A fluorine scan of non-peptidic inhibitors of neprilysin: Fluorophobic and fluorophilic regions in an enzyme active site

Article abstract of DOI:10.1016/j.jfluchem.2008.02.004

This article describes the synthesis and in vitro biological affinities of (poly)fluorinated neprilysin inhibitors. Two series of inhibitors with F-substitution of the central benzimidazole platform of the ligands and the benzylic vector to fill the S1' pocket of NEP were investigated. The S1' pocket was found to be highly fluorophobic, and F-substitution led to significantly decreased binding affinities of inhibitors. This result is explained by electrostatically unfavorable close contacts of organic fluorine with the negatively polarized π-surfaces of surrounding aromatic amino acid side chains. In contrast, the protein environment around the benzimidazole platform, with three electropositive guanidinium side chains of Arg residues, was found to provide a fluorophilic environment. Overall, the data support that organic fluorine, with its high negative charge density prefers to orient into electropositive regions of receptor sites. pK_a measurements of fluorinated ligands provided several simple patterns for the prediction of pK_a values of benzimidazoles, important building blocks in medicinal chemistry.

Full text of DOI:10.1016/j.jfluchem.2008.02.004

Journal of Fluorine Chemistry 129 (2008) 852–865
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
A fluorine scan of non-peptidic inhibitors of neprilysin: Fluorophobic and
fluorophilic regions in an enzyme active site
Martin Morgenthaler a, Johannes D. Aebi b, Fiona Gru¨ninger b, Daniel Mona b, Bjo¨rn Wagner b,
Manfred Kansy b, Franc¸ois Diederich a,
*
a Laboratorium fu¨r Organische Chemie, ETH Zu¨rich HCI, Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland
b Pharmaceuticals Division, Discovery Chemistry, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history:
This article describes the synthesis and in vitro biological affinities of (poly)fluorinated neprilysin
inhibitors. Two series of inhibitors with F-substitution of the central benzimidazole platform of the
ligands and the benzylic vector to fill the S1’ pocket of NEP were investigated. The S1’ pocket was found to
be highly fluorophobic, and F-substitution led to significantly decreased binding affinities of inhibitors.
This result is explained by electrostatically unfavorable close contacts of organic fluorine with the
negatively polarized p-surfaces of surrounding aromatic amino acid side chains. In contrast, the protein
environment around the benzimidazole platform, with three electropositive guanidinium side chains of
Arg residues, was found to provide a fluorophilic environment. Overall, the data support that organic
fluorine, with its high negative charge density prefers to orient into electropositive regions of receptor
sites. pKa measurements of fluorinated ligands provided several simple patterns for the prediction of pKa
values of benzimidazoles, important building blocks in medicinal chemistry.
Received 26 January 2008
Received in revised form 19 February 2008
Accepted 19 February 2008
Available online 23 February 2008
Keywords:
Neprilysin
Fluorine interactions
Medicinal chemistry
pKa value
Benzimidazole
ß 2008 Elsevier B.V. All rights reserved.
1. Introduction
Since NEP cleaves ANP and enkephalins, it is a potential drug
target. ANP reduces blood pressure by sodium excretion in the
Neprilysin (NEP, EC 3.4.24.11) is a mammalian ZnII-dependent,
membrane-bound endopeptidase (type-II integral membrane
protein), related to other mammalian metalloproteases such as
the endothelin-converting enzymes (ECE-1 and ECE-2), KELL, and
PEX [1,2]. It is involved in the metabolism of a number of
regulatory peptides of the nervous, cardiovascular, inflammatory,
and immune systems.
The enzyme was extracted for the first time in 1974 from rabbit
kidney brush border [3]. It is found in various tissues, but most
abundantly in the kidney [4]. Besides the wide distribution, it also
has broad substrate specificity. The first substrates discovered
were the pentapeptidic endorphins Leu- and Met-enkephalin and
Substance P [5,6]. Among numerous other substrates [4,7], the
cardiovascular peptide atrial natriuretic peptide (ANP) is the most
familiar [8]. Furthermore, NEP plays an important role in various
cancers. The downregulation of NEP in the lung by cigarette smoke
may be casually related to the development of small-cell
carcinomas [9,10]. Recently, it has been reported that NEP
degrades both amyloid b-peptides 1–40 and 1–42, which play a
pivotal role in Alzheimer’s disease [11,12].
kidneys [8]. Selective NEP inhibitors are, therefore, possible anti-
hypertensive agents, either alone or in combination with selective
ACE (angiotensin-converting enzyme) inhibitors or as dual NEP/ACE
inhibitors, so-called vasopeptidase inhibitors [13–16]. Enkephalins
act as endogenous painkillers in the central nervous system by
binding tom- and d-opiatereceptors [17]. Blocking NEP bya selective
inhibitorleadstoenhancedenkephalinconcentrationsinvitroandto
antinociceptive activity in mice [18]. This suggests that selective
inhibitors of NEP can act as new analgesic therapeutics [4].
After the structure of the extracellular domain (residues 52–
749) of human NEP complexed with the metalloprotease inhibitor
˚
phosphoramidon was solved by X-ray crystallography at 2.1 A
resolution (PDB: 1DMT) [19], our group started the development of
novel non-peptidic inhibitors of NEP by X-ray structure-based de
novo design using the molecular modeling program MOLOC [20].
This strategy has been successfully pursued by us in a variety of
medicinal chemistry projects [21–26]. These investigations
[27–29] led to ligands featuring central benzimidazole- and
imidazo[4,5-c]pyridine-type platforms, with inhibitory activities
(IC50 values; concentration at which 50% maximal initial velocity is
observed) in the low nanomolar potency range. Here, we report on
systematic introduction of fluorine into these classes of inhibitors
with the aim to explore fluorine interactions at the active site of
neprilysin [30]. A similar ‘‘fluorine scan’’ had previously been
* Corresponding author. Tel.: +41 44 632 2992; fax: +41 44 632 1109.
E-mail address: diederich@org.chem.ethz.ch (F. Diederich).
0022-1139/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2008.02.004

M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
853
conducted for inhibitors of thrombin, a serine protease from the
blood coagulation cascade [25,26,31,32]. The aims of such efforts
are the identification of fluorophilic and fluorophobic regions in
protein environments and the development of predictive skills for
gaining binding potency and selectivity through selective intro-
duction of organic fluorine into lead compounds [33–36].
1a/(+)-1b) to biphenyl (in (+)-2a/(+)-2b) does not generate
additional binding potency [29]. The imidazole ring of the central
platform acts as peptide isoster [27] and forms H-bonds to the side
chains of Arg717 and Asp542, while the annulated benzene/
pyridine ring undergoes aromatic interactions with the side chains
of His711 and Trp693. The crystal structure provides a good
explanation for the measured enhanced binding affinity of the
imidazo[4,5-c]pyridine-based ligands as compared to the benzi-
midazole derivatives (Table 1). In the complexes of (+)-1b/(+)-2b,
two positively polarized H-atoms of the annulated benzene ring
point to the positively charged side chain of Arg102, while in those
of (+)-1a/(+)-2a, the N-atom of the annulated pyridine ring
interacts with the guanidinium residue. However, at an Nꢀ ꢀ ꢀN
2. Results and discussion
2.1. The active site of neprilysin complexed to a non-peptidic ligand
Compounds (+)-1a/(+)-2a with a central imidazo[4,5-c]pyridine
platform and (+)-1b/(+)-2b with a benzimidazole scaffold were
found to be potent inhibitors of NEP with IC50 values in the
nanomolar range (Table 1) [29]. In fact, their activities are actually
higher than previously determined when optimized assay condi-
tions, preventing oxidative thiol deterioration, are applied as in the
present study (see below). Thus, ligand (+)-1a binds to NEP with an
IC50 value of 8 nM (Table 1). The predicted binding mode of (+)-1a
was confirmed by X-ray crystallography (PDB code 1Y8J) and is
shown in Fig. 1.
The thiolate residue of (+)-1a ligates to the Zn2+ ion,
participating together with the side chains of Glu646, His587,
and His583 in an ideal tetrahedral coordination pattern. The benzyl
group fills the deep hydrophobic S1’ pocket which is lined by
Phe106, Ile558, Phe563, Met579, Val580, Val692, and Trp693. This
pocket has been shown to accommodate large substituents, such as
biphenyl residues [6,7,37,38]; however, changing phenyl (in (+)-
˚
distance ꢁ3.7 A, a possibly formed H-bond will only be very weak.
2.2. Exploring the S1’ pocket: a distinct fluorophobic environment
Based on the finding that the S1’ pocket of NEP accommodates a
biphenyl substituent, as in (+)-2a/(+)-2b, albeit at no gain in
binding potency [29], we wanted to further explore size and depth
of this hydrophobic sub-site and in particular its affinity for organic
fluorine. For this purpose, we prepared a series of enantiomerically
pure inhibitors featuring alkyl and cycloalkyl ((+)-4a–d) besides
trifluoromethyl ((+)-3a/(+)-3b) substituents in para-position of a
benzyl ring to fill the pocket. Additionally, the benzylic ring was
mono-, di-, and perfluorinated ((+)-5a–j).
The synthesis of all inhibitors followed the same route and is,
with minor improvements, largely based on previously reported
Table 1
Biological activities of NEP inhibitors
Cmpd.
X
R
IC50 (nM)
(+)-1a
(+)-1b
(+)-2a
(+)-2b
(+)-3a
(+)-3b
(+)-4a
(+)-4b
(+)-4c
(+)-4d
N
H
40a/8
280a/28
34a
CH
N
H
Ph
CH
N
Ph
300a
175
CF3
CH
N
CF3
520
CH3
90
N
Cyclobutyl
Cyclobutyl
Cyclobutyl
120
N
320
N
170
Cmpd.
R1
R2
R3
R4
R5
IC50 (nM)
(+)-5a
(+)-5b
(+)-5c
(+)-5d
(+)-5e
(+)-5f
(+)-5g
(+)-5h
(+)-5i
(+)-5j
H
H
F
H
F
F
H
H
H
H
H
H
F
H
H
H
H
H
H
H
F
160
170
180
340
220
210
220
260
170
430
H
H
F
H
F
H
F
H
F
F
F
H
H
H
H
F
F
H
H
F
F
H
F
H
F
H
F
F
F
IC50 values (uncertainties ꢂ10%) measured under optimized assay conditions are given in nM.
a
IC50 values previously obtained using non-optimized assay conditions (see [29]).

854
M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
˚
Fig. 1. Crystal structure of ligand (+)-1a complexed to NEP at a resolution of 2.25 A [50]. PDB code 1Y8J [29]. Distances between N(5) and C(6) of the imidazo[4,5-c]pyridine
˚
platform to the N-atoms of the side chain of Arg102 are shown in A. The bound ligand is shown as 3H-imidazo[4,5-c]pyridine tautomer; the other 1H-tautomer or a mixture of
both could also be present. Color code—protein C-skeleton: grey; ligand C-skeleton: green; N-atoms: blue; O-atoms: red; S-atom: yellow; Zn2+ ion: pink ball. This color code
also applies to the other figures where F-atoms are shown in pink.
Scheme 1. General synthesis sequence for fluorinated NEP inhibitors examplified for (+)-3a. All other inhibitors were synthesized in an identical way. (a) (1) PivCl, Et3N, THF,
ꢃ30 8C, 90 min; (2) LiCl, (S)-7, r.t., 18 h, 68%. (b) (1) TiCl4, (iPr)2EtN, CH2Cl2, 0 8C, 1 h; (2) BOMCl, 0 8C, 3 h, 55%. (c) H2, Pd/C, EtOAc, r.t., 24 h, 91%. (d) TBDMSCl, DMAP, CH2Cl2,
r.t., 18 h, 99%. (e) H2O2, LiCl.H2O, THF, H2O, 0 8C, 3 h, r.t., 16 h, 90%. (f) (1) (COCl)2, CH2Cl2, r.t., 12 h; (2) 3,4-diaminopyridine, Et3N, THF, r.t., 12 h, 95%. (g) (1) AcOH, 65 8C, 24 h;
(2) nBu4NF, THF, r.t., 2 h, 61%. (h) (1) PPh3, DIAD, THF, 0 8C, 30 min; (2) AcSH, 0 8C, 1 h, r.t., 2 h, 75%. (i) NaOMe, H2, MeOH, r.t., 2 h, 49%. Piv: pivaloyl; BOM: benzyloxymethyl;
TBDMS: t-butyldimethylsilyl; DMAP: 4-(N,N-dimethylamino)pyridine; THF: tetrahydrofuran; Ac: acetyl; DIAD: diisopropylazodicarboxylate; r.t.: room temperature.

