Exploration of pipecolate sulfonamides as binders of the FK506-binding proteins 51 and 52

Article abstract of DOI:10.1021/jm201747c

FK506-binding proteins (FKBP) 51 and 52 are cochaperones that modulate the signal transduction of steroid hormone receptors. Single nucleotide polymorphisms in the gene encoding FKBP51 have been associated with a variety of psychiatric disorders. Rapamycin and FK506 are two macrocyclic natural products, which tightly bind to most FKBP family members, including FKBP51 and FKBP52. A bioisosteric replacement of the α-ketoamide moiety of rapamycin and FK506 with a sulfonamide was envisaged with the retention of the conserved hydrogen bonds. A focused solid support-based synthesis protocol was developed, which led to ligands with submicromolar affinity for FKBP51 and FKBP52. The molecular binding mode for one sulfonamide analogue was confirmed by X-ray crystallography.

Full text of DOI:10.1021/jm201747c

Article
pubs.acs.org/jmc
Exploration of Pipecolate Sulfonamides as Binders of the
FK506-Binding Proteins 51 and 52
Ranganath Gopalakrishnan,† Christian Kozany,† Yansong Wang,† Sabine Schneider,§
Bastiaan Hoogeland,† Andreas Bracher,‡ and Felix Hausch*,†
†Max Planck Institute of Psychiatry, Kraepelinstrasse 2, 80804 Munich, Germany
‡Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
§Department Chemie, Technical University Munich, Lichtenbergstrasse 4, 85747 Garching, Germany
S
* Supporting Information
ABSTRACT: FK506-binding proteins (FKBP) 51 and 52 are cochaperones that modulate the signal transduction of steroid
hormone receptors. Single nucleotide polymorphisms in the gene encoding FKBP51 have been associated with a variety of
psychiatric disorders. Rapamycin and FK506 are two macrocyclic natural products, which tightly bind to most FKBP family
members, including FKBP51 and FKBP52. A bioisosteric replacement of the α-ketoamide moiety of rapamycin and FK506 with
a sulfonamide was envisaged with the retention of the conserved hydrogen bonds. A focused solid support-based synthesis
protocol was developed, which led to ligands with submicromolar affinity for FKBP51 and FKBP52. The molecular binding
mode for one sulfonamide analogue was confirmed by X-ray crystallography.
hydrogen bonds shown in Figure 1 are required for binding to
FKBPs. The electrophilicity of the α-ketoamide moiety present
in most of the nonimmunosuppressive FK506 analogues is an
undesired reactive liability that could result in metabolic
instability or potential toxicity. For FKBP12, it has been shown
that the α-ketoamide can be bioisosterically replaced by a
sulfonamide moiety to yield compounds that retain binding to
FKBP12.2,13−15
However, these compounds have not been tested for their
binding profile with the larger FKBPs. Until very recently,
compound 2 has been the only synthetic ligand tested for its
binding affinity for FKBP51. In the quest for finding improved
inhibitors of FKBP51 or FKBP52, we envisaged a solid-phase
synthesis methodology for the synthesis of pipecolate sulfonamide
compounds to gain insight into the structure−activity relationship
(SAR) of this series for the larger FKBP isoforms.
INTRODUCTION
■
Members of the FKBP (FK506-binding protein) family display
peptidyl prolyl isomerase (PPIase) activity and bind to the
immunosuppressive natural products FK506 and rapamycin.
The prototypical FKBP12 is the most widely studied member
of this family. In complex with FKBP12, FK506 and rapamycin
also interact with and inhibit calcineurin (CaN) and mTOR,
respectively, thereby mediating their immunosuppressive
action. Prior studies led to analogues devoid of immunosup-
pressive activity,1−3 as exemplified by compound 2 (Figure 1).4
The high molecular weight, multidomain FKBPs FKBP51 and
FKBP52 act as cochaperones for the heat shock protein 90
(Hsp90) and modulate the signal transduction of the
glucocorticoid receptor in a mutually antagonistic direction.5−7
Human genetic studies have shown intronic single nucleotide
polymorphisms in the gene encoding FKBP51 to be associated
with FKBP51 expression and with various stress-related
psychiatric disorders.8 Recent characterization of FKBP51
knockout mice has further validated these findings,9−12
suggesting that inhibition of FKBP51 might be of therapeutic
benefit for psychiatric disorders.32 To further dissect the role of
larger FKBPs and to better understand the underlying biology,
selective inhibitors targeting FKBP51 are required. Neither
FK506 nor rapamycin can be used as tools as they have nearly
equipotent affinities for all FKBPs.
