H2O); 1H NMR(the free base)
(methanol-d4, 250 MHz) d 3.72 (s, 3H), 4.52 (s, 2H),
4.70 (m, 2H), 5.19 (s, 1H), 5.24 (s, 1H), 5.54 (s, 1H),
7.20–7.32 (m, 5H), 7.50 (d, J=7.9 Hz, 2H), 7.75 (d,
J=7.9 Hz, 2H); 13C NMR(D 2O, 62.9 MHz) d 25.9,
45.1, 56.0, 59.6, 80.1, 80.2, 99.4, 129.5, 130.3, 130.4,
130.7, 131.9, 137.2, 137.3, 146.7, 169.1, 173.2, 174.0,
174.7, 184.2. Anal. calcd for C22H24N4O60.58H2O1.48-
HOAc: C, 55.53; H, 5.76; N, 10.38. Found: C, 55.50; H,
5.71; N, 10.19.
trans-(4R,5R,20R)-N1 -{4-[Amino(imino)methyl]benzyl}-
N2-[20-(methyl)carboxylphenylethyl]-1,3-dioxolane-4,5-di-
carboxamide (6k). Compound 6k (32 mg as the acetate
salt, 24%) was prepared from 4 (118 mg, 0.26 mmol)
and (R)-phenylalanine methyl ester according to Proce-
22
1
dure C. 6k: aD À14.9 (c 0.8, MeOH); H NMR(metha-
nol-d4, 250 MHz) d 1.94 (s, 3H), 3.03 (dd, J=13.9, 9.4
Hz, 1H), 3.27 (dd, J=13.9, 5.2 Hz, 1H), 3.75 (s, 3H),
4.47 (d, J=3.8 Hz, 1H), 4.62 (d, J=3.8 Hz, 1H), 4.78
(dd, J=9.4, 5.2 Hz, 1H), 4.91 (s, 1H), 5.14 (s, 1H), 7.17–
7.33 (m, 5H), 7.51(d, J=8.4 Hz, 2H), 7.77 (d, J=8.4
Hz, 2H); 13C NMR(methanol- d4, 62.9 MHz) d 24.0,
38.0, 43.4, 52.9, 54.7, 79.1, 79.2, 97.8, 128.0, 128.2,
trans-(4R,5R,20R)-N1 -{4-[Amino(imino)methyl]benzyl}-
N2-(20-carboxylbenzyl)-1,3-dioxolane-4,5-dicarboxamide
(6h). Compound 6h (210 mg as the acetate salt, 100%)
was prepared from benzyloxycarbonyl protected 6g
(248 mg, 0.43 mmol) according to the method for the

1576
A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
128.5, 129.2, 129.5, 130.3, 138.0, 146.6, 168.1, 171.7,
172.0, 172.9, 181.7. Anal. calcd for C23H26N4O62.00-
H2O1.28HOAc: C, 54.10; H, 6.24; N, 9.88. Found: C,
54.10; H, 6.09; N, 9.94.
NMR(the free base) (methanol- d4, 62.9 MHz) d 38.4,
43.1, 76.9, 79.2, 98.2, 128.3, 128.7, 129.1, 129.3, 129.5,
131.0, 143.7, 146.7, 170.0, 172.1, 176.3. HRMS m/z calcd
for C20H23N4O4 (MH+): 383.1719. Found: 383.1721.
trans-(4R,5R,20S)-N1-{4-[Amino(imino)methyl]benzyl}-N2
-(20 -methylbenzyl)-1,3-dioxolane-4,5-dicarboxamide (6l).
Compound 6l (48 mg as the acetate salt, 23%) was pre-
pared from 4 (213 mg, 0.47 mmol) and (S)-1-phenyl-
ethyl amine according to Procedure C. 6l: a2D2À45.7 (c
trans-(4R,5R)-N1-{4-[Amino(imino)methyl]benzyl} - N2 -
(20 -phenylbenzyl)-1,3-dioxolane-4,5-dicarboxamide (6p).
Compound 6p (77 mg, 64%) was prepared from 4 (85
mg, 0.19 mmol) and 1,1-diphenyl methylamine accord-
ing to Procedure E. 6p: aD À11.1 (c 0.2, MeOH); H
NMR(the free base) (methanol- d4, 250 MHz) d 4.50 (s,
2H), 4.68 (d, J=4.3 Hz, 1H), 4.73 (d, J=4.3 Hz, 1H),
5.20 (s, 1H), 5.21 (s, 1H), 6.22 (s, 1H), 7.20–7.39 (m, 10H),
7.48 (d, J=8.5 Hz, 2H), 7.74 (d, J=8.5 Hz, 2H); 13C
NMR(the free base) (methanol- d4, 62.9MHz) d 43.4,
58.1, 79.2, 79.4, 98.0, 128.5, 128.6, 128.6, 128.8, 129.0,
129.4, 129.6, 130.4, 142.4, 142.5, 145.5, 167.9, 170.7,
172.1. Anal. calcd for C26H26N4O42.10HOAc: C, 62.04;
H, 5.93; N, 9.59. Found: C, 62.08; H, 5.53; N, 9.86.
