Discovery of N-[5-(6-Chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide, a Cortisol-Sparing CYP11B2 Inhibitor that Lowers Aldosterone in Human Subjects

Article abstract of DOI:10.1021/acs.jmedchem.5b01545

Human clinical studies conducted with LCI699 established aldosterone synthase (CYP11B2) inhibition as a promising novel mechanism to lower arterial blood pressure. However, LCI699's low CYP11B1/CYP11B2 selectivity resulted in blunting of adrenocorticotropic hormone-stimulated cortisol secretion. This property of LCI699 prompted its development in Cushing's disease, but limited more extensive clinical studies in hypertensive populations, and provided an impetus for the search for cortisol-sparing CYP11B2 inhibitors. This paper summarizes the discovery, pharmacokinetics, and pharmacodynamic data in preclinical species and human subjects of the selective CYP11B2 inhibitor 8.

Full text of DOI:10.1021/acs.jmedchem.5b01545

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Article
Discovery of N-[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-
pyridin-3-ylmethyl]-ethanesulfonamide, a Cortisol-Sparing
CYP11B2 Inhibitor that Lowers Aldosterone in Human Subjects
Julien P.N. Papillon, Changgang Lou, Alok Singh, Christopher M. Adams, Gary M. Ksander,
Michael Beil, Wei Chen, Jennifer Leung-Chu, Fumin Fu, Lu Gan, Chii-Whei Hu, Arco Jeng, Daniel
LaSala, Guiqing Liang, Dean F Rigel, Kerry S Russell, John A Vest, and Catherine Watson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01545 • Publication Date (Web): 05 Nov 2015
Downloaded from http://pubs.acs.org on November 12, 2015
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Discovery of Nꢀ[5ꢀ(6ꢀchloroꢀ3ꢀcyanoꢀ1ꢀmethylꢀ1Hꢀ
indolꢀ2ꢀyl)ꢀpyridinꢀ3ꢀylmethyl]ꢀethanesulfonamide,
a CortisolꢀSparing CYP11B2 Inhibitor that Lowers
Aldosterone in Human Subjects
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,ǂ
ǂ,
ǂ
ǂ
⊥
Julien P.N. Papillon,* Changgang Lou, Alok K. Singh, Christopher M. Adams, Gary M.
Ksander,ǂ,○ Michael E. Beil,ꢀ Wei Chen, ꢀ Jennifer LeungꢀChu,ꢀ Fumin Fu,ꢀ Lu Gan, ◊,⊥ Chiiꢀ
,
Whei Hu,ꢀ ,∞ Arco Y. Jeng,ꢀ ⊥ Daniel LaSala,ꢀ Guiqing Liang,ǁ Dean F. Rigel,ꢀ Kerry S. Russell,
§ John A. Vest, §,⊥ Catherine Watson.§
ǂ Global Discovery Chemistry, ǁ Metabolism and Pharmacokinetics, § Translational Medicine, ◊
Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, 250
Massachusetts Avenue, Cambridge, MA 02139; ꢀ Cardiovascular and Metabolism, Novartis
Institutes for BioMedical Research, One Health Plaza, East Hanover, NJ 07936.
Abstract
Human clinical studies conducted with LCI699 established aldosterone synthase (CYP11B2)
inhibition as a promising novel mechanism to lower arterial blood pressure. However, LCI699’s
low CYP11B1/CYP11B2 selectivity resulted in blunting of adrenocorticotropic hormoneꢀ
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stimulated cortisol secretion. This property of LCI699 prompted its development in Cushing’s
disease, but limited more extensive clinical studies in hypertensive populations, and provided an
impetus for the search for cortisolꢀsparing CYP11B2 inhibitors. This paper summarizes the
discovery, pharmacokinetics, and pharmacodynamic data in preꢀclinical species and human
subjects of the selective CYP11B2 inhibitor 8.
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Introduction
Aldosterone is the principal mineralocorticoid in man and is produced in the zona glomerulosa
(outer zone) of the adrenal cortex by aldosterone synthase (CYP11B2). Aldosterone is a key
component of the reninꢀangiotensinꢀaldosterone system (RAAS) and acts primarily at the renal
distal convoluted tubules as a critical regulator of fluid and electrolyte homeostasis.
Consequently, increases in aldosterone can result in elevated blood pressure and fluid retention.
Elevated serum aldosterone has been linked to resistant hypertension,1 which represents a
significant medical need.2 Patients with an aldosterone excess are more susceptible to premature
vascular disease, cardiac fibrosis, vessel wall inflammation, left ventricular hypertrophy, atrial
fibrillation, stroke, myocardial infarction, and metabolic syndrome.3 Aldosterone escape4 due to
inadequate suppression of aldosterone levels during longꢀterm blockade of RAAS with either
angiotensin converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) is also
associated with end organ damage. Suppression of aldosterone’s untoward effects, therefore,
represents an attractive therapeutic approach to reduce blood pressure and cardiovascular disease
burden. Mineralocorticoid receptor antagonists (MRAs) spironolactone and eplerenone have
demonstrated efficacy in hypertension, primary aldosteronism, obstructive sleep apnea or renal
disease. In addition MRAs have shown morbidity and mortality benefits in patients with both
mild and severe heart failure on standard therapy,5 in patients with left ventricular dysfunction
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after myocardial infarction,6 and in heart failure patients with a preserved ejection fraction and
elevated brain natriuretic peptide.7 However, there are significant limitations associated with
MRAs. The use of spironolactone is associated with hormonal side effects including breast pain,
gynecomastia, impotence in males, and menstrual irregularities in females. Eplerenone is more
selective for MR receptors and consequently has a lower incidence of sex hormoneꢀrelated
adverse events. However, eplerenone is less potent than spironolactone, and due to an increased
risk of hyperkalemia, is contraindicated in diabetic hypertensive patients with microalbuminuria
and various degrees of renal impairment. Importantly, treatment with MRAs is associated with a
reactive elevation of circulating aldosterone levels, which is postulated to potentially limit
efficacy by activating central mineralocorticoid receptors and increasing sympathetic neural
drive in the heart failure setting.8 Moreover, substantial evidence exists suggesting a pathological
role of aldosterone via nonꢀMR mediated pathways.9 Therefore, therapeutic agents that inhibit
the production of aldosterone may confer additional benefits over MRAs.
