Convergent and enantioselective syntheses of cytosolic phospholipase A 2α inhibiting N-(1-indazol-1-ylpropan-2-yl)carbamates

Article abstract of DOI:10.1039/c4ob00535j

Cytosolic phospholipase A₂α (cPLA₂α) is an important enzyme of the inflammation cascade. Therefore, inhibitors of cPLA₂α are assumed to be promising drug candidates for the treatment of inflammatory disorders. Recently we have found that indole-5-carboxylic acid with a 3-(4-octylphenoxy)-2-(phenoxycarbonylamino) propyl substituent in position 1 is an inhibitor of cPLA₂α. We have now synthesized a corresponding derivative with the indole heterocycle replaced by an indazole (4) employing an analogous reaction sequence as for the synthesis of the indole derivative. Besides, a more convergent synthesis for 4 was established using an aziridine as central intermediate. Furthermore, a chiral-pool based enantioselective synthesis was developed for the synthesis of (R)- and (S)-4. Starting compound for both enantiomers was the (R)-serine derived oxazolidine (R)-25. Compound 4 proved to be a moderate inhibitor of cPLA₂α, with the S-enantiomer being twice as active as the R-enantiomer. The racemate 4 and the enantiomers (R)- and (S)-4 showed a high in vitro metabolic stability in rat liver S9 fractions. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00535j

Organic &
Biomolecular Chemistry
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Convergent and enantioselective syntheses
of cytosolic phospholipase A2α inhibiting
N-(1-indazol-1-ylpropan-2-yl)carbamates†
Cite this: Org. Biomol. Chem., 2014,
12, 4021
Tom Sundermann, Martina Arnsmann, Julian Schwarzkopf, Walburga Hanekamp and
Matthias Lehr*
Cytosolic phospholipase A2α (cPLA2α) is an important enzyme of the inflammation cascade. Therefore,
inhibitors of cPLA2α are assumed to be promising drug candidates for the treatment of inflammatory dis-
orders. Recently we have found that indole-5-carboxylic acid with a 3-(4-octylphenoxy)-2-(phenoxycar-
bonylamino)propyl substituent in position 1 is an inhibitor of cPLA2α. We have now synthesized a
corresponding derivative with the indole heterocycle replaced by an indazole (4) employing an analogous
reaction sequence as for the synthesis of the indole derivative. Besides, a more convergent synthesis for 4
was established using an aziridine as central intermediate. Furthermore, a chiral-pool based enantio-
selective synthesis was developed for the synthesis of (R)- and (S)-4. Starting compound for both enantio-
mers was the (R)-serine derived oxazolidine (R)-25. Compound 4 proved to be a moderate inhibitor of
cPLA2α, with the S-enantiomer being twice as active as the R-enantiomer. The racemate 4 and the
enantiomers (R)- and (S)-4 showed a high in vitro metabolic stability in rat liver S9 fractions.
Received 10th March 2014,
Accepted 28th April 2014
DOI: 10.1039/c4ob00535j
www.rsc.org/obc
Introduction
Cytosolic phospholipase A2α (cPLA2α) is an esterase that selec-
tively cleaves the sn-2 position of arachidonoyl-glycerophos-
pholipids of biomembranes to generate free arachidonic acid
and lysophospholipids.1,2 Subsequent metabolism of these
products leads to a variety of inflammatory mediators includ-
ing prostaglandins, leukotrienes and platelet activating factor
(PAF). Mice with cPLA2α deficiency display a reduced eicosa-
noid production and are resistant to disease in a variety of
models of inflammation.3 Therefore, cPLA2α is considered as a
target for the treatment of inflammatory diseases, such as
rheumatoid arthritis, atopic dermatitis and Alzheimer’s
disease.4–6 Today several potent inhibitors of cPLA2α are
known,7,8 which show activity in diverse animal models of
inflammation after systemic or local application. However,
none of these agents is actually in clinical development.
Fig. 1 Structures of known and designed inhibitors of cPLA2α.
substances is the activated electrophilic ketone moiety present
in the middle part of the molecules. This structural element is
supposed to form reversible covalent binding interactions with
a serine residue of the active site of cPLA2α.
Recent studies have shown that 1-(indol-1-yl)propan-2-ones
are extensively metabolized in vitro as well as in vivo.11,12
Especially, the activated ketone is reduced to an alcohol result-
ing in compounds, which do not inhibit cPLA2α any more.
Therefore, we have replaced the ketone by metabolically more
stable polar moieties such as acyloxy, acylamino, urea and car-
bamate.13 These variations led to a more or less pronounced
drop of inhibitory potency. One of the most active compounds
of the investigated substances was the carbamate substituted
indole-5-carboxylic acid 2, which possessed an IC50 against
cPLA2α in the micromolar range.
We have found that certain 1-(indol-1-yl)propan-2-ones,
such as compound 1 (Fig. 1), inhibit cPLA2α with high
potency.9 The latter compound shows structural similarities to
the propan-2-one inhibitor ARC-70484XX developed by Astra-
Zeneca.10 An important part of the pharmacophore of these
Because structure–activity relationship studies on the
1-(indol-1-yl)propan-2-ones have revealed that replacement of
the indole scaffold of 1 by an indazole (3) led to an about five-
fold increase of activity,14 we wanted to synthesize and evaluate
the corresponding carbamate-substituted indazole derivative 4.
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster,
Corrensstrasse 48, D-48149 Münster, Germany. E-mail: lehrm@uni-muenster.de
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob00535j
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In contrast to the ketones 1 and 3, the carbamates 2 and 4 base followed by hydrolysis of the tert-butyl ester with trifluoro-
possess a stereogenic centre resulting in R- and S-configurated acetic acid gave the desired target compound 4.
products. A further aim of this study, therefore, was to develop
In Scheme 2, an alternative approach for the synthesis of 4
enantioselective syntheses for the enantiomers of 4 and to is outlined, which is more convergent in respect of the vari-
determine the influence of the configuration of this com- ation of the octylphenoxy-part of 4 during the course of
pound on cPLA2α inhibition.
structure–activity studies. This synthetic route started from
indazole-5-carboxylic acid, which was converted into its allyl
ester (15) by reaction with allyl bromide in presence of K2CO3.
Using Cs2CO3 as base, the indazole ester 15 was alkylated with
epichlorohydrin in position 1 yielding the oxiranylmethylinda-
zole derivative 16. The oxiranyl ring of 16 was opened to a
Results and discussion
Chemistry
For the preparation of the indazole 4 the synthetic route deve- azidoalcohol by reaction with sodium azide.16 Then the azido
loped for the synthesis of the corresponding indole 213 was group of 17 was transformed to an amine (18) by treatment
used (Scheme 1). Starting material was indazole-5-car- with triphenylphosphine, which in turn was protected with
boxylic acid (5), which first was converted into its tert-butyl- BOC using di-tert-butyl dicarbonate. The alcohol group of
ester 8 employing a reaction sequence published for the obtained compound 19 was reacted with tosyl chloride in pres-
synthesis of tert-butyl benzotriazole-5-carboxylate.15 Treatment ence of 4-dimethylaminopyridine to give the tosylate 20. Cycli-
of 8 with epichlorohydrin in presence of KOH and tetrabutyl- zation to the aziridine 21 was accomplished by treatment of 20
ammonium bromide led to 9, which was reacted with 4-octyl- with powdered KOH and tetrabutylammonium hydrogen-
phenol to obtain the secondary alcohol 10. The alcohol sulfate in CH2Cl2. Ring opening of the BOC-protected aziridine
functionality of this compound was converted to a mesylate 21 with 4-octylphenol was achieved in methylene chloride with
(11) by reaction with methanesulfonyl chloride. From the catalytical amounts of BF3-etherate.17 The BOC group of 22
latter intermediate the azide 12 was obtained by treatment was removed by trifluoroacetic acid. Reaction of the generated
with trimethylsilyl azide and tetrabutylammonium fluoride. amine 23 with phenyl chloroformate led to the carbamate 24.
