Identification of highly efficacious glucocorticoid receptor agonists with a potential for reduced clinical bone side effects

Article abstract of DOI:10.1021/jm4019178

Synthesis and structure-activity relationship (SAR) of a series of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain "diazaindole" moieties and display different transcriptional regulatory profiles in vitro and are

Full text of DOI:10.1021/jm4019178

Article
pubs.acs.org/jmc
Identification of Highly Efficacious Glucocorticoid Receptor Agonists
with a Potential for Reduced Clinical Bone Side Effects
Christian Harcken,* Doris Riether, Daniel Kuzmich, Pingrong Liu, Raj Betageri, Mark Ralph,
Michel Emmanuel, Jonathan T. Reeves, Angela Berry, Donald Souza, Richard M. Nelson, Alison Kukulka,
Tazmeen N. Fadra, Ljiljana Zuvela-Jelaska, Roger Dinallo, Jorg Bentzien, Gerald H. Nabozny,
̈
and David S. Thomson
Department of Medicinal Chemistry, Department of Immunology and Inflammation, Department of Chemical Development,
Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, Connecticut 06877, United States
ABSTRACT: Synthesis and structure−activity relationship (SAR) of a series of nonsteroidal
glucocorticoid receptor (GR) agonists are described. These compounds contain “diazaindole”
moieties and display different transcriptional regulatory profiles in vitro and are considered
“dissociated” between gene transrepression and transactivation. The lead optimization effort described
in this article focused in particular on limiting the transactivation of genes which result in bone side
effects and these were assessed in vitro in MG-63 osteosarcoma cells, leading to the identification of
(R)-18 and (R)-21. These compounds maintained anti-inflammatory activity in vivo in collagen induced arthritis studies in
mouse but had reduced effects on bone relevant parameters compared to the widely used synthetic glucocorticoid prednisolone 2
in vivo. To our knowledge, we are the first to report on selective glucocorticoid ligands with reduced bone loss in a preclinical in
vivo model.
the nuclear receptor super family. When the GR binds an
agonist ligand in the cytosol, it sheds chaperone proteins and
translocates to the nucleus.4 Here the receptor−ligand complex
regulates transcription either positively or negatively.5 The
precise molecular mechanism of GC-mediated effects are highly
complex and only partially understood.6 Since the early 2000s, a
possibly oversimplistic hypothesis has been widely accepted
among researchers aiming at safer alternatives to classical GCs.
This hypothesis describes two distinct pathways of the
receptor−ligand complex: (1) As a homodimer the receptor−
ligand complex acts itself as transcription factor by interacting
directly with Glucocorticoid response elements (GRE) on the
DNA, upregulating transcription of respective genes. This
pathway has been called transactivation (TA) and has been
attributed to many of the GC mediated side effects. (2) The
receptor−ligand complex indirectly affects transcription,
interacting in a monomeric form with transcription factors
such as NF-κB and AP-1, downregulating the transcription of
genes.7 This pathway has, in a broader sense, been called
transrepression (TR) and is considered primarily responsible
for the anti-inflammatory effects of GCs as it results in down-
regulation of key cytokine inflammatory mediators such as
TNF-α, IL-1, IL-2, and IL-6. This hypothesis of the two distinct
pathways owes its genesis to a series of experiments described
elsewhere and has been the basis of “dissociated” GCs.8
Evidence has continued to accumulate to support the existence
of transrepression as a means of downregulating pro-
inflammatory cells.9 However, data generated more recently
do not support and partially even contradict the hypothesis and
INTRODUCTION
■
Glucocorticoids (GCs) such as dexamethasone (1) and
prednisolone (2) are being extensively used to treat
inflammatory diseases (Figure 1).1 However, the dose selection
Figure 1. Synthetic glucocorticoid agonists.
and duration of treatment of these drugs is limited by their side
effects such as weight gain, hypertension, muscle weakness, skin
thinning, diabetes, and GC-induced osteoporosis.2 For diseases
such as rheumatoid arthritis (RA), perhaps the most trouble-
some and dose-limiting side effect upon chronic treatment with
low dose GCs is GC-induced osteoporosis, leading to a
weakening of trabecular bone and a significant increase in the
risk of spine, hip, and rib fractures.3
Glucocorticoids exert their pharmacological effect through
interaction with the glucocorticoid receptor (GR), a member of
Received: December 13, 2013
Published: February 7, 2014
© 2014 American Chemical Society
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Scheme 1. General Synthesis of Diazaindoles
Figure 2. Halogenated aminoarenes.
a
Scheme 2. Synthesis of Diazaindole Isomers
a
Reagents and conditions: (a) arylbromide, Mg (1.2 equiv), THF, reflux, 3 h, then CuI (1.1 equiv), −10 °C, 1 h; then 14 (1.1 equiv), room
temperature, 16 h; (b) Al (3 equiv), HgCl2 (0.02 equiv), THF, 0 °C, 10 min, propargylbromide (3 equiv), 50 °C, 1 h, then 16, −78 °C to room
temperature, 2 h, yield 99%; (c) 8−13 (1 equiv), Pd(PPh3)2Cl2 (0.05 equiv), CuI (0.1 equiv), DMF, NEt3, room temperature, 15 h, yield 32−75%;
(d) NMP, KOtBu (2.2 equiv), room temperature, 17 h, yield 15−36% over 2 steps; (e) pyridine (3 equiv), AcCl (2 equiv), CH2Cl2, 0 °C, 1 h;
tetramethylguanidine (5 equiv), DMSO, 140 °C, μwave, 10 min, yield 18% over 2 steps; (f) Pd(PPh3)4 (0.1 equiv), PhB(OH)2 (2 equiv), K2CO3 (3
equiv), DMF/MeOH/DME, 80 °C, 3 h, yield 71%.
left it experimentally poorly supported.6 Nevertheless, for an
industrial drug discovery program, the hypothesis has served as
a highly appealing working model throughout the past decade10
that has sparked the search for a functionally selective, so-called
“dissociated” synthetic glucocorticoid that could potentially
offer a therapeutic advantage over currently marketed GCs.11
In addition, many of the clinically used GCs also interact
with other nuclear receptors, especially the progesterone
receptor (PR) and the mineralocorticoid receptor (MR).
Therefore, an improved selectivity over other nuclear receptors
is also desirable.
demonstrated excellent nuclear receptor selectivity, potent GR
binding, potent GR agonism (as indicated by their in vitro
suppression of IL-1 induced IL-6 production in human foreskin
fibroblasts), and excellent in vivo activity in acute models of
inflammation (such as inhibition of LPS induced TNF-α
production in mouse) and in chronic disease models (such as
inhibition of collagen-induced arthritis in mouse) (Figure 1). In
addition, these compounds showed reduced transactivation (as
indicated in vitro by their reduced potency and maximum
efficacy in an MMTV-promoter transfected HeLa cell and their
reduced incidence of metabolic side effects upon chronic dosing
in vivo). However, for a bone relevant dissociation marker
(such as suppression of vitamin D stimulated osteocalcin
production in MG-63 osteosarcoma cells) these compounds
did show only a moderately attenuated effect compared to
Previously, we have disclosed nonsteroidal GC mim-
etics,12−14 including a series of trifluoromethylcarbinol derived
compounds.15−17 Therein we described the identification and
profile of azaindole compounds such as 3 and 4 that have
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a
Scheme 3. Synthesis of 5-Substituted 4,6-Diazaindoles
a
Reagents and conditions: (a) Pd(PPh3)2Cl2 (0.05 equiv), CuI (0.1 equiv), DMF, NEt3, room temperature, 15 h, yield 36−57%; (b)
tetramethylguanidine (5 equiv), dioxane, 90 °C, 15 min, yield 33−64%; (c) Pd(PPh3)2Cl2 (0.1 equiv), CuI (0.1 equiv), MeCN, NEt3, 70 °C, 2 h,
yield 57−72%.
prednisolone. Encouraged by the results achieved thus far, we
next decided to tackle the question whether an even more
desirable profile is possible, i.e., achieving dissociation for bone
side effect relevant markers.
subjected to a Suzuki coupling to give the 5-phenyl-substituted
derivative 24b.
5-Substituted 4,6-diazaindoles 26−31 were prepared analo-
gously by Sonogashira coupling of alkyne 17a with appropri-
ately substituted brominated trifluoroacetylamino pyrimidines
25a−f (Scheme 3). 25a,19 25b,19 and 25c were prepared in six
steps from acyclic precursors. 25d and 25e were prepared in
five steps from acyclic precursors. 25f was prepared in four
steps from 5-chloro-2-nitropyrimidine. The coupling products
were cyclized using tetramethylguanidine in dioxane to give the
5-substituted 4,6-diazaindoles 26−31.
Previous studies had revealed that the position of the
pyridine nitrogen atom in the azaindole moiety of compounds
such as 3 was of utmost importance.16 Whereas the 4-, 5-, 6-,
and 7-azaindole are all potent GR ligands, they showed anti-
inflammatory potency to a different degree: the 5- and 6-
azaindole were potent agonists, whereas the 4- and 7-azaindole
displayed almost no anti-inflammatory efficacy in vitro.16 It was
speculated that the pyridine nitrogen in the 5- and 6-azaindole
can form an H-bonding interaction with GLU570 and ARG611
of the GR, mimicking the interaction of the steroid A-ring. It is
conceivable that this may result in a conformational change
required to achieve an agonist conformation of the ligand−
receptor complex. This publication describes the synthesis and
biological evaluation of azaindole derived heteroaryl moieties as
A-ring mimetics that contain two nitrogen atoms in the six-
membered ring (“diazaindoles”) with a particular focus on
optimization for bone dissociation.
4,6-Diazaindoles with additionally substituted right-hand side
rings 32−35 were prepared from the aminobromopyrimidine
12 and alkynes 17b−e (Scheme 4). Alkynes 17b,16 17d, and
a
Scheme 4. Synthesis of 4,6-Diazaindoles
CHEMISTRY
■
The general strategy to synthesize the isomers of diazaindoles 7
consists of the Sonogashira coupling of an alkyne 6 with an
appropriately halogenated aminoheteroarene 5 and subsequent
base catalyzed cyclization (Scheme 1).18 The desired
halogenated heteroarylamines 8−13 were prepared the
following way (Figure 2): 8, 9, 10, and 12 were prepared by
bromination or iodination of the corresponding aminoarenes.19
11 was prepared in four steps from acyclic precursors.20 13a
was prepared in one step from 2,3-dichloropyrazine.21 13b was
commercially available.
The synthesis of alkynes 17 begins with the addition of
appropriately substituted arylmagnesium bromide reagents 15
to the trifluoromethylenone 14 (Scheme 2).16 The resulting
ketones 16 are then treated with propargyl aluminum to yield
the racemic alkynes 17. Alternatively, the ketones can be
transformed in four steps to the enantiopure (S)-isomers of
17.22 The alkynes 17 and halogenated aminoarenes 8−13 were
treated with a Pd catalyst to afford the Sonogashira coupling.
The resulting coupling product was then either treated with
KOtBu in N-methylpyrrolidinone or N-acylated and sub-
sequently treated with tetramethylguanidine in dioxane to
effect the cyclization to the diazaindole 18−23 and 24a
(cyclization procedure A and B, respectively). 24a was
a
Reagents and conditions: (a) 12 (1 equiv), Pd(PPh3)2Cl2 (0.05
equiv), CuI (0.1 equiv), DMF, NEt3, room temperature, 15 h, yield
53−71%; (b) NMP, KOtBu (2.2 equiv), room temperature, 17 h, yield
25−63%.
17e were prepared in analogy to 17a. Alkyne 17c was prepared
in eight steps from 4′-fluoro-2′-hydroxy-acetophenone. Dia-
zaindoles 36 and 37 were synthesized from alkynes 17f−g,
which were prepared in five and six steps respectively from
ethyl trifluoropyruvate16 (Scheme 5). Diazaindole 40 was
prepared through acylation and subsequent cyclization of 5-
amino-4-methylpyrimidine 38 with the ester 39 (Scheme 6).
Diazaindoles 41 and 42 were prepared by demethylation of the
corresponding methoxy analogue 32 and 33 with BBr3,
respectively (Scheme 7).
The pure enantiomers (R)-18 and (R)-21 (>98% ee) were
synthesized following the same sequence outlined in Scheme 1
using enantiopure alkynes 6, which have been described
elsewhere.16,22
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with varying degree of dissociation remained largely empirical.
The 5-phenyl-substituted 4,7-diazaindole 24b showed agonism,
albeit with an IC50 of 170 nM at a reduced potency. To assess
the potential dissociation of these analogues, the selective
agonist compounds were counterscreened for their ability to
activate the MMTV promoter in HeLa cells transfected with an
MMTV luciferase construct. The 6,7-diazaindole 20 showed
potent transactivation in this marker, whereas the 4,5- and 4,6-
diazaindoles 18 and 21 showed no or minimal activity in this
assay. It has been described before in the literature that
increased potency can be achieved upon increasing the steric
demand of the A-ring mimic above a certain threshold.25
To further assess the SAR in this class of compounds,
additional racemic analogues were prepared with the 4,6-
diazaindole A-ring mimetic. Table 2 shows the effect of
substitution in the 5-position of the 4,6-diazaindole.