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Early studies with FAD286, an aldosterone synthase inhibitor (ASI), suggested that
pharmacological inhibition of CYP11B2 might indeed be clinically useful. Chronic
administration of FAD286 in rat models showed positive effects on inflammatory markers,
fibrosis, and progression of heart failure.10 Later studies with LCI69911 provided additional
support for the development of ASIs. Treatment of doubleꢀtransgenic rats prevented the
development of cardiac and renal functional abnormalities and prolonged survival, independent
of blood pressure.12 LCI699 doseꢀdependently lowered aldosterone in healthy males,12 and
lowered blood pressure in patients with primary hyperaldosteronism,13 essential hypertension14
and resistantꢀhypertension.15 LCI699 was also found to blunt cortisol secretion in response to
adrenocorticotropic hormone (ACTH) stimulation,16 a finding that is most likely related to potent
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inhibition of CYP11B1 by LCI699 (human rCYP11B2 IC50 = 0.7 nM, rCYP11B1 IC50 = 2.5
nM).11,12 Although this finding led to cessation of further exploration of the relationship between
aldosterone lowering, electrolytes modulation and blood pressure regulation with LCI699, they
created an opportunity to address an unmet medical need in Cushing’s disease. LCI699 was
shown to normalize urinary free cortisol in a 10ꢀweek proofꢀofꢀconcept study in 12 patients.16
The results from this study may spur further exploration of CYP11B1 selective inhibitors.17
However, antihypertensive proofꢀofꢀconcept with a CYP11B2ꢀselective ASI is still needed,18 and
much effort has been devoted to identifying such compounds.19 Herein, we describe the
discovery, pharmacokinetics and pharmacodynamics data in preꢀclinical species and human
subjects of the cortisolꢀsparing ASI Nꢀ[5ꢀ(6ꢀchloroꢀ3ꢀcyanoꢀ1ꢀmethylꢀ1Hꢀindolꢀ2ꢀyl)ꢀpyridinꢀ3ꢀ
ylmethyl]ꢀethanesulfonamide (8).
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Results and Discussion
The lead compound identified from the Novartis archive was pyridinyl indole 1, which had
been previously described in 1969 as useful in the treatment of hyperaldosteronism by Geigy
Chemical Corporation.20 As shown in Table 1, despite its low molecular weight, 1 showed
excellent inhibition of aldosterone secretion in an H295R cellular assay21 (IC50 = 21 nM) and
potent inhibition of recombinant CYP11B222 (IC50 = 3 nM). Most importantly, the selectivity
over CYP11B1 was an order of magnitude superior to that of LCI699 (S = 27 vs. 3), which led us
to optimize 1. One area of focus was the unacceptable level of aromatase (CYP19) inhibition,
namely 94% at 10 µM and 59% at 1 µM. Capping the indole hydrogen with methyl, as in 2, did
not impact aromatase inhibition but had a positive impact on biochemical and cellular potency,
as well as selectivity over CYP11B1. We quickly identified that nitrile substitution at the 3ꢀ
position of the indole, such as in 3, gave further improvement in selectivity over both CYP11B1
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and aromatase, while also improving stability in rat liver microsomes (LM), but at the expense of
onꢀtarget potency. Further substitution of the indole at the 6ꢀposition with chlorine, as in 4,
regained potency and dramatically improved stability in human liver microsomes. While 4 had
an attractive combination of potency, CYP selectivity and in vitro metabolic stability, more
extensive profiling revealed potent inhibition of monoamine oxidase A (MAOꢀA), with an IC50 =
0.21 µM. MAOꢀA regulates neurotrasmitter levels by oxidation of biogenic amines, and, like
CYP11B2, is expressed in mitochondrial membranes. Intestinal MAOꢀA inhibition can lead to
hypertensive crisis, when foods containing tyramine are consumed (soꢀcalled "cheese
reaction")23 or hyperserotonemia, if foods containing tryptophan are consumed.24 We found that
introduction of polar groups at the 5ꢀposition of the pyridine ring effectively eliminated MAOꢀA
inhibition. A sulfamate group, as in 5, not only removed inhibition of MAOꢀA (IC50 >10 µM),
but also increased selectivity over CYP11B1 and aromatase. However, 5 showed inadequate in
vitro metabolic stability. We surmised that a sulfonamide might afford improved stability.
Although sulfonamide 6, the matched pair of 5, was a weak ASI, adding the chlorine back to the
indole ring afforded 7, which exhibited improved potency against CY11B2, excellent
microsomal stability, and was also devoid of aromatase and MAOꢀA activity. However, 7
possessed only moderate selectivity over CYP11B1 (S = 43). Further gains in potency and
selectivity were achieved with 8, albeit at the expense of selectivity over aromatase.
Nevertheless, aromatase inhibition by 8 (IC50 = 3.4 µM) was still nearly 1000ꢀfold weaker than
CYP11B2.25 We also found that the indole core could be replaced with a 7ꢀazaꢀ, but not 5ꢀ
azabenzimidazole core. Compound 10 showed comparable cellular potency and selectivity over
CYP11B1 as 8, albeit with improved selectivity over aromatase, despite lacking the nitrile at the
3ꢀposition of the azaindole ring.
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Although much of the early literature around ligand efficiency metrics was being published at
the time these studies were conducted and did not impact our decisions, it is interesting to
consider a retrospective analysis (Table 1). As optimization proceeded from 1 to 8, LEI gradually
decreased as functionalities were appended to dial out offꢀtarget activities (0.53 to 0.32), but
LipE remained remarkably constant (5.4 to 5.7).
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Table 1. Optimization of potency, selectivity and microsomal stability of lead compound 1
IC50 (nM)
CYP11B2 CYP11B2 CYP19
LM t1/2 (min)
Rat Human
LEId
LipEe
%i at 10
Compd Aldoa hB2b Sc
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82f
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a Aldosterone secretion in H295R cells; b Human recombinant CYP11B2; c Ratio of CYP11B1
IC50 over CYP11B2 IC50; d LEI = ꢀlog(IC50)/HA; e LipE = ꢀlog(IC50) − clogP; f IC50 = 3.4 µM.
Equilibrium solubility and pharmacokinetic data for 8 and 10 are summarized in Table 2. The
7ꢀazabenzimidazole afforded much improved solubility over the indole core, however, 10
suffered from high clearance and very short halfꢀlife. Despite its low equilibrium solubility, 8
showed good oral exposure and excellent oral bioavailability, which, combined with its cellular
potency and favorable selectivity profile, prompted us to select this compound for further
evaluation.26
Table 2. Equilibrium Solubility and Rat Pharmacokinetics of 8 and 10.
Compd
Equilibrium
solubility (mM) in
buffer pH 6.8
In vivo SD rat PKa ꢀ Dose (mg/kg): 3 (po), 1 (iv)
CL
T1/2
AUC(0ꢀ24h)
(nM•h) p.o
F (%)
(mL/min/kg)
<0.005
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18
68
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0.4
5785
750b
84
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100
10
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Dosing vehicle for 8: NMP (10%), PEG400 (30%), cremophorꢀEL (10%), ETPGS (5%), and
10: NMP (5%), PEG 300 (10%), CremophorꢀEL (5%), WFI. b po dose: 1 mg/kg
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Up to this point, we had relied on a continuous angiotensin II infusion SpragueꢀDawley (SD)
rat model27 to evaluate PK/PD relationships, a model successfully used to support the discovery
of LCI699,12 as well as profile followꢀup candidates in vivo.17a However, profiling of 1 and
subsequent analogs in rat recombinant CYP11B2 and CYP11B112 revealed a profound
disconnect between inhibition of human and rat enzymes (Table 3). Indeed plotting rat CYP11B2
IC50 against human CYP11B1/CYP11B2 IC50 ratio for 786 internal compounds possessing a
human CYP11B2 IC50 < 20 nM shows a positive correlation between increased human
selectivity and loss of potency against rat CYP11B2 (Figure 1).
