Evaluation of synthetic FK506 analogues as ligands for the FK506-binding proteins 51 and 52

Article abstract of DOI:10.1021/jm201746x

The FK506-binding proteins (FKBP) 51 and 52 are cochaperones that modulate the signal transduction of steroid hormone receptors. Both proteins have been implicated in prostate cancer. Furthermore, 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 that bind to these proteins indiscriminately but with nanomolar affinity. We here report the cocrystal structure of FKBP51 with a simplified α-ketoamide analogue derived from FK506 and the first structure-activity relationship analysis for FKBP51 and FKBP52 based on this compound. In particular, the tert-pentyl group of this ligand was systematically replaced by a cyclohexyl ring system, which more closely resembles the pyranose ring in the high-affinity ligands rapamycin and FK506. The interaction with FKBPs was found to be surprisingly tolerant to the stereochemistry of the attached cyclohexyl substituents. The molecular basis for this tolerance was elucidated by X-ray cocrystallography.

Full text of DOI:10.1021/jm201746x

Article
pubs.acs.org/jmc
Evaluation of Synthetic FK506 Analogues as Ligands for the FK506-
Binding Proteins 51 and 52
Ranganath Gopalakrishnan,† Christian Kozany,† Steffen Gaali,† Christoph Kress,† 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
S
* Supporting Information
ABSTRACT: The FK506-binding proteins (FKBP) 51 and
52 are cochaperones that modulate the signal transduction of
steroid hormone receptors. Both proteins have been
implicated in prostate cancer. Furthermore, 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 that bind to
these proteins indiscriminately but with nanomolar affinity. We
here report the cocrystal structure of FKBP51 with a simplified
α-ketoamide analogue derived from FK506 and the first
structure−activity relationship analysis for FKBP51 and
FKBP52 based on this compound. In particular, the tert-pentyl group of this ligand was systematically replaced by a cyclohexyl
ring system, which more closely resembles the pyranose ring in the high-affinity ligands rapamycin and FK506. The interaction
with FKBPs was found to be surprisingly tolerant to the stereochemistry of the attached cyclohexyl substituents. The molecular
basis for this tolerance was elucidated by X-ray cocrystallography.
coping behavior.9−12 These findings have rendered FKBP51 as
INTRODUCTION
Immunosuppressant natural products like FK506 (Figure 1a)
■
a novel target for treatment of psychiatric disorders.49 However,
neither FK506 nor rapamycin can be used as a tool to
investigate the roles of individual FKBPs in mammalian systems
due to strong off-target effects and lack of selectivity. Thus,
nonimmunosuppressive and selective inhibitors for the large
FKBP homologues FKBP51 and FKBP52 are required.
and rapamycin bind with high affinity to immunophilins of the
FKBP (FK506 binding protein) family, which often also
possess peptidyl-propyl isomerase (PPIase) activity. The best-
characterized member of the FKBP family is FKBP12, a 12 kDa
protein, which consists only of the FK506-binding domain.
FKBP12−FK506 and FKBP12−rapamycin complexes create
binding surfaces for binding to calcineurin (CaN) and mTOR,
respectively.1 The inhibition of the latter proteins mediates the
immunosuppressive action of the two natural products.
FKBP12 has also been shown to modulate the ryanodine
receptor (RyR) channels and to bind to the transforming
growth factor β receptor I. FK506 inhibits these interactions
consistent with a shared common binding site.2
The higher molecular weight FKBP homologues FKBP51
and FKBP52 act as cochaperones for the heat shock protein 90
(Hsp90). In the Hsp90 heterocomplex, FKBP51 and FKBP52
have been shown to modulate signal transduction by the
glucocorticoid receptor in a mutually antagonistic direction.3−5
FK506 was shown to inhibit the proliferation of prostate cancer
cells. This was attributed to blockade of the enhancing effect of
FKBP51 on the androgen receptor in these cells.6,7 Numerous
human genetic studies have shown that single nucleotide
polymorphisms in the gene encoding FKBP51 are associated
with a variety of psychiatric disorders.8 Very recently, several
independent studies using knockdown and knockout mice
strongly supported an important role of FKBP51 in stress-
At the end of the last millennium, various subclasses of high-
affinity FKBP12 ligands were described, which were devoid of
the immunosuppressive activity present in FK506 and
rapamycin.13,14 α-Ketoamide derivatives without the effector
region were the most widely studied series exemplified by
compound 2a15 (Figure 1). For FKBP12, the tert-pentyl group
in 2a was found to be a good surrogate for the pyranose group
in FK506 and rapamycin.16 While the high affinity of the
natural products FK506 and rapamycin was retained for the
larger FKBPs, the binding affinity of 2a for the larger FKBPs
was substantially weaker.17 We thus first set out for a basic
characterization of the structure−activity relationship of 2a. To
analyze the interactions with the 80s loop in more detail, we
then substituted the tert-pentyl group in 2a with cyclohexyl
analogues, which more closely mimic the pyranose group in the
high-affinity natural product ligands (Figure 1c).