22
1
1
0.5, MeOH); H NMR(methanol- d4, 250 MHz) d 1.49
(d, J=7.0 Hz, 3H), 1.97 (s, 3H), 4.48 (d, J=15.7 Hz,
1H), 4.57 (d, J=15.7 Hz, 1H), 4.63 (d, J=4.1 Hz, 1H),
4.67 (d, J=4.1 Hz, H), 5.07 (q, J=7.0 Hz, 1H), 5.17 (s,
1H), 5.21 (s, 1H), 7.21–7.35 (m, 6H), 7.51 (d, J=8.2 Hz,
2H), 7.75 (d, J=8.2 Hz, 2H); 13C NMR(methanol- d4,
62.9 MHz) d 20.9, 22.2, 43.4, 50.2, 79.3, 79.4, 98.0,
127.0, 128.2, 129.2, 129.3, 129.4, 129.6, 144.8, 146.7,
168.1, 170.6, 172.2, 175.4. Anal. calcd for
C21H24N4O40.75H2O1.66HOAc: C, 57.31; H, 6.36; N,
11.00. Found: C, 57.31; H, 6.34; N, 11.00.
trans-(4R,5R)-N1 -{4-[Amino(imino)methyl]benzyl}-N2 -
cyclohexyl-1,3-dioxolane-4,5-dicarboxamide (6q). Com-
pound 6q (42 mg as the acetate salt, 88%) was prepared
from 4 (51 mg, 0.11 mmol) and cyclohexylamine
according to Procedure C. 6q: a2D2À16.2 (c 0.7, MeOH)
1H NMR(the free base) (methanol- d4, 250 MHz) d
1.11–1.50 (m, 5H), 1.59–1.92 (m, 5H), 3.68–3.72 (m,
1H), 4.50 (d, J=15.7 Hz, 1H), 4.58 (d, J=15.7 Hz, 1H),
4.58 (d, J=4.1 Hz, 1H), 4.65 (d, J=4.1 Hz, 1H), 5.18 (s,
1H), 5.21 (s, 1H), 7.55 (d, J=8.3 Hz, 2H), 7.79 (d,
J=8.3 Hz, 2H); 13C NMR(the free base) (methanol- d4,
62.9 MHz) d 26.2, 26.5, 33.4, 33.5, 43.4, 49.9, 79.3, 79.4,
97.9, 128.3, 129.2, 129.3, 146.7, 168.1, 170.4, 172.2.
HRMS m/z calcd for C19H27N4O4 (MH+): 375.2032.
Found: 375.2034.
trans-(4R,5R,20R)-N1-{4-[Amino(imino)methyl]benzyl} -
N2-(20 -methylbenzyl)-1,3-dioxolane-4,5-dicarboxamide
(6m). Compound 6m (80 mg as the acetate salt, 47%)
was prepared from 4 (170 mg, 0.37 mmol) and (R)-1-
phenylethylamine according to Procedure A. 6m: aD22
1
+5.2 (c 0.3, MeOH); H NMR(D O, 250 MHz) d 1.49
2
(d, J=7.0 Hz, 3H), 1.94 (s, 3H), 4.55 (s, 2H), 4.63 (d,
J=4.3 Hz, 1H), 4.71 (d, J=4.3 Hz, 1H), 5.0 (q, J=7.0
Hz, 1H), 5.25 (s, 1H), 5.26 (s, 1H), 7.30–7.43 (m, 5H),
7.50 (d, J=8.2 Hz, 2H), 7.73 (d, J=8.2 Hz, 2H); 13C
NMR(D O, 62.9 MHz) d 23.6, 25.5, 44.9, 52.0, 80.1,
2
80.2, 99.2, 128.4, 129.2, 130.0, 130.3, 130.6, 131.4, 145.4,
146.7, 168.8, 172.3, 173.8, 183.3. Anal. calcd for
C21H24N4O48.0H2O: C, 46.65; H, 7.46; N, 10.37.
Found: C, 46.87; H, 7.50; N, 10.48.
trans-(4S,5S)-N1-{4-[Amino(imino)methyl]benzyl} - N2 -
cyclohexyl-1,3-dioxolane-4,5-dicarboxamide [(ent)-6q].
Compound (ent)-6q (109 mg as the acetate salt,
51%) was prepared from 5 (134 mg, 0.49 mmol) and
4-(benzyloxycarbonyl)amidinobenzylamine hydrochlo-
ride [Pab(Z)HCl] according to Procedure C. (ent)-6q:
a2D2: +20.0 (c 0.9, MeOH). HRMS m/z calcd for
C19H27N4O4 (MH+): 375.2032. Found: 375.2035.
trans-(4R,5R)-N1-{4-[Amino(imino)methyl]benzyl} - N2 -
phenyl - 1,3 - dioxolane - 4,5 - dicarboxamide (6n). Com-
pound 6n (26 mg as the acetate salt, 70%) was prepared
from 4 (39 mg, 0.085 mmol) and aniline according to
22
1
Procedure E. 6n: aD À27.8 (c 0.4, MeOH); H NMR
(the free base) (methanol-d4, 250 MHz) d 4.52 (d,
J=15.7 Hz, 1H), 4.61 (d, J=15.7 Hz, 1H), 4.76 (d,
J=4.2 Hz, 1H), 4.81 (d, J=4.2 Hz, 1H), 5.27 (s, 1H),
5.29 (s, 1H), 7.15–7.20 (m, 1H), 7.28–7.38(m, 2H) 7.53–
7.62 (m, 4H), 7.79 (d, J=8.4 Hz, 2H); 13C NMR(the
free base) (methanol-d4, 62.9 MHz) d 43.4, 79.3, 79.8,
98.1, 121.9, 126.0, 128.3, 129.0, 129.2, 129.8, 138.8,
146.7, 168.2, 169.8, 172.2. Anal. calcd for
C19H20N4O44.86H2O0.36HOAc: C, 49.59; H, 6.58; N,
11.73. Found: C, 49.61; H, 6.49; N, 11.77.
trans-(4R,5R)-N-{4-[Amino(imino)methyl]benzyl}-4-(1-
piperidinylcarbonyl)-1,3-dioxolane-5-carboxamide (6r).
