Lead Optimization Generates CYP11B1 Inhibitors of Pyridylmethyl Isoxazole Type with Improved Pharmacological Profile for the Treatment of Cushing's Disease

Article abstract of DOI:10.1021/acs.jmedchem.7b00437

Cushing's disease, characterized by elevated plasma cortisol levels, can be controlled by inhibition of 11β-hydroxylase (CYP11B1). The previously identified selective and potent CYP11B1 inhibitor 5-((5-methylpyridin-3-yl)methyl)-2-phenylpyridine Ref 7 (IC₅₀= 2 nM) exhibited promutagenic potential as well as very low oral bioavailability in rats (F = 2%) and was therefore modified to overcome these drawbacks. Successful lead optimization resulted in similarly potent and selective 5-((5-methoxypyridin-3-yl)methyl)-3-phenylisoxazole 25 (IC₅₀ = 2 nM, 14-fold selectivity over CYP11B2), exhibiting a superior pharmacological profile with no mutagenic potential. Furthermore, compound 25 inhibited rat CYP11B1 (IC₅₀ = 2 μM) and showed a high oral bioavailability (F = 50%) and sufficient plasma concentrations in rats, providing an excellent starting point for a proof-of-principle study.

Full text of DOI:10.1021/acs.jmedchem.7b00437

Article
Lead optimization generates CYP11B1 inhibitors of
pyridylmethyl isoxazole type with improved pharmacological
profile for the treatment of Cushing’s disease
Juliette Emmerich, Chris J van Koppen, Jens L. Burkhart, Qingzhong Hu, Lorenz Siebenbürger,
Carsten Boerger, Claudia Scheuer, Mathias W. Laschke, Michael D. Menger, and Rolf W. Hartmann
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017
Downloaded from http://pubs.acs.org on June 1, 2017
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Lead Optimization Generates CYP11B1 Inhibitors
of Pyridylmethyl Isoxazole Type with Improved
Pharmacological Profile for the Treatment of
Cushing’s disease
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Juliette Emmerich,† Chris J. van Koppen,‡ Jens L. Burkhart,‡ Qingzhong Hu,§ Lorenz
Siebenbürger,ǁ Carsten Boerger,ǁ Claudia Scheuer,┴ Matthias W. Laschke,┴ Michael D.
Menger,┴ Rolf W. Hartmann†,§,*
†Pharmaceutical and Medicinal Chemistry, Saarland University, Campus E8.1, 66123
Saarbrücken, Germany
‡Elexopharm GmbH, Campus A1, 66123 Saarbrücken, Germany
ǁPharmBioTec GmbH, Science Park 1, 66123 Saarbrücken, Germany
┴Institute for Clinical & Experimental Surgery, Saarland University, 66421 Homburg/Saar,
Germany
§Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical
Research Saarland (HIPS), Campus E8.1, 66123 Saarbrücken, Germany
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ABSTRACT
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Cushing’s disease, characterized by elevated plasma cortisol levels, can be controlled by
inhibition of 11βꢀhydroxylase (CYP11B1). The previously identified selective and potent
CYP11B1 inhibitor 5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)ꢀ2ꢀphenylpyridine Ref 7 (IC50= 2 nM)
exhibited promutagenic potential as well as very low oral bioavailability in rats (F= 2%) and
was, therefore, modified to overcome these drawbacks. Successful lead optimization resulted
in similarly potent and selective 5ꢀ((5ꢀmethoxypyridinꢀ3ꢀyl)methyl)ꢀ3ꢀphenylisoxazole 25
(IC50= 2 nM, 14ꢀfold selectivity over CYP11B2), exhibiting a superior pharmacological
profile with no mutagenic potential. Furthermore, compound 25 inhibited rat CYP11B1
(IC50= 2 ꢁM), showed a high oral bioavailability (F= 50%) and sufficient plasma
concentrations in rats, providing an excellent starting point for a proofꢀofꢀprinciple study.
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INTRODUCTION
Cushing’s disease, an endogenous rare syndrome with an incidence of 1–2 cases per million
population1a, is mainly caused by a pituitary adenoma overproducing adrenocorticotropic
hormone (ACTH).1b As a consequence, plasma cortisol levels are abnormally elevated and
patients suffer from diabetes mellitus, hypertension, osteoporosis or psychological
dysfunctions.1a Usually, surgical removal of the tumor is the firstꢀline treatment with
remission rates of 65–90%.1c However, recurrence rate is about 10ꢀ20%1c and surgery bears
the risk of pituitary gland damage. In addition, tumors are not always accessible. Alternatively
used radiation therapy exhibits a delayed response (2–5 years) and high risk of
hypopituitarism.1d Medications which are targeting steroid 11βꢀhydroxylase (CYP11B1,
catalyzes the last step of cortisol biosynthesis, scheme 1) such as inhibitors metyrapone and
ketoconazole (figure 1), are widely used in the clinic.1e Furthermore, the potent 11βꢀ
hydroxylase inhibitor osilodrostat (LCI699, figure 1) was investigated in a phase 2 study as
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potential new treatment for Cushing’s disease.1f However, all of them induce various sideꢀ
effects (e.g. hirsutism, acne, hypertension, hypokalemia, hypogonadism, rash) caused by
additional inhibition of other steroidogenic CYP enzymes like aromatase (CYP19A1), 17αꢀ
hydroxylaseꢀ17,20ꢀlyase (CYP17A1) or aldosterone synthase (CYP11B2).1d,f,g In fact,
selective inhibition is challenging especially towards CYP11B2 (catalyzes last steps of
aldosterone biosynthesis, scheme 1) since CYP11B1 exhibits a sequence identity of 94% to its
isoenzyme.2 Nevertheless, beside successful development of selective CYP17A13 and
CYP19A1 inhibitors4, we identified highly potent and selective CYP11B2 inhibitors5 which
are active in vivo.5a In addition, we recently published the first selective human CYP11B1
inhibitors.6 In the class of imidazolylmethyl pyridine type of compounds, Ref 1–6 (chart 1,
IC50= 47–122 nM, SF (IC50CYP11B2/ IC50CYP11B1)= 6–15)6b,c were the most active and
selective ones, which were subsequently optimized regarding potency and selectivity.6d
Thereby, exchange of the hemeꢀcomplexing imidazolꢀ1ꢀyl ring for 5ꢀMeꢀ or 5ꢀOMeꢀpyridinꢀ
3ꢀyl led to improved compounds regarding potency, Ref 7, 8 (chart 1, IC50= 2–4 nM, SF= 6–
17).6d Inhibitor Ref 7 exhibited selectivities over CYP17A1, CYP19A1 and hepatic CYP
enzymes (CYP2A6, CYP3A4) as well as negligible cytotoxicity and satisfying metabolic
stability in human and rat liver S9 fraction.6d
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SUBSEQUENT BIOLOGICAL RESULTS OF REF 7
Despite the previously shown good in vitro pharmacological profile of Ref 7,6d in subsequent
studies it became apparent that Ref 7 is a promutagen in TA98 strain (frameshift mutation) of
Salmonella typhimurium and, therefore, a potential carcinogen (see SI). Furthermore, a
pharmacokinetic study in rats revealed a very low oral bioavailability (F= 2%) of Ref 7.
Associated calculated pharmacokinetic parameters of Ref 7 after a single dose in rats are
summarized in table S4.
DESIGN CONCEPT FOR LEAD OPTIMZATION
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Due to the low oral bioavailability in rats of Ref 7, the inhibitor is a poor candidate for
preclinical development. This encouraged us to perform lead optimization of Ref 7 to obtain
other candidates for in vivo studies with the challenge to retain CYP11B1 selectivity and
potency. There are several possible reasons for low oral bioavailability in vivo such as low
aqueous solubility, low permeability, high first pass metabolism in the liver or high renal
clearance. The calculated log D (pH= 7.4) value for Ref 7 is 3.77 and, therefore, structural
optimization aiming for more polar compounds should balance solubility and permeability
properties of the inhibitors under in vivo conditions. Furthermore, modification of electronic
properties and molecular geometry of Ref 7 was expected to influence CYP11B1 potency,
selectivity over CYP11B2 and in vivo pharmacokinetics. Previously, the influence of
substitution at the central pyridine ring of the imidazolylmethyl pyridine class was explored
extensively.6c Thereby, substituents in 2ꢀposition of pyridine exemplified in Ref 1–6 (R1,
chart 1) improved CYP11B1 inhibition and exhibited the best effect on selectivity over
CYP11B2 (table 1). However, a distinct trend within these inhibitors could not be identified
as introduced substituents vary in terms of polarity, electronic properties and size.
Consequently, the phenyl moiety in Ref 7 was substituted with the most promising of these
substituents (2ꢀFꢀPh, furanꢀ3ꢀyl). The 3ꢀNH2ꢀPh, benzo[b]thiophene and naphthaleneꢀ1ꢀyl
compounds were not synthesized due to anticipated toxicity (carcinogenicity)8 and solubility
(lipophilicity) issues. As possible carcinogen Ref 3 (3ꢀNH2ꢀPh) exhibited highest selectivity
in this series further hydrophilic substituents differing in size and electronic properties such as
Nꢀphenylmethanesulfonamide, pyrazoles or polar nonꢀaromatic cycles were introduced. To
investigate the influence of the molecular shape on selectivity heteroatom linkers (ꢀSꢀ, ꢀSO2ꢀ)
were used. In the imidazolylmethyl pyridine class exchange of the pyridine core by furan or
thiophene caused an increase in CYP11B1 potency, but a loss of selectivity towards
CYP17A1 or CYP19A1.6b Hence, it could be assumed that the aromatic N is essential for
selectivity and change of the molecular geometry by introduction of 5ꢀmembered heterocycles
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is beneficial for CYP11B1 potency. For further investigation, several azoles differing in
electronic and steric properties were introduced into selective lead compounds Ref 1 and Ref
7 with the phenyl substituent next to the aromatic N. The optimized central core was
subsequently incorporated into very potent and moderate selective Ref 8 to get a variety of
candidates for further biological evaluation. Thus, 27 structurally diverse novel compounds
were obtained and evaluated for inhibition of CYP11B1 and selectivity towards CYP11B2.