Received: December 28, 2011
Published: March 29, 2012
© 2012 American Chemical Society
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Figure 1. Natural and synthetic FKBP ligands: (a) Structure of FK506 (1), (b) prototypic synthetic ligand of FKBPs 2a, which is devoid of
immunosuppressive activity (hydrophobic contacts with FKBP51 are indicated in green, and hydrogen bonds are represented as pink dotted lines),
(c) prototypic cyclohexyl-substituted ligand 3, and (d and e) binding mode of 2a with FKBP51. (d) Surface representation of FKBP51 in complex
with 2a (green). FK506 bound to FKBP51 (3O5R) is superimposed in yellow. (e) Ribbon representation of FKBP51 showing the conserved H-
bonds between O1-2a and HN-Ile87 (dark blue) and between O8-2a and HO-Tyr113 (red) as black dotted lines. Leu119 and Pro120 at the top of the
80s loop are colored in cyan. The dipolar interaction between OH-Tyr113 and C1-carbonyl is shown as a dotted line in magenta.
no longer conserved because of the absence of the
corresponding hydroxyl group in compound 2a. The tert-
pentyl group of compound 2a sits in pocket formed by the 80s
loop (Ser118-Ile122), which is occupied by the pyranose group of
FK506 in the FK506−FKBP51 complex. As compared to a
similar compound (SB3) in a complex with FKBP12 (1FKG16),
the ethyl of the tert-pentyl group is rotated by 180° and faces
the 80s loop. The dimethoxyaryl group (ring A) of 2a sits in a
cradle formed by residues Gly84-Ile87 and Tyr113 and engages in
van der Waals contacts with Glu20 from a neighboring FKBP51
molecule in the crystal. The acetyloxyaryl group (ring B) stacks
on top of the edge of Phe77, and its carboxyl moiety forms
electrostatically enhanced hydrogen bonds with Lys108 and
Arg31 from a neighboring molecule.
RESULT AND DISCUSSION
■
Crystal Structure of the 2a−FKBP51 Complex. As a
structural starting point for a rational design the cocrystal
structure of 2a, the only synthetic ligand known for FKBP51,
was solved in complex with the FK506-binding domain of
FKBP51 at 1.5 Å resolution (Figure 1d,e). Upon binding of
compound 2a, FKBP51 adopts a very similar conformation as
found in the FK506 complex18 (Figure 1d). Most active site
residues are virtually superimposable in the two cocrystal
structures. As compared to the FK506 complex (3O5R), Phe77
moves into the binding pocket, while Asp68 and the tip of the
80s loop (Leu119-Lys122) move outward in the FKBP51−2a
complex, the latter in part due to crystal contacts with a
neighboring FKBP51 molecule.
Structure−Activity Relationship (SAR) of the Pipeco-
late Core and Ester Substituent. So far, virtually nothing is
known about the interaction of the large FKBPs with small
molecule ligands. To the best of our knowledge, only one and
three synthetic ligands have been described for FKBP51 and
FKBP52, respectively.17,20,21
As a first characterization of the recognition properties of
FKBP51 and FKBP52, we engaged on a basic SAR analysis of
the prototypic ligand 2a. The analogues of 2a (Table 1) were
synthesized by esterification or by alkylation of the C1
carboxylate of the building blocks 4a−d as outlined in Scheme
The core interactions of FK506 are conserved for 2a with the
common pipecolate ring sitting atop the indole of Trp90, which
forms the floor of the FKBP binding pocket. The C1-carbonyl
of the pipecolate forms a hydrogen bond with the backbone
amide of Ile87 (d = 2.92 Å), while the C8-carbonyl of the α-
ketoamide engages in a hydrogen bond with the hydroxyl group
of Tyr113 (d = 2.65 Å). The latter approaches the C1-carbonyl at
an angle of 107° and below van der Waals distance (3.17 Å),
consistent with an attractive dipolar interaction.19 The C9-keto
oxygen of 2a occupies a position similar to the keto group of
FK506, while the hydrogen bond with Asp68 seen in 3O5R is
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block 5a was obtained in 98% enantiomeric excess and 94%
yield by a Noyori-catalyzed enantioselective reduction of the
known keto precursor 13a (Scheme S1 in the Supporting
Information).15 Building blocks 5b (Scheme S1 in the
Supporting Information) and 5c were synthesized as
described.23
Table 1. FKBP Affinities of Synthetic FK506 Analogues
In an initial SAR analysis, we explored the contributions of
individual substructures in 2a by first focusing on the pipecolate
core. Replacement by a proline (2b) or a 4,5-dehydropipeco-
linic acid (2c) decreased the affinity for FKBPs 4−6-fold, while
thiomorpholine-3-carboxylic acid (2d) abrogated detectable
binding to the large FKBPs. Because even small changes at the
core diminished affinity, we kept the pipecolate core constant in
all further derivatives. We then replaced the pipecolate C1 ester
by an amide (2e), which completely abolished binding to larger
FKBPs. This was anticipated since the additional hydrogen
bond donor would point to the hydrophobic tert-pentyl group
of 2e when bound in a homologous binding mode as 2a.
We next explored the requirements of the ester “top” group.
Smaller substituents like in 6a−d resulted in analogues with 7−
100-fold lower affinity for FKBP12 and no activity for larger
FKBPs as compared to 2a. Compound 6b (also called GPI-
104624) has been reported as one of the most potent and
advanced inhibitors for FKBP12. Similar to the corresponding
pipecolate analogue 6c, GPI-1046 (6b) had no binding to
larger FKBPs and micromolar affinity to FKBP12 in the
fluorescence polarization assay, which is consistent with the
discrepancies previously observed for GPI-1046 by others.1 To
eliminate the negative charge in 2a, we exchanged the free acid
moiety by a morpholine group (6e), which increased affinity
2−4-fold and induced a slight preference for FKBP52 vs
FKBP51. A similar trend was also observed in a related
sulfonamide pipecolate series.48 In contrast to the carboxylic
acid analogue 2b, the morpholine-containing proline derivative
6f retained detectable but 3-fold reduced binding. Replacement
of the oxyacetyl group in 6g by an amine resulted in a
compound having similar affinity.