Scheme 5. Synthesis of Dihydrobenzofuran Containing
Analogues
a
a
Reagents and conditions: (a) 8 (1 equiv), Pd(PPh3)2Cl2 (0.05 equiv),
CuI (0.1 equiv), DMF, NEt3, room temperature, 15 h, yield 24−47%;
(b) NMP, KOtBu (2.2 equiv), room temperature, 17 h, yield 41−50%.
a
Scheme 6. Synthesis of Diazaindole 40
Substitution at the 5-position did not affect the GR binding
potency significantly. The analogues 26−31 show excellent
nuclear receptor selectivity, with the exception of 30, that for
unknown reasons shows increased MR activity. The isopropyl
analogue 26 and the pyrrolidinyl analogue 29 show significantly
reduced transrepression potency with IC50 values of 560 and
130 nM, respectively. The sterically more demanding phenyl,
pyridyl, and 1,1-dioxo-thiomorpholin-4-yl analogues 27, 28, and
31 retain their transrepression potency, however, they show
increased activity in the MMTV reporter assay with 36%, 32%,
and 22% maximum efficacy, respectively, indicating a less
dissociated profile. Again, it has been described before in the
literature that increased potency can be achieved upon
increasing the steric demand of the A-ring mimic above a
certain threshold.25
a
Reagents and conditions: (a) 38 (3.3 equiv), nBuLi (8.2 equiv), THF,
−20 °C to room temperature, 2.5 h; 39, THF, room temperature, 20
min, HCl (6 N), 2 h, yield 47%.
a
Scheme 7. Synthesis of Phenole Containing Analogues
Table 3 shows the effect of changes in substitution pattern on
the right-hand side phenyl group. Replacement of the methyl
group in 21 with a methoxy group in compound 32 or a
hydroxy group as in 41 is well tolerated: 32 and 41 retain the
nuclear receptor selectivity as well as the agonist potency of 21.
Moving the fluoro substituent from the 5-position to the 4-
position as in 42 results in reduced nuclear receptor selectivity.
This has been observed before in related series.16 Complete
removal of the fluoro substituent from 21 as in 40 results in a
slight loss in potency and maximum efficacy. It has been
demonstrated before that complete removal of 2-substitution
also results in a dramatic loss of transrepression potency.16
Docking of compound 21 suggests that the phenyl group
resides in a space occupied by the steroid D-ring of
dexamethasone. Formal cyclization of an alkoxy group with
the 3-position of the right-hand side phenyl ring results in the
formation of a dihydrofuran moiety. Dihydrofuran compounds
36 and 37 show a slightly increased affinity to the progesterone
receptor, however, they retain potent transrepression activity.
The replacement of the hydroxy group with a larger hydrogen
bond acceptor group such as the methylsulfonyl group in 34
also maintains selectivity and agonist potency. Moving the
sulfonyl group to the 3-position is not tolerated: compound 35
shows significantly reduced binding affinity and no trans-
repression activity. This is presumed to be due to increased
steric clashes in the D-ring binding pocket. Large substituents
to access an expanded D-ring pocket can be accessed by 3-aryl
substitution on the D-ring mimic as described previously.16
To assess the dissociation profile of compounds with regard
to potential bone side effects, compounds with the appropriate
selectivity and potency profile were tested for suppression of
vitamin D induced osteocalcin production in human MG-63
a
Reagents and conditions: (a) BBr3 (1 equiv), DCM, 0 °C, 18−60 h,
yield 68−70%.
RESULTS AND DISCUSSION
■
All possible isomers of the racemic diazaindoles 18−23 were
synthesized. Because of synthetic accessibility, the 6,7-
diazaindole 20 was synthesized bearing an additional 5-phenyl
substituent. The 5,7-diazaindole 22 was prepared with a
methoxy substituted phenyl ring on the right-hand side of the
molecule instead of the methyl substituted phenyl group. These
two groups were previously shown to be equivalent in their in
vitro biological profile.16 The prepared diazaindole isomers
were all potent GR binders, indicated by their ability to
compete for receptor binding with tetramethylrhodamine
labeled dexamethasone (Table 1). It was previously hypothe-
sized that the nitrogen atoms of 5-azaindoles and 6-azaindoles
interact with the arginine−glutamine pair of the steroid A-ring
binding pocket.16 Diazaindole compounds such as 21 dock the
same way into the GR-LBD using the GR-LBD/dexamethasone
co-complex X-ray structure23 (Figure 3).
The 5,6-diazaindole 19 and the 5,7-diazaindole 22 showed
potent affinity to the progesterone receptor (PR) and the
mineralocorticoid receptor (MR), the MR/GR selectivity for
19 and 22 was merely 28- and 40-fold, respectively. Except for
the 5,7-diazaindole 23, all compounds were shown to be GR
agonists, indicated by inhibiting IL-1 stimulated IL-6
production in human foreskin fibroblast (HFF), with a lower
maximum efficacy compared to dexamethasone.24 The SAR to
identify partial agonists of varying degree and/or compounds
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Table 1. Azaindole Isomers
a
b
IC50 values are the mean of at least two values, each determined from duplicate 11-point concentration response curves Maximum efficacy at the
c
highest tested concentration compared to dexamethasone, defined at 100%; maximum concentration tested is 2 μM. NT = not tested.
3 had not shown statistically significantly reduced GC-mediated
bone side effects in a chronic model of mouse collagen-induced
arthritis (CIA).16 Therefore, a maximal efficacy lower than that
of 3 in this assay was desirable, assuming osteocalcin is a viable
biomarker for bone formation rate related side effects.
Compounds 21, 18, 42, 37, 41, 36, and 34 all showed maximal
efficacies <50%. Compound 21 and 18, as potent and NR-
selective, and additionally osteocalcin-dissociated analogues
were selected for further study.
It should be noted that while these compounds show the
desired pharmacology profile and are suitable tools to assess the
dissociation in vivo, their drug-like properties are suboptimal.
Compounds 21 and 18 show low aqueous crystalline solubility
of 11 and 2 μg/mL in buffer at pH 7, respectively. Both
compound are predicted to be rapidly metabolized in human
Figure 3. Docking results for (R)-21 (yellow) into the GR-LBD/
dexamethasone (orange) co-complex X-ray structure.23 Potential H-
bonds of (R)-21 with the protein are indicated by white lines. Docking
was performed using Glide (Small-Molecule Drug Discovery Suite
with a human liver microsome clearance rate of >89% Qh. Both
compounds show good membrane permeability of 11 and 13 ×
10−6 cm/s in Caco-2 cells, respectively, and no detectable
efflux.
The active enantiomers (R)-18 and (R)-21 were selected for
profiling in vivo to assess how well the in vitro osteocalcin
marker predicts attenuated GC-mediated side effects in vivo
(Table 5). It has been demonstrated previously that all activity
of the racemic mixtures in this class of GR agonists resides in
the (R)-enantiomer.16 As predicted by their in vitro ADME
profile, the pharmacokinetic profile of both compounds is
2013−2: Glide, version 6.0, Schrodinger, LLC, New York). The
̈
picture was generated with MOE (Molecular Operating Environment
2012, Chemical Computing Group Inc., Montreal, QC, Canada).
osteosarcoma cells. Table 4 shows the results for selected
compounds. All tested compounds showed significantly
reduced maximal efficacy for the suppression in osteocalcin
production in this assay compared to dexamethasone. However,
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Table 2. 5-Substituted 4,6-Diazaindoles
a
b
IC50 values are the mean of at least two values, each determined from duplicate 11-point concentration response curves. Maximum efficacy at the
c
highest tested concentration compared to dexamethasone, defined at 100%; maximum concentration tested is 2 μM. NT = not tested.
suboptimal: (R)-18 showed high clearance (>100% Qh) and
moderate volume of distribution (Vss = 3.7 L/kg), resulting in a
short half-life (t1/2 = 0.5 h) and correspondingly low
bioavailability (9%F) in rat PK (2 mg/kg iv in 80/20 PEG/
water; 30 mg/kg po in 80/18/2 PEG/water/Tween). (R)-21
also showed high clearance (61% Qh) and moderate volume of
= 3, 4320 nM at cmax). Prednisolone reached 43% and 78%
disease score reduction at 3 and 30 mg/kg doses, respectively.
(R)-18 was tested at 100 mg/kg in a separate experiment and
showed 53% reduction in disease score, comparable to
prednisolone at 3 mg/kg (53% inhibition in this study).
Plasma concentrations were determined after a single dose of
100 mg/kg in satellite animals (B10.RIII mice, n = 3, 38900 nM
at cmax). Prednisolone at 30 mg/kg showed 90% inhibition in
this study. The difference in prednisolone response between
the two studies is assumed to be caused by differences in the
exposure of prednisolone in the utilized vehicles.
Table 6 lists the side-effect markers for (R)-18 and (R)-21.
Both compounds clearly showed reduced effects on metabolic
side effect markers such as % body fat, free fatty acids, and
insulin. (R)-21 was also tested for the effect on triglycerides.
Both test compounds showed statistically significant reduction
in free fatty acids compared to the approximately equi-
efficacious dose of 3 mg/kg prednisolone. The other metabolic
side effect markers did not reach statistical significance
comparing the compound treated group to the group treated
with prednisolone at 3 mg/kg, however, all of them trended in a
favorable direction. This profile had been demonstrated before
with 3 and is believed to be predicted by in vitro dissociation
for the MMTV marker.16 To assess the in vivo bone
dissociation profile, the study was repeated with nonimmunized
mice because the diseased state of the CIA study mice interferes
with bone-relevant readouts. Nondiseased, age-matched mice
were dosed with (R)-18 or (R)-21 at the same doses and for
the same duration as in the prior study of immunized mice.
MicroCT analysis for femur cortical thickness demonstrated
significantly reduced bone loss over the treatment course. (R)-
21 showed only 6% reduction in mean femur cortical thickness
at the 100 mg/kg dose (57% CIA disease score reduction)
compared to the less efficacious 3 mg/kg dose of prednisolone
distribution (Vss = 2.1 L/kg) resulting in a short half-life (t1/2
=
0.6 h) and correspondingly low bioavailability (13%F) in rat PK
(5 mg/kg iv in 80/20 PEG/water; 30 mg/kg po in 80/18/2
PEG/water/Tween). No PK/PD analysis was performed.
Both compounds showed potent inhibition of LPS-
stimulated TNF-α production in mouse in vivo consistent
with potent anti-inflammatory transrepression activity (dosed
po in 30% cremophor; (R)-21 ED50 1−3 mg/kg; (R)-18 ED50
3−10 mg/kg; data not shown). Furthermore, these compounds
were tested in a collagen-induced arthritis model, a chronic
model of inflammatory polyarthritis that shares many features
with human rheumatoid arthritis. B10.RIII mice were immu-
nized with porcine type II collagen and complete Freund’s
adjuvant and then monitored for signs of arthritis. Animals were
enrolled into the study at the first signs of disease (paw/joint
swelling) and treated daily with compound, prednisolone, or
vehicle for a period of five weeks. The animals were monitored
regularly and arthritis severity was assessed through clinical
scoring of all four paws based on a score of 1−4. The clinical
score per paw was summed to give a maximal severity score of
16 for each animal. (R)-21 was evaluated in this model at once
daily oral doses of 100 mg/kg for a period of five weeks versus
prednisolone at 30 and 3 mg/kg. At the end of the study, serum
was collected for biomarker analysis to assess the side effect
profile. (R)-21 significantly inhibited disease progression (57%
inhibition by AUC vs vehicle; Mann−Whitney test: p < 0.05)
(Figure 4). Plasma concentrations were determined after a
single dose of 100 mg/kg in satellite animals (B10.RIII mice, n
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Table 3. 4,6-Diazaindole Analogues
a
b
IC50 values are the mean of at least two values, each determined from duplicate 11-point concentration response curves. Maximum efficacy at the
c
highest tested concentration compared to dexamethasone, defined at 100%; maximum concentration tested is 2 μM. NT = not tested.
(43% CIA disease score reduction) that caused 9% reduction in
mean femur cortical thickness (Table 7). This difference does
not reach statistical significance, however, this is not
unexpected because the two groups being compared are not
equi-efficacious (57% vs 43% reduction in CIA disease score).
(R)-18 showed only 8% reduction at the 100 mg/kg dose
compared to the equi-efficacious 3 mg/kg dose of prednisolone
that caused 13% reduction. This difference reaches statistical
significance. This difference was maintained after 10 weeks of
treatment (data not shown). The difference in prednisolone
response between the two studies is assumed to be caused by
differences in the exposure of prednisolone in the utilized
vehicles (not measured).
steroid-A-ring mimetic portion and the right-hand-side
substituents of these analogues, compounds with reduced in
vitro osteocalcin production suppression were identified. These
compounds demonstrated reduced GC-mediated osteoporosis
in chronic treatment in mouse in vivo. This constitutes a
significant milestone in the development of dissociated GCs.
