Multipolar interactions in the D pocket of thrombin: Large differences between tricyclic imide and lactam inhibitors

Article abstract of DOI:10.1039/b602585d

Two series of tricyclic inhibitors of the serine protease thrombin, imides (±)-1-(±)-8 and lactams (±)-9-(±)-13, were analysed to evaluate contributions of orthogonal multipolar interactions with the backbone CO moiety of Asn98 to the free enthalpy of pro

Full text of DOI:10.1039/b602585d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Multipolar interactions in the D pocket of thrombin: large differences between
tricyclic imide and lactam inhibitors†
Eliane Schweizer,a Anja Hoffmann-Ro¨der,b Jacob A. Olsen,a Paul Seiler,a Ulrike Obst-Sander,c Bjo¨rn Wagner,c
Manfred Kansy,c David W. Banner*c and Franc¸ois Diederich*a
Received 21st February 2006, Accepted 12th April 2006
First published as an Advance Article on the web 10th May 2006
DOI: 10.1039/b602585d
Two series of tricyclic inhibitors of the serine protease thrombin, imides ( )-1–( )-8 and lactams
( )-9–( )-13, were analysed to evaluate contributions of orthogonal multipolar interactions with the
=
backbone C O moiety of Asn98 to the free enthalpy of protein–ligand complexation. The lactam
derivatives are much more potent and more selective inhibitors (Ki values between 0.065 and 0.005 lM,
selectivity for thrombin over trypsin between 361- and 1609-fold) than the imide compounds (Ki values
between 0.057 and 23.7 lM, selectivity for thrombin over trypsin between 3- and 67-fold). The increase
in potency and selectivity is explained by the favorable occupancy of the P-pocket of thrombin by the
additional isopropyl substituent in the lactam derivatives. The nature of the substituent on the benzyl
ring filling the D pocket strongly influences binding potency in the imide series, with Ki values
increasing in the sequence: F < OCH2O < Cl < H < OMe < OH < Npyr ꢀ Br. This sequence can be
explained by both steric fit and the occurrence of orthogonal multipolar interactions with the backbone
=
C O moiety of Asn98. In contrast, the substituent on the benzyl ring hardly affects the ligand potency
in the lactam series. This discrepancy was clarified by the comparison of X-ray structures solved for
co-crystals of thrombin with imide and lactam ligands. Whereas the benzyl substituents in the imide
˚
=
inhibitors are sufficiently close (≤3.5 A) to the C O group of Asn98 to allow for attractive orthogonal
˚
multipolar interactions, the distances in the lactam series are too large (≥4 A) for attractive dipolar
contacts to be effective.
( )-2 (inhibitory constant Ki = 0.31 lM) to F-substituted ( )-
Introduction
1 (Ki = 0.057 lM). Subsequently, we quantified the attractive
5
Over recent years, we have pursued the systematic substitu-
tion of H- for F-atoms (“fluorine scan”) in nonpeptidic, tri-
cyclic inhibitors1 of thrombin in order to map the fluorophilic-
ity/fluorophobicity of an entire enzyme active site.2,3 During
H/F substitution of the benzyl ring, which fills the hydrophobic
distal (D) pocket (Fig. 1) of the serine protease from the blood
coagulation cascade,4 we had observed by X-ray analysis of a co-
=
nature of orthogonal C–F · · · C O interactions in model studies.
Furthermore, we documented in a comprehensive review the
general occurrence of such orthogonal electrostatic interactions
between dipoles in structural chemistry and biology.6
Here, we report the synthesis and biological activity of a series of
new thrombin inhibitors, consisting of tricyclic imides ( )-5–( )-8
and tricyclic lactams ( )-12 and ( )-13. Their inhibitory potencies
and physicochemical properties (pKa, log D) are compared to
those of the previously described imides ( )-1–( )-4 and lactams
( )-9–( )-11.1,2a,b,16 By this comparison, we intended to explore
how binding affinity changes when different dipoles such as C–
crystal a short orthogonal contact between the C–F residue of ( )-
˚
=
=
1 and the backbone C O of Asn98 (d(F · · · C O) = 3.5 A, angle
◦
2a,b
=
a(F · · · C O) = 96 ). Database mining subsequently revealed
the frequent occurrence of similar electrostatic contacts both in
small-molecule crystal structures (Cambridge Structural Database
(CSD)) and in co-crystal structures of protein–ligand complexes
(Protein Databank (PDB)). Therefore, we proposed that mul-
=
=
F, C–Br, C–O and C Npyridyl interact with the backbone C O of
Asn98 in the D pocket. The results of this study are quite surpris-
ing: whereas large differences in binding affinity are observed in the
series of the tricyclic imide inhibitors, the substituted lactams are
nearly equipotent. The structures of co-crystals of thrombin with
the active (3aS,4R,8aS,8bR)-configured enantiomers of lactams
( )-12 and ( )-13 were solved, and X-ray structural comparisons
finally provided an explanation for the unexpected differences in
binding behaviour between the two classes of ligands.
=
tipolar orthogonal C–F · · · C O interactions are a significant
contributor to the gain in binding free enthalpy (DDG(
=
)-2 → ( )-1
−4.4 0.7 kJ mol−1) measured upon changing from H-substituted
aLaboratorium fu¨r Organische Chemie, ETH-Zu¨rich, Ho¨nggerberg HCI,
CH-8093, Zu¨rich, Switzerland. E-mail: diederich@org.chem.ethz.ch;
Fax: +41 44 6321109; Tel: +41 44 6322992
bInstitut fu¨r Organische Chemie, Johannes Gutenberg-Universita¨t, Duesberg-
weg 10-14, D-55128, Mainz, Germany
Results and discussion
cPharma Division, Pra¨klinische Forschung, F. Hoffmann–La Roche AG,
CH-4070, Basel, Switzerland
Synthesis of the tricyclic thrombin inhibitors
† Electronic supplementary information (ESI) available: Synthesis of
precursors to the new inhibitors, crystal packing and overlay of enzyme–
ligand co-crystal structures. See DOI: 10.1039/b602585d
The synthesis of the new inhibitors followed earlier published
protocols.2b,7 The 1,3-dipolar cycloadditions between maleimides
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Fig. 1 Left: Schematic representation of the binding mode of the tricyclic inhibitors in the active site of thrombin. The active site is defined by the
catalytic centre with the nucleophilic Ser195 and the oxanion hole, the selectivity (S1) pocket, the large hydrophobic distal (D) pocket and a small proximal
(P) pocket. Right: Inhibitors ( )-1–( )-8 and ( )-9–( )-13 investigated in this study. Only the (3aS,4R,8aS,8bR)-configured enantiomer is bound.1,2
14/158 (Scheme 1) and the azomethine ylide9 formed from L-
proline and 4-bromobenzaldehyde furnished in each case two of
the possible four diastereoisomers (( )-16/( )-18 starting from
14, and ( )-17/( )-19 from 15) which were separated chro-
matographically. Subsequent conversion of the desired endo,trans-
configured diastereoisomers (see the caption to Scheme 1 for
definitions) into nitriles ( )-20/( )-21 and Pinner reaction10
afforded the tricyclic imide inhibitors ( )-6 and ( )-7, respectively.
11
Ether cleavage of ( )-20 with BBr3 to ( )-22, followed by the
Pinner reaction, yielded ligand ( )-8. Ligand ( )-5 was obtained
by 1,3-dipolar cycloaddition between 4-bromobenzylmaleimide
2312 and the azomethine ylide formed from 4-formylbenzonitrile
and L-proline, followed by separation of the diastereoisomeric
cycloadducts ( )-24/( )-25 and Pinner reaction (Scheme 1).
The synthesis of the tricyclic lactam inhibitors ( )-12 and
( )-13, directing an additional Pri substituent into the P pocket
of thrombin, also followed previously established protocols.2b,7,16
=
Regio- and diastereoselective reduction of the ‘upper’ C O group
in ( )-26 and ( )-16, using superhydride (Li[Et3BH]) in THF,
Fig.
2 X-Ray crystal structure of the (C1S,C4S,C5R,C6R,C8S)-
afforded the hydroxylactams ( )-27 and ( )-28, respectively. We
recently solved the X-ray crystal structure of the previously
reported2a,b fluorinated hydroxylactam ( )-29, obtained by re-
duction of the corresponding imide under similar conditions.
It shows that hydride attack occurred from the less hindered
exo-side of the bicyclic perhydropyrrolo[3,4-c]pyrrole scaffold,
leading to a cis orientation of the hydroxyl group with regard
to the bromophenyl substituent (Fig. 2). We therefore assign to
the newly created stereogenic centre in ( )-27 and ( )-28 the
same configuration as seen for ( )-29. Similar diastereofacial
selectivities in nucleophilic additions to N-benzylated tricyclic
imide scaffolds, e.g. addition of CF3SiMe3,13 have been observed
before.
configured hydroxylactam 29. Being a racemic mixture, an equal number
of molecules with opposite stereochemistry are present in the crystal.
Numbering is arbitrary. Atomic displacement parameters obtained at
173 K are drawn at the 30% probability level (for the crystal packing,
see ESI).