Selected inhibitors were further tested for CYP17A1 and CYP19A1 inhibition, metabolic
stability, toxicity and inhibition of hepatic CYP enzymes (CYP1A2, 2B6, 2C9, 2C19, 2D6
and 3A4). Candidates which exhibited a good pharmacological profile were also tested for
inhibition of rat CYP11B1 and metabolic stability in rat liver S9 fraction. Preliminary
pharmacokinetic experiments were performed with the best inhibitors to find a suitable
candidate for clinical use.
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CHEMISTRY
The synthesis of the (5ꢀMeꢀpyridinꢀ3ꢀyl)pyridine derivatives 1–11 (scheme 2) was performed
using the common building block 29, a 3:1 mixture of 2ꢀchloro and 2ꢀbromo compounds. 29
was accessible from racemic alcohol 28, which was converted into the chloride by thionyl
chloride and reductively dehalogenated to obtain 29. Aromatically substituted target
compounds 1–5 were synthesized using 29 and the corresponding organoboranes under cross
coupling conditions. In terms of the Nꢀsubstituted compounds 6 and 7 BuchwaldꢀHartwig
amination of 29 was applied, whereas thioethers 8 and 10 were synthesized via nucleophilic
attack of corresponding thiolate at 29. Oxidation of thioethers 8 and 10 with potassium
peroxymonosulfate resulted in the final sulfone compounds 9 and 11.
The synthesis of 4ꢀ and 5ꢀ substituted 3ꢀphenylisothiazoles 12–15 (scheme 3) was performed
from ester substituted isothiazoles 31 and 32, which were obtained by decarboxylation of
oxathiazolone 30 and subsequent 1,3ꢀdipolar cycloaddition with ethyl propiolate.9a
Subsequently, the esters 31 and 32 were reduced to the alcohols, transferred into the bromines
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33, 34 and reacted to the target compounds 12–15 via nucleophilic substitution or Suzuki
reaction.
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The 5ꢀmembered heteroaromatic derivatives 16–27 (scheme 4) were synthesized applying
different routes, in which corresponding halogenmethyl or hydroxymethyl compounds 41–46
were common intermediates. Bromomethyl thiazole 41 was prepared from 2ꢀbromoꢀ5ꢀ
methylthiazole 35 by coupling with phenylboronic acid and subsequent bromination under
WohlꢀZiegler conditions. Intramolecular oxidative cyclization of Nꢀallylbenzamide 36 using
Nꢀbromosuccinimide (NBS)9b led to a chloride and bromide (3:1) oxazole mixture 42,
whereas 1,3ꢀdipolar cycloaddition of (E)ꢀbenzaldehyde oxime 37 and propargyl bromide9c
resulted in chloride and bromide (1:3) isoxazole mixture 43. In terms of the bromomethyl
selenazole 44, propargyl selenoamide was first cycloisomerized to methyl selenazole 38 and
then brominated with NBS.9d Introduction of bromine to methyl 1ꢀmethylꢀ1Hꢀimidazoleꢀ5ꢀ
carboxylate (39) under WohlꢀZiegler conditions, Suzuki coupling and subsequent reduction
led to hydroxymethyl imidazole 45. Furthermore, reduction of 1ꢀmethylꢀ3ꢀphenylꢀ1Hꢀ
pyrazoleꢀ5ꢀcarboxylic acid 40 to the alcohol and further reaction with phosphorus tribromide
resulted in bromomethyl pyrazole 46. Finally, the desired imidazolꢀ1ꢀyl and pyridinꢀ3ꢀyl
derivatives 16–26 were obtained either by substitution of the halogen or hydroxyl with an
imidazolꢀ1ꢀyl or Suzuki reaction of intermediates 41–46. Moreover, Nꢀdemethylation of 1ꢀ
Meꢀpyrazole 26 using pyridine hydrochloride resulted in pyrazole 27.
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IN VITRO BIOLOGICAL RESULTS AND DISCUSSION
Inhibition of Human CYP11B1 and CYP11B2
Inhibitory potencies of target compounds 1–27 were determined using V79MZ cells
expressing either recombinant human CYP11B1 or CYP11B2, with [3H]ꢀlabeled 11ꢀ
deoxycorticosterone as substrate. Obtained IC50ꢀvalues are shown in tables 1–2 in comparison
to metyrapone and osilodrostat. Orthoꢀsubstitution of phenyl moiety in Ref 7 (IC50= 2 nM, SF
(IC50CYP11B2/ IC50CYP11B1)= 17) with electron withdrawing and lipophilic fluorine led to
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a similarly potent, but less selective compound 1 (table 1, IC50= 3 nM, SF= 9). In case of
substitution in 3ꢀposition with an electron withdrawing, but hydrophilic methylsulfonamide a
very similar result was obtained (2, IC50= 2 nM, SF= 8). Exchange of phenyl for πꢀelectron
rich and hydrophilic furanꢀ3ꢀyl (3, IC50= 4 nM, SF= 7) was not appropriate to increase
selectivity towards CYP11B2 either. Introduction of πꢀelectron rich and hydrophilic 1ꢀMeꢀ
pyrazoles resulted in a loss of potency towards both enzymes (4, 5, IC50= 16–42 nM, SF= 4–
6). Interestingly, exchange of the phenyl by nonꢀaromatic, hydrophilic, Hꢀbond acceptor
morpholinꢀ4ꢀyl (6) or hydrophilic, Hꢀbond donor cyclopropylamine (7) did not change the
inhibitory potency for CYP11B1 and CYP11B2 much (Table 1, IC50= 4–22 nM, SF= 4–8).
Moreover, introduction of a ꢀSꢀ or ꢀSO2ꢀ linker (8, 9) between central pyridine and phenyl
moiety led to similar findings (Table 1). Orthoꢀfluorine inserted into the phenyl moiety of 8
and 9 enhanced inhibitory potency for CYP11B1, but did not improve selectivity (10, 11,
IC50= 2–3 nM, SF= 8–9). Hence, structural diverse substituents in 2ꢀposition of the central
pyridine core exhibiting various profiles concerning electronic properties, bulkiness and Hꢀ
bond acceptor or donor properties were tolerated by both enzymes. Nevertheless, six
compounds (1–3, 7, 10, 11) inhibited CYP11B1 with IC50ꢀvalues less than 5 nM and
exhibited good selectivity over CYP11B2 (SF= 7–9) thus exceeding the references
metyrapone (IC50= 15 nM, SF= 5) and osilodrostat (IC50= 3 nM, SF= 0.07).
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Replacement of the central pyridine in imidazolylmethyl pyridine Ref 1 (IC50= 107 nM, SF=
13) by πꢀelectron rich 5ꢀmembered heterocycles led to differing results (table 2). Pyridine
isosteres isothiazoles 12, 14 and thiazole 16 caused increased CYP11B1 inhibitory potency
(IC50= 14–62 nM), but also a loss of selectivity towards CYP11B2. Thereby, the 3,5ꢀ
disubstituted isothiazole 14 (IC50= 14 nM, SF= 9) demonstrated a 4ꢀfold increased potency for
the target enzyme with a better selectivity compared to the 3,4ꢀdisubstituted compound 12
(IC50= 57 nM, SF= 3). Interestingly, bioisosteric selenazole 19 (IC50= 104 nM, SF= 6) was
not as potent and selective as thiazole 16. Exchange of the thiazole for the more
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electronegative and less bulkier oxazole (17) or isoxazole (18) led to decreased CYP11B1
inhibition (IC50= 148–150 nM). Thereby, differences regarding electronegativity (O > S, Se)
or molecular geometry (size: Se, S > O) might influence interaction with the target enzyme.
Furthermore, introduction of 1ꢀMeꢀimidazole (20) led to a significant decrease in CYP11B1
inhibition (IC50= 516 nM) with improved potency for CYP11B2 (SF=1). Obviously, the Nꢀ
methyl group is not tolerated by the CYP11B1 enzyme in this position, in contrast to the Nꢀ
methylated pyrazole 21 (IC50= 58 nM). But 21 is less active than the corresponding
isothiazole compound 14, which showed an 8ꢀfold improvement of CYP11B1 inhibition
compared to Ref 1, but a slight loss of selectivity over CYP11B2.