M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
855
protocols [29]. The preparation of (+)-3a is exemplarily shown in
Scheme 1. Hydrocinnamic acid derivative 6 was converted to a
mixed anhydride with pivaloyl chloride and coupled in situ with
the chiral Evans auxiliary (S)-7 [39] to the acylated species (+)-8.
Transformation into the corresponding Ti-enolate and diaster-
eoselective alkylation with BOMCl gave (+)-9 (for abbreviations,
see scheme caption). Debenzylation to (+)-10 and subsequent
protection of the primary alcohol gave (+)-11. The chiral auxiliary
was then removed by LiOOH to give carboxylic acid (ꢃ)-12,
which was converted into the corresponding acid chloride and
reacted in situ with 3,4-diaminopyridine to give (+)-13.
Cyclocondensation in warm AcOH and deprotection with nBu4NF
yielded primary alcohol (+)-14, which was then converted to (ꢃ)-
15 using Mitsunobu conditions with AcSH as nucleophile.
Methanolysis of the thioacetate gave the free thiol (+)-3a, which
was prone to oxidation to the corresponding disulfide. To prevent
the formation of the disulfide, the reaction was carried out under
an atmosphere of H2. After workup, the crude thiol was
immediately purified by reverse-phase (RP)-HPLC and isolated
as its trifluoroacetate salt, which did not oxidize even after
several weeks being exposed to air.
The in vitro activity of this series of inhibitors towards NEP was
determined in a fluorimetric assay (Table 1, for details see [27,40]).
In addition, the previously reported inhibitors (+)-1a and (+)-1b
(R = H) were retested [29]. The assay conditions were optimized by
eliminating the previously used preincubation (1 h at 37 8C) of the
prepared mixtures of inhibitor and NEP, before the substrate was
added. It was reasoned that preincubation in neutral buffer
solution facilitates the oxidation of the inhibitors to the
corresponding disulfides. Using these optimized conditions, the
IC50 values of (+)-1a and (+)-1b were found to be significantly
lower than previously reported (9 nM vs. 40 nM for (+)-1a, and
28 nM vs. 280 nM for (+)-1b) [29]. These findings demonstrate the
ease with which our class of inhibitors is oxidized, and a rapid
handling of them in neutral solution is highly beneficial.
The IC50 values of inhibitors (+)-4a–d (90–320 nM), with 4-
alkylbenzyl vectors to fill the S1’ pocket, are all higher than
expected, espacially when compared to the previously published
Fig. 2. Energy-minimized structures of (+)-3a (a) and (+)-5j (b) modeled in the active site of NEP. Short contacts between F atoms and side chains are indicated with dotted
˚
lines, the distances are given in A. For the color code, see caption to Fig. 1.

856
M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
˚
between F- and aromatic C-atoms are 2.7 and 3.0 A (with Trp693),
values for the corresponding biphenyl substituted inhibitor (+)-2a
(34 nM). The results suggest that the lower part of the S1’ pocket is
only filled without overall energetic losses by an aromatic
substituent such as in biphenyl derivative (+)-2a. Molecular
modeling indicates that the bottom of the S1’ pocket actually is
not that wide. Thus, the additional phenyl ring in the biphenyl
derivatives (+)-2a/b undergoes repulsive contacts (as indicated by
the program MOLOC) with the aromatic ring of Phe563 and the
methyl group of the side chain of Met579. Additionally, a bound
water molecule seen at the bottom of the pocket in the crystal
structure of (+)-1a is displaced which could be energetically
unfavorable. This water molecule is engaged in three hydrogen-
˚
and 2.8 and 3.6 A (Phe563), respectively. The only attractive
interaction could be a weak, H-bond-type contact [30] between a
˚
fluorine of the ligand and NH of Trp693 (distance Fꢀ ꢀ ꢀN of 2.9 A).
However, this interaction may not be very strong since the
orientation of the N–H bond towards the F atom is not optimal. The
IC50 value of the CF3 derivative (+)-3a (175 nM) is by a factor of 2
higher than that of CH3-substituted (+)-4a. The actual loss in
receptor–ligand binding affinity however is actually substantial,
given the fact that introduction of the CF3 moiety strongly
enhances the lipophilicity (as expressed by the logarithmic
coefficient log D for distribution (D) of a compound between
octanol and water at pH 7.4) and thus promotes the transfer of the
ligand from the aqueous solution into the protein, resulting in
lower IC50 values [30,36,44]. A single H/F exchange usually renders
log D more positive by 0.25 units.
bonding interactions with the indole nitrogen of Trp693
˚
(d(Nꢀ ꢀ ꢀO) = 2.9 A), with the backbone
C
O
of Phe689
˚
˚
(d(Oꢀ ꢀ ꢀO) = 3.0 A) and a second water molecule (d(Oꢀ ꢀ ꢀO) = 2.7 A).
This water molecule however does not seem to be conserved, as it is
absent in other crystal structures (PDB codes 1R1I, 1R1J, 1DMT), and
the energetics of its displacement are difficult to be estimated. On
the other hand, the terminal phenyl ring in (+)-2a/b benefits from
SMet579ꢀ ꢀ ꢀp interactions [41] and in particular from a favorable N–
Hꢀ ꢀ ꢀp interaction with the NH residue of the indole ring of Trp693
[42]. Overall, energeticgainsandlossesseemtobalanceoutsincethe
biphenyl derivatives (+)-2a/b are not more active than the benzyl
derivatives (+)-1a/b (Table 1). In contrast, the alkylated derivatives
(+)-4a–d do not benefit from specific aromatic interactions such as
the NH/p interactions, but rather undergo several repulsive contacts
only, which destabilize their complexes.
Similarly, the F-atoms of (+)-5a–j undergo electrostatically
unfavorable contacts with the surrounding electron-rich protein
environment, as illustrated by the modeling shown in Fig. 2b. Thus,
the fluorine substituents in pentafluorinated (+)-5j are in close
contact with the p-electron surfaces of Phe106, Phe563, and
His583 as well as with the electron-rich heteroatoms of the side
chains of Asp650 and Met579.
It should be noted that the models shown in Fig. 2 are readily
and reproducibly obtained by starting from the crystal structure of
benzyl derivative (+)-1a (PDB code 1Y8J), adding the fluorine
substituents to the NEP-bound ligand, and performing an energy
minimization of the ligand while keeping the geometry of the
protein fixed.
A remarkable destabilization of the complexes was also observed
when organic fluorine was introduced into (+)-1a/b. Both the
introduction of a CF3 group into the para-position of the benzyl ring
(in (+)-3a/b) as well as exchanging the hydrogen substituents of this
ring by fluorine (in (+)-5a–j) led to substantially weaker binding,
with IC50 values between 160 and 520 nM (as compared to 9 and
28 nM for (+)-1a and (+)-1b, respectively). Clearly, the S1’ pocket of
NEP constitutes a pronounced fluorophobic environment. The origin
of the incompatibility for fluorine was further investigated by
molecular modeling (Fig. 2).
This analysis suggests that the loss in binding potency
originates from a too close proximity between the strongly
negatively polarized F-atoms and electron-rich aromatic p-
surfaces [43]. Thus, the CF3 group in (+)-3a is surrounded by the
aromatic side chains of Trp693 and Phe563 and the F-atoms point
into the p-surfaces of the rings (Fig. 2a). The shortest distances
2.3. Exploring the surrounding of the benzimidazole platform: Arg
residues provide a fluorophilic environment
The X-ray crystal structure of (+)-1a bound to NEP (Fig. 1) had
suggested that the advantage of the imidazo[4,5-c]pyridine over
the benzimidazole platform (e.g. (+)-1a vs. (+)-2a) resulted from
favorable interactions of the N-lone pair of the annellated pyridine
with the flexible side chain of Arg102. Cd+–Fdꢃ bonds can be viewed
as bioisosteric replacements for heteroaromatic N-atoms with lone
pairs [45]. Therefore, we explored whether F-substitution of the
benzimidazole platform would enhance ligand affinity to NEP,
since we expected the resulting C–F bonds to undergo favorable
polar interactions with the electropositive guanidinium side
Table 2
Inhibitors of NEP with a fluorinated benzimidazole platform
a
a
Cmpd.
R1
R2
R3
R4
IC50 (nM)
pKa1
pKa2
(+)-16a
(+)-16b
(+)-16c
(+)-16d
(+)-16e
(+)-16f
(+)-16g
(+)-16h
(+)-16i
(+)-3b
F
H
F
H
H
H
F
H
H
H
H
F
550
310
320
360
950
170
380
910
640
520
4.2
4.8
3.8
3.8
3.3
4.4
3.4
2.9
3.0
5.4
10.9
11.0
10.8
10.8
10.4
10.9
10.6
10.0
9.6
H
F
F
F
H
H
F
F
H
F
H
F
H
H
F
F
F
F
H
F
F
F
F
F
H
H
H
H
>12.0
IC50 values (uncertainties ꢂ10%) obtained under optimized assay conditions are given in nM.
a
The pKa values were measured for precursor compounds with a CH2OH instead of a CH2SH group, in order to prevent interference of a third ionization step, involving thiol
deprotonation. They are referred to in the text as (+)-18 and (+)-17a–i.

M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
857
Fig. 3. Energy-minimized structure of (+)-16i modeled in the active site of NEP. Short contacts between F atoms and amino acid side chains are indicated with dotted lines, the
˚
distances are given in A. For the color code, see caption to Fig. 1.
chains of Arg102, Arg110, and Arg717. While the position of
Arg717 is highly conserved in the various X-ray crystal structures,
the side chains of Arg102 and Arg110 are highly flexible and adopt
various geometries in the presence of different classes of bound
ligands [19,29,46]. For this purpose, we prepared inhibitors (+)-
16a–i (Table 2), following the protocol shown in Scheme 1, which
all showed IC50 values in the upper nanomolar range for binding to
NEP. It should be noted that this study and the earlier reported
exploration of the S1’ pocket were conducted in parallel, i.e.
knowledge of the unfavorable effect of the para-CF3 group on the
benzyl ring on protein–ligand affinity was not yet available. It can
be assumed with great confidence that similar ligands without the
Fig. 4. DpKa1 effects observed upon introduction of a fluorine substituent in ortho-position to a nitrogen atom in substituted benzimidazoles. R = –CH(CH2OH)–CH2–C6H4-p-
CF3. Accuracy of pKa measurements: W0.1 units.