The hydroxylactams ( )-27 and ( )-28 were converted into the
corresponding sulfones ( )-30 and ( )-31, respectively, upon reac-
tion with p-toluenesulfinic acid in the presence of CaCl2. Introduc-
tion of the desired isopropyl group in compounds ( )-32 and ( )-
33 was achieved by displacement of the toluenesulfonyl group with
PriMgCl in the presence of ZnCl2.14 In both steps, the nucleophile
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Scheme 1 Synthesis of the tricyclic imide inhibitors. Reagents and conditions: (i) CH3CN, 80 ◦C, 14–16 h; ( )-16 (44%)/( )-18 (53%), ( )-17 (35%)/( )-19
(18%), ( )-24 (41%)/( )-25 (38%); (ii) CuCN, DMF, 165 ◦C, 24 h, or [Pd2(dba)3] dppf, Zn(CN)2, DMF, 120 ◦C, 24 h; ( )-20 (78%), ( )-21 (32%);
(iii) MeOH, HCl(g), CH2Cl2, 4 ◦C, 28–35 h; (iv) NH3, MeOH, 65 ◦C, 3 h; ( )-5 (49%), ( )-6 (21%), ( )-7 (28%), ( )-8 (40% from ( )-20); (v) BBr3,
CH2Cl2, −50 ◦C → 25 ◦C, 8 h. DMF = dimethylformamide, dba = dibenzylideneacetone, dppf = diphenylphosphinoferrocene. Exo and endo refer to the
orientation of the 4-bromophenyl substituent at C(4) with respect to the bicyclic perhydropyrrolo[3,4-c]pyrrole scaffold, and cis and trans to the position
of this 4-bromophenyl ring with respect to the configuration of C(8a) at the fusion of the two pentagons in the perhydropyrrolizidine bicycle (for atom
numbering, see Fig. 1).
adds to the presumed acyliminium ion intermediate from the exo
face of the bicyclic perhydropyrrolo[3,4-c]pyrrole scaffold. The
targeted ligands ( )-12 and ( )-13 were finally obtained via nitriles
( )-34 and ( )-35, again using the Pinner reaction (Scheme 2).
the r-inductive effect of the phenylamidinium ring in the a-
position to the N-atom and (ii) the large r-inductive effects
=
of the two imide C O moieties in the b-position. The latter
explanation is nicely corroborated by the new results: upon
changing from the imides (pKa1 4.4–4.6) to the lactams (pKa1
6.2–6.5), one of these r-accepting pathways is removed and the
acidity decreases substantially. The pKa3 values of the pyridine
and phenol substituents in ( )-7 and ( )-8 are in the expected
range. The log D values (log D is the logarithmic coefficient of the
distribution of a compound between octanol and water at pH 7.4)
of all compounds (−0.4 to −1.6) are quite negative due to the
phenylamidinium moiety.2c They expectedly increase slightly upon
changing from the imide to the lactam inhibitors (e.g. compare the
Cl-substituted imide ( )-4 (log D −1.0) and lactam ( )-12 (log D
−0.4)). However, this increase in lipophilicity cannot explain the
large differences in potency between imide- and lactam-based
ligands, discussed below.
Biological results
All newly prepared compounds were subjected to biological
assays15 to determine the inhibition constants for thrombin
(Ki/lM) and the selectivity for thrombin over the digestive serine
protease trypsin (Ki[Try]/Ki[Thr]). Furthermore, physicochemical
properties (log D and pKa) of the ligands were also determined
following protocols previously described in great detail.2c Whereas
the pKa2 values of the phenylamidinium substituent are in the
expected range (10.7 to 11.1), the pKa1 values for the tertiary
amine centres (4.4 to 4.6) in the tricyclic imides are remarkably
low. As previously discussed,2c these low values are due to (i)
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Scheme 2 Synthesis of the tricyclic lactam inhibitors ( )-12 and ( )-13. Reagents and conditions: (i) Li[Et3BH], CH2Cl2, −78 ◦C → 0 ◦C, 2 h; (ii)
4-toluenesulfinic acid, CaCl2, CH2Cl2, 25 ◦C, 5–7 d; ( )-30 (32% from ( )-26), ( )-31 (97% from ( )-16); (iii) PriMgCl, ZnCl2, CH2Cl2, 25 ◦C, 16–24 h;
(
)-32 (61%), ( )-33 (46%); (iv) CuCN, DMF, 165 ◦C, 36 h or [Pd2(dba)3], dppf, Zn(CN)2, DMF, 120 ◦C, 24 h; ( )-34 (43%) ( )-35 (62%); (v) MeOH,
HCl(g), CH2Cl2, 4 ◦C, 28–35 h; (vi) NH3, MeOH, 65 ◦C, 3 h; ( )-12 (55%), ( )-13 (25%).
The chromogenic binding assay confirmed that the lactam
derivatives are both more potent and more selective thrombin
inhibitors than the imide-based ligands. In the imide series,
inhibitory strength varies between Ki = 0.057 and 23.7 lM,
whereas the lactam derivatives show Ki values between 0.065
and 0.005 lM. The large extra binding free enthalpy of the
lactam inhibitors is gained by occupancy of the tight lipophilic
P pocket of thrombin by the Pri substituent. Note that natural
substrates of thrombin also prefer directing a valine side-chain
into this pocket.4 The selectivity for thrombin over trypsin is also
dramatically increased upon introduction of the Pri substituent:
while Ki[Try]/Ki[Thr] in the imide series amounts to a moderate
3–67-fold, the selectivity in the lactam series is excellent and varies
between 361–1609-fold. Again, this greatly enhanced selectivity
originates from the favorable occupancy of the P pocket of
thrombin by the Pri substituent, since this pocket is absent in
trypsin.1,7 The extremely high, 1609-fold selectivity of the 4-
chlorophenyl derivative ( )-12 is by far the largest measured for
all tricyclic inhibitors prepared so far.1,2,16
The inhibitory constants in the series of imide inhibitors ( )-
1–( )-8 vary greatly. If the substituent on the benzyl ring in the
D pocket – the only variable in the series – is considered, Ki/M
increases in the sequence F < OCH2O < Cl < H < OMe <
OH < Npyr ꢀ Br (Table 1). An overlay of the crystal structures
of the thrombin complexes with ( )-1 (F),2a ( )-3 (OCH2O)1a
and (+)-36 (OCH2O)17 (Fig. 3) shows that the inhibitors adopt
a nearly identical position in the active site of thrombin while
the surrounding protein stucture, in particular the residues lining
the D pocket,18 i.e. the loop segment Glu97A–Leu99, as well as
Tyr60A and Trp215, are geometrically highly conserved. While we
are well aware that a variety of factors such as lipophilicity and
solvation (as expressed by log D) and van der Waals interactions
(such as with Ca–H of Asn98) certainly influence the measured Ki
=
values, we propose that multipolar interactions with the C O
group of Asn98 together with steric effects make important
contributions to the observed differences in potency. In the halide
series, potency decreases in the sequence F (( )-1) > Cl (( )-4) ꢁ
Br (( )-5). While the F- and Cl-substituted derivatives undergo
=
efficient, nearly orthogonal multipolar interactions with the C O
group,2a,b,6 making them better inhibitors than unsubstituted ( )-
2, the Br substituent is clearly too bulky to fit. When the F-atom in
the co-crystal structure of ( )-1 (PDB-code: 1OYT) is replaced by
a Br-atom, the latter and the carbonyl C-atom of Asn98 are at a
˚
repulsive van der Waals distance of 3.2 A; relaxing the ligand
in the computer modeling19 induces a substantial shift of the
tricyclic inhibitor away from Asn98. The potency of the piperonyl-
substituted ligand ( )-3, similar to that of ( )-1, is more difficult to
compare, since its upper O-atom undergoes additional H-bonding
to the HO residue of Tyr60A.1a Nevertheless, the distance of the
˚
second O-atom to the C-atom of Asn98 (d = 3.4 A) suggests
also a favorable contribution from multipolar interactions. Such
interactions should also contribute to the binding affinity of the
MeO- (( )-6) and HO- (( )-8) substituted ligands,2d although this
favorable contribution is presumably compensated by unfavorable
steric interactions of the MeO residue (modeling) in the complex of
( )-6 and unfavorable desolvation of the HO group in the complex
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Table 1 Inhibitory activities and physicochemical properties of the tricyclic thrombin inhibitors
b
b
b
Ki/lMa
Ki[Try]/Ki[Thr]
pKa1
pKa2
pKa3
log Dc
Imides:
(
(
(
(
(
(
(
(
)-1d,e
)-2d,f
)-3d,f
)-4d,h
)-5d
0.057
0.31
0.09
0.19
23.7
0.621
1.54
0.89
67
15
7.8
30
>2.9
4.1
15.4
9.7
4.47
n.d.g
n.d.
4.60
4.56
4.58
4.38
4.62
11.14
n.d.
n.d.
10.98
11.11
11.00
10.97
10.72
—
—
—
—
—
—
3.75
9.32
−1.16
−0.99
n.d.
−0.95
−0.69
−1.6
)-6d
)-7d
< −1
)-8d
−1.22
Lactams:
(
(
(
(
(
)-9d,e
)-10d,i
)-11d,j
)-12d
)-13d
0.005
0.065
0.013
0.008
0.015
413
385
760
1609
361
6.52
n.d.
n.d.
6.16
6.25
n.d.
n.d.
n.d.
n.d.
10.87
—
—
—
—
—
−0.81
−0.99
n.d.
−0.37
−0.92
a The uncertainty of the measured Ki values is 20%. b pKa1: tertiary amine in the tricyclic core; pKa2: phenylamidinium; pKa3: pyridine and phenol,
respectively; accuracy of the pKa measurements: 0.1 pKa units. c Accuracy of the log D measurements: 0.1 log D units. d Only the (3aS,4R,8aS,8bR)-
configured enantiomer is bound, as determined from the crystal structure analysis in refs. 1 and 2. e From ref. 2a. f From ref. 1a. g n.d. = not determined.
h From ref. 2b. i From ref. 16. j From ref. 1b.
of ( )-8. The N-atom of the pyridine ligand is too far away (>3.9
Conclusions
˚
=
A) from the C O group (modeling) to undergo any significant,
strongly distance-dependent6 multipolar interaction.
Two series of tricyclic inhibitors of the serine protease thrombin,
imides ( )-1–( )-8 and lactams ( )-9–( )-13, were analysed
in order to evaluate the importance of orthogonal multipolar
interactions in the hydrophobic D pocket of the enzyme. Physic-
ochemical property analysis showed that the log D values become
less negative upon changing from the imide to the lactam series.