Compound 6r (56 mg as the acetate salt, 71%) was
prepared from 4 (83 mg, 0.18 mmol) and piperidine
according to Procedure E. 6r: a2D2À33.3 (c 0.7, MeOH);
1H NMR(the free base) (D O, 250 MHz) d 1.41–1.70
2
(m, 6H), 3.40–3.58 (m, 4H), 4.49 (s, 2H), 4.78 (d, J=3.7
Hz, 1H), 5.16 (d, J=3.7 Hz, 1H), 5.19 (s, 1H), 5.22 (s,
1H), 7.46 (d, J=8.4 Hz, 2H), 7.72 (d, J=8.4 Hz, 2H);
13C NMR(the free base) (methanol- d4, 62.9 MHz) d
25.3, 26.7, 27.4, 43.3, 44.7, 47.7, 77.8, 78.5, 98.1, 128.2,
129.2, 129.3, 146.8, 168.1, 168.2, 172.9. Anal. calcd for
C18H24N4O41.65H2O1.24HOAc: C, 52.94; H, 7.00; N,
12.06. Found: C, 53.00; H, 6.72; N, 12.20.
trans-(4R,5R)-N1-{4-[Amino(imino)methyl]benzyl} - N2 -
methyl-N2-phenyl-1,3-dioxolane-4,5-dicarboxamide (6o).
Compound 6o (25 mg as the acetate salt, 49%) was
prepared from 4 (49 mg, 0.11 mmol) and N-methyl ani-
line according to Procedure E. 6o: a2D2À45.3 (c 0.9,
MeOH); 1H NMR(the free base) (methanol- d4,
250 MHz) d 3.31 (s, 3H), 4.40 (s, 2H), 4.47 (d, J=4.7
Hz, 1H), 4.79 (d, J=4.7 Hz, 1H), 5.10 (s, 1H), 5.20 (s,
1H), 7.25–7.50 (m, 7H), 7.72 (d, J=8.3 Hz, 2H); 13C
trans-(4R,5R)-N-{4-[Amino(imino)methyl]benzyl} - 4 - (1 -
indolinylcarbonyl) - 1,3 - dioxolane - 5 - carboxamide (6s).
Compound 6s (46 mg as the acetate salt, 61%) was

A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
1577
prepared from 4 (66 mg, 0.14 mmol) and indoline
according to Procedure E. 6s: a2D2À45.1 (c 0.5, MeOH);
1H NMR(the free base) (D 2O, 250 MHz) d 2.97 (t,
J=8.1 Hz, 2H), 3.85–4.08 (m, 2H), 4.49 (s, 2H), 4.78 (d,
J=3.5 Hz, 1H), 4.98 (d, J=3.5 Hz, 1H), 5.27 (s, 1H),
5.29 (s, 1H), 7.00–7.20 (m, 3H), 7.45 (d, J=8.3 Hz, 2H),
7.70 (d, J=8.3 Hz, 2H), 7.89 (d, J=7.7 Hz, 1H); 13C
NMR(the free base) (D 2O, 62.9 MHz) d 27.2, 42.1,
47.5, 76.7, 77.1, 97.0, 116.7, 124.9, 125.1, 126.3, 126.9,
127.5, 127.8, 132.6 141.2, 144.0, 165.8, 167.1, 171.1.
HRMS m/z calcd for C21H23N4O4 (MH+): 395.1719.
Found: 395.1710.
at 0 ꢁC for 3 h. After addition of water (5 mL) the sol-
vents were evaporated. The crude residue was dissolved
in dichloromethane and washed with aqueous 1 M
hydrogen chloride and saturated aqueous sodium
hydrogen carbonate. The organic phase was dried, fil-
tered and concentrated to give the mesylate as a color-
less solid which was used without further purification.
To a solution of the mesylate in dimethyl formamide (20
mL) was added sodium azide (2.62 g, 40.3 mmol) and
the mixture was stirred at room temperature for 48 h.
Ethyl acetate was added and the solution was washed 5
times with water. The organic phase was dried, filtered,
concentrated and purified by flash column chromato-
graphy (toluene/ethyl acetate 3:1) to give 2.32 g (88%)
of the azide as a colorless oil. To a solution of the azide
in methanol (100 mL) was added triphenylphosphine
(5.60 g, 21.3 mmol) and Boc anhydride (1.86 g, 8.54
mmol) and the resulting mixture was stirred at room
temperature overnight. The solvent was evaporated and
the crude residue was purified by flash column chroma-
tography (toluene/ethyl acetate 3:1). Recrystallization
trans-(4R,5R)-N1-{4 - [Amino(imino)methyl]benzyl} - 1,3 -
dioxolane-4,5-dicarboxamide (6t). Compound 4 (83 mg,
0.18 mmol) was dissolved in methanol saturated with
ammonia (10 mL) and the resulting solution was stirred
at room temperature for 2 h. Volatile matters were eva-
porated, and the residue was applied on to a short col-
umn of silica gel and eluted with ethyl acetate/methanol
(9:1). The appropriate fractions were combined and
evaporated. The product was then hydrogenated
according to the method described in Procedure C to
from dichloromethane/hexane gave 740 mg (26%) of 9
1
as colorless crystals. 9: H NMR(CDCl , 250 MHz) d
3
give 6t (62 mg as the acetate salt, 96%). 6t: a2D2À9.6 (c
1.42 (s, 9H), 2.84 (t, J=7.0 Hz, 2H), 3.37 (t, J=7.0 Hz,
2H), 4.55 (bs, 1H), 5.23 (s, 2H), 6.50 (bs, 1H), 7.20–7.50
(m, 7H), 7.81–7.92 (m, 2H), 9.50 (bs, 1H); 13C NMR
(CDCl3, 62.9 MHz) d 28.4, 36.1, 41.5, 67.2, 79.4, 127.5,
127.9, 128.2, 128.4, 129.2, 132.7, 136.7, 143.9, 164.7,
168.0. Anal. calcd for C23H27N3O4: C, 66.48; H, 6.85;
N, 10.57. Found: C, 66.62; H, 6.87; N, 10.44.