Catalytic hydrogenation of the azide group of 12 with Pd on Finally, the allyl ester of the indazole heterocycle of 24 was
charcoal led to the amine-substituted compound 13. Reaction cleaved with tetrakis(triphenylphosphine)palladium(0) to
of this with phenyl chloroformate in presence of an amine afford the desired target compound 4. By this way, derivatives
Scheme 1 Reagents and conditions: (a) acetic anhydride, reflux, 2 h; (b) tert-butyl 2,2,2-trichloroacetimidate, cyclohexane, THF, BF3-etherate, room
temp., 2 h; (c) 1 M aqueous NaOH, ethanol, room temp., 1 h; (d) epichlorohydrin, powdered KOH, tetrabutylammonium bromide, room temp., 4 h;
(e) 4-octylphenol, 4-dimethylaminopyridine, 120 °C, 2 h; (f) methanesulfonyl chloride, pyridine, room temp., 3.5 h; (g) trimethylsilyl azide, tetra-
butylammonium fluoride, THF, reflux, 72 h; (h) Pd/C, H2, THF, room temp., 6 h; (i) phenyl chloroformate, ethyl(diisopropyl)amine, THF, room temp.,
45 min; ( j) trifluoroacetic acid, CH2Cl2, room temp., 12 h.
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Scheme 2 Reagents and conditions: (a) allyl bromide, K2CO3, DMF, room temp., 6 h; (b) epichlorohydrin, Cs2CO3, DMF, room temp., 14 h; (c) NaN3,
NH4Cl, methanol, H2O, 80 °C, 18 h; (d) triphenylphosphine, acetonitrile, reflux, overnight; (e) di-tert-butyl dicarbonate, triethylamine, diethyl ether,
methanol, 0 °C, 2 h; (f) p-toluenesulfonyl chloride, powdered KOH, THF, room temp., 4 h; (g) KOH powder, tetrabutylammonium hydrogen sulfate,
CH2Cl2, room temp., 6 h; (h) 4-octylphenol, BF3-etherate, CH2Cl2, room temp., 1 min; (i) trifluoroacetic acid, CH2Cl2, room temp., 2 h; ( j) phenyl
chloroformate, triethylamine, THF, room temp., 2 h; (k) tetrakis(triphenylphosphine)palladium(0), acetic acid, THF, room temp., 6 h.
with varying substituents at the phenoxy-residue can be introduced at first into the molecule. After removing the
obtained from a common intermediate (here 21) in only four acetone protection group of obtained compound (S)-32, the
steps instead of six necessary in the synthesis described above free alcohol moiety was esterified with tosyl chloride and
(Scheme 1).
the tosylate group substituted by a 4-octylphenoxy residue
Due to the chiral centre present in 4, this compound exists to afford (S)-22. From this compound (S)-4 was synthesized
as two enantiomers. For the determination of their eudismic employing the same procedure as for the synthesis of (R)-4
ratio, additionally an enantioselective, chiral-pool based syn- from (R)-22. Chiral HPLC on a Chiralpak® IA column revealed
thetic approach was established. The synthesis of the R-con- that (R)-4 and (S)-4 were formed with 93% ee and 100% ee,
figurated enantiomer of 4 started from the commercially respectively (for chromatograms see ESI†).
availably, (R)-serine derived oxazolidine (R)-25,18 which was
Biological evaluation
converted to the tosylate (R)-27 as previously described
(Scheme 3).19,20 This central intermediate was reacted with
The target compounds were evaluated for cPLA2α inhibition in
an assay using cPLA2α isolated from porcine platelets.21 When
4-octylphenol to yield the phenol ether (S)-29. The acetone pro-
tecting group was removed by reaction with p-toluenesulfonic
acid and the alcohol moiety of (R)-30 was converted to a tosyl
ester with p-toluenesulfonyl chloride. Treatment of obtained
compound (R)-31 with allyl indazole-5-carboxylate in DMF in
presence of NaH afforded the BOC protected 1-(2-amino-
propyl)indazole derivative (R)-22. After deprotection of the
BOC group, the amine moiety of resulting compound (R)-23
was reacted with phenyl chloroformate in presence of an
amine base. Subsequent cleavage of the allyl ester group of
resulting compound (R)-24 using Pd(0) catalysis led to the
desired target compound (R)-4.
The synthesis of the S-enantiomer of 4 is outlined in
Scheme 4. With (R)-27, the identical central chiral intermedi-
ate was used as for the synthesis of the R-enantiomer. To get
the antipodal configuration in the target compound, here the
indazole heterocycle and not the 4-octylphenoxy residue was
measuring the inhibitory potency of 4, (S)-4 and (R)-4 it
became evident that the data were strongly influenced by small
impurities of the extreme potent indazolylpropan-2-one 3
(IC50: 0.0045 µM) present in the substances. The highest
amounts of 3 (about 1% measured by HPLC/UV and MS) were
found in 4 synthesized via the route shown in Scheme 1. The
spectral data of the intermediates of the synthesis revealed
that the ketone group was introduced into the molecule
during the reduction of the azide 12 to the amine 13, and that
the ketone impurity could not be separated in the following
steps. The compounds synthesized by the sequences outlined
in Schemes 2–4 contained smaller amounts of 3 (less than
0.1%). However, their inhibition values were also impaired by
this substance. Therefore, all target compounds were purified
by reversed phase HPLC. By this way, impurity 3 could be
totally removed.
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Scheme 3 Reagents and conditions: (a) LiBH4, THF, 0 °C, 3 h; (b) p-toluenesulfonyl chloride, 4-dimethylaminopyridine, triethylamine, CH2Cl2,
room temp., overnight; (c) NaH (dispersion in mineral oil), DMF, 70 °C, 3 h; (d) p-toluenesulfonic acid, room temp., 4 h; (e) p-toluenesulfonyl chlor-
ide, 4-dimethylaminopyridine, triethylamine, CH2Cl2, room temp., overnight; (f) allyl indazole-5-carboxylate (15), NaH (dispersion in mineral oil),
DMF, 80 °C, 3 h; (g) trifluoroacetic acid, CH2Cl2, room temp., 2 h; (h) phenyl chloroformate, triethylamine, THF, room temp.; (i) tetrakis(triphenylphos-
phine)palladium(0), acetic acid, THF, room temp., 6 h.
Scheme 4 Reagents and conditions: (a) NaH, DMF, 80 °C, 3 h; (b) 1. 1 M HCl, THF, 70 °C, 3 h; 2. di-tert-butyl dicarbonate, triethylamine, methanol,
room temp., 12 h; (c) p-toluenesulfonyl chloride, 4-dimethylaminopyridine, triethylamine, CH2Cl2, room temp., 12 h; (d) 4-octylphenol, NaH, DMF,
80 °C, 3 h; (e) trifluoroacetic acid, CH2Cl2, room temp., 2 h; (f) phenyl chloroformate, triethylamine, THF, room temp., 2 h; (g) tetrakis(triphenylphos-
phine)palladium(0), acetic acid, THF, room temp., 6 h.
The previously published lead compound 2 was newly syn- IC50 value against cPLA2α of 26 µM was evaluated. In contrast
thesized from racemic 25 and allyl indole-5-carboxylate follow- to the ketones 1 and 3, the replacement of the indole hetero-
ing the route outlined in Scheme 4 for the synthesis of (S)-4 cycle by an indazole did not increase inhibitory potency in
(see ESI†). After cleaning up by reversed phase HPLC, for 2 an case of the carbamates. With an IC50 of 29 µM 4 was as active
4024 | Org. Biomol. Chem., 2014, 12, 4021–4030
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measured on
a
Varian Mercury Plus 400 spectrometer
Table 1 Inhibition of cPLA2α and metabolic stability
(101 MHz) or an Agilent VNMRS-600 spectrometer (151 MHz).