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M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
Fig. 5. DpKa1 effects observed upon introduction of a fluorine substituent in meta-position to a nitrogen atom in substituted benzimidazoles. R = –CH(CH2OH)–CH2–C6H4-p-
CF3. Accuracy of pKa measurements: W0.1 units.
CF3 group would have affinities in the lower nanomolar range.
Nevertheless, interesting hints on favorable interactions of C–F
residues with Arg side chains were obtained and they provided the
starting point for subsequent database mining in the Protein
Databank (PDB) to conclusively show that these side chains
provide a fluorophilic environment [30].
Organic fluorine clearly encounters a more favorable protein
environment around the central platform than in the S1’ pocket.
While fluorination of the benzyl ring to fill the S1’ pocket increased
the IC50 values from 9 nM ((+)-1a to 160–430 nM (+)-5a–j, Table 1),
H/F substitution of the benzimidazole maintained most of the
binding potency or even increased it as shown by the comparison
between (+)-3b (520 nM) and (+)-16a–i (170–950 nM) (Table 2).
The F-substituents in (+)-16a–i can affect binding affinity both
through favorable/unfavorable interactions with the protein and
by altering the pKa values of the imidazole moiety [47], which were
also measured (Table 2).
chain of Arg102 pointing orthogonally into the positively charged
guanidinium residue. Thereduction ofthe pKa1 valueisweaker upon
meta- than ortho-F-substitution and the H-bond between the
imidazole N-atom and the side chain of Arg717 will not be
weakened as much. Clearly, any loss in H-bonding efficiency is
overcompensated by the additional C–Fꢀ ꢀ ꢀArg102 interactions.
Following these findings, we performed a search in the PDB for
C–F interactions with the guanidinium group of arginine residues
and observed a high number of such interactions, which according
to available structure–activity relationships add to protein–ligand
binding affinities [30]. Rather than undergoing linear C–Fꢀ ꢀ ꢀH–N
interactions, which is in agreement with the inefficiency of poorly
polarizable organic fluorine to undergo H-bonding interactions
[48], C–F bonds preferentially orient either parallel or more
orthogonally to the guanidinium plane with its delocalized
positive charge. The latter orientation is supported by the
modeling for the C–Fmetaꢀ ꢀ ꢀArg102 interaction in the complexes
of NEP. The model shown in Fig. 3 is obtained starting from the
crystal structure of (+)-1a complexed to NEP (PDB code 1Y8J) by
substituting the pyridine ring by an annulated tetrafluorobenzene
ring, followed by energy-minimization of the ligand while keeping
the protein fixed, except for the flexible side chain of Arg102.
Since all IC50 values are within an order of magnitude, strong
conclusions cannot be drawn. Nevertheless, roughly speaking, F-
substituents in ortho-position to the imidazole ring lead to low
affinity ((+)-16a, (+)-16e, (+)-16h, IC50 values: 500–1000 nM). The
pKa values of the imidazole N-atoms are lowered (see below), and
this could lead to a weakened H-bond between the imidazole N-
atom and the side chain of Arg717. Furthermore, molecular
modeling, as shown in Fig. 3 for the tetrafluoro derivative (+)-16i,
indicates that ortho-F-atoms undergo unfavorable interactions with
the side chains of Phe106 and Trp693(shortest distances Fꢀ ꢀ ꢀCarom of
2.4. Trends in pKa values of fluorinated benzimidazoles
Benzimidazoles are common scaffolds in medicinal chemistry
and therefore it was of interest to investigate fluorine effects on
their pKa values. Table 2 gives the pKa values for the protonation
(pKa1) and deprotonation (pKa2) of precursor compounds to (+)-3b
and (+)-16a–i in which the thiomethyl (CH2SH) residue is
exchanged for hydroxymethyl (CH2OH). We refer to them in the
following as (+)-18 and (+)-17a–i. This change enables cleaner
determination of the pKa1 and pKa2 values without interference of
an additional ionization step by thiol deprotonation.
˚
3.2 and 3.1 A, respectively). These two unfavorable effects seem to
become compensated by electrostatically favorable interactions of
the C–F bonds with the side chains of Arg110 and Arg717.
The introduction of F-atoms in meta-position is more favorable:
binding potency increases from 520 nM ((+)-3b, no F) to 310 nM
((+)-16b, 1 meta-F), and to 170 nM ((+)-16f, 2 meta-F). The modeling
suggests that the two meta-F-atoms interact favorably with the side

M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
859
Fig. 6. DpKa1 effects observed for the shift of a fluorine substituent from a meta- to ortho-position to a nitrogen atom in substituted benzimidazoles. R = –CH(CH2OH)–CH2–
C6H4-p-CF3. Accuracy of pKa measurements: W0.1 units.
Interesting correlations between the pKa values and the degree
of fluorination at the benzene ring were observed. They are
explicitly shown in Figs. 4–6. Note that for non-symmetrical cases
such as (+)-17a, two different values for each pKa1 and pKa2 would
have to be considered in principle if tautomerization were slow
relative to pKa measurements. However, we have no experimental
evidence for ‘‘split’’ pKa values.
Upon initial inspection of the data, increasing fluorination of the
phenyl ring strongly affects both pKa values, as expected, which
shift in the case of pKa1 from 5.4 ((+)-18) to 3.0 ((+)-17i), and in the
case of pKa2 from >12 ((+)-18) to 9.6 ((+)-17i) (Table 2). At equal
degrees of fluorination, remarkable variation in the pKa values is
observed which corroborates the importance of the position of the
F-atoms. Introduction of fluorine ortho to an N-atom in the
benzimidazole system has a larger effect than meta-substitution.
Thus, ortho-derivative (+)-17a features a pKa1 value of 4.2, whereas
for meta-derivative (+)-17b, a value of 4.8 is measured. Similarly, in
the difluorinated series, (+)-17e with two ortho-fluorines features
pKa 3.3, whereas the value for bis-meta-substituted (+)-17f is 1.1
units higher (4.4).
Similarly, introduction of an additional fluorine in the meta-
position decreases the pKa1 values by 0.5 ꢂ 0.1 units for all pairs of
compounds (Fig. 5).
Shifting a fluorine from meta to ortho leads to a decrease of
0.6 ꢂ 0.2 units for each series of mono- to tri-fluorinated benzimi-
dazoles, again corroborating the weaker effect of meta-fluorination
(Fig. 6). Similar, but less consistent patterns are observed for pKa2
values (Figs. 4–6).
The only compound not fitting the pattern is tetrafluorinated
(+)-17i with an experimental pKa1 value of 3.0, which is 0.1 units
higher than the tri-fluorinated derivative (+)-17h (2.9). According
to the pattern derived in Fig. 4, this compound should have pKa1 of
ca. 2.3 (introducing an ortho-fluorine into (+)-17g). When using the
pattern of Fig. 5, a pKa1 of ca. 2.4 can be calculated (introducing a
meta-fluorine into (+)-17h). At this point, we do not have a good
explanation for the special acid/base properties of tetrafluorinated
(+)-17i. Overall, the trends of pKa shifts upon F-introduction in (+)-
17a–i are in good agreement with those reported for simple
fluorinated benzimidazoles [47].
3. Conclusions
A more detailed analysis of the data shows remarkably
consistent correlations. When comparing pairs of compounds that
differ only by one additional fluorine at the ortho-position to an
imidazole nitrogen, a decrease in pKa1 of 1.1 ꢂ 0.2 units is observed,
independent of the number of fluorine atoms in the starting
compound (Fig. 4).
While the introduction of organic fluorine into drugs and
agrochemicals has for decades been a popular and successful
approach to optimize physical (pKa, log D) and ADME (adsorption,
distribution, metabolism, and excretion) properties, recent

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M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
research explores how binding potency and selectivity can be
gained by direct interactions of C–F bonds in bound ligands with
the surrounding protein environment. This study confirms the
observed trend, that organic fluorine, with its high negative charge
density and its low polarizability, prefers to orientate into
electropositive protein environments [30]. Thus introduction of
fluorine into the benzyl ring, used by our ligands to fill the S1’
pocket, leads to substantial losses in binding potency. This narrow
pocket is lined by several aromatic rings, and high-confidence
modeling suggests that electronegative F-substituents closely
approach their electron-rich p-surfaces. The resulting electrostatic
repulsion makes the S1’ pocket a highly fluorophobic environment.
Of course, there are many reports in medicinal chemistry on filling
hydrophobic cavities with CF3 and other fluorinated residues.
Partitioning becomes more favorable (positive log D), desolvation
entropy is gained, and C–Hꢀ ꢀ ꢀF contacts are established, which all
should enhance the measurable binding affinity. However, these
gains in affinity can become compensated by losses in affinity
resulting from close, electrostatically repulsive contacts between
organic fluorine, with its high negative charge density, and the
negatively polarized p-clouds of the side chains of Phe, Tyr, and
Trp. We had previously also observed the fluorophobicity of the
electron-rich indole ring of a Trp residue in our fluorine scan of
thrombin inhibitors [25].
in 96 well-plates. Each inhibitor was prepared as a 4 mM stock
solution in DMSO which was used to prepare 12 diluted solutions
per inhibitor with concentrations between 100 and 0.001 mM in
DMSO. Each well was loaded with 155 mL of aqueous buffer
solution (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), 100 mM NaCl, 0.2% of a 5% bovine serum albumin
(BSA) solution in water, pH 7.4), 5 mL of the respective inhibitor
solution in DMSO, 20 mL of 18.75 nM NEP solution in aqueous
buffer (25 mM 2-amino-2-(hydroxymethyl)propane-1,3-diol
(Tris), 200 mM NaCl, 2 mM MgCl2, pH 8.0), and 20 mL of substrate
in buffer solution (0.8% solution of substrate Abz-GGfLRRVQ-
EDDnp in MeOH, diluted to 62.5 mM with aqueous buffer solution
(50 mM HEPES, 100 mM NaCl, containing 0.2% of a 5% BSA
solution in water, pH 7.4)). Cleavage of the substrate was measured
without preincubation of the prepared mixtures at lexc = 320 nm
and lem = 410 nm. The affinity of each inhibitor was determined by
two measurements per concentration. The determination of the
respective IC50 value was performed in Microsoft Excel. The
percentage of inhibition was determined by comparing the
averaged slopes of the fluorescence lines to a reference value of
a measurement containing no inhibitor.
The reproducibility of the IC50 values is ꢂ10%. Thiorphan as
reference ligand gave an IC50 value of 6.0 ꢂ 0.3 (n = 4) in this assay
([18]: 4 nM).
In contrast, binding potency is maintained or even increased,
when the central benzimidazole platform of the ligands is
fluorinated. Here, the highly reproducible modeling suggests that
C–F bonds point orthogonally on the positively charged guanidi-
nium side chains of surrounding Arg residues. The resulting
electrostatically favorable interaction is quite general, as was
shown in subsequent database searches [30]. Finally, pKa
measurement of the fluorinated ligands provided several simple
patterns for the prediction of pKa values of benzimidazoles,
important building blocks in medicinal chemistry.
4.3. Molecular modeling
All modeling was done on a PC using the modeling package
MOLOC for Windows and the all atom force field MAB [20]. All
simulations started from the crystal structure of (+)-1a complexed
to NEP (PDB code 1Y8J). The pyridine ring or the benzyl ring of (+)-
1a were substituted by the newly introduced residues, and the new
bound ligands were energy-minimized. The protein coordinates
were kept fixed, except for the flexible side chain of Arg102. The
modeling shown in Figs. 2 and 3 is highly reproducible when
repeated by other co-workers in the laboratory.
4. Experimental
4.1. General
4.4. Experimental procedures
Characterization. Melting points were obtained with a Bu¨ chi B-
540 and are uncorrected. IR spectra were obtained with a
PerkinElmer Spectrum BX FTIR Spectrometer from the neat
substances. NMR spectra were recorded on Varian Gemini 300,
Varian Mercury 300, or Bruker ARX 300 spectrometers. Chemical
shifts in 1H and 13C NMR spectra are recorded as parts per million
(ppm) referenced to the residual solvent resonances (CDCl3 = 7.26,
CH3OD = 3.31). 19F NMR spectra are recorded against CFCl3 as
internal standard (CFCl3 = 0). Coupling constants are given in
Hertz. High-resolution mass spectra were recorded on VG-
TRIBRID (EI) or IonSpec Ultima (MALDI) spectrometers. 2,5-
Dihydroxybenzoic acid (DHB) or 3-hydroxypicolinic acid (3-HPA)
were used as matrix for MALDI-MS measurements. Elemental
analyses were performed in the Laboratory of Organic Chemistry,
ETH Zurich. Optical rotations were measured at 25 8C on a
PerkinElmer 241 Polarimeter at 589 nm (Na-D line). The
concentration c is given in g/100 mL. RP-HPLC was performed
with Knauer ProntoSil columns (analytical: 120–5 C18 AQ,
250 mm ꢄ 5.0 mm; preparative: 120–5 C18 AQ, 250 mm ꢄ 20
mm). The nomenclature follows suggestions of the computer
program ACD/Name from ACD/Labs.
All inhibitors were synthesized in identical reaction sequences,
with minor improvements to those already described with full
experimental protocols in Ref. [29]. A detailed synthesis of
inhibitor (+)-3a is given in the following. Spectroscopic data only
is given for all other inhibitors. Full experimental protocols can be
found in a doctoral dissertation [49].
4.4.1. (+)-(4S)-4-Isopropyl-3-{3-[4-
(trifluoromethyl)phenyl]propanoyl}-1,3-oxazolidin-2-one ((+)-8)
A solution of 6 (8.36 g, 38.3 mmol, 1.1 equiv.) in THF (190 mL)
was cooled to ꢃ30 8C. Et3N (12.6 mL, 90.6 mmol, 2.6 equiv.) and
pivaloyl chloride (3.13 mL, 36.6 mmol, 1.05 equiv.) were added,
and the suspension was stirred for 90 min. After addition of LiCl
(1.70 g, 40.1 mmol, 1.15 equiv.) and oxazolidinone (S)-7 (4.50 g,
34.8 mmol, 1.0 equiv.), the mixture was allowed to reach r.t. and
stirred for 18 h. H2O was added and the aqueous layer extracted
with EtOAc. The combined organic layers were dried over MgSO4,
concentrated, and the residue was purified by column chromato-
graphy (CC) (SiO2, pentane/EtOAc 7:1).
25
Yield: 7.77 g, 23.6 mmol, 68%. White solid. mp: 59 8C. [a]D
:
+53.8 (c = 1.00, CHCl3). IR (neat): 2966, 1777, 1703, 1618, 1484,
1384, 1325, 1301, 1210, 1170, 1135, 1103, 1065, 1021, 998, 868,
831, 772, 750, 707. 1H NMR (300 MHz, CDCl3): 0.82 (d, J = 6.9, 3H),
0.90 (d, J = 7.1, 3H), 2.28–2.38 (m, 1H), 3.02–3.07 (m, 2H), 3.17–
3.41 (m, 2H), 4.18–4.28 (m, 2H), 4.39–4.44 (m, 1H), 7.36/7.54
(AA0BB0, J = 8.3, 4H). 13C NMR (75 MHz, CDCl3): 14.6, 18.0, 28.4,
4.2. Enzyme assay
The affinities of inhibitors towards NEP were measured in a
FRET-assay performed on a BMG Labtechnologies FLUOstar Optima