1
0.5, MeOH); H NMR(D O, 250 MHz) d 1.84 (s, 3H),
2
4.44 (s, 2H), 4.61 (d, J=4.2 Hz, 1H), 4.68 (d, J=4.2 Hz,
1H), 5.16 (s, 1H), 5.17 (s, 1H), 7.43 (d, J=8.3 Hz, 2H),
7.68 (d, J=8.3 Hz, 2H); 13C NMR (D2O, 62.9MHz) d
25.0, 44.3, 79.2, 79.4, 98.4, 128.6, 129.5, 129.9, 146.0,
168.2, 173.2, 175.7, 183.0. HRMS m/z calcd for
C13H17N4O4 (MH+): 293.1250. Found: 293.1249.
trans-(4R,5R)-5-{4 - [Amino(benzyloxycarbonylimino)me-
thyl]phenylethylaminocarbonyl} - 1,3 - dioxolane - 4 - car-
boxylic acid ethyl ester (10). Compound 10 (184 mg,
50%) was prepared from (À)-3 (149 mg, 0.78 mmol)
and Boc-deprotected 9 according to Procedure B. The
Boc-group in 9 was removed prior to the coupling
reaction by stirring in trifluoroacetic acid/methylene
choride 1:1 for 1 h at room temperature, after which the
solvents were evaporated. The crude product 10 was
purified by flash column chromatography (ethyl acetate/
toluene 3:1). 10: aD22À38.4 (c 0.4, MeOH); 1H NMR
(CDCl3, 250 MHz) d 1.30 (t, J=7.1 Hz, 3H), 2.85 (t,
J=6.9 Hz, 2H), 3.55 (m, 2H), 4.28 (q, J=7.1 Hz, 2H),
4.59 (d, J=3.4 Hz, 1H), 4.73 (d, J=3.4 Hz, 1H), 5.09 (s,
1H), 5.12 (s, 1H), 5.20 (s, 2H), 6.70 (br s, 1H), 7.20 (d,
J=8.2 Hz, 2H), 7.82 (d, J=8.2 Hz, 2H), 7.26–7.48 (m,
2-[4-Amino(benzyloxycarbonylimino)methyl]ethanol (8).
To an ice cooled solution of acetyl chloride (70 mL) in
ethanol (140 mL) was added 2-(4-cyanophenyl)-ethanol
(7) (4.00 g, 27.2 mmol) in portions. The solution was
stirred at 0 ꢁC for 1 h and then at room temperature for
48 h. The solvent was evaporated and residual hydrogen
chloride was removed under reduced pressure. The resi-
due was dissolved in methanol saturated with ammonia
(150 mL) and stirred at room temperature overnight.
The solvent was evaporated and the residue was dis-
solved in tetrahydrofuran/water (1:1, 100 mL). Benzyl
chloroformate (4.24 mL, 29.9 mmol) and potassium
carbonate (11.41 g, 81.6 mmol) were added and the
resulting mixture was stirred at room temperature
overnight. Diethyl ether was added, the phases were
separated, the water phase was extracted with diethyl
ether, and the combined organic phases were washed
with water, dried, filtered, concentrated and purified by
flash column chromatography (toluene/ethyl acetate
5H), 9.51 (br s, 1H); 13C NMR(CDCl , 62.9 MHz) d
3
14.1, 35.4, 40.0, 62.1, 67.2, 77.1, 77.6, 96.8, 127.8, 128.2,
128.4, 128.6, 129.0, 132.9, 136.7, 143.1, 164.6, 168.0,
169.0, 169.5. Anal. calcd for C24H27N3O7: C, 61.40; H,
5.80; N, 8.95. Found: C, 61.26; H, 5.84; N, 8.82.
1
1:1) to give 8 (6.02 g, 74%) as a colorless solid. 8: H
NMR(CDCl , 250 MHz) d 2.72 (m, 2H), 3.30 (br s,
1H), 3.67 (m, 2H), 5.18 (s, 2H), 7.0–7.7 (m, 9H), 9.3 (br
3
trans-(4R,5R)-N1-{4 -[Amino(imino)methyl]phenylethyl} -
N2-benzyl-1,3-dioxolane-4,5-dicarboxamide (11). Com-
pound 11 (49 mg as the acetate salt, 86%) was prepared
from 10 (68 mg, 0.14 mmol) and benzylamine according
s, 1H); 13C NMR(CDCl , 62.9 MHz) d 38.8, 62.7, 67.1,
3
127.5, 128.0, 128.2, 128.5, 129.2, 132.3, 136.6, 143.9,
164.5, 168.6. Anal. calcd for C17H18N2O3: C, 68.44; H,
6.08; N, 9.39. Found: C, 68.32; H, 6.13; N, 9.37.