RESULTS AND DISCUSSION
■
Chemistry. Strategy. A three-dimensional alignment of
the FK506-binding domains of FKBP51 (3O5E),16 FKBP52
(to be published), and FKBP12 (2PPN)17 revealed the largest
structural divergences close to the binding pocket at the 80s
loop (Ser118-Ile122 of FKBP51/52). The 80s loop of FKBP51
contains Leu119, which is replaced by Pro119 in FKBP52 possibly
Extensive medicinal chemistry campaigns on analogues of
Received: December 28, 2011
FK506 and rapamycin have shown that the two conserved
© XXXX American Chemical Society
A
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Figure 1. Natural and synthetic ligands that bind to large FKBPs. (a) Structure of FK506 (1). (b) The prototypic synthetic ligand 2, which is devoid
of immunosuppressive activity. Key hydrogen bonds with FKBP51 are indicated by dotted lines; the position of the 80s loop interacting with the
tert-pentyl moiety is indicated in cyan.
a
Scheme 1. Synthesis of the Pipecolate Framework
a
Reagents and conditions: (a) DCC, DMAP, 0 °C to rt, 20 h (for 3a); HATU, DIPEA, rt, 2 h (for 3b). (b) 20% TFA in CH2Cl2, rt, 6 h. (c) DIPEA,
rt, 4−16 h. (d) 20% 4-methyl-piperidine, CH2Cl2, rt, 1 h. (e) DIPEA, CH2Cl2, rt, 16 h. (f) 1% TFA in CH2Cl2, rt, 1 h.
contributing for the structural difference in this region.
Importantly, the residue at position 119 was shown to be a
major functional determinant for the effect on steroid hormone
receptor.18 Hence, an optimization of interactions with this part
of the protein has a higher probability of achieving selectivity
and functional relevance within the FKBP family. The X-ray
structures of FK506 with the FK1 domain of FKBP51 (3O5R)
and FKBP52 (unpublished) confirmed that the pyranose group
in FK506 (1) contacts the 80s loop. Sulfonamide substituents
as replacements of the pyranose group have been shown to
have contact with the 80s loop in FKBP12.2,13,14,19 Compound
2, until very recently the only known synthetic FKBP51 ligand,
was chosen as a starting point for the synthesis of sulfonamide
analogues. For a rapid derivatization of compounds targeting
the 80s loop, we envisaged a solid-phase strategy for synthesis
of a focused sulfonamide library.
Solid-Phase Synthesis of a Focused Sulfonamide Library.
The precursor 3 was synthesized as described.20 This was
further coupled with the pipecolic acid 4 followed by liberation
of the acid to give the building block 5. The latter was anchored
on a 2-chloro trityl resin (Scheme 1).
The immobilized building block was deprotected to give 6 and
was further reacted with a library of commercial sulfonyl chlorides 7.
Cleavage form the solid support under mild acidic conditions yielded
compounds 8−37, 49−79, and 46 and 47. This solid support
protocol was used for the synthesis of a small focused library
followed by primary screening as well as for the resynthesis of hits for
further characterization. The best sulfonamide analogues of this
B
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a
Scheme 2. Synthesis of the Various Top Groups Containing Sulfonamide
a
Reagent and conditions: (a) DIPEA, RSO2Cl, CH2Cl2, rt, 16 h.
series were further attached to a pipecolate core where the free
acid containing “top” group in 8−37 and 49−79 was truncated
(38−39), exchanged by a morpholine (40−45), or replaced by a
pyridine-containing “top” group (Scheme 2).
Biology. The binding of the sulfonamide ligands to
FKBP12, FKBP51, and FKBP52 were determined using a
competitive fluorescence polarization assays.21 The sulfonyl
chloride building blocks were designed to initially probe a
variety of aliphatic and aromatic sulfonyl moieties as well as
substituents around the aromatic rings. Out of 36 compounds
in the medium-throughput screening, 28 compounds inhibited
tracer binding to FKBP12 by more than 15%, while eight
compounds displayed inhibition of more than 85% at 5 μM
(Supporting Information and Table 1). Seven compounds
α-ketoamides in the context of this scaffold but that efficient
binding critically depended on the nature of the sulfonamide
substituent, at least for the larger FKBPs. In general, the
inhibitory activity was much higher for FKBP12 than for the
larger FKBPs. This could be related to the core structure 6,
which was designed and optimized for FKBP12 as well as to the
more concave 80s loop of FKBP12.
The most promising compounds 8−12 from the primary
screening were selected, resynthesized in larger scale, and
characterized in more detail. The binding affinities of all
sulfonamide hits were weaker for all tested FKBPs as compared
to the reference compound 2. This was most pronounced for
FKBP12 for which affinities were reduced by at least 10-fold. It
was less apparent for FKBP51, which was only affected by a
factor of 2 for compound 10. Compounds 9 and 10 turned out
to have the highest binding affinity for FKBP51 and -52 and
were further evaluated in two series (Table 2).
Table 1. Binding Affinities for the Primary Hits towards
FKBP Paralogues
Exploration of the SAR. The first series of derivatives probed
the influence of the substituent at the meta position. The
compounds in this series included m-CN 13, m-NO2 14, m-
NH2 15, m-(2-methylpyrimidin-4-yl) 16, m-(pyrimidin-4-yl)
17, m-F 18, and m-Br 19 aromatic sulfonamides. All of these
compounds had binding affinity between 0.3 and 10 μM for
FKBP12. The meta-substituted halogen derivatives had better
binding affinity to the larger FKBPs, among these 19 being the
best. Compound 15 was synthesized by reduction of the nitro
group in 14. The m-CN 13, m-NO2 14, and m-NH2 15
analogues had slightly reduced binding affinities to the FKBPs
as compared to the m-Cl aromatic sulfonamide 9, while
compounds 16 and 17 were inactive for the larger FKBPs.
We next set out to explore multisubstituted aromatic
sulfonamide groups. The dichloro-substituted aromatic sulfo-
namide 20 had slightly better binding affinity than the mono
meta-chloro-substituted aromatic sulfonamide 9 (Table 2). In
contrast, an additional chloro-substituent in the para-position
21 was found to substantially reduce binding to the FKBPs.
