Second-generation inhibitors for the metalloprotease neprilysin based on bicyclic heteroaromatic scaffolds: Synthesis, biological activity, and X-ray crystal-structure analysis

Article abstract of DOI:10.1002/hlca.200590051

A new class of nonpeptidic inhibitors of the Zn^II-dependent metalloprotease neprilysin with IC₅₀ values in the nanomolar activity range (0.034-0.30 μM) were developed based on structure-based de novo design (Figs. 1 and 2). The inhibitors feature benzimidazole and imidazo[4,5-c] pyridine moieties as central scaffolds to undergo H-bonding to Asn542 and Arg717 and to engage in favorable π-π stacking interactions with the imidazole ring of His711. The platform is decorated with a thiol vector to coordinate to the Zn^II ion and an aryl residue to occupy the hydrophobic S1′ pocket, but lack a substituent for binding in the S2′ pocket, which remains closed by the side chains of Phe106 and Arg110 when not occupied. The enantioselective syntheses of the active compounds (+)-1, (+)-2, (+)-25, and (+)-26 were accomplished using Evans auxiliaries (Schemes 2,4, and 5). The inhibitors (+)-2 and (+)-26 with an imidazo[4,5-c]pyridine core are ca. 8 times more active than those with a benzimidazole core ((+)-1 and (+)-25) (Table 1). The predicted binding mode was established by X-ray analysis of the complex of neprilysin with (+)-2 at 2.25-A resolution (Fig. 4 and Table 2). The ligand coordinates with its sulfanyl residue to the Zn^II ion, and the benzyl residue occupies the S1′ pocket. The 1H-imidazole moiety of the central scaffold forms the required H-bonds to the side chains of Asn542 and Arg717. The heterobicyclic platform additionally undergoes π-π stacking with the side chain of His711 as well as edge-to-face-type interactions with the side chain of Trp693. According to the X-ray analysis, the substantial advantage in biological activity of the imidazopyridine inhibitors over the benzimidazole ligands arises from favorable interactions of the pyridine N-atom in the former with the side chain of Arg102. Unexpectedly, replacement of the phenyl group pointing into the deep S1′ pocket by a biphenyl group does not enhance the binding affinity for this class of inhibitors.

Full text of DOI:10.1002/hlca.200590051

Helvetica Chimica Acta ± Vol. 88 (2005)
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Second-Generation Inhibitors for the Metalloprotease Neprilysin Based on
Bicyclic Heteroaromatic Scaffolds: Synthesis, Biological Activity, and X-Ray
Crystal-Structure Analysis
by Stefan Sahli, Brian Frank, W. Bernd Schweizer, and François Diederich*
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule, ETH-Hönggerberg, HCI,
8093 Zürich (e-mail: diederich@org.chem.ethz.ch)
and
Denise Blum-Kaelin, Johannes D. Aebi, and Hans-Joachim Böhm
Pharma Research Basel, Discovery Chemistry, F. Hoffmann-La Roche Ltd., 4070 Basel
and
Christian Oefner and Glenn E. Dale
Morphochem AG, 4058 Basel
A new class of nonpeptidic inhibitors of the ZnII-dependent metalloprotease neprilysin with IC50 values in
the nanomolar activity range (0.034 ± 0.30 mm) were developed based on structure-based de novo design (Figs. 1
and 2). The inhibitors feature benzimidazole and imidazo[4,5-c]pyridine moieties as central scaffolds to undergo
H-bonding to Asn542 and Arg717 and to engage in favorable p-p stacking interactions with the imidazole ring of
His711. The platform is decorated with a thiol vector to coordinate to the ZnII ion and an aryl residue to occupy
the hydrophobic S1' pocket, but lack a substituent for binding in the S2' pocket, which remains closed by the side
chains of Phe106 and Arg110 when not occupied. The enantioselective syntheses of the active compounds (‡)-1,
(‡)-2, (‡)-25, and (‡)-26 were accomplished using Evans auxiliaries (Schemes 2, 4, and 5). The inhibitors (‡)-2
and (‡)-26 with an imidazo[4,5-c]pyridine core are ca. 8 times more active than those with a benzimidazole core
((‡)-1 and (‡)-25) (Table 1). The predicted binding mode was established by X-ray analysis of the complex of
neprilysin with (‡)-2 at 2.25-Š resolution (Fig. 4 and Table 2). The ligand coordinates with its sulfanyl residue to
the ZnII ion, and the benzyl residue occupies the S1' pocket. The 1H-imidazole moiety of the central scaffold
forms the required H-bonds to the side chains of Asn542 and Arg717. The heterobicyclic platform additionally
undergoes p-p stacking with the side chain of His711 as well as edge-to-face-type interactions with the side chain
of Trp693. According to the X-ray analysis, the substantial advantage in biological activity of the imidazo-
pyridine inhibitors over the benzimidazole ligands arises from favorable interactions of the pyridine N-atom in
the former with the side chain of Arg102. Unexpectedly, replacement of the phenyl group pointing into the deep
S1' pocket by a biphenyl group does not enhance the binding affinity for this class of inhibitors.
1. Introduction. ± In the preceding paper [1], we described a new class of inhibitors
of the metalloprotease neprilysin with a central 1H-imidazole platform, featuring IC50
values (IC50: concentration of inhibitor at which 50% Vmax is observed) in the low
micromolar range. The de novo design of these compounds was based on the X-ray
crystal structure of NEP complexed with phosphoramidon (Protein Data Bank (PDB)
file name 1DMT) [2]. For the design of the second-generation inhibitors, we reverted
to an unpublished X-ray crystal structure [3] of NEP complexed with the inhibitor
thiorphan [4]. During the analysis, we carefully compared the active sites of the two
ꢀ 2005 Verlag Helvetica Chimica Acta AG, Zürich

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different crystal structures (Fig. 1). The larger phosphoramidon occupies both the S1'
and the S2' pockets of the enzyme (blue structure in Fig. 1), whereas the smaller
thiorphan fills only the S1' pocket (green structure in Fig. 1). In the thiorphan complex,
the S2' pocket is closed by the side chains of Arg110 and Phe106.
Fig. 1. Superimposed active-site residues seen in the X-ray crystal structures of NEP complexed with
phosphoramidon (blue) and thiorphan (green). The inhibitors are omitted for clarity. The conformational
changes leading to the closure of the unoccupied S2' pocket in the thiorphan complex are indicated by arrows.
Although thiorphan does not occupy the S2' pocket, it is a very potent neprilysin
inhibitor (Ki ˆ 4.7 Æ 1.2 nm [4]). Clearly, the occupancy of the S2' pocket is not
necessary for good inhibitor binding [5]. For instance, De Lombaert et al. developed
very potent inhibitors without substituents in the S2' pocket [6]. Also, our
investigations of the first generation, 1H-imidazole-based inhibitors [1] had shown
that increasing the size of the S2' substituent from a Ph to a naphthalenyl residue did

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733
not translate into enhanced binding, which reflects that this pocket is not well-defined
(actually, it has been shown that the P2' and P1' residues depend on each other: smaller
P1' substituents allow for the presence of both, either smaller or larger P2' residues
[7]). Based on these observations, we designed the two potential inhibitors (‡)-1 and
(‡)-2 without a substituent pointing into this pocket (Fig. 2,a). The two compounds
feature the same vectors to coordinate to the ZnII ion (sulfanyl residue) and to fill the
S1' (Ph residue) as the inhibitors described in the preceding paper [1]. As platforms,
which should anchor by H-bonding to the side chains of Asn542 and Arg717, we
introduced more-expanded heterocyclic systems, compared with the 1H-imidazole core
in the first-generation inhibitors. According to modeling studies with the program
MOLOC [8], benzimidazole and imidazo[4,5-c]pyridine should be able to form the
above-mentioned obligatory H-bonds and, additionally, undergo attractive p-p
stacking with the side chain of His711 and edge-to-face interactions with the indole
ring of Trp693 [9]. Furthermore, we expected formation of an H-bond between the
pyridine N-atom in (‡)-2 and the side chain of Arg102 (Fig. 2,b). Here, we report the
synthesis of (‡)-1 and (‡)-2, and some analogs, their biological activities, and the X-ray
crystal structure of NEP bound to inhibitor (‡)-2 (for a preliminary communication on
parts of this work, see [10]).
Fig. 2. a) Structures of the new inhibitors (‡)-1 and (‡)-2. b) Schematic representation of the predicted
interactions of (‡)-2 at the active site of NEP. A similar binding mode is predicted for ligand (‡)-1. Potential H-
bonds are shown as dashed lines.

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2. Results and Discussion. ± 2.1. Synthesis of Inhibitors (‡)-1 and (‡)-2. Stereo-
selective synthesis started from oxazolidinone (À)-3 derived from commercially
available (S)-valinol and diethyl carbonate. According to a procedure of Evans et al.
[11], (À)-3 was acylated to give (‡)-4, which, by diastereoselective alkylation with
BnSCH2Br (5), provided (À)-6 (Scheme 1). The a-bromo thioether was synthesized
according to Reich et al. from benzenemethanethiol, HBr, and s-trioxane [12].
Oxazolidinone (À)-6 was subsequently converted via benzyl ester (À)-7 to carboxylic
acid (À)-8 [11].
Scheme 1. Diastereoselective Synthesis of the Carboxylic Acid (À)-8
a) BuLi, 3-phenylpropanoyl chloride, THF, À 788 ! 08, 35 min; 65%. b) 1. (i-Pr)2NH, BuLi, THF, 08 ! r.t.,
15 min; 2. 5, À 788 ! À 158, 14 h; 62%, diastereoisomeric ratio dr > 95 :5. c) BnOH, BuLi, THF, À 108 ! 08,
2 h; 64%. d) HBr, AcOH, 508, 15 min; 82%.
One method to build up 2-substituted benzimidazoles is the condensation of
carboxylic acids with arene-1,2-diamines [13]. Maekawa and Ohtani developed a mild
protocol consisting of amide coupling between carboxylic acid and amine, followed by
condensation to the heteroaromatic bicycle in AcOH [14]. We used a similar protocol
to build up the desired 2-substituted benzimidazole and imidazo[4,5-c]pyridine
scaffolds, respectively. Amide coupling of carboxylic acid (À)-8 with benzene-1,2-
diamine or pyridine-3,4-diamine was accomplished by forming in situ a mixed
anhydride with i-BuOCOCl, followed by addition of the amine to give (‡)-9 and
(‡)-10, respectively (Scheme 2; for the constitutional assignment, see Sect. 2.4)
[15][16]. Unfortunately, the yields were low (see Sect. 2.2 for optimized coupling
conditions). The intramolecular condensation of (‡)-9 and (‡)-10 according to Chen
et al. yielded bicycles (À)-11 and (‡)-12 in high yields [17]. To prepare control
compound (‡)-13, (À)-11 was N-methylated with NaH and MeI to give (‡)-14. Final S-
debenzylation of (À)-11, (‡)-12, and (‡)-14 was accomplished with Na in liquid NH3 to
afford (‡)-1, (‡)-2, and (‡)-13, respectively. The crude products were purified by

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Scheme 2. Synthesis of the Inhibitors (‡)-1 and (‡)-2 Starting from (À)-8
a) Benzene-1,2-diamine, N-methylmorpholine, i-BuOCOCl, THF, À 208, 16 h; 25%. b) Pyridine-3,4-diamine,
N-methylmorpholine, i-BuOCOCl, THF, À 208, 16 h; 51%. c) AcOH, 658, 2.5 h; 80% (À)-11, 93% (‡)-12. d)
Na, NH3, THF, À 788, 30 min; 33% (‡)-1, 10% (‡)-2, 61% (‡)-13. e) NaH, MeI, THF, 08 ! r.t., 55 min; 95%.
reversed-phase (RP) HPLC with 0.1% aq. CF3COOH/MeCN as eluent to give the
corresponding trifluoroacetate salts. Disappointingly, the yields of (‡)-1 and (‡)-2
were low to very low, presumably due to isolation problems.
2.2. Modified Synthesis of Inhibitor (‡)-2. Since the synthesis of (‡)-2 described in
Sect. 2.1 was unsatisfactory in terms of yields, a modified synthesis was planned in which
the sulfanyl residue is introduced at the end by a Mitsunobu reaction. The synthesis
started from oxazolidinone (‡)-15, which was obtained from l-phenylalanine in two
steps [18][19]. Treatment of (‡)-15 with BuLi and 3-phenylpropanoyl chloride
afforded (‡)-16 (Scheme 3) [20]. The direct conversion to (‡)-17 via the titanium
enolate of (‡)-16, with s-trioxane as electrophile, gave only modest yields. Therefore,
the indirect path via Bn-protected (‡)-18 [20] was pursued. Debenzylation to (‡)-17
was accomplished by using H2 and Pd/C [21]. The overall yield of the two-step
conversion was substantially higher than that of the one-step protocol. Alcohol (‡)-17
was readily silylated with (t-Bu)Me2SiCl to give (‡)-19.