M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
861
30.2, 36.7, 58.4, 63.4, 124.1 (q, J = 271.3), 125.2 (q, J = 3.6), 128.4 (q,
J = 32.2), 128.8, 144.5, 153.9, 171.7. 19F NMR (282 MHz, CDCl3):
ꢃ62.8 (s, 3F). EI-HRMS: 329.1234 ([M]+, C16H18F3NO3+, calcd.
329.1233). Anal. calcd for C16H18F3NO3: C 58.36, H 5.51, N 4.25.
Found: C 58.27, H 5.55, N 4.30.
were dried over MgSO4 an concentrated. The residue was purified
by CC (SiO2, pentane/EtOAc 8:1).
25
Yield: 1.31 g, 2.77 mmol, 99%. White solid. mp: 61 8C. [a]D
:
+61.7 (c = 0.99, MeOH). IR (neat): 2952, 2930, 2883, 2857, 1764,
1701, 1617, 1465, 1387, 1371, 1323, 1301, 1259, 1243, 1228, 1216,
1162, 1110, 1092, 1067, 1018, 963, 939, 836, 823, 777, 724, 697,
682, 666, 636. 1H NMR (300 MHz, CDCl3): 0.01 (s, 3H), 0.02 (s, 3H),
0.84 (d, J = 6.9, 3H), 0.86 (s, 9H), 0.88 (d, J = 7.1, 3H), 2.22–2.37 (m,
1H), 2.87 (dd, J = 6.9, 13.6, 1H), 3.03 (dd, J = 8.3, 13.6, 1H), 3.80 (dd,
J = 4.6, 9.8, 1H), 3.90 (dd, J = 6.4, 9.8, 1H), 4.08–4.15 (m, 2H), 4.31–
4.40 (m, 2H), 7.33/7.51 (AA0BB0, J = 8.2, 4H. 13C NMR (75 MHz,
CDCl3): ꢃ5.61, ꢃ5.55, 14.8, 17.9, 18.2, 25.8, 28.5, 34.2, 47.4, 58.4,
63.2, 63.7, 124.2 (q, J = 271.8), 125.3 (q, J = 3.7), 128.4 (q, J = 32.4),
129.5, 143.2, 153.7, 173.3. 19F NMR (282 MHz, CDCl3): ꢃ62.8 (s,
3F). MALDI-HRMS (3-HPA): 496.2091 ([MNa]+, C23H34F3NO4NaSi+,
calcd 496.2101). Anal. calcd for C23H34F3NO4Si: C 58.33, H 7.24, N
2.96. Found: C 58.27, H 7.30, N 2.91.
4.4.2. (+)-(4S)-3-{(2R)-3-(Benzyloxy)-2-[4-
(trifluoromethyl)benzyl]propanoyl}-4-isopropyl-1,3-oxazolidin-2-
one ((+)-9)
A solution of (+)-8 (7.69 g, 23.3 mmol, 1.0 equiv.) in CH2Cl2
(115 mL) was cooled to 0 8C, mixed with TiCl4 (4.82 mL, 24.5 mmol,
1.05 equiv.), and stirred for 5 min. Diisopropylethylamine
(3.30 mL, 23.3 mmol, 1.0 equiv.) was added, and stirring was
continued for 1 h before BOMCl (6.50 mL, 46.7 mmol, 2.0 equiv.)
was added. Stirring was continued for 3 h, then saturated aqueous
NH4Cl solution was added, and the aqueous layer was extracted
with CH2Cl2. The combined organic layers were dried over MgSO4
and concentrated. The residue was purified by CC (SiO2, pentane/
EtOAc 9:2).
Yield: 5.79 g, 12.9 mmol, 55%. Colorless oil. [a]D25: +43.5
(c = 1.12, CHCl3). IR (neat): 2963, 2873,1771, 1699, 1617, 1453,
1385, 1362, 1322, 1300, 1202, 1161, 1114, 1065, 1017, 990, 846,
823, 735, 697. 1H NMR (300 MHz, CDCl3): 0.79 (d, J = 6.9, 3H), 0.87
(d, J = 6.9, 3H), 2.26–2.36 (m, 1H), 2.90 (dd, J = 7.2, 13.6, 1H), 3.08
(dd, J = 8.1, 13.6, 1H), 3.65 (dd, J = 4.8, 9.3, 1H), 3.76 (dd, J = 6.8, 9.3,
1H), 4.05–4.16 (m, 2H), 4.35–4.40 (m, 1H), 4.45–4.56 (m, 3H),
7.25–7.37 (m, 7H), 7.51 (d, J = 8.3, 2H). 13C NMR (75 MHz, CDCl3):
14.6, 17.9, 28.4, 34.4, 45.2, 58.4, 63.2, 70.5, 73.1, 124.1 (q, J = 271.3),
125.1 (q, J = 3.5), 127.5, 128.1, 128.5 (q, J = 32.2), 129.3, 137.7,
142.8, 153.5, 173.2. 19F NMR (282 MHz, CDCl3): ꢃ62.7 (s, 3F).
MALDI-HRMS (3-HPA): 472.1703 ([MNa]+, C24H26F3NO4Na+, calcd
472.1706). Anal. calcd for C24H26F3NO4: C 64.13, H 5.83, N 3.12.
Found: C 64.01, H 5.75, N 3.15.
4.4.5. (ꢃ)-(2R)-3-{[tert-Butyl(dimethyl)silyl]oxy}-2-[4-
(trifluoromethyl)benzyl]propanoic acid ((ꢃ)-12)
To a degassed solution of (+)-11 (2.81 g, 5.93 mmol, 1.0 equiv.)
in THF (80 mL) and H2O (25 mL), H2O2 (30% in H2O, 4.04 mL,
35.6 mmol, 6.0 equiv.) and a degassed solution of LiOH.H2O
(0.498 g, 11.9 mmol, 2.0 equiv.) in H2O (12 mL) were added at
0 8C. The mixture was stirred for 3 h at 0 8C, then 16 h at r.t. A
solution of aqueous Na2SO3 (1.5 M, 35 mL) was added, stirring was
continued for 15 min, then THF was removed in vacuo. The residue
was diluted with Et2O, and aqueous HCl solution (1 M) was added
until a pH value of 1–2 was reached. The mixture was then quickly
extracted with Et2O, the organic layer dried over MgSO4 and
concentrated. The residue was purified by flash chromatography
(SiO2, pentane/EtOAc 10:1, with 0.5% AcOH added).
25
Yield: 1.46 g, 4.04 mmol, 90%. White solid. mp: 41 8C. [a]D
:
ꢃ1.54 (c = 1.01, CHCl3). IR (neat): 2926, 2855, 1706, 1619, 1469,
1460, 1419, 1360, 1321, 1252, 1218, 1159, 1107, 1087, 1065, 1035,
1018, 1004, 976, 952, 939, 866, 832, 815, 805, 771, 731, 676. 1H
NMR (300 MHz, CDCl3): 0.04 (s, 3H), 0.04 (s, 3H), 0.89 (s, 9H), 2.81–
2.90 (m, 1H), 2.95 (dd, J = 6.9, 13.7, 1H), 3.06 (dd, J = 7.6, 13.7, 1H),
3.74 (dd, J = 5.9, 10.0, 1H), 3.80 (dd, J = 5.1, 10.0, 1H), 7.32/7.54
(AA0BB0, J = 8.0, 4H). 13C NMR (75 MHz, CDCl3): ꢃ5.42, 18.3, 25.8,
33.5, 49.3, 62.6, 124.1 (q, J = 271.3), 125.3 (q, J = 3.6), 128.7 (q,
J = 32.2), 129.2, 142.8, 178.4. 19F NMR (282 MHz, CDCl3): ꢃ62.2 (s,
3F). EI-HRMS: 305.0816 ([M–C4H9]+, C13H16F3O3Si+, calcd
305.0815). Anal. calcd for C17H25F3O3Si: C 56.33, H 6.95. Found:
C 56.05, H 6.95.
4.4.3. (+)-(4S)-3-{(2R)-3-Hydroxy-2-[4-
(trifluoromethyl)benzyl]propanoyl}-4-isopropyl-1,3-oxazolidin-2-
one (+)-10
To a solution of (+)-9 (5.36 g, 11.9 mmol, 1.0 equiv.) in EtOAc
(100 mL), Pd/C (10% Pd, 1.27 g, 0.1 equiv.) was added and the
mixture was stirred under an atmosphere of H2 for 24 h. Filtration
through Celite and concentration of the solution gave crude
product which was purified by CC (SiO2, pentane/EtOAc 2:1).
25
Yield: 3.89 g, 10.8 mmol, 91%. White solid. mp: 108 8C. [a]D
:
+93.7 (c = 1.01, CHCl3). IR (neat): 3475, 2962, 2940, 2877, 1747,
1700, 1616, 1483, 1418, 1399, 1373, 1325, 1303, 1291, 1221, 1161,
1124, 1109, 1063, 1018, 982, 942, 861, 829, 777, 759, 705, 641. 1H
NMR (300 MHz, CDCl3): 0.87 (d, J = 6.9, 3H), 0.90 (d, J = 7.0, 3H),
2.29–2.42 (m, 1H), 2.45 (dd, J = 4.2, 8.1, 1H), 2.90 (dd, J = 7.6, 13.4,
1H), 3.07 (dd, J = 7.6, 13.4, 1H), 3.74 (ddd, J = 6.7, 8.1, 11.1, 1H), 3.86
(td, J = 4.2, 11.1, 1H), 4.09–4.20 (m, 2H), 4.25–4.37 (m, 2H), 7.37/7.52
(AA0BB0, J = 8.0, 4H). 13C NMR (75 MHz, CDCl3): 14.6, 17.8, 28.4, 33.9,
46.9, 58.7, 63.1, 63.5, 124.2 (q, J = 271.8), 125.3 (q, J = 3.7), 128.8 (q,
J = 32.2), 129.5, 142.7, 154.0, 174.5. 19F NMR (282 MHz, CDCl3):
ꢃ62.2 (s, 3F). MALDI-HRMS (3-HPA): 382.1236 ([MNa]+,
4.4.6. (+)-(2R)-N-(3-Amino-4-pyridinyl)-3-{[tert-
butyl(dimethyl)silyl]oxy}-2-[4-(trifluoromethyl)benzyl]propanamide
((+)-13)
To
a
0 8C cold solution of (ꢃ)-12 (0.300 g, 0.828 mmol,
1.0 equiv.) in CH2Cl2 (8.3 mL), oxalylchloride (2 M solution in
CH2Cl2, 0.44 mL, 0.869 mmol, 1.05 equiv.) was added and the
mixture stirred at r.t. for 12 h. The solvent was removed in vacuo
and the residue redissolved in THF (8.3 mL). After addition of 3,4-
diaminopyridine (0.181 g, 1.66 mmol, 2.0 equiv.) and Et3N
(0.23 mL, 1.66 mmol, 2.0 equiv.), the mixture was stirred for
12 h. H2O was added and THF removed in vacuo. The aqueous layer
was extracted with CH2Cl2 and the organic layer dried over MgSO4.
C
17H20F3NO4Na+, calcd 382.1237). Anal. calcd for C17H20F3NO4: C
56.82, H 5.61, N 3.90. Found: C 56.59, H 5.66, N 3.84.
4.4.4. (+)-(4S)-3-{(2R)-3-{[tert-Butyl(dimethyl)silyl]oxy}-2-[4-
(trifluoromethyl)benzyl]propanoyl}-4-isopropyl-1,3-oxazolidin-2-
one (+)-11
The concentrated residue was then purified by CC (SiO2, EtOAc).
25
Yield: 0.357 g, 0.787 mmol, 95%. White solid. mp: 161 8C. [a]D
:
To a solution of (+)-10 (1.00 g, 2.78 mmol, 1.0 equiv.) in CH2Cl2
(3.5 mL), TBDMSCl (0.629 g, 4.17 mmol, 1.5 equiv.) and DMAP
(0.578 g, 4.73 mmol, 1.7 equiv.) were added. After stirring for 18 h,
the mixture was diluted with H2O and CH2Cl2 and the aqueous
layer was extracted with CH2Cl2. The combined organic layers
+11.0 (c = 0.55, CHCl3). IR (neat): 3163, 2950, 2929, 2857, 1698,
1682, 1617, 1579, 1532, 1470, 1427, 1387, 1323, 1296, 1247, 1196,
1186, 1166, 1105, 1064, 1017, 1004, 835, 822, 776, 733, 667. 1H
NMR (300 MHz, CDCl3): 0.06 (s, 3H), 0.07 (s, 3H), 0.89 (s, 9H), 2.79–
2.89 (m, 2H), 3.11–3.20 (m, 1H), 3.63 (s br., 2H), 3.83 (d, J = 5.3, 2H),

