Discovery of Novel Indazole Derivatives as Highly Potent and Selective Human β3-Adrenergic Receptor Agonists with the Possibility of Having No Cardiovascular Side Effects

Article abstract of DOI:10.1021/acs.jmedchem.5b00638

Novel indazole derivatives were prepared and evaluated for their biological activity and cardiovascular safety profile as human β₃-adrenergic receptor (AR) agonists. Although the initial hit compound 5 exhibited significant β₃-AR agonistic activity (EC₅₀ = 21 nM), it also exhibited agonistic activity at the α_1A-AR (EC₅₀ = 219 nM, selectivity: α_1A/β₃ = 10-fold). The major metabolite of 5, which was an oxidative product at the indazole 3-methyl moiety, gave a clue to a strategy for improvement of the selectivity for β₃-AR agonistic activity versus α_1A-AR agonistic activity. Thus, modification of the 3-substituent of the indazole moiety effectively improved the selectivity to develop compound 11 with potent β₃-AR agonistic activity (EC₅₀ = 13 nM) and high selectivity (α_1A/β₃ = >769-fold). Compound 11 was also inactive toward β₁ and β₂-ARs and showed dose dependent β₃-AR mediated relaxation of marmoset urinary bladder smooth muscle, while it did not obviously affect heart rate or blood pressure (iv, 3 mg/kg) in anesthetized rats.

Full text of DOI:10.1021/acs.jmedchem.5b00638

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Article
Discovery of Novel Indazole Derivatives as Highly Potent
3
and Selective Human #-Adrenergic Receptor Agonists with
the Possibility of Having No Cardiovascular Side-Effects
Yasuhiro Wada, Hiromitsu Shirahashi, Taisuke Iwanami, Masami Ogawa, Seiji Nakano, Akifumi Morimoto,
Ken-ichi Kasahara, Eiichi Tanaka, Yoshio Takada, Shigeki Ohashi, Mutsuhiro Mori, and Satoshi Shuto
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00638 • Publication Date (Web): 30 Jun 2015
Downloaded from http://pubs.acs.org on July 5, 2015
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Discovery of Novel Indazole Derivatives as Highly Potent and Selective Human
β3ꢀAdrenergic Receptor Agonists with the Possibility of Having No
Cardiovascular SideꢀEffects
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Yasuhiro Wada*,a,b Hiromitsu Shirahashi,a Taisuke Iwanami,a Masami Ogawa,a Seiji Nakano,a Akifumi
Morimoto,a Kenꢀichi Kasahara,a Eiichi Tanaka,a Yoshio Takada,a Shigeki Ohashi,a Mutsuhiro Mori,a and
Satoshi Shuto*b,c
aPharmaceutical Research Center, Asahi Kasei Pharma Corporation, 632ꢀ1, Mifuku, Izunokuniꢀshi,
Shizuoka 410ꢀ2321, Japan
bFaculty of Pharmaceutical Sciences and cCenter for Research and Education on Drug Discovery, Hokkaido
University, Kitaꢀ12, Nishiꢀ6, Kitaꢀku, Sapporo 060ꢀ0812, Japan
Running Title: Structurally Novel Human β3ꢀAdrenergic Receptor Agonists
Corresponding Author:
Satoshi Shuto
Phone & Fax: +81ꢀ11ꢀ706ꢀ3769. Eꢀmail: shu@pharm.hokudai.ac.jp
Yasuhiro Wada
Phone: +81ꢀ558ꢀ76ꢀ8494. Fax: +81ꢀ558ꢀ76ꢀ5755. Eꢀmail: wada.yp@om.asahiꢀkasei.co.jp
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Abstract
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Novel indazole derivatives were prepared and evaluated for their biological activity and cardiovascular
safety profile as human β3ꢀadrenergic receptor (AR) agonists. Although the initial hit compound 5 exhibited
significant β3ꢀAR agonistic activity (EC50 = 21 nM), it also exhibited agonistic activity at the α1AꢀAR (EC50
= 219 nM, selectivity:α1A/β3 = 10ꢀfold). The major metabolite of 5, which was an oxidative product at the
indazole 3ꢀmethyl moiety, gave a clue to a strategy for improvement of the selectivity for β3ꢀAR agonistic
activity versus α1AꢀAR agonistic activity. Thus, modification of the 3ꢀsubstituent of the indazole moiety
effectively improved the selectivity to develop compound 11 with potent β3ꢀAR agonistic activity (EC50 =
13 nM) and high selectivity (α1A/β3 = >769ꢀfold). Compound 11 was also inactive towards β1 and β2ꢀARs
and showed dose dependent β3ꢀAR mediated relaxation of marmoset urinary bladder smooth muscle, while
it did not obviously affect heart rate or blood pressure (iv, 3 mg/kg) in anesthetized rats.
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Introduction
Overactive bladder (OAB) is a urological disorder that is defined as denoting urgency with or without urge
incontinence, usually with frequency and nocturia.1 OAB symptoms can be highly distressing to the patient,
and patients with OAB have been shown to have significantly lower qualityꢀofꢀlife.2 Populationꢀbased
estimates in five countries suggest that OAB affects 11.8% of adults.3 Muscarinic receptor antagonists are
widely used pharmacological agents for the symptoms of OAB.4 These agents cause reductions in urinary
frequency, urgency and urge urinary incontinence, all of which are primary symptoms of OAB.5 However,
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despite the proven efficacy of muscarinic receptor antagonists, their mechanistic side effects of dry mouth,
constipation, blurred vision and occasional but rare urinary retention have been demonstrated.5 A recent
study found that 12 months after the initial prescription, the persistence rates of muscarinic receptor
antagonists were ≦35% in patients with OAB.6 Such adverse effects limit their tolerability and negatively
affect longꢀterm patient adherence and satisfaction in the clinical treatment of OAB symptoms.7 In the
search for alternative drugs for OAB treatment that have a novel target and that can improve the efficacy
and tolerability profile, β3ꢀadrenergic receptor (AR) agonists have emerged as a promising class of drugs.8
β3ꢀAR, which is one of the three βꢀAR subtypes; β1, β2 and β3ꢀARs,9 is a G proteinꢀcoupled receptor
(GPCR) that was identified by human genomic cloning in the late 1980s.10 The β3ꢀAR mediates lipolysis in
human brown adipose tissue and also mediates evoked muscle relaxation in gall bladder, stomach, small
intestine, prostate, colon, and bladder.11 The β3ꢀAR is vitally important for bladder smooth muscle, and 97%
of the total βꢀAR messenger RNA in human bladder tissue is β3ꢀAR mRNA12, which is likely to mediate
mainly relaxation of the bladder smooth muscle in humans.13 Because β3ꢀAR agonists increased bladder
capacity in anesthetized rats,14 pharmaceutical industries have been attempting to discover potent and
selective β3ꢀAR agonists for the treatment of OAB, with the possibility that they might have fewer liabilities
in terms of adverse effects compared to muscarinic receptor antagonists.15 Several β3ꢀAR agonists such as 1
(mirabegron, YMꢀ178)16, 2 (solabegron, GW427353)17, and 3 (ritobegron, KUCꢀ7483)18, which are shown
in Figure 1, have been advanced into clinical studies, and 1 has been approved for the treatment of OAB.
Treatment with mirabegron demonstrated significant efficacy for symptoms of OAB and resulted in less
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frequent incidence of dry mouth than muscarinic receptor antagonists.8 However, mirabegron has been
shown to increase the heart rate (HR) in animals and healthy subjects.19 We hypothesized that, because the
increased HR resulting from treatment with mirabegron is partly mediated by activation of the β1ꢀAR,19a
highly selective novel β3ꢀAR agonists would be effective drugs for the treatment of OAB without
cardiovascular side effects including an increased HR. We here describe identification of indazole
derivatives as a new class of highly selective β3ꢀAR agonists with the possibility of having no
cardiovascular sideꢀeffects.