A similar result was found for the meta,para-dimethoxy-
substituted sulfonamide 22, but interestingly, compound 23
having an m-Cl, p-OMe substitution had better binding affinity.
This series of compounds indicated the following SAR m-di-Cl
> m-Cl > m,p-di-Cl ≫ p-Cl ≫ o-Cl for the aryl sulfonamide
substituents.
a
Binding affinities to FKBP12, FKBP51 (FK1 domain), and FKBP52
(FK1 domain) were determined by a fluorescence polarization assay.21
inhibited the tracer binding to the FK1 domain of FKBP52 by
more than 15% at 5 μM, whereas five hits were identified for
the FK1 domain of FKBP51. The initial screening assay
results indicated that sulfonamides can be surrogates of the
To further explore the acceptable nature of the groups at the
meta positions, the derivatives m-difluoro 24, 3,5-bis(trifluoromethyl)
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Table 2. Meta-Substituted Analogues Synthesized for SAR Extrapolation
a
b
Purity of the compounds was confirmed by using HPLC. Binding affinities to FKBP12, FKBP51 (FK1 domain), and FKBP52 (FK1 domain) were
determined by a fluorescence polarization assay.21
D
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Figure 2. X-ray crystal structure of 20 in complex with the FK1 domain of FKBP51. (a) Chemical structure of 20. Hydrophobic contacts with
FKBP51 are indicated in green, hydrogen bonds are shown as dotted lines in pink, aromatic hydrogen bonds are indicated in blue, and the
unresolved groups are in gray. (b) Compound 20 bound to the FK1 domain of FKBP51. The three hydrogen bonds between O1-20 and HN-Ile87,
between O8-20 and HO-Tyr113, and between Cl11 and O-S118 are shown as dotted red lines. The dipolar interaction between the C1-carbonyl and
HO-Tyr113 and the halogen bond between Cl11 and O-S118 are shown in black. Aromatic hydrogen bonds between ring A and Gln85, C10-H and
OH-Tyr113, C14-H and OH-Asp68 are shown in blue. Leu119 and Pro120 at the top of the 80s loop are colored in cyan, and the conserved water below
the 80s loop is shown in yellow.
25, 3-bromo-5-(trifluoromethyl) 26, and 3,5-bis(carboxy-methyl) 27
were synthesized. Compound 24 had reduced affinity as compared
to the monofluoro substituted analogue 18, while the three other
compounds were inactive for FKBP51 or FKBP52. This series led us
to conclude that a halogen is a preferred substituent at the meta-
position for the larger FKBPs (Table 2).
and 20 engage in a number of aromatic CH···O−acceptor
interactions, for example, the oxygen of the sulfonamide and
the ε-hydrogens of Tyr57, Phe67, and Phe130. These interactions
correspond to the contacts formed by the C9-keto group of
FK506 with the same residues of FKBP51, thereby confirming
that the sulfonamide is a bioisosteric mimic of the α-ketoamide
moiety. As expected, the dichloro aryl ring sits below the 80s
The second series of sulfonamide derivatives was designed to
probe the substitution pattern at the fused thiazole ring of
compound 10. A substitution by methyl at C-2 resulted in
compound 34, which had equivalent binding to 10. Conversion
to the corresponding benzothiazol-2(3H)-one 35 resulted in
nanomolar affinity for FKBP12, low micromolar binding for
FKBP51, and high micromolar affinity to FKBP52. The reasons
for this striking selective preference for FKBP12 are currently unknown.
However, the sulfur in the meta-position seems to be extremely
important since substitution by a methylene as in 36 or oxygen
37 resulted in a dramatic loss of affinity for all FKBPs (Table 2).
X-ray Crystal Structure. The X-ray crystal structure of the
FK506-binding domain of FKBP51 complexed with ligand 20
was solved to 1.0 Å resolution. In this complex, FKBP51 adopts
the same folding topology as found in FKBP51 complexed with
1 and 2. As compared to the latter structures, Asp68 moves into
the binding pocket, while Tyr113 and Ser118 move out. The
ligand adopts a similar binding mode as compared to that of 1
or 2 with the common pipecolate ring being nearly super-
imposable (Figure 2b). The pipecolyl ring of each ligand sits
atop the indole of Trp90, which forms the floor of the FKBP
binding pocket. Similar to FK506, the C1-carbonyl of the
pipecolate forms a hydrogen bond with the backbone amide of
Ile87. One oxygen of the sulfonamide engages the ε-hydrogen of
Phe130 and the hydroxyl group of Tyr113. This latter contact is
substantially longer (3.37 Å) as compared to the correspond-
ing hydrogen bonds formed between the Tyr113 and the C8-
carbonyl groups of α-ketoamides like FK506, 2, or analogues
thereof. The p-oxygen of Tyr113 engages in a rather short
dipolar contact with the C1-carbonyl of 20 (3.06 Å). Similar
although less intense dipolar interactions have also been
observed in FKBP51 complexes with FK506 and 2. FKBP51
loop and packs on Ile122. The tip of the 80s loop (Ser118-Lys121
)
diverges from the conformations observed with FK50616 or
synthetic FK506 analogues,20 confirming the conformational
flexibility of this region. The two ortho-hydrogens of the
sulfonylphenyl ring form close contacts (2.95 Å) with the
p-oxygen of Tyr113 and with carboxylate of Asp68, respectively.