Electron ionization (EI) mass spectra were obtained on a Finni-
gan GCQ apparatus. The high resolution mass spectra (HRMS)
were recorded on a Bruker micrOTOF-Q II spectrometer using
electro spray chemical ionization (ESI) or atmospheric pressure
chemical ionization (APCI). Preparative reversed phase HPLC
was performed using a Knauer pump P2.1L and a Shimazu
SPD-6A UV detector. Chromatograms were recorded with
MacDAcq32 Control Software from Bischoff. As stationary
phase a Knauer RP18 Eurospher II 5 µm column (20 mm (I.D.)
× 250 mm) with a RP18 Eurospher II 5 µm guard column
(20 mm (I.D.) × 30 mm) was used. The mobile phase consisted
of acetonitrile–water–formic acid (80 : 20 : 0.1, v/v/v). The flow
rate was 25 mL min−1. The compounds were dissolved in
DMSO and the injected sample volume was 0.5–1 mL. The
detection wavelength was set to 254 nm. The substances were
obtained as solids after distilling off the organic solvent and
freeze-drying the remaining aqueous phase using a Christ
alpha 1-2 LD plus apparatus. Chiral HPLC-analysis was per-
formed on a Daicel Chiralpak® IA 5 µm column (4.6 mm (I.D.)
× 250 mm) using isohexane–methanol–isopropanol–formic
acid (90 : 8.75 : 1.25 : 0.1, v/v/v/v) as mobile phase at a flow rate
of 1 mL min−1. The detection wavelength was 254 nm.
Allyl indazole-5-carboxylate (15). To a stirring suspension of
potassium carbonate (0.52 g, 3.76 mmol) in dry DMF (4 mL)
were added allyl bromide (2.2 mL, 13 mmol) and indazole-
5-carboxylic acid (0.150 g, 0.93 mmol) and the obtained
mixture was stirred at room temperature for 3 h. After addition
of water (15 mL), the reaction mixture was exhaustively
extracted with ethyl acetate. The combined organic phases
were dried with Na2SO4, concentrated and chromatographed
on silica gel (hexane–ethyl acetate, 8 : 2) to yield 15 as a solid
(0.175 g, 94%). Mp: 112–113 °C; 1H NMR (400 MHz, CDCl3):
δ 4.87 (dt, J = 5.6 Hz and 1.4 Hz, 2H), 5.31 (dq, J = 10.4 Hz and
1.3 Hz, 1H), 5.44 (dq, J = 17.2 Hz and 1.6 Hz, 1H), 6.07 (ddt, J =
17.2 Hz, 10.5 Hz and 5.6 Hz, 1H), 7.55 (dd, J = 8.9 Hz and 0.8
Hz, 1H), 8.12 (dd, J = 8.8 Hz and 1.5 Hz, 1H), 8.22 (s, 1H), 8.59
(dd, J = 1.4 Hz and 0.8 Hz, 1H); 13C NMR (101 MHz, CDCl3):
δ 65.8, 109.8, 118.4, 123.1, 123.7, 124.7, 128.1, 132.4, 136.3,
142.1, 166.5; HRMS (APCI, direct probe) [M + H]+ calculated:
203.0815, found: 203.0819.
Inhibition of
Metabolic
Compound
cPLA2α IC50 a (µM)
stabilityb (%)
3
0.0045
29
34
8
84
89
81
4 (racemate)
(R)-4
(S)-4
18
a Values are the means of at least two independent determinations,
errors are within 20%; IC50 value of reference inhibitor ARC-70484XX:
0.0065 µM.10,21 b Percentage of parent compound remaining after
metabolism by rat liver S9 fractions in presence of NADPH; values are
the means of at least two independent determinations. Metabolic
stability of reference inhibitor 3-(5-carboxypentanoyl)-1-[3-(4-
octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid:12 63 6.7% (n = 9).
Calculated log P values for 3 and 4 using Advanced Chemistry
Development (ACD/Labs) Software V11.02: 6.5 and 8.4, respectively.
as 2 (Table 1). The two enantiomers of 4 showed a slight but
significant difference in their IC50 values: the S-enantiomer
was about two-fold more active than the R-enantiomer.
As mentioned above, a drawback of the indazolylpropan-
2-one 3 is its metabolic liability. In in vitro experiments it
could be shown that the carbamate 4 is much more stable
against metabolism by rat liver homogenate than 3. After
incubation of 3 in presence of the co-factor NADPH only 8% of
the parent compound could be recovered, under the same con-
ditions still 80–90% of 4, (S)-4 and (R)-4 were present in the
incubation mixture.
Conclusions
Taken together, we have developed new effective synthetic
approaches for the synthesis of 4 as well as for its enantiomers
(S)-4 and (R)-4. The biological evaluation of these compounds
revealed that the increase of metabolic stability achieved by
replacement of the ketone function of 3 by a carbamate moiety
is accompanied by a drastic loss of cPLA2α inhibitory potency.
The aim of further studies will be to improve cPLA2α inhibitory
potency of 4 by structural variations.
Allyl 1-(oxiran-2-ylmethyl)indazole-5-carboxylate (16). To a
solution of 15 (0.070 g, 0.35 mmol) in DMF (5 mL) was added
cesium carbonate (0.337 g, 1.03 mmol) followed by epichloro-
hydrin (0.190 mL, 1.74 mmol). The mixture was stirred at
room temperature for 14 h. After addition of water (10 mL),
the reaction mixture was exhaustively extracted with ethyl
acetate. The combined organic phases were dried with Na2SO4,
Experimental section
Chemistry
General. Column chromatography was performed on silica concentrated and chromatographed on silica gel (hexane–ethyl
gel 60, particle size 0.040–0.063 mm, from Macherey & Nagel. acetate, 9 : 1) to yield 16 as an oil (0.063 g, 70%). 1H NMR
Melting points were determined on a Büchi B-540 apparatus (400 MHz, CDCl3): δ 2.55 (dd, J = 4.7 Hz and 2.6 Hz, 1H), 2.84
and are uncorrected. 1H NMR spectra were recorded on a (dd, J = 4.7 Hz and 4.0 Hz, 1H), 3.37 (dddd, J = 5.7 Hz, 3.9 Hz,
Varian Mercury Plus 400 spectrometer (400 MHz), a Varian 3.2 Hz and 2.5 Hz, 1H), 4.44 (dd, J = 15.2 Hz and 5.6 Hz, 1H),
Unity Plus 600 spectrometer (600 MHz) or an Agilent 4.72 (dd, J = 15.2 Hz and 3.3 Hz, 1H), 4.84 (dt, J = 5.6 Hz and
VNMRS-600 spectrometer (600 MHz). 13C NMR spectra were 1.4 Hz, 2H), 5.29 (dq, J = 10.5 Hz and 1.3 Hz, 1H), 5.42 (dq, J =
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17.2 Hz and 1.6 Hz, 1H), 6.05 (ddt, J = 17.2 Hz, 10.4 Hz and 5.6 trated and chromatographed on silica gel (hexane–ethyl
1
Hz, 1H), 7.50 (dt, J = 8.9 Hz and 0.9 Hz, 1H), 8.07 (dd, J = 8.9 acetate, 9 : 1 to 6 : 4) to afford 19 as an oil (0.256 g, 97%). H
Hz and 1.5 Hz, 1H), 8.10 (d, J = 1.0 Hz, 1H), 8.52 (dd, J = 1.5 Hz NMR (400 MHz, CDCl3): δ 1.44 (s, 9H), 2.59 (s, 1H), 3.16 (dd,
and 0.8 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 45.4, 50.7, 51.3, J = 14.8 Hz and 4.2 Hz, 1H), 3.40 (d, J = 14.4 Hz, 1H), 4.18–4.27
65.6, 109.3, 118.2, 123.3, 123.9, 124.7, 127.5, 132.5, 135.4, (m, 1H), 4.40 (dd, J = 14.2 Hz and 6.7 Hz, 1H), 4.50 (dd, J =
142.1, 166.4; HRMS (APCI, direct probe) [M + H]+ calculated: 14.3 Hz and 4.6 Hz, 1H), 4.86 (dt, J = 5.7 Hz and 1.4 Hz, 2H),
259.1077, found: 259.1093.