Compounds with a biological profile like 18 and 21 have
tremendous potential to show a significant clinical benefit over
existing GC treatment options. However, identification of a
viable clinical candidate will require further optimization of
drug-like properties of these compounds and will be disclosed
in due course.
EXPERIMENTAL SECTION
■
Analytical Methods. 1H NMR spectra were recorded on a Bruker
UltraShield 400 MHz spectrometer operating at 400 MHz in solvents,
as noted. Proton coupling constants (J values) are rounded to the
nearest Hz. All solvents were HPLC grade or better. The reactions
were followed by TLC on precoated Uniplate silica gel plates
purchased from Analtech. The developed plates were visualized using
254 nm UV illumination or by PMA stain. Flash column
CONCLUSION
■
The described nonsteroidal trifluoromethylcarbinol scaffold has
provided multiple glucocorticoid mimetics with potent GR
binding affinity and favorable nuclear receptor selectivity.
Compounds from this series have been established as having
excellent anti-inflammatory in vivo activity. By optimizing the
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(A) over 20 min. All compounds were 95% or greater purity by HPLC
with the exception of 22 (no HPLC data), 37 (94%), and 42 (92%).
Chemistry Experimental. General Sonogashira Coupling
Procedure. To a solution of halogeno diazine 8−13 (1 mmol) in
anhydrous DMF (1 mL) and NEt3 (3 mL) wais added CuI (0.10
mmol), Pd(PPh3)2Cl2 (0.05 mmol) and the alkyne 17 (1 mmol) under
argon and the reaction stirred at room temperature for 15 h. The
reaction was diluted with EtOAc (10 mL) and washed with satd
aqueous NH4Cl solution (2 × 5 mL), and the combined aqueous
layers were extracted with EtOAc (3 × 20 mL). The combined organic
layers are washed with brine (5 mL), dried over MgSO4, and
concentrated. Flash silica gel chromatography using 15−30% EtOAc in
hexanes gives the coupling product as a white solid.
General Cyclization Procedure A. To a solution of the Sonogashira
coupling product (1 mmol) in anhydrous NMP (5 mL) was added
KOtBu (3.0 mmol), and the reaction was stirred under argon at room
temperature for 17 h. The reaction mixture was diluted with Et2O (30
mL) and washed with water (15 mL). The aqueous layer was extracted
with Et2O (3 × 20 mL), and the combined organic layers were washed
with water (2 × 5 mL) and brine (5 mL), dried over MgSO4, and
concentrated. Flash silica gel chromatography using 0−10% MeOH in
CH2Cl2 gives the diazaindole as a white solid.
Table 4. Inhibition of Osteocalcin Production
a,b
compd no.
osteocalcin suppression max efficacy at 2 μM [%]
1 (dex)
2 (pred)
3
100
78
68
45
45
81
73
32
72
74
80
43
26
35
42
28
4
18
19
20
21
27
28
31
34
36
37
41
42
a
IC50 values are the mean of at least two values, each determined from
b
duplicate 11-point concentration response curves. Maximum efficacy
at the highest tested concentration compared to dexamethasone,
defined at 100%; maximum concentration tested is 2 μM.
General Cyclization Procedure B. To a solution of trifluoroacetyl
protected aminopyridine (Sonogashira coupling product) (0.2 mmol)
in anhydrous dioxane (0.8 mL) was added tetramethylguanidine (0.6
mmol) and the reaction mixture heated to 100 °C for 45 min. The
reaction mixture was concentrated and purified using flash silica gel
chromatography using 0−10% MeOH in CH2Cl2 or 0−100% EtOAc
in hexanes to give the azaindole as a white solid.
Table 5. In Vitro Profile of Active Enantiomers (R)-18 and
(R)-21
IL-6 IC50
[nM] and
max.
MMTV
max.
Osteocalcin
suppression
max. efficacy
at 2 μM
5-Amino-4-bromopyrimidine (12). A solution of 5-amino-
pyrimidine (3.00 g, 31.5 mmol) in CH2Cl2 (150 mL) and MeOH
(30 mL) was treated with benzyltrimethylammonium tribromide (13.5
g, 34.7 mmol) in portions over a period of 10 min at 0 °C. The
mixture was stirred for 15 min at 0 °C and then 90 min at room
temperature. The mixture was treated with satd aqueous NaHCO3
solution until the pH was approximately 8. The layers were separated.
The aqueous layer was extracted with EtOAc (3 × 50 mL). The
combined organic layers were dried over MgSO4. The solvent was
removed to give 5-amino-4-bromopyrimidine as an off-white solid
GR
IC
MR
efficacy
compd
no.
PR IC50 IC50
efficacy
at 2 μM
50a
b
b
b,c
[nM]
[nM]
[nM]
[%]
[%]
[%]
(R)-18
(R)-21
5
4
>2000
>2000
260
475
9 (86)
0
0
34
32
13 (72)
a
IC50 values are the mean of at least two values, each determined from
b
duplicate 11-point concentration response curves. Maximum
c
concentration tested is 2 μM. Maximum efficacy at the highest
tested concentration compared to dexamethasone, defined at 100%.
1
which was used without further purification (51% yield). H NMR
(400 MHz, CHCl3-d) δ 8.35 (s, 1H), 8.13 (s, 1H), 4.15 (br s, 2H).19
1,1,1-Trifluoro-4-methyl-pent-3-en-2-one (14). Step 1: To a
0−5 °C mixture of N,O-dimethylhydroxylamine hydrochloride (15.8
g) in CH2Cl2 (400 mL) was added dropwise trifluoroacetic anhydride
(21.7 mL). Pyridine (37 mL) was added to the mixture dropwise. The
resulting mixture was stirred at 0−5 °C for 0.5 h and diluted with
water. The organic layer was washed with water, 1 N aqueous HCl,
water, and brine and dried over MgSO4. Removal of the volatiles in
vacuo for 5 min provided 2,2,2-trifluoro-N-methoxy-N-methylaceta-
mide as colorless oil.
chromatography on silica gel was performed on Redi Sep prepacked
disposable silica gel columns using an Isco Combiflash or on
traditional gravity columns. Reactions were carried out under an
atmosphere of argon. Purity was evaluated by analytical HPLC using a
Varian Dynamax SD-200 pump coupled to a Varian Dynamax UV-1
detector: (A) water + 0.05% TFA and (B) acetonitrile + 0.05% TFA,
flow 1.2 mL/min. Column Vydac RP-18, 5 m, 250 mm × 4.6 mm,
photodiode array detector at 220 or 254 nm; from 95% to 20% solvent
Figure 4. Collagen induced arthritis.
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Table 6. Side Effect Markers
CIA disease score
AUC SE
(% change vs vehicle)
body fat [%fat]
mean SE
(% change vs vehicle)
triglycerides [mEq/L]
mean SE
(% change vs vehicle)
free fatty acids [mEq/L]
mean SE
(% change vs vehicle)
insulin [ng/mL]
mean SE
compd and dose
(% change vs vehicle)
c
a
b
100 mg/kg (R)-21
12.0 2.0 (−57%)*
15.8 1.5 (−43%)*
6.2 1.3 (−78%)*
10.2 1.2 (−53%)*
9.9 1.5 (−55%)*
2.1 0.5 (−90%)*
23.0 4.4 (+32%)
12.8 6.5 (−14%)
18.0 9.4 (+21%)
1.45 0.31 (+14%)**
1.77 0.19 (+39%)*
1.83 0.54 (+44%)*
1.08 0.24 (−2%)**
0.68 0.43 (+17%)
c
a
a
a
a
3 mg/kg 2 (pred)
25.7 2.8 (+48%)*
29.3 6.9 (+68%)*
18.6 2.6 (+8%)
22.8 7.6 (+30%)
0.94 0.22 (+62%)*
1.18 0.94 (+103%)
0.25 0.18 (−36%)
0.55 0.72 (+36%)
3.17 2.84 (+703%)*
c
a
a
a
a
30 mg/kg 2 (pred)
48.6 41.5 (+226%)*
d
a
b
100 mg/kg (R)-18
NT
NT
NT
d
a
a
3 mg/kg 2 (pred)
30 mg/kg 2 (pred)
1.43 0.25 (+30%)*
d
a
a
a
a
29.9 6.1 (+71%)*
1.70 0.44 (+54%)*
a
b
c
d
*p < 0.05 vs vehicle. **p < 0.05 vs pred at 3 mg/kg group. From experiment 1: 1% methylcellulose vehicle. From experiment 2: 30% cremophor
vehicle.
allowed to warm to room temperature, at which time it was stirred for
12 h. The reaction mixture was then poured into ice water (20 mL)
and extracted with Et2O (4 × 30 mL). The combined extracts are
washed with brine (20 mL), dried over MgSO4, and concentrated. The
residual oil was subjected to flash silica gel chromatography using 5−
15% EtOAc in hexanes to give 6-(5-fluoro-2-methyl-phenyl)-6-methyl-
4-trifluoromethyl-hept-1-yn-4-ol (99% yield) which was carried on to
the next step without further purification.
Table 7. Bone Side Effects
CIA disease score
femur cortical thickness [mm]
mean SE
AUC SE
(% change vs vehicle)
compd and dose
(% change vs vehicle)
vehicle (1%
27.8 2.0
0.191 0.002
methylcellulose)
c
a
a
100 mg/kg (R)-21 12.0 2.0 (−57%)*
3 mg/kg 2 (pred)
0.179 0.002 (−6%)*
c
a
a
15.8 1.5 (−43%)*
6.2 1.3 (−78%)*
0.175 0.002 (−9%)*
The synthesis of enantiomerically pure 17a (>98% ee) has been
c
a
a
described elsewhere.16,22
30 mg/kg 2 (pred)
0.156 0.002 (−18%)*
vehicle (30%
cremophor)
21.9 1.6
0.189 0.005
6-(4-Fluoro-2-methoxy-phenyl)-6-methyl-4-trifluoromethyl-
hept-1-yn-4-ol (17c). Step 1: A mixture of 1-(4-fluoro-2-hydroxy-
phenyl)-ethanone (20 g, 0.13 mol), iodomethane (16 mL, 0.26 mol),
K2CO3 (25 g, 0.18 mmol), and DMF (100 mL) was stirred at room
temperature overnight. The mixture was poured into cold water (500
mL) and stirred for 15 min. The precipitate was collected by filtration,
washed with water (3 × 500 mL), and dried under vacuum for 1 h to
give 1-(4-fluoro-2-methoxy-phenyl)-ethanone (89% yield).
Step 2: A solution of 1-(4-fluoro-2-methoxy-phenyl)-ethanone
(19.4 g, 0.12 mol) in toluene (170 mL) was treated with methyl
cyanoacetate (15.2 mL, 0.17 mol), benzylamine (1.4 mL, 12 mmol),
and acetic acid (6.6 mL, 0.10 mol). The mixture was heated to reflux
with removal of water by Dean−Stark trap for 16 h. The solvent and
remaining cyanoacetate were distilled off under high vacuum. The
residue was flittered through a pad of silica gel using 10% EtOAc in a
1:1 mixture of CH2Cl2 and hexanes to give (E)-2-cyano-3-(4-fluoro-2-
methoxy-phenyl)-but-2-enoic acid methyl ester (90% yield).
Step 3: A slurry of CuI (20 g, 0.11 mol) in Et2O (350 mL) was
stirred by overhead stirring and cooled to 0 °C. Methyl lithium (1.6 M
in Et2O, 120 mL, 0.19 mol) was added over 15 min, and the resulting
mixture was allowed to stir at 0 °C for 10 min and then cooled to −15
°C. (E)-2-Cyano-3-(4-fluoro-2-methoxy-phenyl)-but-2-enoic acid
methyl ester (12.1 g, 0.05 mol) in Et2O (150 mL) was added over
20 min, and the reaction mixture was allowed to warm to room
temperature. After 2 h, the reaction mixture was cautiously poured into
aqueous NH4Cl solution. Then 1 N HCl was added and the mixture
was filtered through Celite and the layers separated. The aqueous layer
was extracted with Et2O (3 × 100 mL), and the combined organics
were washed with 1 N HCl (100 mL), brine (100 mL), and aqueous
NaHCO3 (3 × 75 mL), dried over anhydrous MgSO4, and
concentrated to give 2-cyano-3-(4-fluoro-2-methoxy-phenyl)-3-meth-
yl-butyric acid methyl ester (93% yield).
d
a
ab
,
100 mg/kg (R)-18 10.2 1.2 (−53%)*
3 mg/kg 2 (pred)
0.174 0.002 (−8%)*,**
0.165 0.003 (−13%)*
0.147 0.003 (−22%)*
d
a
a
a
9.9 1.5 (−55%)*
2.1 0.5 (−90%)*
a
30 mg/kg 2
d
(pred)
a
b
c
*p < 0.05 vs vehicle. **p < 0.05 vs pred at 3 mg/kg group. From
d
experiment 1: 1% methylcellulose vehicle. From experiment 2: 30%
cremophor vehicle.