One of the aromatic chlorines might form a van der Waals
contact with Lys121, while the other chlorine engages Ser118. For
the latter, two conformations seem possible, one compatible
with a hydrogen bond to the aromatic chlorine, the other with a
linear C−Cl···O geometry consistent with a halogen bond.22,23
The dimethoxyphenyl and acetoxyphenyl rings were poorly
resolved in the electron density map, indicating strong disorder.
In the most populated conformer, the top group is rotated by
120° as compared to compound 2, most likely stabilized by
π−π stacking interactions between the acetoxyphenyl ring B
and the dichloro aryl substituent of the sulfonamide. The
dimethoxyphenyl ring A stacks on the edge of Phe77 and points
into a solvent channel. Its ortho-hydrogen forms an aromatic
hydrogen bond (d = 2.97 Å) to the backbone carbonyl of Gln85.
SAR Extension. The X-ray cocrystal structure of the
sulfonamide 20 revealed a water molecule engaged in a
hydrogen bond with the amide of Lys121 that is situated close to
the para-position of the sulfonamide aromatic ring (Figure 2b).
Water molecules below Pro120 of the 80s loop have been
observed in several FKBP51 crystal structures16 (unpublished
observations). We therefore explored whether this conserved
water molecule could be engaged by substituents in the para-
position of the sulfonamide aromatic ring. Introduction of a p-OH
substituent in compound 28 did not affect the affinity for FKBP51,
while improving the selectivity vs FKBP52 3-fold (Table 2).
E
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Table 3. FKBP Binding Affinity of Sulfonamides with Different Pipecolate Ester Substituents
a
Low solubility impaired binding measurements.
A similar trend was observed for the p-NHAc-substituted
analogue 30 but not in the p-OMe-substituted analogue 29.
The trisubstituted analogue 33 had similar affinities as
compared to the corresponding monosubstituted derivative
15, whereas for 32, the affinities were slightly increased as
compared to 14. The trisubstituted analogue 31 was inactive,
similar to the disubstituted analogue 22.
(para-hydrogen or para-methoxy instead of para-hydroxy) and
45 (para-NH-acetyl). A similar unexpected activity was observed
for the morpholine-containing benzothiazol-2(3H)-one analogue
44, which was much more active than thiazole-containing
analogue 41 or the carboxyl derivative 35. The affinity of 44
for FKBP12 rivaled those of the natural product FK506 (Table 2).
The molecular underpinnings for the extraordinary activities
of 42 and 44 remain to be established. The physiological
consequences of inhibition of FKBP12 are still matter of
debate. However, a phase I clinical trial with FK1706, a high-
affinity, pan-selective FKBP ligand, did not lead to any obvious
adverse side effects, indicating that blockade of FKBPs would
not be grossly toxic.31
Modification of the Pipecolate Ester Substituents
(Top Group). The charged carboxylic acid attached to ring B
in the above series is likely to reduce the cell permeability of
these compounds. To remove this undesired property, the free
acid moiety was replaced by various groups as shown in
Scheme 2 to yield compounds 38−48. Simplified substituents
at C-1 as in compounds 38 and 39 resulted in complete loss of
activity for the large FKBPs. The next series of compounds
included the substitution of the free acid moiety with a
morpholine group. Surprisingly, in the morpholine series,
phenyl sulfonamides substituted with meta-dichloro 40, with
meta-dichloro, p-OMe 41, and the benzothiazole analogue 42
were inactive for all FKBPs including FKBP12 (Table 3). This
could be attributed in part to a detection limit imposed by the
lower solubility of these compounds. In striking contrast, the
meta-dichloro, para-hydroxy-substituted analogue 42 displayed
submicromolar affinities for all tested FKBPs. This potency and
the almost equal affinity for the large FKBPs versus FKBP12 is
remarkable, especially when compared to the very close
analogues 22 (carboxyl group instead of morpholine), 40, 43
Last but not least, we replaced the pipecolate C1 ester by an
amide (46 and 47), which completely abolished the binding to
larger FKBPs. Compound 47 retained substantial binding to
FKBP12 in line with the preference of this substituent for
FKBP12 observed with 44. The loss of binding affinity of 46
and 47 can be attributed to the additional hydrogen bond
donor that would point in the direction of the aromatic ring
when bound in a homologous binding mode as 20. Finally, in
48, the top group was replaced by a symmetric top group as
present in Biricodar,3 which resulted in equivalent affinity as 28.
CONCLUSION
■
Using a bioisosteric replacement strategy, we converted the
α-ketoamide motif derived from FK506 and rapamycin into a
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to yield a free-flowing resin immobilized with compound 5a. The
loading of the free acid was calculated using Fmoc deprotection and
measurement of the Fmoc absorbance.
Coupling of Free Acid 5b to the Trityl Resin. A loading reaction
was carried out as described previously.
sulfonamide motif with conservation of the hydrogen bond
pattern as confirmed by the cocrystal structure of 20. By using a
solid-phase synthesis protocol, we were able to generate, screen,
and optimize a focused library of these compounds. This led to
the identification of aromatic sulfonamides with soft sub-
stituents in the meta-position as preferred binders of the larger
FKBPs. In combination with the morpholine-modified top
group, this resulted in a 15−20-fold enhancement in affinity for
FKBP51 or FKBP52 as compared to the starting compound 2.