5.10 (s, 1H), 5.31 (dq, J = 10.4 Hz and 1.3 Hz, 1H), 5.43 (dq, J =
Allyl 1-(3-azido-2-hydroxypropyl)indazole-5-carboxylate (17). 17.2 Hz and 1.6 Hz, 1H), 6.07 (ddt, J = 17.2 Hz, 10.5 Hz and
Sodium azide (0.076 g, 1.17 mmol) and ammonium chloride 5.6 Hz, 1H), 7.47 (dt, J = 8.9 Hz and 0.9 Hz, 1H), 8.07–8.13 (m,
(0.025 g, 0.47 mmol) were added to a suspension of 16 2H), 8.54 (dd, J = 1.5 Hz and 0.8 Hz, 1H); 13C NMR (101 MHz,
(0.060 g, 0.23 mmol) in methanol–water (9 : 1) (4.5 mL). The CDCl3): δ 28.5, 44.2, 51.8, 65.7, 71.0, 80.3, 109.2, 118.4, 123.5,
mixture was heated at 80 °C for 18 h. After cooling, the suspen- 123.6, 124.9, 127.8, 132.5, 135.4, 142.1, 157.4, 166.4; HRMS
sion was exhaustively extracted with diethyl ether. The com- (APCI, direct probe) [M + H]+ calculated: 376.1867, found:
bined organic phases were dried with Na2SO4 and 376.1875.
concentrated to give 17 as a solid (0.067 g, 96%). Mp:
Allyl 1-{3-[(tert-butoxycarbonyl)amino]-2-(tosyloxy)propyl}-
1
76–77 °C; H NMR (400 MHz, CDCl3): δ 2.72 (s, 1H), 3.38 (dd, indazole-5-carboxylate (20). To a solution of 19 (1.70 g,
J = 5.5 Hz and 1.9 Hz, 2H), 4.31 (dtd, J = 6.4 Hz, 5.5 Hz and 4.53 mmol) in dry THF (40 mL) were added tosyl chloride
4.2 Hz, 1H), 4.45 (dd, J = 14.2 Hz and 6.2 Hz, 1H), 4.50 (dd, J = (1.76 g, 9.23 mmol) and freshly powdered potassium hydroxide
14.2 Hz and 4.3 Hz, 1H), 4.85 (dq, J = 5.6 Hz and 1.4 Hz, 2H), (88%) (1.08 g, 17 mmol). The mixture was stirred at room
5.31 (dq, J = 10.5 Hz and 1.3 Hz, 1H), 5.43 (dq, J = 17.2 Hz and temperature for 4 h, concentrated and chromatographed on
1.5 Hz, 1H), 6.07 (ddtd, J = 17.2 Hz, 10.4 Hz, 5.6 Hz and silica gel (hexane–ethyl acetate, 8 : 2) to yield 20 as an oil
1
1.2 Hz, 1H), 7.47 (dt, J = 8.9 Hz and 1.0 Hz, 1H), 8.11 (dd, J = (2.25 g, 94%). H NMR (400 MHz, CDCl3): δ 1.44 (s, 9H), 2.32
8.9 Hz and 1.5 Hz, 1H), 8.13 (d, J = 1.0 Hz, 1H), 8.54 (dd, J = (s, 3H), 3.51 (t, J = 4.6 Hz, 2H), 4.55 (dd, J = 15.5 Hz and
1.5 Hz and 0.8 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 51.3, 7.2 Hz, 1H), 4.61 (dd, J = 15.1 Hz and 3.8 Hz, 1H), 4.87 (dt, J =
53.9, 65.8, 70.1, 109.0, 118.4, 123.5, 123.6, 124.9, 127.9, 132.4, 5.7 Hz and 1.4 Hz, 2H), 4.94–5.01 (m, 1H), 5.07 (tbroad, J =
135.7, 142.2, 166.4; HRMS (APCI, direct probe) [M + H]+ calcu- 6.4 Hz, 1H), 5.33 (dq, J = 10.4 Hz and 1.3 Hz, 1H), 5.45 (dq, J =
lated: 302.1248, found: 302.1269.
17.2 Hz and 1.5 Hz, 1H), 6.09 (ddt, J = 17.3 Hz, 10.5 Hz and 5.6
Allyl 1-(3-amino-2-hydroxypropyl)indazole-5-carboxylate Hz, 1H), 6.97 (d, J = 8.3 Hz, 2H), 7.29–7.37 (m, 3H), 7.90 (s,
hydrochloride (18). A solution of 17 (1.50 g, 4.98 mmol) in 1H), 8.00 (d, J = 8.9 Hz, 1H), 8.40 (s, 1H); 13C NMR (101 MHz,
acetonitrile (12 mL) was treated with triphenylphosphine CDCl3): δ 21.7, 28.5, 42.4, 50.2, 65.8, 80.0, 80.2, 109.2, 118.5,
(1.31 g, 4.99 mmol) and stirred at room temperature for about 123.3, 123.8, 124.5, 127.4, 127.6, 129.6, 132.5, 132.5, 135.6,
30 min until the development of nitrogen could be observed. 142.1, 144.9, 156.0, 166.4; HRMS (ESI+) [M + H]+ calculated:
Then the mixture was heated under reflux overnight. After 530.1955, found: 530.1956.
cooling and evaporation of the solvent, the residue was dis-
Allyl 1-{[1-(tert-butoxycarbonyl)aziridine-2-yl]methyl}inda-
solved in 1 M HCl and washed with ethyl acetate. The aqueous zole-5-carboxylate (21). To a solution of 20 (2.25 g, 4.25 mmol)
phase was concentrated to dryness and dissolved in a small in CH2Cl2 (40 mL) were added tetrabutylammonium hydrogen
volume of CH2Cl2. Addition of etheric HCl led to the formation sulfate (0.231 g, 0.68 mmol) and freshly powdered potassium
of a precipitate. Finally, the solvent was removed in vacuo to hydroxide (88%) (1.08 g, 17 mmol). The mixture was stirred at
yield 18 as a solid (1.55 g, 100%). Mp: 55–56 °C; 1H NMR room temperature for 6 h. After addition of diethyl ether
(400 MHz, DMSO-D6): δ 2.71–2.85 (m, 1H), 2.90–3.04 (m, 1H), (100 mL), the mixture was filtered and the filtrate was concen-
4.12–4.30 (m, 1H), 4.53 (d, J = 5.6 Hz, 2H), 4.83 (dt, J = 5.4 Hz trated to dryness. The residue was chromatographed on silica
and 1.5 Hz, 2H), 5.29 (dq, J = 10.6 Hz and 1.5 Hz, 1H), 5.42 gel (hexane–ethyl acetate, 9 : 1) to yield 21 as an oil (1.26 g,
(dq, J = 17.2 Hz and 1.7 Hz, 1H), 5.81 (s, 1H), 6.08 (ddt, J = 83%). 1H NMR (400 MHz, CDCl3): δ 1.27 (s, 9H), 2.12 (d, J =
17.2 Hz, 10.6 Hz and 5.4 Hz, 1H), 7.83 (dt, J = 8.9 Hz and 3.6 Hz, 1H), 2.37 (d, J = 6.2 Hz, 1H), 2.83 (tdd, J = 6.1 Hz, 4.8
0.9 Hz, 1H), 7.97 (dd, J = 8.9 Hz and 1.6 Hz, 1H), 8.05 (s, 3H), Hz and 3.5 Hz, 1H), 4.48 (dd, J = 14.8 Hz and 6.0 Hz, 1H), 4.55
8.31 (d, J = 0.9 Hz, 1H), 8.52 (dd, J = 1.6 Hz and 0.8 Hz, 1H); (dd, J = 14.8 Hz and 4.8 Hz, 1H), 4.86 (dt, J = 5.6 Hz and
13C NMR (101 MHz, DMSO-D6): δ 42.2, 52.2, 64.9, 66.8, 110.5, 1.4 Hz, 2H), 5.30 (dq, J = 10.4 Hz and 1.3 Hz, 1H), 5.43 (dq, J =
117.8, 122.0, 123.2, 124.1, 126.2, 132.8, 135.2, 142.0, 165.6; 17.2 Hz and 1.5 Hz, 1H), 6.07 (ddt, J = 17.1 Hz, 10.5 Hz and
HRMS (APCI, direct probe) [M + H]+ calculated: 276.1343, 5.6 Hz, 1H), 7.54 (dt, J = 8.8 Hz and 0.8 Hz, 1H), 8.08 (dd, J =
found: 276.1372.