Step 2: To a 0−5 °C mixture of 2,2,2-trifluoro-N-methoxy-N-
methylacetamide (3.0 g) in anhydrous Et2O (30 mL) was added 2-
methylpropenylmagnesium bromide in THF (42 mL of a 0.5 M
solution). The reaction was stirred at 0−5 °C for 0.5 h, warmed to
room temperature, and stirred overnight. The reaction was quenched
with satd aqueous NH4Cl and extracted with Et2O three times. The
organic layers were combined and washed with water and brine and
dried over MgSO4. Most of the volatiles were removed at atmospheric
pressure, and the resulting THF solution of 1,1,1-trifluoro-4-methyl-
pent-3-en-2-one was used without further purification.
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-pen-
tan-2-one (16a). To a 0−5 °C cold solution of 1,1,1-trifluoro-4-
methyl-pent-3-en-2-one (158 mmol, 75% in THF) and CuI (158
mmol) in Et2O (300 mL) was added (5-fluoro-2-methyl-phenyl)-
magnesium bromide solution (157 mL, 1 M in THF, 157 mmol). The
mixture was warmed to room temperature, stirred overnight, and
diluted with satd aqueous NH4Cl solution (200 mL) and EtOAc. The
organic layer was washed with water and brine and dried over MgSO4.
Removal of the volatiles in vacuo provided a residue which was
purified by flash silica gel chromatography using 0−5% EtOAc in
hexanes as the eluent to give 1,1,1-trifluoro-4-(5-fluoro-2-methyl-
phenyl)-4-methyl-pentan-2-one (46% yield).
Step 4: To 2-cyano-3-(4-fluoro-2-methoxy-phenyl)-3-methyl-buty-
ric acid methyl ester (12.0 g, 45 mmol) in DMSO (100 mL) were
added NaCl (7.48 g, 130 mmol) and water (6 mL). The mixture was
heated to reflux for 4 h. After cooling, brine (100 mL) was added and
the mixture was extracted with EtOAc (4 × 100 mL). The combined
organic extracts were washed with brine (4 × 100 mL), dried over
anhydrous MgSO4, and concentrated to give 3-(4-fluoro-2-methoxy-
phenyl)-3-methyl-butyronitrile (98% yield).
Step 5: A solution of 3-(4-fluoro-2-methoxy-phenyl)-3-methyl-
butyronitrile (9.2 g, 44 mmol) in CH2Cl2 (180 mL) was cooled to −40
°C. DIBAL-H (1 M in CH2Cl2, 91 mL, 91 mmol) was added over 10
min. The reaction mixture was allowed to warm to room temperature
and stirred for 16 h. The mixture was cautiously added to 1 N HCl and
6-(5-Fluoro-2-methyl-phenyl)-6-methyl-4-trifluoromethyl-
hept-1-yn-4-ol (17a). Aluminum amalgam was prepared from
aluminum foil (1.16 g, 14.4 mmol) and mercuric chloride (12 mg,
catalytic amount) in anhydrous THF (20 mL) by vigorously stirring
the mixture at room temperature for 1 h under an argon atmosphere.
A solution of propargyl bromide (4.80 mL, 80 wt % in toluene, 43.1
mmol) in anhydrous THF (25 mL) was slowly added to a stirred
suspension maintaining a temperature of 30−40 °C, and stirring at 40
°C was continued until a dark-gray solution was obtained (1 h). The
propargyl aluminum sesquibromide solution was added to a solution
of 1,1,1-trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-pentan-2-one
(3.74 g, 14.4 mmol) in anhydrous Et2O (150 mL) at −78 °C. The
reaction mixture was stirred at this temperature for 3 h and then was
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the phases separated. The aqueous layer was extracted with CH2Cl2 (2
× 100 mL), the combined organic extracts were washed with 1 N HCl
(100 mL), brine (2 × 100 mL), and NaHCO3 solution (100 mL),
dried over anhydrous MgSO4, filtered, and concentrated to give 3-(4-
fluoro-2-methoxy-phenyl)-3-methyl-butyraldehyde (88% yield).
Step 6: 3-(4-Fluoro-2-methoxy-phenyl)-3-methyl-butyraldehyde
(8.13 g, 39 mmol) was dissolved in THF (100 mL) and treated
with trifluoromethyl trimethylsilane (7.8 g, 55 mmol). Tetrabutylam-
monium fluoride (1 M in THF, 8.5 mL) was added dropwise. The
reaction mixture was stirred 30 min, and additional tetrabutylammo-
nium fluoride (1 M in THF, 34.5 mL) was added. The reaction
mixture was diluted with water and extracted with EtOAc (3 × 100
mL). The combined organic extracts were washed with 1 N HCl (3 ×
100 mL), brine (3 × 100 mL), and NaHCO3 solution (3 × 100 mL),
dried over MgSO4, filtered, and concentrated to give 1,1,1-trifluoro-4-
(4-fluoro-2-methoxy-phenyl)-4-methyl-pentan-2-ol (96% yield).
Step 7: To 1,1,1-trifluoro-4-(4-fluoro-2-methoxy-phenyl)-4-methyl-
pentan-2-ol (10.5 g, 38 mmol) in CH2Cl2 (180 mL) Dess−Martin
periodinane (22.5 g, 53 mmol) was added and the mixture allowed to
stir for 1 h. The reaction mixture was passed through a pad of silica
eluting with hexane to give 1,1,1-trifluoro-4-(4-fluoro-2-methoxy-
phenyl)-4-methyl-pentan-2-one as a pale-yellow crystalline solid
(81% yield).
Step 8: Aluminum foil (226 mg, 8.4 mmol) was cut into small pieces
and stirred with HgCl2 (5 mg, 0.02 mmol) and THF (5 mL) at room
temperature for 1 h. Propargylbromide (80% in toluene, 0.94 mL, 8.4
mmol) was added dropwise, and the mixture was stirred for 2 h at 40
°C. This suspension was added to a solution of 1,1,1-trifluoro-4-(4-
fluoro-2-methoxy-phenyl)-4-methyl-pentan-2-one (1.00 g, 3.6 mmol)
in Et2O (20 mL) at −78 °C. The mixture was warmed to room
temperature. After 2 h, NH4Cl solution (20 mL) and EtOAc (50 mL)
were added. The organic phase was separated, and the aqueous layer
was extracted with EtOAc (100 mL). The combined organic phases
were dried over MgSO4. The solvent was removed to give 17c as an oil
that was used as such (96% yield).
6-(2-Methanesulfonyl-phenyl)-6-methyl-4-trifluoromethyl-
hept-1-yn-4-ol (17d). Step 1: Magnesium turnings (290 mg, 11.8
mmol) under THF (20 mL) were treated with 1-bromo-2-
methylsulfanyl-benzene (2.00 g, 9.85 mmol) in one portion, and the
resulting mixture was heated to 90 °C for 3 h. The reaction was cooled
to −10 °C. With stirring, CuI (2.06 g, 10.8 mmol) was added and the
resulting mixture was stirred at that temperature for 1 h. 1,1,1-
Trifluoro-4-methyl-pent-3-en-2-one (2.75 g, 60% in THF, 10.8 mmol)
was added, and the mixture was allowed to warm to room temperature
and stirred for 16 h. The reaction was poured into rapidly stirring satd
aqueous NH4Cl solution, diluted with Et2O, and the layers were
separated. The aqueous layer was extracted with Et2O (2 × 50 mL).
The combined organic phases were dried over MgSO4, filtered, and
concentrated. The resulting oil was purified by flash chromatography
on silica gel using 0−10% EtOAc in hexanes to give 1,1,1-trifluoro-4-
methyl-4-(2-methylsulfanyl-phenyl)-pentan-2-one as an orange oil
(42% yield).
Step 2: To a solution of 1,1,1-trifluoro-4-methyl-4-(2-methylsulfan-
yl-phenyl)-pentan-2-one (1.14 g, 4.13 mmol) in CH3CN/water (3:1,
80 mL) was added NaIO4 (2.65 g, 12.4 mmol), followed by the
addition of RuCl3 (17 mg, 0.08 mmol), and the reaction was stirred at
room temperature. After 30 min, the reaction was diluted with water
and extracted with CH2Cl2 (3 × 50 mL). The organic phases were
combined, dried over MgSO4, and concentrated. The black residue
was purified by flash chromatography on silica gel using 30−100%
EtOAc in hexanes to give 1,1,1-trifluoro-4-(2-methanesulfonyl-
phenyl)-4-methyl-pentan-2-one as a colorless oil (74% yield).
Step 3: Aluminum foil (250 mg, 9.2 mmol) was cut into small pieces
and stirred with HgCl2 (17 mg, 0.06 mmol) and THF (6 mL) at 0 °C
for 10 min. Propargylbromide (80% in toluene, 1.03 mL, 9.2 mmol)
was added dropwise, and the mixture was stirred for 1 h at 50 °C. This
suspension was added to a solution of 1,1,1-trifluoro-4-(2-
methanesulfonyl-phenyl)-4-methyl-pentan-2-one (947 mg, 3.07
mmol) in THF (10 mL) at −78 °C. The mixture was warmed to
room temperature. After 2 h, NH4Cl solution (10 mL) and Et2O (50
mL) were added. The organic phase was separated, and the aqueous
layer was extracted with Et2O (3 × 100 mL). The combined organic
phases were dried over Na2SO4 and concentrated. The residue was
purified by flash chromatography using 0−30% EtOAc in hexanes to
give 17d as a yellow oil (94% yield).
6-(3-Methanesulfonyl-phenyl)-6-methyl-4-trifluoromethyl-
hept-1-yn-4-ol (17e). 17e was prepared in analogy to 17d in three
steps in an overall yield of 31% using 1-bromo-3-methylsulfanyl-
benzene instead of 1-bromo-2-methylsulfanyl-benzene in step 1.
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-
(5H-pyrrolo[3,2-c]pyridazin-6-ylmethyl)-pentan-2-ol (18) and
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-(1H-
pyrrolo[2,3-d]pyridazin-2-ylmethyl)-pentan-2-ol (19). Following
the general Sonogashira coupling procedure starting from 17a and a
1:1 mixture of 9 and 1019 gave a 1:1 mixture of 1-(4-amino-pyridazin-
3-yl)-6-(5-fluoro-2-methyl-phenyl)-6-methyl-4-trifluoromethyl-hept-1-
yn-4-ol and 1-(5-amino-pyridazin-4-yl)-6-(5-fluoro-2-methyl-phenyl)-
6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (yield 44%).
Following the general cyclization procedure A starting from a 1:1
mixture of 1-(4-amino-pyridazin-3-yl)-6-(5-fluoro-2-methyl-phenyl)-6-
methyl-4-trifluoromethyl-hept-1-yn-4-ol and 1-(5-amino-pyridazin-4-
yl)-6-(5-fluoro-2-methyl-phenyl)-6-methyl-4-trifluoromethyl-hept-1-
yn-4-ol gave 18 (less polar product) as a white solid (16% yield) and
19 (more polar product) as a white solid (35% yield).
18: 1H NMR (400 MHz, MeOH-d4) δ 8.74 (d, J = 6 Hz, 1H), 7.55
(dd, J = 6 Hz, J = 1 Hz, 1H), 7.18 (dd, J = 12 Hz, J = 1 Hz, 1H), 7.07−
7.10 (m, 1H), 6.78−6.82 (m, 1H), 6.49 (s, 1H), 2.88 (d, J = 15 Hz,
1H), 2.77 (d, J = 15 Hz, 1H), 2.62 (d, J = 16 Hz, 1H), 2.50 (s, 3H),
2.13 (d, J = 16 Hz, 1H), 1.70 (s, 3H), 1.46 (s, 3H).
19: 1H NMR (400 MHz, MeOH-d4) δ 9.18 (d, J = 1 Hz, 1H), 9.11
(s, 1H), 7.17 (dd, J = 12 Hz, J = 2 Hz, 1H), 7.07−7.11 (m, 1H), 6.78−
6.82 (m, 1H), 6.37 (s, 1H), 2.87 (d, J = 15 Hz, 1H), 2.74 (d, J = 15
Hz, 1H), 2.61 (d, J = 15 Hz, 1H), 2.50 (s, 3H), 2.12 (d, J = 16 Hz,
1H), 1.70 (s, 3H), 1.45 (s, 3H).
The pure enantiomer (R)-18 was synthesized following the same
sequence outlined in Scheme 1 using enantiopure alkyne 11a.16,22
Chiral HPLC (Chiral APG 150X column, 4.0 mm, 5 μm; mobile phase
20 nM NH4OAc/acetonitrile 82:18 isocratic) rf 5.07 min, (S)-18 7.62
min; ee 99.6%. HRMS: (M + H)+ 396.1700 (calcd 396.1704).
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-(3-
phenyl-7H-pyrrolo[2,3-c]pyridazin-6-ylmethyl)-pentan-2-ol
(20). Following the general Sonogashira coupling procedure starting
from 17a and 1120 gave 1-(3-amino-6-phenyl-pyridazin-4-yl)-6-(5-
fluoro-2-methyl-phenyl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol
(32% yield).