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OH
H
N
S
O
NH2
N
N
H
1 (mirabegron, YM-178)
OH
O
H
N
N
H
OH
Cl
2 (solabegron, GW427353)
OH
H
N
O
HO
O
O
3 (ritobegron, KUC-7483)
Figure 1. Chemical Structure of 1, 2 and 3
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Results and Discussion
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Hit identification and issue of hit compound 5. A number of papers15b, 15g, 20 and patents21 have described
potent β3ꢀAR agonists with either arylethanolamine or aryloxypropanolamine scaffolds. To discover a novel
class of potent and selective β3ꢀAR agonists, we synthesized a series of phenylethanolamine analogs having
a bicyclic heteroaromatic ring such as indazole, isoquinoline, 1Hꢀpyrazolo[3,4ꢀb]pyridine, benzoisoxazole,
benzoisothiazole or benzoimidazole (Figure 2). Throughout screening of these compounds, we identified a
novel indazole derivative 5 with significant and selective β3ꢀAR agonistic activity (EC50 = 21 nM), whose
selectivity over the β1ꢀ and β2ꢀARs was >476ꢀfold (Table 1). This increased selectivity of compound 5 was
an advantage in terms of its use as a hit compound for the development of useful β3ꢀAR agonists, because
the initial hit β3ꢀAR agonists reported in previous papers15d, 22 had lower selectivity for β3ꢀAR versus the β1ꢀ
and β2ꢀARs.
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Figure 2. Hit identification strategy
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Because we aimed to develop highly potent β3ꢀAR agonists without cardiovascular side effects, we
5
evaluated the effects of compound 5 on the heart rate (HR) and blood pressure (BP) of anesthetized rats. As
shown in Table 2, intravenous (iv) administration of compound 5 resulted in a transient biphasic change in
mean blood pressure (MBP), i.e. there was a clear increase followed by a slight decrease. Within 5 minutes
after dosing of compound 5, MBP was restored to baseline. The maximum increase in MBP induced by 5 (3
mg/kg, iv) was 24.2%, and the maximum decrease was 8.5%. HR was slightly increased by 7.9% at 1 to 5
minutes after dosing of compound 5 (3 mg/kg, iv). To determine how 5 mediated this increase in MBP we
therefore tested the agonistic profile of 5 on αꢀARs, because not only a subfamily of βꢀARs but also αꢀARs
are involved in the control of BP. αꢀARs are subꢀclassified into six types, i.e., α1A, α1B, α1D, α2A, α2B,
α2CꢀARs.9 Among these subꢀclasses, compound 5 showed significant α1AꢀAR agonistic activity with an
EC50 of 219 nM (Table 1). Three subtypes (α1A, α1B, α1D) of the α1ꢀAR are widely distributed in the body
and are highly expressed in the cardiovascular system, other smooth muscles and the brain.23 The agonists
of α1ꢀAR are known to increase blood pressure through vascular contraction in the peripheral tissues,24 and
the hypertension that occurred by treatment of compound 5 was prevented by the preꢀdosed α1AꢀAR
antagonist Silodosin25 (Table 2). Therefore, hypertension caused by compound 5 was suggested to occur via
its α1AꢀAR activation. Accordingly, we next aimed to obtain highly selective β3ꢀAR agonists without
α1AꢀAR activity.
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Table 1. Human adrenergic receptor agonist activity of 5a
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AR
EC50 (nM)a
21 ± 0.67
>10000
I.A. (%)
82b
Selectivity
β3
β1
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4.0b
71c
β1 / β3 > 476
β2 / β3 > 476
α1A / β3 = 10
>10000
β2
219 ± 6.9
α1A
aData are shown as means ± SEM (n=3). 5a exhibited insignificant agonistic activity for α1B, α1D, α2A, α2B
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or α2C (EC50 >10000 nM). I.A. (intrinsic activity): maximum response induced by isoproterenol was
defined as 100%. cI.A.: maximum response induced by norepinephrine was defined as 100%.
Table 2. Effect of 5 on HR and MBP in anesthetized ratsa
HR
MBP
Dose (mg/kg)
n
5
3
4
1
Increase (%)
1.8 ± 0.9
4.2 ± 2.3
7.9 ± 2.9
1.7
Decrease (%)
4.5 ± 0.8
1.3 ± 0.7
8.8 ± 4.5
ND
Increase (%)
1.0 ± 0.5
7.5 ± 0.2
24.2 ± 7.7
ND
Decrease (%)
5.4 ± 1.9
9.9 ± 0.2
8.5 ± 1.1
20.6
Saline
1
3
1b
aCompound 5 was administered intravenously to rats, and, thereafter, HR and MBP were recorded for 30
min. Maximum increase or decrease of HR or MBP is shown as a value (%) relative to the baseline. Data
are shown as means ± SEM. bSilodosin, a selective α1AꢀAR antagonist, was administered intravenously at a
dose of 0.01 mg/kg before administration of compound 5. n: number, ND: not detected.
In order to obtain insight into the hypertension caused by compound 5 in vivo, we examined the
metabolism of 5. In this analysis, compound 6 was identified as a major oxidative metabolite in incubation
of compound 5 with rat liver microsomes. This metabolite 6 provided a clue as to the strategy to be used for
further structure activity relationship (SAR) study of 5, because it showed more potent α1AꢀAR agonist
activity (EC50 = 33 nM, I.A. = 91%) than compound 5 (Figure 3). Introduction of a hydroxyl group to the
3ꢀmethyl group of the indazole moiety had brought about the dramatic change in α1AꢀAR agonist activity.
We therefore hypothesized that modifications of the 3ꢀsubstituent of the indazole moiety would change the
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α1AꢀAR agonistic activity towards improving selectivity for β3ꢀAR versus α1AꢀAR.
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Figure 3. Design of indazole derivatives
SAR of indazole analogs. Agonistic activity of derivatives of 5 that were modified at the indazole 3ꢀmoiety
for the β3ꢀAR is shown in Table 3. Replacement of the methyl group (5) with a methoxy group (7) did not
improve this activity (5, EC50 = 21 nM vs. 7, EC50 = 40 nM), and compound 8 bearing an ethoxy substituent
had further decreased activity (8, EC50 = 71 nM). Changing the methyl group to a chloro, ethyl, isoꢀpropyl,
or cyclopropyl group (9, 10, 11, 13, respectively) did not significantly affect the EC50 value (9, EC50 = 43
nM; 10, EC50 = 14 nM; 11, EC50 = 13 nM; 13, EC50 = 17 nM). However, replacement with a tertꢀbutyl or
phenyl groups (12 or 16) hugely decreased the β3ꢀAR agonistic activity (12, EC50 = 338 nM, 16, EC50 =
>10000 nM). Cyclobutyl (14) and trifluoromethyl (15) derivatives displayed slightly lower β3ꢀAR agonistic
activity than 5 (14, EC50 = 66 nM, 15, EC50 = 64 nM vs. 5, EC50 = 21 nM). Thus, in order of their β3ꢀAR
agonist activity, the compounds were ranked as 5, 7, 9, 10, 11, 13 > 8, 14, 15 > 12 > 16.
Agonistic activity of these compounds for the α1AꢀAR is also presented in Table 3. Replacement with a
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methoxy or ethoxy group (7 or 8) did not change this activity (5, EC50 = 219 nM vs. 7, EC50 = 252 nM, 8,
EC50 = 277 nM). A chloro derivative (9) showed higher potency than 5 (9, EC50 = 61 nM vs. 5, EC50 = 219
nM). On the other hand, ethyl (10), cyclopropyl (13) and trifluoromethyl (15) derivatives displayed
remarkably attenuated agonistic potency for the α1AꢀAR (10, EC50 = 857 nM; 13, EC50 = 1548 nM; 15, EC50
= 812 nM). Finally, changing the methyl group to an isoꢀpropyl, tertꢀbutyl, cyclobutyl or phenyl group (11,
12, 14, 16, respectively) made these compounds inactive in terms of α1AꢀAR activity (EC50 = >10000 nM).
Thus, in order of α1AꢀAR agonist activity, the compounds were ranked as 9 > 5, 7, 8 > 10, 13, 15 >> 11, 12,
14, 16.
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Based on these agonistic activities for β3ꢀAR and α1AꢀAR, these compounds were ranked in terms of their
selectivity for β3ꢀAR over α1AꢀAR as: 11 > 14 > 13 > 10 > 12 > 15 > 5 > 7 > 8 > 9. Thus, compounds 10, 11,
and 13 were identified as potent and highly selective β3ꢀAR agonists, and were next subjected to further
pharmacological evaluation.