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M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
7.23 (d, J = 5.2, 1H), 7.33/7.55 (AA0BB0, J = 8.0, 4H), 7.97 (d, J = 5.3,
1H), 8.07 (s, 1H), 8.49 (s br., 1H). 13C NMR (75 MHz, CDCl3): ꢃ5.38,
ꢃ5.34, 18.3, 25.9, 34.4, 51.4, 64.0, 117.0, 124.0 (q, J = 271.3), 125.4
(q, J = 3.5), 128.8 (q, J = 32.3), 129.2, 131.9, 134.6, 140.4, 141.3,
142.9, 171.9. 19F NMR (282 MHz, CDCl3): ꢃ62.2 (s, 3F). MALDI-
HRMS (3-HPA): 454.2126 ([MH]+, C22H31F3N3O2Si+, calcd
454.2132). Anal. calcd for C22H30F3N3O2Si: C 58.26, H 6.67, N
9.26. Found: C 58.48, H 6.91, N 9.27.
and CH2Cl2 and quickly extracted with CH2Cl2. The combined
organic layers were dried over MgSO4 and concentrated. The
residue was dissolved in a 1:1 mixture of MeCN/H2O (3 mL),
filtered through cotton wool, and immediately purified by
preparative RP-HPLC (gradient 0.1% TFA in H2O/MeCN
99:1 ! 0:100 in 60 min). The fractions containing the product
as TFA salt were combined and lyophilized.
Yield: 0.039 g, 0.086 mmol, 49%. Colorless oil. [a]D25: +44.6
(c = 0.11, MeOH). IR (neat): 3060, 3029, 2996, 2967, 2955, 2870,
1770, 1703, 1603, 1489, 1482, 1453, 1406, 1389, 1379, 1368, 1362,
1306, 1226, 1210, 1141, 1122, 1102, 1066, 1021, 997, 970, 915,
787, 771, 709, 700, 639. 1H NMR (300 MHz, CDCl3): 2.88–3.10 (m,
3H), 3.16–3.24 (m, 1H), 3.38–3.44 (m, 1H), 7.18–7.25 (m, 2H), 7.37
(d, J = 6.4, 1H), 7.73–7.83 (m, 2H), 8.22 (d, J = 6.4, 1H), 9.34 (s, 1H).
13C NMR (75 MHz, CDCl3): 33.5, 37.5, 43.1, 105.2, 124.1 (q,
J = 268.5), 126.5, 129.4 (q, J = 3.4), 130.9 (q, J = 32.8), 133.1, 134.8,
137.2, 142.7, 146.8, 158.2. 19F NMR (282 MHz, CDCl3): ꢃ62.5 (s,
3F). MALDI-HRMS (DHB): 338.0930 ([MH]+, C16H15F3N3S+, calcd
338.0933).
4.4.7. (+)-(2S)-2-(1H-Imidazo[4,5-c]pyridin-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanol ((+)-14
A solution of (+)-13 (0.258 g, 0.569 mmol, 1.0 equiv.) in AcOH
(40 mL) was warmed to 65 8C for 24 h. The solvent was removed in
vacuo, and traces of AcOH were removed by azeotropic distillation
with toluene. The residue was redissolved in THF (2.3 mL) and
nBu4NF (2 M solution in THF, 0.57 mL, 1.14 mmol, 2.0 equiv.) was
added. After stirring for 2 h, the THF was removed in vacuo. The
residue was mixed with H2O and extracted with EtOAc. The
combined organic layers were dried over MgSO4 and concentrated.
The residue was purified by CC (SiO2, EtOAc/MeOH 9:1).
25
Yield: 0.112 g, 0.349 mmol, 61%. White solid. mp: 83 8C. [a]D
:
4.4.10. (+)-(2S)-2-(1H-Benzimidazol-2-yl)-3-[4-
+94.8 (c = 0.22, CHCl3). IR (neat): 3084, 2936, 1620, 1591, 1533,
1460, 1419, 1322, 1285, 1159, 1107, 1065, 1031, 1018, 970, 913,
848, 812, 630. 1H NMR (300 MHz, CDCl3 + 3 drops of TFA): 3.13–
3.24 (m, 2H), 3.51–3.60 (m, 1H), 3.83 (dd, J = 7.0, 11.0, 1H), 3.90 (dd,
J = 5.2, 11.0, 1H), 7.21/7.38 (AA0BB0, J = 8.0, 4H), 7.89 (dd, J = 0.7, 6.5,
1H), 8.26 (dd, J = 0.7, 6.5, 1H), 8.95 (s, 1H). 13C NMR (75 MHz,
CDCl3 + 3 drops of TFA): 35.6, 44.7, 63.2, 111.9, 123.9 (q, J = 276.4),
125.3 (q, J = 3.5), 128.8 (q, J = 32.6), 129.0, 130.6, 132.2, 137.0,
142.2, 147.7, 165.9. 19F NMR (282 MHz, CDCl3 + 3 drops of TFA):
ꢃ62.7 (s, 3F). MALDI-HRMS (3-HPA): 322.1155 ([MH]+,
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-3b)
Colorless oil. [a]D25: +49.7 (c = 0.13, MeOH). IR (neat): 3239,
2929, 2857, 1652, 1620, 1527, 1499, 1461, 1415, 1389, 1362, 1320,
1247, 1165, 1112, 1066, 1017, 905, 831, 783, 739, 667, 628. 1H
NMR (300 MHz, CD3OD): 2.91–3.07 (m, 3H), 3.13–3.19 (m, 1H),
3.42–3.48 (m, 1H), 7.20–7.24 (m, 2H), 7.27–7.32 (m, 2H), 7.73–7.80
(m, 4H). 13C NMR (75 MHz, CDCl3): 33.5, 37.5, 43.1, 114.0, 124.1 (q,
J = 266.3), 127.5, 129.5 (q, J = 3.6), 130.2 (q, J = 32.2), 131.8, 137.0,
142.7, 156.9. 19F NMR (282 MHz, CDCl3): ꢃ62.3 (s, 3F). MALDI-
HRMS (DHB): 337.0984 ([MH]+, C17H16F3N2S+, calcd 337.0981).
C
16H15F3N3O+, calcd 322.1162).
4.4.11. (+)-(2S)-2-(1H-Imidazo[4,5-c]pyridin-2-yl)-3-(4-
methylphenyl)-1-propanethiol trifluoroacetate ((+)-4a)
4.4.8. (ꢃ)-S-{(2S)-2-(1H-imidazo[4,5-c]pyridin-2-yl)-3-[4-
Colorless oil. [a]D25: +48.5 (c = 0.09, CHCl3). IR (neat): 3048,
2922, 1639, 1620, 1596, 1539, 1500, 1450, 1426, 1327, 1237, 1190,
1153, 1109, 1067, 1045, 1018, 872, 954, 935, 848, 820, 783, 742,
658. 1H NMR (300 MHz, CD3OD): 2.20 (s, 3H), 3.12–3.26 (m, 4H),
3.54–3.63 (m, 1H), 6.89–7.00 (m, 4H), 8.00–8.04 (m, 1H), 8.47–8.50
(m, 1H), 9.14–9.17 (m, 1H). 13C NMR (75 MHz, CD3OD): 21.0, 28.3,
40.5, 43.8, 112.0, 114.7, 118.5, 129.8, 130.3, 133.4, 136.3, 137.7,
139.8, 166.9. MALDI-HRMS (3-HPA): 284.1212 ([MH]+, C16H18N3S+,
calcd 284.1216).
(trifluoromethyl)phenyl]propyl} ethanethioate ((ꢃ)-15)
To a 0 8C cold solution of PPh3 (0.123 g, 0.468 mmol, 1.5 equiv.)
in THF (4.7 mL), DIAD (0.093 mL, 0.468 mmol, 1.5 equiv.) was
added and the mixture stirred for 30 min. A solution of (+)-14
(0.100 g, 0.311 mmol, 1.0 equiv.) in THF (3.1 mL) and AcSH
(0.044 mL, 0.624 mmol, 2.0 equiv.) were subsequently added.
The mixture was stirred at 0 8C for 1 h and at r.t. for 2 h, then
H2O was added. The aqueous layer was extracted with Et2O, the
combined organic layers were dried over MgSO4 and concentrated.
The residue was purified by CC (SiO2, EtOAc/MeOH 95:5).
25
Yield: 0.089 g, 0.235 mmol, 75%. White solid. mp: 67 8C. [a]D
:
4.4.12. (+)-(2S)-3-(4-Cyclobutylphenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-4b)
ꢃ78.4 (c = 0.23, MeOH). IR (neat): 3069, 2998, 2965, 2874, 1774,
1702, 1600, 1545, 1509, 1482, 1453, 1406, 1389, 1370, 1318, 1305,
1209, 1157, 1142, 1123, 1101, 1064, 1050, 1019, 999, 971, 864,
827, 787, 749, 733, 696, 637. 1H NMR (300 MHz, CDCl3 + 3 drops of
TFA): 2.28 (s, 3H), 3.29–3.41 (m, 2H), 3.50 (dd, J = 5.2, 14.1, 1H),
3.58 (t, J = 14.1, 1H), 3.79–3.87 (m, 1H), 7.24/7.43 (AA0BB0, J = 8.1,
4H), 8.05 (d, J = 6.5, 1H), 8.32 (d, J = 6.5, 1H), 9.25 (s, 1H). 13C NMR
(75 MHz, CDCl3 + 3 drops of TFA): 30.5, 32.2, 39.4, 42.6, 113.8,
123.9 (q, J = 271.1), 125.4 (q, J = 3.4), 129.0, 129.0 (q, J = 32.4),
130.0, 132.2, 135.1, 141.1, 149.4, 166.1, 194.7. 19F NMR (282 MHz,
CDCl3 + 3 drops of TFA): ꢃ62.3 (s, 3F). MALDI-HRMS (3-HPA):
380.1031 ([MH]+, C18H17F3N3OS+, calcd 380.1039).
Colorless oil. [a]D25: +54.7 (c = 0.12, CHCl3). IR (neat): 3072, 2734,
1698, 1616, 1538, 1489, 1454, 1445, 1425, 1324, 1270, 1190, 1165,
1122, 1107, 1065, 1030, 1016, 958, 851, 828, 766, 751, 731, 617. 1H
NMR (300 MHz, CD3OD): 1.75–1.84 (m, 1H), 1.92–2.10 (m, 3H),
2.20–2.31 (m, 2H), 3.02–3.28 (m, 4H), 3.36–3.47 (m, 1H), 6.96–7.05
(m, 4H), 8.03 (d, J = 7.0, 1H), 8.49 (d, J = 7.0, 1H), 9.17 (s, 1H). 13C NMR
(75 MHz, CD3OD): 19.0, 28.3, 30.8, 40.4, 40.5, 41.4, 112.1, 127.5,
129.7, 133.3, 134.4, 136.7, 139.8, 145.9, 147.9, 167.4. MALDI-HRMS
(3-HPA): 324.1530 ([MH]+, C19H21N3S+, calcd 324.1529).
4.4.13. (+)-(2S)-3-(4-Cyclopentylphenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-4c)
Colorless oil. [a]D25: +46.1 (c = 0.11, CHCl3). IR (neat): 3468,
2935, 1747, 1698, 1481, 1397, 1361, 1298, 1215, 1141, 1125, 1103,
1077, 1054, 985, 944, 847, 820, 773, 754, 700, 638. 1H NMR
(300 MHz, CD3OD): 1.31–2.09 (m, 8H), 2.81–3.07 (m, 4H), 3.50–
3.80 (m, 2H), 6.92–7.06 (m, 4H), 8.01 (d, J = 6.3, 1H), 8.47 (d, J = 6.3,
1H), 9.14 (s, 1H). 13C NMR (75 MHz, CD3OD): 26.4, 35.7, 40.5, 41.1,
43.7, 46.9, 112.2, 128.4, 129.8, 133.4, 134.5, 136.3, 139.8, 146.4,
4.4.9. (+)-(2S)-2-(1H-Imidazo[4,5-c]pyridin-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-3a)
A solution of (ꢃ)-15 (0.067 g, 0.177 mmol, 1.0 equiv.) in MeOH
(3.5 mL) was first degassed with Ar, then with H2, and a degassed
solution of NaOMe (0.5 M solution in MeOH, 3.54 mL, 1.77 mmol,
10.0 equiv.) was added. The solution was stirred for 2 h, then the
solvent was removed in vacuo. The residue was diluted with H2O