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Scheme 3. Diastereoselective Synthesis of (‡)-19
a) BuLi, 3-phenylpropanoyl chloride, THF, À 788 ! 08, 35 min; 77%. b) TiCl4, Et3N, s-trioxane, CH2Cl2, 08,
3.5 h; 48%. c) TiCl4, EtN(i-Pr)2, BnOCH2Cl, CH2Cl2, 08, 4.5 h; 80%. d) H2 (1 atm), Pd/C, AcOEt, HCl, r.t., 6 h;
86%. e) (t-Bu)Me2SiCl, DMAP, CH2Cl2, 16 h, r.t.; 71%. DMAP ˆ 4-(Dimethylamino)pyridine.
The chemoselective hydrolysis of the exocyclic ꢁamideꢂ group of (‡)-19 was
accomplished using LiOOH, generated in situ from H2O2 and LiOH, to give carboxylic
acid (À)-20 and to regain oxazolidinone (‡)-15 (Scheme 4) [22] (for an earlier synthesis
of racemic (Æ)-20, see [23]). Since the amide coupling with pyridine-3,4-diamine
described in Sect. 2.1 gave low yields, we established an alternative protocol according
to a procedure of Pigro et al., who coupled pyridine-2,6-diamines with carboxylic acids
[24]. Acid (À)-20 was transformed in situ into its corresponding acid chloride with
(COCl)2, then the amine was added to produce amide (‡)-21. Treatment with AcOH
gave the 2-substituted imidazo[4,5-c]pyridine (‡)-22. Deprotection with Bu4NF
provided the primary alcohol (‡)-23, which was converted in a Mitsunobu reaction
with AcSH to thioester (À)-24 in excellent yield [1][25]. Final deprotection of the
acetylated sulfanyl group according to Zervas et al. yielded the desired (‡)-2 [26]. The
isolated yield of 73% was much higher than that obtained pursuing the Bn-
deprotection route (see Sect. 2.1). The two pKa-values of (‡)-2 were determined by
potentiometric titrations as 6.19and 10.59(for a full experimental description of the
pKa determinations, see [27]).
2.3. Biological Activity of (‡)-1, (‡)-2, (À)-11, and (‡)-13. The in vitro activity of
(‡)-1, (‡)-2, (À)-11, and (‡)-13 towards neprilysin was determined in a fluorometric
assay (Table 1; for details, see [1]). Gratifyingly, the IC50 values of (‡)-1 and (‡)-2 are
in the nanomolar range; these compounds show ca. 5 ± 25 times higher affinities
towards neprilysin compared to the imidazole-based inhibitors [1]. Interestingly, ligand
(‡)-2 (IC50 ˆ 0.040 mm; the higher value given in the preliminary communication [10] is
wrong) is seven times more active than (‡)-1 (IC50 ˆ 0.29 mm). The enhanced activity
seems to support the modeling-based proposal (Fig. 2,b) that the pyridine N-atom of

Helvetica Chimica Acta ± Vol. 88 (2005)
Scheme 4. Modified Synthesis of (‡)-2
737
a) H2O2 (30%), LiOH ´ H2O, THF/H2O, 08, 3 h; 66%. b) 1. (COCl)2, CH2Cl2, r.t., 12 h; 2. Pyridine-3,4-diamine,
Et3N, r.t., 12 h; 93%. c) AcOH, 658, 2.5 h; 96%. d) Bu4NF, THF, r.t., 2.5 h; 84%. e) DIAD, Ph3P, AcSH, THF,
08 ! r.t., 3 h; 93%. f) MeONa, MeOH, H2, r.t., 75 min; 73%. DIAD ˆ Diisopropyl azodicarboxylate.
Table 1. Activities of the New Bicyclic Inhibitors towards Neprilysin
Inhibitor
X
R1
R2
R3
IC50 [mm]
(‡)-1
CH
N
CH
CH
CH
N
H
H
H
Me
H
H
H
H
H
Ph
Ph
H
H
Bn
H
H
H
0.29
0.040
30a)
5.3
0.30
0.034
(‡)-2
(À)-11
(‡)-13
(‡)-25
(‡)-26
H
a) % Inhibition at 100 mm inhibitor concentration.
(‡)-2 undergoes additional attractive interactions with the enzyme, for instance by H-
bonding to the side chain of Arg102 (see also Sect. 2.6). As a control, the binding
affinity of benzylated (À)-11 is very weak, although the Bn group is well accommo-
dated in the active site according to the modeling. Clearly, a thiol ligand to coordinate
to the ZnII ion is still needed for a good binding. The binding affinity of N-methylated
benzimidazole (‡)-13 (IC50 ˆ 5.3 mm) is reduced by a factor of 20, compared to (‡)-1.
This result confirms the assumption that the 1H-imidazole moiety in the heterocyclic
scaffold of (‡)-1 and (‡)-2 serves as a peptide-bond isoster and undergoes H-bonding
to the side chains of Asn542 and Arg717.
2.4. Synthesis of Inhibitors (‡)-25 and (‡)-26 with 1,1'-Biphenyl Substituents to Fill
the S1' Pocket. The S1' pocket is very deep and can accommodate large substituents as
was shown in different studies [6][7][28][29]. For instance, De Lombaert et al. could
enhance the affinity of their inhibitors by a factor of 30 by replacing Ph by 1,1'-biphenyl

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as the S1'-pocket-filling substituent [28]. To test whether we would score similar gains
in binding free enthalpy in our class of ligands, we prepared compounds (‡)-25 and
(‡)-26 with 1,1'-biphenyl substituents. Their synthesis started from 3-(1,1'-biphenyl-4-
yl)propanoic acid, obtained in two steps from [1,1'-biphenyl]-4-carbaldehyde and
malonic acid [30], and oxazolidinone (‡)-15, which were coupled to yield (‡)-27
(Scheme 5) [31]. Diastereoselective alkylation of the titanium enolate of (‡)-27 with
BnOCH2Cl afforded (‡)-28 in excellent yield and high diastereoselectivity. Chemo-
selective hydrolysis of (‡)-28 with LiOOH gave (À)-29. The amide coupling of
Scheme 5. Synthesis of Inhibitors (‡)-25 and (‡)-26
a) 1. BuLi, À 788, 30 min; 2. 3-[1,1'-biphenyl-4-yl]propanoic acid, Et3N, t-BuCOCl, À 788 ! 08, 1 h; 76%.
b) TiCl4, EtN(i-Pr)2, BnOCH2Cl, CH2Cl2, 08, 5 h; 94%. c) H2O2 (30%), LiOH ´ H2O, THF/H2O, 08, 2.5 h; 73%.
d) 1. (COCl)2, CH2Cl2, r.t., 12 h; 2. Benzene-1,2-diamine, Et3N, r.t., 12 h; 66%. e) 1. (COCl)2, CH2Cl2, r.t., 12 h;
2. pyridine-3,4-diamine, Et3N, r.t., 12 h; 57%. f) AcOH, 658, 2.5 h; 85% (‡)-32, 100% (‡)-33. g) 1. BCl3,
CH2Cl2, 4 h, À 788; 2. MeOH, NH3, 08 ! r.t., 10 min; 34%. h) DIAD, Ph3P, AcSH, THF, 08 ! r.t., 3 h; 44% (‡)-
36, 46% (‡)-37. i) 1. BCl3, CH2Cl2, 3 h, À 788; 2. MeOH, NaHCO3, 08 ! r.t., 12 h; 3. H2SO4, EtOH, 508, 2 h
then r.t. 12 h; 59%. j) MeONa, MeOH, H2, r.t., 75 min; 68% (‡)-25, 57% (‡)-26.

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carboxylic acid (À)-29 with benzene-1,2-diamine and pyridine-3,4-diamine gave (‡)-30
and (‡)-31, respectively, and the subsequent condensations to the heteroaromatic
bicycles (‡)-32 and (‡)-33 was accomplished as described in Sect. 2.2. The lower yields
of the amide couplings are due to formation of the corresponding bisamides.
Crystals of (‡)-31 were obtained by recrystallization from AcOEt and analyzed by
X-ray crystallography (Fig. 3). The compound crystallizes in the chiral space group P21.
The crystal structure led to the constitutional assignment for (‡)-31, whose NH2 group
is in meta-position to the pyridine N-atom. The same constitution was derived for
1
amides (‡)-10 and (‡)-21 by comparison of the H-NMR spectra.
Fig. 3. Crystal structure of (Æ)-31. The ORTEP representation is shown with atomic-displacement-parameter
ellipsoids at the 50% probability level. Arbitrary Numbering.
O-Debenzylation of (‡)-32 and (‡)-33 by hydrogenation with H2 and Pd/C in
MeOH or EtOH was unsuccessful, even at enhanced H2 pressure and catalyst loading.
Deprotection of (‡)-32 was finally accomplished with BCl3 in CH2Cl2 to give (‡)-34 in
34% yield [32]. The low yield is likely due to the problematic chromatographic
separation of B-containing impurities. For the debenzylation of (‡)-33, we also used
BCl3 in CH2Cl2, but after workup, the product could not be separated from all
impurities. Therefore, the crude product was treated with H2SO4 in EtOH to give (‡)-
35 in 59% overall yield. The two primary alcohols (‡)-34 and (‡)-35 were transformed
into their corresponding thioesters (‡)-36 and (‡)-37 as described in Sect. 2.2. Final
hydrolysis of the thioesters afforded the desired inhibitors (‡)-25 and (‡)-26.
2.5. Biological Activity of (‡)-25 and (‡)-26. The IC50 values of the two inhibitors
(Table 1) are in the same range as those of the initial leads (‡)-1 and (‡)-2. This result
shows that, contrary to literature-based expectations, introduction of a 1,1'-biphenyl
substituent to fill the S1' pocket does not lead to higher binding affinities towards
neprilysin. It seems likely that the extended heteroaromatic platform and the 1,1'-
biphenyl moiety cannot be both positioned in an ideal way in the active site. Inhibitor
(‡)-26 is by a factor of ca. 9more potent than ( ‡)-25. This finding further supports the
assumption that the pyridine N-atom in (‡)-26 undergoes H-bonding with the side
chain of Arg102.

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2.6. Crystal Structure of Neprilysin Complexed with (‡)-2. For inhibitor binding
studies, compound (‡)-2 was soaked into NEP crystals at mm concentration (for a
detailed protocol, see Exper. Part and Table 2). The crystal structure (PDB file name
1Y8J) confirms the predicted binding mode (Fig. 4). The inhibitor is nestled in the
Fig. 4. Section of the crystal structure of NEP complexed with (‡)-2. a) 2Fo-Fc omit electron-density map for
(‡)-2 at 2.25 Š, calculated with phases from the refined model. The map, contoured at 1s, is shown in light grey
mesh. The molecular surfaces of the S1' and S2' sub-sites are indicated and colored by electrostatic potential;
blue, positive; red, negative. The ZnII ion is shown as a red sphere. The Figure was generated using PyMOL
[33]. b) Top view of (‡)-2 bound in the active site of NEP. Interesting distances discussed in the text are shown in
red. c) Front view showing H-bonds and coordinate bonds (black dashed lines) and the aromatic interactions
involving the central imidazopyridine scaffold (red) of (‡)-2. A H2O molecule is indicated as small red sphere.
Aromatic contacts are shown as red lines.