Table 3. Human β3ꢀ and α1AꢀAR agonistic activity of indazole derivativesa
Compound
R
α1A
Selectivity
β3
EC50 (nM)
I.A. (%)b
EC50 (nM)
I.A. (%)c
α1A / β3
ꢀMe
21 ± 0.67
40 ± 1.9
82
70
219 ± 6.9
252
71
60
10
5
7
ꢀOMe
6.3
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ꢀOEt
ꢀCl
71 ± 6.1
69
89
78
69
62
277
59
91
31
9.1
19
3.9
1.4
61
8
43 ± 2.9
14 ± 0.58
13 ± 1.5
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9
ꢀEt
857 ± 164
>10000
>10000
10
11
12
ꢀiꢀPr
ꢀtꢀBu
>769
>30
338 ± 159
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17 ± 3.8
66
72
68
1548 ± 419
>10000
19
4
91
13
14
>151
13
ꢀCF3
ꢀPh
64 ± 11
>10000
86 ± 3.7
86
29
812 ± 179
>10000
31
6
15
16
isoproterenol
100
NT
norepinephrine
NT
9.1 ± 0.52
100
aData are shown as means ±SEM (n≧3) or are presented as the average of two experiments. I.A.
b
c
(intrinsic activity): maximum response induced by isoproterenol was defined as 100%. I.A.: maximum
response induced by norepinephrine was defined as 100%. NT: not tested.
The β1ꢀAR and β2ꢀAR agonistic activities of 10, 11, and 13 were investigated. All of these compounds
were inactive towards these two AR subtypes (β1ꢀAR EC50 = >10000 nM; β2ꢀAR EC50 = >10000 nM). Thus,
these three compounds were identified as highly selective agonists of the β3ꢀAR.
Cardiovascular safety profiles and Pharmacological data. We evaluated the cardiovascular safety
profiles of the highly selective β3ꢀAR agonists 10, 11 and 13 on HR and MBP, and compared them with
those of the hit compound 5 and the clinically useful β3ꢀAR agonist mirabegron (1). As summarized in
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Table 4, none of the three optimized compounds (iv, 3 mg/kg) affected MBP in anesthetized rats, while the
prototype hit compound 5 clearly raised the MBP (24.2%). In the same evaluation, mirabegron (1) caused a
significant decrease in MBP (38.3%). Compound 11 did not affect the HR (iv, 3 mg/kg), while mirabegron
(1) clearly increased the HR (10.0%). These results can be attributed to the fact that compounds 10, 11 and
13 are highly active only towards the β3ꢀAR and are inactive towards all of the other αꢀand βꢀARs.
Next, we examined the effect of compound 11 on the relaxation of urinary bladder smooth muscle. Rat
models have been used for the evaluation of compounds in terms of their relaxation effect on urinary
bladder smooth muscle.16 However, because compound 11 showed only weak agonistic activity on the rat
β3ꢀAR (EC50 = 535 nM, I.A. = 95%), other animal species were required to examine its relaxation effect on
urinary bladder smooth muscle. We found that compound 11 acted as a strong agonist for the marmoset
β3ꢀAR (EC50 = 15 nM, I.A. = 75%), and therefore compound 11 was evaluated for its effects on the
relaxation response using urinary bladder smooth muscle strips from marmosets. In this evaluation with
isolated marmoset urinary bladder smooth muscle that was preꢀcontracted with KCl, compound 11 showed
full relaxant activity on the smooth muscle, and this activity was similar to that of a potent but nonꢀselective
βꢀAR agonist isoproterenol (Figure 4).
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Based on the excellent β3ꢀAR selectivity profile, as well as the favorable relaxation effect on marmoset
urinary bladder smooth muscle, the optimized compound 11 would be useful for the treatment of OAB with
the possibility of having no cardiovascular sideꢀeffects.
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Table 4. Effects of Intravenous Administration of Indazole derivatives on HR and MBP in
Anaesthetized Ratsa
5
6
HR
MBP
Compound
N
5
3
4
6
6
3
Increase (%)
1.8 ± 0.9
10.0 ± 2.5
7.9 ± 2.9
9.6 ± 1.3
3.3 ± 0.6
5.7 ± 1.5
Decrease (%)
4.5 ± 0.8
ND
Increase (%)
1.0 ± 0.5
3.5 ± 3.5
24.2 ± 7.7
3.1 ± 0.2
4.7 ± 0.9
2.7 ± 0.3
Decrease (%)
5.4 ± 1.9
7
8
9
Saline
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11
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17
18
19
20
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58
59
60
mirabegron (1)
38.3 ± 3.4
8.5 ± 1.1
8.8 ± 4.5
1.6 ± 2.0
1.8 ± 0.5
3.3 ± 0.7
5
10.8 ± 1.1
7.6 ± 1.7
10
11
13
11.0 ± 3.0
aCompounds were administered intravenously to rats (3 mg/kg), and, thereafter, HR and MBP were
recorded for 30 min. Maximum increase or decrease of HR or MBP is shown as a value (%) relative to the
baseline. Data are shown as means ± SEM. N: Numbers of rats used. ND: Not detected.
Figure 4. Relaxation of marmoset urinary bladder smooth muscle by 11. Each point represents the mean ±
SD (n=3). The marmoset urinary bladder was cut by a midline incision and 6 muscle strips of the bladder
body were prepared. The strips were preꢀcontracted with 40 mmol/L KCl, and, after the tension had
stabilized, test compounds were cumulatively added. When the relaxation response at the maximum
concentration of the test compound was completed, papaverine was added at a final concentration of 10ꢀ4
mol/L, and the maximum relaxation response of each strip was determined. See Experimental Section for
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Synthesis of the indazole analogs 5ꢀ16 is shown in Scheme 1. Mitsunobu reaction of 17 and 18ꢀ28
proceeded to give the corresponding indazole ether products, of which deprotection with 4 M HCl in
1,4ꢀdioxane or ethyl acetate gave the target compounds (5, 7ꢀ16). Similarly, Mitsunobu reaction of 17 and
29 proceeded to give 30. After hydrolysis of the ethyl ether group of 30, reduction of its carboxylic acid
group with borane tetrahydrofuran complex and subsequent deprotection with HCl in ethanol gave the target
compound 6.
Scheme 1. Synthesis of Targets 5ꢀ16a
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R
Et3Si
R
OH
O
Boc
N
H
N
N
N
O
a, b
N
N
Boc
OH
H
+
HO
HN
N
S
Boc
S
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O
O
18: R = Me
19: R = OMe
20: R = OEt
21: R = Cl
22: R = Et
23: R = iso-Pr
24
: R = t-Bu
O
O
25
26
27
28
: R = c-Pr
: R = c-Bu
: R = CF3
: R = Ph
17
5, 7-16
O
OEt
c
N
N
THP
HO
29
O
OEt
OH
Et3Si
O
Boc
N
N
THP
OH
N
d, e, f
H
N
O
N
N
O
H
N
Boc
S
HN
S
O
O
O
O
30
6
aReagents and conditions: (a) TMAD, PPh3, toluene, rt; (b) 4 M HCl in EtOAc or 4 M HCl in 1,4ꢀdioxane,
rt; (c) TMAD, PPh3, THF, rt; (d) 2 M NaOH, MeOH, 40 °C; (e) BH3·THF, THF, rt; (f) conc. HCl aq, EtOH,
rt.
Conclusion
We identified a new series of indazole derivatives as β3ꢀAR agonists. Among the developed β3ꢀAR
agonists, compound 11 had a high selectivity for β3ꢀAR over β1, β2 and α1AꢀARs and showed dose
dependent β3ꢀAR mediated responses in marmoset urinary bladder smooth muscle. Compound 11 did not
obviously affect HR or MBP (iv, 3 mg/kg) in anesthetized rats. Thus, we were successful in identification of
11, a highly selective β3ꢀAR agonist with the possibility of having no cardiovascular sideꢀeffects.
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Experimental Section
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General Methods. All reagents and solvents were purchased from commercial sources and were used as
received. Anhydrous solvents were obtained from commercial sources. Thin layer chromatography (TLC)
was carried out using Merck GmbH Precoated silica gel 60 F254. Chromatography on silica gel was carried
out using prepacked silica gel cartridges (Yamazen HiꢀFlash Column Silicagel or PurifpackꢀSi series).