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Table 3. Potency of compounds 1, 3-5, 7, 8 and 10 against human, rat and monkey CYP11B2,
and comparison of selectivity ratio for human versus monkey
Compd
CYP11B2 IC50 (µM)
Sc
humana
0.003
0.011
0.002
0.002
0.011
0.003
0.001
Rata
3.9
10.9
8
monkeyb
human
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monkey
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3
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0.006
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a Recombinant enzyme; b Adrenal homogenate; c Ratio of CYP11B1 IC50 over CYP11B2 IC50
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Figure 1. Rat CYP11B2 IC50 plotted against human CYP11B2/CYP11B1 IC50 ratio for 786
5
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compounds with human CYP11B2 IC50 < 20 nM.
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Fortunately, a survey of aldosterone secretion from adrenal homogenates of various
mammalian species revealed that the activity of human selective ASIs could be assessed in
cynomolgus monkeys (macaca fascicularis). As shown in Table 3, inhibition of human
recombinant CYP11B2 was comparable to inhibition of aldosterone secretion in monkey adrenal
homogenates. Selectivity for CYP11B2 over CYP11B1 was also observed in both species. Based
on these data, a model was developed using conscious, chairꢀtrained chronically cannulated male
cynomolgus monkeys.12,28 Plasma aldosterone (PAC) and plasma cortisol (PCC) concentrations
were measured over time in response to an ACTH challenge (3 µg/kg iv) given 3 h after dosing
with the test article. Timeꢀcourse data for 8 delivered as a methylcellulose suspension at 0.03,
0.1, 0.3, and 3 mg/kg po are shown in Figure 2. Plasma concentrations of 8 doseꢀdependently
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increased following oral dosing with the compound (top panel). At the 3 lowest doses, the
maximum concentration (Cmax) was not achieved until 4ꢀ6 h after dosing. In contrast, at the
highest dose, the Cmax was reached rapidly. Plasma concentrations remained high at 8 h and
declined by an order of magnitude at 24 h. Group mean baseline PAC ranged from 0.42 to 1.00
nM (Figure 2, middle panel). ACTH injection at 3 h rapidly stimulated an increase in PAC to a
peak of 2.02 nM (peak ∆PAC = 1.52 nM) in the vehicle group. Responses were compared to
those evoked by vehicle administration in the same animals. Oral dosing with 8 doseꢀ
dependently blunted the ACTHꢀstimulated PAC response. At the highest dose, the peak PAC
was 0.14±0.03 nM, which reflects an approximately 95% decrease from the corresponding
monkeys’ vehicleꢀcontrol response. The ED50 and EC50 values of 8 for blunting the 3 h postꢀ
dosing ACTHꢀstimulated PAC response (peak ∆PAC) were 61 µg/kg and 8.5 nM, respectively.
Consistent with the low plasma concentrations of 8 at 24 h, ACTH injection 24 h postꢀdosing
showed that the blunting of the PAC response had largely dissipated (data not shown).
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Figure 2. Time courses of total compound plasma concentration, PAC and PCC following
administration of methylcellulose vehicle (n=6) and 8 at 0.03, 0.1, 0.3 and 3 mg/kg (n=3/dose),
and an ACTH challenge 3 h postꢀdosing (error bars excluded for clarity).
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1.0
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MC Vehicle
0.03 mg/kg
0.1 mg/kg
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3 mg/kg
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Time post-dosing (h)
Group mean baseline plasma cortisol concentrations (PCCs) were approximately 1000 times
higher than the PACs and ranged from 679 to 839 nM (Figure 2, bottom panel). There were no
significant decreases in the baseline or ACTHꢀstimulated PCC values after administration of 8,
as expected from its high (640) in vitro aldosterone/cortisol selectivity in monkey adrenal
homogenate (Table 3). However, LCI699 also had no significant effect on the ACTHꢀstimulated
PCC in this model, despite its poor in vitro selectivity (5).12 On the basis of the in vivo efficacy,
a 30ꢀfold improvement in human in vitro selectivity over LCI699, and a favorable toxicology
profile, compound 8 was progressed to human studies.
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A doubleꢀblind, randomized, placeboꢀcontrolled study in healthy male and female volunteers
18ꢀ63 years old on a restricted sodium/potassium diet was conducted. Subjects were orally
administered bid doses of 30, 60, 100 and 200 mg of 8 or placebo (the ratio of subjects receiving
8 to placebo was 5:2). In these cohorts, median Tmax was 3ꢀ4 h, and plasma exposure, namely
AUCtau and Cmax, both increased underꢀproportionally between 30 and 200 mg (Table 4). A doseꢀ
dependent decrease in plasma aldosterone levels relative to baseline was observed (Figure 3, left
panel), with a maximum decrease of 70% at 200 mg bid. Consistent with plasma aldosterone
measurements, a doseꢀdependent decrease in aldosterone excretion measured in urine was
observed, also with a maximum of 70% at the highest dose (Figure 3, right panel). Gratifyingly,
and in good accord with the 100ꢀfold selectivity for CYP11B2 over CYP11B1 in vitro, no
notable decreases in urinary cortisol (Figure 4, left panel) or increases in urinary 11ꢀ
deoxycortisol (Figure 4, right panel) were observed at any dose. Plasma cortisol, 11ꢀ
deoxycortisol and ACTH levels were also not affected (data not shown). These data suggest that
8 is a potent and selective inhibitor of aldosterone synthase in humans.
10
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Table 4. Pharmacokinetic parameters in healthy volunteers upon bid dosing of 8.
Dose
N
Median
Tmax (h)
AUCtau
Cmax
AUCtau/dose Cmax/dose
(ng*h/mL) (ng/mL) (ng*h/mL)
(ng/mL)
3.9
30
60
10
17
4.0
3.0
4.0
4.0
768
117
173
173
306
25.6
17.1
12.7
10.9
1030
1270
2180
2.8
100 10
200 10
1.7
1.5
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Figure 3. Plasma aldosterone AUC(0ꢀ24h) (left panel) and amount of aldosterone excreted in
urine over 24 h (right panel), expressed as mean placeboꢀadjusted percent of baseline, following
30, 60, 100 and 200 mg bid dosing of 8.
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Figure 4. Amount of cortisol (left panel) and 11ꢀdeoxycortisol (right panel) excreted in urine
over 24 h, expressed as mean placeboꢀadjusted percent of baseline, following 30, 60, 100 and
200 mg bid dosing of 8.