Following the general cyclization procedure A starting from 1-(3-
amino-6-phenyl-pyridazin-4-yl)-6-(5-fluoro-2-methyl-phenyl)-6-meth-
yl-4-trifluoromethyl-hept-1-yn-4-ol gave 20 after recrystallization from
1
Et2O as a white solid (27% yield). H NMR (400 MHz, MeOH-d4) δ
8.08 (s, 1H), 7.96−7.98 (m, 2H), 7.50−7.54 (m, 2H), 7.43−7.47 (m,
1H), 7.21 (dd, J = 12 Hz, J = 2 Hz, 1H), 7.10 (dd, J = 8 Hz, J = 6 Hz,
1H), 6.83 (td, J = 8 Hz, J = 2 Hz, 1H), 6.31 (s, 1H), 2.95 (d, J = 15 Hz,
1H), 2.77 (d, J = 16 Hz, 1H), 2.68 (d, J = 15 Hz, 1H), 2.52 (s, 3H),
2.19 (d, J = 16 Hz, 1H), 1.49 (s, 3H), 1.19 (s, 3H).
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-
(5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pentan-2-ol (21). Fol-
lowing the general Sonogashira coupling procedure starting from 17a
and 12 gave 1-(5-amino-pyrimidin-4-yl)-6-(5-fluoro-2-methyl-phenyl)-
6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (47% yield).
Following the general cyclization procedure A starting from 1-(5-
amino-pyrimidin-4-yl)-6-(5-fluoro-2-methyl-phenyl)-6-methyl-4-tri-
1
fluoromethyl-hept-1-yn-4-ol gave 21 as a white solid (15% yield). H
NMR (400 MHz, MeOH-d4) δ 8.70 (s, 1H), 8.67 (s, 1H), 7.17 (dd, J
= 12 Hz, J = 2 Hz, 1H), 7.06−7.10 (m, 1H), 6.79−6.83 (m, 1H), 6.29
(s, 1H), 2.90 (d, J = 15 Hz, 1H), 2.75 (d, J = 15 Hz, 1H), 2.63 (d, J =
15 Hz, 1H), 2.50 (s, 3H), 2.13 (d, J = 16 Hz, 1H), 1.70 (s, 3H), 1.45
(s, 3H).
The pure enantiomer (R)-21 was synthesized following the same
sequence outlined in Scheme 1 using enantiopure alkyne 11a.16,22
Chiral HPLC (Chiral APG 150X column, 4.0 mm, 5 μm; mobile phase
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20 nM NH4OAc/acetonitrile 82:18 isocratic) rf 4.65 min, (S)-21 7.03
min; ee 99.4%. HRMS: (M + H)+ 396.1704 (calcd 396.1704).
Elemental analysis: C, 60.67% (calcd 60.75%); H, 5.38% (calcd
5.35%); N, 10.35% (calcd 10.63%); F, 19.38% (calcd 19.22%).
1,1,1-Trifluoro-4-(5-fluoro-2-methoxy-phenyl)-4-methyl-2-
(7H-pyrrolo[2,3-d]pyrimidin-6-ylmethyl)-pentan-2-ol (22). Step
1: Following the general Sonogashira coupling procedure starting from
17b16 and 1219 gave 1-(4-amino-pyrimidin-5-yl)-6-(5-fluoro-2-me-
thoxy-phenyl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (50% yield).
Step 2: 1-(4-Amino-pyrimidin-5-yl)-6-(5-fluoro-2-methoxy-phenyl)-
6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (150 mg, 0.37 mmol) was
dissolved in CH2Cl2 (5 mL). At 0 °C, pyridine (0.09 g, 1.1 mmol) was
added, followed by slow addition of acetyl chloride (0.06 g, 0.73
mmol), and the mixture was stirred for 1 h. The reaction mixture was
diluted with CH2Cl2 and washed with brine (3 × 10 mL). The residue
was purified by flash chromatography on silica gel using 3% MeOH in
CH2Cl2 to give slightly impure N-{5-[6-(5-fluoro-2-methoxy-phenyl)-
4-hydroxy-6-methyl-4-trifluoromethyl-hept-1-ynyl]-pyrimidin-4-yl}-
acetamide (75% yield).
7.84−7.86 (m, 2H), 7.38−7.42 (m, 2H), 7.30−7.34 (m, 1H), 7.08 (dd,
J = 12 Hz, J = 3 Hz, 1H), 6.95 (dd, J = 8 Hz, J = 7 Hz, 1H), 6.70 (td, J
= 8 Hz, J = 3 Hz, 1H), 6.26 (s, 1H), 2.85 (d, J = 15 Hz, 1H), 2.62 (d, J
= 16 Hz, 1H), 2.60 (d, J = 15 Hz, 1H), 2.39 (s, 3H), 2.08 (d, J = 16
Hz, 1H), 1.58 (s, 3H), 1.37 (s, 3H).
N-(4-Chloro-2-pyridin-3-yl-pyrimidin-5-yl)-2,2,2-trifluoro-
acetamide (25c). Step 1: Ethylnitro acetate (5.6 mL, 50 mmol) and
N,N-dimethylformamide dimethyl acetal (6.6 mL, 50 mmol) were
stirred for 45 min at room temperature and then heated to 90 °C for
15 min. After cooling, the obtained (Z)-3-dimethylamino-2-nitro-
acrylic acid ethyl ester was used directly in the next step.
Step 2: A mixture of (Z)-3-dimethylamino-2-nitro-acrylic acid ethyl
ester (9.4 g, 50 mmol), 3-pyridyl amidine hydrochloride (4.7 g, 30
mmol), and K2CO3 (4.1 g, 30 mmol) in anhydrous ethanol (30 mL)
was heated to 90 °C for 8 h. A yellowish solid precipitated from the
reaction mixture which was filtered, washed with Et2O, and dried to
give 5-nitro-2-pyridin-3-yl-3H-pyrimidin-4-one as a yellowish solid
(92% yield). This material contained residual potassium salts and was
used as such in the next step.
Step 3: N-{5-[6-(5-fluoro-2-methoxy-phenyl)-4-hydroxy-6-methyl-
4-trifluoromethyl-hept-1-ynyl]-pyrimidin-4-yl}-acetamide (125 mg,
0.28 mmol) was dissolved in DMSO (0.5 mL) and treated with
tetramethylguanidine (0.10 g, 0.83 mmol). The mixture was heated in
the microwave at 140 °C for 10 min. The reaction mixture was then
diluted with EtOAc (10 mL) and washed with water (3 × 5 mL). The
organic phase was separated, dried over MgSO4, filtered, and
concentrated. The residue was purified by reversed phase HPLC
using 35−100% CH3CN in water to give 22 as a white solid (23%
yield). 1H NMR (400 MHz, CHCl3-d) δ 9.31 (br s, 1H), 8.87 (s, 1H),
8.78 (s, 1H), 7.02 (dd, J = 10 Hz, J = 2 Hz, 1H), 6.91−6.94 (m, 1H),
6.80−6.83 (m, 1H), 6.26 (s, 1H), 3.74 (s, 3H), 2.98 (s, 2H), 2.49 (s,
2H), 1.49 (s, 3H), 1.38 (s, 3H). No analytical HPLC data.
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-
(5H-pyrrolo[2,3-b]pyrazin-6-ylmethyl)-pentan-2-ol (23). Follow-
ing the general Sonogashira coupling procedure starting from 17a and
13a21 gave 1-(3-amino-pyrazin-2-yl)-6-(5-fluoro-2-methyl-phenyl)-6-
methyl-4-trifluoromethyl-hept-1-yn-4-ol (54% yield).
Following the general cyclization procedure A starting from 1-(3-
amino-pyrazin-2-yl)-6-(5-fluoro-2-methyl-phenyl)-6-methyl-4-trifluor-
omethyl-hept-1-yn-4-ol gave 23 as a white solid (18% yield). 1H NMR
(400 MHz, MeOH-d4) δ 8.14 (d, J = 3 Hz, 1H), 8.04 (d, J = 3 Hz,
1H), 7.07 (dd, J = 12 Hz, J = 3 Hz, 1H), 6.94 (dd, J = 8 Hz, J = 7 Hz,
1H), 6.69 (td, J = 8 Hz, J = 3 Hz, 1H), 6.21 (s, 1H), 2.82 (d, J = 15 Hz,
1H), 2.60 (d, J = 16 Hz, 1H), 2.59 (d, J = 15 Hz, 1H), 2.38 (s, 3H),
2.06 (d, J = 16 Hz, 1H), 1.57 (s, 3H), 1.36 (s, 3H).
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-(2-
phenyl-5H-pyrrolo[2,3-b]pyrazin-6-ylmethyl)-pentan-2-ol
(24b). Following the general Sonogashira coupling procedure starting
from 17a and 13b gave 1-(3-amino-6-bromo-pyrazin-2-yl)-6-(5-fluoro-
2-methyl-phenyl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (75%
yield).
Following the general cyclization procedure A starting from 1-(3-
amino-6-bromo-pyrazin-2-yl)-6-(5-fluoro-2-methyl-phenyl)-6-methyl-
4-trifluoromethyl-hept-1-yn-4-ol gave 2-(2-bromo-5H-pyrrolo[2,3-b]-
pyrazin-6-ylmethyl)-1,1,1-trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-
methyl-pentan-2-ol (36% yield).
Step 3: A stirred solution of DMF (6.8 mL, 88 mmol) in CH2Cl2
(100 mL) was cooled to 0 °C. POCl3 (8.2 mL, 88 mmol) in CH2Cl2
(50 mL) was added, and stirring was continued for 30 min. 5-Nitro-2-
pyridin-3-yl-3H-pyrimidin-4-one (5.4 g, 70%, 18 mmol) was added
and the mixture stirred for 1 h. The reaction mixture was diluted with
CH2Cl2 (200 mL), treated with ice and solid KHCO3. The organic
layer was separated, washed with water, dried over anhydrous Na2SO4,
and concentrated to give a reddish oil. This material was taken up in
cold water, a solid precipitated which was filtered, washed with water,
and dried to give 4-chloro-5-nitro-2-pyridin-3-yl-pyrimidine as an
orange solid (41% yield).
Step 4: 4-Chloro-5-nitro-2-pyridin-3-yl-pyrimidine (480 mg, 2
mmol) was stirred in concentrated HCl (6 mL) at room temperature
until a clear solution was obtained. Acetic acid (3 mL) was added.
Tin(II) chloride dihydrate (1.8 g, 8.0 mmol) in 3 mL of concentrated
HCl was slowly added over a period of 5 min. The reaction mixture
was stirred at room temperature for 18 h. At 0 °C, the reaction mixture
was treated with 10 N KOH solution until the pH of the reaction
mixture was 8−9. The mixture was extracted with EtOAc (4 × 75 mL).
The combined extracts were dried over anhydrous Na2SO4 and solvent
evaporated to give 4-chloro-2-pyridin-3-yl-pyrimidin-5-ylamine as a
light-yellow thick oil that was used as was in the next step.
Step 5: To a solution of 4-chloro-2-pyridin-3-yl-pyrimidin-5-ylamine
(413 mg, 2 mmol) in CH2Cl2 (8 mL) at room temperature was added
a solution of trifloroacetic anhydride (0.56 mL, 4 mmol) in CH2Cl2 (2
mL). The reaction mixture was stirred for 30 min. The solvent was
removed to give a thick oil that was purified by flash chromatography
on silica gel using 0−100% EtOAc in CH2Cl2 to give 25c as an off-
white crystalline solid (56% yield over two steps).
2,2,2-Trifluoro-N-(4-iodo-2-pyrrolidin-1-yl-pyrimidin-5-yl)-
acetamide (25d). Step 1 is identical to step 1 described for 25c. Step
2: To a stirred solution of (Z)-3-dimethylamino-2-nitro-acrylic acid
ethyl ester (3.5 g, 20 mmol) in trifluoroethanol (50 mL) was added
pyrrolidinoamidine hydrochloride (4.8 g, 20 mmol) followed by
triethylamine (2.0 mL, 20 mmol), and the mixture was heated to 90
°C for 6 h. After cooling, the reaction mixture was concentrated and
the residue was purified by flash chromatography using 5% MeOH in
CH2Cl2 to give 5-nitro-2-pyrrolidin-1-yl-3H-pyrimidin-4-one as a light-
yellow solid (50% yield).
Step 3: 2-(2-Bromo-5H-pyrrolo[2,3-b]pyrazin-6-ylmethyl)-1,1,1-
trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-pentan-2-ol (50 mg,
0.11 mmol) was dissolved in DME/MeOH/DMF (1 mL, 0.5 mL, 1.5
mL). Pd(PPh3)4 (12 mg, 0.01 mmol), phenylboronic acid (26 mg,
0.22 mmol), and K2CO3 (43 mg, 0.33 mmol) were added, and the
mixture was heated for 3 h to 80 °C. NaOH (1 M, 5 mL) was added.