Furthermore, the pKa value of the tertiary amine centre in the
tricyclic scaffold increases by ca. 2 units when passing from the
imide to the lactam ligands, mainly due to the removal of the
While the variation in potency is large in the series of tricyclic
imide inhibitors, the four lactams ( )-9,2a,b ( )-11,1b ( )-12 and
( )-13 bearing substituents on the benzyl ring are nearly equipo-
tent, with Ki values varying between 0.005 and 0.015 lM. Since
only one X-ray crystal structure of a lactam inhibitor (( )-11)
bound to thrombin existed prior to this work, the structures
of the co-crystals formed by the new Cl- and MeO-substituted
˚
ligands were solved to 1.3 A (( )-12; PDB-code: 2CF8) and
˚
=
r-acceptor pathway between the N-atom and one b-C O unit,
1.79 A (( )-13; PDB-code: 2CF9) resolution. Fig. 4 depicts the
thereby confirming earlier interpretations of the remarkably low
pKa values (≈ 4.5) of this centre in the imide inhibitors.2c Biological
assays demonstrated large differences between the potency of
the imide and lactam series of inhibitors. In accordance with
previous results, the lactam derivatives are much more potent and
more selective inhibitors (Ki values between 0.065 and 0.005 lM,
selectivity for thrombin over trypsin between 361- and 1609-fold)
than the imide compounds (Ki values between 0.057 and 23.7 lM,
selectivity for thrombin over trypsin between 3- and 67-fold).
These differences originate from the favorable occupancy of the
tight hydrophobic P pocket by the isopropyl residue of the lactam
inhibitors; this pocket is absent in trypsin. In the imide series,
binding potency is determined by the nature of the substituent on
chloro derivative ( )-12 in the active site of thrombin, whereas the
complex of the methoxy compound ( )-13 is shown in Fig. 5. Both
crystal structures clearly demonstrate that the electronegative O-
and Cl-atoms attached to the 4-position of the benzyl residue in
=
the tricyclic lactams are at much greater distance from the C O
group of Asn98 than in the protein-bound imide derivatives. In the
˚
complex of ( )-12, the distance Cl · · · CC=
amounts to 4.0 A,
O(Asn)
and the same O · · · C distance is measured in the complex of ( )-
13. At such large distances, multipolar interactions are no longer
effective, which explains why the measured binding potencies are
very similar. An overlay of crystal structures of imide and lactam
inhibitors (ESI) provides another nice illustration of the different
distances of the protein–ligand contacts seen in the D pocket.
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Fig. 3 Left: Overlay of the co-crystal structures of the imide-based inhibitors ( )-1 (PDB-Code: 1OYT),2a
(
)-36 (PDB-Code: 1VZQ)17 and ( )-31a with
thrombin, as determined by X-ray crystallographic analysis, showing a nearly perfect superimposition of the protein residues and the inhibitors. Right:
=
Zoom into the D pocket. Noticeable is the exact overlap of the backbone C O group of Asn98, involved in multipolar interactions with the inhibitors.
Color code: C-skeleton of ( )-1: green, ( )-36: blue, ( )-3: brown, C-skeleton of the protein: grey, O-atoms: red, N-atoms: blue, S-atoms: yellow.
Fig. 4 Left: Inhibitor ( )-12 in the active site of thrombin as revealed by X-ray crystal structure analysis. Only the (3aS,4R,8aS,8bR)-configured
enantiomer is bound. Right: Binding mode of the 4-chlorobenzyl moiety in the region of the D pocket of thrombin. Color code: C-skeleton of the
˚
inhibitor: green, C-skeleton of the protein: grey, O-atoms: red, N-atoms: blue, S-atoms: yellow, Cl-atom: dark green. Distances in A.
the benzyl ring filling the D pocket, with Ki values increasing in
the sequence: F < OCH2O < Cl < H < OMe < OH < Npyr ꢀ Br.
This sequence can be explained by both steric fit and the occur-
rence of orthogonal multipolar interactions with the backbone
˚
=
imide inhibitors are sufficiently close (≤3.5 A) to the C O group
of Asn98 to allow for efficient orthogonal multipolar interactions,
˚
the distances in the lactam series are too large (≥4 A) for attractive
dipolar contacts to occur. This study clearly shows that a large
number of biological and structural data are required to map
in detail the molecular recognition properties of a pocket in
an enzyme active site, and to evaluate contributions of weak
interactions such as multipolar contacts to the measured binding
free enthalpies.
=
C O moiety of Asn98. In contrast, the substituent on the
benzyl ring hardly affects the ligand potency in the lactam series.
This discrepancy was clarified by solving two additional crystal
structures of the Cl- and MeO-substituted lactams ( )-12 and
( )-13 bound to thrombin. Whereas the benzyl substituents in the
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Fig. 5 Left: Inhibitor ( )-13 in the active site of thrombin as determined by X-ray crystallographic analysis. Only the (3aS,4R,8aS,8bR)-configured
enantiomer is bound. Right: Binding mode of the 4-methoxybenzyl moiety in the D pocket of thrombin. Hydrophobic contacts between the methyl group
and the protein are also shown. Color code: C-skeleton of the inhibitor: green, C-skeleton of the protein: grey, O-atoms: red, N-atoms: blue, S-atom:
˚
yellow. Distances in A.
Enzymatic assay for the determination of Ki
Experimental
For the enzymatic assay, the release of p-nitroaniline by cleavage
of the chromogenic substrate H–D-Phe–Pip–Arg-p-nitroanilide (S-
2238)15a by means of thrombin was followed spectrophotomet-
rically at 405 nm. The enzyme used for the assay was human
thrombin prepared according to Fenton et al.15b The thrombin
preparation contained 90% a-thrombin as determined by active-
site titration and SDS-PAGE. For each measurement 30 lL of
inhibitor and 20 lL of water were mixed with 180 lL of thrombin
in buffer (2 nM fc; HEPES 100 mM, NaCl 140 mM, PEG 6000
General details
Solvents and reagents were reagent-grade, purchased from com-
mercial suppliers, and used without further purification unless
otherwise stated. 4-Toluenesulfinic acid was prepared according
to a literature procedure.20 The synthesis of ( )-1,2b ( )-2,7 ( )-
3,7 ( )-4,2b ( )-9,2b ( )-10,16 and ( )-117 followed published
procedures. THF was freshly distilled from sodium benzophenone
ketyl, and CH2Cl2 from CaH2. HCl gas was dried with conc.
H2SO4. If not mentioned otherwise, all products were dried
under high vacuum (10−2 Torr) before analytical characterisation.
Column chromatography (CC) was conducted on silica gel 60
(230–400 mesh, 0.040–0.063 mm) from Fluka. Analytical thin
layer chromatography (TLC) was conducted on silica gel 60-F245
(on glass, Merck). Plates were visualised by UV light at 245 nm
and staining with a solution of KMnO4 (1.5 g), K2CO3 (10 g), 5%
NaOH (2.5 cm3) in H2O (150 cm3); a solution of anisaldehyde
(6.8 cm3), conc. H2SO4 (9.2 cm3) and acetic acid (2.8 cm3) in
EtOH (250 cm3); or a solution of ninhydrin (0.3 g) in butanol
(100 cm3) and glacial acetic acid (3 cm3). Melting points (mp) were
determined using a Bu¨chi-510 apparatus and are uncorrected. IR
spectra were recorded on a Perkin–Elmer Spectrum BX FTIR
System spectrometer (ATR-unit, Attenuated Total Reflection,
Golden Gate). NMR spectra were recorded at 25 ◦C on Varian
Gemini-300 and Bruker ARX-300 spectrometers. Chemical shifts
d are reported in ppm using the solvent peak as a reference. J
values are given in Hz. The exchangeable amidinium protons were
◦
0.1%, Tween 80 0.2%, pH 7.8) and incubated at 25 C for 240 s.
50 lL of the substrate S-2238 (50 lM fc, Km 3.33 lM) and 20 lM
of water were added, and the release of p-nitroaniline was recorded
for 60 s at intervals of 10 s. Measurements were carried out at dif-
ferent inhibitor concentrations (in the range of 100–0.0001 lM in
1/10 dilution steps). The Ki value was calculated [Ki = IC50/{1 +
(S/Km)}] from the IC50 value, determined graphically from the
dose response curve of the inhibitor. An exhaustive protocol of
the binding assay used in this study is provided in ref. 15c.
General procedure A for the synthesis of N-alkylated maleimides
To a solution of maleic anhydride (182 mmol) in dry CH2Cl2
(300 cm3) under Ar, 4-substituted benzylamine (182 mmol) was
added over 30 min at 0 ◦C and the reaction stirred for 12 h at
◦
25 C. Under ice cooling, DMF (0.14 cm3) and, over 2 h, oxalyl
chloride (200 mmol) was added. The mixture was stirred for 24 h
and the solvent evaporated. The residue was dried under high
vacuum and dissolved in dry CH2Cl2 (200 cm3) under Ar. Et3◦N
(237 mmol) was added over 30 min, the mixture stirred at 25 C
for 2 h, and washed with 1 N HCl solution (3 × 150 cm3). The org.
phase was dried (Na2SO4), the solvent evaporated in vacuo and the
residue purified by CC (SiO2; cyclohexane–AcOEt 2 : 1).
1
not observed in H NMR spectra recorded in CD3OD. High-
resolution MALDI mass spectra (HR-MS) were recorded at
IonSpec Ultima with 2,5-dihydroxybenzoic acid (DHB) as ma-
trix, EI at VG-TRIBRID; molecular ions (M+) reported for
phenylamidinium salts refer to the corresponding phenylamidine
derivatives. The nomenclature was generated with the computer
programs AUTONOM (Beilstein) and ACD-Name (ACD/Labs).
High-throughput log D screening and pKa determinations by
potentiometric titration were performed as previously described.2c
General procedure B for the 1,3-dipolar cycloaddition
A mixture of L-proline (24.2 mmol), 4-bromobenzaldehyde
(24.2 mmol) or 4-formylbenzonitrile (24.2 mmol) and
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N-substituted maleimide (24.2 mmol) in CH3CN (100 cm3) was
heated to 80 ◦C for 14–16 h. The solvent was evaporated in vacuo
and the residue purified by CC (SiO2; CH2Cl2–AcOEt 4 : 1 or 1 : 1).
solvent was removed in vacuo. The residue was precipitated with
Et2O, filtrated and dried under high vacuum, then dissolved in
a solution of NH3 (2 cm3, 7 M in MeOH) and stirred for 3 h at
65 ◦C. The solvent was evaporated in vacuo and the residue purified
by CC (SiO2; CH2Cl2–MeOH 9 : 1).