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Compared to the imidazolylmethyl class similar results were obtained by exchange of the
central pyridine in Ref 7 with πꢀelectron rich 5ꢀmembered heterocycles (table 2). However, in
the pyridylmethyl class introduction of thiazole did not lead to an enhanced inhibition of the
target enzyme (22, IC50= 9 nM, SF= 3). The isoxazole compound 24 showed only a slight
decrease of potency, but a retained selectivity (IC50= 5 nM, SF= 16). Hence, isoxazole is a
suitable replacement for the central pyridine of Ref 7 and derivatisation according to Ref 8
was performed (exchange of 5ꢀMe by 5ꢀOMe) to obtain another potent and selective
candidate. Thereby, CYP11B1 inhibition could be enhanced (25, IC50= 2 nM, SF= 14). The
pyrazole 27 resulted in a loss of activity towards the target enzyme, indicating that a hydrogen
bond donor is not well tolerated. Modification of the central pyridine in Ref 7 led to four
novel inhibitors with IC50 values of ≤ 5 nM (15, 24–26) with isoxazoles 24 and 25 (IC50= 2–5
nM, SF= 14–16) showing similar selectivity over CYP11B2 as Ref 7 (IC50= 2 nM, SF= 17). It
is worth to mention that 24 and 25 were more potent and selective than the clinically used
inhibitor metyrapone (IC50= 15 nM, SF= 5) or the recently published osilodrostat (IC50= 3
nM, SF= 0.07). Albeit significant improvement of CYP11B1 inhibitory potency was achieved
in the imidazolylmethyl class, the 5ꢀMeꢀpyridinꢀ3ꢀylmethyl analogues showed stronger
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inhibition of the target enzyme. Compounds 24 (IC50= 5 nM) and 25 (IC50= 2 nM) exhibiting
selectivity factors above 10 were chosen for further biological evaluation.
Inhibition of Rat CYP11B1
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Inhibition of rat CYP11B1 by isoxazoles 24 and 25 was determined in V79MZ cells
expressing recombinant rat CYP11B1 as a precondition for in vivo proofꢀofꢀprinciple
experiments. Both inhibitors revealed improved similar potencies towards rat CYP11B1 (24,
IC50= 4.0 ꢁM; 25, IC50= 1.8 ꢁM) compared to metyrapone (IC50= 4.6 ꢁM). The 1.000ꢀfold
lower rat CYP11B1 potency is in accordance with the low sequence identity between human
and rat CYP11B1 (64%).2
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Metabolic Stability
To achieve sufficient plasma concentrations of the parent compounds, metabolic stability in
rat and human liver S9 fraction was determined. Isoxazoles 24 and 25 exhibited good
stabilities in human liver S9 fraction with halfꢀlives of 145 min and 125 min, respectively.
Furthermore, inhibitors 24 and 25 showed increased metabolic stability in rat liver S9 fraction
(24: t1/2= 30 min, 25: t1/2= 23 min) compared to Ref 7 (t1/2= 16 min) and metyrapone (t1/2= 12
min).
IN VITRO TOXICITY EVALUATION
Selectivities over Human CYP17A1 and CYP19A1
Since both 17αꢀhydroxylaseꢀ17,20ꢀlyase (CYP17A1) and aromatase (CYP19A1) are essential
for the production of the sex steroids, their inhibition might cause severe sideꢀeffects such as
hypogonadism10a or hyperandrogenism,10b respectively. Described assays for CYP17A1 and
CYP19A1 inhibition were performed with compound concentrations of 2 ꢁM.11,12 This rather
high concentration was chosen to provide a selectivity factor of at least 400 for highly potent
CYP11B1 inhibitors 24 and 25 showing IC50 values of 5 nM and 2 nM (table 1). Compounds
24 and 25 exhibited no significant inhibition (table S1, ≤ 9% at 2 ꢁM) and are considered as
inactive.
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Selectivities over Human Hepatic CYP Enzymes
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Hepatic CYP enzymes play an eminent role in the metabolism of drugs and their inhibition
could result in adverse drug reactions or toxicity. Thus, derivatives 24 and 25 were tested for
inhibition of a series of relevant metabolizing CYP enzymes, comprising CYP1A2, CYP2B6,
CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Tested at a concentration of 1 ꢁM, compound
24 showed percent inhibition values of 54%, 3%, 28%, 8%, 0% and 61%, compound 25
values of 23%, 19%, 17%, 17%, 8% and 33% for CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4,
respectively. Hence, selectivity over hepatic CYP enzymes should be sufficient for the highly
active compounds.
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Cytotoxicity
Effect of compounds 24 and 25 on cell viability was tested in a MTT assay using HEK293
cells. No effect on cellular viability was observed up to 28 µM and 42 µM (IC20 values) after
66 h of incubation for compounds 24 and 25, respectively. Given the strong CYP11B1
inhibition of these compounds (IC50 ≤ 5 nM), it is expected that no cytotoxic effects will be
observed at therapeutic concentrations.
Mutagenicity
For determination of the mutagenic potential of compounds 24 and 25, AMES II tests were
performed using TA98 (frameshift mutation) or TAMix (baseꢀpair substitution, TA7001ꢀ
TA7006) strains of Salmonella typhimurium in the presence or absence of rat liver S9
fraction. The inhibitors 24 and 25 showed no mutagenic potential up to the highest tested
concentration of 100 ꢁM in the absence or presence of rat liver S9 fraction.
Aryl Hydrocarbon Receptor Activation
Activation of the aryl hydrocarbon receptor (AhR) induces several biochemical responses,
such as expression of CYP1A1 and CYP1A2. Both are responsible for the metabolic
activation of promutagens, which is a potential risk for cancer development.13 Compounds 24
and 25 did not show activation of the AhR in HepG2 hepatocellular carcinoma cells at a
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concentration of 3.16 µM and, therefore, no interference with the receptor is expected at
pharmacologically relevant concentrations.
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Cardiotoxicity
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Drugꢀinduced arrhythmia caused by blocking the cardiac potassium ion channel hERG
(human etherꢀaꢀgoꢀgo related gene) is a potential cause for sudden death among patients.
Compound 25 was tested using a hERG fluorescence polarization assay14 and showed an IC50
value of 23 µM. Due to the high CYP11B1 potency of 25 (IC50= 2 nM), the safety margin can
be considered as sufficient.
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IN VIVO BIOLOGICAL RESULTS AND DISCUSSION
Preliminary Evaluation of Plasma Concentrations of 24 and 25 in Rats
Based on in vitro biological evaluation, inhibitors 24 and 25 are promising candidates for a
proofꢀofꢀprinciple study in rats. Both compounds exhibit different physicochemical properties
due to distinct scaffolds compared to Ref 7. For instance, the calculated log D (pH= 7.4)
value was changed from 3.7 for Ref 7 to 3.1 and 2.8 for 24 and 25, respectively.7 This should
balance solubility and permeability properties of the compounds under in vivo conditions. In
the light of the poor oral bioavailability of Ref 7 (F= 2%) in rats, preliminary pharmacokinetic
experiments for novel inhibitors 24 and 25 were first performed to show their suitability for
extensive in vivo experiments. Both compounds should be soluble under these conditions as
they exhibited good aqueous solubility (>200 µM). Plasma concentrations of 24 and 25 in rats
were determined using a single dose of 100 mg/kg (peroral) per inhibitor (figure S1).
Interestingly, 5ꢀOMe compound 25 showed much higher plasma concentrations of 19.7 ꢁM
and 106.4 ꢁM at 1 h and 4 h, respectively, than 5ꢀMe derivative 24 (1 h, 2.5 ꢁM; 4 h, 0.3 ꢁM).
Required plasma levels for a therapeutic effect in rats on the basis of the in vitro assay (25,
IC50= 2 ꢁM; 24, IC50= 4 ꢁM) were achieved in case of 25 and, therefore, the oral
bioavailability of 25 was determined in rats.
Oral Bioavailability Study of 25 in Rats
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The pharmacokinetic parameters of 25 were determined after intravenous (5 mg/kg, n= 2) and
oral (28 mg/kg, n= 2) application in rats (mean values are listed in table 3). Mean maximal
concentrations of 27.8 µM (i.v.) and 6.4 µM (p.o.) were achieved. As depicted in figure 2,
plasma concentrations above the in vitro IC50 value for rat CYP11B1 (2 µM) were obtained
for a duration of up to 7 hours. The compound exhibited long halfꢀlives of 4.8 hours (i.v.) and
3.3 hours (p.o.) as well as a moderate plasma clearance of 22.6 mL/min·kg. Most importantly,
absolute oral bioavailability (F) was determined as 50%. In a previously published paper,
metyrapone (IC50 in vitro rat= 5 ꢁM) showed an ED50 of 40 mg/kg and EC50 of 3.6 ꢁM for
inhibition of the corticosterone biosynthesis (CYP11B1 catalyzes the last step of
corticosterone biosynthesis in rats) in an 8 h experiment.15 Hence, it can be assumed that
inhibitor 25 is able to reduce corticosterone plasma levels in rats at higher doses due to high
plasma concentrations and, therefore, it is a good candidate for a following proofꢀofꢀprinciple
study.
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CONCLUSION
For the treatment of Cushing’s disease, the steroid 11βꢀhydroxylase (CYP11B1) inhibitor
metyrapone is used in the clinic. Very high (500ꢀ6000 mg per day) and frequent dosing (every
8 hours) of metyrapone is needed and it causes severe sideꢀeffects due to unselective
inhibition of other steroidogenic CYP enzymes.1d,g Previously, we identified the selective and
very potent CYP11B1 inhibitor Ref 7 (IC50= 2 nM, SF= 17), which exceeded metyrapone in
CYP11B1 potency and selectivity (IC50= 15 nM, SF= 5), and exhibited an acceptable in vitro
pharmacological profile.6d However, further determination of mutagenicity identified Ref 7 as
promutagen in TA98 strain (frameshift mutation) of Salmonella typhimurium and a
pharmacokinetic study in rats revealed a poor oral bioavailability of Ref 7 (F= 2%).