The most advanced compound 42 is the best synthetic ligand
known for the large FKBPs. Compound 44 has exceptionally
high affinity for FKBP12, rivaling those of the natural products
FK506 and rapamycin.
Final Library Synthetic Route. Fmoc-protected immobilized
pipecolate 5a was preweighed (approximately 50 mg, 0.019 mmol)
and transferred to each of 36 wells of a 96-well parallel synthesis
reactor platform obtained from FlexChem peptide synthesis system.
The resin was swollen for 1 h in 1 mL of DCM followed by addition of
2 mL of 20% 4-methyl piperdiene in DCM, and the reactor was stirred
for 1 h for the Fmoc deprotection to give 6a. The wells were washed
with DCM (1 × 3 mL) by vacuum-assisted filtration. The resins were
dried, and the sulfonyl chlorides (0.095 mmol) obtained commercially
from Maybridge were weighed (15−40 mg) and added to the wells as
a solution in DIPEA (30 mg, 0.237 mmol) in 0.25 mL of DCM for the
first coupling and 15 mg (0.119 mmol) in 0.25 mL of DCM for the
second coupling with 0.057 mmol (5−12 mg) sulfonyl chloride. The
reaction time was 4 h for first coupling and 20 h for the second
coupling. The wells were subsequently washed with DCM and ethanol
to completely remove excess of unreacted sulfonyl chlorides. The
compounds were finally cleaved in presence of 1 mL of 1% TFA
solution in DCM for 20 min. Each of the solutions were collected by
vacuum filtration and dried by air blowing to give approximately 12 mg
of the crude products. The purity of the above crude products was
analyzed by HPLC using gradient A, and 36 of these compounds were
further purified by preparative HPLC using the gradient B. The
remaining compound 50 was purified using ion exchange column
chromatography to get rid of the traces of unreacted educt. The
purified compounds were characterized using mass spectroscopy and
dried under high vacuum to yield approximately 1−3 mg of the final
desired sulfonamides.
EXPERIMENTAL SECTION
■
Chemistry. All solvents were purchased from Roth, reagents were
bought from Aldrich-Fluka, and the sulfonyl chlorides were obtained
from Maybridge, Sigma Aldrich, ABCR, or AKos, unless otherwise
stated. Chromatographic separations were performed either by manual
flash chromatography or by automated flash chromatography using an
Interchim-Puriflash 430 with a UV detector. Extracts were dried over
MgSO4, and the solvents were removed under reduced pressure.
Merck F-254 commercial plates were used for analytical TLC to follow
the course of reaction and visualized by UV light at either 254 or
365 nm. Silica gel 60 (Merck 70−230 mesh) was used for column
chromatography. NMR spectra of all compounds were obtained from
the Department of Chemistry and Pharmacy, LMU, on a Bruker AC
300, a Bruker XL 400, or a Bruker AMX 600 at room temperature in
deutero-CDCl3 with tetramethylsilane (TMS) as an internal standard,
unless otherwise stated. Mass spectra (m/z) were recorded on a
Thermo Finnigan LCQ DECA XP Plus mass spectrometer at the Max
Planck Institute of Psychiatry, while the high-resolution mass
spectrometry was carried out at the MPI for Biochemistry (Micro-
chemistry Core Facility) on a Varian Mat711 mass spectrometer. The
purity of the compounds was verified by reversed phase HPLC. All of
the final compounds synthesized and tested have a purity of more
than 95%.
Medium Scale Synthesis. Deprotection of Fmoc Resin 6a.
The coupled resin 5a was weighed (210 mg, 0.08 mmol) and added
to syringes and swollen in DCM (4 mL) for 1 h, and the Fmoc
protecting group was removed using 20% 4-methyl piperidine/DCM
(4 mL) for 1 h. After filtration, the resin was washed with DCM
(3 × 5 mL) and used for the next coupling step.
Synthesis of Sulfonamides. To the above resin, i-Pr2EtN (40 mg,
0.317 mmol) in dry DCM (3 mL) was added and stirred for 20 min.
To this solution, the sulfonyl chloride (0.237 mmol) in 500 μL of
DCM was added, and the reaction was stirred for 4 h at room tem-
perature. After the first coupling step, the resins were filtered, washed
with DCM (3 × 10 mL), and then subjected to second coupling with
i-Pr2EtN (30 mg, 0.237 mmol), sulfonyl chloride (0.158 mmol) in
DCM (3 mL), and stirred for 16 h at room temperature. The resins
were washed with DCM (3 × 5 mL) and dried to give the derivatized
resins. These were reswollen in DCM reacted with 1% TFA/DCM
(3 mL) for 1 h and then washed with 1% TFA/DCM (3 × 3 mL)
and further washed with DCM (3 × 5 mL). The combined filtrates
were concentrated in vacuo to yield the compounds 8−37 (crude
weight ∼ 50 mg). The crude compounds were further purified by
preparative HPLC using gradient C. The purified peaks were further
dried by lyophilization.