General procedure C for the reduction of an imide and conversion
of the resulting hydroxylactam into a p-toluenesulfone
4-(3aSR,4RS,8aSR,8bRS)- and 4-(3aSR,4SR,8aRS,8bRS)-(4-
Bromophenyl)-2-(4-methoxybenzyl)hexahydropyrrolo[3,4-a]-
pyrrolizin-1,3-dione (( )-16 and ( )-18)
To a solution of imide (7.2 mmol) in CH2Cl2 (50 cm3) cooled
to −78 ◦C, a solution of Li[Et3BH] (19 mmol, 1 M in THF)
◦
was added. After 2 h, the mixture was warmed to 0 C, sat. aq.
NaHCO3 (30 cm3) was added, and the aqueous phase extracted
with CH2Cl2 (100 cm3). The organic layer was dried (Na2SO4)
and evaporated in vacuo. The crude product, CaCl2 (19.4 mmol)
and 4-toluenesulfinic acid (19.4 mmol) were dissolved in CH2Cl2
(50 cm3), and the mixture was stirred for 5–7 days. Sat. aq.
NaHCO3 (30 cm3) was added and the aqueous phase extracted
with CH2Cl2 (3 × 30 cm3). The organic layer was dried (Na2SO4),
evaporated in vacuo and the residue purified by CC (SiO2; CH2Cl2–
AcOEt 4 : 1) or recrystallized from AcOEt.
General procedure B, starting from 14 (see ESI†) (5.00 g,
23.0 mmol), L-proline (2.79 g, 24.2 mmol) and 4-bromo-
benzaldehyde (4.47 g, 24.2 mmol) in CH3CN (100 cm3), gave endo-
adduct ( )-16 (4.58 g, 44%) and exo-adduct ( )-18 (5.55 g, 53%).
Data for ( )-16. Colorless solid; mp 137–139 ◦C; mmax/cm−1
(neat) 3273, 2951, 2874, 1694, 1612, 1585, 1515, 1486, 1467, 1428,
1393, 1340, 1302, 1249, 1210, 1166, 1115, 1102, 1085, 1070, 1030;
dH(300 MHz; CDCl3) 1.60–1.78 (2 H, m), 1.97–2.13 (2 H, m), 2.58–
2.65 (1 H, m), 2.77–2.85 (1 H, m), 3.24 (1 H, d, J 8.1), 3.44 (1 H, t,
J 8.4), 3.72–3.81 (1 H, m), 3.79 (3 H, s), 3.99 (1 H, d, J 8.7), 4.46
(2 H, s), 6.81, 7.06 (4 H, AAꢂBBꢂ, J 8.6); 7.23, 7.34 (4 H, AAꢂBBꢂ,
J 8.7); dC(75 MHz; CDCl3) 23.4, 29.6, 41.9, 49.1, 50.4, 50.8, 55.3,
67.8, 68.2, 113.7, 121.4, 127.9, 129.6, 130.3, 131.1, 137.0, 159.1,
General procedure D for the introduction of the isopropyl residue
To a solution of ZnCl2 (2.8 mmol, 1 M in Et2O) in dry CH2Cl2
(30 cm3), a solution of PriMgCl (5.2 mmol, 2 M in Et2O) was
added and the mixture stirred under Ar for 30 min. A solution
of sulfone (2.6 mmol) in dry CH2Cl2 (50 cm3) was slowly added
under ice cooling and the mixture stirred for 16–24 h at 25 ◦C. After
addition of 1 M HCl (20 cm3), the mixture was neutralised with
aq. NaHCO3 (50 cm3) and extracted with CH2Cl2 (3 × 80 cm3).
The organic phase was dried (Na2SO4), the solvent evaporated in
vacuo and the residue purified by CC (SiO2; cyclohexane–AcOEt
2 : 1 or CH2Cl2–AcOEt 1 : 2).
+
174.9, 177.7; MALDI-HR-MS calcd for C23H24BrN2O3 ([M +
H]+): 455.0965; found 455.0965.
Data for ( )-18. Colorless solid; mp 102–103 ◦C; mmax/cm−1
(neat) 2934, 1772, 1702, 1611, 1586, 1512, 1483, 1424, 1393, 1336,
1305, 1292, 1239, 1167, 1110, 1099, 1037; dH(300 MHz; CDCl3)
1.49–1.60 (1 H, m), 1.63–1.72 (2 H, m), 1.89–1.95 (1 H, m), 2.36–
2.44 (1 H, m), 2.87–2.95 (1 H, m), 3.25 (1 H, dd, J 9.0 and 5.8),
3.49 (1 H, t, J 9.0), 3.77 (3 H, s), 3.82–3.87 (1 H, m), 4.03 (1 H, d,
J 5.6), 4.57 (2 H, s), 6.82, 7.34 (4 H, AAꢂBBꢂ, J 8.7), 7.43, 7.45 (4
H, AAꢂBBꢂ, J 8.7); dC(75 MHz; CDCl3) 24.4, 26.3, 42.0, 47.9, 52.0,
55.3, 55.5, 66.3, 69.0, 113.8, 121.1, 127.6, 128.5, 130.4, 131.5, 141.1,
General procedure E for the conversion of an aryl bromide into an
aryl nitrile
+
159.2, 176.4, 177.5; MALDI-HR-MS calcd for C23H24BrN2O3
([M + H]+): 455.0965; found: 455.0964.
Method A. A well degassed suspension of CuCN (4.7 mmol)
in dry DMF (5 cm3) was heated to reflux under Ar for 30–60 min,
before a degassed solution of bromide (1.2 mmol) in dry DMF
(3 cm3) was added and the mixture stirred for 24–36 h. The solvent
was evaporated in vacuo, the residue dissolved in CH2Cl2 (10 cm3)
and conc. aq. NH4OH solution (5 cm3) was added. The mixture
was stirred at 25 ◦C for 1 h, the blue aqueous phase removed, the
organic phase washed with conc. aq. NH4OH solution (10 cm3)
and sat. aq. NaCl solution (10 cm3), dried (Na2SO4) and the solvent
removed in vacuo. The residue was purified by CC (SiO2; CH2Cl2–
AcOEt 1 : 1).
4-(3aSR,4RS,8aSR,8bRS)- and 4-(3aSR,4SR,8aRS,8bRS)-(4-
Bromophenyl)-2-pyridin-4-ylmethylhexahydropyrrolo[3,4-
a]pyrrolizin-1,3-dione (( )-17 and ( )-19)
General procedure B, starting from 15 (see ESI†) (1.77 g,
9.4 mmol), L-proline (1.19 g, 10.3 mmol) and 4-bromo-
benzaldehyde (1.91 g, 10.3 mmol) in CH3CN (20 cm3), gave endo-
adduct ( )-17 (1.41 g, 35%) and exo-adduct ( )-19 (730 mg, 18%).
Data for ( )-17. Colorless solid; mp 69–72 ◦C; mmax/cm−1
(neat) 2956, 2877, 1773, 1701, 1601, 1564, 1485, 1417, 1395,
1337, 1317, 1285, 1233, 1197, 1167, 1104, 1087, 1069, 1048, 1009;
dH(300 MHz; CDCl3) 1.57–1.83 (2 H, m), 1.93–2.17 (2 H, m),
2.57–2.67 (1 H, m), 2.79–2.90 (1 H, m), 3.29 (1 H, d, J 8.1), 3.49
(1 H, dd, J 8.4 and 8.1), 3.70–3.77 (1 H, m), 4.02 (1 H, d, J 8.7), 4.49
(2 H, s), 7.07, 7.33 (4 H, AAꢂBBꢂ, J 8.4), 7.13, 8.51 (4 H, AAꢂBBꢂ,
J = 5.9); dC(75 MHz; CDCl3) 23.5, 29.7, 41.4, 49.1, 50.7, 50.8, 68.1,
68.2, 121.8, 123.3, 129.8, 131.5, 136.9, 144.2, 150.3, 175.1, 177.9;
MALDI-HR-MS calcd for C21H21BrN3O2+ ([M + H]+): 426.0812;
found: 426.0803.
Method B. To a suspension of [Pd2(dba)3] (0.04 mmol) and
dppf (0.09 mmol) in degassed, dry DMF (3 cm3) under Ar, a
solution of bromide (0.7 mmol) in degassed, dry DMF (2 cm3)
and Zn(CN)2 (0.7 mmol) were added. The mixture was stirred for
24 h at 120 ◦C, the solvent evaporated in vacuo and the residue
purified by CC (SiO2; cyclohexane–AcOEt 1 : 1).
General procedure F for the preparation of amidinium salts (Pinner
reaction)
◦
Dry HCl gas was bubbled at 0 C for 10 min into a solution of
the nitrile (0.5 mmol) in dry CH2Cl2 (0.6 cm3) and dry MeOH
Data for ( )-19. Brown solid; mp 65–67 ◦C; mmax/cm−1 (neat)
2966, 2879, 1773, 1698, 1601, 1485, 1416, 1393, 1338, 1314, 1241,
◦
(1.2 cm3). The mixture was stored at 4 C for 28–35 h, then the
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1165, 1069, 1009; dH(300 MHz; CDCl3) 1.42–1.55 (1 H, m), 1.67–
1.77 (2 H, m), 1.87–1.99 (1 H, m), 2.40–2.49 (1 H, m), 2.86–2.96
(1 H, m), 3.31 (1 H, dd, J 9.1 and 6.0), 3.57 (1 H, t, J 9.1), 3.81–
3.90 (1 H, m), 4.00 (1 H, d, J 6.1), 4.61 (2 H, s), 7.26, 8.56 (4
H, AAꢂBBꢂ, J 5.8), 7.33, 7.45 (4 H, AAꢂBBꢂ, J 8.5); dC(75 MHz;
CDCl3) 24.3, 26.3, 41.3, 47.7, 51.7, 55.3, 66.1, 68.9, 121.3, 123.4,
128.6, 131.6, 140.7, 143.6, 150.2, 176.2, 177.3; MALDI-HR-MS
dd, J 10.0 and 7.2), 4.10 (1 H, d, J 8.7), 4.41, 4.48 (2 H, AB, J
14.4), 7.14, 7.40 (4 H, AAꢂBBꢂ, J 8.4), 7.29, 7.49 (4 H, AAꢂBBꢂ, J
8.4); dC(75 MHz; CDCl3) 23.6, 29.8, 42.0, 49.2, 50.7, 51.0, 68.2,
68.6, 111.7, 119.1, 122.3, 129.0, 130.9, 131.9, 132.2, 134.8, 143.9,
+
175.1, 177.8; MALDI-HR-MS calcd for C23H21BrN3O2 ([M +
H]+): 450.0812; found: 450.0805.