Optimizations of Ref 7 and less potent, but selective Ref 1 (IC50= 107 nM, SF= 13) were
performed to obtain other candidates for further in vivo studies with at least similar CYP11B1
potency and selectivity. Therefore, phenyl moiety of Ref 7 was exchanged by various
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substituents and the central pyridine cores of selective Ref 1 and Ref 7 were replaced by
several azoles differing in electronic and steric properties. From the 27 synthesized
compounds, derivatives 1–3, 7, 8, 10, 11, 15, 24–26 showed IC50 values ≤ 5 nM. Among
them, isoxazoles 24 and 25 exhibited highest selectivity over CYP11B2 (SF > 10) and
showed, therefore, an improved inhibitory profile compared to the clinically used metyrapone
(IC50= 15 nM, SF= 5) or the recently published CYP11B1 inhibitor osilodrostat (IC50= 3 nM,
SF= 0.07). Both inhibitors showed selectivities over CYP17A1, CYP19A1 and hepatic CYP
enzymes (CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4), metabolic stability in human liver S9
fraction, negligible cytotoxicity, no interference with the aryl hydrocarbon receptor and for 25
negligible inhibition of the cardiac potassium ion channel hERG. Furthermore, both
compounds exhibited increased metabolic stability in rat liver S9 fraction (24: t1/2= 30 min,
25: t1/2= 23 min) compared to Ref 7 (t1/2= 16 min) and metyrapone (t1/2= 12 min) and
moderately inhibited rat CYP11B1 (24, IC50= 4 ꢁM; 25, IC50= 2 ꢁM). Compounds 24 and 25
showed no mutagenic potential and in vivo PK evaluation in rats revealed sufficiently high
plasma concentrations (1 h, 19.7 ꢁM; 4 h, 106.4 ꢁM) for therapeutic effects in case of
inhibitor 25 (100 mg/kg, peroral). Further pharmacokinetic study of orally applied 25 in rats
demonstrated plasma levels above the required concentration for a therapeutic effect up to 7
hours (28 mg/kg, peroral) and, thus, a significantly improved oral bioavailability of 50%. It is
worth mentioning that in spite of the fact that compound 25 is much less active towards the rat
than the human enzyme (factor of 1000), plasma levels are even high enough in rats to expect
biological activity. Hence, a novel selective and very potent CYP11B1 inhibitor was
identified, exhibiting high lipophilic efficiency (LiPE= 5.9) and a good pharmacological
profile with expected ability to reduce corticosterone plasma levels in vivo.
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EXPERIMENTAL SECTION
Biological Test Procedures
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Inhibition of CYP11B1 and CYP11B2
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V79MZ cells expressing human or rat CYP11B1 or CYP11B2 were incubated with [1,2ꢀ3H]ꢀ
11ꢀdeoxycorticosterone (100 nM) as the substrate and the inhibitor at different concentrations.
The assay was performed using ketoconazole as reference and as previously described.16
CYP17A1 Preparation and Assay
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Human CYP17A1 was expressed in E. coli and the assay was performed using the method
previously described with [3H]ꢀprogesterone (25 ꢁM) as substrate and NADPH as cofactor.11
Abiraterone and ketoconazole were used as references.
CYP19A1 Preparation and Assay
Human CYP19A1 was obtained from microsomal preparations of human placenta and the
assay was performed as previously described with [1βꢀ3H]androstenedione (500 nM) as
substrate and fadrozole and aminoglutethimid as references.12
Metabolic Stability Tests in Human and Rat Liver S9 Fraction
For the evaluation of phase I and II metabolic stability, the compound (1 µM) was incubated
with 1 mg/mL pooled mammalian liver S9 fraction (BD Gentest), 2 mM NADPH
regenerating system, 1 mM UDPGA and 0.1 mM PAPS at 37 °C for 0, 5, 15 and 60 min. The
incubation was stopped by precipitation of S9 enzymes with 2 volumes of cold acetonitrile
containing internal standard. Concentration of the remaining test compound at the different
time points was analyzed by LCꢀMS/MS.
Inhibition of Human Hepatic CYP Enzymes
The inhibition of hepatic CYP enzymes CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6
and CYP3A4 was determined in microsomes of baculovirusꢀinfected insect cells expressing
the recombinant human enzyme according to the manufacturer's instruction (BD Gentest).
Cytotoxicity Assay
HEK293 cells (2x105 cells per well) were seeded in 24ꢀwell flatꢀbottom plates. Culturing of
cells, incubations and OD measurements were performed as described previously17 with
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minor modifications. Four hours after seeding the cells, the incubation was started by the
addition of compounds (100, 50, 25, 12.5 and 6.25 µM) in quadruplicate in a final DMSO
concentration of 1%. Fluorescence was measured in a BMG Labtech Pherastar FS reader. The
decrease in fluorescence (at 570 nm) in the presence of the test compound compared to the
fluorescence in the presence of the vehicle control (1% DMSO) was determined after 66 h
followed by the calculation of IC20 values using GraphpadPrism curve fitting.
Mutagenicity Testing
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Mutagenic potential of 24 and 25 were evaluated using Xenometrix AMES II mutagenicity
assay kit containing TA98 and TA7001ꢀTA7006 strains of Salmonella typhimurium in the
presence or absence of rat liver S9 fraction according to manufacturer's instruction.
HERG Cardiotoxicity Evaluation
Compound 25 was tested using the predictorTM hERG fluorescence polarization assay kit
(Invitrogen) according to manufacturer's instruction. High affinity hERG ligands quinidine
and Eꢀ4031 were used as positive controls and atenolol served as the negative control.
Controls and the tested compound were incubated for 3.5 hours.
Aryl Hydrocarbon Receptor Assay
The aryl hydrocarbon receptor agonistic activity of compounds was determined in a human
hepatocellular carcinoma cell line (HepG2) by measuring the CYP1A1 activity. Cells were
splitted on a 24 well plate (each compound in quadruplicate) and incubated for 16ꢀ24 hours
before compounds or vehicle were added to a final DMSO concentration of 0.1%. After 48
hours of incubation with compound (3.16 µM) or vehicle, cells were washed with 1 mL of
warm PBS (37 °C). Then, 500 µL of 3ꢀcyanoꢀ7ꢀethoxycoumarin (CEC, specific CYP1A1
substrate), which forms a fluorescent product, was added to the cells at a final concentration
of 40 µM in DME medium with 10% fetal calf serum + 1% penicillin + streptomycin (37 °C).
After an incubation of 30 minutes, the fluorescence was measured in the BMG Labtech
Clariostar reader (excitation: 409 nm; emission 460 nm). The increase in fluorescence induced
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by test compound was expressed relative to the increase induced by the reference compound,
omeprazole (50 µM).
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Solubility Determination
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Aqueous solubility was evaluated as previously described18. Briefly, final concentrations of 5,
15, 50, 100 and 200 µM of 24 and 25 in an aqueous solution containing 2% DMSO were
prepared and the solution clarity and potential compound precipitation were determined after
1 h and 24 h at room temperature (19ꢀ24 °C).
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In Vivo Rat Pharmacokinetics
All animal procedures were approved by the local government animal care committee and
performed in accordance with the Guide for the Care and Use of Laboratory Animals.
Pharmacokinetic analysis of 24 and 25 was conducted on female SpragueꢀDawley rats (body
weight 244–299 g) purchased from Charles River Laboratory (Sulzfeld, Germany). After an
acclimatization period of 1 week, compounds were administered orally at a dose of 100 mg/kg
(1 animal) or 28 mg/kg (2 animals), and intravenously at a dose of 5 mg/kg (2 animals). For
oral administration, suspensions of the inhibitors in 0.5% porcine gelatin/5% mannitol (w/w)
in water were freshly prepared following 15 min in an ultrasonic bath 60ꢀ90 min before
administration. For iv administration, 25 was dissolved in PEG300/ethanol/water (60/10/30).
Before iv application (1 mL solution/kg body weight) and oral application (4 mL
suspension/kg body weight), rats were anesthetized with 2% isoflurane. Blood samples (50
µL) were taken from the tail vein and collected in 0.2 mL Eppendorf tubes containing 5 µL of
106 mM sodium citrate buffer. After centrifugation, at 5000 rpm, 4 °C, the plasma samples
were first frozen at ꢀ20 °C, and within 24 h, stored at ꢀ80 °C. For bioanalysis, plasma samples
were thawed and 10 ꢁL of plasma were added to 50 ꢁL acetonitrile containing
diphenhydramine (750 nM) as internal standard. Samples and calibration standards (in rat
plasma) were centrifuged at 2400 xg for 5 min at 4 °C. The solutions were transferred to fresh
vials for HPLCꢀMS/MS analysis (Accucore RPꢀMS, TSQ Quantum triple quadrupole mass
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spectrometer, electrospray interface). After injection of 10 ꢁL (performed in duplicate), data
were analyzed based on the ratio of the peak areas of analyst and internal standard. Mean key
pharmacokinetic parameters were estimated by noncompartmental analysis using the
computer software Phoenix WinNonlin. Cmax and Tmax were obtained directly from the
plasma concentrationꢀtime curve. All other parameters (t1/2, Vdss, CL, AUC and F) were
calculated.
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Chemistry
General Experimental
Reagents and solvents were used as obtained from commercial suppliers without further
purification or drying. All reactions were performed under a nitrogen atmosphere unless
otherwise indicated. Yields refer to purified products and are not optimized. Flash
chromatography was performed on silica gel 60 (40−60 ꢁm). Melting points of samples were
determined in open capillaries using a SMP3 Melting Point Apparatus of Bibby Sterilin and
are uncorrected. Microwave irradiation experiments were performed in sealed tubes in a CEM
Discover Explorer 12 microwave reactor. 1H NMR and 13C spectra were recorded on a Bruker
DRXꢀ500 or Bruker Fourier 300 instrument. Chemical shifts are given in parts per million
(ppm) and spectra are obtained from DMSOꢀd6, CDCl3 or Acetonꢀd6 solutions, in which the
hydrogenated residues of deuterated solvents were used as internal standard (CDCl3: δ = 7.27,
77.00. Acetonꢀd6: δ = 2.05, 29.92. DMSOꢀd6: δ = 2.50, 39.51). The following abbreviations
are used to denote signal multiplicities: s= singlet, d= doublet, dd= doublet of doublet, t =
triplet, q = quartet and m = multiplet. All coupling constants (J) are given in Hertz (Hz).