Finally, we replaced the tert-pentyl group with 3,4,5-
trimethoxyphenyl in 6h (Scheme S6 in the Supporting
Information), which led to a 2-fold decrease in affinity for
FKBP51, while having equivalent binding for FKBP12 and
FKBP52. Additionally, two FK506 analogues that had been
evaluated in the clinic were tested for their binding to the larger
FKBPs.1 Biricodar (VX-710, 6i)39 potently bound to FKBP12,
while displaying moderate affinity for the larger FKBPs. In
contrast, the related Timcodar (VX-853, 6j), which lacks the
pipecolate core, had no binding affinity for any FKBPs,
consistent with the SAR data observed above.
Exploration of Pyranose/tert-Pentyl Analogues. A
three-dimensional alignment of FKBP12- and the FK506-
binding domains of FKBP51 and FKBP52 revealed that the
core residues of the binding pockets are highly conserved. The
largest differences were found in the adjacent 40s and 80s loops
(residues 71−76 and 118−122 for FKBP51, respectively). The
80s loop of FKBP51 further contains Leu119, which is replaced
by Pro119 in FKBP52. Cellular studies have shown the residue
at position 119 to be a major functional determinant for the
effect on steroid hormone receptors.25 Optimization of
interactions with this part of the protein thus could impart
selectivity and functional efficacy toward steroid hormone
receptor for the large FKBPs. We therefore decided to
investigate the interaction with this part of the protein in
more detail.
a
Binding affinities to FKBP12, FKBP51 (FK1 domain), and FKBP52
(FK1 domain) were determined by a fluorescence polarization assay.17
1 or Scheme S4 in the Supporting Information. The latter were
prepared from the corresponding pipecolate analogues by N-
oxalylation, introduction of the tert-pentyl moiety followed by
deprotection of the C1 carboxylate (Scheme S3 in the
Supporting Information).16 The 4,5-dehydropipecolate building
block 4c was synthesized from allyl glycine in four steps
(Schemes S2 and S3 in the Supporting Information).22 Building
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a
Scheme 1. General Synthesis Protocol of Compounds 2a−d and 6a−h
a
Reagent and conditions: (a) DCC, DMAP, rt, 12h. (b) (i) DCC, DMAP, rt, 12h. (ii) 20% TFA in DCM, rt, 6 h, (c) DIPEA, toluene, reflux, 40 h.
a
Scheme 2. General Synthesis Protocol of Diketo Acids 11a−e
a
Reagent and conditions: (a) L-Threonine, MgSO4, HCHO, THF, 5 days. (b) MOMCl, DIPEA, DCM, 12 h. (c) TMS acetylene, n-BuLi, −78 °C, 2
h. (d) N-Bromosuccinimide, AgNO3, acetone, 2 h. (e) KMnO4, pH 7 (MgSO4, NaHCO3), MeOH:H2O: 1:1, 0 °C to rt, 1 h. (f) 1 M LiOH, MeOH,
6 h.
The X-ray structure of FK506 with FKBP12 (1FKJ),26 with
the FK1 domains of FKBP51 (PDB code 3O5R)18 and
FKBP52 (manuscript in preparation) revealed that the
pyranose group in FK506 (1) contacts the 80s loop. SAR
studies around the pyranose group have shown that the methyl
group at C11 of FK506 analogues is important, while the
pyranose ring oxygen is dispensable for binding to
FKBP12.27−29 This is consistent with the FK506-FKBP51
cocrystal structure where the C11-methyl fills a small hydro-
phobic cavity, while the pyranose ring oxygen of FK506 does
not seem to act as a hydrogen bond acceptor.18 The pyranose
of FK506 further contains an exocyclic hydroxyl group at C10
that engages in a hydrogen bond with Asp68 of FKBP51. This
could contribute to the higher affinity observed for the natural
product. The 2a−FKBP51 cocrystal structure shows that the
tert-pentyl group in 2a occupies the same subpocket below the
80s loop as the pyranose ring in FK506. We therefore decided
to replace the tert-pentyl group in 2a with cyclohexyl derivatives
that more closely resembled the pyranose in the high-affinity
ligand FK506 (1). The first series of compounds investigated
had a methyl substituent (3a) at C11 as in FK506. The FK506−
FKBP51 crystal structure (3O5R) further revealed that the 80s
subpocket in the large FKBPs is more open and has a potential
hydrogen bond interaction partner (S118) in its vicinity. We
therefore also prepared cyclohexyl analogues with larger or
hydrophilic C11 substituents.
A four-step synthesis scheme for the α-keto acids 11a,b and
11d,e was set up starting from the corresponding racemic
cyclohexanones 8a or 8b (Scheme 2). Alternatively, for 11c, the
enantiopure MOM-protected 2-hydroxymethyl cyclohexanone
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a
Scheme 3. General Synthesis Protocol of Compounds 3a−j
a
Reagent and conditions: (a) (S)-1-Boc-piperidine-2-carboxylic acid, K2CO3, KI, 60 °C, 12 h. (b) 20% TFA in DCM, rt, 2 h. (c) (S)-1-Fmoc-
piperidine-2-carboxylic acid, DCC, DMAP, rt, 12 h. (d) 20% 4-methylpiperidine in DCM, rt, 4h. (e) Compounds 11a−e, HATU, DIPEA, rt, 16 h.