M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
863
147.9, 167.0. MALDI-HRMS (3-HPA): 338.1686 ([MH]+, C20H24N3S+,
calcd 338.1684).
6.96–7.09 (m, 2H), 8.03 (dd, J = 0.7, 6.6, 1H), 8.49 (dd, J = 0.7, 6.6,
1H), 9.17 (t, J = 0.7, 1H). 13C NMR (75 MHz, CD3OD): 31.1, 37.1,
40.4, 112.2, 118.4 (d, J = 17.2), 118.8 (d, J = 17.3), 126.4 (dd, J = 3.6,
6.2), 133.6, 134.6, 136.7 (t, J = 4.8), 139.8, 147.9, 150.5 (dd, J = 12.6,
246.0), 151.4 (dd, J = 12.9, 247.1), 166.3. 19F NMR (282 MHz,
CD3OD): ꢃ139.0 (m, 1F), ꢃ142.0 (m, 1F). MALDI-HRMS (3-HPA):
304.0870 ([MH]+, C15H14F2N3S+, calcd 304.0871).
4.4.14. (+)-(2S)-3-(4-Cyclohexylphenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-4d)
Colorless oil. [a]D25: +44.8 (c = 0.07, CHCl3). IR (neat): 3056,
3002, 2967, 2877, 1773, 1700, 1608, 1515, 1484, 1464, 1451, 1435,
1406, 1389, 1372, 1312, 1280, 1206, 1143, 1116, 1102, 1063, 1049,
1019, 997, 970, 947, 892, 881, 821, 789, 766, 749, 730, 697, 644,
616. 1H NMR (300 MHz, CD3OD): 1.23–1.44 (m, 5H), 1.68–1.87 (m,
5H), 2.35–2.46 (m, 1H), 3.03–3.27 (m, 4H), 3.54–3.63 (m, 1H),
6.92–7.04 (m, 4H), 8.03 (d, J = 6.7, 1H), 8.49 (d, J = 6.7, 1H), 9.17 (s,
1H). 13C NMR (75 MHz, CD3OD): 27.2, 28.0, 28.3, 35.7, 40.4, 45.6,
48.4, 112.1, 128.1, 128.1, 129.8, 133.3, 134.4, 136.7, 139.8, 147.9,
167.4. MALDI-HRMS (3-HPA): 352.1842 ([MH]+, C21H26N3S+, calcd
352.1842).
4.4.19. (+)-(2S)-3-(2,4-Difluorophenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-5e)
Colorless oil. [a]D25: +49.8 (c = 0.11, CHCl3). IR (neat): 3031,
2964, 2927, 2860, 1764, 1703, 1616, 1583, 1493, 1483, 1457,
1397, 1382, 1358, 1296, 1274, 1246, 1228, 1219, 1201, 1173,
1141, 1124, 1104, 1089, 1052, 1028, 1012, 982, 960, 943, 909,
894, 845, 807, 778, 760, 738, 730, 692, 625, 611. 1H NMR
(300 MHz, CD3OD): 3.08 (d, J = 7.1, 2H), 3.20 (dd, J = 8.9, 13.6,
1H), 3.35 (dd, J = 6.6, 13.6, 1H), 3.59–3.68 (m, 1H), 6.74–6.88 (m,
2H), 7.13 (dt, J = 6.5, 8.6, 1H), 8.06 (dd, J = 0.8, 6.6, 1H), 8.51 (dd,
J = 0.8, 6.6, 1H), 9.19 (t, J = 0.8, 1H). 13C NMR (75 MHz, CD3OD):
28.2, 33.6, 46.8, 104.7 (t, J = 26.1), 112.1, 112.4 (dd, J = 3.7, 21.4),
122.2 (dd, J = 3.8, 15.9), 133.4, 134.5, 139.9, 147.9, 162.5 (dd,
J = 12.0, 247.0), 163.5 (dd, J = 12.2, 246.8), 166.7. 19F NMR
(282 MHz, CD3OD): ꢃ112.5 (m, 1F), ꢃ114.3 (dd, J = 8.6, 17.4, 1F).
MALDI-HRMS (3-HPA): 306.0864 ([MH]+, C15H14F2N3S+, calcd
306.0871).
4.4.15. (+)-(2S)-3-(4-Fluorophenyl)-2-(1H-imidazo[4,5-c]pyridin-2-
yl)-1-propanethiol trifluoroacetate ((+)-5a)
Colorless oil. [a]D25: +41.7 (c = 0.08, CHCl3). IR (neat): 3468,
2935, 1747, 1698, 1481, 1397, 1361, 1298, 1215, 1141, 1125, 1103,
1077, 1054, 985, 944, 847, 820, 773, 754, 700, 638. 1H NMR
(300 MHz, CD3OD): 3.12–3.28 (m, 4H), 3.77–3.87 (m, 1H), 6.87–
6.93 (m, 2H), 7.03–7.08 (m, 2H), 8.01 (dd, J = 0.7, 6.7, 1H), 8.49 (dd,
J = 0.7, 6.7, 1H), 9.16 (t, J = 0.7, 1H). 13C NMR (75 MHz, CD3OD):
31.1, 35.2, 40.4, 112.2, 116.4 (d, J = 21.5), 131.7 (d, J = 8.0), 133.6,
134.6, 135.1 (d, J = 3.3), 139.8, 147.8, 163.2 (d, J = 242.8), 166.6. 19
F
4.4.20. (+)-(2S)-3-(2,3-Difluorophenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-5f)
NMR (282 MHz, CD3OD): ꢃ116.8 (m, 1F). MALDI-HRMS (3-HPA):
288.0960 ([MH]+, C15H15FN3S+, calcd 288.0965).
Colorless oil. [a]D25: +47.1 (c = 0.07, CHCl3). IR (neat): 3072,
2734, 1698, 1616, 1538, 1489, 1454, 1445, 1425, 1324, 1270, 1190,
1165, 1122, 1107, 1065, 1030, 1016, 958, 851, 828, 766, 751, 731,
617. 1H NMR (300 MHz, CD3OD): 3.09 (d, J = 7.2, 2H), 3.23–3.28 (m,
1H), 3.39–3.46 (m, 1H), 3.62–3.72 (m, 1H), 6.88–7.00 (m, 2H), 7.07
(dtd, J = 1.9, 8.0, 10.1, 1H), 8.06 (dd, J = 0.7, 6.6, 1H), 8.51 (dd, J = 0.7,
6.6, 1H), 9.19 (t, J = 0.7, 1H). 13C NMR (75 MHz, CD3OD): 28.3, 33.8,
46.7, 112.1, 117.1 (d, J = 17.3), 125.6 (dd, J = 4.8, 6.9), 127.2 (t,
J = 3.3), 128.8 (d, J = 12.3), 133.6, 134.5, 139.9, 147.8, 150.3 (dd,
J = 12.9, 245.7), 151.9 (dd, J = 13.1, 246.8), 166.5. 19F NMR
(282 MHz, CD3OD): ꢃ139.7 (m, 1F), ꢃ144.5 (td, J = 6.8, 20.6, 1F).
MALDI-HRMS (3-HPA): 304.0871 ([MH]+, C15H14F2N3S+, calcd
304.0871).
4.4.16. (+)-(2S)-3-(3-Fluorophenyl)-2-(1H-imidazo[4,5-c]pyridin-2-
yl)-1-propanethiol trifluoroacetate ((+)-5b)
Colorless oil. [a]D25: +52.9 (c = 0.09, CHCl3). IR (neat): 3065,
3030, 2967, 2939, 2870, 2793, 1759, 1702, 1593, 1497, 1474, 1452,
1427, 1400, 1382, 1359, 1298, 1249, 1200, 1125, 1103, 1091, 1054,
1043, 1029, 1013, 983, 945, 882, 825, 772, 737, 711, 692, 621. 1H
NMR (300 MHz, CD3OD): 3.14–3.27 (m, 4H), 3.80–3.90 (m, 1H),
6.81–6.91 (m, 3H), 7.14–7.22 (m, 1H), 8.02 (dd, J = 0.8, 6.6, 1H),
8.49 (dd, J = 0.8, 6.6, 1H), 9.17 (t, J = 0.8, 1H). 13C NMR (75 MHz,
CD3OD): 31.1, 37.1, 40.4, 112.2, 114.8 (d, J = 2.8), 116.6 (d, J = 21.4),
125.8 (d, J = 2.7), 131.5 (d, J = 8.5), 133.6, 134.6, 139.7, 141.9 (d,
J = 7.7), 147.8, 164.3 (d, J = 245.6), 166.5. 19F NMR (282 MHz,
CD3OD): ꢃ113.8 (m, 1F). MALDI-HRMS (3-HPA): 288.0963 ([MH]+,
4.4.21. (+)-(2S)-3-(2,5-Difluorophenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-5g)
C
15H15FN3S+, calcd 288.0965).
Colorless oil. [a]D25: +61.7 (c = 0.11, CHCl3). IR (neat): 3069,
2998, 2965, 2874, 1774, 1702, 1600, 1545, 1509, 1482, 1453,
1406, 1389, 1370, 1318, 1305, 1209, 1157, 1142, 1123, 1101,
1064, 1050, 1019, 999, 971, 864, 827, 787, 749, 733, 696, 637. 1H
NMR (300 MHz, CD3OD): 3.16–3.29 (m, 4H); 3.84–3.94 (m, 1H),
6.83–7.04 (m, 3H), 8.03 (dd, J = 0.8, 6.6, 1H), 8.50 (dd, J = 0.8, 6.6,
1H), 9.17 (t, J = 0.8, 1H). 13C NMR (75 MHz, CD3OD): 31.1, 31.6,
38.0, 112.1, 116.4 (dd, J = 8.5, 24.6), 117.7 (dd, J = 9.4, 26.0),
118.5 (dd, J = 5.2, 18.3), 127.6 (dd, J = 6.1, 16.4), 133.7, 134.6,
139.9, 147.8, 162.0 (dd, J = 2.7, 248.1), 162.7 (dd, J = 2.7, 249.6),
166.0. 19F NMR (282 MHz, CD3OD): ꢃ119.5 (m, 1F), ꢃ124.5 (m,
1F). MALDI-HRMS (3-HPA): 304.0874 ([MH]+, C15H14F2N3S+,
calcd 304.0871).
4.4.17. (+)-(2S)-3-(2-Fluorophenyl)-2-(1H-imidazo[4,5-c]pyridin-2-
yl)-1-propanethiol trifluoroacetate ((+)-5c)
Colorless Oil. [a]D25: +58.3 (c = 0.12, CHCl3). IR (neat): 3048,
2922, 1639, 1620, 1596, 1539, 1500, 1450, 1426, 1327, 1237, 1190,
1153, 1109, 1067, 1045, 1018, 872, 954, 935, 848, 820, 783, 742,
658. 1H NMR (300 MHz, CD3OD): 3.15–3.28 (m, 4H), 3.82–3.92 (m,
1H), 6.93–7.07 (m, 3H), 7.15–7.23 (m, 1H), 8.01 (dd, J = 0.8, 6.6, 1H),
8.49 (dd, J = 0.8, 6.6, 1H), 9.15 (t, J = 0.8, 1H). 13C NMR (75 MHz,
CD3OD): 29.8, 31.1, 38.0, 112.1, 116.4 (d, J = 22.0), 125.5 (d, J = 3.4),
125.8 (d, J = 15.6), 130.2 (d, J = 8.3), 132.4 (d, J = 4.4), 133.6, 134.6,
139.9, 147.8, 162.5 (d, J = 247.3), 166.4. 19F NMR (282 MHz,
CD3OD): ꢃ118.8 (m, 1F). MALDI-HRMS (3-HPA): 288.0961 ([MH]+,
C
15H15FN3S+, calcd 288.0965).
4.4.22. (+)-(2S)-3-(2,6-Difluorophenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-5h)
4.4.18. (+)-(2S)-3-(3,4-Difluorophenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-5d)
Colorless oil. [a]D25: +40.6 (c = 0.08, CHCl3). IR (neat): 3450,
2941, 1682, 1601, 1572, 1500, 1386, 1306, 1169, 1083, 1013, 980,
820, 771, 675, 628. 1H NMR (300 MHz, CD3OD): 3.19–3.33 (m, 4H),
3.83–3.92 (m, 1H), 6.85 (t, J = 8.0, 2H), 7.19–7.29 (m, 1H), 8.03 (dd,
J = 0.8, 6.6, 1H), 8.51 (dd, J = 0.8, 6.6, 1H), 9.15 (t, J = 0.8, 1H). 13C
Colorless oil. [a]D25: +57.8 (c = 0.12, CHCl3). IR (neat): 3484,
3389, 3236, 2933, 2860, 1686, 1661, 1638, 1532, 1487, 1442, 1417,
1390, 1363, 1321, 1257, 1233, 1214, 1165, 1105, 1066, 1044, 1031,
1018, 1006, 954, 910, 851, 833, 753, 671, 628. 1H NMR (300 MHz,
CD3OD): 3.13–3.27 (m, 4H), 3.79–3.89 (m, 1H), 6.80–6.86 (m, 1H),