Chemical shifts in 1H NMR spectra were reported in δ values (ppm) relative to trimethylsilane. Ion
chromatographic analyses were performed under the following conditions: (Anion) ICSꢀ1000 (Dionex),
Dionex IonPac AS14 (Dionex or Thermo Scientific), 30 °C column temperature, 1.2 mL/min flow rate,
electrical conductivity detection, mobile phase of 1.0 mmol/L sodium hydrogen carbonate and 3.5 mmol/L
sodium carbonate in water, standard solution of Mixed Anion Standard Solution IV (Kanto Chemical
Industry Co., Ltd.); (Cation) DX500 (Dionex), Dionex IonPac CS14 (Dionex), 30 °C column temperature,
1.0 mL/min flow rate, electrical conductivity detection, mobile phase of 10 mmol/L methanesulfonic acid in
water, standard solution of Mixed Cation Standard Solution II (Kanto Chemical Industry Co., Ltd.). HPLC
analyses were performed following conditions: (Method A) Shiseido CAPCELL CORE ADME column (2.7
ꢀm, 2.1 x 50 mm), 40 °C column temperature, 1.0 mL/min flow rate, photodiode array detection (254 nm),
linear mobile phase gradient of 10ꢀ95% B over 2 min, holding 1.5 min at 95% B, holding 1.5 min at 10% B
(mobile phase A: 10 mM ammonium acetate in water; mobile phase B: acetonitrile), (Method B) YMC
Meteoric Core C18 column (2.7 ꢀm, 3.0 x 50 mm), 40 °C column temperature, 1.0 mL/min flow rate,
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photodiode array detection (254 nm), linear mobile phase gradient of 10ꢀ95% B over 2 min, holding 2 min
at 95% B, holding 2 min at 10% B (mobile phase A: 10 mM ammonium acetate in water; mobile phase B:
acetonitrile).
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Nꢀ(3ꢀ((1R)ꢀ1ꢀHydroxyꢀ2ꢀ(2ꢀ((3ꢀmethylꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ethyl)phenyl)methanesulfona
mide (5). To a stirred solution of 18 (128 mg, 0.52 mmol), 17 (2 mL, 1.0 mmol, 0.5 M toluene solution),
and triphenylphosphine (261 mg, 1.0 mmol) in anhydrous toluene (5 mL) was added
N,N,N’,N’ꢀtetramethylazodicarboxamide (195 mg, 1.1 mmol) at room temperature, and the solution was
stirred for 4 days. The reaction solution was then purified by flash column chromatography on silica gel
(74:26 to 53:47 nꢀhexane/ethyl acetate) to give 371 mg (90% yield) of the coupling product. 1HꢀNMR (400
MHz, CDCl3, 1:1 rotamers): δ 0.54 (6H, q, J = 8.0), 0.89 (9H, t, J = 8.0), 1.44 (9H, s), 1.48 and 1.52 (9H,
each s), 1.69 and 1.70 (9H, each s), 2.52 and 2.53 (3H, each s), 3.24ꢀ3.26 (7H, m), 4.03ꢀ4.11 (2H, m),
4.94ꢀ4.97 and 5.10ꢀ5.13 (1H, each m), 6.86 (1H, dd, J = 1.7, 8.6), 7.12ꢀ7.16 (1H, m), 7.21ꢀ7.47 (4H, m),
7.54 (1H, s); LC/MS (ESI, [M+H]+, m/z) 819. To the solution of the obtained product (287 mg, 0.35 mmol)
in 1,4ꢀdioxane (0.4 mL) was added 4 M HCl in 1,4ꢀdioxane (4 mL), and the mixture was stirred at room
temperature for 6.5 h. The resultant solid was collected by suction filtration and dried. The crude solid (170
mg) was treated with water (0.5 mL) and ethanol (2.0 mL) to promote crystallization. The crystals were
filtered, and washed with ethanol to afford 61 mg (37% yield) of the title compound as dihydrochloride salt.
1HꢀNMR (400 MHz, DMSOꢀd6): δ 2.46 (3H, s), 3.00 (3H, s), 3.04ꢀ3.10 (1H, m), 3.24ꢀ3.28 (1H, m),
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3.44ꢀ3.47 (2H, m), 4.34ꢀ4.41 (2H, m), 5.02 (1H, dd, J = 2.1, 10.1), 6.80 (1H, dd, J = 2.1, 8.8), 6.92 (1H, d, J
= 2.1), 7.12ꢀ7.17 (2H, m), 7.31 (1H, s), 7.35 (1H, t, J = 7.8), 7.63 (1H, d, J = 8.8), 9.05 (1H, brs), 9.38 (1H,
brs), 9.86 (1H, brs); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 11.3, 39.1, 45.9, 53.6, 63.4, 67.9, 92.1, 111.9, 116.9,
117.0, 118.9, 121.0, 121.1, 129.3, 138.5, 140.7, 141.6, 143.0, 157.4; HRMS calculated for C19H24N4O4S +
H+ 405.1591, found (ESI, [M+H]+) 405.1588; LC/MS (ESI, [M+H]+, m/z) 405; HPLC (Method A): purity
100% RT 1.7 min.
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Nꢀ(3ꢀ((1R)ꢀ1ꢀHydroxyꢀ2ꢀ(2ꢀ((3ꢀ(hydroxymethyl)ꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ethyl)phenyl)metha
nesulfonamide (6). To a stirred solution of 30 (139 mg, 0.16 mmol) in THF (1.2 mL) and methanol (2.0
mL) was added 2 M NaOH (1.6 mL, 3.2 mmol) at 40 °C, and the solution was stirred for 5 h. The reaction
mixture was poured into water (50 mL) and washed twice with diethyl ether. The aqueous layer was added 5
M HCl (5 mL) and extracted three times with ethyl acetate. The combined organic layers were dried over
magnesium sulfate and filtered. The solution was concentrated to give carboxylic acid (95 mg), which was
used without further purification. This material (77 mg, 0.11 mmol) was dissolved in anhydrous THF (4.3
mL) under nitrogen and the solution was added borane tetrahydrofuran complex (0.5 mL, 0.6 mmol, 1.2 M
in THF) at 0 °C. The resulting solution was allowed to warm to room temperature and was then stirred
overnight. The reaction mixture was added methanol (0.5 mL) at 0 °C. The solution was poured into water
and extracted twice with ethyl acetate. The combined organic layers were washed twice with water, dried
over sodium sulfate, and filtered. The organic layer was concentrated to give alcohol (95 mg), which was
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used without further purification. This material (68 mg, 0.1 mmol) was dissolved in ethanol (0.5 mL),
4
5
hydrochloric acid (0.17 mL) was then added at room temperature and the mixture was stirred for 24 h. The
resultant solid was collected by filtration and dried to give 39 mg (80% yield) of the title compound as
dihydrochloride salt. 1HꢀNMR (400 MHz, DMSOꢀd6): δ 3.00 (3H, s), 3.04ꢀ3.11 (1H, m), 3.24ꢀ3.28 (1H, m),
3.41ꢀ3.46 (2H, m), 4.32ꢀ4.41 (2H, m), 4.73 (2H, s), 5.02 (1H, dd, J = 2.1, 10.2), 6.79 (1H, dd, J = 2.1, 8.8),
6.93 (1H, d, J = 2.1), 7.12ꢀ7.17 (2H, m), 7.31 (1H, s), 7.35 (1H, t, J = 7.8), 7.74 (1H, d, J = 8.8), 9.02 (1H,
brs), 9.32 (1H, brs), 9.86 (1H, s), 12.65 (1H, brs); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 39.1, 45.9, 53.6, 56.6,
63.3, 67.9, 92.1, 111.6, 116.5, 116.9, 118.9, 121.2, 121.5, 129.3, 138.5, 141.9, 143.0, 145.4, 156.9; HRMS
calculated for C19H24N4O5S + H+ 421.1540, found (ESI, [M+H]+) 421.1537; LC/MS (ESI, [M+H]+, m/z)
421; HPLC (Method A): purity 100% RT 1.5 min.