(f) (i) Compounds 11a−e, HATU, DIPEA, rt, 16 h; (ii) 20% TFA in DCM, rt, 6 h.
8c was used. The latter was obtained in two steps from
cyclohexanone by an organocatalyzed formylation.30,31 TMS
acetylene was reacted with 8a−c to obtain the cis and trans
diastereomers 9a−f32 (stereochemistry assigned by NMR33) in
nearly equal amounts, which could be separated using column
chromatography. N-Bromosuccinimide was used to cleave the
TMS group and introduce the bromide at the terminal alkynes
(10a−e)34 followed by oxidation of the activated alkynes by
KMnO4 to yield the corresponding α-keto esters.35−38 These
were further hydrolyzed to give the α-keto acids 11a,b and
11d,e, as racemic mixtures, and enantiopure 11c.
time. To further investigate the influence of stereochemistry
and the substitution pattern at the cyclohexyl ring in more
detail, we separated the individual diastereomers 3a-1, 3a-2, 3i-
1, 3i-2, 3f-1, and 3f-2.
Again, these diastereomers had almost equivalent binding to
the proteins. These observations led us to conclude that in
linear FK506 analogues the stereochemistry around the
pyranose group per se is not as important for activity as
previously thought and that the 80s loop is flexible enough to
accommodate the small stereo chemical changes in the active
site.
The α-keto acids (11a−e) were coupled with the pipecolic
acid building blocks 12a−d as outlined in Scheme 3 to give
compounds 3a−g and 3i,j as mixture of diastereomers and 3h
as a single pure diastereomer. The affinities for FKBPs were
either tested as mixture of diastereomers (3a−g and 3i,j) or
after diastereomeric separation using preparative HPLC (Table
2).
Introduction of the FK506-like cyclohexyl moiety in 3a
increased affinity for FKBPs 2-fold as compared to 2a,
indicating that the cyclohexyl moiety might indeed better
interact with the 80s loop than the tert-pentyl group. We next
explored the influence of the ester “top” group in the context of
the cyclohexyl substituent. Removing the acetyloxyaryl ring
(ring B) as in 3c reduced the affinity for FKBPs by 6-fold. This
is in contrast to the results observed for the C11-ethyl analogue
3d and the corresponding tert-pentyl containing substance 6d.
Further shortening of the linker connecting the dimethoxyaryl
moiety (ring A) as in 3b substantially decreased affinity for all
FKBPs. This indicates that the linker length is critical for
optimal positioning of the dimethoxyaryl moiety, at least in the
cyclohexyl series. Similar to the tert-pentyl series, replacement
of the carboxylate by a morpholine in compounds 3e and 3g
increased affinity for FKBPs and induced a slight preference for
FKBP52 as compared to FKBP51.
Cocrystal Structures of 3f-1 and 3f-2. To understand the
unexpected binding of the noncanonical diastereomers, we
solved the cocrystal structure of both 3f-1 and 3f-2 with the
FK506-binding domain of FKBP51 (Figure 2). Depending on
whether the complexes were crystallized or the compounds
were added to preformed crystals, different crystal forms were
obtained. In both cocrystal lattices, the ligands engaged Glu20,
Arg31, and Lys108 of a neighboring FKBP51 molecule, similar to
the crystal contacts observed for 2a (see above).
Upon binding of compound 3f-1 or 3f-2, FKBP51 adopts the
same structure as found in FKBP51 complexed with 1 and 2a.
Likewise, the binding modes for the pipecolate, the ester “top”
group, and the α-keto amide of 3f-1 or 3f-2 were almost
perfectly superimposable to those found for 2a in complex with
FKBP51. In particular, the hydrogen bond network and the
dipolar interaction comprising Ile87-NH, C1O, Tyr113-OH,
and C8O are conserved. In 3f-1, a hydrogen bond of C10-OH
with Asp68 (d = 2.75 Å) is formed similar to the one observed
for the pyranose group of FK506 (PDB code 3O5R). However,
the cyclohexyl group in 3f-1 is slightly lifted out of the binding
pocket and slightly rotated likely to relieve a steric clash of the
larger C11 substituent. For the C11 substituent, two orientations
seem to be possible, which occupy similar positions like the
ethyl group of the tert-pentyl moiety in 2a (Figure 2a). In the
case of 3f-2, the cyclohexyl moiety is rotated by 180°, which
allows the C11 ethyl substituent to occupy almost an identical
position as for 3f-1, indicating that this hydrophobic interaction
might be rather important (Figure 2b). In this conformation,
the hydrogen bond with Asp68 is no longer possible, but the
C10-OH now forms water-mediated hydrogen bonds to Tyr113
and Ser118. This water network might provide the binding
energy to compensate for the loss of the C10-OH···Asp68 H-
bond.
We next investigated the role of the C11 substituent on the
cyclohexyl moiety. The C11-methyl (3a*), C11-ethyl (3f*), and
C11-hydroxylmethyl derivative (3h) had similar binding for the
larger FKBPs, while the affinity for FKBP12 was reduced.