864
M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
NMR (75 MHz, CD3OD): 27.8, 41.3, 41.8, 112.2 (d, J = 9.5), 112.6,
114.4 (t, J = 20.1), 130.6 (t, J = 10.5), 133.7, 134.7, 139.9, 147.9,
162.8 (dd, J = 8.1, 246.8), 166.0. 19F NMR (282 MHz, CD3OD):
ꢃ115.6 (t, J = 6.9, 2F). MALDI-HRMS (3-HPA): 304.0865 ([MH]+,
MALDI-HRMS (3-HPA): 355.0889 ([MH]+, C17H15F4N2S+, calcd
355.0887).
4.4.27. (+)-(2S)-2-(6,7-Difluoro-1H-benzimidazol-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16c)
Colorless oil. [a]D25: +61.3 (c = 0.12, CHCl3). IR (neat): 3049,
2921, 1620, 1592, 1537, 1445, 1419, 1364, 1325, 1273, 1189, 1164,
1109, 1067, 1018, 986, 972, 939, 903, 849, 805, 767, 739, 619. 1H
NMR (300 MHz, CD3OD): 3.04 (dd, J = 9.0, 14.0, 1H), 3.12 (dd,
J = 5.7, 14.0, 1H), 3.27 (dd, J = 9.4, 13.6, 1H), 3.43 (dd, J = 6.2, 13.6,
1H), 3.67–3.77 (m, 1H), 7.32/7.51 (AA0BB0, J = 8.1, 4H), 7.35–7.46
(m, 2H). 13C NMR (75 MHz, CD3OD): 28.1, 39.9, 47.1, 110.7 (d,
J = 3.5), 115.8 (d, J = 16.7), 125.6 (q, J = 271.2), 125.9, 126.7 (q,
J = 3.7), 130.4 (q, J = 32.2), 130.5, 132.6 (d, J = 4.9), 139.6 (dd,
J = 16.1, 253.4), 143.4, 148.6 (dd, J = 9.4, 240.4), 158.5. 19F NMR
(282 MHz, CD3OD): ꢃ62.9 (s, 3F), ꢃ143.3 (m, 1F), ꢃ154.2 (m, 1F).
MALDI-HRMS (3-HPA): 373.0791 ([MH]+, C17H14F5N2S+, calcd
373.0792).
C
15H14F2N3S+, calcd 306.0871).
4.4.23. (+)-(2S)-3-(3,5-Difluorophenyl)-2-(1H-imidazo[4,5-
c]pyridin-2-yl)-1-propanethiol trifluoroacetate ((+)-5i)
Colorless oil. [a]D25: +52.4 (c = 0.10, CHCl3). IR (neat): 3056,
3002, 2967, 2877, 1773, 1700, 1608, 1515, 1484, 1464, 1451,
1435, 1406, 1389, 1372, 1312, 1280, 1206, 1143, 1116, 1102,
1063, 1049, 1019, 997, 970, 947, 892, 881, 821, 789, 766, 749,
730, 697, 644, 616. 1H NMR (300 MHz, CD3OD): 3.05–3.10 (m,
2H), 3.23 (dd, J = 9.3, 13.6, 1H), 3.10–3.37 (m, 1H), 3.60–3.70 (m,
1H), 6.68–6.79 (m, 3H), 8.06 (dd, J = 0.8, 6.6, 1H), 8.50 (dd, J = 0.8,
6.6, 1H), 9.20 (t, J = 0.8, 1H). 13C NMR (75 MHz, CD3OD): 28.5,
40.0, 47.6, 103.0 (t, J = 25.7), 112.2, 112.9 (dd, J = 7.8, 17.1),
133.6, 134.5, 139.9, 144.0 (t, J = 9.2), 147.8, 164.5 (dd, J = 13.1,
247.5), 166.5. 19F NMR (282 MHz, CD3OD): ꢃ110.6 (m, 2F).
MALDI-HRMS (3-HPA): 304.0867 ([MH]+, C15H14F2N3S+, calcd
304.0871).
4.4.28. (+)-(2S)-2-(5,7-Difluoro-1H-benzimidazol-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16d)
Colorless oil. [a]D25: +57.9 (c = 0.10, CHCl3). IR (neat): 3065,
3030, 2967, 2939, 2870, 2793, 1759, 1702, 1593, 1497, 1474, 1452,
1427, 1400, 1382, 1359, 1298, 1249, 1200, 1125, 1103, 1091, 1054,
1043, 1029, 1013, 983, 945, 882, 825, 772, 737, 711, 692, 621. 1H
NMR (300 MHz, CD3OD): 3.02 (dd, J = 9.0, 14.1, 1H), 3.15 (dd,
J = 5.3, 14.1, 1H), 3.27 (dd, J = 9.3, 13.9, 1H), 3.46 (dd, J = 6.2, 13.9,
1H), 3.76 (ddt, J = 5.3, 6.2, 9.0, 1H), 7.25 (ddd, J = 2.1, 9.9, 10.5, 1H),
7.34/7.56 (AA0BB0, J = 8.0, 4H), 7.33–7.37 (m, 1H). 13C NMR
(75 MHz, CD3OD): 27.9, 39.7, 47.0, 97.9 (dd, J = 4.2, 27.2), 102.5
(m), 122.4, 125.6 (q, J = 267.9), 126.9 (q, J = 3.8), 130.6, 130.6 (q,
J = 33.1), 135.6, 143.1, 153.8 (dd, J = 6.3, 243.8), 157.9, 158.2 (dd,
J = 5.8, 244.2). 19F NMR (282 MHz, CD3OD): ꢃ62.9 (s, 3F), ꢃ116.5
(m, 1F), ꢃ126.7 (m, 1F). MALDI-HRMS (3-HPA): 373.0789 ([MH]+,
4.4.24. (+)-(2S)-2-(1H-Imidazo[4,5-c]pyridin-2-yl)-3-
(pentafluorophenyl)-1-propanethiol trifluoroacetate ((+)-5j)
Colorless oil. [a]D25: +48.7 (c = 0.11, CHCl3). IR (neat): 3049, 2921,
1620, 1592, 1537, 1445, 1419, 1364, 1325, 1273, 1189, 1164, 1109,
1067, 1018, 986, 972, 939, 903, 849, 805, 767, 739, 619. 1H NMR
(300 MHz, CD3OD): 3.26–3.44 (m, 4H), 3.83–3.91 (m, 1H), 8.07 (dd,
J = 0.7, 6.5, 1H), 8.52 (dd, J = 0.7, 6.5, 1H), 9.19 (t, J = 0.7, 1H). 13C NMR
(75 MHz, CD3OD): 27.6, 41.5, 48.2, 112.3, 115.4/117.9 (mm), 119.2
(m), 133.9, 134.7, 137.2/140.5 (mm), 139.9, 145.1/148.3 (mm),
147.9, 165.2. 19F NMR (282 MHz, CD3OD): ꢃ143.8 (dd, J = 8.0, 21.6,
2F), ꢃ157.6 (t, J = 20.0, 1F), ꢃ157.6 (m, 2F). MALDI-HRMS (3-HPA):
358.0587 ([MH]+, C15H11F5N3S+, calcd 360.0588).
C
17H14F5N2S+, calcd 373.0792).
4.4.25. (+)-(2S)-2-(7-Fluoro-1H-benzimidazol-2-yl)-3-[4-
4.4.29. (+)-(2S)-2-(4,7-Difluoro-1H-benzimidazol-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16e)
Colorless oil. [a]D25: +47.1 (c = 0.09, CHCl3). IR (neat): 3056,
3002, 2967, 2877, 1773, 1700, 1608, 1515, 1484, 1464, 1451, 1435,
1406, 1389, 1372, 1312, 1280, 1206, 1143, 1116, 1102, 1063, 1049,
1019, 997, 970, 947, 892, 881, 821, 789, 766, 749, 730, 697, 644,
616. 1H NMR (300 MHz, CD3OD): 3.04 (dd, J = 9.1, 14.0, 1H), 3.11
(dd, J = 6.0, 14.0, 1H), 3.26 (dd, J = 9.3, 13.8, 1H), 3.43 (dd, J = 6.4,
13.8, 1H), 3.62–3.72 (m, 1H), 7.17 (t, J = 6.5, 2H), 7.32/7.55 (AA0BB0,
J = 8.2, 4H). 13C NMR (75 MHz, CD3OD): 28.2, 40.1, 47.4, 111.2 (m),
125.7 (q, J = 272.8), 126.8 (q, J = 3.5), 130.5 (q, J = 32.4), 130.6,
135.6, 143.5 (m), 147.6 (d, J = 250.4), 158.2. 19F NMR (282 MHz,
CD3OD): ꢃ62.8 (s, 3F), ꢃ135.8 (m, 2F). MALDI-HRMS (3-HPA):
373.0786 ([MH]+, C17H14F5N2S+, calcd 373.0792).
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16a)
Colorless oil. [a]D25: +63.8 (c = 0.13, CHCl3). IR (neat): 3484,
3389, 3236, 2933, 2860, 1686, 1661, 1638, 1532, 1487, 1442,
1417, 1390, 1363, 1321, 1257, 1233, 1214, 1165, 1105, 1066,
1044, 1031, 1018, 1006, 954, 910, 851, 833, 753, 671, 628. 1H
NMR (300 MHz, CD3OD): 3.06 (dd, J = 9.3, 14.1, 1H), 3.18 (dd,
J = 5.6, 14.1, 1H), 3.30 (dd, J = 9.3, 13.7, 1H), 3.48 (dd, J = 6.4, 13.7,
1H), 3.85 (tt, J = 5.6, 9.3, 1H), 7.28–7.31 (m, 1H), 7.35/7.53
(AA0BB0, J = 8.3, 4H), 7.48–7.58 (m, 2H). 13C NMR (75 MHz,
CD3OD): 27.8, 39.6, 46.7, 111.2 (d, J = 4.5), 112.6 (d, J = 16.2),
121.9 (d, J = 16.9), 125.4 (q, J = 250.6), 126.8 (q, J = 3.6), 128.4 (d,
J = 6.5), 130.5, 130.6 (q, J = 32.4), 135.3 (d, J = 3.6), 142.9, 150.7
(d, J = 250.9), 156.8. 19F NMR (282 MHz, CD3OD): ꢃ62.9 (s, 3F),
ꢃ129.6 (dd, J = 4.5, 10.6, 1F). MALDI-HRMS (3-HPA): 355.0893
([MH]+, C17H15F4N2S+, calcd 355.0887).
4.4.30. (+)-(2S)-2-(5,6-Difluoro-1H-benzimidazol-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16f)
Colorless oil. [a]D25: +45.7 (c = 0.09, CHCl3). IR (neat): 3072,
2734, 1698, 1616, 1538, 1489, 1454, 1445, 1425, 1324, 1270,
1190, 1165, 1122, 1107, 1065, 1030, 1016, 958, 851, 828, 766,
751, 731, 617. 1H NMR (300 MHz, CD3OD): 3.03 (dd, J = 9.1, 14.1,
1H), 3.17 (dd, J = 5.3, 14.1, 1H), 3.28 (dd, J = 9.3, 13.8, 1H), 3.46
(dd, J = 6.3, 13.8, 1H), 3.79–3.89 (m, 1H), 7.34/7.54 (AA0BB0,
J = 8.1, 4H), 7.73 (t, J = 8.1, 2H). 13C NMR (75 MHz, CD3OD): 27.8,
39.5, 46.6, 103.7 (dd, J = 9.4, 15.5), 125.5 (q, J = 271.2), 126.8 (q,
J = 3.7), 128.3, 130.5, 130.6 (q, J = 32.4), 142.9, 151.3 (dd, J = 16.9,
249.4), 157.5. 19F NMR (282 MHz, CD3OD): ꢃ62.9 (s, 3F), ꢃ139.2
(t, J = 8.1, 2F). MALDI-HRMS (3-HPA): 373.0782 ([MH]+,
4.4.26. (+)-(2S)-2-(6-Fluoro-1H-benzimidazol-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16b)
Colorless oil. [a]D25: +64.7 (c = 0.13, CHCl3). IR (neat): 3450,
2941, 1682, 1601, 1572, 1500, 1386, 1306, 1169, 1083, 1013,
980, 820, 771, 675, 628. 1H NMR (300 MHz, CD3OD): 3.03 (dd,
J = 9.3, 14.1, 1H), 3.19 (dd, J = 5.6, 14.1, 1H), 3.28 (dd, J = 9.5, 13.9,
1H), 3.47 (dd, J = 6.3, 13.9, 1H), 3.79–3.89 (m, 1H), 7.34/7.55
(AA0BB0, J = 8.0, 4H), 7.34–7.41 (m, 1H), 7.51–7.56 (m, 1H), 7.75
(ddd, J = 0.5, 4.3, 9.1, 1H). 13C NMR (75 MHz, CD3OD): 27.7, 39.5,
46.6, 101.7 (d, J = 28.5), 116.3 (d, J = 24.0), 116.6 (d, J = 7.8),
125.5 (q, J = 271.1), 126.9 (q, J = 3.7), 130.5, 130.7 (q, J = 32.6),
132.9 (d, J = 13.5), 142.9, 156.8 (d, J = 1.3), 162.6 (d, J = 244.7),
164.2. 19F NMR (282 MHz, CD3OD): ꢃ63.0 (s, 3F), ꢃ114.1 (m, 1F).
C
17H14F5N2S+, calcd 373.0792).