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Nꢀ(3ꢀ((1R)ꢀ1ꢀHydroxyꢀ2ꢀ(2ꢀ((3ꢀmethoxyꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ethyl)phenyl)methanesulfon
amide (7). To a stirred solution of 19 (215 mg, 0.8 mmol), 17 (942 mg, 1.6 mmol), and triphenylphosphine
(448 mg, 1.7 mmol) in anhydrous toluene (12 mL) was added N,N,N’,N’ꢀtetramethylazodicarboxamide (288
mg, 1.7 mmol) at room temperature, and the solution was stirred overnight. The reaction solution was then
purified by flash column chromatography on silica gel (100:0 to 74:26 nꢀhexane/ethyl acetate) to give 587
mg (87% yield) of the coupling product. 1HꢀNMR (300 MHz, CDCl3, 1:1 rotamers): δ 0.53 (6H, q, J = 7.9),
0.87 (9H, t, J = 7.9), 1.42 (9H, s), 1.46 and 1.51 (9H, each s), 1.67 (9H, s), 3.20ꢀ3.60 (7H, m), 3.99ꢀ4.04 (m,
2H), 4.12 (3H, s), 4.92ꢀ4.96 and 5.08ꢀ5.12 (1H, each m), 6.80 (1H, dd, J = 2.1, 8.7), 7.11ꢀ7.47 (6H, m);
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LC/MS (ESI, [M+H]+, m/z) 835. To the solution of the obtained product (580 mg, 0.7 mmol) in tertꢀbutyl
methyl ether (1 mL) was added 4 M HCl in 1,4ꢀdioxane (7 mL), and the mixture was stirred overnight at
room temperature. The resultant solid was collected by suction filtration, washed with tertꢀbutyl methyl
ether, and dried to afford 321 mg (93% yield) of the title compound as dihydrochloride salt. 1HꢀNMR (400
MHz, DMSOꢀd6): δ 3.00 (3H, s), 3.03ꢀ3.10 (1H, m), 3.23ꢀ3.27 (1H, m), 3.44ꢀ3.46 (2H, m), 3.96 (3H, s),
4.33ꢀ4.41 (2H, m), 5.04 (1H, dd, J = 2.1, 10.2), 6.70 (1H, dd, J = 2.0, 8.8), 6.81 (1H, d, J = 2.0), 7.13 (1H, d,
J = 7.8), 7.16 (1H, dd, J = 1.3, 7.8), 7.30 (1H, s), 7.35 (1H, t, J = 7.8), 7.48 (1H, d, J = 8.8), 9.09 (1H, brs),
9.47 (1H, brs), 9.87 (1H, s), 11.83 (1H, brs); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 39.7, 46.4, 54.2, 56.1, 63.9,
66.8, 92.8, 106.5, 111.4, 117.5, 119.5, 120.5, 121.8, 129.8, 139.1, 143.5, 143.6, 156.7, 158.4; HRMS
calculated for C19H24N4O5S + H+ 421.1540, found (ESI, [M+H]+) 421.1538; LC/MS (ESI, [M+H]+, m/z)
421; HPLC (Method A): purity 100% RT 1.8 min.
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Nꢀ(3ꢀ((1R)ꢀ2ꢀ(2ꢀ((3ꢀEthoxyꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ꢀ1ꢀhydroxyꢀethyl)phenyl)methanesulfona
mide (8). To a stirred solution of 20 (51 mg, 0.18 mmol), 17 (212 mg, 0.36 mmol), and triphenylphosphine
(94 mg, 0.36 mmol) in anhydrous toluene (2.7 mL) was added N,N,N’,N’ꢀtetramethylazodicarboxamide (62
mg, 0.36 mmol) at room temperature, and the solution was stirred overnight. The reaction solution was then
purified by flash column chromatography on silica gel (88:12 to 67:33 nꢀhexane/ethyl acetate) to give 137
mg (89% yield) of the coupling product. 1HꢀNMR (300 MHz, CDCl3, 1:1 rotamers): δ 0.54 (6H, t, J = 7.9),
0.89 (9H, q, J = 7.9), 1.44ꢀ1.47 (12H, m), 1.48 and 1.52 (9H, each s), 1.68 (9H, s), 3.22ꢀ3.61 (7H, m),
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4.01ꢀ4.11 (2H, m), 4.51 (2H, q, J = 7.0), 4.93ꢀ4.98 and 5.09ꢀ5.13 (1H, each m), 6.80 (1H, dd, J = 2.1, 8.7),
7.13ꢀ7.50 (6H, m); LC/MS (ESI, [M+H]+, m/z) 849. To the solution of the obtained product (130 mg, 0.15
mmol) in tertꢀbutyl methyl ether (0.2 mL) was added 4 M HCl in 1,4ꢀdioxane (1.5 mL), and the mixture
was shaken (600 minꢀ1) for overnight at room temperature. Nitrogen gas was blown into the reaction
solution to evaporate the solvent. Subsequently, water was added to dissolve the residue and the solution
was freezeꢀdried to give 76 mg (98 % yield) of the title compound as dihydrochloride salt. 1HꢀNMR (400
MHz, DMSOꢀd6): δ 1.39 (3H, t, J = 7.0), 3.00 (3H, s), 3.03ꢀ3.10 (1H, m), 3.23ꢀ3.27 (1H, m), 3.43ꢀ3.49 (2H,
m), 4.33 (2H, q, J = 7.0), 4.34ꢀ4.41 (2H, m), 5.03 (1H, dd, J = 2.1, 10.2), 6.70 (1H, dd, J = 2.0, 8.8), 6.81
(1H, d, J = 2.0), 7.13 (1H, d, J = 7.7), 7.15ꢀ7.18 (1H, m), 7.30 (1H, s), 7.35 (1H, t, J = 7.8), 7.47 (1H, d, J =
8.8), 9.07 (1H, brs), 9.42 (1H, brs), 9.87 (1H, s), 11.78 (1H, s); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 14.7,
39.1, 45.8, 53.6, 63.3, 63.8, 67.9, 92.2, 106.1, 110.8, 117.0, 119.0, 120.0, 121.2, 129.2, 138.5, 142.7, 143.0,
155.5, 157.7; HRMS calculated for C20H26N4O5S + H+ 435.1967, found (ESI, [M+H]+) 435.1696; LC/MS
(ESI, [M+H]+, m/z) 435; HPLC (Method A): purity 100% RT 1.9 min.
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Nꢀ(3ꢀ((1R)ꢀ2ꢀ(2ꢀ((3ꢀChloroꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ꢀ1ꢀhydroxyꢀethyl)phenyl)methanesulfona
mide (9). To a stirred solution of 21 (26 mg, 0.1 mmol), 17 (118 mg, 0.2 mmol), and triphenylphosphine (55
mg, 0.2 mmol) in anhydrous toluene (1.5 mL) was added N,N,N’,N’ꢀtetramethylazodicarboxamide (35 mg,
0.2 mmol) at room temperature, and the solution was stirred overnight. Then the reaction solution was then
purified by flash column chromatography on silica gel (81:19 to 60:40 nꢀhexane/ethyl acetate) to give 71
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mg (85% yield) of the coupling product. 1HꢀNMR (300 MHz, CDCl3, 1:1 rotamers): δ 0.54 (6H, q, J = 7.9),
0.89 (9H, t, J = 7.9), 1.44 (9H, s), 1.49 and 1.52 (9H, each s), 1.69 (9H, s), 3.25ꢀ3.63 (7H, m), 4.03ꢀ4.11 (2H,
m), 4.94ꢀ4.98 and 5.10ꢀ5.14 (1H, each m), 6.93 (1H, dd, J = 2.0, 8.8), 7.12ꢀ7.44 (4H, m), 7.50 (1H, dd, J =
3.6, 8.8), 7.57 (1H, d, J = 1.5); LC/MS (ESI, [M+H]+, m/z) 839. To the solution of the obtained product (71
mg, 0.085 mmol) in tertꢀbutyl methyl ether (0.2 mL) was added 4 M HCl in 1,4ꢀdioxane (1.2 mL), and the
mixture was shaken (600 minꢀ1) overnight at room temperature. The resultant solid was collected by suction
filtration, washed with tertꢀbutyl methyl ether, and dried to afford 41 mg (96% yield) of the title compound
as dihydrochloride salt. 1HꢀNMR (400 MHz, DMSOꢀd6): δ 3.00 (3H, s), 3.04ꢀ3.10 (1H, m), 3.24ꢀ3.28 (1H,
m), 3.46ꢀ3.48 (2H, m), 4.38ꢀ4.43 (2H, m), 5.03 (1H, dd, J = 2.1, 10.2), 6.91 (1H, dd, J = 2.0, 8.9), 7.01 (1H,
d, J = 2.0), 7.12ꢀ7.17 (2H, m), 7.31 (1H, s), 7.35 (1H, t, J = 7.8), 7.56 (1H, d, J = 8.9), 9.08 (1H, brs), 9.43
(1H, brs), 9.86 (1H, s), 13.21 (1H, brs); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 39.1, 45.8, 53.6, 63.5, 67.9,
92.6, 113.8, 114.4, 116.9, 118.9, 119.5, 121.2, 129.2, 132.0, 138.5, 142.1, 143.0, 157.9; HRMS calculated
for C18H21ClN4O4S + H+ 425.1045, found (ESI, [M+H]+) 425.1045; LC/MS (ESI, [M+H]+, m/z) 425; HPLC
(Method A): purity 100% RT 1.9 min.