Data for ( )-25. Brownsolid;mp116–119 ◦C; mmax/cm−1 (neat)
2955, 2876, 2806, 2224, 1771, 1703, 1608, 1489, 1431, 1393, 1332,
1295, 1162, 1107, 1071, 1014; dH(300 MHz; CDCl3) 1.81–1.95 (2
H, m), 1.98–2.23 (3 H, m), 2.69–2.78 (1 H, m), 2.87–2.95 (1 H,
m), 3.12–3.18 (1 H, m), 3.61–3.66 (2 H, m), 4.41, 4.54 (2 H, AB,
J 14.0), 7.10, 7.44 (4 H, AAꢂBBꢂ, J 8.4), 7.19, 7.47 (4 H, AAꢂBBꢂ,
J 8.4); dC(75 MHz; CDCl3) 22.7, 27.2, 41.8, 43.6, 45.8, 56.0, 66.3,
70.8, 111.5, 118.8, 122.0, 128.2, 130.7, 131.5, 131.9, 134.7, 142.4,
calcd for C21H21BrN3O2 ([M + H]+): 426.0812; found: 426.0810.
+
4-[(3aSR,4RS,8aSR,8bRS)-2-(4-Methoxybenzyl)-1,3-
dioxodecahydropyrrolo[3,4-a]pyrrolizin-4-yl]benzonitrile (( )-20)
General procedure E, method B, starting from ( )-16 (2.40 g,
5.25 mmol), [Pd2(dba)3] (300 mg, 0.32 mmol), dppf (350 mg,
0.63 mmol) and Zn(CN)2 (622 mg, 5.25 mmol) in DMF (5 cm3),
gave ( )-20 (1.66 g, 78%) as a colorless solid; mp 161–164 ◦C;
mmax/cm−1 (neat) 2958, 2833, 2224, 2051, 1979, 1767, 1703, 1683,
1652, 1608, 1584, 1514, 1463, 1432, 1393, 1341, 1318, 1299, 1250,
1202, 1169, 1111, 1086, 1036; dH(300 MHz; CDCl3) 1.57–1.87 (2
H, m), 1.95–2.20 (2 H, m), 2.54–2.64 (1 H, m), 2.80–2.91 (1 H,
m), 3.28 (1 H, d, J 8.0), 3.49 (1 H, t, J 8.5), 3.73–3.81 (1 H, m),
3.79 (3 H, s), 4.08 (1 H, d, J 8.8), 4.39, 4.51 (2 H, AB, J 14.0),
6.80, 7.29 (4 H, AAꢂBBꢂ, J 8.4), 7.21, 7.48 (4 H, AAꢂBBꢂ, J 8.4);
dC(75 MHz; CDCl3) 23.6, 29.8, 42.2, 49.3, 50.7; 51.1, 55.5, 68.2,
68.6, 111.6, 114.0, 119.2, 128.1, 129.0, 130.7, 132.1, 144.1, 159.5,
175.2, 177.9; MALDI-HR-MS calcd for C24H24N3O3+ ([M + H]+):
402.1812; found: 402.1817.
+
174.7, 175.3; MALDI-HR-MS calcd for C23H21BrN3O2 ([M +
H]+): 450.0812; found: 450.0805.
4-[(3aSR,4RS,8aSR,8bRS)-2-(4-Bromobenzyl)-1,3-
dioxodecahydropyrrolo[3,4-a]pyrrolizin-4-yl]benzamidine
hydrochloride (( )-5)
General procedure F, starting from ( )-24 (86 mg, 0.19 mmol),
gave ( )-5 (46 mg, 49%) as a colorless solid; mp 198–201 ◦C;
mmax/cm−1 (neat) 3411, 3328, 3253, 3158, 3050, 2960, 2871, 1771,
1683, 1651, 1610, 1535, 1489, 1435, 1415, 1398, 1347, 1299, 1282,
1170, 1094, 1078, 1043, 1016; dH(300 MHz; CD3OD) 1.60–1.78 (2
H, m), 1.89–2.06 (2 H, m), 2.43–2.55 (1 H, m), 2.69–2.81 (1 H,
m), 3.45 (1 H, d, J 8.1), 3.54 (1 H, dd, J 8.1 and 7.8), 3.71 (1 H,
dd, J 8.1 and 8.4), 4.16 (1 H, d, J 8.7), 4.36, 4.44 (2 H, AB, J
15.0), 7.12, 7.55 (4 H, AAꢂBBꢂ, J 8.4), 7.27, 7.73 (4 H, AAꢂBBꢂ, J
8.4); dC(75 MHz; CD3OD) 23.5, 29.7, 41.9, 48.6, 50.6, 51.0, 53.5,
68.0, 121.9, 126.2, 127.8, 129.0, 129.9, 131.7, 134.1, 145.2, 165.4,
4-[(3aSR,4RS,8aSR,8bRS)-1,3-Dioxo-2-pyridin-4-
ylmethyldecahydropyrrolo[3,4-a]pyrrolizin-4-yl]benzonitrile
(( )-21)
General procedure E, method A, starting from ( )-17 (426 mg,
1.0 mmol) and CuCN (358 mg, 4.0 mmol) in DMF (4 cm3), gave
( )-21 (119 mg, 32%) as a colorless solid; mp (AcOEt) 200–202 ◦C;
mmax/cm−1 (neat) 3045, 2968, 2221, 1779, 1705, 1601, 1505, 1424,
1416, 1400, 1361, 1341, 1200, 1174; dH(300 MHz; CDCl3) 1.60–
1.88 (2 H, m), 1.96–2.21 (2 H, m), 2.54–2.65 (1 H, m), 2.81–2.94 (1
H, m), 3.36 (1 H, d, J 7.8), 3.56 (1 H, dd, J 8.4 and 8.1), 3.77 (1 H,
dd, J 9.9 and 6.9), 4.13 (1 H, d, J 8.4), 4.47, 4.53 (2 H, AB, J 14.7),
7.14, 8.53 (4 H, AAꢂBBꢂ, J 5.7), 7.34, 7.51 (4 H, AAꢂBBꢂ, J 8.4);
dC(75 MHz; CDCl3) 23.6, 29.8, 41.5, 49.0, 50.8, 51.0, 68.3, 68.5,
111.8, 119.0, 123.4, 128.9, 132.2, 143.6, 144.2, 150.3, 174.9, 177.7;
+
176.2, 177.4; MALDI-HR-MS calcd for C23H24BrN4O2 ([M +
H]+): 468.1155; found: 468.1147.
4-[(3aSR,4RS,8aSR,8bRS)-2-(4-Methoxybenzyl)-1,3-
dioxodecahydropyrrolo[3,4-a]pyrrolizin-4-yl]benzamidine
hydrochloride (( )-6)
General procedure F, starting from ( )-20 (200 mg, 0.5 mmol),
gave ( )-6 (47 mg, 21%) as a colorless solid; mp 193–196 ◦C;
mmax/cm−1 (neat) 3421, 3264, 3065, 2966, 2873, 1771, 1694, 1613,
1538, 1516, 1484, 1462, 1438, 1404, 1348, 1300, 1256, 1210, 1175,
1095, 1029; dH(300 MHz; CDCl3) 1.57–1.73 (2 H, m), 1.82–1.95 (1
H, m), 2.00–2.12 (1 H, m), 2.29–2.41 (1 H, m), 2.58–2.70 (1 H, m),
3.25 (1 H, d, J 7.7), 3.54 (1 H, dd, J 8.0 and 7.7), 3.64 (1 H, dd, J
8.8 and 8.0), 3.71 (3 H, s), 4.09 (1 H, d, J 7.7), 4.30, 4.43 (2 H, AB,
J 14.0), 6.73, 6.99 (4 H, AAꢂBBꢂ, J 8.4), 7.47, 7.79 (4 H, AAꢂBBꢂ, J
8.1), 8.74 (2H, bs); 9.16 (2H, bs); dC(75 MHz; CDCl3) 23.6, 29.9,
42.2, 48.8, 50.6, 51.1, 55.6, 68.3, 114.2, 126.9, 127.5, 128.3, 129.4,
130.0, 145.6, 159.5, 166.1, 176.8, 177.8; MALDI-HR-MS calcd
MALDI-HR-MS calcd for C22H21N4O2 ([M + H]+): 373.1665;
+
found: 373.1653.
4-[(3aSR,4RS,8aSR,8bRS)- and 4-[(3aSR,4SR,8aRS,8bRS)-2-
(4-Bromobenzyl)-1,3-dioxodecahydropyrrolo[3,4-a]pyrrolizin-4-
yl]benzonitrile (( )-24 and ( )-25)
General procedure B, starting from 23 (1.0 g, 3.8 mmol), L-proline
(459 mg, 4.0 mmol) and formylbenzonitrile (523 mg, 4.0 mmol)
in CH3CN (20 cm3), gave endo-adduct ( )-24 (701 mg, 41%) and
exo-adduct ( )-25 (650 mg, 38%).
for C24H27N4O3 ([M + H]+): 419.2078; found: 419.2071.