Chemistry
Various methodologies were used to synthesize 3ꢀpyridyl indoles 1ꢀ10. A classic Fischer indole
synthesis was employed to prepare 1, which was converted into 2 using standard conditions
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(Scheme 1). 3ꢀCyanoindoles paired with unsubstituted pyridines were accessed from a Madelung
reaction as described in Scheme 2. The product of the Madelung reaction, 14,29 was methylated
to give 3, which could be chlorinated with Nꢀchlorosuccinimide to give 4. For more complex
pyridines, we relied on the Suzuki reaction as shown in Scheme 3. Either Nꢀmethyl or NꢀBocꢀ
indolꢀ2ꢀyl boronic acid was used to couple with 5ꢀsubstituted 3ꢀbromopyridines using anhydrous
sꢀPhos/ Pd2dba3 conditions, with the exception of 22, for which Pd(PPh3)4 was used. Oneꢀpot
electrophilic substitution of indole 19, 21, 22 and 25 with chlorosulfonyl isocyanate, followed by
treatment with DMF was used as a mild method to introduce the nitrile group at the 3ꢀposition of
the indole ring.30 Also of note is the use of mild heating and silica gel under vacuum to cleave
Boc groups in compounds 23 and 27. 5ꢀAzaindole 34 was prepared using a Sonogashira/ tBuOKꢀ
mediated electrophilic cyclization sequence, and was then converted into indole 9 using standard
procedures (Scheme 4). As for 7ꢀazaindole 10, we reverted back to using Suzuki couplings,
given the commercial availability of precursor 37 (Scheme 5).
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Scheme 1. Synthesis of ASIs 1 and 2
a
N
R
N
N
O
11
R=H
R=Me 2
1
b
(a) Phenylhydrazine hydrochloride (0.95 equiv), ethanol, reflux, 1 h, then catalytic HCl, reflux, 3
h, 63%; (b) NaH (60%, 2.2 equiv), DMF, 0 °C, 30 min, then MeI (1.1 equiv), 1.5 h, then NaH
(60%, 1.4 equiv), 16 h, 16%.
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Scheme 2. Synthesis of ASIs 3 and 4
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(a) Nicotinoyl chloride (1.1 equiv), diisopropylethylamine (2.5 equiv), DCM, r.t., 16 h, 31%; (b)
NaH (60%, 1.0 equiv), DMF, 130 °C, 18 h, 45%; (c) NaH (60%, 3.0 equiv), DMF, 30 min, then
MeI (1.5 equiv), r.t., 1 h, 65%. (d) Nꢀchlorosuccinimide (2.5 equiv), dichloroethane, reflux, 2
days, 10%.
Scheme 3. Synthesis of ASIs 5-8.
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9
R"
OH
OH
R"
a
B
N
R
Br
R'
N
R
N
R'
N
10
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15 R"=OSO2NMe2 19 R=Me, R'=H, R"=OSO2NMe2
b
c
16 R"=NH2
17 R"=N(Boc)2
18 R"=CHO
21 R"=N(SO2Et)2
24 R=H
20 R=Me, R'=H, R"=NH2
22 R=Me, R'=Cl, R"=N(Boc)2
23 R=Boc, R'=Cl, R"=CHO
d
25 R=Me
N
R"
5 R=Me, R'=H, R"=OSO2NMe2
26 R=Me, R'=H, R"=N(SO2Et)2
e
f
6 R"=NHSO2Et
7 R"=NHSO2Et
N
R
N
R'
c,b,f
g
27 R=Me, R'=Cl, R"=N(Boc)2
28 R=Me, R'=Cl, R"=CHO
8 R"=CH2NHSO2Et
(a) K3PO4 (2.0 equiv), sꢀPhos (0.05 equiv), Pd2dba3 (0.02 equiv), toluene, 85 °C, 45ꢀ90 min,
87% (19), 72% (20) [Pd(PPh3)4 (0.05 equiv) in DMF was used for 22, 89%]; (b) ethanesulfonyl
chloride (3ꢀ5 equiv), diꢀisopropylethylamine (3ꢀ4 equiv), DCM, r.t., 1ꢀ16 h, 98% (21); (c) silica
gel, 60ꢀ65 °C under high vacuum, 2ꢀ48 h, 73% (2 steps, 24); (d) MeI (1.4 equiv), NaH (60%, 1.6
equiv), DMF, 0 °C to r.t., 1 h, quant. (25); (e) chlorosulfonyl isocyanate (3ꢀ5 equiv), DCM or
acetonitrile, r.t., 10ꢀ45 min, then DMF, 10ꢀ120 min, 19% (5); (f) aqueous NaOH, methanol, r.t.,
1 h, 51% (2 steps, 6), 53% (4 steps, 7); (g) ethanesulfonamide (2 equiv), Ti(OiPr)4 (1.5 equiv),
toluene, reflux, 16 h then NaBH4 (2 equiv), DCM, MeOH, 0 °C, 30 min, 37% (2 steps, 8).
Scheme 4. Synthesis of ASI 9
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(a) trimethylsilylacetylene (3.0 equiv), CuI (0.05 equiv), Pd(PPh3)2Cl2 (0.05 equiv), TEA, 50 °C,
1 h; (b) K2CO3 (0.25 equiv), MeOH, r.t., 30 min, 67% (2 steps); (c) ICl (1.05 equiv), NaOAc (6
equiv), acetic acid, 60 °C, 2 h, 45%; (d) 30 (1.0 equiv), CuI (0.05 equiv), Pd(PPh3)2Cl2 (0.05
equiv), TEA, 100 °C, 3 h; (e) KOtBu (3.0 equiv), NMP, r.t., 16 h, then MeI (3.0 equiv), 0 °C, 30
min; (f) LAH (2.0 equiv), THF, 0 °C to r.t., 45 min, 11% (3 steps); (g) MnO2 (10 equiv),
dioxane, reflux, 16 h; (h) ethanesulfonamide (2 equiv), Ti(OiPr)4, (1.5 equiv), toluene, reflux, 16
h then NaBH4 (4 equiv), DCM, MeOH, 0 °C, 30 min, 17% (2 steps).
Scheme 5. Synthesis of ASI 10
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(a) NaH (2.0 equiv), DMF, 0 °C, 20 min then MeI (1.2 equiv), 0 °C to r.t., 2 h; (b) tBuLi (2.5
equiv), THF, −78 °C, 2 h, then B(OMe)3 (1.5 equiv), −78 °C to r.t., 16 h; (c) 5ꢀbromoꢀ3ꢀpyridine
carboxaldehyde (0.66 equiv), 2M aqueous K2CO3 (2 equiv), PSꢀPd(PPh3)4 (0.03 equiv), dioxane,
85 °C, 2 h, 31% (3 steps); (d) ethanesulfonamide (2 equiv), Ti(OiPr)4, (1.5 equiv), toluene,
reflux, 16 h then NaBH4 (4 equiv), DCM, MeOH, 0 °C, 30 min, 20%.