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Nꢀ(3ꢀ((1R)ꢀ2ꢀ(2ꢀ((3ꢀEthylꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ꢀ1ꢀhydroxyꢀethyl)phenyl)methanesulfonami
de (10). To a stirred solution of 22 (1.326 g, 5 mmol), 17 (10 mL, 10 mmol, 1 M toluene solution), and
triphenylphosphine (2.905 g, 11 mmol) in anhydrous toluene (15 mL) was added
N,N,N’,N’ꢀtetramethylazodicarboxamide (1.953 g, 11 mmol) at room temperature, and the solution was
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stirred overnight. Triphenylphosphine (1.344 g) and N,N,N’,N’ꢀtetramethylazodicarboxamide (0.973 g) were
further added to the reaction solution, and the mixture was stirred for 2 h at room temperature.
Triphenylphosphine (1.24 g) and N,N,N’,N’ꢀtetramethylazodicarboxamide (0.923 g) were further added to
the reaction solution, and the mixture was stirred for 0.5 h at room temperature. The reaction solution was
then purified by flash column chromatography on silica gel (88:12 to 67:33 nꢀhexane/ethyl acetate) to give
3.71 g (88% yield) of the coupling product. 1HꢀNMR (300 MHz, CDCl3, 1:1 rotamers): δ 0.54 (6H, q, J =
7.9), 0.89 (9H, t, J = 7.9), 1.38 (3H, t, J = 7.6), 1.43 (9H, s), 1.48 and 1.52 (9H, each s), 1.70 (9H, s), 2.95
(2H, q, J = 7.6), 3.22ꢀ3.62 (7H, m), 4.02ꢀ4.11 (2H, m), 4.93ꢀ4.97 and 5.09ꢀ5.13 (1H, each m), 6.77 (1H, dd,
J = 2.0, 8.7), 7.13ꢀ7.60 (6H, m); LC/MS (ESI, [M+H]+, m/z) 833. The obtained product (3.56 g, 4.3 mmol)
and 4 M HCl in ethyl acetate (70 mL) were stirred overnight at room temperature. The resultant solid was
collected by suction filtration, washed with diethyl ether, and dried to afford 2.056 g (97% yield) of the title
compound as dihydrochloride salt. 1HꢀNMR (400 MHz, DMSOꢀd6): δ 1.31 (3H, t, J = 7.6), 2.93 (2H, q, J =
7.6), 3.00 (3H, s), 3.04ꢀ3.11 (1H, m), 3.23ꢀ3.27 (1H, m), 3.43ꢀ3.48 (2H, m), 4.37ꢀ4.43 (2H, m), 5.05 (1H, dd,
J = 2.1, 10.2), 6.83 (1H, dd, J = 2.1, 8.8), 6.95 (1H, d, J = 2.1), 7.13 (1H, d, J = 7.8), 7.16ꢀ7.18 (1H, m),
7.31 (1H, s), 7.35 (1H, t, J = 7.8), 7.70 (1H, d, J = 8.8), 9.13 (1H, brs), 9.54 (1H, brs), 9.88 (1H, s);
13CꢀNMR (100 MHz, DMSOꢀd6): δ 13.3, 19.2, 39.1, 45.8, 53.7, 63.4, 67.9, 92.1, 112.4, 115.8, 117.0, 119.0,
121.2, 121.3, 129.2, 138.5, 141.6, 143.0, 145.9, 157.8; HRMS calculated for C20H26N4O4S + H+ 419.1748,
found (ESI, [M+H]+) 419.1747; LC/MS (ESI, [M+H]+, m/z) 419; HPLC (Method A): purity 100% RT 1.8
min.
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Nꢀ(3ꢀ((1R)ꢀ1ꢀHydroxyꢀ2ꢀ(2ꢀ((3ꢀisopropylꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ethyl)phenyl)methanesulfon
amide (11). To a stirred solution of 23 (146 mg, 0.5 mmol), 17 (1.19 g, 2.0 mmol), and triphenylphosphine
(574 mg, 2.2 mmol) in anhydrous toluene (5 mL) was added N,N,N’,N’ꢀtetramethylazodicarboxamide (371
mg, 2.1 mmol) at room temperature and the solution was stirred overnight. The reaction solution was then
purified by flash column chromatography on silica gel (100:0 to 67:33 nꢀhexane/ethyl acetate) to give 400
mg (95% yield) of the coupling product. 1HꢀNMR (300 MHz, CHCl3, 1:1 rotamers): δ 0.54 (6H, q, J = 7.9),
0.89 (9H, t, J = 7.9), 1.43ꢀ1.45 (15H, m), 1.47 and 1.52 (9H, each s), 1.70 (9H, s), 3.22ꢀ3.62 (8H, m),
4.02ꢀ4.11 (2H, m), 4.94ꢀ4.98 and 5.10ꢀ5.14 (1H, each m), 6.85 (1H, dd, J = 1.6, 8.8), 7.12ꢀ7.44 (4H, m),
7.51 (1H, s), 7.57 (1H, dd, J = 2.4, 8.8); LC/MS (ESI, [M+H]+, m/z) 847. To the solution of the obtained
product (400 mg, 0.47 mmol) in THF (2.4 mL) was added 4 M HCl in 1,4ꢀdioxane (1.5 mL) and the mixture
was stirred overnight at room temperature. The resultant solid was collected by suction filtration, washed
with diethyl ether, and dried under reduced pressure. The solid was dissolved with water, and the solution
was freezeꢀdried to give 191 mg (93 % yield) of the title compound as dihydrochloride salt. 1HꢀNMR (400
MHz, DMSOꢀd6): δ 1.38 (6H, d, J = 7.0), 3.00 (3H, s), 3.05ꢀ3.12 (1H, m), 3.24ꢀ3.28 (1H, m), 3.40 (1H,
septet, J = 7.0), 3.45ꢀ3.49 (2H, m), 4.37ꢀ4.47 (2H, m), 5.07 (1H, dd, J = 2.0, 10.1), 6.86 (1H, dd, J = 2.0,
8.9), 6.97 (1H, d, J = 2.0), 7.15 (1H, d, J = 7.8), 7.17ꢀ7.19 (1H, m), 7.32 (1H, s), 7.35 (1H, t, J = 7.8), 7.78
(1H, d, J = 8.9), 9.20 (1H, brs), 9.62 (1H, brs), 9.90 (1H, s); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 22.1, 26.6,
39.3, 45.9, 53.9, 63.6, 68.1, 92.4, 113.0, 115.0, 117.2, 119.2, 121.4, 121.9, 129.4, 138.7, 141.9, 143.2, 149.8,
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158.1; HRMS calculated for C21H28N4O4S + H+ 433.1904, found (ESI, [M+H]+) 433.1903; LC/MS (ESI,
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[M+H]+, m/z) 433; HPLC (Method A): purity 100% RT 1.9 min.