+
4-[(3aSR,4RS,8aSR,8bRS)-1,3-Dioxo-2-pyridin-4-
ylmethyldecahydropyrrolo[3,4-a]pyrrolizin-4-yl]benzamidine
hydrochloride (( )-7)
Data for ( )-24. Brown solid;mp 160–162 ◦C; mmax/cm−1 (neat)
2959, 2886, 2842, 2223, 1771, 1702, 1607, 1489, 1429, 1393, 1333,
1299, 1166, 1087, 1071, 1014; dH(300 MHz; CDCl3) 1.59–1.85 (2
H, m), 1.96–2.18 (2 H, m), 2.54–2.63 (1 H, m), 2.81–2.91 (1 H,
m), 3.30 (1 H, d, J 7.8), 3.52 (1 H, dd, J 8.4 and 8.1), 3.75 (1 H,
General procedure F, starting from ( )-21 (87 mg, 0.23 mmol),
gave ( )-7 (27 mg, 28%) as a colorless solid; mp 204–207 ◦C;
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mmax/cm−1 (neat) 3322, 3204, 3059, 2972, 1772, 1695, 1675, 1608,
1561, 1538, 1484, 1419, 1399, 1338, 1317, 1174, 1082, 1048, 1017,
1008; dH(300 MHz; CD3OD) 1.76–1.93 (2 H, m), 2.05–2.19 (2 H,
m), 2.62–2.71 (1 H, m), 2.88–2.97 (1 H, m), 3.53 (1 H, d, J 7.8),
3.75 (1 H, dd, J 9.0 and 7.8), 3.83 (1 H, t, J 8.4), 4.36 (1 H, d,
J 8.4,), 4.54, 4.59 (2 H, AB, J 15.6), 7.29, 8.49 (4 H, AAꢂBBꢂ, J
6.0), 7.54, 7.67 (4 H, AAꢂBBꢂ, J 8.1); dC(75 MHz; CD3OD) 23.9,
30.2, 41.7, 49.9, 51.7, 51.8, 68.9, 69.3, 123.9, 128.0, 128.2, 129.8,
146.3, 147.0, 149.8, 167.7, 176.5, 179.3; MALDI-HR-MS calcd
(3.04 g, 19.4 mmol) and CaCl2 (2.15 g, 19.4 mmol) in CH2Cl2
(100 cm3), gave ( )-30 (1.55 g, 32%) as a colorless solid; mp 183–
186 ◦C; mmax/cm−1 (neat) 2982, 2940, 2877, 1711, 1595, 1488, 1386,
1292, 1199, 1136, 1083, 1007; dH(300 MHz; CDCl3) 1.52–1.72 (2
H, m), 1.85–2.04 (2 H, m), 2.42–2.50 (1 H, m), 2.47 (3 H, s), 2.52–
2.58 (1 H, m), 2.81–2.88 (1 H, m), 2.95–2.98 (1 H, m), 3.02–3.08
(1 H, m), 3.90 (1 H, d, J 6.3), 4.14, 5.09 (2 H, AB, J 15.0), 4.26
(1 H, s), 7.15, 7.34 (4 H, AAꢂBBꢂ, J 8.4), 7.18, 7.40 (4 H, AAꢂBBꢂ,
J 8.4), 7.39, 7.71 (4 H, AAꢂBBꢂ, J 8.4); dC(75 MHz; CDCl3) 22.0,
24.6, 32.0, 43.1, 44.7, 51.1, 51.9, 69.7, 71.6, 81.3, 121.3, 125.4,
129.2, 129.6, 129.9, 130.0, 130.7, 131.1, 132.2, 134.0, 137.2, 146.5,
172.8; MALDI-HR-MS calcd for C29H29BrClN2O3S+ ([M + H]+):
599.0765; found 599.0755.
for C22H24N5O2 ([M + H]+): 390.1925; found: 390.1920.
+
4-[(3aSR,4RS,8aSR,8bRS)-2-(4-Hydroxybenzyl)-1,3-
dioxodecahydropyrrolo[3,4-a]pyrrolizin-4-yl]benzamidine
hydrochloride (( )-8)
4-(1RS,3aSR,4RS,8aSR,8bRS)-(4-Bromophenyl)-2-(4-
methoxybenzyl)-1-(toluene-4-sulfonyl)octahydropyrrolo[3,4-
a]pyrrolizin-3-one (( )-31)
A solution of methyl ether ( )-20 (220 mg, 0.55 mmol) in dry
CH2Cl2 (5 cm3) under Ar was cooled to −50 ◦C, then BBr3 (1.2 cm3,
1.2 mmol, 1 M in CH2Cl2) was added and the mixture stirred for
8 h at 25 ◦C. The reaction mixture was poured onto ice and stirred
for 30 min, before being extracted with CH2Cl2 (3 × 10 cm3). The
organic phase was dried (Na2SO4) and concentrated in vacuo. The
residue showed decomposition upon purification by CC, therefore
the crude product was used directly following general procedure
F, giving ( )-8 (31 mg, 40% over two steps) as a brown solid; mp
173–175 ◦C; mmax/cm−1 (neat) 3344, 3204, 3122, 2961, 2873, 1771,
1674, 1612, 1538, 1515, 1486, 1431, 1398, 1339, 1287, 1232, 1204,
1171, 1090, 1045, 1018; dH(300 MHz; CD3OD) 1.72–1.86 (2 H, m),
2.00–2.15 (2 H, m), 2.57–2.66 (1 H, m), 2.77–2.87 (1 H, m), 3.40
(1 H, d, J 7.8), 3.38–3.73 (2 H, m), 4.20 (1 H, d, J 9.0), 4.37, 4.45
(2 H, AB, J 14.4), 6.71, 7.08 (4 H, AAꢂBBꢂ, J 8.7), 7.25, 7.70 (4 H,
AAꢂBBꢂ, J 8.4); dC(75 MHz; CD3OD) 23.8, 30.0, 42.3, 49.9, 51.5,
69.1, 115.7, 116.6, 127.9, 128.1, 130.0, 130.57, 133.8, 146.5, 157.8,
167.9, 176.9, 179.7. In the 13C NMR spectrum of compound ( )-8,
two resonances are buried under the solvent peak. MALDI-HR-
MS calcd for C23H26N4O3+ ([M + H]+): 405.1921; found: 405.1917.
General procedure C, starting from ( )-16 (3.51 g, 7.70 mmol),
Li[Et3BH] solution (8.34 cm3, 8.34 mmol), 4-toluenesulfinic acid
(3.53 g, 20.8 mmol) and CaCl2 (2.31 g, 20.8 mmol) in CH2Cl2
(100 cm3), gave ( )-31 (4.45 g, 97%) as a colorless solid; mp 206–
207 ◦C; mmax/cm−1 (neat) 2940, 2874, 1713, 1612, 1584, 1512, 1486,
1443, 1386, 1365, 1328, 1302, 1289, 1251, 1198, 1173, 1135, 1082,
1032, 1007; dH(300 MHz; CDCl3): 1.51–1.70 (2 H, m), 1.82–2.01
(2 H, m), 2.37–2.56 (2 H, m), 2.47 (3 H, s), 2.80–2.89 (1 H, m),
2.94 (1 H, dd, J 7.7 and 3.6), 2.99–3.05 (1 H, m), 3.82 (3 H, s),
3.89 (1 H, d, J 6.3), 4.00, 5.07 (2 H, AB, J 14.6), 4.27 (1 H, s),
6.88, 7.14 (4 H, AAꢂBBꢂ, J 8.5), 7.16, 7.40 (4 H, AAꢂBBꢂ, J 8.5),
7.39, 7.71 (4 H, AAꢂBBꢂ, J 8.5); dC(75 MHz; CDCl3) 22.1, 24.8,
32.1, 43.3, 44.9, 51.4, 52.2, 55.5, 70.0, 71.4, 81.1, 114.4, 121.3,
127.3, 129.6, 129.9, 130.1, 130.7, 131.1, 132.5, 137.5, 146.4, 159.5,
172.7; MALDI-HR-MS calcd for C30H32BrN2O4S+ ([M + H]+):
595.1254; found: 595.1261.
4-(1RS,3aSR,4RS,8aSR,8bRS)-(4-Bromophenyl)-2-(4-
chlorobenzyl)-1-isopropyloctahydropyrrolo[3,4-a]pyrrolizin-3-one
(( )-32)
4-(3aSR,4RS,8aSR,8bRS)-(4-Bromophenyl)-2-(4-chlorobenzyl)-
hexahydropyrrolo[3,4-a]-pyrrolizin-1,3-dione (( )-26)
General procedure B, starting from 37 (see ESI†) (3.50 g,
15.8 mmol), L-proline (1.91 g, 16.6 mmol) and 4-bromo-
benzaldehyde (3.07 g, 16.6 mmol) in CH3CN (50 cm3), gave endo-
adduct ( )-26 (3.34 g, 46%) as a colorless solid; mp 161–163 ◦C;
mmax/cm−1 (neat) 2963, 2873, 1698, 1695, 1486, 1423, 1393, 1337,
1168, 1089, 1069, 1042, 1006; dH(300 MHz; CDCl3) 1.57–1.85
(2 H, m), 1.95–2.17 (2 H, m), 2.57–2.61 (1 H, m), 2.77–2.89 (1
H, m), 3.27 (1 H, d, J 7.8), 3.46 (1 H, dd, J 8.3 and 8.1), 3.75
(1 H, dd, J 10.0 and 7.2), 4.01 (1 H, d, J 8.7), 4.48 (2 H, s),
7.06, 7.35 (4 H, AAꢂBBꢂ, J 8.4), 7.20–7.29 (4 H, m); dC(75 MHz;
CDCl3) 23.5, 29.7, 41.8, 49.1, 50.5, 50.8, 67.9, 68.2, 121.5, 128.6,
129.6, 130.3, 131.2, 133.7, 134.1, 136.9, 174.9, 177.6; MALDI-
General procedure D, starting from ( )-30 (1.55 g, 2.58 mmol),
ZnCl2 solution (2.84 cm3, 2.84 mmol) and PriMgCl solution
(2.58 cm3, 5.16 mmol) in CH2Cl2 (50 cm3), gave ( )-32 (768 mg,
61%) as a yellowish solid; mp 195–198 ◦C; mmax/cm−1 (neat) 2963,
2900, 2859, 2821, 2774, 1672, 1488, 1446, 1431, 1251, 1151, 1128,
1086, 1068, 1017, 1008; dH(300 MHz; CDCl3) 0.70 (3 H, d, J 6.8),
0.91 (3 H, d, J 6.8), 1.50–1.81 (2 H, m), 1.88–2.10 (3 H, m), 2.49
(1 H, dt, J 6.2 and 2.5), 2.56–2.67 (1 H, m), 2.85–2.98 (1 H, m),
3.18–3.27 (2 H, m), 3.30 (1 H, dd, J 8.4 and 8.1), 3.78, 4.82 (2 H,
AB, J 14.9), 4.08 (1 H, d, J 7.5), 7.10, 7.44 (4 H, AAꢂBBꢂ, J 8.4),
7.29 (4 H, d, J 8.4); dC(75 MHz; CDCl3) 14.9, 18.6, 24.7, 28.2,
31.5, 41.6, 43.5, 52.5, 52.9, 67.6, 70.2, 73.5, 121.0, 128.9, 129.6,
130.0, 131.1, 133.3, 135.4, 138.9, 172.8; MALDI-HR-MS calcd
for C25H29BrClN2O+ ([M + H]+): 487.1146; found: 487.1139.