Allyl
8.8 Hz and 1.5 Hz, 1H), 8.11 (d, J = 0.9 Hz, 1H), 8.53 (dd, J =
1-{3-[(tert-butoxycarbonyl)amino]-2-hydroxypropyl}- 1.5 Hz and 0.8 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 27.8,
indazole-5-carboxylate (19). A solution of 18 (0.220 g, 30.3, 36.4, 51.4, 65.7, 81.6, 109.7, 118.3, 123.2, 124.0, 124.7,
0.71 mmol) in diethyl ether (10 mL) and methanol (6 mL) was 127.4, 132.5, 135.4, 142.0, 161.6, 166.6; HRMS (APCI, direct
treated at 0 °C with triethylamine (0.11 mL, 0.79 mmol). Then probe) [M + H]+ calculated: 358.1761, found: 358.1749.
di-tert-butyl dicarbonate (0.174 g, 0.80 mmol) was added and
Allyl
1-{2-[(tert-butoxycarbonyl)amino]-3-(4-octylphenoxy)-
the mixture was stirred at room temperature for 2 h, concen- propyl}indazole-5-carboxylate (22). A solution of 21 (0.050 g,
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0.14 mmol) in CH2Cl2 (10 mL) was treated with 4-octylphenol 9.0 Hz, 1H), 8.04 (dd, J = 8.9 Hz and 1.4 Hz, 1H), 8.15 (d, J =
(0.044 g, 0.21 mmol). After addition of 2 drops of BF3-etherate, 0.7 Hz, 1H), 8.54 (d, J = 0.7 Hz, 1H); 13C NMR (101 MHz,
the mixture was stirred at room temperature for 1 min, con- CDCl3): δ 14.3, 22.8, 29.4, 29.6, 31.8, 32.0, 35.2, 48.6, 51.0,
centrated and chromatographed on silica gel (hexane–ethyl 65.7, 66.2, 109.2, 114.5, 118.3, 121.7, 123.5, 123.7, 124.9, 125.7,
acetate, 9 : 1 to 8 : 2) to yield 22 as an oil (0.016 g, 20%). 1H 127.9, 129.5, 129.6, 132.5, 135.9, 136.3, 142.3, 150.9, 154.4,
NMR (300 MHz, CDCl3): δ 0.88 (t, J = 6.6 Hz, 3H), 1.22–1.35 (m, 156.1, 166.4; HRMS (APCI, direct probe) [M + H]+ calculated:
10H), 1.41 (s, 9H), 1.50–1.57 (m, 2H), 2.49–2.58 (m, 2H), 3.78 584.3119, found: 584.3344.
(dd, J = 9.5 Hz and 5.6 Hz, 1H), 3.95 (dd, J = 9.5 Hz and 3.3 Hz,
1-{3-(4-Octylphenoxy)-2-[(phenoxycarbonyl)amino]propyl}-
1H), 4.36–4.50 (m, 1H), 4.66 (dd, J = 14.2 Hz and 5.8 Hz, 1H), indazole-5-carboxylic acid (4). To a solution of 24 (0.038 g,
4.76 (dd, J = 14.3 Hz and 5.2 Hz, 1H), 4.85 (d, J = 5.6 Hz, 2H), 0.065 mmol) in dry THF (3 mL) was added under nitrogen
5.31 (dd, J = 10.4 Hz and J = 1.2 Hz, 1H), 5.37–5.49 (m, 2H), tetrakis(triphenylphosphine)palladium(0) (0.008 g, 0.007 mmol).
6.07 (ddt, J = 17.1 Hz, 10.8 Hz and 5.6 Hz, 1H), 6.79 (d, J = Then nitrogen was bubbled through the solution for 10 min.
8.5 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.9 Hz, 1H), After addition of acetic acid (0.1 mL), the mixture was stirred
8.03 (dd, J = 8.9 Hz and 1.3 Hz, 1H), 8.13 (s, 1H), 8.53 (s, 1H); at room temperature for 6 h, concentrated and chromato-
13C NMR (101 MHz, CDCl3): δ 14.2, 22.8, 28.4, 29.4, 29.6, 31.8, graphed on silica gel (hexane–ethyl acetate–acetic acid,
32.0, 35.2, 49.1, 50.3, 65.7, 66.7, 80.1, 109.2, 114.5, 118.3, 8 : 2 : 0.1) to yield 4 as a solid (0.036 g, 98%). Mp: 189–190 °C;
123.3, 123.7, 124.7, 127.6, 129.5, 132.5, 135.6, 136.1, 142.3, 1H NMR (400 MHz, DMSO-D6): δ 0.83 (t, J = 6.5 Hz, 3H),
155.3, 156.2, 166.5; HRMS (APCI, direct probe) [M + H]+ calcu- 1.16–1.30 (m, 10H), 1.44–1.55 (m, 2H), 2.49 (m, 2H), 4.02–4.12
lated: 564.3432, found: 564.3505.
(m, 2H), 4.23–4.34 (m, 1H), 4.61 (dd, J = 14.4 Hz and 8.0 Hz,
Allyl 1-[2-amino-3-(4-octylphenoxy)propyl]indazole-5-carboxy- 1H), 4.72 (dd, J = 14.5 Hz and 5.1 Hz, 1H), 6.76 (d, J = 7.8 Hz,
late (23). A solution of 22 (0.108 g, 0.19 mmol) in CH2Cl2 2H), 6.85 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.1 Hz, 2H), 7.13 (t, J =
(8 mL) was treated with several drops of trifluoroacetic acid 7.8 Hz, 1H), 7.26 (dd, J = 8.3 Hz and 7.3 Hz, 2H), 7.73 (d, J =
(0.57 mL, 7.4 mmol) and stirred at room temperature for 2 h. 9.1 Hz, 1H), 7.92 (dd, J = 9.0 Hz and 1.2 Hz, 1H), 8.03 (d, J =
Then the reaction mixture was evaporated. The residue was 8.7 Hz, 1H), 8.27 (s, 1H), 8.43 (s, 1H), 12.76 (sbroad, 1H); 13C
treated with ethyl acetate and washed with 2 M aqueous NaOH NMR (101 MHz, DMSO-D6): δ 14.0, 22.1, 28.6, 28.7, 28.9, 31.2,
solution. The aqueous layer was extracted three times with 31.3, 34.3, 49.2, 51.3, 67.1, 109.6, 114.4, 121.5, 123.3, 123.4,
ethyl acetate. The combined organic layers were dried over 124.0, 125.0, 126.6, 129.1, 129.2, 134.8, 135.2, 141.7, 153.9,
Na2SO4 and concentrated to yield 23 as an oil (0.089 g, 100%). 155.9, 156.2, 167.6; HRMS (ESI+) [M
1H NMR (400 MHz, DMSO-D6): δ 0.83 (t, J = 6.6 Hz, 3H), 544.2806, found: 544.2853.