Helvetica Chimica Acta ± Vol. 88 (2005)
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Fig. 4 (cont.)
interior of the enzyme cavity and binds near the conserved residues of the consensus
sequences 583HExxH587 and 646ExxxD650 and the residues of the 542NAFY545 motif.
Fig. 4,a, shows the final difference-omit electron-density map for (‡)-2 at 2.25-Š
resolution, calculated with phases from the refined model [33]. The inhibitor
coordinates the ZnII ion via its sulfanyl moiety with an interatomic distance d(S ´´´
Zn) ˆ 2.2 Š (Fig. 4,b). The Bn residue expectedly occupies the S1' pocket, encom-
passed by Phe106, Ile558, Phe563, Met579, Val580, Val692, and Trp693 (Fig. 4,c) [1].
The annellated imidazole ring forms the required two H-bonds to the side chains of
Asn542 (d(N ´´´ O) ˆ 2.8 Š) and Arg717 (d(N ´´´ N) ˆ 3.3 Š; Fig. 4,b). Additionally, it
is nicely sandwiched between the side chains of His711 and Trp693, undergoing p-p
interactions at short distance (2.9Š) with the imidazole ring of the former and edge-to-
face-type interactions (shortest C ´´´ C distance: 3.3 Š) with the indole ring of the latter.
The distances between N(5) and C(6) of the imidazo[4,5-c]pyridine and the N-atoms of
the side chain of Arg102 are 4.1 Š and 3.7 Š, respectively. At a resolution of 2.25 Š, it
is not clearly apparent, which position the N-atom takes in the pyridine ring. Thus, it is
also possible that the other tautomeric form (1H-imidazo[4,5-c]pyridine instead of 3H-
imidazo[4,5-c]pyridine) binds to the enzyme or that both tautomers are present.
Indeed, we suggest that the scaffold is complexed as 1H-imidazo[4,5-c]pyridine
tautomer, since this binding mode establishes the short distance (3.7 Š) between the
attracting N(pyridine) and NÀH(Arg) and the long distance (4.1 Š) between the
repulsive CÀH(pyridine) and NÀH(Arg) fragments.
3. Conclusions. ± This paper reports the successful development of new neprilysin
inhibitors with heteroaromatic bicyclic scaffolds. The stereoselective synthesis of the

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chiral, nonracemic ligands was accomplished using Evans auxiliaries. Two different
synthetic routes were explored with the most efficient one (Schemes 4 and 5) featuring
the introduction of the thiol residue (for coordination to the ZnII ion of the
metalloprotease) in a Mitsunobu reaction at the end of the multi-step sequence. The
binding affinities towards neprilysin of the new inhibitors with the heterobicyclic
scaffolds were strongly improved (by a factor of 5 ± 25) over the first-generation
imidazole-based inhibitors [1]. Inhibitors (‡)-2 and (‡)-26 with an imidazo[4,5-
c]pyridine core proved to be more active than the corresponding benzimidazole-
derived ligands (‡)-1 and (‡)-25 by a factor of ca. 8. The binding mode predicted by
modeling studies was established by the X-ray-analysis of the complex of neprilysin
with (‡)-2 at 2.25-Š resolution (Fig. 4). The ligand coordinates with its thiol residue to
the ZnII ion, and the Bn residue occupies the S1' pocket. The 1H-imidazole moiety of
the central scaffold forms the required H-bonds to the side chains of Asn542 and
Arg717. At the same time, the heterobicyclic platform undergoes aromatic interactions
with the side chains of His711 and Trp693. According to the X-ray analysis, the
substantial advantage in biological activity of the imidazo-pyridine inhibitors over the
benzimidazole ligands arises from favorable interactions of the pyridine N-atom in the
former with the side chain of Arg102. In future work, we intend to further explore
favorable contacts between suitably decorated heterocyclic scaffolds and residues
above the S2' pocket of the protein. Contrary to literature expectations, introduction of
1,1'-biphenyl substituents for occupation of the deep S1' pocket did not lead to
enhanced binding activities and further optimization of the residues for filling this
pocket will be required.
We are grateful to F. Hoffmann-La Roche, Chugai Pharmaceuticals, and the ETH Research Council for
support of this work. We thank Dr. B. Wagner (Roche, Basel) for the pKa determinations and Dr. C. Thilgen for
help with the nomenclature.
Experimental Part
General. See [1]. The following compounds were prepared according to literature procedures: (À)-3 [11],
(‡)-4 [11], 5 [12], (À)-7 [11], (À)-8 [11], (‡)-15 [18], (‡)-16 [20], (‡)-18 [20], 3-[1,1'-biphenyl-4-yl]propanoic
acid [30]. Optical rotations: Perkin-Elmer 241 polarimeter; 1-dm cell, l ˆ 589nm (Na-D-line). Prep. RP-
HPLC: Merck Hitachi HPLC with HPLC pump L-7150; Vydac 201TP C18 column (2501  4 mm, 5 mm, 100 Š).
Biological Assay. See [1]. General Procedure for the Amide Coupling with Aromatic Amines (GP 1).
Method A: To a soln. of carboxylic acid (1 equiv.) in THF (0.05m), 4-methylmorpholine (1.5 equiv.) and isobutyl
chloroformate (1.1 equiv.) were added dropwise at À 208. After stirring for 15 min, the arom. amine (2 equiv.)
was added, and the mixture was stirred for additional 16 h at À 208. The solvent was removed in vacuo, the
residue was dissolved in AcOEt and washed with half-sat. aq. NH4Cl soln., half-sat. aq. NaHCO3 soln., and sat.
aq. NaCl soln. The org. phases were dried (MgSO4) and concentrated in vacuo.
Method B: To a soln. of carboxylic acid (1 equiv.) in CH2Cl2 (0.1m), (COCl)2 (1 equiv.) was added at 08, and
the mixture was stirred for 12 h at r.t. The solvent was removed in vacuo, and the residue was dissolved in THF
(0.1m). The arom. amine (2 equiv.) and NEt3 (2 equiv.) were added, and the mixture was stirred for 12 h at r.t.
After addition of H2O, THF was removed in vacuo, and the residual aq. mixture was extracted (CH2Cl2). The
org. phases were washed with sat. aq. NaCl soln., dried (Na2SO4), and concentrated in vacuo.
General Procedure for the Condensation to Bicyclic Heteroaromatics (GP 2). The amide (1 equiv.) was
dissolved in AcOH, and the mixture was stirred at 658 for 2.5 h. AcOH was removed by adding PhMe (3Â),
followed by concentration in vacuo. The residue was dissolved in CH2Cl2, and the soln. was washed with sat. aq.
NaHCO3 soln. The aq. phases were extracted (CH2Cl2), and the combined org. phases were dried (MgSO4) and
concentrated in vacuo.
General Procedure for the Benzyl Thioether Cleavage with Na/NH3 (GP 3). To a soln. of thioether (1 equiv.)
in THF (0.05m), condensed NH3 (NH3/THF 2 :1) was added at À 788. The NH3 (g) was dried before

Helvetica Chimica Acta ± Vol. 88 (2005)
743
condensation by streaming through soda lime. Na (6 equiv.) was added in 3 portions, and the mixture was stirred
until a deep blue color persisted. After stirring for additional 30 min at À 788, NH4Cl (10 equiv.) was added and
the suspension turned yellow. The NH3 was removed by bubbling N2 (g) through the suspension at 08. Et2O was
added to the residue, and the resulting suspension was washed with sat. aq. NH4Cl soln. The aq. phases were
extracted (Et2O), and the combined org. phases were dried (MgSO4) and concentrated in vacuo.
General Procedure for the Replacement of a OH Group with AcSH under Mitsunobu Conditions (GP 4). To
a soln. of Ph3P (1.5 equiv.) in dry THF (0.1m), DIAD (1.5 equiv.) was added at 08, and the mixture was stirred for
30 min, until a white solid precipitated. A soln. of the alcohol (1 equiv.) in dry THF (0.16m) was added dropwise
to the mixture, followed by addition of freshly distilled AcSH (2 equiv.). The mixture was stirred for 1 h at 08
and for 2 h at r.t. After addition of Et2O, and washing with H2O and sat. aq. NaCl soln., the aq. phases were
extracted (CH2Cl2), and the combined org. phases were dried (MgSO4) and concentrated in vacuo.
General Procedure for Thioester Cleavage (GP 5). The thioester (1 equiv.) was dissolved in MeOH (0.05m),
and the soln. was degassed under Ar for 30 min. The soln. was added to a suspension of MeONa (10 equiv.) in
MeOH (0.03m) that was degassed for 30 min under Ar and for 10 min under H2. The mixture was stirred for 2 h
at r.t. After addition of sat. aq. NH4Cl soln. and extraction (CH2Cl2), the org. phases were dried (MgSO4) and
concentrated in vacuo.
(2S)-N-(2-Aminophenyl)-2-benzyl-3-(benzylsulfanyl)propanamide ((‡)-9). GP 1, Method A, starting from
(À)-8 (1.00 g, 3.49mmol) and diamine, gave ( ‡)-9 (330 mg, 25%) after purification by CC (SiO2-60; CH2Cl2/
AcOEt 91:9). Colorless oil. [a]2D5 ˆ ‡6.7 (c ˆ 1, CHCl3). IR (neat): 3251, 3028, 2916, 1651, 1622, 1495, 1453,
1381, 1304, 1155, 1077. 1H-NMR (300 MHz, CDCl3): 2.06 ± 2.48 (m, 1 H); 2.65 (dd, J ˆ 13.4, 4.7, 1 H); 2.81 (dd,
J ˆ 13.1, 5.3, 1 H); 2.91 (dd, J ˆ 13.1, 10.0, 1 H); 2.92 (dd, J ˆ 13.4, 10.0, 1 H); 3.51 (br. s, 2 H); 3.75 (s, 2 H); 6.53
(br. s, 1 H); 6.67 ± 6.73 (m, 2 H); 6.9 0 (dd, J ˆ 7.5, 1.6, 1 H); 7.01 (dt, J ˆ 7.5, 1.6, 1 H); 7.13 ± 7.35 (m, 10 H).
13C-NMR (75 MHz, CDCl3): 34.1; 37.3; 39.0; 50.9; 117.0; 118.8; 123.0; 126.1; 126.5; 127.1; 127.3; 128.5; 128.8;
‡
‡
128.7; 128.9; 138.5; 138.9; 141.1; 171.8. MALDI-MS (DHB): 399.2 (76, [ M ‡ Na] ), 377.2 (100, MH ), 359.2
‡
‡
(15), 331.2 (12). MALDI-HR-MS (DHB): 377.1687 (MH , C23H25N2OS ; calc. 377.1682).
2-[(1S)-1-Benzyl-2-(benzylsulfanyl)ethyl]-1H-benzimidazole ((À)-11). GP 2, starting from (‡)-9 (200 mg,
0.53 mmol), gave (À)-11 (152 mg, 80%) after purification by CC (SiO2-60; pentane/AcOEt 80 :20). Colorless
solid. M.p. 161 ± 1628. [a]2D5 ˆ À4.0 (c ˆ 1, EtOH). IR (CHCl3): 3451, 3064, 2952, 2364, 1622, 1600, 1527, 1494,
1
1455, 1424, 1329, 1273. H-NMR (300 MHz, (CD3)2SO, 5 drops of CF3COOH added): 2.93 (dd, J ˆ 13.8, 5.4,
1 H); 3.00 (dd, J ˆ 13.8, 9.5, 1 H); 3.14 ± 3.26 (m, 2 H); 3.72 (s, 2 H); 3.82 ± 3.92 (m, 1 H); 7.11 ± 7.27 (m, 10 H);
7.51 ± 7.57 (m, 2 H); 7.77 ± 7.83 (m, 2 H). 13C-NMR (75 MHz, CDCl3): 35.3; 37.1; 40.2; 43.0; 122.6; 126.8; 127.4;
‡
128.8; 129.1; 129.3; 138.5; 139.1; 156.0 (3 arom. signals overlapping). MALDI-MS (DHB): 359.2 (100, MH ),
‡
‡
307.1 (6), 179.6 (7). MALDI-HR-MS (DHB): 359.1581 (MH , C23H23N2S ; calc. 359.1582). Anal. calc. for
C23H22N2S (358.50): C 77.06, H 6.19, N 7.81, S 8.94; found C 76.80, H 6.40, N 7.73, S 8.96.
2-[(1S)-1-Benzyl-2-sulfanylethyl]-1H-benzimidazol-3-ium Trifluoroacetate ((‡)-1). GP 3, starting from
(À)-11 (100 mg, 0.28 mmol), gave a residue that was purified by CC (SiO2-60; CH2Cl2/MeOH 98 :2) and RP-
HPLC (RP-18 SiO2; 0.1% aq. CF3COOH/MeCN 99 :1 ! 0 :100 in 60 min) to give (‡)-1 (35 mg, 33%).
Colorless oil. [a]2D5 ˆ ‡15.2 (c ˆ 1.17, EtOH). IR (KBr): 3423, 3120, 3053, 2840, 2678, 1941, 1891, 1813, 1620,
1538, 1491, 1454, 1426, 1328, 1309, 1270. 1H-NMR (300 MHz, CDCl3): 1.35 (t, J ˆ 8.7, 1 H); 2.85 ± 2.94 (m, 1 H);
3.00 ± 3.11 (m, 1 H); 3.19( dd, J ˆ 13.7, 8.1, 1 H); 3.25 (dd, J ˆ 13.7, 7.8, 1 H); 3.79± 3.89( m, 1 H); 7.02 ± 7.12 (m,
5 H); 7.28 ± 7.33 (m, 2 H); 7.59± 7.65 ( m, 2 H). 13C-NMR (75 MHz, CDCl3): 26.9; 39.1; 44.8; 114.0; 125.7; 127.0;
‡
‡
128.5; 128.7; 131.0; 136.3; 154.5. MALDI-MS (DHB): 291.1 (12, [M ‡ Na] ), 269.1 (100, MH ). MALDI-HR-
‡
‡
MS (DHB): 269.1103 (MH , C16H17N2S ; calc. 269.1107).
(2S)-N-(3-Aminopyridin-4-yl)-2-benzyl-3-(benzylsulfanyl)propanamide ((‡)-10). GP 1, Method A, start-
ing from (À)-8 (751 mg, 2.63 mmol) and pyridine-3,4-diamine, gave (‡)-12 (502 mg, 51%) after purification by
CC (SiO2-60; CH2Cl2/AcOEt 91 :9). Colorless foam. M.p. 458. [a]2D5 ˆ ‡4.4 (c ˆ 1, CHCl3). IR (neat): 3028,
2919, 2361, 1661, 1582, 1511, 1455, 1421, 1331, 1300, 1219, 1168. 1H-NMR (300 MHz, CDCl3): 2.44 ± 2.54 (m,
1 H); 2.66 (dd, J ˆ 13.4, 4.7, 1 H); 2.80 ± 2.94 (m, 3 H); 3.32 (br. s, 2 H); 3.75 (s, 2 H); 6.99 (br. s, 1 H); 7.10 ± 7.34
(m, 11 H); 7.9 6 (d, J ˆ 5.0, 1 H); 8.04 (s, 1 H). 13C-NMR (75 MHz, CDCl3): 33.8; 37.4; 38.9; 51.1; 117.3; 126.6;
127.2; 128.6; 128.7; 128.8; 128.8; 131.5; 134.5; 138.3; 138.7; 140.0; 141.3; 171.9. MALDI-MS (DHB): 400.1 (2,
‡
‡
‡
‡
[M ‡ Na] ), 378.2 (100, MH ). MALDI-HR-MS (DHB): 378.1629( MH , C22H24N3OS ; calc. 378.1635). Anal.
calc. for C22H23N3OS (377.50): C 70.00, H 6.14, N 11.13; found C 70.08, H 6.22, N 10.90.
2-[(1S)-1-Benzyl-2-(benzylsulfanyl)ethyl]-1H-imidazo[4,5-c]pyridine ((‡)-12). GP 2, starting from (‡)-10
(400 mg, 1.06 mmol), gave (‡)-12 (356 mg, 93%) after purification by CC (SiO2-60; AcOEt). Colorless oil,
which solidified upon standing. M.p. 638. [a]2D5 ˆ ‡14.9( c ˆ 1, CHCl3). IR: 3027, 2920, 1734, 1621, 1587, 1533,
1494, 1454, 1424, 1283, 1238, 1210, 1167. 1H-NMR (300 MHz, CHCl3): 2.91 (dd, J ˆ 13.4, 5.3, 1 H); 2.97 (dd, J ˆ