+
HR-MS calcd for C22H21BrClN2O2 ([M + H]+): 459.0469;
found: 459.0464.
4-(1RS,3aSR,4RS,8aSR,8bRS)-(4-Bromophenyl)-2-(4-
chlorobenzyl)-1-(toluene-4-sulfonyl)octahydropyrrolo[3,4-
a]pyrrolizin-3-one (( )-30)
4-(1RS,3aSR,4RS,8aSR,8bRS)-(4-Bromophenyl)-1-isopropyl-2-
(4-methoxybenzyl)octahydropyrrolo[3,4-a]pyrrolizin-3-one
(( )-33)
General procedure C, starting from ( )-26 (3.30 g, 7.2 mmol),
General procedure D, starting from ( )-31 (4.45 g, 7.47 mmol),
Li[Et3BH] solution (13.7 cm3, 13.7 mmol), 4-toluenesulfinic acid
ZnCl2 solution (11.2 cm3, 11.2 mmol) and PriMgCl solution
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(9.34 cm3, 18.7 mmol) in CH2Cl2 (50 cm3), gave ( )-33 (1.64 g,
46%) as a brown solid; mp 72–75 ◦C; mmax/cm−1 (neat) 2957, 1683,
1612, 1586, 1511, 1485, 1435, 1418, 1289, 1243, 1174, 1101, 1070,
1033, 1009; dH(300 MHz; CDCl3) 0.70 (3 H, d, J 6.9), 0.90 (3 H,
d, J 6.9), 1.53–1.62 (1 H, m), 1.66–1.77 (1 H, m), 1.87–2.11 (3 H,
m), 2.46 (1 H, dt, J 9.0 and 3.0), 2.56–2.65 (1 H, m), 2.88–2.96 (1
H, m), 3.16–3.26 (2 H, m), 3.30 (1 H, dd, J 8.4 and 7.8), 3.71, 4.82
(2 H, AB, J 14.8), 3.81 (3 H, s), 4.07 (1 H, d, J 7.8), 6.84, 7.28 (4
H, AAꢂBBꢂ, J 8.4), 7.05, 7.43 (4 H, AAꢂBBꢂ, J 8.4); dC(75 MHz;
CDCl3) 14.8, 18.6, 24.6, 27.9, 31.4, 41.7, 43.4, 52.5, 52.9, 55.3,
67.1, 70.2, 73.1, 113.8, 120.6, 128.5, 129.3, 129.8, 130.7, 138.7,
1445, 1407, 1390, 1289, 1245, 1090, 1015; dH(300 MHz; CD3OD)
0.74 (3 H, d, J 6.8), 0.97 (3 H, d, J 6.8), 1.67–1.83 (2 H, m), 1.97–
2.10 (2 H, m), 2.11–2.22 (1 H, m), 2.57–2.73 (2 H, m), 2.90–3.02 (1
H, m), 3.26–3.37 (2 H, m), 3.48 (1 H, dd, J 8.1 and 7.8), 3.97, 4.69
(2 H, AB, J 15.3), 4.33 (1 H, d, J 7.5), 7.27, 7.36 (4 H, AAꢂBBꢂ, J
8.4), 7.62, 7.72 (4 H, AAꢂBBꢂ, J 8.4); dC(75 MHz; CD3OD) 14.8,
18.4, 25.1, 29.1, 31.7, 42.0, 44.0, 53.1, 54.3, 69.3, 71.1, 74.8, 127.4,
127.9, 129.3, 129.9, 130.3, 134.0, 136.1, 147.7, 164.3, 167.8, 174.3;
MALDI-HR-MS calcd for C26H32ClN4O+ ([M + H]+): 451.2259;
found: 451.2261.
+
158.7, 172.2; MALDI-HR-MS calcd for C26H32BrN2O2 ([M +
4-[(1RS,3aSR,4RS,8aSR,8bRS)-1-Isopropyl-2-(4-
methoxybenzyl)-3-oxodecahydropyrrolo[3,4-a]pyrrolizin-4-
yl]benzamidine hydrochloride (( )-13)
H]+): 483.1642; found: 483.1636.
4-[(1RS,3aSR,4RS,8aSR,8bRS)-2-(4-Chlorobenzyl)-1-isopropyl-
3-oxodecahydropyrrolo[3,4-a]pyrrolizin-4-yl]benzonitrile
(( )-34)
General procedure F, starting from ( )-35 (205 mg, 0.45 mmol),
gave ( )-13 (54 mg, 25%) as a colorless solid; mp 215–218 ◦C;
mmax/cm−1 (neat) 3355, 2958, 2833, 2224, 1767, 1703, 1699, 1694,
1666, 1611, 1584, 1513, 1452, 1432, 1393, 1341, 1300, 1253, 1202,
1172, 1111, 1086, 1035; dH(300 MHz; (CD3)2SO) 0.64 (3 H, d,
J 6.5), 0.87 (3 H, d, J 6.9), 1.53–1.97 (2 H, m), 1.83–1.97 (2
H, m), 2.03–2.13 (1 H, m), 2.38–2.60 (2 H, m), 2.74–2.86 (1 H,
m), 3.09–3.17 (2 H, m), 3.20–3.28 (1 H, m), 3.64, 4.55 (2 H,
AB, J 14.8), 4.17 (1 H, d, J 7.2), 6.90, 7.12 (4 H, AAꢂBBꢂ, J
8.4), 7.55, 7.75 (4 H, AAꢂBBꢂ, J 7.8), 8.99 (2 H, bs), 9.31 (2
H, bs); dC(75 MHz; CD3OD) 15.3, 18.7, 25.3, 28.5, 31.9, 41.4,
51.0, 52.2, 53.9, 55.5, 67.9, 70.6, 73.7, 114.4, 126.1, 128.2, 128.4,
128.6, 129.4, 146.9, 159.3, 166.0, 172.6; MALDI-HR-MS calcd
General procedure E, method A, starting from ( )-32 (570 mg,
1.17 mmol) and CuCN (419 mg, 4.7 mmol) in DMF (8 cm3), gave
( )-34 (217 mg, 43%) as a brown solid; mp 124–125 ◦C; mmax/cm−1
(neat) 2963, 2900, 2823, 2229, 1671, 1608, 1490, 1447, 1434, 1412,
1390, 1349, 1266, 1251, 1126, 1087, 1017; dH(300 MHz; CDCl3)
0.69 (3 H, d, J 6.7), 0.90 (3 H, d, J 6.7), 1.50–1.68 (1 H, m), 1.65–
1.81 (1 H, m), 1.87–2.09 (3 H, m), 2.50 (1 H, ddd, J 8.7, 2.8 and
2.5), 2.53–2.61 (1 H, m), 2.88–2.98 (1 H, m), 3.17–3.26 (2 H, m),
3.33 (1 H, dd, J 8.1 and 7.8), 3.79, 4.77 (2 H, AB, J 15.3), 4.15
(1 H, d, J 7.5), 7.11, 7.27 (4 H, AAꢂBBꢂ, J 8.4,), 7.52, 7.58 (4 H,
AAꢂBBꢂ, J 8.4); dC(75 MHz; CDCl3) 15.0, 18.6, 24.8, 28.3, 31.5,
41.4, 43.4, 52.5, 53.0, 67.6, 70.4, 73.5, 110.4, 119.3, 128.6, 128.8,
129.3, 131.5, 133.1, 135.1, 145.7, 172.2; MALDI-HR-MS calcd
for C26H29ClN3O+ ([M + H]+): 434.1994; found: 434.1987.
for C27H35N4O2 ([M + H]+): 447.2755; found: 447.2749.