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Several signals were assigned with the help of H, HꢀCOSY, H, 13CꢀHSQC and H, 13Cꢀ
HMBC experiments. Mass spectra (LC/MS) were measured on an MSQ® electro spray mass
spectrometer (ThermoFisher, Dreieich, Germany). The purity of all compounds was ≥95% as
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measured by LC/MS, monitored at 254 nm. The final compounds do not belong to known
classes of assay interference compounds.19
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Method A: SuzukiꢀCoupling using Pd(PPh3)4
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The corresponding brominated aromatic compound (1.0 eq) and the boronic acid (1.5 eq)
were dissolved in toluene (10 mL/mmol), ethanol (10 mL/mmol) and aq. Na2CO3 (2.0 M,
2.5 mL/mmol). The mixture was deoxygenated under reduced pressure and flushed with N2.
After having repeated this cycle three times, Pd(PPh3)4 (5 mol%) was added. The resulting
suspension was heated under reflux for 4–12 h. After cooling, the phases were separated and
the aqueous phase was extracted two times with EtOAc. The combined organic extracts were
dried over MgSO4 and concentrated under reduced pressure. The purification was performed
by flash chromatography using SiO2. After flash chromatography the product was dissolved in
ethyl acetate and a few drops of conc. HCl and water were added. Mixture was stirred for
5 min followed by separation of the phases and neutralization of the aqueous phase with aq.
Na2CO3 solution (2.0 M). After extraction with ethyl acetate and drying over MgSO4 the
solvent was removed under reduced pressure.
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Method B: SuzukiꢀReaction using Palladium(II) acetate
The corresponding brominated aromatic compound (1.0 eq) was dissolved in degassed
1,4ꢀdioxane (7 mL/mmol) under N2. Subsequently, the boronic acid or boronic acid
pinacolester (1.7 eq), palladium(II) acetate (0.7 mol%), SPhos (2 mol%) and 2 M aqueous,
degassed LiOH solution (3.5 eq) were added. The reaction mixture was stirred at 90 °C for
20 h. After cooling to room temperature water and ethyl acetate were added and the
suspension was filtered over celite. The phases were separated and the aqueous phase was
extracted with ethyl acetate thrice. The combined organic layers were washed with 1 M
aqueous NaOH solution, water and brine, dried over Na2SO4 and concentrated in vacuum.
The purification was performed by flash chromatography using SiO2.
Method C: Wohl Ziegler Bromination
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The methyl heteroaromatic compound was dissolved in dry carbon tetrachloride
(10 mL/mmol). To this solution Nꢀbromsuccinimide (NBS) (1.1 eq) and benzoyl peroxide
(5 mol%) were added and the mixture was refluxed over night. After cooling, the succinimide
was removed by filtration and the filtrate was concentrated under reduced pressure. The crude
product was purified by flash chromatography on silicaꢀgel.
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Method D: SNꢀReaction
K2CO3 (5.0 eq), imidazole (4.0 eq) and the corresponding methyl heteroaromatic bromide
were suspended in DMF (28 mL/mmol). The resulting mixture was heated to 120 °C for 2 h.
After cooling, water was added and the aqueous layer was extracted with EtOAc thrice. The
combined organic layers were washed with brine, dried over MgSO4 and evaporated in vacuo.
The crude product was purified by column chromatography using SiO2. After flash
chromatography the product was dissolved in ethyl acetate and a few drops of conc. HCl and
water were added. Mixture was stirred for 5 min followed by separation of the phases and
neutralization of the aqueous phase with aq. Na2CO3 solution (2.0 M). After extraction with
ethyl acetate and drying over MgSO4 the solvent was removed under reduced pressure.
2ꢀ(2ꢀFluorophenyl)ꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)pyridine (1). Synthesized using
compound 29 (3:1 mixture of chloride and bromide, 150 mg, 0.65 mmol),
2ꢀfluorophenylboronic acid (160 mg, 1.20 mmol), Palladium(II) acetate (1.00 mg, 4.45 µmol),
SPhos (5.00 mg, 12.2 µmol) and 2 M aqueous LiOH (1.15 mL, 2.30 mmol) according to
method B. As the reaction was not completed after 12 h the same amount of boronic acid,
SPhos , Pd(OAc)2 and LiOH was added and the reaction mixture was stirred again for 2 h at
90 °C. Crude product was purified by flash chromatography on silicaꢀgel using ethyl acetate
1
as eluent. White solid. Yield: 113 mg, 62%. Mp 71–72 °C. H NMR (Acetonꢀd6, 500 MHz):
δH (ppm) = 8.67 (d, J = 1.6 Hz, 1 H), 8.40 (d, J = 2.5 Hz, 1 H), 8.30 (d, J = 1.6 Hz, 1 H), 8.05
(td, J = 8.0, 1.7 Hz, 1 H), 7.78 (ddd, J = 8.2, 2.2, 1.0 Hz, 1 H), 7.73 (dd, J = 8.2, 2.2 Hz, 1 H),
7.50 (m, 1 H), 7.45 (m, 1 H), 7.30 (td, J = 7.6, 1.3 Hz, 1 H), 7.23 (ddd, J = 11.7, 8.3, 1.4 Hz, 1
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H), 4.07 (s, 2 H), 2.28 (s, 3 H). 13C NMR (Acetonꢀd6, 125 MHz): δC (ppm) =
161.5 (d, JC,F = 248.4 Hz), 152.1 (d, JC,F = 1.8 Hz), 151.0, 149.3, 148.2, 137.6, 137.4, 136.3,
133.9, 132.0 (2 C), 131.4 (d, JC,F = 9.2 Hz), 128.1 (d, JC,F = 11.9 Hz), 125.5 (d, JC,F = 2.8 Hz),
124.9 (d, JC,F = 10.1 Hz), 117.0 (d, JC,F = 23.8 Hz), 36.1, 18.3. (ESI): m/z = 279 [M+H]+.
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Nꢀ(3ꢀ(5ꢀ((5ꢀMethylpyridinꢀ3ꢀyl)methyl)pyridinꢀ2ꢀyl)phenyl)methanesulfonamide
(2).
Synthesized using compound 29 (3:1 mixture of chloride and bromide, 100 mg, 0.44 mmol)
and (3ꢀ(methylsulfonamido)phenyl)boronic acid (120 mg, 0.56 mmol) according to Method
A. The reaction mixture was stirred for 18 hours at 100 °C. Crude product was purified by
flash chromatography on silicaꢀgel using a mixture of dichloromethane/ methanol (95:5) as
eluent. Afterwards the product was washed with diethylether. Beige solid. Yield: 101 mg,
1
65%. Mp 204–206 °C. H NMR (CDCl3, 500 MHz): δH (ppm) = 9.84 (br s, NH), 8.62 (d, J =
1.3 Hz, 1 H), 8.37 (br s, 1 H), 8.27 (br s, 1 H), 7.95 (m, 1 H), 7.83 (d, J = 7.8 Hz, 1 H), 7.74
(d, J = 7.8 Hz, 1 H), 7.50 (br s, 1 H), 7.43 (t, J = 7.9 Hz, 1 H), 7.27 (d, J = 8.0 Hz, 1 H), 4.00
13
(s, 2 H), 3.01 (s, 3 H), 2.25 (s, 3 H). C NMR (CDCl3, 125 MHz): δC (ppm) = 153.5, 149.6,
147.9, 146.8, 139.5, 138.9, 137.3, 136.5, 135.4, 135.2, 132.8, 129.6, 121.8, 120.1, 120.1,
117.6, 39.2, 34.5, 17.7. (ESI): m/z = 354 [M+H]+.
2ꢀ(Furanꢀ3ꢀyl)ꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)pyridine (3). Synthesized using compound
29 (3:1 mixture of chloride and bromide, 200 mg, 0.87 mmol) and furanꢀ3ꢀylboronic acid
(128 mg, 1.14 mmol) according to method A. The reaction mixture was stirred for 20 h at 100
°C. Crude product was purified by flash chromatography on silicaꢀgel using a mixture of
dichloromethane/methanol (98:2) as eluent. White solid. Yield: 168 mg, 77%. Mp 69–71 °C.
1H NMR (CDCl3, 500 MHz): δH (ppm) = 8.45 (m, 1 H), 8.30–8.31 (m, 2 H),
7.98 (dd, J = 1.5, 0.9 Hz, 1 H), 7.46 (t, J = 1.7 Hz, 1 H), 7.43 (dd, J = 8.1, 2.3 Hz, 1 H),
7.37 (dd, J = 8.1, 0.8 Hz, 1 H), 7.24 (m, 1 H), 6.89 (dd, J = 1.9, 0.9 Hz, 1 H), 3.92 (s, 2 H),
2.26 (d, J = 0.5 Hz, 3 H). 13C NMR (CDCl3, 125 MHz): δC (ppm) = 150.1, 149.8, 148.5,
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(ESI): m/z = 251 [M+H]+.
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Nꢀcyclopropylꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)pyridinꢀ2ꢀamine
(7).