M. Morgenthaler et al. / Journal of Fluorine Chemistry 129 (2008) 852–865
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865
4.4.31. (+)-(2S)-2-(5,6,7-Trifluoro-1H-benzimidazol-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16g)
Colorless oil. [a]D25: +41.7 (c = 0.09, CHCl3). IR (neat): 3056,
3002, 2967, 2877, 1773, 1700, 1608, 1515, 1484, 1464, 1451, 1435,
1406, 1389, 1372, 1312, 1280, 1206, 1143, 1116, 1102, 1063, 1049,
1019, 997, 970, 947, 892, 881, 821, 789, 766, 749, 730, 697, 644,
616. 1H NMR (300 MHz, CD3OD): 2.97–3.09 (m, 2H), 3.23 (dd,
J = 9.3, 13.6, 1H), 3.37 (dd, J = 6.1, 13.6, 1H), 3.54–3.63 (m, 1H),
7.28/7.49 (AA0BB0, J = 8.1, 4H), 7.32–7.36 (m, 1H). 13C NMR
(75 MHz, CD3OD): 28.4, 40.2, 47.4, 97.9 (dd, J = 4.1, 23.3), 124.1
(m), 125.6 (q, J = 271.1), 126.6 (q, J = 3.7), 130.2 (q, J = 32.3), 130.5,
131.4 (m), 137.2/140.3 (mm), 139.3/142.6 (mm), 143.9, 148.8/
152.1 (mm), 159.2. 19F NMR (282 MHz, CD3OD): ꢃ62.8 (s, 3F),
ꢃ141.7 (dd, J = 9.6, 19.3, 1F), ꢃ152.5 (d, J = 19.3, 1F), ꢃ170.1 (dt,
J = 6.1, 19.3, 1F). MALDI-HRMS (3-HPA): 391.0699 ([MH]+,
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(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16h)
Colorless oil. [a]D25: +49.7 (c = 0.08, CHCl3). IR (neat): 3048,
2922, 1639, 1620, 1596, 1539, 1500, 1450, 1426, 1327, 1237, 1190,
1153, 1109, 1067, 1045, 1018, 872, 954, 935, 848, 820, 783, 742,
658. 1H NMR (300 MHz, CD3OD): 3.03 (d, J = 7.2, 2H), 3.22 (dd,
J = 9.4, 13.5, 1H), 3.36 (dd, J = 6.1, 13.5, 1H), 3.47–3.57 (m, 1H), 7.07
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125.0 (m), 125.7 (q, J = 271.1), 126.5 (q, J = 4.0), 130.1 (q, J = 31.5),
130.6, 135.2/138.3 (mm), 144.2, 145.0/148.3 (mm), 159.6. 19F NMR
(282 MHz, CD3OD): ꢃ62.8 (s, 3F), ꢃ132.6 (dd, J = 9.9, 20.1, 1F),
ꢃ145.6 (dd, J = 11.5, 19.8, 1F), ꢃ160.8 (dt, J = 5.5, 19.8, 1F). MALDI-
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4.4.33. (+)-(2S)-2-(4,5,6,7-Tetrafluoro-1H-benzimidazol-2-yl)-3-[4-
(trifluoromethyl)phenyl]-1-propanethiol trifluoroacetate ((+)-16i)
Colorless oil. [a]D25: +40.7 (c = 0.11, CHCl3). IR (neat): 3069, 2998,
2965, 2874, 1774, 1702, 1600, 1545, 1509, 1482, 1453, 1406, 1389,
1370, 1318, 1305, 1209, 1157, 1142, 1123, 1101, 1064, 1050, 1019,
999, 971, 864, 827, 787, 749, 733, 696, 637. 1H NMR (300 MHz,
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J = 6.1, 13.6, 1H), 3.45–3.54 (m, 1H), 7.26/7.49 (AA0BB0, J = 8.0, 4H).
13C NMR (75 MHz, CD3OD): 28.7, 40.6, 47.8, 124.6 (m), 125.7 (q,
J = 271.1), 126.5 (q, J = 3.5), 130.1 (q, J = 32.4), 130.6, 135.3/138.6
(mm), 136.8/140.1 (mm), 144.3, 160.2. 19F NMR (282 MHz, CD3OD):
ꢃ62.9(s, 3F), ꢃ157.8 (m, 2F), ꢃ167.2(m, 2F). MALDI-HRMS(3-HPA):
409.0608 ([MH]+, C17H11F7N2S+, calcd 409.0604).
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