744
Helvetica Chimica Acta ± Vol. 88 (2005)
13.4, 7.8, 1 H); 3.09± 3.16 ( m, 1 H); 3.21 ± 3.31 (m, 2 H); 3.60 (s, 2 H); 7.02 ± 7.27 (m, 10 H); 7.40 (br. s, 1 H); 8.36
(d, J ˆ 5.6, 1 H); 8.90 (s, 1 H). 13C-NMR (75 MHz, CDCl3): 35.0; 36.9; 40.3; 43.2; 110.0; 126.6; 127.1; 128.5;
128.7; 128.8; 137.9; 138.4; 140.8 (5 arom. signals not observable due to exchange). MALDI-MS (DHB): 360.2
‡
‡
‡
(100, MH ). MALDI-HR-MS (DHB): 360.1527 (MH , C22H22N3S ; calc. 360.1529).
2-[(1S)-1-Benzyl-2-sulfanylethyl]-1H-imidazo[4,5-c]pyridin-5-ium Trifluoroacetate ((‡)-2). GP 3, Meth-
od 1, starting from (‡)-12 (400 mg, 1.11 mmol), gave a residue that was purified by CC (SiO2-60; AcOEt/MeOH
95 :5) and RP-HPLC (RP-18 SiO2; 0.1% aq. CF3COOH/MeCN 99 :1 ! 0 :100 in 60 min) to give (‡)-2 (42 mg,
10%). GP 5, Method 2, starting from (À)-24 (100 mg, 0.32 mmol), gave (‡)-2 (90 mg, 73%) after purification by
RP-HPLC (RP-18 SiO2; 0.1% aq. CF3COOH/MeCN 99 :1 ! 0 :100 in 60 min). Colorless oil. [a]2D5 ˆ ‡23.0 (c ˆ
1, CHCl3). IR: 3687, 3088, 3035, 2927, 2733, 2372, 2097, 1667, 1526, 1454, 1422, 1363, 1312, 1269. 1H-NMR
(300 MHz, CDCl3): 1.49( t, J ˆ 8.6, 1 H); 2.99 ± 3.14 (m, 2 H); 3.24 (dd, J ˆ 13.8, 7.9, 1 H); 3.31 (dd, J ˆ 13.8, 7.3,
1 H); 3.66 ± 3.76 (m, 1 H); 7.14 ± 7.25 (m, 5 H); 7.9 7 d(, J ˆ 6.5, 1 H); 8.23 (d, J ˆ 6.5, 1 H); 9.27 (s, 1 H).
13C-NMR (75 MHz, CDCl3): 27.4; 39.6; 46.3; 113.8; 126.8; 128.6; 128.8; 129.4; 131.6; 131.9; 137.2; 137.5; 167.3.
‡
‡
‡
MALDI-MS (DHB): 270.1 (100, MH ), 236.1 (3). MALDI-HR-MS (DHB): 270.1064 (MH , C15H16N3S ; calc.
270.1059).
2-[(1S)-1-Benzyl-2-(benzylsulfanyl)ethyl]-1-methyl-1H-benzimidazole ((‡)-14). A soln. of (‡)-11 (55 mg,
0.15 mmol) in THF (3 ml) was added to NaH (12 mg) at 08, and the mixture was stirred for 15 min. After
addition of MeI (11 ml, 0.17 mmol) at 08, the soln. was stirred for 10 min at 08 and for 30 min at r.t. H2O was
added, the mixture was extracted (CH2Cl2), the org. phases were dried (MgSO4) and concentrated in vacuo. The
residue was purified by CC (SiO2-60; pentane/AcOEt 71:29) to give (‡)-14 (53 mg, 95%). Colorless oil. [a]D25
ˆ
‡78.5 (c ˆ 1, CHCl3). IR (CHCl3): 2949, 1946, 1887, 1809, 1750, 1696, 1602, 1492, 148, 1452, 1320, 1282, 1153,
1
1073. H-NMR (300 MHz, CDCl3): 2.97 ± 3.25 (m, 5 H); 3.10 (s, 3 H); 3.56 (d, J ˆ 13.5, 1 H); 3.61 (d, J ˆ 13.5,
1 H); 6.87 ± 6.92 (m, 2 H); 7.11 ± 7.31 (m, 11 H); 7.76 ± 7.80 (m, 1 H). 13C-NMR (75 MHz, CDCl3): 29.1; 36.5;
41.4; 41.6; 109.2; 119.2; 121.8; 121.9; 126.4; 126.9; 128.3; 128.5; 128.8; 128.8; 135.0; 138.6; 139.0; 142.4; 156.2.
‡
‡
‡
MALDI-MS (DHB): 395.2 (4, [M ‡ Na] ), 373.2 (100, MH ). MALDI-HR-MS (DHB): 373.1730 (MH ,
‡
C24H25N2S ; calc. 373.1733).
2-[(1S)-1-Benzyl-2-sulfanylethyl]-1-methyl-1H-benzimidazol-3-ium Trifluoroacetate ((‡)-13). GP 3, start-
ing from (‡)-14 (94 mg, 0.25 mmol), gave a residue that was purified by CC (SiO2-60; pentane/AcOEt 80 :20)
and RP-HPLC (RP-18 SiO2-60; 0.1% aq. CF3COOH/MeCN 99 :1 ! 0 :100 in 60 min) to give (‡)-15 (60 mg,
61%). Colorless oil. [a]2D5 ˆ ‡44.8 (c ˆ 0.5, CHCl3). IR (CHCl3): 3003, 1669, 1522, 1495, 1454, 1417, 1347, 1191,
1
1139, 1075. H-NMR (300 MHz, CDCl3): 1.68 (br. s, 1 H); 3.16 ± 3.23 (m, 1 H); 3.35 (d, J ˆ 6.9, 2 H); 3.42 (s,
3 H); 3.54 ± 3.66 (m, 2 H); 6.88 ± 7.03 (m, 2 H); 7.15 ± 7.19( m, 3 H); 7.35 ± 7.38 (m, 1 H); 7.45 ± 7.55 (m, 2 H);
8.03 ± 8.06 (m, 1 H); 10.49(br. s, 1 H). 13C-NMR (75 MHz, CDCl3): 27.7; 30.7; 40.7; 46.1; 110.8; 116.9; 125.7;
126.3; 127.5; 128.8; 129.2; 132.2; 137.5; 154.6 (1 arom. signal overlapping). MALDI-MS (DHB): 283.1 (100,
‡
‡
‡
MH ), 281.1 (55). MALDI-HR-MS (DHB): 283.1262 (MH , C17H19N2S ; calc. 283.1262).
(4S)-4-Benzyl-3-[(2R)-2-benzyl-3-hydroxypropanoyl]-1,3-oxazolidin-2-one ((‡)-17). Method 1: To a soln.
of (‡)-16 (6.79g, 21.95 mmol) in CH 2Cl2 (100 ml), TiCl4 (2.65 ml, 24.14 mmol) and Et3N (3.35 ml, 24.14 mmol)
were added at 08. After stirring the mixture for 30 min at 08, a soln. of s-trioxane (2.17 g, 24.14 mmol) in CH2Cl2
(15 ml) and then TiCl4 (2.65 ml, 24.14 mmol) were added, and the mixture was stirred for 3 h at 08. After
addition of half-sat. aq. NH4Cl soln., the mixture was extracted (AcOEt). The org. phases were washed with sat.
aq. NaHCO3 soln., H2O, and sat. aq. NaCl soln., dried (MgSO4), and concentrated in vacuo. The residue was
purified by CC (SiO2-60; hexanes/AcOEt 40 :60) to give (‡)-17 (3.61 g, 48%).
Method 2: To soln. of (‡)-18 (50 mg, 0.12 mmol) in AcOEt (1 ml), Pd/C (10%, 15 mg) and conc. HCl
(37%, 1 drop) were added, and the mixture was stirred for 6 h under H2 (1 atm). The suspension was filtered
over Celite and concentrated in vacuo to give (‡)-17 (34 mg, 86%). Colorless oil. [a]2D0 ˆ ‡115.7 (c ˆ 1, CHCl3).
IR (neat): 3514, 2925, 1773, 1692, 1494, 1452, 1385, 1349, 1210, 1109, 1055, 1013, 962. 1H-NMR (300 MHz,
CDCl3): 2.29(br. s, 1 H); 2.79( dd, J ˆ 13.4, 9.7, 1 H); 2.89 (dd, J ˆ 13.4, 7.8, 1 H); 3.02 (dd, J ˆ 13.4, 7.2, 1 H);
3.27 (dd, J ˆ 13.4, 3.4, 1 H); 3.82 (dd, J ˆ 11.2, 6.5, 1 H); 3.89( dd, J ˆ 11.2, 4.0, 1 H); 3.98 (dd, J ˆ 8.9, 7.8, 1 H);
4.11 (dd, J ˆ 8.9, 2.2, 1 H); 4.25 ± 4.34 (m, 1 H); 4.51 ± 4.58 (m, 1 H); 7.17 ± 7.36 (m, 10 H). 13C-NMR (75 MHz,
CDCl3): 34.8; 38.0; 47.1; 55.6; 63.1; 66.2; 126.5; 127.3; 128.4; 128.9; 129.1; 129.4; 135.0; 138.1; 153.2; 175.1. EI-
‡
MS: 339.1 (1, M ), 321.1 (17), 131.0 (78), 91.0 (100). Anal. calc. for C20H21NO4 (339.39): C 70.78, H 6.24, N 4.13;
found C 70.93, H 6.26, N 3.94.
(4S)-4-Benzyl-3-((2R)-2-benzyl-3-{[(tert-butyl)(dimethyl)silyl]oxy}propanoyl)-1,3-oxazolidin-2-one ((‡)-
19). To a soln. of (‡)-17 (3.6 g, 10.6 mmol) in dry CH2Cl2 (25 ml), (t-Bu)Me2SiCl (2.4 g, 15.9mmol) and DMAP
(2.2 g, 18.1 mmol) were added, and the mixture was stirred for 16 h at r.t. After addition of Et2O, and washing