22
1
to Procedure C. 11: aD À3.8 (c 1.1, MeOH); H NMR
(methanol-d4, 250 MHz) d 1.90 (s, 3H), 2.96 (t, J=7.1
Hz, 2H), 3.54 (t, J=7.1 Hz, 2H), 4.43 (s, 2H), 4.55 (s,
2H), 5.11 (s, 1H), 5.15 (s, 1H), 7.20–7.35 (m, 5H), 7.47
(d, J=8.3 Hz, 2H), 7.75 (d, J=8.3 Hz, 2H); 13C NMR
(D2O, 62.9 MHz) d 25.9, 37.2, 42.3, 45.4, 80.1, 80.3,
N-Butyloxycarbonyl - 2 - [4 - amino(benzyloxycarbonyl-
imino)methyl]ethylamine (9). To a solution of 8 (2.39 g,
8.05 mmol) in pyridine (50 mL) was added mesyl chlo-
ride (0.73 mL, 9.66 mmol) and the mixture was stirred

1578
A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
99.1, 128.6, 129.8, 130.2, 130.4, 131.5, 132.5, 140.2,
148.5, 169.1, 173.4, 173.5, 184.4. HRMS m/z calcd for
C21H25N4O4 (MH+): 397.1876. Found: 397.1874.
1.50 (s, 3H), 1.52 (s, 3H), 4.31 (q, J=7.1 Hz, 2H), 4.82
(d, J=5.5 Hz, 1H), 4.88 (d,J=5.1 Hz, 1H); 13C NMR
(CDCl3, 62.9 MHz) d 14.1, 26.3, 62.3, 76.7, 77.2, 114.2,
169.9, 174.2. Anal. calcd for C9H14O60.86HOAc: C,
47.71; H, 6.51. Found: C, 47.61; H, 6.75.
trans-(4R,5R)-5-(2,5-Dichlorobenzylaminocarbonyl)-1,3-
dioxolane-4-carboxylic acid ethyl ester (12). Compound
12 (100 mg, 61%) was prepared from (À)-3 (90 mg, 0.47
mmol) and 2,5-dichloro-benzylamine according to Pro-
cedure B. Chromatography mobile phase: Toluene/ethyl
acetate 3:1. 12: aD22À25.03 (c 1.0, MeOH); 1H NMR
(CDCl3, 250 MHz) d 1.33 (t, J=7.1 Hz, 3H), 4.29 (q,
J=7.1 Hz, 2H), 4.40–4.60 (m, 2H), 4.69 (d, J=3.4 Hz,
1H), 4.83 (d, J=3.4 Hz, 1H), 5.18 (s, 1H), 5.27 (s, 1H),
trans-(4R,5R)-5-{4-[Amino(benzyloxycarbonylimino)me-
thyl]benzylaminocarb-onyl}-2,2-dimethyl - 1,3 - dioxolane-
4-carboxylic acid ethyl ester (16). Compound 16 (750
mg, 87%, a colorless solid) was prepared from 15 (387
mg, 1.78 mmol) and 4-(benzyloxycarbonyl)amidino-
benzylamine hydrochloride [Pab(Z)HCl] according to
Procedure B. Chromatography mobile phase: Toluene/
ethyl acetate 1:1. 16: a2D2À23.8 (c 0.6, MeOH); 1H NMR
(CDCl3, 250 MHz) d 1.32 (t, J=7.1 Hz, 3H), 1.45 (s,
3H), 1.46 (s, 3H), 4.29 (q, J=7.1 Hz, 2H), 4.50 (d,
J=6.2 Hz, 2H), 4.71 (d, J=5.4 Hz, 1H), 4.81 (d, J=5.4
Hz, 1H), 5.20 (s, 2H), 7.00 (t, J=6.2 Hz, 1H), 7.20–7.45
7.04 (br s, 1H), 7.18–7.33 (m, 3H); 13C NMR(CDCl ,
3
62.9 MHz) d 14.1, 40.9, 62.1, 76.8, 76,9, 97.0, 129.2,
129.9, 130.7, 131.8, 133.0, 136.6, 169.1, 169.5. HRMS
m/z calcd for C14H16Cl2NO5 (MH+): 348.0406. Found:
348.0419.
(m, 7H), 7.82 (d, J=8.3 Hz, 2H). 13C NMR(CDCl ,
3
trans-(4R,5R)-N1-(2,5 - Dichlorobenzyl) - N2 - cyclohexyl -
1,3 - dioxolane - 4,5 - dicarboxamide (13). Compound 13
(90 mg, 0.26 mmol) was dissolved in dioxane (5 mL) and
sodium hydroxide (1 M, 0.50 mL) was added dropwise.
The solution was stirred for 30 min at room tempera-
ture after which it was acidified with 1 M aqueous
hydrogen chloride to pH 2–3 and extracted several times
with dichloromethane. The organic layer was dried, fil-
tered and concentrated. The resulting carboxylic acid
was coupled with cyclohexylamine according to Proce-
dure B to give 13 (79 mg, 76%) as a colorless solid.