Conclusion
Nꢀ[5ꢀ(6ꢀchloroꢀ3ꢀcyanoꢀ1ꢀmethylꢀ1Hꢀindolꢀ2ꢀyl)ꢀpyridinꢀ3ꢀylmethyl]ꢀethanesulfonamide (8) is
an orally available CYP11B2 inhibitor with 100ꢀfold selectivity over CYP11B1 that reduces
plasma and urinary aldosterone in healthy volunteers. The lack of concomitant effect on cortisol,
11ꢀdeoxycortisol or ACTH in humans effectively validates the selectivity for CYP11B2 over
CYP11B1 observed in biochemical assays using recombinant proteins.
Experimentals
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Pharmacology Studies. Measurement of cellular aldosterone synthase inhibition,17a aromatase
inhibition,11 recombinant human CYP11B1 and human and rat CY11B2 inhibition22,12,17a were
conducted as previously described. The monkey in vitro assays were conducted similarly, using
adrenal homogenates and 11ꢀDOC as a substrate.12 The microsomal stability assay was
previously described.17a
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The study protocols for the monkey model have been described.12 Briefly, thirty minutes
before the start of the experiment, a Huber needle was inserted transdermally into the vascular
access port for the collection of blood samples and injection of ACTH. Blood samples (0.3 mL in
15 U/mL heparin) for baseline pharmacokinetic and pharmacodynamic assessments were
collected at 0.5 h, 0.25 h and immediately before dosing. Compound 8 or vehicle was
administered followed 3 h later by ACTH(1–24) (Cortrosyn; Amphastar Pharmaceuticals, Inc.,
Rancho Cucamonga, CA, USA) 3000 ng/kg i.v. in 0.1 mL/kg (over ~2 min). Blood samples were
collected at 0.125, 0.25, 0.5, 0.75 and 1 h after ACTH injection to assess the time course of
plasma aldosterone and cortisol stimulation. Further blood samples were collected up to 8 h and
at 23.5 and 24 h after 8/vehicle administration. Plasma aldosterone and cortisol were determined
by radioimmunoassay.22 Plasma concentration of 8 was measured by a liquid chromatography
separation coupled with tandem mass spectrometric detection (LC/MS/MS) method (LLOQ 0.26
nmol/L). Samples with compound 8 concentrations below the LLOQ were treated as zero.
Rat Pharmacokinetic Studies. Male Sprague Dawley rats (200−300 g) (Harlan Laboratories
Inc., Indianapolis, IN, USA) were used in the experiments. All animal experiments were
performed in accordance with IACUC protocol. Two rats received 0.3 mg/1 mL/kg (free base
equivalents) by slow intravenous injection via the jugular vein, and three rats received 1 mg/5
mL/kg or 3 mg/5 mL/kg in solution via oral gavage. Approximately 0.2 mL of venous whole
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blood was collected from the jugular vein catheter of each animal at 5 min (iv dose only) and
0.25, 0.5, 1, 2, 4, 6, and 8 h postdose and transferred to a EDTA tube. The blood was centrifuged
at 3000 rpm, and the plasma was transferred to a polypropylene tube, capped, and stored frozen
(−20 °C) for parent compound analysis. Protein precipitation was employed for sample
preparation. A 25 ꢁL aliquot of sample was subjected to protein precipitation using 150 ꢁL of
acetonitrile containing 100 ng/mL of internal standard (Glyburide). After vortex and
centrifugation for 5 min at 4000 rpm, the supernatant (125 ꢁL) was transferred to a 1 mL 96ꢀwell
plate, followed by the addition of 50 ꢁL of water. The analysis was conducted by using HPLC
separation coupled with mass spectrometric detection. All pharmacokinetic (PK) parameters
were derived from concentration−time data by noncompartmental analyses. All pharmacokinetic
parameters were calculated with the computer program WinNonlin (Enterprise, version 5.2)
purchased from Pharsight Corporation (St. Louis, MO).
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General Chemical Methods. Starting materials, reagents, and solvents were obtained from
commercial sources and used as received. THF and diethyl ether were anhydrous grade. Progress
of the reactions was monitored by thinꢀlayer chromatography (TLC) analysis (Merck, 0.2 mm
silica gel 60 F254 on glass plates) or by analytical LC−MS using an Agilent 1100 series with UV
detection at 214 and 254 nm and an electrospray mode (ESI) coupled with a Waters ZQ single
quad mass detector. Purification of intermediates and final products was carried out on normal
phase using an ISCO CombiFlash system or an Analogix Intelliflash 280 or a Biotage SP1
system and prepacked SiO2 cartridges eluted with optimized gradients of either heptane−ethyl
acetate mixture or dichloromethane−methanol as described. Preparative high pressure liquid
chromatography (HPLC) was performed on Waters or Gilson instruments. Systems were run
with either a 5−95% or 10−90% acetonitrile/water gradient with either a 0.1% TFA or 0.1%
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NH4OH modifier. All target compounds had purity of >95% as established by analytical HPLC.
NMR spectra were recorded on a Bruker AV400 (Avance 400 MHz) instrument. Chemical shifts
(δ) are reported in parts per million (ppm) relative to deuterated solvent as the internal standard
(CDCl3 7.26 ppm, DMSOꢀd6 2.50 ppm, CD3OD 3.31), and coupling constants (J) are in hertz
(Hz). Peak multiplicities are expressed as follows: singlet (s), doublet (d), doublet of doublets
(dd), triplet (t), quartet (q), multiplet (m), and broad singlet (br s).
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3-Methyl-2-pyridin-3-yl-1H-indole (1). A flask was charged with 3ꢀpropionylpyridine (1.004
g, 7.279 mmol), phenylhydrazine hydrochloride (1.010 g, 6.915 mmol) and ethanol (15 mL). The
mixture was heated to reflux for 1 h and cooled to room temperature. To a portion of the reaction
mixture (2.5 mL) was added HCl (4M in dioxane, 1 mL, 4 mmol) and the mixture was heated to
reflux. After 3 h, the mixture was concentrated in vacuo. The solid was dissolved in the
minimum amount of boiling methanol (17 mL) and allowed to cool slowly to room temperature.
A solid precipitated and after cooling to 0 °C for 30 min, the solid was filtered off, washed with
portions of cold methanol and dried under high vacuum to give 1 as the hydrochloride salt (0.177
g, 0.723 mmol, 63%) as yellow needles. 1H NMR (400 MHz, MeOD) δ ppm (HCl salt) 2.56 (s, 3
H), 7.09 ꢀ 7.13 (m, 1 H), 7.23 ꢀ 7.26 (m, 1 H), 7.43 (d, J=8.1 Hz, 1 H), 7.63 (d, J=8.1 Hz, 1 H),
8.14 ꢀ 8.18 (m, 1 H), 8.74 (d, J=5.8 Hz, 1 H), 8.83ꢀ8.85 (m, 1 H), 9.08 (s, 1 H).