Helvetica Chimica Acta ± Vol. 88 (2005)
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with H2O and sat. aq. NaCl soln., the aq. phases were extracted (CH2Cl2), and the combined org. phases were
dried (MgSO4) and concentrated in vacuo. The residue was purified by CC (SiO2-60; hexanes/AcOEt 83 :17) to
give (‡)-19 (3.45 g, 71%). Colorless oil. [a]2D5 ˆ ‡70.0 (c ˆ 1, CHCl3). IR (neat): 2930, 2858, 1778, 1760, 1700,
1605, 1494, 1452, 1390, 1349, 1212, 1184, 1099, 1003, 972. 1H-NMR (300 MHz, CDCl3): 0.05 (s, 6 H); 0.88 (s,
9H); 2.67 ( dd, J ˆ 13.2, 9.8, 1 H); 2.87 (dd, J ˆ 13.5, 7.3, 1 H); 2.94 (dd, J ˆ 13.5, 8.2, 1 H); 3.26 (dd, J ˆ 13.2, 3.3,
1 H); 3.80 (dd, J ˆ 9.6, 5.0, 1 H); 3.89 (dd, J ˆ 9.0, 8.4, 1 H); 3.96 (dd, J ˆ 9.6, 7.2, 1 H); 4.03 (dd, J ˆ 9.0, 2.5, 1 H);
4.37 ± 4.46 (m, 1 H); 4.48 ± 4.56 (m, 1 H); 7.15 ± 7.35 (m, 10 H). 13C-NMR (75 MHz, CDCl3): À 5.3; 18.4; 26.0;
35.0; 38.1; 47.5; 55.4; 63.6; 65.8; 126.3; 127.2; 128.2; 128.8; 129.0; 129.3; 135.3; 138.6; 152.9; 174.0. EI-MS: 438.3
‡
‡
(1, [M À Me] ), 396.2 (32, [M À Me3C] ), 219.1 (37), 145.0 (36), 117.0 (100). Anal. calc. for C26H35NO4Si
(453.65): C 68.84, H 7.78, N 3.09; found C 68.91, H 7.83, N 3.29.
(2R)-2-Benzyl-3-{[(tert-butyl)(dimethyl)silyl]oxy}propanoic Acid ((À)-20). To a soln. of (‡)-19 (2.63 g,
5.80 mmol) in a mixture of THF/H2O (75 ml/25 ml), aq. H2O2 (30%, 3.55 ml, 34.78 mmol) was added. After
addition of a soln. of LiOH ´ H2O (487 mg, 11.59mmol) in H 2O (15 ml) at 08, the mixture was stirred for 3 h at 08,
then 1.5n aq. Na2SO3 soln. (30 ml) was added, and the THF was removed in vacuo. The aq. phases were acidified
by addition of 1n HCl to a pH of 1 ± 2 and extracted (CH2Cl2). The org. phases were dried (MgSO4),
concentrated in vacuo, and the residue was purified by CC (SiO2-60; pentane/AcOEt 83 :17) to give (À)-20
(1.13 g, 66%). Colorless oil. [a]2D5 ˆ À5.3 (c ˆ 1, CHCl3). IR (CHCl3): 3067, 2954, 1948, 1879, 1806, 1709, 1602,
1470, 1406, 1390, 1360, 1258. 1H-NMR (300 MHz, CDCl3): 0.04 (s, 3 H); 0.04 (s, 3 H); 0.89( s, 9H); 2.80 ± 2.89
(m, 1 H); 2.86 (dd, J ˆ 16.2; 7.2, 1 H); 3.02 (dd, J ˆ 16.2, 10.0, 1 H); 3.73 (dd, J ˆ 10.0, 5.6, 1 H); 3.78 (dd, J ˆ 10.0,
4.8, 1 H); 7.16 ± 7.34 (m, 5 H). 13C-NMR (75 MHz, CDCl3): À 5.3; 18.4; 26.0; 34.0; 49.6; 62.9; 126.7; 128.7; 129.2;
‡
‡
138.9; 178.7. MALDI-MS (DCTB): 339.1 (100, [ M À H ‡ 2 Na] ), 317.2 (59, [M ‡ Na] ), 309.2 (11), 261.1 (18).
‡
‡
MALDI-HR-MS (DCTB): 317.1539([ M ‡ Na] , C16H26NaO3Si ; calc. 317.1543). Anal. calc. for C16H26O3Si
(294.46): C 65.26, H 8.90; found C 65.02, H 8.88.
(2R)-N-(3-Aminopyridin-4-yl)-2-benzyl-3-{[(tert-butyl)(dimethyl)silyl]oxy}propanamide ((‡)-21). GP 1,
Method B, starting from (À)-20 (100 mg, 0.34 mmol) and pyridine-3,4-diamine, gave (‡)-21 (122 mg, 93%) after
purification by CC (SiO2-60; pentane/AcOEt 33 :67). Colorless solid. M.p. 1078. [a]2D5 ˆ ‡7.4 (c ˆ 1, CHCl3). IR
(CHCl3): 3412, 3299, 2954, 2857, 1951, 1882, 1806, 1691, 1621, 1586, 1511, 1470, 1422, 1298, 1258. 1H-NMR
(300 MHz, CDCl3): 0.06 (s, 3 H); 0.07 (s, 3 H); 0.89( s, 9H); 2.78 ± 2.87 ( m, 2 H); 3.03 ± 3.11 (m, 1 H); 3.50 (br. s,
2 H); 3.84 (d, J ˆ 5.6, 2 H); 7.21 ± 7.34 (m, 6 H); 8.00 (d, J ˆ 5.3, 1 H); 8.04 (br. s, 1 H); 8.09( s, 1 H). 13C-NMR
(75 MHz, CDCl3): À 5.3; 18.4; 26.0; 34.8; 52.0; 63.3; 117.0; 126.6; 128.6; 128.9; 132.0; 134.5; 138.8; 140.3; 141.4;
‡
172.4. MALDI-MS (DCTB): 386.2 (100, MH ), 368.2 (3), 254.1 (4). MALDI-HR-MS (DCTB): 386.2254
‡
‡
(MH , C21H32N3O2Si ; calc. 386.2258). Anal. calc. for C21H31N3O2Si (385.58): C 65.42, H 8.10, N 10.90; found C
65.46, H 7.92, N 10.84.
2-((1S)-1-Benzyl-2-{[(tert-butyl)(dimethyl)silyl]oxy}ethyl)-1H-imidazo[4,5-c]pyridine ((‡)-22). GP 2,
starting from (‡)-21 (72 mg, 0.19mmol), gave ( ‡)-22 (66 mg, 96%). Colorless oil. [a]2D5 ˆ ‡52.6 (c ˆ 1,
CHCl3). IR (CHCl3): 3391, 2954, 2857, 1954, 1857, 1618, 1583, 1529, 1460, 1401, 1277, 1258. 1H-NMR (300 MHz,
CDCl3): 0.03 (s, 3 H); 0.03 (s, 3 H); 0.9 3 (s, 9H); 3.08 ( dd, J ˆ 13.7, 9.3, 1 H); 3.32 (dd, J ˆ 13.7, 6.2, 1 H); 3.40 ±
3.48 (m, 1 H); 3.84 (dd, J ˆ 10.1, 5.4, 1 H); 3.93 (dd, J ˆ 10.1, 3.9, 1 H); 7.16 ± 7.31 (m, 6 H); 8.39( d, J ˆ 5.6, 1 H);
9.02 (br. s, 1 H). 13C-NMR (75 MHz, CDCl3, 2 drops of CF3COOH added): À 5.5; 18.3; 25.8; 36.2; 44.6; 63.8;
113.6; 127.5; 128.8; 129.1; 131.6; 134.4; 136.8; 147.2; 167.4 (1 arom. signal overlapping). MALDI-MS (DHB):
‡
‡
‡
390.2 (5, [M ‡ Na] ), 368.2 (100, MH ), 352.2 (4), 254.1 (8). MALDI-HR-MS (DHB): 368.2148 (MH ,
‡
C21H30N3OSi ; calc. 368.2153).
(2S)-2-(1H-Imidazo[4,5-c]pyridin-2-yl)-3-phenylpropan-1-ol ((‡)-23). To a soln. of (‡)-22 (740 mg,
2.01 mmol) in dry THF (25 ml), Bu4NF soln. (1m in THF, 4 ml, 4.00 mmol) was added dropwise at r.t. The
mixture was stirred for 2.5 h at r.t., and the reaction was quenched by addition of H2O. After extraction
(CH2Cl2), the org. phases were dried (MgSO4) and concentrated in vacuo. Purification by CC (SiO2-60; AcOEt/
MeOH 93 :7) afforded (‡)-23 (428 mg, 84%). Colorless foam. M.p. 858. [a]2D5 ˆ ‡92.4 (c ˆ 1, CHCl3). IR (neat):
2929, 1621, 1589, 1532, 1495, 1455, 1418, 1283, 1201, 1167, 1057. 1H-NMR (300 MHz, CDCl3): 3.13 (dd, J ˆ 13.7,
8.1, 1 H); 3.26 (dd, J ˆ 13.7, 7.5, 1 H); 3.39± 3.49 ( m, 1 H); 3.99 (dd, J ˆ 10.9, 6.2, 1 H); 4.08 (dd, J ˆ 10.9, 3.7,
1 H); 7.16 ± 7.32 (m, 5 H); 7.39 (d, J ˆ 5.8, 1 H); 8.33 (d, J ˆ 5.8, 1 H); 8.89( s, 1 H). 13C-NMR (75 MHz, CDCl3):
37.1; 45.0; 63.8; 109.5; 126.7; 128.7; 129.1; 137.5; 138.0; 138.9; 140.5; 143.0; 160.2. ESI-MS: 276. 1 (28, [M ‡
‡
‡
‡
‡
Na] ), 254.1 (100, MH ). ESI-HRMS: 254.1286 (MH , C15H16N3O ; calc. 254.1288).
S-[(2S)-2-(1H-Imidazo[4,5-c]pyridin-2-yl)-3-phenylpropyl] Ethanethioate ((À)-24). GP 4, starting from
(‡)-23 (140 mg, 0.55 mmol), afforded (À)-24 (160 mg, 93%) after purification by CC (SiO2-60; AcOEt/MeOH/
NEt3 94 :5 :1). Colorless oil. [a]2D5 ˆ À30.8 (c ˆ 1, CHCl3). IR (neat): 3028, 2922, 1688, 1621, 1587, 1532, 1495,
1455, 1423, 1353, 1282, 1210. 1H-NMR (300 MHz, CDCl3, 4 drops of CF3COOH added): 2.28 (s, 3 H); 3.25 (dd,