+
H]+ calculated:
1.29–1.14 (m, 10H), 1.49 (t, J = 7.9 Hz, 2H), 2.38–2.45 (m, 2H),
(S)-tert-Butyl 2,2-dimethyl-4-[(4-octylphenoxy)methyl]oxazoli-
3.96 (dd, J = 10.3 Hz and 4.9 Hz, 1H), 4.06 (s, 1H), 4.15 (dd, J = dine-3-carboxylate ((S)-29). To a solution of 4-octylphenol
10.5 Hz and 3.5 Hz, 1H), 4.85–4.75 (m, 4H), 5.27 (dq, J = (0.400 g, 1.94 mmol) in dry DMF (7 mL) was added sodium
10.5 Hz and 1.5 Hz, 1H), 5.40 (dq, J = 17.2 Hz and 1.7 Hz, 1H), hydride (60% dispersion in mineral oil) (0.085 g, 2.13 mmol).
6.05 (ddt, J = 17.6 Hz, 10.5 Hz and 5.3 Hz, 1H), 6.83 (d, J = The mixture was stirred at room temperature for about 30 min
8.6 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 8.8 Hz, 1H), until no further development of hydrogen could be observed.
7.97 (dd, J = 8.8 Hz and 1.6 Hz, 1H), 8.39 (s, 1H), 8.53 (d, J = A solution of (R)-tert-butyl 2,2-dimethyl-4-[(tosyloxy)methyl]oxa-
1.6 Hz, 1H); 13C NMR (101 MHz, DMSO-D6): δ 14.4, 22.5, 29.0, zolidine-3-carboxylate ((R)-27)19,20 (0.897 g, 2.33 mmol) in
29.1, 29.3, 31.6, 31.7, 34.7, 51.4, 51.9, 65.4, 70.3, 110.6, 114.7, DMF (7 mL) was added dropwise and the mixture was heated
118.2, 122.4, 123.6, 124.6, 126.6, 129.5, 133.3, 134.9, 135.5, at 70 °C for about 3 h. After addition of water (50 mL), the reac-
142.2, 156.8, 166.0; MS (EI 70 eV) m/z (%): 463 (3) M+, 258 (16), tion mixture was exhaustively extracted with ethyl acetate. The
248 (50), 107 (100).
combined organic phases were dried with Na2SO4, concen-
Allyl 1-{3-(4-octylphenoxy)-2-[(phenoxycarbonyl)amino]propyl}- trated and chromatographed on silica gel (hexane–ethyl
1
indazole-5-carboxylate (24). To a solution of 23 (0.044 g, acetate, 19 : 1) to yield (S)-29 as an oil (0.661 g, 81%). H NMR
0.095 mmol) in dry THF (4 mL) were added dropwise at 0 °C (400 MHz, DMSO-D6): δ 0.84 (t, J = 6.7 Hz, 3H), 1.16–1.32 (m,
triethylamine (0.016 mL, 0.11 mmol) followed by phenyl chloro- 10H), 1.36–1.55 (m, 17H), 2.44–2.55 (m, 2H), 3.81 (t, J = 9.2 Hz,
formate (0.015 mL, 0.12 mmol). The mixture was stirred at 1H), 3.91 (dd, J = 9.1 Hz and 1.5 Hz, 1H), 3.96–4.06 (m, 2H),
room temperature for 2 h, concentrated and chromatographed 4.08–4.16 (m, 1H), 6.87 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 8.5 Hz,
on silica gel (hexane–ethyl acetate, 9 : 1 to 8 : 2) to yield 24 as 2H); 13C NMR (101 MHz, DMSO-D6): δ 13.9, 22.0, 22.9, 24.1,
1
an oil (0.041 g, 74%). H NMR (400 MHz, CDCl3): δ 0.87 (t, J = 26.4, 27.2, 28.0, 28.5, 28.6, 28.8, 31.2, 31.2, 34.2, 55.2, 55.6,
6.9 Hz, 3H), 1.20–1.34 (m, 10H), 1.52–1.55 (m, 2H), 2.50–2.57 64.6, 64.9, 66.0, 66.7, 79.3, 79.7, 92.9, 93.2, 114.3, 114.3, 129.2,
(m, 2H), 3.82 (dd, J = 9.7 Hz and 6.0 Hz, 1H), 4.08 (dd, J = 134.6, 134.7, 151.0, 151.4, 156.2; HRMS (APCI, direct probe)
9.7 Hz and 3.7 Hz, 1H), 4.48–4.58 (m, 1H), 4.74 (dd, J = 14.4 Hz [M + H]+ calculated: 420.3108, found: 420.3148.
and 5.9 Hz, 1H), 4.81–4.90 (m, 3H), 5.30 (dq, J = 10.5 Hz and
(R)-tert-Butyl [1-hydroxy-3-(4-octylphenoxy)propan-2-yl]car-
1.3 Hz, 1H), 5.43 (dq, J = 17.2 Hz and 1.6 Hz, 1H), 5.98–6.13 bamate ((R)-30). To a solution of (S)-29 (0.550 g, 1.31 mmol)
(m, 2H), 6.82 (d, J = 8.6 Hz, 2H), 7.04–7.11 (m, 4H), 7.21 (t, J = in methanol (10 mL) was added p-toluenesulfonic acid mono-
7.5 Hz, 1H), 7.35 (dd, J = 8.1 Hz and 7.6 Hz, 2H), 7.56 (d, J = hydrate (0.113 g, 0.59 mmol). The mixture was stirred at room
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temperature for about 4 h. After neutralization with saturated 0.064 mmol) was reacted with phenyl chloroformate (8 µL,
sodium bicarbonate solution (10 mL), methanol was removed 0.064 mmol) in the same manner as described for the synthesis
1
under reduced pressure and the aqueous residue was extracted of 24 to yield (R)-24 as an oil (0.030 g, 79%). The H NMR and
with ethyl acetate (3 × 10 mL). The combined organic phases 13C NMR spectral data were identical with those of 24. HRMS
were dried with Na2SO4, concentrated and chromatographed (APCI, direct probe) [M + H]+ calculated: 584.3119, found: 584.3187.
on silica gel (hexane–ethyl acetate, 8 : 2) to yield (R)-30 as an
(R)-1-{3-(4-Octylphenoxy)-2-[(phenoxycarbonyl)amino]propyl}-
1
oil (0.302 g, 61%). H NMR (300 MHz, CDCl3): δ 0.87 (t, J = 6.7 indazole-5-carboxylic acid ((R)-4). (R)-4 was obtained from (R)-
Hz, 3H), 1.15–1.37 (m, 10H), 1.46 (s, 9H), 1.53–1.57 (m, 2H), 24 (0.026 g, 0.045 mmol) in the same manner as described for
2.33 (s, 1H), 2.46–2.62 (m, 2H), 3.74–3.85 (m, 1H), 3.86–3.95 (m, the synthesis of 4. Yield: 0.022 g, 91%. Mp: 173–174 °C. The
1H), 3.96–4.16 (m, 3H), 5.20 (d, J = 6.8 Hz, 1H), 6.82 (d, J = 8.6 1H NMR and 13C NMR spectral data were identical with those
Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H); 13C NMR; MS (EI 70 eV) m/z of 24. HRMS (APCI, direct probe) [M + H]+ calculated:
(%): 379 (2) M+, 206 (75), 118 (100), 107 (64).