Importantly, however, we also found that the diastereomeric
mixtures 3i* and 3j* had almost equivalent binding to FKBPs
as their FK506-like counterparts 3a* and 3f*. This was
somewhat surprising since in the “unnatural” diastereomers 3i*
and 3j*, the Asp68-HO10 hydrogen bond and hydrophobic 80s
loop contacts of the C11 substituent are not possible at the same
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Table 2. FKBP affinities of Pyranose Analogs
Figure 2. X-ray crystal structure of 3f-1 and 3f-2 in the FK506-binding
domain of FKBP51. The hydrogen bonds between O1 and HN-Ile87
(shadowed blue) and between O8 and HO-Tyr113 (shadowed red) are
represented as dotted black lines. The dipolar interaction between
OH-Tyr113 and C1 carbonyl is indicated by a dotted pink line. Leu119
and Pro120 of the 80s loop are indicated in cyan. (a) Binding mode of
3f-1 in the active site of FKBP51. The additional hydrogen bond
between HO10-3f-1 and O-Asp68 (shadowed magenta) is shown as
dotted black line. (b) Binding mode of 3f-2 in the active site of
FKBP51. The hydrogen bond network formed by a water molecule
(green) with Tyr113 and Ser118 (yellow) of FKBP51 and with C10-OH
of 3f-2 complex is indicated by a dotted black line.
CONCLUSION
■
This study for the first time describes a detailed SAR of ligands
for the larger FKBPs 51 and 52. Although SAR of α-ketoamides
for FKBP12 has been extensively documented, this is the first
instance where a direct comparison of binding trends between
FKBP12 and larger FKBPs have been studied. The X-ray
cocrystal structure of 2a was obtained as the starting point,
followed by a systematic exploration of the contributions of
each substituent on affinity to FKBP51 or FKBP52. Larger top
groups as in 2a and 6e were found to have better binding
affinity, while the pipecolic core (2a) was found to be essential.
The tert-pentyl group in 2a was further substituted by a
cyclohexyl group, which mimicked the pyranose in FK506 and
rapamycin. From the binding studies and X-ray cocrystal
structure of the diasteromers (3f-1 and 3f-2), we can conclude
that the FKBPs are tolerant toward change of the stereo-
chemistry around the cyclohexyl (pyranose) substituents, at
least in a linear, unconstrained context. These cocrystal
structures also suggest that multiple molecular binding modes
are possible for the 80s loop interaction, which is in line with
the high flexibility of this region.
EXPERIMENTAL SECTION
■
Chemistry. Chromatographic separations were performed either
by manual flash chromatography or by automated flash chromatog-
raphy using an Interchim Puriflash 430 with an UV detector. Organic
phases were dried over MgSO4, and the solvents were removed under
reduced pressure. Merck F-254 (thickness 0.25 mm) commercial
plates were used for analytical TLC to follow the progress of reactions.
Silica gel 60 (Merck 70−230 mesh) was used for manual column
chromatography. Unless otherwise specified, H NMR spectra, 13C
1
NMR spectra, 2D HSQC, HMBC, and COSY of all intermediates
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. Chemical shifts for 1H or 13C are given in ppm (δ)
relative to tetramethylsilane (TMS) as an internal standard. 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 MPI
for Biochemistry (Microchemistry Core facility) on Varian Mat711
mass spectrometer. The purity of the compounds was verified by
reversed phase HPLC. All of the final compounds synthesized and
a
Binding affinities to FKBP12, FKBP51 (FK1 domain), and FKBP52
(FK1 domain) were determined by a fluorescence polarization assay.17
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tested have a purity of more than 95%. The optical rotation for the
compounds was obtained from the Department of Chemistry and
Pharmacy, LMU, on a Perkin-Elmer 241 polarimeter.
HPLC Conditions for Product Analysis. Column: Jupiter 4 μm
Proteo 90 A, 250 mm × 4.6 mm, Phenomenex, Torrance, CA;
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 elution with 100% buffer A, linear gradient of
0−100% buffer B for 30 min.
General Procedure for the Synthesis of 2-Alkyl-1-(bromoethynyl)-
cyclohexanol (10). To a solution of 9a−e (1.3 mmol), N-
bromosuccinimide (1.5 mmol) and AgNO3 (0.5 mmol) in acetone
(10 mL) were added, and the resulting solution was stirred in darkness
for 2 h at room temperature. Acetone was evaporated under reduced
pressure, and the solids were removed by filtration through a Celite
pad (washing with ether). The combined organic phase were
concentrated and subjected to purification by column chromatography
using hexane:EtOAc 9:1 to yield 10a−e as yellow liquids.
(1S,2R)-1-(Bromoethynyl)-2-methylcyclohexanol and (1R,2S)-1-
(Bromoethynyl)-2-methylcyclohexanol (10a). Compound 10a (256
mg, 91%) was obtained from 9a (273 mg) as a yellow liquid. TLC
(hexane:EtOAc 9:1): Rf = 0.30. 1H NMR (300 MHz, CDCl3) δ = 1.06
(d, 3H, J = 6.9 Hz), 1.29−1.73 (m, 8H), 1.97−2.04 (m, 1H). 13C
NMR (75 MHz, CDCl3) δ = 16.05, 20.94, 24.95, 29.09, 39.17, 40.52,
43.04, 70.79, 84.60. HRMS: m/z 199.0193, 201.0169 [M − OH]+;
calculated, 199.0122, 201.0102 [M − OH]+.