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Helvetica Chimica Acta ± Vol. 88 (2005)
J ˆ 13.9, 8.6, 1 H); 3.32 (dd, J ˆ 13.9, 7.6, 1 H); 3.36 (dd, J ˆ 14.0, 8.4, 1 H); 3.46 (dd, J ˆ 14.0, 5.3, 1 H); 3.81 ±
3.88 (m, 1 H); 7.05 ± 7.23 (m, 5 H); 8.19( d, J ˆ 6.5, 1 H); 8.50 (d, J ˆ 6.5, 1 H); 9.45 (s, 1 H). 13C-NMR (75 MHz,
CDCl3; 4 drops of CF3COOH added): 30.4; 31.9; 39.6; 43.0; 113.1; 127.3; 128.5; 128.8; 131.6; 133.8; 134.3; 136.0;
‡
146.6; 166.0; 197.0. MALDI-MS (DCTB): 312.1 (100, MH ), 236.1 (48), 156.1 (1). MALDI-HR-MS (DCTB):
‡
‡
312.1165 (MH , C17H18N3OS ; calc. 312.1165).
(4S)-4-Benzyl-3-(3-[1,1'-biphenyl-4-yl]propanoyl)-1,3-oxazolidin-2-one ((‡)-27). To a soln. of 3-(1,1'-
biphenyl-4-yl)propanoic acid (6.88 g, 30.42 mmol) and Et3N (5.13 ml, 36.79mmol) in dry THF (100 ml),
pivaloyl chloride (3.91 ml, 31.81 mmol) was added at À 788. The mixture was stirred for 1 h at 08. To a soln. of
(‡)-15 (4.90 g, 27.66 mmol) in dry THF (60 ml), BuLi (1.6m in hexanes, 19.01 ml, 30.42 mmol) was slowly added
at À 788. The latter soln. was added via canula to the anhydride soln. at À 788, and the mixture was stirred for
30 min at À 788. The mixture was warmed to 08, and CH2Cl2 and a phosphate-buffer soln. (pH 7) were added.
The org. phases were washed with sat. aq. NaHCO3 soln., dried (MgSO4), and concentrated in vacuo.
Purification by recrystallization from AcOEt afforded (‡)-27 (8.12 g, 76%). Colorless crystals. M.p. 1828.
[a]2D5 ˆ ‡48.3 (c ˆ 1, CHCl3). IR (CHCl3): 2922, 1780, 1699, 1487, 1452, 1384, 1352, 1108, 1048. 1H-NMR
(300 MHz, CDCl3): 2.77 (dd, J ˆ 13.4, 9.7, 1 H); 3.04 ± 3.10 (m, 2 H); 3.23 ± 3.42 (m, 3 H); 4.11 ± 4.22 (m, 2 H);
4.64 ± 4.72 (m, 1 H); 7.17 ± 7.59( m, 14 H). 13C-NMR (75 MHz, CDCl3): 30.0; 37.2; 37.9; 55.2; 66.3; 126.9; 127.0;
127.1; 127.3; 128.6; 128.9; 129.3; 135.1; 139.1; 139.4; 140.8; 172.2 (1 arom. and 1 CO signal overlapping).
‡
‡
MALDI-MS (DCTB): 408.2 (33, [M ‡ Na] ), 386.2 (10, MH ), 235.1 (87), 224.1 (100). MALDI-HR-MS
‡
‡
(DCTB): 408.1568 ([M ‡ Na] , C25H23NNaO3 ; calc. 408.1517). Anal. calc. for C25H23NO3 (385.46): C 77.90, H
6.01, N 3.63; found C 77.66, H 5.93, N 3.64.
(4S)-4-Benzyl-3-[(2R)-3-(benzyloxy)-2-([1,1'-biphenyl-4-yl]methyl)propanoyl]-1,3-oxazolidin-2-one ((‡)-
28). To a soln. of (‡)-27 (386 mg, 1.00 mmol) in dry CH2Cl2 (10 ml), TiCl4 (0.12 ml, 1.05 mmol) was slowly
added at 08. After stirring for 5 min, EtN(i-Pr)2 (0.19ml, 1.1 mmol) was added, and the mixture was stirred for
1 h at 08. After addition of BnOCH2Cl (0.28 ml, 2 mmol), the mixture was stirred for additional 4 h at 08. The
reaction was cautiously quenched with sat. aq. NH4Cl soln., and the mixture was extracted (CH2Cl2). The org.
phases were washed with H2O, dried (MgSO4), and concentrated in vacuo. Purification by CC (SiO2-60;
pentane/AcOEt 83 :17) afforded (‡)-32 (470 mg, 94%). Colorless crystals. M.p. 728. [a]2D5 ˆ ‡77.2 (c ˆ 1,
CHCl3). IR (CHCl3): 3019, 2954, 1777, 1694, 1599, 1484, 1384, 1349, 1261, 1099, 1008. 1H-NMR (300 MHz,
CDCl3): 2.69( dd, J ˆ 13.5, 9.2, 1 H); 2.93 (dd, J ˆ 13.5, 7.4, 1 H); 3.03 (dd, J ˆ 13.5, 8.3, 1 H); 3.20 (dd, J ˆ 13.5,
3.3, 1 H); 3.70 (dd, J ˆ 9.2, 4.9, 1 H); 3.89 (dd, J ˆ 9.2, 7.6, 1 H); 3.90 (dd, J ˆ 9.0, 8.4, 1 H); 4.03 (dd, J ˆ 9.0, 2.8,
1 H); 4.52 ± 4.64 (m, 4 H); 7.16 ± 7.58 (m, 19 H). 13C-NMR (75 MHz, CDCl3): 35.0; 37.9; 45.4; 55.5; 66.1; 70.8;
73.4; 127.2; 127.3; 127.4; 127.5; 127.9; 128.0; 128.6; 129.0; 129.1; 129.7; 129.8; 135.4; 137.8; 138.3; 139.6; 141.0;
‡
‡
153.3; 174.4. EI-MS: 506.2 (2, MH ), 505.2 (8, M ), 397.2 (42), 220.1 (75), 219.1 (83), 91.1 (100), 69.0 (96).
Anal. calc. for C33H31NO4 (505.63): C 78.39, H 6.18, N 2.77; found C 78.26, H 6.04, N 2.78.
(2R)-3-(Benzyloxy)-2-([1,1'-biphenyl-4-yl]methyl)propanoic Acid ((À)-29). To a soln. of (‡)-28 (7.41 g,
14.65 mmol) in a mixture of THF/H2O (225 ml/75 ml), aq. H2O2 soln. (30%, 2.69ml, 26.37 mmol) and aq. LiOH
soln. (0.8m, 35 ml, 29.31 mmol) were added at 08. The mixture was stirred for 2.5 h at r.t, then 1.5n aq. Na2SO3
soln. (90 ml) was added, and the THF was removed in vacuo. The aq. phases were acidified by addition of 1n
HCl to a pH of 1 ± 2 and extracted (CH2Cl2). The org. phases were dried (MgSO4), concentrated in vacuo, and
the residue was purified by CC (SiO2-60; CH2Cl2/pentane/AcOH 66 :33 :1) to give (À)-33 (3.71 g, 73%).
Colorless solid. M.p. 978. [a]2D5 ˆ À7.4 (c ˆ 1, CHCl3). IR (CHCl3): 2927, 2857, 1712, 1602, 1487, 1449, 1258, 1099.
1H-NMR (300 MHz, CDCl3): 2.92 (dd, J ˆ 12.2, 6.9, 1 H); 2.99 ± 3.07 (m, 1 H); 3.11 (dd, J ˆ 12.2, 6.4, 1 H); 3.65
(d, J ˆ 5.3, 2 H); 4.52 (d, J ˆ 12.1, 1 H); 4.57 (d, J ˆ 12.1, 1 H); 7.23 ± 7.58 (m, 14 H). 13C-NMR (75 MHz, CDCl3):
34.0; 47.5; 69.5; 73.5; 127.2; 127.4; 127.4; 128.0; 128.0; 128.7; 129.0; 129.6; 137.7; 137.9; 139.7; 141.0; 178.8. ESI-
‡
MS: 345.1 (100, M ), 193.1 (16). Anal. calc. for C23H22O3 (345.55): C 79.74, H 6.40; found C 79.78, H 6.69.
(2R)-N-(2-Aminophenyl)-3-(benzyloxy)-2-([1,1'-biphenyl-4-yl]methyl)propanamide ((‡)-30). GP 1,
Method B, starting from (À)-29 (1.00 g, 2.89mmol) and benzene-1,2-diamine, afforded ( ‡)-30 (0.83 g, 66%)
after purification by CC (SiO2-60; CH2Cl2/AcOEt 91:9). Yellow solid. M.p. 1098. [a]2D5 ˆ ‡30.1 (c ˆ 1, CHCl3).
1
IR (CHCl3): 3412, 3019, 2857, 1675, 1621, 1503, 1481, 1258, 1099. H-NMR (300 MHz, CDCl3): 2.88 ± 2.96 (m,
2 H); 3.10 ± 3.19( m, 1 H); 3.48 (br. s, 2 H); 3.72 ± 3.76 (m, 2 H); 4.57 (s, 2 H); 6.68 (dd, J ˆ 7.8, 1.6, 1 H); 6.74 (dt,
J ˆ 7.8, 1.3, 1 H); 7.00 (dt, J ˆ 7.8, 1.6, 1 H); 7.06 (dd, J ˆ 7.8, 1.3, 1 H); 7.26 ± 7.60 (m, 15 H). 13C-NMR (75 MHz,
CDCl3): 34.8; 50.1; 70.6; 73.7; 117.1; 118.9; 123.6; 125.6; 126.9; 127.0; 127.2; 127.2; 127.9; 128.5; 128.7; 129.4; 137.4;
‡
138.0; 139.4; 140.7; 171.9 (2 arom. signals overlapping). MALDI-MS (DCTB): 459.2 (15, [M ‡ Na] ), 437.2 (21,
‡
‡
‡
MH ), 420.2 (31), 419.2 (100). MALDI-HR-MS (DCTB): 459.2050 ([M ‡ Na] , C29H28N2NaO2 ; calc.
459.2043). Anal. calc. for C29H28N2O2 (436.55): C 79.79, H 6.46, N 6.42; found C 79.81, H 6.52, N 6.29.