Chromatography mobile phase: Toluene/ethyl acetate
62.9 MHz) d 14.1, 26.2, 26.7, 42.7, 62.1, 67.2, 77.8,
113.4, 127.7, 128.0, 128.2, 128.4, 129.0, 133.9, 136.6,
142.1, 164.6, 167.7, 169.8, 170.0. Anal. calcd for
C25H29N3O7: C, 62.10; H, 6.05; N, 8.69. Found: C,
62.14; H, 5.89; N, 8.53.
trans-(4R,5R)-N1-{4-[Amino(imino)methyl]benzyl} - N2 -
benzyl - 2,2 - dimethyl - 1,3-dioxolane-4,5-dicarboxamide
(17). Compound 17 (112 mg as the acetate salt, 35%)
was prepared from 16 (290 mg, 0.60 mmol) and benzyl-
amine according to Procedure C. 17: a2D2À21.0 (c 0.9,
1
MeOH); H NMR(methanol- d4, 250 MHz) d 1.45 (s,
3:1. 13: a2D2À25.6 (c 3.2, MeOH); 1H NMR(CDCl
,
3H), 1.49 (s, 3H), 1.99 (s, 3H), 4.39 (d, J=14.9 Hz, 1H),
4.50 (d, J=14.9 Hz, 1H), 4.53 (s, 2H), 4.67 (s, 2H),
7.22–7.35 (m, 5H), 7.55 (d, J=8.4 Hz, 2H), 7.78 (d,
J=8.4 Hz, 2H); 13C NMR(methanol- d4, 62.9 MHz) d
20.8, 26.5, 26.6, 43.4, 43.8, 79.3, 79.4, 114.2, 128.3,
128.5, 129.2, 129.3, 129.5, 129.6, 139.7, 146.8, 168.1,
171.9, 172.2, 175.2. Anal. calcd for C22H26N4O40.85-
H2O1.57HOAc: C, 58.06; H, 6.59; N, 10.78. Found: C,
58.10; H, 6.31; N, 10.92.
3
250 MHz) d 1.08–1.47 (m, 5H), 1.58–1.80 (m, 3H), 1.81–
1.98 (m, 2H), 3.78 (m, 1H), 4.51 (d, J=5.7 Hz, 1H), 4.59
(d, J=5.7 Hz, 1H), 4.54 (d, J=6.2 Hz, 2H), 5.14 (s,
1H), 5.17 (s, 1H), 6.63 (d, J=8.2 Hz, 1H), 7.13–7.35 (m,
3H), 7.53 (t, J=6.2 Hz, 1H); 13C NMR(CDCl
,
3
62.9 MHz) d 24.7, 25.4, 32.8, 32.9, 40.9, 48.2, 77.4, 77.5,
96.3, 129.0, 129.7, 130.7, 131.7, 132.9, 136.8, 167.9, 169.1.
HRMS m/z calcd for C18H22Cl2NaN2O4 (MNa+):
423.0854. Found: 423.0862.
trans-(4R,5R)-2-Methyl-1,3-dioxolane-4,5-dicarboxylic
acid diethyl ester (18). To a solution of (+)-diethyl l-
tartrate [(+)-1] (3.00 g, 14.5 mmol) in toluene (35 mL)
were added 1,1-diethoxyethane (11.8 mL, 82.5 mmol)
and camphorsulfonic acid (790 mg, 3.40 mmol). The
mixture was stirred at 95 ꢁC for 24 h, after which it was
washed with saturated aqueous sodium hydrogen car-
bonate. The organic phase was dried, filtered and con-
centrated and the crude product was purified by flash
column chromatography (toluene/ethyl acetate 10:1) to
give 18 in 80% yield (2.68 g) as a slightly brown oil. 18:
a2D2À66.3 (c 0.9, MeOH) (lit:13bÀ72.2); 1H NMR
(CDCl3, 250 MHz) d 1.32 (t, J=7.1 Hz, 6H), 1.54 (d,
J=4.9 Hz, 3H), 4.31 (q, J=7.1 Hz, 4H), 4.69 (d, J=4.1
Hz, 1H), 4.78 (d, J=4.1 Hz, 1H), 5.42 (q, J=4.9 Hz,
trans-(4R,5R)-2,2-Dimethyl-1,3 - dioxolane - 4,5 - dicar-
boxylic acid diethyl ester (14). (+) - Diethyl l-tartrate
[(+)-1] (2.727 g, 13.2 mmol) was dissolved in toluene
(20 mL), 2-methoxy propene (2.0 mL, 21 mmol) and
camphorsulfonic acid (70.0 mg, 0.30 mmol) were added
and the mixture was stirred at room temperature for 5
h. The solution was washed with saturated sodium
hydrogen carbonate and the organic phase was dried,
filtered and concentrated. The crude product was pur-
ified by flash column chromatography (toluene/ethyl
acetate 19:1) to give 14 (2.40 g, 74%) as a colorless
liquid. 14: a2D2À52.5 (c 3.6, MeOH) (lit:
À51.2); H
23a
1
NMR(CDCl 3, 250 MHz) d 1.32 (t, J=7.1 Hz, 6H), 1.50
(s, 6H), 4.28 (q, J=7.1 Hz, 4H), 4.78 (s, 2H); 13C NMR
(CDCl3, 62.9 MHz) d 14.1, 26.4, 61.9, 77.2, 113.8, 169.7.
1H); 13C NMR(CDCl , 62.9 MHz) d 14.2, 19.7, 61.9,
3
62.0, 77.4, 77.6, 104.8, 169.2, 169.9. Anal. calcd for
C10H16O6: C, 51.72; H, 6.94. Found: C, 51.81; H, 6.98.
trans-(4R,5R)-2,2 - Dimethyl - 1,3 - dioxolane - 4,5 - dicar-
boxylic acid monoethyl ester (15). Compound 15 (691
mg, 47%) was prepared from 14 (1.678 g, 6.82 mmol)
according to Procedure A. 15: a2D2À17.3 (c 2.1, MeOH);
trans-(4R,5R)-2-Methyl-1,3-dioxolane-4,5-dicarboxylic
acid monoethyl ester (diastereomeric mixture) (19).