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Nꢀ(3ꢀ((1R)ꢀ2ꢀ(2ꢀ((3ꢀtertꢀButylꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ꢀ1ꢀhydroxyꢀethyl)phenyl)methanesulfo
namide (12). To a stirred solution of 24 (141 mg, 0.5 mmol), 17 (0.92 mL, 0.9 mmol, 1 M toluene solution),
and triphenylphosphine (401 mg, 1.5 mmol) in anhydrous toluene (5 mL) was added
N,N,N’,N’ꢀtetramethylazodicarboxamide (284 mg, 1.6 mmol) at room temperature, and the solution was
stirred overnight. The reaction solution was then purified by flash column chromatography on silica gel
(95:5 to 74:26 nꢀhexane/ethyl acetate) to give 354 mg (82% yield) of the coupling product. 1HꢀNMR (300
MHz, CDCl3, 1:1 rotamers): δ 0.54 (6H, t, J = 7.9), 0.89 (9H, t, J = 7.9), 1.43 (9H, s), 1.47 and 1.51 (9H,
each s), 1.50 (9H, s), 1.69 (9H, s), 3.22ꢀ3.62 (7H, m), 4.01ꢀ4.10 (2H, m), 4.93ꢀ4.97 and 5.1ꢀ5.13 (1H, each
m), 6.83 (1H, d, J = 8.8), 7.13ꢀ7.44 (4H, m), 7.51 (1H, d, J = 1.7), 7.67 (1H, dd, J = 2.4, 8.8); LC/MS (ESI,
[M+H]+, m/z) 861. To the solution of the obtained product (232 mg, 0.27 mmol) in 1,4ꢀdioxane (0.45 mL)
was added 4 M HCl in 1,4ꢀdioxane (1.5 mL), and the mixture was shaken (600 minꢀ1) overnight at room
temperature. The reaction mixture was added ethanol and was shaken (600 minꢀ1) for 4 h at room
temperature. Nitrogen gas was blown into the reaction solution to evaporate the solvent. Subsequently,
water was added to dissolve the residue and the solution was freezeꢀdried to give 140 mg (quantitative
yield) of the title compound as dihydrochloride salt. 1HꢀNMR (400 MHz, DMSOꢀd6): δ 1.44 (9H, s), 3.00
(3H, s), 3.06ꢀ3.08 (1H, m), 3.24ꢀ3.28 (1H, m), 3.45ꢀ3.49 (2H, m), 4.36ꢀ4.39 (2H, m), 5.03 (1H, dd, J = 2.0,
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10.1), 6.77 (1H, dd, J = 2.0, 8.9), 6.93 (1H, d, J = 2.0), 7.13 (1H, d, J = 7.8), 7.15ꢀ7.17 (1H, m), 7.31 (1H, s),
7.35 (1H, t, J = 7.8), 7.80 (1H, d, J = 8.9), 9.08 (1H, brs), 9.45 (1H, brs), 9.87 (1H, s); 13CꢀNMR (100 MHz,
DMSOꢀd6): δ 29.9, 33.1, 39.1, 45.8, 53.6, 63.4, 67.9, 92.4, 111.3, 114.7, 117.0, 119.0, 121.2, 122.4, 129.2,
138.5, 142.5, 143.0, 152.1, 156.6; HRMS calculated for C22H30N4O4S + H+ 447.2061, found (ESI, [M+H]+)
447.2061; LC/MS (ESI, [M+H]+, m/z) 447; HPLC (Method A): purity 100% RT 2.0 min.
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Nꢀ(3ꢀ((1R)ꢀ2ꢀ(2ꢀ((3ꢀCyclopropylꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ꢀ1ꢀhydroxyꢀethyl)phenyl)methanesul
fonamide (13). To a stirred solution of 25 (1.415 g, 5 mmol), 17 (10 mL, 10 mmol, 1 M toluene solution),
and triphenylphosphine (2.910 g, 11 mmol) in anhydrous toluene (50 mL) was added
N,N,N’,N’ꢀtetramethylazodicarboxamide (1.915 g, 11 mmol) at room temperature, and the solution was
stirred overnight. The reaction solution was purified by flash column chromatography on silica gel (95:5 to
74:26 nꢀhexane/ethyl acetate) to give 3.779 g (89% yield) of the coupling product. 1HꢀNMR (300 MHz,
CDCl3, 1:1 rotamers): δ 0.54 (6H, q, J = 7.9), 0.89 (9H, t, J = 7.9), 1.03ꢀ1.05 (2H, m), 1.15ꢀ1.20 (2H, m),
1.44 (9H, s), 1.48 and 1.52 (9H, each s), 1.68 (9H, s), 2.13ꢀ2.21 (1H, m), 3.22ꢀ3.63 (7H, m), 4.02ꢀ4.13 (2H,
m), 4.94ꢀ4.98 and 5.10ꢀ5.14 (1H, each m), 6.84 (1H, dd, J = 2.0, 8.7), 7.13ꢀ7.53 (6H, m); LC/MS (ESI,
[M+H]+, m/z) 845. To the solution of the obtained product (3.770 g, 4.5 mmol) in 1,4ꢀdioxane (9 mL) was
added 4 M HCl in 1,4ꢀdioxane (20 mL), and the mixture was stirred overnight at room temperature. Then, 4
M HCl in 1,4ꢀdioxane (14 mL) was further added to the reaction solution, and the mixture was stirred for 2
h at room temperature. The resultant solid was collected by suction filtration, washed with diethyl ether, and
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dried under reduced pressure. The solid was dissolved with water, and the solution was freezeꢀdried to give
2.033 g (90 % yield) of the title compound as dihydrochloride salt. 1HꢀNMR (400 MHz, DMSOꢀd6): δ
0.96ꢀ1.02 (4H, m), 2.23ꢀ2.30 (1H, m), 3.00 (3H, s), 3.04ꢀ3.10 (1H, m), 3.24ꢀ3.28 (1H, m), 3.44ꢀ3.47 (2H,
m), 4.34ꢀ4.42 (2H, m), 5.04 (1H, dd, J = 2.1, 10.2), 6.80 (1H, dd, J = 2.1, 8.8), 6.91 (1H, d, J = 2.1), 7.13
(1H, d, J = 8.0), 7.17 (1H, dd, J = 1.3, 8.0), 7.31 (1H, s), 7.34 (1H, t, J = 8.0), 7.70 (1H, d, J = 8.8), 9.11 (1H,
brs), 9.50 (1H, brs), 9.87 (1H, s); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 7.6, 7.7, 39.1, 45.8, 53.7, 63.4, 67.9,
92.2, 112.1, 116.2, 117.0, 119.0, 121.0, 121.2, 129.2, 138.5, 141.7, 143.0, 146.1, 157.6; HRMS calculated
for C21H26N4O4S + H+ 431.1748, found (ESI, [M+H]+) 431.1743; LC/MS (ESI, [M+H]+, m/z) 431; HPLC
(Method B): purity 96% RT 1.9 min.
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Nꢀ(3ꢀ((1R)ꢀ2ꢀ(2ꢀ((3ꢀCyclobutylꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ꢀ1ꢀhydroxyꢀethyl)phenyl)methanesulf
onamide (14). To a stirred solution of 26 (1.426 g, 5 mmol), 17 (10 mL, 10 mmol, 1 M toluene solution),
and triphenylphosphine (2.914 g, 11 mmol) in anhydrous toluene (25 mL) was added
N,N,N’,N’ꢀtetramethylazodicarboxamide (1.911 g, 11 mmol) at room temperature, and the solution was
stirred overnight. The reaction solution was purified by flash column chromatography on silica gel (88:12 to
67:33 nꢀhexane/ethyl acetate) to give 3.848 g (90% yield) of the coupling product. 1HꢀNMR (300 MHz,
CDCl3, 1:1 rotamers): δ 0.54 (6H, q, J = 7.9), 0.89 (9H, t, J = 7.9), 1.44 (9H, s), 1.48 and 1.52 (9H, each s),
1.70 (9H, s), 1.95ꢀ2.02 (1H, m), 2.11ꢀ2.20 (1H, m), 2.39ꢀ2.59 (4H, m), 3.22ꢀ3.63 (7H, m), 3.87 (1H, qu, J =
8.8), 4.02ꢀ4.08 (2H, m), 4.94ꢀ4.98 and 5.10ꢀ5.14 (1H, each m), 6.84 (1H, dd, J = 1.7, 8.6), 7.13ꢀ7.54 (6H,
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m); LC/MS (ESI, [M+H]+, m/z) 859. The obtained product (3.840 g, 4.3 mmol) and 4 M HCl in ethyl
acetate (80 mL) were stirred overnight at room temperature. The resultant solid was collected by suction
filtration, washed with diethyl ether, and dried to afford 2.166 g (93% yield) of the title compound as
dihydrochloride salt. 1HꢀNMR (400 MHz, DMSOꢀd6): δ 1.90ꢀ1.98 (1H, m), 2.02ꢀ2.13 (1H, m), 2.35ꢀ2.43
(4H, m), 3.00 (3H, s), 3.04ꢀ3.11 (1H, m), 3.23ꢀ3.28 (1H, m), 3.45ꢀ3.48 (2H, m), 3.90 (1H, qu, J = 8.7),
4.37ꢀ4.44 (2H, m), 5.05 (1H, dd, J = 2.0, 10.2), 6.82 (1H, dd, J = 2.1, 8.8), 6.95 (1H, d, J = 2.1), 7.14 (1H, d,
J = 7.8), 7.16ꢀ7.18 (1H, m), 7.31 (1H, s), 7.35 (1H, t, J = 7.8), 7.69 (1H, d, J = 8.8), 9.15 (1H, brs), 9.56 (1H,
brs), 9.89 (1H, s); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 18.4, 27.9, 32.0, 39.1, 45.8, 53.7, 63.4, 67.9, 92.2,
112.4, 115.3, 117.0, 119.0, 121.2, 121.3, 129.2, 138.5, 141.8, 143.0, 147.6, 157.7; HRMS calculated for
C22H28N4O4S + H+ 445.1904, found (ESI, [M+H]+) 445.1897; LC/MS (ESI, [M+H]+, m/z) 445; HPLC
(Method A): purity 96% RT 2.0 min.