The reaction mixture was diluted with EtOAc (20 mL). The organic
phase was separated, and the aqueous phase was extracted with EtOAc
(3 × 20 mL). The combined organic extracts were dried over MgSO4
and the solvent evaporated. The residue was purified by flash
chromatography on silica gel using 0−50% hexanes in EtOAc to give
impure product as a solid. The material was repurified by preparative
TLC (50% hexanes in EtOAc) to give pure product 24b as a white
Step 3: A stirred solution of DMF (1.6 mL, 20 mmol) in CH2Cl2
(20 mL) was cooled to 0 °C. POCl3 (1.9 mL, 20 mmol) was added,
and stirring was continued for 30 min. 5-Nitro-2-pyrrolidin-1-yl-3H-
pyrimidin-4-one (2.1 g, 10 mmol) was added and the mixture stirred
for 10 min. The reaction mixture was diluted with CH2Cl2 (100 mL)
and treated with ice and solid KHCO3. The organic layer was
separated, washed with water, dried over anhydrous Na2SO4, and
concentrated to give a brown oil that was purified by flash
chromatography on silica gel using 25% EtOAc in hexanes to give
4-chloro-5-nitro-2-pyrrolidin-1-yl-pyrimidine as an off-white solid
(36% yield).
1
solid (71% yield). H NMR (400 MHz, MeOH-d4) δ 8.48 (s, 1H),
1593
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Step 4: To 4-chloro-5-nitro-2-pyrrolidin-1-yl-pyrimidine (0.82 g, 3.6
mmol) in EtOH (15 mL) and glacial acetic acid (7 mL) was added
iron powder (2.0 g, 36 mmol), and the mixture was heated to 90 °C
for 20 min. After cooling to room temperature, the reaction mixture
was diluted with CH2Cl2 (250 mL) and filtered through Celite. The
filtrate was washed with NaHCO3 solution (50 mL) and water (50
mL), dried over anhydrous Na2SO4, and the solvent was evaporated to
give 4-chloro-2-pyrrolidin-1-yl-pyrimidin-5-ylamine as a bluish thick oil
that was used for the next reaction as is (84% yield).
Step 5: To a stirred mixture of 4-chloro-2-pyrrolidin-1-yl-pyrimidin-
5-ylamine (0.60 g, 3.0 mmol) in 40% aqueous hydroiodic acid (15 mL,
120 mmol) was added NaI (2.26 g, 15 mmol). The mixture was stirred
at room temperature for 4 h and then poured into EtOAc (200 mL)
and washed with aqueous Na2CO3 solution, followed by sodium
bisulfite solution and water. The organic layer was dried over
anhydrous Na2SO4 and concentrated to give 4-iodo-2-pyrrolidin-1-yl-
pyrimidin-5-ylamine as an oil which slowly solidified (97% yield).
Step 6: To a stirred solution of 4-iodo-2-pyrrolidin-1-yl-pyrimidin-5-
ylamine (850 mg, 2.9 mmol) in CH2Cl2 (5 mL) at room temperature
was added a solution of trifluoroacetic anhydride (0.62 mL, 4.4 mmol)
in CH2Cl2 (2 mL) After 15 min, the solvent was evaporated to give
25d as a brownish solid that was used as is in the next reaction (93%
yield).
2,2,2-Trifluoro-N-(4-chloro-2-morpholin-4-yl-pyrimidin-5-
yl)-acetamide (25e). 25e was prepared in analogy to 25c in five steps
in 15% overall yield from ethylnitroacetate and dimethyl formamide
dimethyl acetal using morpholinoamidine hydrochloride instead of 3-
pyridyl amidine hydrochloride in step 2.
N-[4-Chloro-2-(1,1-dioxo-1λ6-thiomorpholin-4-yl)-pyrimi-
din-5-yl]-2,2,2-trifluoro-acetamide (25f). Step 1: 2-Chloro-5-
nitro-pyrimidine (4.00 g, 25 mmol) was stirred in CH3CN (40 mL)
and THF (10 mL) until a clear solution was obtained. Thiomorpho-
line (2.06 g, 25 mmol) in CH3CN (5 mL) was added. A yellowish
solid precipitated. After 10 min, a solution of NaHCO3 (26 mmol) in
water was added and the reaction mixture was stirred at room
temperature for 45 min. The solvent was removed and the residue
taken up in cold water. The solid was collected by filtration and dried
to give 4-(5-nitro-pyrimidin-2-yl)-thiomorpholine as a yellowish solid
(99% yield).
Step 2: 4-(5-Nitro-pyrimidin-2-yl)-thiomorpholine (2.7 g, 12
mmol) was stirred in concentrated HCl (10 mL) at room temperature
until a clear solution was obtained. Tin(II) chloride (10.8 g, 48 mmol)
in concentrated HCl (10 mL) was added over a period of 15 min. A
yellow solid precipitated. The reaction mixture was diluted with water
(300 mL) and EtOAc (400 mL). The stirred mixture was neutralized
with solid NaHCO3 until the pH was 6−7 and filtered. The organic
fraction of the filtrate was separated. The aqueous fraction was
extracted with EtOAc (2 × 150 mL). The combined organic fractions
were washed with brine, dried over anhydrous Na2SO4, and the solvent
evaporated to give a reddish thick oil which was purified by flash
chromatography on silica gel using 0−100% EtOAc in hexanes to give
4-chloro-2-thiomorpholin-4-yl-pyrimidin-5-ylamine (20% yield) and 2-
thiomorpholin-4-yl-pyrimidin-5-ylamine (14% yield).
Step 3: To a solution of 4-chloro-2-thiomorpholin-4-yl-pyrimidin-5-
ylamine (540 mg, 2.3 mmol) in CH2Cl2 (7 mL) at 0 °C was added a
solution of the trifloroacetic anhydride (0.42 mL, 3.0 mmol) in
CH2Cl2 (3 mL). The reaction mixture was allowed to stand at room
temperature. After 30 min, the solvent was removed to give a thick oil
that was purified by flash chromatography on silica gel using 0−30%
EtOAc in hexanes to give N-(4-chloro-2-thiomorpholin-4-yl-pyrimi-
din-5-yl)-2,2,2-trifluoro-acetamide as a light-yellow solid (75% yield).
Step 4: To a solution of N-(4-chloro-2-thiomorpholin-4-yl-
pyrimidin-5-yl)-2,2,2-trifluoro-acetamide (570 mg, 1.7 mmol) in
CH3CN (10 mL) was added a solution of NaIO4 (0.93 g, 4.4
mmol) in water (3 mL), and the reaction mixture was stirred at room
temperature. After 1 h, RuCl3 hydrate (15 mg, 0.1 mmol) was added
and the reaction mixture was stirred for 15 min. The reaction was
diluted with water and extracted with CH2Cl2. The combined organic
extracts were dried over Na2SO4 and concentrated. The residue was
filtered through Celite and concentrated to give 25f as a thick oil (67%
yield).
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-2-(2-isopropyl-
5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-4-methyl-pentan-2-ol
(26). Following the general Sonogashira coupling procedure starting
from 17a and 25a19 gave 2,2,2-trifluoro-N-{4-[6-(5-fluoro-2-methyl-
phenyl)-4-hydroxy-6-methyl-4-trifluoromethyl-hept-1-ynyl]-2-isoprop-
yl-pyrimidin-5-yl}-acetamide (38% yield).
Following the general cyclization procedure B starting from 2,2,2-
trifluoro-N-{4-[6-(5-fluoro-2-methyl-phenyl)-4-hydroxy-6-methyl-4-
trifluoromethyl-hept-1-ynyl]-2-isopropyl-pyrimidin-5-yl}-acetamide
gave 27 as a white solid (33% yield). 1H NMR (400 MHz, DMSO-d6)
δ 11.17 (s, 1H), 8.68 (d, J = 1 Hz, 1H), 7.09−7.14 (m, 2H), 6.91 (td, J
= 8 Hz, J = 3 Hz, 1H), 6.22 (s, 1H), 6.15 (s, 1H), 3.12 (sept, J = 7 Hz,
1 H), 2.84 (d, J = 15 Hz, 1H), 2.65 (d, J = 15 Hz, 1H), 2.48 (d, J = 15
Hz, 1H), 2.43 (s, 3H), 2.15 (d, J = 15 Hz, 1H), 1.57 (s, 3H), 1.40 (s,
3H), 1.26 (d, J = 7 Hz, 6H).
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-(2-
phenyl-5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pentan-2-ol
(27). Following the general Sonogashira coupling procedure starting
from 17a and 25b19 gave 2,2,2-trifluoro-N-{4-[6-(5-fluoro-2-methyl-
phenyl)-4-hydroxy-6-methyl-4-trifluoromethyl-hept-1-ynyl]-2-phenyl-
pyrimidin-5-yl}-acetamide (56% yield).
Following the general cyclization procedure B starting from 2,2,2-
trifluoro-N-{4-[6-(5-fluoro-2-methyl-phenyl)-4-hydroxy-6-methyl-4-
trifluoromethyl-hept-1-ynyl]-2-phenyl-pyrimidin-5-yl}-acetamide gave
1
27 as a white solid (60% yield). H NMR (400 MHz, DMSO-d6) δ
11.51 (s, 1H), 8.98 (s, 1H), 8.46−8.48 (m, 2H), 7.45−7.55 (m, 3H),
7.16−7.22 (m, 2H), 6.97 (td, J = 8 Hz, J = 3 Hz, 1H), 6.44 (s, 1H),
6.31 (s, 1H), 2.96 (d, J = 15 Hz, 1H), 2.77 (d, J = 15 Hz, 1H), 2.56−
2.60 (m, 1H), 2.50 (s, 3H), 2.23 (d, J = 15 Hz, 1H), 1.64 (s, 3H), 1.48
(s, 3H).
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-(2-
pyridin-3-yl-5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pentan-2-
ol (28). A solution of the alkyne 17a (151 mg, 0.50 mmol) and the
chloropyrimidine 25c (91 mg, 0.30 mmol) in CH3CN (0.7 mL) and
triethylamine (0.2 mL) was degassed by bubbling argon through for 5
min. Pd(PPh3)2Cl2 (21 mg, 0.03 mmol) and CuI (5.7 mg, 0.03 mmol)
were added. The reaction mixture was heated to 70 °C for 2 h. The
reaction mixture was subjected to flash chromatography on silica gel
using 0−100% EtOAc in CH2Cl2 to give 28 as an off-white solid (57%
yield). 1H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 9.53 (s, 1H),
8.91 (s, 1H), 8.67 (td, J = 4 Hz, J = 2 Hz, 1H), 8.61 (d, J = 4 Hz, 1H),
7.50 (dd, J = 8 Hz, J = 5 Hz, 1H), 7.11−7.16 (m, 2H), 6.91 (td, J = 8
Hz, J = 3 Hz, 1H), 6.42 (s, 1H), 6.24 (s, 1H), 2.89 (d, J = 15 Hz, 1H),
2.71 (d, J = 15 Hz, 1H), 2.50−2.54 (m, 1H), 2.44 (s, 3H), 2.16 (d, J =
15 Hz, 1H), 1.58 (s, 3H), 1.41 (s, 3H).
1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-(2-
pyrrolidin-1-yl-5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pen-
tan-2-ol (29). Following the general Sonogashira coupling procedure
starting from 17a and 25d gave 2,2,2-trifluoro-N-{4-[6-(5-fluoro-2-
methyl-phenyl)-4-hydroxy-6-methyl-4-trifluoromethyl-hept-1-ynyl]-2-
pyrrolidin-1-yl-pyrimidin-5-yl}-acetamide (52% yield).
Following the general cyclization procedure B starting from 2,2,2-
trifluoro-N-{4-[6-(5-fluoro-2-methyl-phenyl)-4-hydroxy-6-methyl-4-
trifluoromethyl-hept-1-ynyl]-2-pyrrolidin-1-yl-pyrimidin-5-yl}-acet-
1
amide gave 29 as a (36% yield). H NMR (400 MHz, DMSO-d6) δ
10.54 (s, 1H), 8.40 (s, 1H), 7.08−7.13 (m, 2H), 6.89−6.92 (m, 1H),
6.04 (s, 1H), 5.96 (s, 1H), 3.44−3.47 (m, 4H), 2.79 (d, J = 15 Hz,
1H), 2.64 (d, J = 15 Hz, 1H), 2.41 (s, 3H), 2.38−2.41 (m, 1H), 2.15
(d, J = 16 Hz, 1H), 1.89−1.91 (m, 4H), 1.52 (s, 3H), 1.40 (s, 3H).
(R)-1,1,1-Trifluoro-4-(5-fluoro-2-methyl-phenyl)-4-methyl-2-
(2-morpholin-4-yl-5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-
pentan-2-ol (30). Following the general Sonogashira coupling
procedure starting from 17a and 25e gave 2,2,2-trifluoro-N-{4-[(S)-
6-(5-fluoro-2-methyl-phenyl)-4-hydroxy-6-methyl-4-trifluoromethyl-
hept-1-ynyl]-2-morpholin-4-yl-pyrimidin-5-yl}-acetamide (46% yield).