Synthesis of 2-(3-((R)-1-((S)-1-(3,5-Dichlorophenylsulfonyl)-
piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)-
phenoxy)acetic Acid (20). TLC (hexane:EtOAc:TFA 5.5:4.5:0.2):
Rf = 0.58, yield = 22.6 mg (44%). HPLC (gradient A) retention time =
26.8−27.3 min. 1HNMR (600 MHz, CDCl3) δ = 1.03−1.10 (m, 1H),
1.40−1.47 (m, 1H), 1.60−1.63 (m, 2H), 1.71−1.77 (m, 1H), 1.99−
2.06 (m, 1H), 2.16−2.23 (m, 2H), 2.49−2.59 (m, 2H), 3.20 (dt, 1H,
J = 2.4 Hz, 12.6 Hz), 3.71 (td, 1H, J = 1.2, 12.6 Hz), 3.85 (s, 3H), 3.86
(s, 3H), 4.66 (s, 2H), 4.78 (d, 1H, J = 4.8 Hz), 5.63 (dd, 1H, J = 6
Hz), 6.66−6.68 (m, 2H), 6.79 (d, 1H, J = 7.8 Hz), 6.83−6.84 (m, 1H),
6.85 (t, 1H, J = 2.4 Hz), 6.90 (d, 1H, J = 7.8 Hz), 7.26 (dd, 1H, J = 7.8
Hz), 7.49 (t, 1H, J = 1.8 Hz), 7.66 (d, 1H, J = 1.8 Hz). 13C NMR
(150 MHz, CDCl3) δ = 19.74, 24.61, 27.74, 31.28, 37.84, 42.89,
55.32, 55.92, 64.78, 76.65, 111.33, 111.75, 112.95, 114.17, 120.00,
120.17, 125.45, 129.87, 132.42, 133.21, 135.73, 141.63, 142.89, 147.40,
148.86, 157.53, 169.57, 172.53. MS (ESI) m/z: found Rt 14.33 min.
HPLC analysis was carried out using a Jupiter 4 μm Proteo column
(250 mm × 4.6 mm, 5 μm particle size); wavelength: 224 nm, 280 nm;
flow rate: 1 mL/min; buffer A: 0.1% TFA in 5% MeCN/water; buffer
B: 0.1% TFA in 95% MeCN/water; gradient A: after 1 min of elution
with 100% buffer A, linear gradient of 0−100% buffer B for 30 min.
Method LCMS: YMC Pro C-8 (100 mm × 4.6 mm, 3 μm particle
size) column; wavelength: 224 nm, 280 nm; flow rate: 1 mL/min;
buffer A: 0.1% HCOOH in 5% MeCN/water; buffer B: 0.1% HCOOH
in 95% MeCN/water; gradient B: 1 min 100% buffer A, then linear
gradient of 0−100% buffer B for 11 min.
The final compounds were purified using a preparative HPLC
Jupiter 10 μm Proteo (250 mm × 21.7 mm, 10 μm particle size)
column. Compounds were dissolved in 40% buffer B, and the
purification was carried out with an injection loop volume of 2 mL.
Wavelength: 224 nm; flow rate: 25 mL/min; buffer A: 0.1% TFA in
5% MeOH/water; buffer B: 0.1% TFA in 95% MeOH/water; gradient
C: 40% B, then a linear gradient of 60−70% B for 15 min.
Coupling of Free Acid 5a to the Trityl Resin. 2-Chloro trityl resin
(6.1 g, 7.9 mmol, Novabiochem) was swollen in DCM for 1 h and
added to a mixture of 2-(3-((1R)-1-(1-(((9H-fluoren-9-yl)methoxy)-
carbonyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)-
phenoxy) acetic acid 5a (2.7 g, 3.97 mmol) and i-Pr2EtN (2 g,
15.89 mmol) in 20 mL of dry DCM. The reaction was monitored by
removing aliquots of the reactant mixture (10 μL) over a period of
1−24 h, which were filtered and dried. The analytes were then
redissolved in buffer A subjected to HPLC analysis and checked for
the disappearance of compound 5a. The slurry was agitated for 16 h
and then filtered. The resin was washed with DCM (3 × 50 mL),
DCM:MeOH:i-Pr2EtN 17:2:1 (3 × 50 mL), DCM (3 × 20 mL), DMF
(3 × 25 mL), MeOH (3 × 50 mL), CHCl3 (3 × 50 mL), and diethyl
ether (3 × 50 mL). The resin was dried under high vacuum overnight
G
dx.doi.org/10.1021/jm201747c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Method LCMS, 688.19, 690.19 [M + Na]+. HRMS: 666.1753,
668.1713 [M + H]+; calculated, 666.1753 [M + H]+.
130.46, 133.10, 133.39, 138.88, 142.22, 147.47, 148.93, 156.99, 169.62.
MS (ESI) m/z: found Rt 9.47 min. Method LCMS, 726.29 [M + H]+.
HRMS: 726.3111 [M + H]+; calculated, 726.3091 [M + H]+.
Deprotection of Fmoc (Resin 6b). The coupled resin 5b was
weighed (190 mg, 0.05 mmol) and added to syringes and swollen in
DCM (4 mL) for 1 h, and the Fmoc protecting group was removed using
20% 4-methyl piperidine/DCM (4 mL) for 1 h. After filtration, the resin
was washed with DCM (3 × 5 mL) and used for the next coupling step.
Synthesis of Sulfonamides. To the above resin, i-Pr2EtN (25 mg,
0.20 mmol) in dry DCM (3 mL) was added and stirred for 20 min. To
this solution, the sulfonyl chloride (0.15 mmol) in 500 μL of DCM
was added, and the reaction was stirred for 4 h at room temperature.