Compound 19 (183 mg, 69%, a slightly yellow oil)
1H NMR(CDCl , 250 MHz) d 1.35 (t, J=7.1 Hz, 3H),
3

A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
1579
(diastereomeric mixture 1:1) was prepared from 18 (300
mg, 1.29 mmol) according to Procedure A. 19: a2D2À65.7
3. Rang, H. P.; Dale, M. M. In Pharmacology, 2nd ed.;
Churchill Livingstone: 1991, pp. 377–398.
4. Uzan, A. Emerging Drugs 1998, 3, 189.
1
(c 0.7, MeOH); H NMR(CDCl , 250 MHz) d 1.38 (t,
3
5. Das, J.; Kimball, S. D. Bioorg. Med. Chem. 1995, 3, 999.
6. Diaz-Ricart, M. Drug News Perspect. 2000, 13, 363.
7. (a) Semple, J. E.; Rowley, D. C.; Brunck, T. K.; Ripka,
W. C. 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. Bioorg. Med. Chem. Lett.
1998, 8, 1249. (c) Hart, S. A.; Sabat, M.; Etzkorn, F. A. J.
Org. Chem. 1998, 63, 7580. (d) Noteberg, D.; Branalt, J.;
Kvarnstrom, I.; Classon, B.; Samuelsson, B.; Nillroth, U.;
Danielsson, U. H.; Karlen, A.; Hallberg, A. Tetrahedron 1997,
53, 7975. (e) Turbanti, L.; Cerbai, G.; Di Bugno, C.; Giorgi,
R.; Garzelli, G.; Criscuoli, M.; Renzetti, A. R.; Subissi, A.;
Bramati, G.; DePriest, S. A. J. Med. Chem. 1993, 36, 699.
8. Noteberg, D.; Branalt, J.; Kvarnstrom, I.; Linschoten, M.;
Musil, D.; Nystrom, J-E.; Zuccarello, G.; Samuelsson, B. J.
Med. Chem. 2000, 43, 1705.
J=7.1 Hz, 3H), 1.48–1.62 (m, 3H), 4.32 (q, J=7.1 Hz,
2H), 4.71–4.91 (m, 2H), 5.45 (q, J=4.8 Hz, 1H); 13C
NMR(CDCl , 62.9 MHz) d 14.1, 19.6, 62.3, 76.6, 77.6,
3
105.1, 169.1, 169.6, 174.1, 175.0. Anal. calcd for
C8H12O60.32HOAc: C, 46.45; H, 5.99. Found: C, 46.45;
H, 5.96.
trans-(4R,5R) - 5 - {4 - [Amino(benzyloxycarbonylimino)-
methyl]benzylaminocarbonyl}-2-methyl-1,3-dioxolane-4-
carboxylic acid ethyl ester (diastereomeric mixture 1:1)
(20). Compound 20 (375 mg, 61%, a white solid) (dia-
stereomeric mixture 1:1) was prepared from 19 (267 mg,
1.31 mmol) and 4-(benzyloxycarbonyl)amidinobenzyl-
amine hydrochloride [Pab(Z)HCl] according to Proce-
dure B. Chromatography mobile phase: Ethyl acetate/
toluene 20:1. 20: aD22À18.8 (c 0.5, MeOH); 1H NMR
(CDCl3, 250 MHz) d 1.31 (t, J=7.1 Hz, 3H), 1.46 (d,
J=4.8 Hz, 3H), 4.18–4.31 (m, 2H), 4.37–4.50 (m, 2H),
4.61–4.81 (m, 2H), 5.13 (s, 2H), 5.35 (q, J=4.8 Hz, 1H),
7.10–7.50 (m, 7H), 7.71–7.87 (m, 2H); 13C NMR
(CDCl3, 62.9 MHz) d 14.6, 19.6, 19.9, 42.6, 62.0, 67.1,
77.3, 77.9, 78.2, 104.2, 104.3, 127.3, 127.4, 127.9, 128.0,
128.4, 133.7, 136.6, 142.0, 164.4, 167.8, 169.4, 169.6, 169.7,
170.0. Anal. calcd for C24H27N3O70.12toluene: C, 62.08;
H, 5.86; N, 8.75. Found: C, 62.11; H, 5.77; N, 8.82.
9. To the best of our knowledge, 1,3-dioxolane-4,5-dicarbox-
amides have not previously been used as N-acylproline isosteres.
10. Antonsson, T.; Bylund, R.; Gustafsson, N.; Nilsson, N.
World Patent, 94 29336, 1994.
11. (a) Tucker, T. J.; Brady, S. F.; Lumma, W. C.; Dale
Lewis, S.; Gardell, S. J.; Naylor-Olsen, A. M.; Yan, Y.; Sisko,
J. T.; Stauffer, K. J.; Lucas, B. J.; Lynch, J. J.; Cook, J. J.;
Stranieri, M. T.; Holahan, M. A.; Lyle, E. A.; Baskin, E. P.;
Chen, I-W.; Dancheck, K. B.; Krueger, J. A.; Cooper, C. M.;
Vacca, J. P. J. Med. Chem. 1998, 41, 3210. (b) Sanderson, P.
E.; Lyle, T. A.; Vacca, J. P.; Lumma, W. C.; Brady, S. F.;
Tucker, T. J. US Patent 05792779 (c) Sanderson, P. E.; Lyle,
T. A.; Vacca, J. P.; Lumma, W. C.; Brady, S. F.; Tucker, T. J.
World Patent 09730708.