(1S,2R)-1-(Bromoethynyl)-2-ethylcyclohexanol and (1R,2S)-1-
(Bromoethynyl)-2-ethylcyclohexanol (10b). Compound 10b (264
mg, 88%) was obtained from 9b (292 mg) as a yellow liquid. TLC
(hexane:EtOAc 9:1): Rf = 0.36. 1H NMR (300 MHz, CDCl3) δ = 0.96
(d, 3H, J = 7.2 Hz), 1.13−1.30 (m, 3H), 1.36−1.45 (m, 1H), 1.50−
1.74 (m, 5H), 1.83−2.02 (m, 2H). 13C NMR (75 MHz, CDCl3) δ =
12.17, 21.10, 23.03, 24.81, 25.35, 39.55, 43.17, 47.78, 71.39, 84.74.
HRMS: m/z 213.0268, 215.0245 [M − OH]+; calculated, 213.0279,
215.0259 [M − OH]+.
General Procedure for the Synthesis of 2-(1-Hydroxy-2-
alkylcyclohexyl)-2-oxoacetic Acid (11a−e). To the above synthesized
α-ketoesters was added 1 M LiOH in MeOH:H2O (1:1), and the
reaction was stirred for 6 h at room temperature. The reaction was
acidified to pH 2 by the addition of 1 M HCl. The aqueous layer was
extracted with ethyl acetate (3 × 20 mL). The combined organic
phases were washed with brine (30 mL) and dried over MgSO4. The
solution was concentrated under reduced pressure to furnish the free
acid 11a−e as an oily liquid.
2-((1S,2R)-1-Hydroxy-2-methylcyclohexyl)-2-oxoacetic Acid and
2-((1R,2S)-1-Hydroxy-2-methylcyclohexyl)-2-oxoacetic Acid (11a).
Compound 11a (105 mg, overall yield for two steps 52%) was
obtained from 10a (235 mg) as a oily liquid. TLC (hexane:EtOAc:T-
FA 9:1:0.1): Rf = 0.28. 1H NMR (400 MHz, CDCl3) δ = 0.78 (d, 3H, J
= 6.8 Hz), 1.33−1.95 (m, 8H), 2.15−2.24 (m, 1H). 13C NMR (100
MHz, CDCl3) δ = 16.32, 20.32, 25.46, 29.41, 35.12, 36.57, 81.55,
162.81, 200.53.
LCMS Conditions for Product Analysis. Column: YMC Pack
Pro C8, 100 mm × 4.6 mm, 3 μm; 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 A: 1 min 100%
buffer A, then linear gradient of 0−100% buffer B for 11 min.
Preparative HPLC for Diasteromer Separation. The com-
pounds were dissolved in 40% buffer B, and the purification was
carried out with a injection loop volume of 2 mL. Column: Jupiter 10
μm Proteo 90 A, 250 mm × 21.7 mm, 10 μm, Phenomenex;
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.
Synthesis of (S)-Methyl 1-(3,3-Dimethyl-2-oxopentanoyl)-
piperidine-2-carboxylate (4a). The compound was prepared as
described previously.16
Synthesis of (S)-Methyl 1-(3,3-Dimethyl-2-oxopentanoyl)-
pyrrolidine-2-carboxylate (4b). The compound was prepared from
the methyl ester of L-proline in an analogous manner to 4a.
General Method A. A solution of alcohol 5a−c, carboxylic acid 4a−
d, and DMAP in DCM at room temperature was treated with DCC.
After it was stirred for 12 h, the mixture was diluted with EtOAc and
filtered through a plug of Celite. The filtrate was concentrated, and the
crude material was flash chromatographed to afford the product.
General Method B. A solution of bromide 7a−c and carboxylic acid
4a or 4b was treated with DIPEA in toluene at reflux for 40 h.
Afterwards, the mixture was diluted with EtOAc (30 mL) and filtered
through a plug of Celite. The filtrate was concentrated, and the crude
material was flash chromatographed to afford the product.
Synthesis of 2-(3-((R)-3-(3,4-Dimethoxyphenyl)-1-((S)-1-(3,3-di-
methyl-2-oxopentanoyl)piperidine-2-carbonyloxy)propyl)phenoxy)-
acetic Acid (2a). The compound was prepared as described
previously.17
General Procedure for the Synthesis of 2-Alkyl-1-((trimethylsilyl)-
ethynyl)cyclohexanol (9). The THF solution of the lithium reagent
was generated by treating trimethylsilylacetylene (3 mL, 21.4 mmol)
with n-BuLi (2 M in hexane, 11.6 mL) at −78 °C. The solution was
stirred for 0.5 h at that temperature. To this, a solution of 2-
alkylcyclohexanone (8a−c) (17.8 mmol) in THF (5 mL) was added
and stirred for an additional 2 h. Then, the solution was quenched by
addition of a saturated aqueous NH4Cl solution. The organic phase
was separated, and the aqueous phase was extracted with ethyl acetate
(3 × 100 mL). The combined organic phases were washed with brine
(30 mL) and dried over MgSO4. The solution was concentrated and
then flash chromatographed using hexane:EtOAc 9:1 to afford each of
the two diastereomers 9a−f.
2-((1S,2R)-2-Ethyl-1-hydroxycyclohexyl)-2-oxoacetic Acid and 2-
((1R,2S)-2-Ethyl-1-hydroxycyclohexyl)-2-oxoacetic Acid (11b). Com-
pound 11b (141 mg, overall yield for two steps 65%) was obtained
from 10b (250 mg) as a oily liquid. TLC (hexane:EtOAc:TFA
1
9:1:0.1): Rf = 0.26. H NMR (400 MHz, CDCl3) δ = 0.83 (t, 3H, J =
7.6 Hz), 1.13−1.36 (m, 4H), 1.57−1.62 (m, 2H), 1.73−1.96 (m, 5H).
13C NMR (100 MHz, CDCl3) δ = 11.82, 20.39, 23.95, 25.08, 25.46,
35.32, 43.14, 82.20, 164.12, 201.23.