After the first coupling step, the resins were filtered, washed with
DCM (3 × 10 mL), and then subjected to second coupling with i-
Pr2EtN (30 mg, 0.237 mmol) and sulfonyl chloride (0.158 mmol) in
DCM (3 mL) and stirred for 16 h at room temperature. The resins
were washed with DCM (3 × 5 mL) and dried to give the derivatized
resins. These were reswollen in DCM reacted with 1% TFA/DCM
(3 mL) for 1 h and then washed with 1% TFA/DCM (3 × 3 mL) and
further washed with DCM (3 × 5 mL). The combined filtrates were
concentrated in vacuo to yield the compounds 46 and 47. (crude
weight ∼ 50 mg). The crude compounds were further purified by
preparative HPLC using gradient C. The purified peaks were further
dried by lyophilization.
X-ray Crystallography. Crystals and cocrystals of the FKBP51
Fk1 domain construct comprising residues 16−140 and containing
mutation A19T were obtained as previously described.16 Diffraction
data were collected at beamline X06DA of the Swiss Light Source
(SLS) synchrotron in Villigen, Switzerland. The data were processed
with MOSFLM24 and XDS,25 SCALA,26 and TRUNCATE.27 The
crystal structure was isomorphous with the apo structure (PDB code
3O5R). The dictionaries for the ligand compounds were generated
with the PRODRG server.28 The structures were refined with
REFMAC.29 Manual model building was performed with COOT.30
Molecular graphic figures were generated using PyMOL (http://www.
pymol.org).
Synthesis of 2-(3-((R)-1-((S)-1-(3,5-Dichloro-4-hydro-
xyphenylsulfonyl)piperidine-2-carbonyloxy)-3-(3,4-
dimethoxyphenyl)propyl)phenoxy)acetic Acid (28). TLC (hexane:E-
tOAc:TFA 4.8:5:0.2): Rf = 0.50, yield = 12.3 mg (23%). HPLC
1
(gradient A) retention time = 26.8−27.1 min. HNMR (300 MHz,
CDCl3) δ = 1.00−1.10 (m, 1H), 1.38−1.53 (m, 1H), 1.60−1.68 (m,
2H), 1.74−1.87 (m, 1H), 2.01−2.12 (m, 1H), 2.16−2.26 (m, 2H),
2.53−2.62 (m, 2H), 3.21 (t, 1H, J = 12 Hz), 3.72 (d, 1H, J = 12 Hz),
3.87 (s, 6H), 4.65 (s, 2H), 4.77 (d, J = 2.7 Hz), 5.63 (t, J = 6 Hz),
6.69−6.71 (m, 2H), 6.80−6.93 (m, 4H), 7.28 (s, 1H), 7.78 (s, 1H),
7.99 (s, 1H). 13C NMR (75 MHz, CDCl3) δ = 19.66, 24.66, 27.85,
31.34, 37.88, 43.01, 55.4, 55.95, 64.82, 77.21, 111.44, 111.83, 113.02,
114.23, 119.84, 120.21, 122.20, 127.86, 128.97, 129.89, 139.94, 141.75,
145.83, 147.22, 148.89, 153.52, 157.56, 169.51, 172.35. MS (ESI) m/z:
found Rt 13.66 min. Method LCMS, 704.68 [M + Na]+. HRMS:
682.1962, 684.1949 [M + H]+; calculated, 682.1902 [M + H]+.
Synthesis of 2-(3-((R)-3-(3,4-Dimethoxyphenyl)-1-((S)-1-(2-oxo-
2,3-dihydrobenzo[d]thiazol-6-ylsulfonyl)piperidine-2-carbonyloxy)-
propyl)phenoxy)acetic Acid (35). TLC (DCM:MeOH 9.7:0.3): Rf =
0.37, yield = 24.20 mg (46.8%). HPLC (gradient A) retention time =
1
22.74−22.94 min. HNMR (600 MHz, CDCl3) δ = 1.38−1.45 (m,
1H), 1.59−1.66 (m, 1H), 1.73−1.75 (m, 2H), 1.89−1.99 (m, 2H),
2.09−2.16 (m, 2H), 2.41−2.46 (m, 1H), 2.52−2.57 (m, 1H), 3.23
(t, 1H, J = 10.2 Hz), 3.78 (d, 1H, J = 7.8 Hz), 3.82 (s, 3H), 3.83 (s, 3H),
4.72−4.82 (m, 3H), 5.41−5.43 (m, 1H), 6.18 (d, 1H, J = 8.4 Hz),
6.58−6.65 (m, 3H), 6.76 (d, 1H, J = 8.4 Hz), 6.92 (d, 1H, J = 6.6 Hz),
7.01 (d, 1H, J = 8.4 Hz), 7.09 (d, 1H, J = 8.4 Hz), 7.41 (t, 1H, J = 7.8
Hz), 7.66 (d, 1H, J = 1.8 Hz). 13C NMR (150 MHz, CDCl3) δ =
19.52, 24.83, 28.47, 31.44, 38.63, 42.16, 54.77, 55.85, 55.92, 64.28,
75.54, 111.34, 111.60, 111.69, 113.56, 118.85, 120.12, 121.10, 124,32,
125.93, 130.27, 133.04, 133.23, 138.48, 142.51, 147.43, 148.89, 157.44,
169.46, 172.87, 173.00. MS (ESI) m/z: found Rt 11.08 min. Method
LCMS, 693.26 [M + Na]+. HRMS: 671.2268 [M + H]+; calculated,
671.2255 [M + H]+.