1,3-Dimethyl-2-(pyridin-3-yl)-1H-indole (2). To a suspension of 1 hydrochloride (205 mg,
0.838 mmol) in DMF (10 mL) at 0 °C was added NaH (60% in mineral oil, 73 mg, 1.84 mmol)
and the mixture was stirred at 0 °C for 30 min. MeI (131 mg, 0.92 mmol) was added and after
1.5 h, another portion of NaH (60% in mineral oil, 50 mg, 1.22 mmol) was added. The mixture
was stirred overnight, whereupon it was quenched with methanol (2 mL). The mixture was
1
purified by reverse phase HPLC to give 2 (30 mg, 0.134 mmol, 16%) as a beige solid. H NMR
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(400 MHz, DMSOꢀd6) δ ppm 2.23 (s, 3 H), 3.62 (s, 3 H), 7.06 ꢀ 7.12 (m, 1 H), 7.22 (td, J=7.6,
1.1 Hz, 1 H), 7.48 (d, J=8.3 Hz, 1 H), 7.55 ꢀ 7.61 (m, 2 H), 7.93 (dt, J=7.9, 2.0 Hz, 1 H), 8.66
(dd, J=4.8, 1.5 Hz, 1 H), 8.69 (dd, J=2.3, 0.8 Hz, 1 H). HRMS: (ESI) m/z 223.1236 [(M+H)+
Calcd for C15H15N2: 223.1230].
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N-(2-Cyanomethyl-phenyl)-nicotinamide (13). 2ꢀaminophenylacetonitrile (5.0 g, 37.1 mmol)
and nicotinoyl chloride (7.5 g, 40.8 mmol) were taken up in dry dichloromethane (200 mL) and
diisopropylethylamine (12.1 g, 92.7 mmol) was added while cooling the mixture with a cold
water bath. The mixture was stirred overnight at room temperature, whereupon it was washed
twice with saturated aqueous sodium bicarbonate. The combined aqueous layer was backꢀ
extracted with ethyl acetate. The combined organic phase was dried over MgSO4 and
concentrated in vacuo to give a residue, which was purified by silica gel flash chromatography
(dichloromethaneꢀmethanol, 1:0 to 19:1) to give 13 (2.70 g, 11.37 mmol, 31%) as an offꢀwhite
solid. 1H NMR (400 MHz, DMSOꢀd6) δ ppm 4.02 (s, 2 H), 7.32 ꢀ 7.37 (m, 1 H), 7.39 ꢀ 7.44 (m,
2 H), 7.50 (d, J=7.1 Hz, 1 H), 7.59 (dd, J=8.1, 4.8 Hz, 1 H), 8.30 ꢀ 8.33 (m, 1 H), 8.78 (dd,
J=4.8, 1.5 Hz, 1 H), 9.14 (d, J=1.8 Hz, 1 H), 10.36 (s, 1 H).
2-Pyridin-3-yl-1H-indole-3-carbonitrile (14). 13 (0.095 g, 0.384 mmol) was dissolved in
DMF (3 mL). NaH (60%, 0.015 g, 0.384 mmol) was added and the mixture was heated to 130
°C. After 18 h, the mixture was cooled down, diluted with ethyl acetate, and washed with 1M
aqueous sodium hydroxide. The combined washings were backꢀextracted with ethyl acetate. The
combined organic phase was dried over magnesium sulfate, filtered and concentrated in vacuo.
The residue was purified by silica gel flash chromatography (dichloromethaneꢀmethanol, 99:1 to
1
19:1) to give 14 (0.038 mg, 0.17 mmol, 45%) as a brown solid. H NMR (400 MHz, MeOD) δ
ppm 7.29 ꢀ 7.32 (m, 1 H), 7.34 ꢀ 7.39 (m, 1 H), 7.57 (d, J=8.0 Hz, 1 H), 7.66 (dd, J=8.0, 4.9 Hz,
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1 H), 7.70 (d, J=7.8 Hz, 1 H), 8.39 ꢀ 8.44 (m, 1 H), 8.68 (dd, J=4.8, 1.5 Hz, 1 H), 9.15 (d, J=1.5
Hz, 1 H).
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1-Methyl-2-pyridin-3-yl-1H-indole-3-carbonitrile (3). To 14 (1.0 g, 4.56 mmol) in DMF (17
mL) was added 60% sodium hydride in mineral oil (547 mg, 13.68 mmol) and the suspension
was stirred for 30 min. Iodomethane (971 mg, 6.84 mmol) was then added to the reaction
mixture, which was stirred at ambient temperature for 1 h. Aqueous NaHCO3 (3 mL) was added
and the mixture was concentrated in vacuo. The residue was purified by silica gel flash
chromatography (dichloromethaneꢀmethanol gradient) to give 3 (0.70 g, 3.00 mmol, 65%) as a
white solid. 1H NMR (400 MHz, MeOD) δ ppm 3.83 (s, 3 H), 7.35 (t, J=7.5 Hz, 1 H), 7.40 ꢀ 7.46
(m, 1 H), 7.64 (d, J=8.3 Hz, 1 H), 7.67 ꢀ 7.74 (m, 2 H), 8.16 (dt, J=8.0, 2.0, 1.9 Hz, 1 H), 8.76
(dd, J=5.1, 1.5 Hz, 1 H), 8.86 (d, J=1.5 Hz, 1 H). HRMS (ESI) m/z 234.1029 [(M+H)+ Calcd for
C15H12N3: 234.1031].
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6-Chloro-2-pyridin-3-yl-1-methyl-1H-indole-3-carbonitrile (4). Nꢀchlorosuccinimide (0.086
g, 0.644 mmol) was added to 3 (150 mg, 0.644 mmol) in dichloroethane (10 mL) and the mixture
was refluxed for 2 days. Three additional portions of Nꢀchlorosuccinimide (0.043 g, 0.322 mmol)
were added at 1 hour intervals, whereupon the volatiles were removed in vacuo. Purification by
1
reverse phase HPLC afforded 4 (18 mg, 0.067 mmol, 10%). H NMR (400 MHz, DMSOꢀd6) δ
ppm 3.79 (s, 3 H), 7.37 (dd, J=8.5, 1.9 Hz, 1 H), 7.69 (dd, J=7.5, 4.4 Hz, 1 H), 7.73 (d, J=8.6 Hz,
1 H), 7.98 (d, J=1.5 Hz, 1 H), 8.17 (dt, J=8.1, 2.0, 1.8 Hz, 1 H), 8.82 (dd, J=4.9, 1.6 Hz, 1 H),
8.90 (d, J=1.5 Hz, 1 H). HRMS (ESI) m/z 268.0653 [(M+H)+ Calcd for C15H11ClN3: 268.0642].