Compound
9
29 (3:1 mixture of chloride and bromide, 800 mg, 3.48 mmol) was dissolved under N2 in 40
mL dry toluene and cyclopropylamine (1.27 mL, 1.04 g, 18.2 mmol), sodium ethoxide (556
mg, 8.17 mmol), Pd(OAc)2 (8.00 mg, 35.6 µmol) and SPhos (27.9 mg, 68 µmol) were added.
The reaction mixture was stirred under reflux overnight and subsequently quenched with
water. After extraction of the water phase with EtOAc thrice the combined organic layers
were washed with water, brine, dried over Na2SO4, filtered and concentrated in vacuum.
Crude product was purified by flash chromatography on silicaꢀgel using a mixture of ethyl
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acetate/ethanol (9:1) as eluent. Pale yellow solid. Yield: 182 mg, 22%. Mp 81–83 °C. H
NMR (CDCl3, 300 MHz): δH (ppm) = 8.31 (s, 1 H), 8.28 (s, 1 H), 7.81 (dd, J = 2.1, 0.6 Hz, 1
H), 7.43 (dd, J = 8.8, 2.2 Hz, 1 H), 7.24 (m, 1 H), 6.88 (d, J = 8.8 Hz, 1 H), 6.45 (br s, 1 H),
3.81 (s, 2 H), 2.52 (m, 1 H), 2.29 (d, J = 0.6 Hz, 3 H), 0.82 (m, 2 H), 0.62 (m, 2 H). 13C NMR
(CDCl3, 75 MHz): δC (ppm) = 156.8, 148.6, 147.0, 142.3, 140.9, 136.7, 134.7, 133.2, 124.7,
108.3, 34.9, 23.9, 18.3, 7.5. (ESI): m/z = 240 [M+H]+.
2ꢀ((2ꢀFluorophenyl)thio)ꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)pyridine
(10).
Compound
29 (3:1 mixture of chloride and bromide, 100 mg, 0.44 mmol) was dissolved under N2 in
dimethylacetamid (2 mL), followed by addition of caesium carbonate (371 mg, 1.14 mmol)
and 2ꢀfluorothiophenol (45 ꢁL, 4.20 mmol). The reaction mixture was stirred at 90 °C
overnight, quenched with sat. NaHCO3ꢀsolution afterwards and extracted with EtOAc thrice.
The combined organic layers were washed with water, brine, dried over Na2SO4, filtered and
concentrated in vacuum. Crude product was purified by preparative thin layer
chromatography using a mixture of hexane/ethyl acetate (2:8) as eluent. Pale yellow oil.
1
Yield: 40 mg, 29%. H NMR (CDCl3, 500 MHz): δH (ppm) = 8.26–8.29 (m, 3 H),
7.57 (td, J = 7.4, 1.6 Hz, 1 H), 7.41 (m, 1 H), 7.28 (m, 1 H), 7.25 (dd, J = 8.2, 2.5 Hz, 1 H),
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7.19–7.13 (m, 2 H), 6.87 (d, J = 8.3 Hz, 1 H), 3.86 (s, 2 H), 2.28 (s, 3 H). C NMR (CDCl3,
125 MHz): δC (ppm) = 162.6 (d, JC,F = 249.3 Hz), 157.4, 149.6, 147.5, 146.0, 137.7, 137.1,
136.9, 135.1, 133.6, 131.7, 131.6, 124.9 (d, JC,F = 3.7 Hz), 121.1, 117.9 (d, JC,F = 18.3 Hz),
116.3 (d, JC,F = 22.0 Hz), 35.3, 18.2. (ESI): m/z = 311 [M+H]+.
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2ꢀ((2ꢀFluorophenyl)sulfonyl)ꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)pyridine (11). To
a
solution of thioether 10 (30 mg, 96.7 µmol) in ethyl acetate (2 mL) was added water (2 mL)
and potassium peroxymonosulfate (119 mg, 0.39 mmol) at room temperature and the reaction
mixture was vigorously stirred for 4 h at room temperature. Saturated NaHCO3ꢀsolution was
added and the mixture was extracted with DCM. The combined organic layers were dried
over Na2SO4 and concentrated in vacuum. Crude product was purified by flash
chromatography on silicaꢀgel using ethyl acetate as eluent. Beige solid. Yield: 21 mg, 64%.
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Mp 144–146 °C. H NMR (CDCl3, 300 MHz): δH (ppm) = 8.49 (s, 1 H), 8.33 (s, 1 H),
8.28 (s, 1 H), 8.16–8.23 (m, 2 H), 7.73 (d, J = 8.1 Hz, 1 H), 7.62 (m, 1 H), 7.36 (m, 1 H),
7.24 (br s, 1 H), 7.10 (dd, J = 9.6, 8.6 Hz, 1 H), 4.01 (s, 2 H), 2.29 (s, 3 H). 13C NMR (CDCl3,
75 MHz): δC (ppm) = 159.5 (d, JC,F = 257.2 Hz), 156.5, 150.5, 149.1, 147.2, 140.2, 138.1,
136.9, 136.5, 136.4, 133.4 (d, JC,F = 13.8 Hz), 131.1, 127.0 (d, JC,F = 13.8 Hz),
124.7 (d, JC,F = 3.8 Hz), 122.8 (d, JC,F = 1.9 Hz), 117.1 (d, JC,F = 20.9 Hz), 35.9, 18.3.
(ESI): m/z = 343 [M+H]+.
5ꢀ((1HꢀImidazolꢀ1ꢀyl)methyl)ꢀ3ꢀphenylisothiazole (14). Synthesized using compound 34
(46 mg, 0.18 mmol), imidazole (49 mg, 0.72 mmol) and K2CO3 (124 mg, 0.90 mmol) in DMF
according to method D. Crude product was purified by flash chromatography on silicaꢀgel
1
using ethyl acetate as eluent. White solid. Yield: 17 mg, 40%. Mp 92–94 °C. H NMR
(CDCl3, 500 MHz): δH (ppm) = 7.91–7.86 (m, 2H, Ph H), 7.64 (s, 1H, imidazolyl Hꢀ2), 7.47–
7.38 (m, 3H, Ph H), 7.37 (t, J = 1.0 Hz, 1H, isothiazole Hꢀ4), 7.16 (s, 1H, imidazolyl Hꢀ4),
7.02 (s, 1H, imidazolyl Hꢀ5), 5.46–5.41 (m, 2H, CH2). 13C NMR (CDCl3, 125 MHz): δC
(ppm) = 168.1 (isothiazole Cꢀ3), 163.5 (isothiazole Cꢀ5), 137.2 (imidazolyl Cꢀ2), 134.2 (Ph
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Cq), 130.4 (imidazolyl Cꢀ4), 129.5 (Ph C), 128.8 (Ph C), 126.8 (Ph C), 120.5 (isothiazole Cꢀ
4), 119.0 (imidazolyl Cꢀ5), 43.6 (CH2). (ESI): m/z = 242 [M+H]+.
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5ꢀ((5ꢀMethylpyridinꢀ3ꢀyl)methyl)ꢀ3ꢀphenylisothiazole (15). Synthesized using compound
34 (180 mg, 0.71 mmol) and (5ꢀmethylpyridinꢀ3ꢀyl)boronic acid (146 mg, 1.07 mmol)
according to method A. Crude product was purified by flash chromatography on silicaꢀgel
using ethyl acetate as eluent. Colorless oil. Yield: 40 mg, 21%. 1H NMR (CDCl3, 500 MHz):
δH (ppm) = 8.40 (s, 1H, pyridinyl Hꢀ2), 8.38 (s, 1H, pyridinyl Hꢀ6), 7.94–7.87 (m, 2H, Ph H),
7.44–7.39 (m, 3H, Ph H), 7.39–7.37 (m, 1H, pyridinyl Hꢀ4), 7.31 (t, J = 1.0 Hz, 1H,
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isothiazole Hꢀ4), 4.23 (s, 2H, CH2), 2.35–2.31 (m, 3H, CH3). C NMR (CDCl3, 125 MHz):
δC (ppm) = 168.1 (isothiazole Cꢀ3), 167.6 (isothiazole Cꢀ5), 149.4 (pyridinyl Cꢀ6), 147.1
(pyridinyl Cꢀ2), 136.8 (Ph C), 134.9 (Ph Cq), 133.7 (pyridinyl Cꢀ3), 133.5 (pyridinyl Cꢀ5),
129.4 (pyridinyl Cꢀ4), 128.9 (Ph C), 127.0 (Ph C), 120.9 (isothiazole Cꢀ4), 31.4 (CH2), 18.5
(CH3). (ESI): m/z = 267 [M+H]+.
5ꢀ((5ꢀMethylpyridinꢀ3ꢀyl)methyl)ꢀ3ꢀphenylisoxazole (24). Synthesized using compound
43 (1:3 mixture of chloride and bromide, 127 mg, 0.56 mmol) and
(5ꢀmethylpyridinꢀ3ꢀyl)boronic acid (110 mg, 0.80 mmol) according to method A. Crude
product was purified by flash chromatography on silicaꢀgel using ethyl acetate as eluent.
1
Yellow solid. Yield: 58 mg, 41%. Mp 62–64 °C. H NMR (CDCl3, 500 MHz): δH (ppm) =
8.42–8.40 (m, 1H, pyridinyl Hꢀ2), 8.40–8.38 (m, 1H, pyridinyl Hꢀ6), 7.79–7.74 (m, 2H, Ph
H), 7.47–7.40 (m, 4H, Ph H, pyridinyl Hꢀ4), 6.26 (t, J = 0.8 Hz, 1H, isoxazol Hꢀ4), 4.11 (s,
13
2H, CH2), 2.34 (s, 3H CH3). C NMR (CDCl3, 125 MHz): δC (ppm) = 171.6 (isoxazol Cꢀ5),
162.8 (isoxazol Cꢀ3), 149.5 (pyridinyl Cꢀ6), 147.3 (pyridinyl Cꢀ2), 137.1 (pyridinyl Cꢀ4),
133.6 (pyridinyl Cꢀ5), 131.3 (pyridinyl Cꢀ3), 130.2 (Ph C), 129.2 (Ph Cq), 129.1 (Ph C), 127.0
(Ph C), 100.5 (isoxazol Cꢀ4), 30.6 (CH2), 18.5 (CH3). (ESI): m/z = 251 [M+H]+.