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Nꢀ(3ꢀ((1R)ꢀ1ꢀHydroxyꢀ2ꢀ(2ꢀ((3ꢀ(trifluoromethyl)ꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ethyl)phenyl)metha
nesulfonamide (15). To a stirred solution of 27 (1.511 g, 5 mmol), 17 (10 mL, 10 mmol, 1 M toluene
solution), and triphenylphosphine (2.600 g, 9.9 mmol) in anhydrous toluene (15 mL) was added
N,N,N’,N’ꢀtetramethylazodicarboxamide (1.733 g, 10 mmol) at room temperature, and the solution was
stirred overnight. The reaction solution was then purified by flash column chromatography on silica gel
(82:18 to 61:39 nꢀhexane/ethyl acetate) to give 3.544 g (81% yield) of the coupling product. 1HꢀNMR (300
MHz, CDCl3, 1:1 rotamers): δ 0.54 (6H, q, J = 8.1), 0.89 (9H, t, J = 8.1), 1.44 (9H, s), 1.49 and 1.53
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(9H,each s), 1.71 (9H, s), 3.22ꢀ3.64 (7H, m), 4.04ꢀ4.13 (2H, m), 4.94ꢀ4.99 and 5.10ꢀ5.14 (1H, each m), 6.98
(1H, dd, J = 2.2, 8.8), 7.14ꢀ7.45 (4H, m) 7.59 (1H, d, J = 1.8), 7.64(1H, dd, J = 4.0, 8.8); LC/MS (ESI,
[M+H]+, m/z) 873. The obtained product (3.514 g, 4 mmol) and 4 M HCl in ethyl acetate (80 mL) were
stirred overnight at room temperature. The resultant solid was collected by suction filtration, washed with
tertꢀbutyl methyl ether, and dried to afford 1.930 g (93% yield) of the title compound as dihydrochloride salt.
1HꢀNMR (400 MHz, DMSOꢀd6): δ 3.00 (3H, s), 3.05ꢀ3.12 (1H, m), 3.24ꢀ3.28 (1H, m), 3.49 (2H, brs),
4.40ꢀ4.47 (2H, m), 5.02 (1H, d, J = 9.8), 6.28 (1H, brs), 7.03 (1H, dd, J = 2.1, 8.8), 7.13ꢀ7.18 (3H, m) 7.31
(1H, s), 7.35 (1H, t, J = 7.8), 7.71 (1H, d, J = 8.8), 9.11 (1H, brs), 9.46 (1H, brs), 9.87 (1H, s), 13.96 (1H, s);
13CꢀNMR (100 MHz, DMSOꢀd6): δ 39.2, 45.8, 53.6, 63.6, 67.9, 92.8, 113.7, 115.4, 117.0, 119.0, 119.6,
121.2, 122.2 (q, J = 268), 129.2, 133.0 (q, J = 36), 138.5, 141.9, 143.0, 157.7; HRMS calculated for
C19H21F3N4O4S + H+ 459.1308, found (ESI, [M+H]+) 459.1307; LC/MS (ESI, [M+H]+, m/z) 459; HPLC
(Method A): purity 99% RT 2.0 min.
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Nꢀ(3ꢀ((1R)ꢀ1ꢀHydroxyꢀ2ꢀ(2ꢀ((3ꢀphenylꢀ1Hꢀindazolꢀ6ꢀyl)oxy)ethylamino)ethyl)phenyl)methanesulfona
mide (16). To a stirred solution of 28 (34 mg, 0.13 mmol), 17 (0.5 mL, 0.20 mmol, 0.4 M toluene solution),
and triphenylphosphine (56 mg, 0.21 mmol) in anhydrous toluene (1 mL) was added
N,N,N’,N’ꢀtetramethylazodicarboxamide (40 mg, 0.23 mmol) at room temperature, and the solution was
stirred overnight. The reaction solution was then purified by flash column chromatography on silica gel
(88:12 to 67:33 nꢀhexane/ethyl acetate) to give 77 mg (87% yield) of the coupling product. 1HꢀNMR (300
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Journal of Medicinal Chemistry
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MHz, CDCl3, 1:1 rotamers): δ 0.55 (6H, q, J = 7.9), 0.89 (9H, t, J = 7.9), 1.44 (9H, s), 1.46 and 1.49 (9H,
each s), 1.73 (9H, s), 3.24ꢀ3.64 (7H, m), 4.06ꢀ4.15 (2H, m), 4.95ꢀ4.99 and 5.10ꢀ5.15 (1H, each m), 6.93 (1H,
dd, J = 1.9, 8.8), 7.13ꢀ7.52 (7H, m), 7.64 (1H, d, J = 1.9), 7.79 (1H, dd, J = 3.0, 8.8), 7.96 (2H, dd, J = 1.5,
8.0); LC/MS (ESI, [M+H]+, m/z) 881. To the solution of the obtained product (75 mg, 0.09 mmol) in
tertꢀbutyl methyl ether (0.2 mL) was added 4 M HCl in 1,4ꢀdioxane (1 mL) and the mixture was stirred
overnight at room temperature. The resultant solid was collected by suction filtration, washed with
tertꢀbutyl methyl ether, and dried to afford 48 mg (quantitative yield) of the title compound as
dihydrochloride salt. 1HꢀNMR (400 MHz, DMSOꢀd6): δ 3.00 (s, 3H), 3.08ꢀ3.12 (1H, m), 3.25ꢀ3.30 (1H, m),
3.47ꢀ3.50 (2H, m), 4.42ꢀ4.44 (2H, m), 5.04 (1H, dd, J = 2.0, 10.2), 6.91 (1H, dd, J = 2.0, 8.9), 7.05 (1H, d, J
= 2.0), 7.13ꢀ7.18 (2H, m), 7.32 (1H, s), 7.35 (1H, t, J = 7.9), 7.38ꢀ7.42 (1H, m), 7.49ꢀ7.53 (2H, m),
7.96ꢀ7.99 (3H, m), 9.08 (1H, brs), 9.41 (1H, brs), 9.87 (1H, s); 13CꢀNMR (100 MHz, DMSOꢀd6): δ 39.1,
45.9, 53.6, 63.4, 67.9, 92.5, 113.0, 115.1, 116.9, 118.9, 121.2, 121.5, 126.5, 127.6, 128.7, 129.2, 133.6,
138.5, 142.6, 142.9, 143.0, 156.9; HRMS calculated for C24H26N4O4S + H+ 467.1748, found (ESI, [M+H]+)
467.1746; LC/MS (ESI, [M+H]+, m/z) 467; HPLC (Method A): purity 99% RT 2.0 min.
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Ethyl
6ꢀ(2ꢀ(tertꢀbutoxycarbonylꢀ((2R)ꢀ2ꢀ(3ꢀ(tertꢀbutoxycarbonyl(methylsulfonyl)amino)phenyl)ꢀ2ꢀtriethylsily
loxyꢀethyl)amino)ethoxy)ꢀ1ꢀtetrahydropyranꢀ2ꢀylꢀindazoleꢀ3ꢀcarboxylate (30). To a stirred solution of
17 (506 mg, 0.86 mmol), 29 (124 mg, 0.43 mmol), and triphenylphosphine (229 mg, 0.87 mmol) in
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