Dimethyl-sulfamic acid 5-bromo-pyridin-3-yl ester (15). A flask was charged with 5ꢀ
bromoꢀpyridinꢀ3ꢀol (0.200 g, 1.126 mmol), potassium phosphate (0.631 g, 2.884 mmol) and
acetone (5 mL) and cooled to 0 °C. Dimethylsulfamoyl chloride (0.261 g, 1.802 mmol) was then
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added dropwise and the cooling bath was removed. After 2 h, the mixture was diluted with
acetone, filtered and the filtrate was concentrated in vacuo. The residue was dissolved in THF
(30 mL) and polymerꢀsupported trisamine (3.85 mmol/g, 0.7 g, 2.7 mmol) was added. After 1 h,
the mixture was filtered. Concentration in vacuo gave a residue which was purified by silica gel
flash chromatography (heptaneꢀethyl acetate, 9:1 to 4:1) to give 15 (0.228 g, 0.811 mmol, 72%)
as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 3.05 (s, 3 H), 7.86 (m, 1 H), 8.50 (d, J=2.3
Hz, 1 H), 8.62 (d, J=1.9 Hz, 1 H).
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Dimethyl-sulfamic acid 5-(1-methyl-1H-indol-2-yl)-pyridin-3-yl ester (19). A flask was
charged with 15 (0.225 g, 0.76 mmol), Nꢀmethylꢀindole boronic acid (0.200 g, 1.14 mmol), sꢀ
Phos (0.016 g, 0.038 mmol), finely crushed potassium phosphate (0.323 g, 1.52 mmol) and
toluene (5 mL). After sparging with nitrogen for 15 min, Pd2dba3 (0.014 g, 0.015 mmol) was
added, the flask was flushed with nitrogen and the mixture was heated to 85 °C. After 45 min,
the mixture was allowed to cool to r.t., diluted with ethyl acetate and filtered through a plug of
silica gel (elution with ethyl acetate). The volatiles were removed in vacuo and the residue was
purified by silica gel flash chromatography (heptaneꢀethyl acetate, 3:7 to 0:1) to give to give 19
(220 mg, 0.664 mmol, 87%); MS (ESI) m/z 332.1 (M+H)+.
Dimethyl-sulfamic acid 5-(3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-yl ester (5). A flask
was charged with 19 (220 mg, 0.664 mmol) and dichloromethane (10 mL). Chlorosulfonyl
isocyanate (0.282 g, 1.99 mmol) was added and the reaction was stirred for 45 min, whereupon
DMF (3 mL) was added. After another 2 h, the reaction was concentrated in vacuo and the
residue was purified by reverse phase HPLC to give 5 (46 mg, 0.129 mmol, 19%). 1H NMR (400
MHz, DMSOꢀd6) δ ppm 2.99 (s, 6 H), 3.82 (s, 3 H), 7.35 ꢀ 7.40 (m, 1 H), 7.43 ꢀ 7.48 (m, 1 H),
7.74 (d, J=7.8 Hz, 1 H), 7.79 (d, J=8.3 Hz, 1 H), 8.24 (dd, J=2.5, 1.8 Hz, 1 H), 8.85 (d, J=2.5 Hz,
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1 H), 8.91 (d, J=1.8 Hz, 1 H). HRMS (ESI) m/z 357.1018 [(M+H)+ Calcd for C17H17N4O3S:
357.1021].
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2-(5-Amino-pyridin-3-yl)-1-methyl-1H-indole (20). A flask was charged with 3ꢀaminoꢀ5ꢀ
bromopyridine (4.05 g, 22.71 mmol), Nꢀmethylꢀindoleꢀ2ꢀboronic acid (5.27 g, 29.52 mmol), sꢀ
Phos (0.625 g, 1.476 mmol), finely crushed potassium phosphate (9.74 g, 45.41 mmol) and
toluene (75 mL). After sparging with nitrogen for 60 min, Pd2dba3 (0.420 g, 0.454 mmol) was
added, the flask was flushed with nitrogen and the flask was lowered into a preheated oil bath at
85 °C. After 1.5 h, the mixture was allowed to cool to r.t., diluted with ethyl acetate and filtered
through a plug of silica gel (elution with ethyl acetate). The residue was purified by silica gel
flash chromatography (heptaneꢀethyl acetate, 3:7 to 0:1) to give 20 (3.64 g, 16.30 mmol, 72%) as
an offꢀwhite powder. 1H NMR (400 MHz, DMSOꢀd6) δ ppm 3.74 (s, 3 H), 5.49 (s, 2H), 6.56 (s,
1H), 7.05ꢀ7.09 (m, 1H), 7.09ꢀ7.10 (m, 1H), 7.17ꢀ7.21 (m, 1H), 7.49 (d, J=7.6 Hz, 1 H), 7.57 (d,
J=7.6 Hz, 1 H), 7.94 (d, J=2.0 Hz, 1 H), 7.99 (d, J=2.5 Hz, 1 H). MS (ESI) m/z 224 (M+H)+.
Ethanesulfonic acid [5-(1-methyl-1H-indol-2-yl)-pyridin-3-yl]-N-ethanesulfonyl-amide
(21). To a solution of 20 (223 mg, 1.0 mmol) in dichloromethane (20 mL) was added
ethanesulfonyl chloride (386 mg, 3.0 mmol) and diisopropylethylamine (517 mg, 4.0 mmol). The
mixture was stirred at room temperature for 1 h. Saturated NaHCO3 in water (0.5 mL) and silica
gel (10 g) were added and the mixture was concentrated in vacuo. The residue was purified by
silica gel chromatography (heptaneꢀethyl acetate, 1:0 to 1:9) to give 21 (400 mg, 0.98 mmol,
98%) as a white solid. MS (ESI) m/z 408.1 (M+H)+.
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Ethanesulfonic
acid
[5-(3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-yl]-amide
(6)
Chlorosulfonyl isocyanate (0.162 g, 1.15 mmol) was added to 21 (96 mg, 0.23 mmol) in
acetonitrile (10 mL) and the mixture was stirred for 20 min before adding DMF (1 mL). After 10
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min, the solvent was removed and the crude mixture was used with no further purification; MS
(ESI) m/z 432.96 (M+H)+. The crude mixture in DMF (2 mL), methanol (10 mL) and 5 M NaOH
in water (1 mL) was stirred at room temperature for 1 h. The mixture was purified by reverse
phase HPLC on Xbridge C18 eluting with a 1:9 to 9:1 acetonitrileꢀwater gradient to give 6 (40
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mg, 0.117 mmol, 51%). H NMR (400 MHz, DMSOꢀd6) δ ppm 1.25 (t, J=7.3 Hz, 3 H), 3.24 ꢀ
3.32 (m, 2 H), 3.80 (s, 3 H), 7.32 ꢀ 7.38 (m, 1 H), 7.43 (td, J=7.8, 1.1 Hz, 1 H), 7.70 (d, J=7.8
Hz, 1 H), 7.75 (d, J=8.3 Hz, 1 H), 7.90 (t, J=2.3 Hz, 1 H), 8.58 ꢀ 8.62 (m, 2 H), 10.42 (s, 1 H).
HRMS (ESI) m/z 341.1078 [(M+H)+ Calcd for C17H17N4O2S: 341.1072].