Following the general cyclization procedure B starting from 2,2,2-
trifluoro-N-{4-[(S)-6-(5-fluoro-2-methyl-phenyl)-4-hydroxy-6-methyl-
4-trifluoromethyl-hept-1-ynyl]-2-morpholin-4-yl-pyrimidin-5-yl}-acet-
1
amide gave 30 as a (64% yield). H NMR (400 MHz, DMSO-d6) δ
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10.83 (s, 1H), 8.44 (s, 1H), 7.08−7.12 (m, 2H), 6.89 (td, J = 8 Hz, J =
3 Hz, 1H), 6.06 (s, 1H), 5.99 (s, 1H), 3.57−3.66 (m, 8H), 2.79 (d, J =
15 Hz, 1H), 2.62 (d, J = 15 Hz, 1H), 2.41 (d, J = 15 Hz, 1H), 2.41 (s,
3H), 2.13 (d, J = 15 Hz, 1H), 1.51 (s, 3H), 1.39 (s, 3H).
Hz, 2H), 2.86 (d, J = 16 Hz, 2H), 2.59 (d, J = 15 Hz, 1H), 2.59 (d, J =
15 Hz, 1H), 1.50 (s, 3H), 1.27 (s, 3H).
4-(5-Chloro-2,3-dihydro-benzofuran-7-yl)-1,1,1-trifluoro-4-
methyl-2-(5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pentan-2-ol
(37). Following the general Sonogashira coupling procedure starting
from 17g16 and 819 gave 1-(5-amino-pyrimidin-4-yl)-6-(5-chloro-2,3-
dihydro-benzofuran-7-yl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol
(24% yield).
2-[2-(1,1-Dioxo-1λ6-thiomorpholin-4-yl)-5H-pyrrolo[3,2-d]-
pyrimidin-6-ylmethyl]-1,1,1-trifluoro-4-(5-fluoro-2-methyl-
phenyl)-4-methyl-pentan-2-ol (31). A solution of the alkyne 17a
(151 mg, 0.50 mmol), the chloro pyrimidine 25f (90 mg, 0.25 mmol),
and triethylamine (0.17 mL, 1.25 mmol) in CH3CN (0.8 mL) was
degassed by bubbling argon through for 5 min. Pd(PPh3)2Cl2 (18 mg,
0.03 mmol) and CuI (4.7 mg, 0.03 mmol) were added. The reaction
mixture was heated to 70 °C for 2 h. The reaction mixture was
subjected to flash chromatography on silica gel using 0−100% EtOAc
in CH2Cl2 to give 31 as an off-white solid (72% yield). 1H NMR (400
MHz, DMSO-d6) δ 11.01 (s, 1H), 8.55 (s, 1H), 7.16−7.20 (m, 2H),
6.97 (td, J = 8 Hz, J = 3 Hz, 1H), 6.18 (s, 1H), 6.10 (s, 1H), 4.25 (br s,
4H), 3.13 (br s, 4H) 2.86 (d, J = 15 Hz, 1H), 2.67 (d, J = 15 Hz, 1H),
2.54−2.56 (m, 1H), 2.49 (s, 3H), 2.20 (d, J = 15 Hz, 1H), 1.62 (s,
3H), 1.47 (s, 3H).
Following the general cyclization procedure A starting from 1-(5-
amino-pyrimidin-4-yl)-6-(5-chloro-2,3-dihydro-benzofuran-7-yl)-6-
methyl-4-trifluoromethyl-hept-1-yn-4-ol gave 37 as a white solid (50%
1
yield). H NMR (400 MHz, MeOH-d4) δ 8.70 (s, 1H), 8.68 (s, 1H),
7.04−7.05 (m, 2H), 6.32 (s, 1H), 4.55 (t, J = 8 Hz, 2H), 3.11 (t, J = 9
Hz, 2H), 2.93 (2d, J = 16 Hz, 2H), 2.71 (d, J = 15 Hz, 1H), 2.08 (d, J
= 15 Hz, 1H), 1.59 (s, 3H), 1.36 (s, 3H). HPLC purity 94%.
1,1,1-Trifluoro-4-methyl-2-(5H-pyrrolo[3,2-d]pyrimidin-6-yl-
methyl)-4-(2-methyl-phenyl)-pentan-2-ol (40). Step 1: A mixture
1,1,1-trifluoro-4-methyl-pent-3-en-2-one (800 mL, ∼0.45 M in THF,
0.35 mol) and CuI (33.3 g, 0.18 mol) was cooled to −20 °C. 2-
Methylphenylmagnesum bromide (175 mL, 2 M in THF, 0.35 mol)
was added, and the mixture was allowed to warm to room temperature
and stirred for 2 h. The reaction was quenched with HCl (6 N in
water, 500 mL) and filtered through Celite, and the layers were
separated. The organic phase was dried over MgSO4, filtered, and
concentrated to give 1,1,1-trifluoro-4-methyl-4-(2-methylphenyl)-
pentan-2-one as an oil (70% yield).
Step 2: To a solution of LHMDS (1 M in THF, 778 mL, 0.82 mol)
at −65 °C was added methyl acetate (59 mL, 0.74 mol) over 15 min.
1,1,1-Trifluoro-4-methyl-4-(2-methylphenyl)-pentan-2-one (99 g, 0.41
mol) was added over 20 min. After 10 min, NaOH (4 M, 200 mL, 0.80
mol) was added and the mixture allowed to warm to room
temperature. The reaction was stirred for 70 h at room temperature.
The mixture was concentrated to remove THF, acidified with conc
HCl to pH 1 and extracted with CH2Cl2 (1000 mL). The organic
phase was separated, dried over MgSO4, and concentrated. The
residue was recrystallized from hexane to give 3-hydroxy-5-methyl-5-o-
tolyl-3-trifluoromethyl-hexanoic acid as a white solid (53% yield).
Step 3: 3-Hydroxy-5-methyl-5-o-tolyl-3-trifluoromethyl-hexanoic
acid (8.0 g, 17.6 mmol) in MeOH (40 mL) was treated with conc
H2SO4 (2.0 mL) and heated to reflux for 16 h. The mixture was
concentrated and then diluted with heptane and water. The organic
layer was separated, and the aqueous layer was extracted with heptane
(200 mL). The combined organic phases were washed with satd
NaHCO3 solution, dried over MgSO4, and concentrated to give 3-
hydroxy-5-methyl-5-o-tolyl-3-trifluoromethyl-hexanoic acid methyl
ester as a yellow oil (100% yield).
1,1,1-Trifluoro-4-(5-fluoro-2-methoxy-phenyl)-4-methyl-2-
(5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pentan-2-ol (32). Fol-
lowing the general Sonogashira coupling procedure starting from
17b16 and 819 gave 1-(5-amino-pyrimidin-4-yl)-6-(5-fluoro-2-methoxy-
phenyl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (57% yield).
Following the general cyclization procedure A starting from 1-(5-
amino-pyrimidin-4-yl)-6-(5-fluoro-2-methoxy-phenyl)-6-methyl-4-tri-
1
fluoromethyl-hept-1-yn-4-ol gave 32 as a white solid (63% yield). H
NMR (400 MHz, MeOH-d4) δ 8.69 (s, 1H), 8.67 (s, 1H), 7.10 (d, J =
8 Hz, 1H), 6.90−6.92 (m, 2H), 6.27 (s, 1H), 3.83 (s, 3H), 3.10 (d, J =
15 Hz, 1H), 2.89 (d, J = 15 Hz, 1H), 2.63 (d, J = 15 Hz, 1H), 2.09 (d,
J = 15 Hz, 1H), 1.63 (s, 3H), 1.40 (s, 3H).
1,1,1-Trifluoro-4-(2-methanesulfonyl-phenyl)-4-methyl-2-
(5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pentan-2-ol (34). Fol-
lowing the general Sonogashira coupling procedure starting from 17d
and 819 gave 1-(5-amino-pyrimidin-4-yl)-6-(2-methanesulfonyl-phe-
nyl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (53% yield).
Following the general cyclization procedure A starting from 1-(5-
amino-pyrimidin-4-yl)-6-(2-methanesulfonyl-phenyl)-6-methyl-4-tri-
1
fluoromethyl-hept-1-yn-4-ol gave 34 as a white foam (25% yield). H
NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 8.75 (s, 1H), 8.70 (s,
1H), 8.17 (dd, J = 8 Hz, J = 1 Hz, 1H), 7.75 (d, J = 7 Hz, 1H), 7.62
(ddd, J = 8 Hz, J = 7 Hz, J = 1 Hz, 1H), 6.28 (s, 1H), 6.07 (s, 1H),
3.28 (s, 3H), 3.00 (d, J = 15 Hz, 1H), 2.87 (d, J = 16 Hz, 1H), 2.68 (d,
J = 16 Hz, 1H), 2.43 (d, J = 15 Hz, 1H), 1.74 (s, 3H), 1.61 (s, 3H).
ES-MS m/z = 442 (M + H)+.
1,1,1-Trifluoro-4-(3-methanesulfonyl-phenyl)-4-methyl-2-
(5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pentan-2-ol (35). Fol-
lowing the general Sonogashira coupling procedure starting from 17e
and 819 gave 1-(5-amino-pyrimidin-4-yl)-6-(3-methanesulfonyl-phe-
nyl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (71% yield).
Step 4: 3-Hydroxy-5-methyl-5-o-tolyl-3-trifluoromethyl-hexanoic
acid methyl ester (66.0 g, 207 mmol) was dissolved in DMF (350
mL). At room temperature, imidazole (35.2 g, 518 mmol) was added,
followed by TMSCl (39.7 mL, 311 mmol). After 20 h, NH4Cl solution
and Et2O were added. The organic layer was separated, and the
aqueous layer was extracted with Et2O (200 mL). The combined
organic phases were washed with satd brine, dried over MgSO4, and
concentrated to give 5-methyl-5-o-tolyl-3-trifluoromethyl-3-trimethyl-
silanyloxy-hexanoic acid methyl ester as a colorless oil (95% yield).
Step 5: To a solution of 5-amino-4-methylpyrimidine (4.72 g, 43.3
mmol) in THF (210 mL) at −20 °C was added n-BuLi (2.5 M in
hexane; 42.1 mL, 105 mmol). The reaction was allowed to warm to
room temperature over 2.5 h. A solution of 5-methyl-5-o-tolyl-3-
trifluoromethyl-3-trimethylsilanyloxy-hexanoic acid methyl ester (5.0
g, 13 mmol) in THF (5 mL) was added at room temperature. After 20
min, MeOH (42 mL) was added, followed by water (21 mL). After 18
h, HCl (6 N; 25 mL) was added and the mixture stirred for 2 h. The
mixture was neutralized with NaOH (2 N) to pH 7. CH2Cl2 was
added. The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2. The combined organic phases were dried over
Following the general cyclization procedure A starting from 1-(5-
amino-pyrimidin-4-yl)-6-(3-methanesulfonyl-phenyl)-6-methyl-4-tri-
1
fluoromethyl-hept-1-yn-4-ol gave 35 as a white solid (30% yield). H
NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.78 (s, 1H), 8.73 (s,
1H), 7.92 (s, 1H), 7.69−7.75 (m, 2H), 7.45−7.49 (m, 1H), 6.36 (s,
1H), 6.11 (s, 1H), 3.19 (s, 3H), 2.89 (d, J = 15 Hz, 1H), 2.89 (d, J =
14 Hz, 1H), 2.31 (d, J = 16 Hz, 1H), 2.23 (d, J = 15 Hz, 1H), 1.50 (s,
3H), 1.37(s, 3H). ES-MS m/z = 442 (M + H)+.
1,1,1-Trifluoro-4-(5-fluoro-2,3-dihydro-benzofuran-7-yl)-4-
methyl-2-(5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-pentan-2-ol
(36). Following the general Sonogashira coupling procedure starting
from 17f16 and 819 gave 1-(5-amino-pyrimidin-4-yl)-6-(5-fluoro-2,3-
dihydro-benzofuran-7-yl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol
(47% yield).
Following the general cyclization procedure A starting from 1-(5-
amino-pyrimidin-4-yl)-6-(5-fluoro-2,3-dihydro-benzofuran-7-yl)-6-
methyl-4-trifluoromethyl-hept-1-yn-4-ol gave 36 as a white solid (41%
1
MgSO4 and concentrated to give 40 as a tan foam (47% yield). H
1
yield). H NMR (400 MHz, MeOH-d4) δ 8.60 (s, 1H), 8.58 (s, 1H),
NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1H), 8.75 (s, 1H), 8.70 (s,
1H), 7.40−7.38 (m, 1H), 7.14−7.06 (m, 3H), 6.27 (s, 1H), 6.17 (s,
6.71−6.75 (m, 2H), 6.21 (s, 1H), 4.45 (t, J = 9 Hz, 2H), 3.02 (t, J = 9
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1H), 2.86−2.82 (m, 1H), 2.63−2.56 (m, 2H), 2.49 (s, 3H), 2.13−2.10
(m, 1H), 1.62 (s, 3H), 1.42 (s, 3H).