General Procedure for the Synthesis of Compounds 40−45.
To 80b (19 mg, 0.037 mmol), DIPEA (9.2 mg, 0.055 mmol) and the
corresponding sulfonyl chloride (0.055 mmol) were added. The
reaction was stirred at room temperature overnight and was purified by
preparative HPLC using gradient C to yield compounds 40−45.
Synthesis of (S)-((R)-3-(3,4-Dimethoxyphenyl)-1-(3-(2-
morpholinoethoxy)phenyl)propyl)1-(3,5-dichloro-4-
hydroxyphenylsulfonyl)piperidine-2-carboxylate (42). TLC
(DCM:MeOH 9.7:0.3): Rf = 0.45, yield = 15.96 mg (58%). HPLC
ASSOCIATED CONTENT
* Supporting Information
Purity and activity data for compounds 47−77 from the high-
throughput synthesis and assay. Experimental data of 5a,b, 8−
19, 21−27, 29−34, 36−41, 43, and 45−48. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
1
(gradient A) retention time = 19.28−19.61 min. HNMR (300 MHz,
AUTHOR INFORMATION
Corresponding Author
*Tel: +49(89)30622640. Fax: +49(89)30622610. E-mail:
hausch@mpipsykl.mpg.de.
■
CDCl3) δ = 1.54−2.06 (m, 6H), 2.14−2.30 (m, 2H), 2.44−2.64 (m,
2H), 2.99−3.27 (m, 3H), 3.51 (s, 2H), 3.74−3.80 (m, 3H), 3.86
(s, 3H), 3.87 (s, 3H), 4.06 (s, 4H), 4.43 (s, 2H), 4.72 (d, 1H, J = 4.2 Hz),
5.53−5.59 (m, 1H), 6.65−6.69 (m, 3H), 6.78−6.85 (m, 2H), 6.92 (d,
1H, J = 7.8 Hz), 7.29−7.33 (m, 1H), 7.49 (s, 2H). 13C NMR (75
MHz, CDCl3) δ = 20.15, 24.94, 28.07, 31.45, 37.87, 42.86, 53.05,
55.25, 55.87, 55.95, 56.81, 62.50, 63.96, 76.25, 111.39, 111.71, 112.71,
114.02, 119.57, 120.15, 121.53, 127.30, 130.22, 132.31, 133.18, 141.75,
147.47, 148.95, 151.71, 157.01, 169.52. MS (ESI) m/z: found Rt 8.76
min. Method LCMS, 737.04, 739.13 [M + H]+. HRMS: 737.2532,
739.2506 [M + H]+; calculated, 737.2501, 739.2542 [M + H]+.
Synthesis of (S)-((R)-3-(3,4-Dimethoxyphenyl)-1-(3-(2-
morpholinoethoxy)phenyl)propyl) 1-(2-oxo-2,3-dihydrobenzo[d]-
thiazol-6-ylsulfonyl)piperidine-2-carboxylate (44). TLC
(DCM:MeOH 9.7:0.3): Rf = 0.39, yield = 6.4 mg (24.6%). HPLC
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Gerd Ruhter and the Lead Discovery Center
̈
(Dortmund) for providing the precursor for the synthesis of
morpholine analogues and compound 46. We thank Prof.
Florian Holsboer and the CIPSM for financial support. We are
indebted to Mrs. E. Weyher and Dr. S. Uebel (MPI of
Biochemistry) and to Mrs. C. Dubler (Ludwig-Maximilians-
1
(gradient A) retention time = 18.92−19.16 min. HNMR (300 MHz,
CDCl3) δ = 1.71−1.81 (m, 2H), 1.84−2.01 (m, 2H), 2.06−2.22 (m,
2H), 2.39−2.62 (m, 2H), 3.01−3.12 (m, 2H), 3.26−3.37 (m, 2H),
3.54 (s, 2H), 3.80−3.91 (m, 12H), 4.06 (s, 4H), 4.26−4.50 (m, 2H),
4.74 (d, 1H, J = 4.8 Hz), 5.36−5.41 (m, 1H), 6.55−6.64 (m, 4H), 6.77
(d, 1H, J = 7.5 Hz), 6.89 (d, 1H, J = 6.9 Hz), 6.99 (d, 1H, J = 7.5 Hz),
7.19 (d, 1H, J = 8.4 Hz), 7.38 (t, 1H, J = 7.6 Hz), 7.62 (d, 1H, J = 1.8
Hz). 13C NMR (75 MHz, CDCl3) δ = 24.98, 28.29, 31.43, 38.45,
42.48, 53.47, 54.79, 55.87, 55.94, 57.28, 62.02, 63.93, 75.83, 111.03,
111.37, 111.69, 112.73, 113.79, 119.18, 120.17, 121.54, 124.28, 125.73,
Universitat Munich) for HRMS and NMR measurements. This
̈
research project has been supported by the European
Commission under the seventh Framework Program: Research
Infrastructures. Grant Agreement Number 226716.
ABBREVIATIONS USED
FKBP, FK506-binding protein; Hsp90, heat shock protein 90;
SAR, structure−activity relationship
■
H
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Journal of Medicinal Chemistry
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