3-[Bis[[(1,1-dimethylethyl)oxy]carbonyl]amino]-5-bromo-pyridine (17). To 5ꢀaminoꢀ3ꢀ
bromoꢀpyridine (1.73 g, 10 mmol) and Boc2O (4.8 g, 22 mmol) in acetonitrile (100 mL) at
ambient temperature was added DMAP (212 mg, 1 mmol) and the reaction mixture was heated
to 60 °C and stirred overnight. The reaction mixture was then cooled to room temperature. The
solvent was removed in vacuo and the residue was purified by silica gel flash chromatography
(heptaneꢀethyl acetate, 1:0 to 7:3) to afford 17 (3.0 g, 8.04 mmol, 80%) as a white solid. MS
(ESI) m/z 374.9 (M+H)+.
3-[Bis[[(1,1-dimethylethyl)oxy]carbonyl]amino]-5-(6-chloro-1-methyl-1H-indol-2-yl)-
pyridine (22). A flask was charged with 17 (3.0 g, 8.0 mmol), Nꢀmethylꢀ6ꢀchloroindoleꢀ2ꢀ
boronic acid (2.0 g, 9.65 mmol), finely crushed potassium phosphate (3.4 g, 16.0 mmol) and
DMF (40 mL). After sparging with nitrogen for 15 min, Pd(PPh3)4 (465 mg, 0.402 mmol) was
added. The flask was flushed with nitrogen and the mixture was heated to 90 °C and stirred
overnight. The mixture was then cooled to room temperature and poured into water (300 mL).
The mixture was extracted with EtOAc three times and the combined organic phase was then
dried over Na2SO4 and concentrated. The residue was purified by silica gel flash chromatography
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(ethyl acetateꢀheptane, 0:1 to 1:9) to give 22 (3.3 g, 7.2 mmol, 89%). MS (ESI) m/z 458.1
(M+H)+.
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Ethanesulfonic acid [5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-yl]-amide
(7). Chlorosulfonyl isocyanate (4.08 g, 28.8 mmol) was added to a solution of 22 (3.3 g, 7.2
mmol) in acetonitrile (200 mL) and the reaction mixture was stirred for 10 min. DMF (3 mL)
was added and the reaction mixture was stirred for 2 h. 20 g silica gel was added to the mixture
and the solvent was removed in vacuo. The resulting solid was heated to 65 °C under high
vacuum for 2 h. The mixture was cooled to room temperature and then purified by silica gel flash
chromatography (dichloromethaneꢀmethanolꢀtriethylamine, 83:8:9) to afford 2ꢀ(5ꢀaminoꢀpyridinꢀ
3ꢀyl)ꢀ6ꢀchloroꢀ1ꢀmethylꢀ1Hꢀindoleꢀ3ꢀcarbonitrile mixed with triethylammonium chloride. The
combined fractions were concentrated and taken up in DCM. After washing with water and
drying over Na2SO4, a residue was obtained (1.8 g), which was used with no further purification;
MS (ESI) m/z 283.0 (M+H)+. A portion (512 mg) was dissolved in DCM (10 mL). Diꢀ
isopropylethylamine (0.898 g, 6.88 mmol) and ethanesulfonyl chloride (0.68 g, 5.16 mmol) were
added and the reaction mixture was stirred overnight. Another portion of ethanesulfonyl chloride
(0.68 g, 5.16 mmol) was added, and after 2 h, the mixture was concentrated and purified by silica
gel flash chromatography (heptaneꢀethyl acetate 1:1) to give a residue (260 mg) which was used
with no further purification. A portion (200 mg) was redissolved in methanol (5 mL). Aqueous
1M NaOH (4 mL) was added and the reaction mixture was stirred for 1 h. The pH was adjusted
to 5 with 1M aqueous HCl and the mixture was extracted with DCM twice. The organic phase
was dried over Na2SO4. The solvent was removed in vacuo and the residue was purified by silica
gel flash chromatography (methanolꢀdichloromethane, 1:19) to give 7 (115 mg, 0.306 mmol,
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53%, 4 steps) as a white solid. H NMR (400 MHz, DMSOꢀd6) δ ppm 1.25 (t, J=7.3 Hz, 3 H),
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3.23 ꢀ 3.33 (m, 2 H), 3.79 (s, 3 H), 7.36 (dd, J=8.6, 1.8 Hz, 1 H), 7.72 (d, J=8.6 Hz, 1 H), 7.90 (t,
J=2.3 Hz, 1 H), 7.95 (d, J=1.5 Hz, 1 H), 8.61 (d, J=2.3 Hz, 2 H), 10.43 (s, 1 H). HRMS (ESI)
m/z 375.0692 [(M+H)+ Calcd for C17H16ClN4O2S: 375.0682].
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5-(6-Chloro-1H-indol-2-yl)-pyridine-3-carbaldehyde (24). A flask was charged with Nꢀ
(tertꢀbutoxycarbonyl)ꢀ6ꢀchloroꢀ1Hꢀindolꢀ2ꢀylboronic acid (10 g, 33.8 mmol), 5ꢀ
bromonicotinaldehyde (5.25 g, 28.2 mmol), K PO (11.97 g, 56.4 mmol), sꢀPhos (0.579 g, 1.410
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mmol) and Pd (dba) (0.516 g, 0.564 mmol), and the flask was flushed with N . Toluene (200
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mL) was added, and the mixture was heated to 90 ºC for 1 h. The mixture was cooled to room
temperature. Ethyl acetate (250 mL) was added and the mixture was filtered through a pad of
silica gel, which was washed with EtOAc. Silica gel was added to the combined filtrate, which
was concentrated in vacuo. The residue was placed under high vacuum at 60 ºC for 2 days, and
after elution with ethyl acetate, 24 (5.6 g, 20.7 mmol, 73%) was obtained. MS (ESI) m/z 257.0
and 258.9 (M+H)+.
6-Chloro-2-(5-formyl-pyridin-3-yl)-1-methyl-1H-indole (25). A flask was charged with 24
(5.6 g, 17.3 mmol), MeI (3.41 g, 24.0 mmol) and DMF (200 mL), and 60% NaH in mineral oil
(1.134 g, 28.4 mmol) was added at 0 ºC. The mixture was stirred for 1 h. Water (200 mL) was
added. The mixture was extracted with EtOAc (2 × 300 mL) and the combined organic phase
was washed with water (3 × 100 mL) and brine (50 mL), dried over Na SO , filtered and
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4
concentrated in vacuo. The residue was purified by silica gel chromatography (heptaneꢀethyl
acetate, 3:2 to 1:1) to give 25 (5.2 g, 17.3 mmol, quant.). MS (ESI) m/z 271.0, 272.9 (M+H)+.
N-[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide
(8). A flask was charged with 25 (3.00 g, 11.1 mmol) and acetonitrile (300 mL), and
chlorosulfonyl isocyanate (4.73 g, 33.4 mmol) was added at 0 ºC. The mixture was stirred for 10
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