5ꢀ((5ꢀMethoxypyridinꢀ3ꢀyl)methyl)ꢀ3ꢀphenylisoxazole (25). A mixture of compound 43
(1:3 mixture of chloride and bromide, 315 mg, 1.39 mmol), 3ꢀmethoxyꢀ5ꢀ(4,4,5,5ꢀ
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tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)pyridine (373 mg, 1.59 mmol), Cs2CO3 (1.29 g,
3.96 mmol) and PdCl2(dppf) (48.0 mg, 0.07 mmol) were dissolved in DME/H2O/EtOH
(3 mL/ 3 mL/ 3 mL). The reaction mixture was stirred for 20 min at 150 °C, 150 W and 18
bar in the microwave oven. After addition of H2O and extraction with ethyl acetate three
times, the combined organic phases were dried over MgSO4 and concentrated under reduced
pressure. Crude product was purified by flash chromatography on silicaꢀgel using a mixture of
hexane/ethyl acetate (1:2) as eluent. After flash chromatography the product was dissolved in
ethyl acetate and a few drops of conc. HCl and water were added. After stirring for 30 min the
phases were separated and aqueous phase was neutralized with aqueous Na2CO3 solution
(2M). After extraction with ethyl acetate and drying over MgSO4 the solvent was removed
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under reduced pressure. White solid. Yield: 107 mg, 29%. Mp: 71–73 °C. H NMR (CDCl3,
500 MHz): δH (ppm) = 8.27 (s, 1H, pyridinyl Hꢀ6)), 8.21 (s, 1H, pyridinyl Hꢀ2), 7.73–7.80 (m,
2H, Ph H), 7.41–7.48 (m, 3H, Ph H), 7.13–7.17 (m, 1H, pyridinyl Hꢀ4), 6.28 (t, J = 0.8 Hz,
1H, isoxazol Hꢀ4), 4.13 (s, 2H, CH2), 3.86 (s, 3H, CH3). 13C NMR (CDCl3, 125 MHz): δC
(ppm) = 171.1 (isoxazol Cꢀ5), 162.6 (isoxazol Cꢀ3), 155.8 (pyridinyl Cꢀ5), 142.1 (pyridinyl
Cꢀ2), 136.5 (pyridinyl Cꢀ6), 132.1 (pyridinyl Cꢀ3), 130.0 (Ph C), 128.9 (Ph Cq), 128.9 (Ph C),
126.7 (Ph C), 120.9 (pyridinyl Cꢀ4), 100.3 (isoxazol Cꢀ4), 55.6 (CH3), 30.4 (CH2). MS (ESI):
m/z = 267 [M+H]+.
3ꢀMethylꢀ5ꢀ((1ꢀmethylꢀ3ꢀphenylꢀ1Hꢀpyrazolꢀ5ꢀyl)methyl)pyridine (26). Synthesized using
compound 46 (122 mg, 0.49 mmol) and (5ꢀmethylpyridinꢀ3ꢀyl)boronic acid (100 mg,
0.73 mmol) according to method A. Crude product was purified by flash chromatography on
silicaꢀgel using ethyl acetate as eluent. White solid. Yield: 65 mg, 50%. Mp 41–43 °C.
1H NMR (CDCl3, 500 MHz): δH (ppm) = 8.39–8.37 (m, 1H), 8.36–8.35 (m, 1H), 7.78–7.75
(m, 2H), 7.40–7.36 (m, 2H), 7.31–7.26 (m, 2H), 6.31 (s, 1H), 3.99 (s, 2H), 3.79 (s, 3H),
2.32 (d, J = 0.6 Hz, 3H). 13C NMR (CDCl3, 125 MHz): δC (ppm) = 150.2, 148.9, 146.9, 141.5,
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136.5, 133.3, 133.3, 132.4, 128.6, 127.5, 125.4, 103.4, 36.5, 29.1, 18.3.
(ESI): m/z = 264 [M+H]+.
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(6ꢀBromopyridinꢀ3ꢀyl)(5ꢀmethylpyridinꢀ3ꢀyl)methanol (28). To a stirred solution of
3ꢀbromoꢀ5ꢀmethylpyridine (6.30 mL, 9.39 g, 54.6 mmol) in dry diethyl ether (760 mL) was
added nꢀButyllithium (2.5 M in hexane, 20.6 mL, 51.5 mmol) dropwise at –78 °C and the
clear solution was stirred for additional 5 min at this temperature. Subsequently a solution of
6ꢀbromonicotinaldehyde (10.1 g, 54.3 mmol) in dry toluene (20 mL) and dry THF (30 mL)
was added dropwise and the pale brown suspension was stirred for additional 30 min
at ꢀ78 °C. The reaction mixture was quenched with sat. aqueous NH4Cl solution, diluted with
water at room temperature and extracted with diethyl ether thrice. The combined organic
layers were dried over Na2SO4 and concentrated in vacuum. Crude product was purified by
flash chromatography on silicaꢀgel using a mixture of ethyl acetate/ethanol (9:1) as eluent.
Yellow solid. Yield: 9.76 g, 64%. Mp 126–128 °C. 1H NMR (Acetonꢀd6, 500 MHz): δH (ppm)
= 8.46–8.47 (m, 2 H), 8.32 (d, J = 1.7 Hz, 1 H), 7.74 (ddd, J = 8.2, 2.5, 0.6 Hz, 1 H), 7.60 (m,
1 H), 7.56 (d, J = 8.2 Hz, 1 H), 5.98 (s, 1 H), 5.41 (br s, 1 H), 3.16 (s, 3 H). 13C NMR
(Acetonꢀd6, 125 MHz): δC (ppm) = 151.2, 150.4, 147.2, 142.7, 141.9, 140.8, 139.1, 136.0,
134.9, 129.7, 72.3, 19.3.
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Mixture
of 2ꢀChloroꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)pyridine and 2ꢀBromoꢀ
5 ((5 methylpyridinꢀ3ꢀyl)methyl)pyridine (29). Ice cooled alcohol 28 (9.76 g, 35.0 mmol)
was dissolved under nitrogen atmosphere in fresh distilled thionyl chloride (43.0 mL,
595 mmol). The reaction mixture was stirred for 3 h at 60 °C. After cooling to room
temperature, the thionyl chloride was removed in vacuum, the residue neutralized with sat.
aqueous NaHCO3ꢀsolution and extracted with ethyl acetate thrice. The combined organic
layers were washed successively with sat. NaHCO3 solution, water, brine and dried over
Na2SO4. The solvent was removed in vacuum and the crude chloride was dissolved under
nitrogen atmosphere in degassed glacial acetic acid (90 mL). Zincꢀpowder was added in small
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portions and the reaction mixture was stirred for 24 h at room temperature. Afterwards the
solvent was evaporated in vacuum, saturated aqueous NaHCO3 solution and ethyl acetate
were added to the residue and the mixture was filtered over celite. Subsequently, the phases
were separated and the aqueous phase was extracted twice with ethyl acetate. The combined
organic layers were washed with water and brine, dried over Na2SO4 and concentrated in
vacuum. Crude product was purified by flash chromatography on silicaꢀgel using ethyl acetate
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as
eluent
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an
inseparable
mixture
of
2ꢀChloroꢀ5ꢀ((5ꢀmethylꢀ
pyridinꢀ3ꢀyl)methyl)pyridine and 2ꢀBromoꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)pyridine (3:1).
White solid. Yield: 6.25 g, 78%. 2ꢀChloroꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀyl)methyl)pyridine.
1H NMR (CDCl3, 300 MHz): δH (ppm) = 8.31 (s, 1 H), 8.27 (s, 1 H), 8.25 (d, J = 2.5 Hz, 1 H),
7.40 (dd, J = 8.2, 2.5 Hz, 1 H), 7.20–7.24 (m, 2 H), 3.90 (s, 2 H), 2.27 (s, 3 H). 13C NMR
(CDCl3, 75 MHz): δC (ppm) = 149.7, 149.6, 148.8, 147.0, 139.1, 136.6, 134.3, 134.1, 133.2,
124.2, 35.2, 18.2. (ESI): m/z = 219 [M+H]+. 2ꢀBromoꢀ5ꢀ((5ꢀmethylpyridinꢀ3ꢀ
1
yl)methyl)pyridine. H NMR (CDCl3, 300 MHz): δH (ppm) = 8.31 (d, J = 1.6 Hz, 1 H),
8.28 (d, J = 1.7 Hz, 1 H), 8.24 (d, J = 2.0 Hz, 1 H), 7.39 (d, J = 7.9 Hz, 1 H),
13
7.30 (dd, J = 8.2, 2.5 Hz, 1 H), 7.21 (m, 1 H), 3.88 (s, 2 H), 2.28 (s, 3 H). C NMR (CDCl3,
75 MHz): δC (ppm) = 150.2, 148.8, 147.1, 140.2, 138.9, 136.7, 134.8, 134.0, 133.2, 128.0,
35.2, 18.2. (ESI): m/z = 263 [M+H]+.