544.2806, found: 544.2816. Enantiomeric excess determined
(S)-2-[(tert-Butoxycarbonyl)amino]-3-(4-octylphenoxy)propyl by chiral HPLC: 93.1% ee. Specific rotation [α]2D0 = +5.4 (c =
4-methylbenzenesulfonate ((S)-31). A solution of (R)-30 0.20 g per 100 mL; ethyl acetate).
(0.280 g, 0.74 mmol) in CH2Cl2 (15 mL) was added dropwise
(S)-tert-Butyl 4-({5-[(allyloxy)carbonyl]indazole-1-yl}methyl)-
to a solution of tosyl chloride (0.282 g, 1.48 mmol) and 2,2-dimethyloxazolidine-3-carboxylate ((S)-32). To a solution of
4-dimethylaminopyridine (0.045 g, 0.37 mmol) in CH2Cl2 allyl indazole-5-carboxylate (15) (0.913 g, 4.52 mmol) in dry
(15 mL). The mixture was treated with triethylamine (204 µL, DMF (15 mL) was added sodium hydride (60% dispersion in
1.46 mmol) and stirred for 12 h. The reaction mixture was mineral oil) (0.199 g, 4.98 mmol). The mixture was stirred at
washed three times with 1 M KHSO4 and with brine, dried room temperature for about 30 min until no further develop-
over Na2SO4, concentrated and chromatographed on silica gel ment of hydrogen could be observed. A solution of (R)-tert-
(hexane–ethyl acetate, 9 : 1 to 8 : 2) to yield (S)-31 as an oil butyl 2,2-dimethyl-4-[(tosyloxy)methyl]oxazolidine-3-carboxy-
(0.275 g, 70%). 1H NMR (400 MHz, CDCl3): δ 0.88 (t, J = 6.9 Hz, late ((R)-27)19,20 (2.09 g, 5.42 mmol) in DMF (15 mL) was
3H), 1.21–1.34 (m, 10H), 1.43 (s, 9H), 1.53–1.57 (m, 2H), 2.37 added dropwise and the mixture was heated at 70 °C for about
(s, 3H), 2.50–2.57 (m, 2H), 3.84 (dd, J = 9.3 Hz and 5.4 Hz, 1H), 3 h. After addition of water (50 mL), the reaction mixture
3.97 (dd, J = 9.3 Hz and 3.3 Hz, 1H), 4.14–4.27 (m, 3H), 4.94 (d, was exhaustively extracted with ethyl acetate. The combined
J = 7.1 Hz, 1H), 6.66 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H), organic phases were dried with Na2SO4, concentrated and
7.22 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.3 Hz, 2H); 13C NMR chromatographed on silica gel (hexane–ethyl acetate, 9 : 1 to
1
(101 MHz, CDCl3): δ 14.2, 21.8, 22.8, 28.4, 29.4, 29.6, 31.9, 8 : 2) to yield (S)-32 as an oil (1.20 g, 64%). H NMR (400 MHz,
32.0, 35.2, 48.7, 65.5, 68.0, 80.3, 114.3, 128.0, 129.4, 130.0, CDCl3): δ 1.20–1.59 (m, 15H), 3.80–4.68 (m, 5H), 4.85 (dt, J =
132.5, 135.9, 145.1, 155.1, 156.1; HRMS (ESI+) [M + H]+ calcu- 5.6 Hz and 1.5 Hz, 2H), 5.30 (d, J = 10.6 Hz, 1H), 5.42 (dq, J =
lated: 534.2884, found: 534.2867.
17.2 Hz and 1.5 Hz, 1H), 5.98–6.14 (m, 1H), 7.48–7.67 (m, 1H),
(R)-Allyl 1-{2-[(tert-butoxycarbonyl)amino]-3-(4-octylphenoxy)- 8.03–8.15 (m, 2H), 8.50–8.55 (m, 1H); 13C NMR (151 MHz,
propyl}indazole-5-carboxylate ((R)-22). To a solution of allyl CDCl3): δ 23.1, 24.2, 27.0, 27.2, 28.5, 28.7, 49.6, 50.3, 56.8,
indazole-5-carboxylate (15) (0.114 g, 0.56 mmol) in dry DMF 57.3, 65.3, 65.5, 65.6, 65.7, 80.7, 80.9, 93.9, 94.7, 108.7, 109.5,
(5 mL) was added sodium hydride (60% dispersion in mineral 118.2, 118.3, 123.2, 123.3, 123.8, 123.9, 124.7, 124.9, 127.4,
oil) (0.024 g, 0.60 mmol). The mixture was stirred at room 128.0, 132.5, 132.6, 135.1, 135.5, 141.8, 142.1, 151.7, 152.6,
temperature for about 30 min until no further development of 166.4, 166.6; HRMS (APCI, direct probe) [M + H]+ calculated:
hydrogen could be observed. Then a solution of (S)-31 (0.200 g, 416.2180, found: 416.2174.
0.37 mmol) in DMF (5 mL) was added dropwise and the
(S)-Allyl 1-{2-[(tert-butoxycarbonyl)amino]-3-hydroxypropyl}-
mixture was heated at 80 °C for about 3 h. After addition of indazole-5-carboxylate ((S)-33). A solution of (S)-32 (0.900 g,
water (10 mL), the reaction mixture was exhaustively extracted 2.17 mmol) in THF (10 mL) and 1 M HCl (10 mL) was heated
with ethyl acetate. The combined organic phases were dried at 70 °C for 3 h. After evaporation of the solvent in vacuo, the
with Na2SO4, concentrated and chromatographed on silica gel residue was dissolved in methanol (20 mL) and triethylamine
(hexane–ethyl acetate, 9 : 1) to yield (R)-22 as an oil (0.121 g, (1.04 mL, 7.46 mmol) and di-tert-butyl dicarbonate (0.543 g,
1
57%). The H NMR and 13C NMR spectral data were identical 2.49 mmol) were added at 0 °C. The mixture was stirred
with those of 22. HRMS (APCI, direct probe) [M + H]+ for 12 h, concentrated and chromatographed on silica gel
calculated: 564.3432, found: 564.3473.
(hexane–ethyl acetate, 8 : 2) to yield (S)-33 as a solid (0.753 g,
1
(R)-Allyl 1-[2-amino-3-(4-octylphenoxy)propyl]indazole-5-car- 93%). Mp: 124–125 °C; H NMR (400 MHz, CDCl3): δ 1.41 (s,
boxylate ((R)-23). (R)-22 (0.080 g, 0.14 mmol) was reacted with 9H), 3.11 (sbroad, 1H), 3.54 (dd, J = 11.7 Hz and 4.8 Hz, 1H),
trifluoroacetic acid in the same manner as described for the syn- 3.69 (dd, J = 11.6 Hz and 3.6 Hz, 1H), 3.99–4.10 (m, 1H), 4.63
thesis of 23 to yield (R)-23 as an oil (0.065 g, 99%). The 1H NMR (dd, J = 14.4 Hz and 4.5 Hz, 1H), 4.71 (dd, J = 14.5 Hz and
and 13C NMR spectral data were identical with those of 23. MS 6.5 Hz, 1H), 4.85 (dt, J = 5.7 Hz and 1.4 Hz, 2H), 5.13 (s, 1H),
(EI 70 eV) m/z (%): 463 (12) M+, 258 (38), 248 (100), 107 (31).