IL-1 Induced IL-6 Assay (Transrepression). The fibroblast-IL-6
assay measures the ability of test compounds to inhibit the elaboration
of IL-6 by human foreskin fibroblasts following stimulation by IL-1 in
vitro. Human foreskin fibroblasts (ATCC no. CRL-2429) were grown
in Iscove’s media supplemented with 10% (v/v) charcoal/dextran-
treated fetal bovine serum and plated at 20000 cells/well two days
prior to assay. On the day of the assay, the media was replaced,
followed by the addition of test compound (0.4% DMSO final assay
concentration) and recombinant human IL-1 (final concentration = 1
ng/mL). After incubation at 37 °C for 18−24 h, IL-6 in the tissue
culture media was quantitated using standard electrochemilumines-
cence, DELFIA, or ELISA methods. Test compound IC50 values were
determined by fitting the data from duplicate 11-point concentration−
response curves to a four-parameter logistic equation. In this assay, IL-
6 inhibition is considered a measure of agonist response of the
glucocorticoid receptor, and dexamethasone is considered to possess
100% efficacy.
MMTV Reporter Gene Assay (Transactivation). HeLa MMTV
transactivation assay measures the ability of test compounds to activate
MMTV promoter in HeLa cells stably transfected with MMTV
luciferase construct. HeLa MMTV cells were grown in DMEM media
supplemented with 10% (v/v) fetal bovine serum (Hyclone), 100 U/
mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and 300
μg/mL genetecin. The day prior to assay the cells were plated at 25000
cells/well in 96-well white clear-bottom cell culture plates in DMEM
media supplemented with 3% (v/v) FBS, 100 U/mL penicillin, 100
μg/mL streptomycin, and 2 mM L-glutamine. On the day of the assay,
the media was replaced, followed by addition of test compound (0.2%
DMSO final assay concentration). Positive control wells received 1 μM
dexamethasone and represent 100% induction, while negative control
wells received vehicle only and represent background. After final
addition of the compounds, plates were incubated for 6 h at 37 °C and
5% CO2. After incubation period, luminescence was detected upon cell
lysis with Steady Glow Luciferase Reagent (Promega no. E2520).
Activation of MMTV promoter by test compounds is expressed in
percentage relative to positive control. Test compound EC50 values
were determined by nonlinear curve fitting of the data from duplicate
eight-point concentration−response curve.
Osteocalcin Production Assay. Osteocalcin is a marker of bone
formation in bone growth and remodeling. It is produced by
osteoblasts and is the most abundant noncollagenous bone matrix
protein. Human MG-63 cells, an osteosarcoma cell line of osteoblast
lineage, produce osteocalcin upon stimulation with vitamin D. The
addition of dexamethasone effectively inhibits the vitamin D-induced
osteocalcin production in MG-63. Compounds that bind to the
glucocorticoid receptor and inhibit IL-1-induced IL-6 expression in
human foreskin fibroblast cells were evaluated for their ability to
inhibit osteocalcin production in MG-63 cells.
Human osteosarcoma MG-63 cells (ATCC) were plated on 96-well
plates at 20000 cells per well on day 1 in 200 μL of media of 99%
DMEM/F-12 (Gibco-Invitrogen), supplemented with 1% penicillin
and streptomycin (Gibco-Invitrogen), 10 μg/mL vitamin C (Sigma),
and 1% charcoal filtered fetal bovine serum (HyClone). On day 2,
wells were replaced with fresh media. Cells were treated with vitamin
D (Sigma) to a final concentration of 10 nM and with the test
compounds in concentrations of 2 × 10−6 M to 10−10 M in a total
volume of 200 μL per well. Samples were tested in duplicates.
Background control wells did not receive vitamin D or compounds.
Positive control wells received vitamin D only, without compounds,
and represented maximum (100%) amount of osteocalcin production.
Plates were incubated at 37 °C incubator for 48 h, and supernatants
were harvested at the end of incubation. Amounts of osteocalcin in the
supernatants were determined by the Gla-type osteocalcin ELISA kit
(Zymed) according to manufacturer’s protocol. Inhibition of
osteocalcin by test compounds is expressed in percentage relative to
positive controls. IC50 values of the test compounds were derived by
nonlinear curve fitting.
4-Fluoro-2-[4,4,4-trifluoro-3-hydroxy-1,1-dimethyl-3-(5H-
pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-butyl]-phenol (41). A sol-
ution of 32 (50 mg, 0.12 mmol) in CH2Cl2 (2 mL) at −15 °C was
treated with a solution of BBr3 (1 M in CH2Cl2, 1.2 mL, 1.2 mmol) in
a dropwise fashion. The reaction mixture was stirred at 0 °C for 60 h.
The reaction was quenched with satd aqueous NaHCO3 solution until
pH 7 was reached. The mixture was extracted with EtOAc (4 × 10
mL). The combined organic layers were dried over Na2SO4, filtered,
and concentrated. The residue was purified by preparative TLC using
1
12% MeOH in CH2Cl2 to give 41 as a white solid (68% yield). H
NMR (400 MHz, MeOH-d4) δ 8.71 (s, 1H), 8.67 (s, 1H), 7.03 (dd, J
= 11 Hz, J = 2 Hz, 1H), 6.70−6.76 (m, 2H), 6.27 (s, 1H), 3.13 (d, J =
15 Hz, 1H), 2.96 (d, J = 15 Hz, 1H), 2.76 (d, J = 15 Hz, 1H), 2.12 (d,
J = 15 Hz, 1H), 1.66 (s, 3H), 1.47 (s, 3H).
5-Fluoro-2-[4,4,4-trifluoro-3-hydroxy-1,1-dimethyl-3-(5H-
pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-butyl]-phenol (42). Fol-
lowing the general Sonogashira coupling procedure starting from
17c and 819 gave 1-(5-amino-pyrimidin-4-yl)-6-(4-fluoro-2-methoxy-
phenyl)-6-methyl-4-trifluoromethyl-hept-1-yn-4-ol (70% yield).
Following the general cyclization procedure A starting from 1-(5-
amino-pyrimidin-4-yl)-6-(4-fluoro-2-methoxy-phenyl)-6-methyl-4-tri-
fluoromethyl-hept-1-yn-4-ol gave 1,1,1-trifluoro-4-(4-fluoro-2-me-
thoxy-phenyl)-4-methyl-2-(5H-pyrrolo[3,2-d]pyrimidin-6-ylmethyl)-
pentan-2-ol (33) as a solid (41% yield).
Step 3: 33 (110 mg, 0.27 mmol) was dissolved in CH2Cl2 (5 mL)
and treated with BBr3 (1 M in CH2Cl2, 2.5 mL, 2.5 mmol) at 0 °C.
The mixture was allowed to stand at 4 °C for 18 h. The mixture was
quenched by the dropwise addition of MeOH. The solvent was
evaporated. EtOAc (10 mL) and water (10 mL) were added. Solid
Na2CO3 was added until pH > 7. The organic phase was separated,
and the aqueous layer was extracted with EtOAc (3 × 50 mL). The
combined organic phases were dried over MgSO4. The solvent was
removed. The residue was purified by flash chromatography using 50−
1
100% EtOAc in hexanes to yield 42 as a white solid (70% yield). H
NMR (400 MHz, MeOH-d4) δ 8.59 (s 1H), 8.56 (s, 1H), 7.17 (dd, J =
9 Hz, J = 7 Hz, 1H), 6.38−6.42 (m, 2H), 6.15 (s, 1H), 3.14 (d, J = 15
Hz, 1H), 2.84 (d, J = 15 Hz, 1H), 2.55 (d, J = 15 Hz, 1H), 1.92 (d, J =
15 Hz, 1H), 1.56 (s, 3H), 1.34 (s, 3H). HPLC purity 92%.
GR, PR, and MR Binding Assays. GR, PR, and MR binding assays
were performed in a fluorescence polarization microplate format that
measures competition for binding to the nuclear receptor between a
test compound and a fluorescently labeled receptor ligand or probe.
The probes used for the assays were as follows. GR and MR assays, 5
nM tetramethylrhodamine-labeled dexamethasone; PR assay, 5 nM
tetramethylrhodamine-labeled RU-486; for GR and MR assays,
receptors present in lysates of baculovirus-infected insect cells
coinfected with receptor, HSP-70, HSP-90, and p23 were used. For
PR binding assays, lysates were generated from insect cells infected
with receptor-containing baculovirus alone. Binding reactions
consisted of lysate, test compound, and probe combined in assay
buffer containing 10 mM TES, 50 mM KCl, 20 mM Na2MoO4·2H2O,
1.5 mM EDTA, 0.04% w/v CHAPS, 10% v/v glycerol, 1 mM
dithiothreitol, pH = 7.4. Lysates containing GR, PR, and MR were
diluted into the assay 300-fold, 13-fold, and 17-fold into the respective
binding reactions. The DMSO concentration in all binding reactions
was 1% (v/v). Following one hour incubation in the dark at room
temperature, fluorescence polarization signal was measured using a
Molecular Devices Analyst plate reader with excitation and emission
filters of 550 and 580 nm selected and with a 561 nm dichroic mirror
installed. Positive control FP signal was measured in assay wells
containing binding reactions without inhibitor. The fluorescence
polarization signal produced by maximal inhibition (background
control) was determined using the signals from wells containing 2 μM
concentrations of a standard inhibitor for each receptor: dexametha-
sone for GR and MR and RU-486 for PR. Fluorescence polarization
signals from duplicate 11-point concentration−response curves were
fitted to a four-parameter logistic equation to determine IC50 values.
In Vivo LPS-Challenge Assay. Female Balb/c mice, weighing
approximately 20 g, were administered the test compound in
cremophor (po) approximately 60 min prior to LPS/D-gal
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administration. The volume of oral gavage was 0.15 mL. Then mice
were administered LPS (Escherichia coli LPS 055:85, 1.0 μg/mouse)
plus D-gal (50 mg/kg) intravenously in 0.2 mL of pyrogen-free saline.
One hour after LPS/D-gal, each mouse was anesthetized, bled by
cardiac puncture, and collected for serum TNF-α and compound
levels. Blood samples were centrifuged at 2500 rpm for 10−15 min,
the serum was decanted, and samples were stored frozen at −70 °C
until transfer either for TNF-α determination or for plasma
concentration analysis by HPLC. The concentration of TNF-α in
the serum was measured by a commercially available ELISA kit (R&D
Systems, Minneapolis, MN). ELISA was performed according to the
manufacturers assay procedure. All samples are assayed in duplicate.
Collagen Induced Arthritis. Female B10.RIII mice (Jackson
Laboratories) were immunized intradermally at 12−15 weeks of age
with lyophilized native porcine type II collagen (CII lot P9603,
purchased from Dr. Marie Griffiths, Utah University) dissolved
overnight at 4 °C in 0.01 N acetic acid at a concentration of 2 mg/
mL. The collagen solution was then emulsified at a 1:1 ratio with
complete Freund’s adjuvant (containing 2 mg/mL Mycobacterium
tuberculosis strain H37Ra). On day 0, animals received a single
intradermal injection of 100 μL (containing 100 μg type II collagen) of
cold emulsion in the base of the tail. Beginning on day 13 post
immunization, all immunized animals were monitored daily for signs
of clinical arthritis. Arthritic animals were randomly enrolled into
treatment groups until a total of 10 animals per group was achieved.
Upon enrollment, the animal was anesthetized with 50 mg/kg (10 mg/
mL) ketamine/15 mg/kg (2 mg/mL) zylazine. A whole body
noninvasive density scan was performed to assess body fat content.
(PIXImus densitometer, Lunar Corp.). Three to four h after
recovering from anesthesia, the animal received the appropriate
treatment once a day for a period of 35 days. Disease progression and
severity were assessed every Monday, Wednesday, and Friday for 5
weeks. A clinical scoring system was used whereby each limb was
graded as follows: 0, normal; 1, erythema and edema in 1−2 digits; 2,
erythema and edema in >2 digits or severe edema and erythema
encompassing the tarsal and metatarsal joints; 3, edema and erythema
encompassing the tarsal and metatarsal joints along with joint
deformity. At termination (day 35), a serum sample was collected
for biomarker analysis. Triglyceride levels were determined with the
Wako L-Type TG-H kit (Wako Diagnostics, Richmond, VA). Free
fatty acids in serum were measured using the Wako NEFA-C
microtiter procedure (Wako Diagnostics, Richmond, VA, catalogue no.
994−75409). For the serum insulin assay, the Ultra Sensitive Rat
Insulin ELISA Kit (Crystal Chem Inc., Chicago, IL, catalogue no.
90060) and Mouse Insulin Standard 1.28 ng (catalogue no. 90090)
was used.
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Corresponding Author
*Phone: (203) 778 7924. Fax: (203) 791 6072. E-mail:
christian.harcken@boehringer-ingelheim.com.
Notes
The authors declare no competing financial interest.
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■
AP-1, activator protein 1; CHAPS, 3-[(3-cholamidopropyl)-
dimethylammonio]-1-propanesulfonate; CIA, collagen induced
arthritis; DMEM, Dulbecco’s Modified Eagle Medium; FBS,
fetal bovine serum; GC, glucocorticoid; GR, glucocorticoid
receptor; GRE, glucocorticoid response element; IL-1,
interleukin 1; MMTV, mouse mammary tumor virus; MR,
mineralocorticoid receptor; PR, progesterone receptor; Qh,
hepatic blood flow; RA, rheumatoid arthritis; TA, trans-
activation; TR, transrepression
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