General Procedure for Coupling of 12a−d with 11a−e To Yield
3a*−3j*. To a stirred solution of the free amines (12a−d) in
acetonitrile under argon was added sequentially N,N-diisopropyl-
ethylamine (DIPEA), HATU, and the diketoacids (11a−e). The
reaction mixture was stirred for 16 h at room temperature. Saturated
NH4Cl solution was added to the reaction, and the solution was stirred
for 10 min. The organic phase was separated, and the aqueous phase
was extracted with ethyl acetate (3 × 100 mL). The combined organic
phases were washed with brine (10 mL) and dried over MgSO4, and
the residual solid was purified by column chromatogrpahy.
Synthesis of 2-(3-((R)-3-(3,4-Dimethoxyphenyl)-1-((S)-1-(2-
((1S,2R)-1-hydroxy-2-methylcyclohexyl)-2-oxoacetyl)piperidine-2-
carbonyloxy)propyl)phenoxy)acetic Acid and 2-(3-((R)-3-(3,4-Dime-
thoxyphenyl)-1-((S)-1-(2-((1R,2S)-1-hydroxy-2-methylcyclohexyl)-2-
oxoacetyl)piperidine-2-carbonyloxy)propyl)phenoxy)acetic Acid
(3a*). Compound 3a* ester (46 mg, 0.067 mmol) was treated with
20% TFA in DCM at room temperature. The mixture was allowed to
stir for 6 h. TFA and DCM were evaporated under reduced pressure to
yield the free acid 3a* (32 mg, 0.051 mmol, 77%).
(1S,2R)-2-Methyl-1-((trimethylsilyl)ethynyl)cyclohexanol and
(1R,2S)-2-Methyl-1-((trimethylsilyl)ethynyl)cyclohexanol (9a). Com-
pound 9a (1.2 g, 33%) was obtained from 8a (2 g) as a colorless
1
liquid. TLC (hexane:EtOAc 9:1): Rf = 0.36. H NMR (300 MHz,
CDCl3) δ = 0.17 (s, 9H), 1.06 (d, 3H, J = 6.9 Hz), 1.23−1.33 (m, 1H),
1.48−1.71 (m, 7p), 1.95−2.02 (m, 1P). 13C NMR (75 MHz, CDCl3) δ
= 0.01, 16.00, 21.06, 25.02, 29.15, 39.15, 40.48, 69.77, 86.95, 110.61.
HRMS: 193.1390 [M − OH]+; calculated, 193.1413 [M − OH]+.
(1S,2R)-2-Ethyl-1-((trimethylsilyl)ethynyl)cyclohexanol and
(1R,2S)-2-Ethyl-1-((trimethyl silyl)ethynyl)cyclohexanol (9b). Com-
pound 9b (1.65 g, 46%) was obtained from 8b (2.2 g) as a colorless
1
liquid. TLC (hexane:EtOAc 9:1): Rf = 0.40. H NMR (300 MHz,
CDCl3) δ = 0.17 (s, 9H), 0.86−0.96 (m, 6H), 1.12−2.42 (m, 22H).
13C NMR (75 MHz, CDCl3) δ = 0.024, 11.68, 12.30, 21.23, 22.88,
24.80, 25.46, 28.00, 33.28, 39.47, 41.94, 47.47, 52.31, 70.32, 87.09,
110.85. HRMS: 208.2069 [M − OH]+; calculated, 208.1569 [M −
OH]+.
4120
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Journal of Medicinal Chemistry
Article
TLC (hexane:EtOAc:TFA 6:3.9: 0.1): Rf = 0.33. HPLC (gradient
A) retention time = 24.6−25.1 min. 1H NMR (600 MHz, CDCl3) δ =
0.82−0.88 (m, 3H), 1.36−1.92 (m, 13H), 2.03−2.13 (m, 2H), 2.23−
2.38 (m, 2H), 2.50−2.67 (m, 2H), 3.24−3.31 (m, 1H), 3.48−3.55 (m,
1H), 3.85 (s, 3H), 3.86 (s, 3H), 4.67 (s, 2H), 5.25−5.27 (m, 2H),
5.74−5.77 (m, 1H), 6.56−6.70 (m, 2H), 6.77−6.80 (m, 1H), 6.82−
6.87 (m, 1H), 6.89−6.96 (m, 2H), 7.26−7.29 (m, 1H). 13C NMR
(150 MHz, CDCl3) δ = 16.3, 20.16, 20.87, 24.79, 25.29, 26.55, 29.30,
31.35, 35.59, 37.60, 39.45, 44.28, 51.92, 55.87, 55.92, 65.07, 76.86,
82.24, 111.37, 111.70, 115.71, 116.21, 119.71, 120.20, 129.90, 133.21,
141.51, 147.41, 148.89, 157.74, 167.39, 169.20, 171.63, 205.23. MS
(ESI) m/z: found Rt 13.88 min. Method LCMS, 648.45 [M + Na]+.
HRMS: 626.2902 [M + H]+; calculated, 626.2887 [M + H]+. The
diasteromeric mixture was further separated using preparative HPLC
gradient 62−77% B for 35 min to yield diasteromer 3a-1 (6 mg) and
3a-2 (9 mg).
program MOLREP.44 The dictionaries for the ligand compounds were
generated with the PRODRG server.45 The structures were refined
with REFMAC.46 Manual model building was performed with
COOT.47 Molecular graphics figures were generated using PyMOL
(http://www.pymol.org).