Helvetica Chimica Acta ± Vol. 88 (2005)
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2-[(1S)-2-(Benzyloxy)-1-([1,1'-biphenyl-4-yl]methyl)ethyl]-1H-benzimidazole ((‡)-32). GP 2, starting
from (‡)-30 (777 mg, 1.78 mmol), afforded (‡)-32 (638 mg, 85%). Yellow foam. M.p. 608. [a]2D5 ˆ ‡40.3 (c ˆ
1, CHCl3). IR (CHCl3): 3418, 3013, 2954, 2863, 2356, 1677, 1621, 1484, 1452, 1411, 1266, 1099. 1H-NMR
(300 MHz, CDCl3): 3.17 (dd, J ˆ 13.6, 10.0, 1 H); 3.37 (dd, J ˆ 13.6, 5.8, 1 H); 3.49± 3.58 ( m, 1 H); 3.73 (dd, J ˆ
9.2, 5.5, 1 H); 3.82 (dd, J ˆ 9.2, 3.6, 1 H); 4.55 (d, J ˆ 12.0, 1 H); 4.60 (d, J ˆ 12.0, 1 H); 7.17 ± 7.61 (m, 18 H).
13C-NMR (75 MHz, CDCl3, 2 drops of CF3COOH added): 36.6; 39.8; 67.7; 74.1; 113.9; 126.9; 127.3; 127.4; 127.5;
128.3; 128.7; 128.8; 128.9; 129.1; 129.2; 129.8; 134.7; 136.1; 140.3; 154.6. MALDI-MS (DCTB): 419.21 (100,
‡
‡
‡
MH ), 209.6 (4). MALDI-HR-MS (DCTB): 419.2123 (MH , C29H27N2O ; calc. 419.2118). Anal. calc for
C29H26N2O (418.53): C 83.22, H 6.26, N 6.69; found C 83.32, H 6.54, N 6.98.
(2S)-2-(1H-Benzimidazol-2-yl)-3-[1,1'-biphenyl-4-yl]propan-1-ol ((‡)-34). To a soln. of (‡)-32 (577 mg,
1.38 mmol) in dry CH2Cl2 (60 ml), BCl3 (1m in CH2Cl2, 13.8 ml, 13.8 mmol) was slowly added at À 788, and the
mixture was stirred for 4 h at À 788. At 08, MeOH was slowly added, and the solvent was removed in vacuo. This
was repeated twice. After addition of sat. methanolic NH3 soln., the mixture was concentrated in vacuo, and the
residue was purified by CC (SiO2-60; AcOEt) to give (‡)-34 (155 mg, 34%). Colorless solid. M.p. 2108. [a]D25
ˆ
‡165.8 (c ˆ 0.82, EtOH). IR (neat): 3053, 3027, 2871, 2050, 1980, 1916, 1651, 1626, 1600, 1538, 1520, 1486, 1455,
1372, 1276, 1223, 1074, 1006. 1H-NMR (300 MHz, CD3OD): 3.14 (dd, J ˆ 13.7, 9.0, 1 H); 3.23 (dd, J ˆ 13.7, 5.6,
1 H); 3.36 ± 3.45 (m, 1 H); 3.91 (dd, J ˆ 10.8, 5.9, 1 H); 3.95 (dd, J ˆ 10.8, 7.2, 1 H); 7.14 ± 7.53 (m, 13 H).
13C-NMR (75 MHz, CD3OD): 37.3; 46.5; 64.7; 122.7; 127.3; 127.4; 127.6; 129.3; 129.9; 139.2; 140.0; 141.6; 156.8;
‡
164.3; 173.5 (1 arom. signal overlapping). MALDI-MS (DHB): 329.2 (100, MH ), 164.6 (7). MALDI-HR-MS
‡
‡
(DHB): 329.1645 (MH , C22H21N2O ; calc. 329.1648).
S-[(2S)-2-(1H-Benzimidazol-2-yl)-3-[1,1'-biphenyl-4-yl]propyl] Ethanethioate ((‡)-36). GP 4, starting
from (‡)-34 (148 mg, 0.45 mmol), gave (‡)-36 (79mg, 44%) after double purification by CC (SiO 2-60;
CH2Cl2/AcOEt 91:9 and pentane/AcOEt 83 :17). Colorless solid. M.p. 1838. [a]2D5 ˆ ‡20.5 (c ˆ 1, CHCl3). IR
(neat): 2745, 1694, 1539, 1486, 1445, 1271, 1130, 1008, 957. 1H-NMR (300 MHz, CDCl3): 2.32 (s, 3 H); 3.20 (dd,
J ˆ 13.4, 5.3, 1 H); 3.33 (dd, J ˆ 13.4, 7.8, 1 H); 3.42 ± 3.46 (m, 3 H); 7.09± 7.56 ( m, 12 H); 7.77 (d, J ˆ 7.2, 1 H);
8.91 (br. s, 1 H). 13C-NMR (75 MHz, CDCl3): 30.3; 30.6; 39.2; 41.1; 113.9; 126.8; 126.9; 127.4; 127.6; 128.7; 129.1;
‡
‡
130.3; 134.2; 140.0; 140.4; 153.5; 199.4. MALDI-MS (DHB): 409.1 (17, [M ‡ Na] ); 387.2 (100, MH ).
‡
‡
MALDI-HR-MS (DHB): 387.1522 (MH , C24H23N2OS ; calc. 387.1526).
2-[(1S)-2-[1,1'-Biphenyl-4-yl]-1-(sufanylmethyl)ethyl]-1H-benzimidazol-3-ium Trifluoroacetate ((‡)-25).
GP 5, starting from (‡)-36 (25 mg, 0.064 mmol), afforded (‡)-25 (20 mg, 68%) after purification by RP-HPLC
(RP-18 SiO2; 0.1% aq. CF3COOH/MeCN 99 :1 ! 0 :100 in 60 min). Colorless solid. M.p. 798. [a]2D5 ˆ ‡52.9
(c ˆ 1, CHCl3). IR (neat): 2635, 1657, 1622, 1567, 1520, 1487, 1461, 1435, 1410, 1300, 1183, 1131. 1H-NMR
(300 MHz, CDCl3): 1.26 (br. s, 1 H); 2.81 (dd, J ˆ 13.9, 4.8, 1 H); 3.02 (dd, J ˆ 13.9, 9.8, 1 H); 3.13 (dd, J ˆ 14.0,
7.8, 1 H); 3.24 (dd, J ˆ 14.0, 8.1, 1 H); 3.76 ± 3.86 (m, 1 H); 7.08 ± 7.11 (d, J ˆ 8.1, 2 H); 7.24 ± 7.40 (m, 9H); 7.57 ±
7.62 (m, 2 H). 13C-NMR (75 MHz, CDCl3): 27.0; 38.9; 44.7; 114.1; 125.5; 126.7; 127.2; 127.3; 128.6; 128.9; 131.5;
‡
‡
135.4; 139.8; 140.1; 154.6. MALDI-MS (DHB): 367.1 (8, [M ‡ Na] ), 345.1 (100, MH ). MALDI-HR-MS
‡
‡
(DHB): 345.1424 (MH , C22H20N2S , calc. 345.1420).
(2R)-N-(3-Aminopyridin-4-yl)-3-(benzyloxy)-2-([1,1'-biphenyl-4-yl]methyl)propanamide ((‡)-31). GP 1,
Method B, starting from (À)-29 (1.00 g, 2.89mmol) and 3,4-diaminopyridine, afforded ( ‡)-31 (719mg, 57%)
after purification by CC (SiO2-60; AcOEt). Colorless crystals. M.p. 1488. [a]2D5 ˆ ‡12.6 (c ˆ 1, CHCl3). IR
(CHCl3): 3407, 3326, 2868, 1688, 1618, 1583, 1511, 1484, 1422, 1306. 1H-NMR (300 MHz, CDCl3): 2.89± 2.99( m,
2 H); 3.13 ± 3.23 (m, 3 H); 3.72 (dd, J ˆ 9.7, 6.5, 1 H); 3.76 (dd, J ˆ 9.7, 3.7, 1 H); 4.56 (s, 2 H); 7.26 ± 7.59( m,
15 H); 8.01 (d, J ˆ 5.3, 1 H); 8.03 ± 8.08 (m, 2 H). 13C-NMR (75 MHz, CDCl3): 34.7; 50.2; 70.2; 74.1; 116.9; 127.2;
127.6; 128.4; 128.5; 128.9; 129.1; 129.7; 132.6; 134.1; 137.4; 138.0; 139.8; 140.4; 140.8; 142.1; 172.5 (1 arom. signal
‡
overlapping). MALDI-MS (DCTB): 438.2 (100, MH ), 330.2 (9), 219.1 (5). MALDI-HR-MS (DCTB):
‡
‡
438.2170 (MH , C28H28N3O2 ; calc. 438.2176). Anal. calc. for C28H27N3O2 (437.53): C 76.86, H 6.22, N 9.60; found
C 76.84, H 6.29, N 9.64. X-Ray: see Fig. 3.
2-[(1S)-2-(Benzyloxy)-1-([1,1'-biphenyl-4-yl]methyl)ethyl]-1H-imidazo[4,5-c]pyridine ((‡)-33). GP 2,
starting from (‡)-31 (842 mg, 1.92 mmol), afforded (‡)-33 (805 mg, 100%). Colorless foam. M.p. 698. [a]D25
ˆ ‡48.8 (c ˆ 0.91, CHCl3). IR (CHCl3): 3396, 3024, 2965, 1621, 1487, 1452, 1403, 1277, 1102, 903. 1H-NMR
(300 MHz, CDCl3): 3.15 (dd, J ˆ 13.5, 9.5, 1 H); 3.37 (dd, J ˆ 13.5, 5.5, 1 H); 3.54 ± 3.57 (m, 1 H); 3.75 (dd, J ˆ
9.5, 5.8, 1 H); 3.84 (dd, J ˆ 9.5, 3.6, 1 H); 4.56 (d, J ˆ 11.7, 1 H); 4.61 (d, J ˆ 11.7, 1 H); 7.16 ± 7.58 (m, 15 H); 8.40
(d, J ˆ 5.6, 1 H); 9.02 (br. s, 1 H). 13C-NMR (75 MHz, CDCl3, 2 drops of CF3COOH added): 36.4; 42.2; 68.4;
73.9; 113.5; 126.8; 127.4; 127.4; 128.3; 128.5; 128.6; 128.7; 129.1; 131.5; 133.2; 134.2; 135.2; 136.1; 140.1; 140.2;
‡
‡
146.8; 166.6. MALDI-MS (DCTB): 420.21 (100, MH ), 390.2 (6). MALDI-HR-MS (DCTB): 420.2065 (MH ,

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Helvetica Chimica Acta ± Vol. 88 (2005)
‡
C28H26N3O ; calc. 420.2070). Anal. calc. for C28H25N3O (419.50): C 80.16, H 6.01, N 10.02; found C 80.18, H 6.19,
N 9.88.
(2S)-3-[1,1'-Biphenyl-4-yl]-2-(1H-imidazo[4,5-c]pyridin-2-yl)propan-1-ol ((‡)-35). To a soln. of (‡)-33
(477 mg, 1.14 mmol) in dry CH2Cl2 (50 ml), BCl3 (1m in CH2Cl2, 11.4 ml, 11.4 mmol) was slowly added at À 788,
and the mixture was stirred for 3 h at À 788. At 08, MeOH and NaHCO3 (ca. 5 g) were added, and the mixture
was stirred for 12 h. The suspension was filtered, the precipitate was washed with CH2Cl2/MeOH, and the
combined org. phases were concentrated in vacuo. The residue was purified by CC (SiO2-60; AcOEt/MeOH
91:9). Since not all impurities could be removed, the crude product was dissolved in EtOH (12.3 ml) and 96%
H2SO4 (0.18 ml), and the mixture was stirred for 2 h at 508 and for 12 h at r.t. The mixture was slowly neutralized
with sat. aq. NaHCO3 soln. and extracted (CH2Cl2). The org. phases were dried (MgSO4) and concentrated in
vacuo to afford (‡)-35 (220 mg, 59%). Colorless solid. M.p. 1138. [a]2D5 ˆ ‡138.9( c ˆ 1, EtOH). IR (neat): 3027,
1
2925, 2362, 2340, 1622, 1589, 1532, 1487, 1422, 1283, 1202, 1169, 1029. H-NMR (300 MHz, CD3OD): 3.16 (dd,
J ˆ 13.7, 9.0, 1 H); 3.24 (dd, J ˆ 13.7, 6.2, 1 H); 3.45 ± 3.52 (m, 1 H); 3.95 ± 3.98 (m, 2 H); 7.15 ± 7.54 (m, 10 H);
8.25 (d, J ˆ 5.6, 1 H); 8.78 (s, 1 H). 13C-NMR (75 MHz, CD3OD): 36.2; 45.7; 63.7; 126.6; 126.8; 127.0; 128.6;
129.2; 138.1; 139.5; 140.5; 140.6; 140.8; 160.1 (3 arom. signals overlapping). MALDI-MS (DHB): 330.2 (100,
‡
‡
‡
MH ), 322.3 (22), 165.1 (8). MALDI-HR-MS (DHB): 330.1607 (MH , C21H20N3O ; calc. 330.1601).
S-[(2S)-3-[1,1'-Biphenyl-4-yl]-2-(1H-imidazo[4,5-c]pyridin-2-yl)propyl] Ethanethioate ((‡)-37). GP 4,
starting from (‡)-35 (400 mg, 1.21 mmol), gave (‡)-37 (218 mg, 46%) after purification by CC (SiO2-60;
AcOEt/MeOH 99 :1). Colorless oil. [a]2D5 ˆ ‡23.0 (c ˆ 1, CHCl3). IR (neat): 2923, 1979, 1687, 1620, 1587, 1520,
1487, 1422, 1353, 1282, 1130, 1107. 1H-NMR (300 MHz, CDCl3, 4 drops of CF3COOH added): 2.31 (s, 3 H);
3.32 ± 3.49( m, 4 H); 3.87 ± 3.96 (m, 1 H); 7.16 (d, J ˆ 8.1, 2 H); 7.29± 7.56 ( m, 7 H); 8.24 (d, J ˆ 6.5, 1 H); 8.53 (d,
J ˆ 6.5, 1 H); 9.59 (s, 1 H). 13C-NMR (75 MHz, CDCl3, 4 drops of CF3COOH added): 30.5; 32.0; 39.1; 43.0;
112.6; 113.9; 126.7; 127.4; 127.5; 127.6; 128.7; 128.8; 128.9; 131.4; 134.9; 140.0; 166.1; 185.0 (1 arom. signal
‡
‡
overlapping). MALDI-MS (DHB): 410.1 (11, [M ‡ Na] ); 388.1 (100, MH ); 312.1 (27). MALDI-HR-MS
‡
‡
(DHB): 388.1478 (MH , C23H22N3OS ; calc. 388.1478).
2-[(1S)-2-[1,1'-Biphenyl-4-yl]-1-(sulfanylmethyl)ethyl]-1H-imidazo[4,5-c]pyridin-5-ium Trifluoroacetate
((‡)-26). GP 5, starting from (‡)-37 (40 mg, 0.103 mmol), afforded (‡)-26 (27 mg, 57%) after purification
by RP-HPLC (RP-18 SiO2; 0.1% aq. CF3COOH/MeCN 99 :1 ! 0 :100 in 60 min). Colorless solid. M.p. 828.
[a]2D5 ˆ ‡71.0 (c ˆ 0.5, CHCl3). IR (neat): 3029, 2657, 2111, 1667, 1644, 1522, 1487, 1475, 1411, 1309, 1177, 1128,
1007. 1H-NMR (300 MHz, CDCl3): 1.52 (t, J ˆ 8.4, 1 H); 3.07 ± 3.16 (m, 2 H); 3.28 ± 3.35 (m, 2 H); 3.70 ± 3.79( m,
1 H); 7.20 ± 7.51 (m, 9 H); 7.94 (d, J ˆ 6.4, 1 H); 8.16 (d, J ˆ 6.4, 1 H); 9.26 (s, 1 H). 13C-NMR (75 MHz, CDCl3):
27.6; 39.4; 46.4; 114.1; 127.1; 127.5; 128.9; 129.5; 131.9; 136.8; 139.8; 140.7; 167.6 (4 arom. signals overlapping).
‡
‡
MALDI-MS (DHB): 368.1 (4, [M ‡ Na] ), 346.1 (100, MH ), 322.3 (24). MALDI-HR-MS (DHB): 346.1377
‡
‡
(MH , C21H20N3S ; calc. 346.1372).
X-Ray Crystal Structure of (‡)-31. Crystal data at 298 K for C28H27N3O2 (Mr 437.54): monoclinic, space
group P21, Dc ˆ 1.269g cm À3, Z ˆ 2, a ˆ 4.79580(10), b ˆ 9.8396(2), c ˆ 24.2769(6) Š, b ˆ 91.5860(11)8, Vˆ
1145.16(4) Š3. Bruker-Nonius Kappa CCD diffractometer, MoKa radiation, l ˆ 0.71073, linear crystal
dimensions 0.44 Â 0.2 Â 0.08 mm. The structure was solved by direct methods (SIR97) [34]. The non-H-atoms
were refined anisotropically (SHELXL-97) [35]. The H-atoms were calculated at idealized positions and
refined with constrained isotropic displacement parameters. Final R(F) ˆ 0.0495, wR(F2) ˆ 0.1314 for 299
parameters, 1 restraint, and 5213 reflections with I > 2s(I) and 0.10 < q < 27.498. Deposition No. CCDC-258179.
Copies of the data can be obtained free of charge on application to Cambridge Crystallographic Data Centre
(CCDC), 12 Union Road, Cambridge CB21EZ, UK (fax: (‡44)1223-336-033; e-mail: deposit@ccdc.cam.
ac.uk).
X-Ray Crystal Structure of Inhibitor (‡)-2 Bound to Neprilysin. Crystals of the soluble extracellular domain
(residues 52 ± 749) of human NEP were obtained by vapor diffusion as described by Oefner et al. [2]. A binary
complex of the glycosidase-treated sNEP was formed with (‡)-2 by soaking experiment. Crystals were
transferred into the stabilization soln. for 30 min, which consists of 200 mm ammonium acetate, 25% PEG 3350,
100 mm Hepes (pH 7.5), and 30% ethylene glycol and containing inhibitor at a concentration of 10 mm. A
crystal was flash-frozen in liquid N at 100 K and belongs to the trigonal space group P3221 with one molecule in
2
the asymmetric unit. Diffraction intensities were measured with CuKa radiation provided from a NONIUS
FR591 rotating anode generator equipped with an OSMIC mirror system and were recorded on a MAR-
Research image plate area detector. All diffraction data were processed and scaled with DENZO and
SCALEPACK [36], and further analyzed with the CCP4 program suite [37]. The structure of the binary
complex was solved by using the refined protein coordinates of human sNEP present in a complex with
phosphoramidon (PDB file name 1DMT, [2]). Prior to refinement, the phosphoramidon inhibitor and all