+
X-Ray crystal structure of ( )-29
Crystal data at 173(2) K for C22H22BrFN2O2: Mr = 445.33,
−3
¯
triclinic, space group P1 (no. 2), Dc = 1.550 g cm , Z = 2, a =
4-[(1RS,3aSR,4RS,8aSR,8bRS)-1-Isopropyl-2-(4-
methoxybenzyl)-3-oxodecahydropyrrolo[3,4-a]pyrrolizin-4-
yl]benzonitrile (( )-35)
◦
˚
˚
˚
9.4567(2) A, b = 10.5163(2) A, c = 10.6619(2) A, a = 78.900(1) ,
◦
◦
3
˚
b = 89.899(1) , c = 66.981(1) , V = 954.45(3) A . Bruker–Nonius
˚
Kappa-CCD diffractometer, MoKa radiation, k = 0.7107 A, l =
General procedure E, method B, starting from ( )-33 (351 mg,
0.73 mmol), [Pd2(dba)3] (41 mg, 0.04 mmol), dppf (49 mg,
0.09 mmol) and Zn(CN)2 (86 mg, 0.73 mmol) in DMF (5 cm3),
gave ( )-35 (205 mg, 62%) as a brown solid; mp 166–168 ◦C;
mmax/cm−1 (neat) 2960, 2873, 2224, 1684, 1610, 1584, 1512, 1437,
1414, 1390, 1367, 1339, 1300, 1245, 1175, 1101, 1036; dH(300 MHz;
CDCl3) 0.70 (3 H, d, J 6.9), 0.90 (3 H, d, J 6.9), 1.52–1.60 (1 H,
m), 1.66–1.77 (1 H, m), 1.90–2.14 (3 H, m), 2.47 (1 H, dt, J 9.0
and 2.7), 2.54–2.61 (1 H, m), 2.90–2.98 (1 H, m), 3.19–3.26 (2 H,
m), 3.34 (1 H, t, J 8.4), 3.72, 4.80 (2 H, AB, J 15.0), 3.81 (3 H, s),
4.14 (1 H, d, J 7.8), 6.84, 7.08 (4 H, AAꢂBBꢂ, J 8.7), 7.52, 7.59 (4
H, AAꢂBBꢂ, J 8.4); dC(75 MHz; CDCl3) 14.9, 18.6, 24.8, 28.0, 31.5,
41.5, 43.4, 52.6, 53.1, 55.3, 67.1, 70.5, 73.3, 110.4, 113.8, 119.3,
128.3, 128.8, 129.2, 131.5, 145.7, 158.7, 171.9; MALDI-HR-MS
2.184 mm−1. Crystal dimensions ca. 0.18 × 0.16 × 0.15 mm.
The numbers of measured and unique reflections were 7417
and 4368, respectively (Rint = 0.016). The structure was solved
by direct methods (SIR-97)21 and refined by full-matrix least-
squares analysis (SHELXL-97),22 using an isotropic extinction
correction. All non H-atoms were refined anisotropically, H-atoms
isotropically, whereby H-positions are based on stereochemical
considerations. Final R(F) = 0.029, wR(F2) = 0.064 for 276
parameters and 3942 reflections with I > 2r(I) and 3.76 < h <
27.50◦ (corresponding R values based on all 4368 reflections are
0.034 and 0.067 respectively).
Crystallographic data (excluding structure factors) for the struc-
ture reported in this paper have been deposited with the Cambridge
Crystallographic Data Centre: CCDC reference number 297366.
For crystallographic data in CIF or other electronic format see
DOI: 10.1039/b602585d
+
calcd for C27H30N3O2 ([M − H]+): 428.2339; found: 428.2333.
4-[(1RS,3aSR,4RS,8aSR,8bRS)-2-(4-Chlorobenzyl)-1-isopropyl-
3-oxodecahydropyrrolo[3,4-a]pyrrolizin-4-yl]benzamidine
hydrochloride (( )-12)
X-Ray crystal structure of the complex of thrombin with (+)-13
Diffraction data were measured on a Bruker FR591 X-ray gener-
ator, with 0.2 mm focus, run at 50 kV/60 mA and equipped with
an Osmic focussing monochromator and an Oxford Cryostream
cooler run at 100 K. The Marresearch300 image plate detector
General procedure F, starting from ( )-34 (68 mg, 0.16 mmol),
gave ( )-12 (42 mg, 55%) as a colorless solid; mp 200–203 ◦C;
mmax/cm−1 (neat) 3251, 3061, 2959, 2868, 1659, 1613, 1539, 1489,
2374 | Org. Biomol. Chem., 2006, 4, 2364–2375
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©

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was placed 120 mm from the crystal and scanned with 0.15 mm
2 (a) J. A. Olsen, D. W. Banner, P. Seiler, U. Obst Sander, A. D’Arcy,
M. Stihle, K. Mu¨ller and F. Diederich, Angew. Chem., Int. Ed., 2003,
42, 2507–2511; (b) J. A. Olsen, D. W. Banner, P. Seiler, B. Wagner,
T. Tschopp, U. Obst-Sander, M. Kansy, K. Mu¨ller and F. Diederich,
ChemBioChem, 2004, 5, 666–675; (c) J. Olsen, P. Seiler, B. Wagner,
H. Fischer, T. Tschopp, U. Obst-Sander, D. W. Banner, M. Kansy,
K. Mu¨ller and F. Diederich, Org. Biomol. Chem., 2004, 2, 1339–1352;
(d) E. Schweizer, A. Hoffmann-Ro¨der, K. Scha¨rer, J. Olsen, C. Fa¨h,
P. Seiler, U. Obst-Sander, B. Wagner, M. Kansy and F. Diederich,
ChemMedChem, 2006, DOI: 10.1002/cmdc.200600015.
pixel size. Exposure times were 600 s for 0.5◦ frames. Data from
23
˚
456 frames were processed to 1.79 A resolution using XDS. The
˚
space group is C2 with unit cell dimensions a = 71.29 A, b =
◦
˚
˚
71.84 A, c = 73.06 A, b = 100.56 . For 141 897 observations
˚
of 31 281 reflections (with ice rings excluded 2.28–2.22 A and
˚
1.92–1.89 A), the merging R factor on intensities was 4.1%
˚
(25.3% in the outermost shell, 1.79–1.9 A), with completeness
3 For a similar approach, see: (a) C.-Y. Kim, J. S. Chang, J. B. Doyon,
T. T. Baird, Jr., C. A. Fierke, A. Jain and D. W. Christianson, J. Am.
Chem. Soc., 2000, 122, 12125–12134; (b) R. D. Madder, C.-Y. Kim, P. P.
Chandra, J. B. Doyon, T. A. Baird, Jr., C. A. Fierke, D. W. Christianson,
J. G. Voet and A. Jain, J. Org. Chem., 2002, 67, 582–584.
4 For thrombin inhibitors, see: (a) J. B. M. Renwinkel and A. E. P. Adang,
Curr. Pharm. Des., 1999, 5, 1043–1075; (b) J. A. Huntington and T. P.
Baglin, Trends Pharmacol. Sci., 2003, 24, 589–595; (c) S. Srivastava,
L. N. Goswami and D. K. Dikshit, Med. Res. Rev., 2005, 25, 66–92;
(d) M. Di Nisio, S. Middeldorp and H. R. Bu¨ller, New Engl. J. Med.,
2005, 353, 1028–1040.
91.4% (86.6%) and I/r 22.9 (5.4). Data reduction used the
CCP4 package.24 Starting from 1OYT.pdb, model building with
MOLOC19 and refinement with Refmac525 to 1.79 A resolution
˚
gave for 29 702 (working) and 1582 (free) reflections, final overall
crystallographic R factors of 16.0% and 19.8%, with values in
˚
the outer shell (1.83–1.79 A) of 20.5% and 24.8%, respectively,
for 2827 non-hydrogen atoms, including one Na+ ion, one Ca2+
ion and 493 water molecules. The inhibitor density is very clear.
Coordinates have been deposited at the Protein Data Bank,
PDB code: 2CF9.
5 F. Hof, D. M. Scofield, W. B. Schweizer and F. Diederich, Angew.
Chem., Int. Ed., 2004, 43, 5056–5059.
6 R. Paulini, K. Mu¨ller and F. Diederich, Angew. Chem., Int. Ed., 2005,
44, 1788–1805.
7 U. Obst, P. Betschmann, C. Lerner, P. Seiler, F. Diederich, V. Gramlich,
L. Weber, D. W. Banner and P. Scho¨nholzer, Helv. Chim. Acta, 2000,
83, 855–909.
8 (a) T. F. Braish and D. E. Fox, Synlett, 1992, 979–980; (b) C. J. Walter,
H. L. Anderson and J. K. M. Sanders, J. Chem. Soc., Chem. Commun.,
1993, 458–460.
9 (a) R. Huisgen, J. Org. Chem., 1976, 41, 403–419; (b) R. Grigg, J. Idle,
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X-Ray crystal structure of the complex of thrombin with (+)-12
Diffraction data were measured at the Swiss Light Source (SLS)
˚
on beamline PXII. The wavelength was 0.97001 A, the detector
MarCCD225 at 120 mm, and exposure times were 1 s for 360
frames of 0.5◦. The images showed no ice rings and ∼10 overloads
˚
per image. The data were processed to 1.3 A resolution using
XDS, including the ‘zero dose’ radiation damage correction with
default values. The space group is C2 with unit cell dime◦nsions
˚
˚
˚
a = 71.07 A, b = 71.51 A, c = 72.40 A, b = 100.25 . For
307621 observations of 85919 reflections, the merging R factor
on intensities was 3.9% (30.2% in the outermost shell, 1.37–
˚
1.3 A), with completeness 98.2% (95.9%) and I/r 17.8 (4.2).
Data reduction was as above. Refinement with Refmac5 used
the TLS option and hydrogens were inserted at riding positions,
where unique. The final overall crystallographic R factors are
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˚
(1.334–1.30 A) of 26.3% and 29.6%, respectively, for 81 614 (4308)
reflections and 2789 non-hydrogen atoms, including one Na+ ion,
one Ca2+ ion and 397 water molecules. Seven residues were given
alternative conformations. The inhibitor density is very clear.
Coordinates have been deposited at the Protein Data Bank, PDB
code: 2CF8.
16 P. Betschmann, S. Sahli, F. Diederich, U. Obst and V. Gramlich, Helv.
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Acknowledgements
Support from F. Hoffmann–La Roche Ltd, Chugai Pharmaceuti-
cals and the ETH Research Council is gratefully acknowledged.
We thank Olivier Kuster for the measurement of enzyme inhibitor
constants and Andri Schu¨tz and Tobias Platen for their help in the
preparation of some compounds. This work was further supported
by postdoctoral fellowships to A. H.-R. (Emmy Noether-Program,
DFG) and J. A. O. (Carlsberg Foundation).
22 G. M. Sheldrick, SHELXL-97, Program for refinement of crystal
structures, University of Go¨ttingen, Germany, 1997.
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