12. Beck, A. K.; Bastani, B.; Plattner, D. A.; Petter, W.; See-
bach, D.; Braunschweiger, H.; Gysi, P.; La Vecchia, L. Chimia
1991, 45, 238.
13. (a) Seebach, D.; Beck, A. K.; Imwinkelried, R.; Roggo, S.;
Wonnacott, A. Helv. Chim. Acta 1987, 70, 954. (b) Tsuzuki, X.
Bull. Chem. Soc. Jpn. 1936, 11, 362. (c) Dulphy, H.; Gras, J.;
Lejon, T. Tetrahedron 1996, 52, 8517.
trans-(4R,5R)-N1-{4 - [Amino(imino)methyl]benzyl} - N2 -
benzyl-2-methyl-1,3-dioxolane-4,5-dicarboxamide (dia-
stereomeric mixture 1:1) (21). Compound 21 (146 mg as
the acetate salt, 66%) (diastereomeric mixture mixture
1:1) was prepared from 20 (228 mg, 0.49 mmol) and
benzylamine according to Procedure C. 21: a2D2À16.4 (c
1
0.9, MeOH); H NMR(methanol- d4, 250 MHz) d 1.46
(d, J=4.8 Hz, 3H from diastereomer 1), 1.48 (d, J=4.8
Hz, 3H from diastereomer 2), 1.92 (s, 3H), 4.37–4.62
(m, 4H), 4.62–4.70 (m, 2H), 5.31 (m, 1H), 7.20–7.32 (m,
5H), 7.58 (d, J=8.3 Hz, 2H), 7.81 (d, J=8.3 Hz, 2H);
13C NMR(the free base) (methanol- d4, 62.9 MHz) d
19.9, 23.6, 43.4, 43.8, 79.7, 80.0, 105.4, 128.3, 128.4,
128.5, 128.7, 129.2, 129.6, 139.7, 146.7, 168.2, 171.8,
172.0, 172.2, 172.4. HRMS m/z calcd for C21H25N4O4
(MH+): 397.1876. Found: 397.1867.
14. Initially several standard reaction conditions were exam-
ined, but in our hands they all failed. The reaction of (+)-1
with diethoxymethane and 4-toluenesulfonic acid was exam-
ined, resulting in the formation of diethyl 2,3-di-O-ethoxy-
methyl-l-tartrate instead of the desired product. Numerous
other attempts also failed, including the use of (a) diethoxy-
methane, 4-toluenesulfonic acid and lithium bromide15 and (b)
N-bromosuccinimide and dimethyl sulfoxide.16
˜
15. Rodrıguez-Galan, A.; Bou, J. J.; Munoz-Guerra, S. J.
Polym. Sci. Part A: Polym. Chem. 1992, 30, 713.
16. Hanessian, S.; Yang-Chung, G.; Lavellee, P.; Pernet, A. G.
J. Am. Chem. Soc. 1972, 94, 8929.
17. Since it proved impossible to extract the afforded acid into
an organic solvent, the reaction mixture was neutralized with
hydrochloric acid and evaporated, and the crude acid was used
directly in the coupling reaction.
18. By first coupling (+)-3 with cyclohexylamine instead of
Pab(Z) we avoided the extraction problems (following the
subsequent hydrolysis step) described in ref 17.
19. Reers, M.; Koschinsky, R.; Dickneite, R.; Hoffmann, D.;
Czech, J.; Stuber, W. J. Enzyme Inhib. 1995, 9, 61.
20. The Boc group was introduced since its presence proved to
simplify the purification of the desired amine.
Acknowledgements
We would like to thank AstraZeneca R&D Molndal for
financial support. We would also like to thank Erika
Gyzander and the Biochemistry Group, AstraZeneca
R&D Molndal, for providing the biological testings,
and Karl-Erik Karlsson, AstraZeneca R&D Molndal,
for performing the HRMS measurements.
References and Notes
21. Teger-Nilsson, A. C.; Bylund, R.; Gustafsson, D.;
Gyzander, E.; Eriksson, U. Thromb. Res. 1997, 85, 133.
22. Lipshutz, B. H.; Barton, J. C. J. Org. Chem. 1988, 53,
4495.
1. Ripka, A. S.; Rich, D. H. Curr. Opin. Chem. Biol. 1998, 2,
441.
2. Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem.
2000, 43, 305.
23. (a) Tsuzuki, X. Bull. Chem. Soc. Jpn. 1935, 10, 255. (b)
Lucas, B. J. Am. Chem. Soc. 1941, 63, 1653. (c) Onoda, T.;

1580
A. Dahlgren et al. / Bioorg. Med. Chem. 10 (2002) 1567–1580
Shirai, R.; Kawai, N.; Iwasaki, S. Tetrahedron 1996, 52,
13327.
27. (a) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997,
276, 307.
24. Bode, W.; Mayr, I.; Baumann, U.; Uber, R.; Stone, S. R.;
Hofskeenge, J. EMBO. J. 1989, 8, 3467.
25. Musil, D., Nilsson, I. Experimental results; AstraZeneca
R&D.
26. Skrzypczak-Jankun, E.; Carperos, V. E.; Ravichandran,
K. G.; Tulinsky, A. J. Mol. Biol. 1991, 221, 1379.
28. Collaborative Computational Project, Number 4. The
CCP4 suite: Programs for protein crystallography. Acta Cryst.
1994, D50, 760.
29. Computational results for the crystallographic refinement
were obtained using the CNX program from Molecular
Simulation Inc.