Helvetica Chimica Acta ± Vol. 88 (2005)
749
solvent molecules were removed from the model. Iterative rounds of model building were performed with
MOLOC [8c], stereochemically restrained positional and temp.-factor refinement was done with REFMAC
[38], using parameters for ideal stereochemistry as described by Engh and Huber [39]. A difference Fourier
map revealed a residual electron density located in the active site corresponding to the bound small molecule.
Progressive introduction of solvent molecules with good geometry lead to the binary complex, lacking the N-
terminal two residues D52 and D53 of human sNEP. Data collection and refinement statistics are summarized in
Table 2. PDB file name 1Y8J.
Table 2. Data Collection and Refinement Statistics for the Complex of NEP with (‡)-2
Cell axes a, c
Resolution range
107.4 Š, 112.5 Š
20.0 ± 2.25 Š
93.629
No. of obs. reflections
No. of unique reflections
Rsym overall/outer shella)
I/s(I) overall/outer shell
Completeness overall/outer shell
34.323
7.8%/47.7% (2.39± 2.25 Š)
11.9/1.8
95.4%/84.9%
Refinement statistics
Resolution range [Š]
20 ± 2.25
22.6 (29.6)
5595 (29.7)
212
20 (27.3)
42 (44.1)
0.007
Rcryst (Rfree) [%]b)
No. of protein atoms (mean B in Š2)
No. of H2O molecules
No. of ligand atoms (mean B in Š2)
No. of NAG atoms (mean B in Š2)
Rmsd bonds [Š2]c)
Rmsd angles [8]
0.89
a) Rsym ˆ ShSi j Ii(h) À hI(h)i j /ShSi(h), where Ii(h) and hI(h)i are the ith and mean measurement of the intensity
of reflection h. b) Sh j j Fobs j À j Fcalc jj /Sh j Fobs j , where j Fobs j and j Fcalc j are the observed and calculated
structure factor amplitudes for the reflection h, applied to the working (Rcryst) and test (Rfree) sets, respectively.
c) Rmsd: root mean-square deviation from mean.
REFERENCES
[1] S. Sahli, B. Stump, T. Welti, W. B. Schweizer, F. Diederich, D. Blum-Kaelin, J. D. Aebi, H.-J. Böhm, Helv.
Chim. Acta. 2005, 88, 707.
[2] C. Oefner, A. DꢂArcy, M. Henning, F. K. Winkler, G. E. Dale, J. Mol. Biol. 2000, 296, 341.
[3] C. Oefner, F. Hoffmann-La Roche, Basel, unpublished results.
Â
[4] B. P. Roques, M.-C. Fournie-Zaluski, E. Soroca, J. M. Lecomte, B. Malfroy, C. Llorens, J. C. Schwartz,
Nature 1980, 288, 286.
[5] R. Bohacek, S. De Lombaert, C. McMartin, J. Priestle, M. Grütter, J. Am. Chem. Soc. 1996, 118, 8231.
[6] S. De Lombaert, L. Blanchard, J. Tan, Y. Sakane, C. Berry, R. D. Ghai, Bioorg. Med. Chem. Lett. 1995, 5,
145.
Â
[7] C. Oefner, B. P. Roques, M.-C. Fournie-Zaluski, G. E. Dale, Acta Crystallogr. Sect. D 2004, 60, 392.
[8] a) P. R. Gerber, K. Müller, J. Comput.-Aided Mol. Des. 1995, 9, 251; b) Gerber Molecular Design (http://
www.moloc.ch); c) P. R. Gerber, Biopolymers 1992, 32, 1003.
[9] E. A. Meyer, R. K. Castellano, F. Diederich, Angew. Chem. 2003, 115, 1244; Angew. Chem., Int. Ed. 2003,
42, 1210.
[10] S. Sahli, B. Stump, T. Welti, D. Blum-Kaelin, J. D. Aebi, C. Oefner, H.-J. Böhm, F. Diederich,
ChemBioChem 2004, 5, 996.
[11] D. A. Evans, D. J. Mathre, W. L. Scott, J. Org. Chem. 1985, 50, 1830.
[12] H. J. Reich, C. P. Jasperse, J. M. Renga, J. Org. Chem. 1986, 51, 2981.
[13] P. N. Preston, ꢁBenzimidazoles and Congeneric Tricyclic Compoundsꢂ, John Wiley & Sons, New York, 1981.

750
Helvetica Chimica Acta ± Vol. 88 (2005)
[14] K. Maekawa, J. Ohtani, Agric. Biol. Chem. 1977, 41, 811.
[15] A. K. Forrest, P. J. Oꢂ Hanlon, G. Walker, J. Chem. Soc., Perkin Trans. 1 1994, 2657.
[16] M. C. Bagley, K. E. Bashford, C. L. Hesketh, C. J. Moody, J. Am. Chem. Soc. 2000, 122, 3301.
[17] J. J. Chen, Y. Zhang, S. Hammond, N. Dewdney, T. Ho, X. Lin, M. F. Browner, A. L. Castelhano, Bioorg.
Med. Chem. Lett. 1996, 6, 1601.
[18] D. A. Evans, A. E. Weber, J. Am. Chem. Soc. 1986, 108, 6757.
[19] J. Granander, R. Sott, G. Hilmersson, Tetrahedron 2002, 58, 4717.
[20] M. K. Edmonds, A. D. Abell, J. Org. Chem. 2001, 66, 3747.
[21] J. M. Crawforth, J. Fawcett, B. J. Rawlings, J. Chem. Soc., Perkin Trans. 1 1998, 1721.
[22] D. A. Evans, T. C. Britton, J. A. Ellman, Tetrahedron Lett. 1987, 28, 6141.
[23] N. P. Peet, N. L. Lentz, M. W. Dudley, A. M. L. Ogden, D. R. McCarty, M. M. Racke, J. Med. Chem. 1993,
36, 4015.
[24] M. C. Pigro, G. Angiuoni, G. Piancatelli, Tetrahedron 2002, 58, 5459.
[25] O. Mitsunobu, Synthesis 1981, 1.
[26] L. Zervas, I. Photaki, N. Ghelis, J. Am. Chem. Soc. 1963, 85, 1337.
[27] J. Olsen, P. Seiler, B. Wagner, T. Tschopp, H. Fischer, U. Obst-Sander, D. W. Banner, M. Kansy, K. Müller,
F. Diederich, Org. Biomol. Chem. 2004, 2, 1339.
[28] S. De Lombaert, L. B. Stamford, L. Blanchard, J. Tan, D. Hoyer, C. G. Diefenbacher, D. Wei, E. M.
Wallace, M. A. Moskal, P. Savage, A. Y. Jeng, Bioorg. Med. Chem. Lett. 1997, 7, 1059.
Â
[29] N. Inguimbert, P. Coric, H. Poras, H. Meudal, F. Teffot, M.-C. Fournie-Zaluski, B. P. Roques, J. Med. Chem.
2002, 45, 1477.
[30] G. Cavallini, E. Massarini, D. Nardi, R. DꢂAmbrosio, J. Am. Chem. Soc. 1957, 79, 3514.
[31] D. A. Evans, T. C. Britton, R. L. Dorow, J. F. Daellaria Jr., Tetrahedron 1988, 44, 5525.
[32] B. Brown, L. S. Hegedus, J. Org. Chem. 2000, 65, 1865.
[33] W. L. DeLano, ꢁThe PyMOL Userꢂs Manualꢂ, DeLano Scientific, San Carlos, CA, USA, 2001.
[34] A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, M. C. Burla, G. Polidori, M. Camalli, J. Appl.
Crystallogr. 1994, 27, 435.
[35] G. M. Sheldrick, ꢀSHELX-97, program for the refinement of crystal structuresꢂ, University of Göttingen,
Germany, 1997.
[36] Z. Otwinowski, ꢁOscillation data reduction programꢂ in ꢁProceedings of the CCP4 study weekend: data
collection and processingꢂ, L. Wawyey, N. Isaacs, S. Bailey (Eds.), SERC Daresbury Laboratory,
Daresbury, 1993, p. 56 ± 62.
[37] Collaborative Computational Project No. 4, Acta Crystallogr., Sect. D 1994, 50, 760.
[38] G. N. Murshudov, A. A. Vagin, E. J. Dodson, Acta Crystallogr., Sect. D 1997, 53, 240.
[39] R. Engh, R. Huber, Acta Crystallogr., Sect. A 1991, 47, 961.
Received December 20, 2004