Ethyl 3ꢀPhenylisothiazoleꢀ5ꢀcarboxylate (32). A mixture of 30 (877 mg, 4.89 mmol) and
ethyl propiolate (4.90 mL, 4.80 g, 48.9 mmol) in CHCl3 (3 mL/mmol) was irradiated for
25 min at 160 °C and 300 W in the microwave oven. The solvent was evaporated and the
regioisomers 31 and 32 were obtained. The mixture was purified by flash chromatography on
silicaꢀgel using a mixture of hexane/ethyl acetate (25:1) as eluent. White solid. Yield: 427 mg,
37%. Mp 70–72 °C. 1H NMR (500 MHz, CDCl3): δH (ppm) = 8.12 (s, 1H), 7.99–
7.95 (m, 2H), 7.50–7.41 (m, 3H), 4.43 (q, J = 7.0 Hz, 2H), 1.43 (t, J = 7.3 Hz, 3H). 13C NMR
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(125 MHz, CDCl3,): δC (ppm) = 168.3, 160.4, 158.0, 134.3, 129.9, 129.2, 127.1, 125.1, 62.3,
14.5. (ESI): m/z = 234 [M+H]+.
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5ꢀ(Bromomethyl)ꢀ3ꢀphenylisothiazole (34). To a solution of 32 (360 mg, 1.54 mmol) in dry
THF (10 mL/mmol) was added lithiumaluminiumhydrid (117 mg, 3.08 mmol) in portions
at ꢀ40 °C. After 1 h stirring saturated NaHCO3ꢀsolution (10 mL/mmol) was added slowly
at ꢀ40 °C and the mixture was extracted with ethyl acetate twice. The combined organic
phases were dried over MgSO4 and concentrated under reduced pressure resulting in (3ꢀ
phenylisothiazolꢀ5ꢀyl)methanol as a white solid (291 mg). Without further purification
(3ꢀphenylisothiazolꢀ5ꢀyl)methanol (202 mg, 1.06 mmol) was dissolved in CH2Cl2
(15 mL/mmol) and triphenylphosphine (433 mg, 1.65 mmol) and tetrabrommethane (701 mg,
2.11 mmol) were added under stirring at 0 °C. The reaction mixture was then stirred for 1 h at
room temperature, concentrated under reduced pressure and purified by flash chromatography
on silicaꢀgel using a mixture of hexane/ethyl acetate (16:1) as eluent. Brown solid. Yield:
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208 mg, 77%. Mp 67–70 °C. H NMR (500 MHz, CDCl3): δH (ppm) = 7.97–7.90 (m, 2H),
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7.60 (t, J = 0.8 Hz, 1H), 7.49–7.40 (m, 3H), 4.75 (d, J = 1.0 Hz, 2H). C NMR (125 MHz,
CDCl3,): δC (ppm) = 168.1, 164.9, 134.7, 129.6, 129.1, 127.1, 122.2, 21.9. (ESI):
m/z = 254 [M+H]+.
Mixture of 5ꢀ(Bromomethyl)ꢀ3ꢀphenylisoxazole and 5ꢀ(Chloromethyl)ꢀ3ꢀphenylisoxazole
(43). To a stirred solution of (E)ꢀbenzaldehyde oxime (38, 1.80 mL, 2.00 g, 16.5 mmol),
propargyl bromide (80% in toluene, 2.20 mL, 2.35 g, 19.8 mmol) and triethylamine (2.30 mL,
1.68 g, 16.6 mmol) in CH2Cl2 (30 mL) was added 12% aqueous sodium hypochlorite
(49.5 mL, 79.8 mmol) dropwise. After 8 h stirring at room temperature phases were separated
and aqueous phase was extracted with CH2Cl2 twice. The combined organic phases were
washed with water, dried over MgSO4, filtered and concentrated in vacuum. Crude product
was purified by flash chromatography on silicaꢀgel using a mixture of hexane/ethyl acetate
(9:1) as eluent to yield an inseparable mixture of 5ꢀ(Chloromethyl)ꢀ3ꢀphenylisoxazole and 5ꢀ
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(Bromomethyl)ꢀ3ꢀphenylisoxazole (1:3). Orange solid. Yield: 1.48 g, 40%. H NMR (CDCl3,
500 MHz): δH (ppm) = 7.79–7.84 (m, 9H), 7.45–7.50 (m, 12H), 6.63–6.66 (m, 3H),
4.67 (CH2Cl, d, J = 0.95 Hz, 2H), 4.52 (CH2Br, d, J = 0.63 Hz, 6H). 13C NMR (CDCl3,
125 MHz): δC (ppm) = 167.9, 162.7, 130.2, 129.9, 129.0, 128.5, 128.5, 126.8, 101.9, 101.8,
34.5, 18.6.
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5ꢀ(Bromomethyl)ꢀ1ꢀmethylꢀ3ꢀphenylꢀ1Hꢀpyrazole (46). To a stirred suspension of
1ꢀMethylꢀ3ꢀphenylꢀ1Hꢀpyrazoleꢀ5ꢀcarboxylic acid (40, 927 mg, 4.58 mmol) in dry THF (35
mL) was added lithium aluminium hydride (348 mg, 9.17 mmol) at 0 °C. Reaction mixture
was allowed to warm to room temperature and was stirred for 12 h. Afterwards ice water (20
mL) was added slowly and the obtained mixture was extracted with ethyl acetate twice. The
combined organic phases were dried over MgSO4 and concentrated under reduced pressure.
Crude product was purified by flash chromatography on silicaꢀgel using ethyl acetate as
eluent. (1ꢀMethylꢀ3ꢀphenylꢀ1Hꢀpyrazolꢀ5ꢀyl)methanol was obtained as white solid. Yield: 634
1
mg, 74%. Mp 124–126 °C. H NMR (CDCl3, 500 MHz): δH (ppm) = 7.78–7.73 (m, 2H),
7.42–7.36 (m, 2H), 7.32–7.28 (m, 1H), 6.44 (s, 1H), 4.65 (s, 2H), 3.90 (s, 3H), 2.39 (br s, 1H).
13C NMR (CDCl3, 125 MHz): δC (ppm) = 150.1, 142.6, 133.3, 128.6, 127.6, 125.4, 102.9,
55.6, 36.6. (ESI): m/z = 189 [M+H]+. To a solution of (1ꢀMethylꢀ3ꢀphenylꢀ1Hꢀpyrazolꢀ5ꢀ
yl)methanol (719 mg, 3.82 mmol) in dry THF (50 mL) was added phosphorus tribromide
(0.72 mL, 2.07 g, 7.65 mmol) dropwise at 0 °C. Reaction mixture was allowed to warm to
room temperature and was stirred for 20 h. Afterwards ice water (30 mL) was added slowly
and the obtained mixture was extracted with ethyl acetate twice. The combined organic
phases were dried over MgSO4 and concentrated under reduced pressure. Crude product was
purified by flash chromatography on silicaꢀgel using a mixture of hexane/ethyl acetate (6:1) as
1
eluent. White solid. Yield: 775 mg, 81%. Mp 127–130 °C. H NMR (CDCl3, 500 MHz): δH
(ppm) = 7.78–7.72 (m, 2H, Ph H), 7.43–7.37 (m, 2H, Ph H), 7.34–7.29 (m, 1H, Ph H), 6.60
(s, 1H, pyrazole Hꢀ4), 4.59 (s, 2H, CH2), 4.05 (s, 3H, CH3). 13C NMR (CDCl3, 125 MHz): δC
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(ppm) = 150.2 (pyrazole Cꢀ3), 138.8 (pyrazole Cꢀ5), 132.9 (Ph Cq), 128.6 (Ph C), 127.7 (Ph
C), 125.4 (Ph C), 104.0 (pyrazole Cꢀ4), 36.6 (CH3), 20.6 (CH2). (ESI): m/z = 251 [M+H]+.
ASSOCIATED CONTENT
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Supporting Information. HPLC purity of final compounds, synthetic procedures and
characterizations of the final compounds 4–6, 8, 9, 12, 13, 16–23, 27 and intermediates 31,
33, 36, 38, 41, 42, 45. Inhibition of CYP17A1 and CYP19A1 by compounds 24 and 25 and
plasma concentrations in rat versus time after oral application of compounds 24 and 25. This
material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* For R.W. H.: Phone: +(49) 681 98806 2000. EꢀMail: rolf.hartmann@helmholtzꢀhzi.de.
ACKNOWLEDGMENTS
The authors thank Dr. Nina Hanke, Martina Jankowski, Jeannine Jung and Isabella Mang for
their help in performing in vitro tests, Tobias Bernard for his help in performing synthesis, Dr.
Franck Lach for the synthesis of compounds 3 and 6, and Julia Parakenings for her help in the
PK experiments. We thank prof. Gerald Thiel (Dept of Medicinal Biochemistry and
Molecular Biology) for providing the HepG2 cell line.
ABBREVIATIONS
CYP11B1, 11βꢀhydroxylase; CYP11B2, aldosterone synthase; CYP17A1, 17αꢀhydroxylaseꢀ
17,20ꢀlyase; CYP19A1, aromatase; ACTH, adrenocorticotropic hormone; SF, selectivity
factor = IC50
/ IC50 CYP11B1; t1/2, half–life; Ref, reference; CDI, 1,1'ꢀ
CYP11B2
carbonyldiimidazole; NBS, Nꢀbromosuccinimide; SPhos, 2ꢀDicyclohexylphosphinoꢀ2',6'ꢀ
dimethoxybiphenyl; AIBN, Azobisisobutyronitrile; NMP, NꢀMethylꢀ2ꢀpyrrolidone
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