(R)-Allyl 1-{3-(4-octylphenoxy)-2-[(phenoxycarbonyl)amino]- 1.5 Hz, 1H), 6.06 (ddt, J = 17.2 Hz, 10.4 Hz and 5.6 Hz, 1H),
5.30 (dq, J = 10.4 Hz and 1.3 Hz, 1H), 5.43 (dq, J = 17.2 Hz and
propyl}indazole-5-carboxylate
((R)-24). (R)-23
(0.030
g, 7.56 (d, J = 8.9 Hz, 1H), 8.10 (dd, J = 8.9 Hz and 1.5 Hz, 1H),
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8.14 (d, J = 0.9 Hz, 1H), 8.54 (dd, J = 1.5 Hz and 0.8 Hz, 1H); 1H NMR and 13C NMR spectral data were identical with those of
13C NMR (151 MHz, CDCl3): δ 28.5, 48.9, 51.9, 62.3, 65.8, 80.1, 24. HRMS (ESI+) [M + H]+ calculated: 584.3119, found: 584.3125.
109.4, 118.4, 123.4, 123.6, 124.9, 128.0, 132.5, 135.5, 142.4,
155.7, 166.4; HRMS (APCI, direct probe) [M + H]+ calculated: indazole-5-carboxylic acid ((S)-4). Following the procedure
376.1867, found: 376.1870. described for the synthesis of 4, (S)-24 (0.120 g, 0.21 mmol)
(S)-1-{3-(4-Octylphenoxy)-2-[(phenoxycarbonyl)amino]propyl}-
(S)-Allyl 1-{2-[(tert-butoxycarbonyl)amino]-3-(tosyloxy)propyl}- was converted to (S)-4 (0.104 g, 93%); mp: 184–185 °C. The
indazole-5-carboxylate ((S)-34). A solution of (S)-33 (0.370 g, 1H NMR and 13C NMR spectral data were identical with those
0.99 mmol) in CH2Cl2 (10 mL) was added dropwise to a solu- of 4. HRMS (ESI+) [M + H]+ calculated: 544.2806, found:
tion of tosyl chloride (0.376 g, 1.97 mmol) and 4-dimethyl- 544.2796. Enantiomeric excess determined by chiral HPLC:
aminopyridine (0.060 g, 0.49 mmol) in CH2Cl2 (10 mL). The 100% ee. Specific rotation [α]2D0 = −5.4 (c = 0.20 g per 100 mL;
mixture was treated with triethylamine (273 µL, 1.96 mmol) ethyl acetate).
and stirred at room temperature for 12 h. The reaction mixture
Biological evaluation
was washed three times with 1 M KHSO4 and with brine, dried
over Na2SO4, concentrated and chromatographed on silica gel
(hexane–ethyl acetate, 9 : 1 to 8 : 2) to yield (S)-34 as an oil
Inhibition of cytosolic phospholipase A2α (cPLA2α). Inhi-
bition of cPLA2α was measured according to a recently pub-
lished procedure.21 Briefly, cPLA2α isolated from porcine
platelets was incubated with co-vesicles consisting of the sub-
1
(0.490 g, 94%). H NMR (400 MHz, CDCl3): δ 1.37 (s, 9H), 2.41
(s, 3H), 3.93 (dd, J = 10.3 Hz and 5.5 Hz, 1H), 4.02 (dd, J =
10.2 Hz and 4.6 Hz, 1H), 4.28–4.38 (m, 1H), 4.49 (dd, J =
14.3 Hz and 5.8 Hz, 1H), 4.59 (dd, J = 14.0 Hz and 5.6 Hz, 1H),
4.86 (dt, J = 5.7 Hz and 1.4 Hz, 2H), 5.28–5.35 (m, 2H), 5.44
(dq, J = 17.2 Hz and 1.5 Hz, 1H), 6.07 (ddt, J = 17.2 Hz, 10.4 Hz
and 5.7 Hz, 1H), 7.24–7.29 (m, 2H), 7.45 (d, J = 8.8 Hz, 1H),
7.69 (d, J = 8.3 Hz, 2H), 8.03–8.09 (m, 2H), 8.50 (dd, J = 1.6 Hz
and 0.8 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 21.8, 28.3, 48.4,
49.8, 65.8, 68.4, 80.4, 109.1, 118.4, 123.5, 123.6, 124.8, 127.9,
128.1, 130.0, 132.2, 132.4, 135.7, 142.1, 145.3, 155.0,
166.4; HRMS (ESI+) [M + H]+ calculated: 530.1955, found:
530.1994.
(S)-Allyl 1-{2-[(tert-butoxycarbonyl)amino]-3-(4-octylphenoxy)-
propyl}indazole-5-carboxylate ((S)-22). To a solution of 4-octyl-
phenol (0.210 g, 1.02 mmol) in dry DMF (5 mL) was added
sodium hydride (60% dispersion in mineral oil) (0.041 g,
1.03 mmol). The mixture was stirred at room temperature for
about 30 min until no further development of hydrogen could
be observed. Then a solution of (S)-34 (0.450 g, 0.85 mmol) in
DMF (5 mL) was added dropwise and the mixture was heated
at 80 °C for about 3 h. After addition of water (10 mL), the reac-
tion mixture was exhaustively extracted with ethyl acetate. The
combined organic phases were dried over Na2SO4, concen-
trated and chromatographed on silica gel (hexane–ethyl
acetate, 9 : 1) to yield (S)-22 as a solid (0.311 g, 65%). Mp:
70–71 °C. The 1H NMR and 13C NMR spectral data were identi-
cal with those of 22. HRMS (APCI, direct probe) [M + H]+ calcu-
lated: 564.3432, found: 564.3470.
strate
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine
(200 µM) and 1,2-dioleoyl-sn-glycerol (100 µM). Enzyme reac-
tions were terminated after 60 min and cPLA2α activity was
determined by measuring the arachidonic acid released by the
enzyme in absence and presence of a test compound with
reversed phase HPLC and UV-detection at 200 nm after on-line
solid phase extraction. Because under the published con-
ditions (RP18 column with mobile phase acetonitrile–water–
phosphoric acid (85%), 77 : 23 : 0.1, v/v/v) the test compounds
2 and 4 co-eluted with the analyte arachidonic acid, the separ-
ation conditions had to be modified. Separation could be
achieved using an Phenomenex Aqua 3 µ C18 column (4.6 mm
(I.D.) × 150 mm) protected with a Phenomenex C18 guard
column (3 mm (I.D.) × 4 mm) using acetonitrile–10 mM
aqueous Na2HPO3 × 12 H2O, pH 7.4 (60 : 40, v/v) as mobile
phase at a flow rate of 0.7 mL.
Metabolic stability in rat liver S9 fractions. The metabolic
stability was tested using S9 fractions of rat liver homogen-
ate.22 Briefly, test compounds were incubated under aerobic
conditions in absence and presence of the co-factor NADPH.
The metabolic reactions were terminated after 30 min. The
extent of metabolism was evaluated with reversed phase HPLC
and UV-detection at 240 nm.
Notes and references
(S)-Allyl 1-[2-amino-3-(4-octylphenoxy)propyl]indazole-5-car-
boxylate ((S)-23). (S)-22 (0.200 g, 0.35 mmol) was treated with
trifluoroacetic acid in a similar manner as described for the
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synthesis of 23 to yield (S)-23 as an oil (0.160 g, 97%). The H
NMR and 13C NMR spectral data were identical with those of
23. HRMS (APCI, direct probe) [M + H]+ calculated: 464.2908,
found: 464.2906.
(S)-Allyl 1-{3-(4-octylphenoxy)-2-[(phenoxycarbonyl)amino]-
propyl}indazole-5-carboxylate ((S)-24). Following the procedure
described for the synthesis of 24, (S)-23 (0.140 g, 0.30 mmol)
was reacted with phenyl chloroformate (42 µL, 0.33 mmol) to
yield (S)-24 as a solid (0.144 g, 82%); mp: 115–116 °C. The
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