ASSOCIATED CONTENT
* Supporting Information
■
S
Reaction schemes and spectroscopic details of intermediates.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +49(89)30622640. Fax: +49(89)30622610. E-mail:
hausch@mpipsykl.mpg.de.
■
3a-1. HPLC (gradient A) retention time = 24.6−24.8 min. 1H
NMR (600 MHz, CDCl3) δ = 0.82 (d, 3H, J = 5.4 Hz), 1.38−1.43 (m,
2H), 1.44−1.48 (m, 2H), 1.53−1.58 (m, 2H), 1.64−1.70 (m, 3H),
1.74−1.81 (m, 2H), 2.04−2.12 (m, 2H), 2.22−2.28 (m, 1H), 2.52−
2.67 (m, 2H), 2.98 (d, 1 h, J = 5.4 Hz), 3.08 (s, 1H), 3.12 (s, 1H), 3.25
(dt, 1H, J = 2.4, 13.2 Hz), 3.53 (d, 1H, J = 13.2 Hz), 3.64−3.67 (m,
1H), 3.72 (s, 1H), 3.85 (s, 3H), 3.86 (s, 3H), 4.63 (s, 2H), 5.24 (d,
1H, J = 4.8 Hz), 5.74−5.80 (m, 1H), 6.66−6.69 (m, 2H), 6.77−6.79
(m, 1H), 6.83−6.94 (m, 3H), 7.26−7.28 (m, 1H). 13C NMR (150
MHz, CDCl3) δ = 16.15, 20.18, 21.06, 24.79, 25.27, 26.52, 29.68,
31.41, 35.57, 36.61, 37.64, 44.18, 51.88, 55.86, 55.92, 63.81, 81,38,
111.35, 111.68, 115.65, 115.66, 119.54, 120.16, 129.85, 133.19, 141.53,
147.45, 148.93, 157.92, 167.57, 169.26, 169.26, 205.46. MS (ESI) m/z:
found Rt 13.87 min. Method LCMS, 648.40 [M + Na]+; calculated,
648.45 [M + Na]+. 21αD = −2.62°.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Gerd Ruhter and the Lead Discovery Center
̈
(Dortmund) for providing building block 5b and 5c and Drs. B.
Gold and E. R. Sanchez for providing a sample of Timcodar.
We are indebted to Claudia Dubler (LMU, Munich, Germany)
and Elisabeth Weyher (MPI of Biochemistry, Martinsried,
Germany) for NMR spectroscopy and HRMS measurements,
respectively. We thank Prof. Florian Holsboer for continuous
and generous financial support. Support by the Joint Structural
Biology Group at the ESRF beamlines is gratefully acknowl-
edged.
3a-2. HPLC (gradient A) retention time = 24.9−25.1 min. 1H
NMR (600 MHz, CDCl3) δ = 0.84 (d, 3H, J = 6.6 Hz), 1.38−1.85 (m,
10H), 2.06 (s, 2H), 2.20−2.31 (m, 1H), 2.49−2.65 (m, 2H), 2.97 (d,
1H, J = 6.6 Hz), 3.05 (s, 1H), 3.12 (s, 1H), 3.25 (t, 1H, J = 12.6 Hz),
3.48 (d, 1H, J = 10.8 Hz), 3.65 (s, 1H), 3.72 (s, 2H), 3.84 (s, 3H), 3.85
(s, 3H), 4.81 (s, 2H), 5.26 (s, 1H), 5.74 (s, 1H), 6.66−6.68 (m, 2H),
6.77−6.94 (m, 4H), 7.21−7.24 (m, 1H). 13C NMR (150 MHz,
CDCl3) δ = 16.15, 20.21, 20.94, 24.82, 25.31, 26.40, 29.68, 31.35,
35.31, 36.72, 37.15, 42.16, 43.25, 44.25, 44.54, 46.53, 48.81, 51.75,
55.86, 55.92, 56.79, 63.84, 81.66, 111.34, 111.70, 115.51, 119.59,
120.17, 129.82, 133.32, 141.58, 147.41, 148.91, 157.91, 167.37, 169.34,
205.95. MS (ESI) m/z: found Rt 13.91 min. Method LCMS, 648.31
[M + Na]+; calculated, 648.45 [M + Na]+. 21αD = +0.31°.
Synthesis of 2-(3-((R)-3-(3,4-Dimethoxyphenyl)-1-((S)-1-(2-
((1S,2R)-2-ethyl-1-hydroxycyclohexyl)-2-oxoacetyl)piperidine-2-
carbonyloxy)propyl)phenoxy)acetic Acid and 2-(3-((R)-3-(3,4-Dime-
thoxyphenyl)-1-((S)-1-(2-((1R,2S)-2-ethyl-1-hydroxycyclohexyl)-2-
oxoacetyl)piperidine-2-carbonyloxy)propyl)phenoxy)acetic Acid
(3f*). Compound 3f* ester (62 mg, 0.089 mmol) was treated with
20% TFA in DCM at room temperature. The mixture was allowed to
stir for 6 h. TFA and DCM were evaporated under reduced pressure to
yield the free acid 3f* (40 mg, 0.062 mmol, 80%).
ABBREVIATIONS USED
FKBP, FK506 binding protein; Hsp90, heat shock protein 90;
SAR, structure−activity relationship
■
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