Efficient synthesis of mibefradil analogues: An insight into in vitro stability

Article abstract of DOI:10.1039/c4ob00504j

This article describes the synthesis and biological evaluation of a chemical library of mibefradil analogues to investigate the effect of structural modification on in vitro stability. The construction of the dihydrobenzopyran structure in mibefradil derivatives 2 was achieved through two efficient approaches based on a diastereoselective intermolecular Reformatsky reaction and an intramolecular carbonyl-ene cyclization. In particular, the second strategy through the intramolecular carbonyl-ene reaction led to the formation of a key intermediate 3 in a short and highly stereoselective way, which has allowed for practical and convenient preparation of analogues 2. Using this protocol, we could obtain 22 new mibefradil analogues 2, which were biologically tested for in vitro efficacies against T-type calcium channels and metabolic stabilities. Among the synthesized compounds, we found that analogue 2aa containing a dihydrobenzopyran ring and a secondary amine linker showed high % remaining activities of the tested CYP enzymes retaining the excellent T-type calcium channel blocking activity. These findings indicated that the structural modification of 1 was effective for improving in vitro stability, i.e., reducing CYP inhibition and metabolic degradation. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00504j

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Efficient synthesis of mibefradil analogues:
an insight into in vitro stability†
Ji Eun Lee,‡a,b Tae Hui Kwon,‡a,c Su Jin Gu,a Duck-Hyung Lee,b B. Moon Kim,c
Jae Yeol Lee,d Jae Kyun Lee,a Seon Hee Seo,a Ae Nim Pae,a,e Gyochang Keum,a,e
Yong Seo Cho*a,e and Sun-Joon Min*a,e
Cite this: Org. Biomol. Chem., 2014,
12, 5669
This article describes the synthesis and biological evaluation of a chemical library of mibefradil analogues
to investigate the effect of structural modification on in vitro stability. The construction of the dihydro-
benzopyran structure in mibefradil derivatives 2 was achieved through two efficient approaches based on
a diastereoselective intermolecular Reformatsky reaction and an intramolecular carbonyl–ene cyclization.
In particular, the second strategy through the intramolecular carbonyl–ene reaction led to the formation
of a key intermediate 3 in a short and highly stereoselective way, which has allowed for practical and con-
venient preparation of analogues 2. Using this protocol, we could obtain 22 new mibefradil analogues 2,
which were biologically tested for in vitro efficacies against T-type calcium channels and metabolic stabi-
lities. Among the synthesized compounds, we found that analogue 2aa containing a dihydrobenzopyran
ring and a secondary amine linker showed high % remaining activities of the tested CYP enzymes retaining
the excellent T-type calcium channel blocking activity. These findings indicated that the structural modifi-
cation of 1 was effective for improving in vitro stability, i.e., reducing CYP inhibition and metabolic
degradation.
Received 6th March 2014,
Accepted 6th June 2014
DOI: 10.1039/c4ob00504j
www.rsc.org/obc
Introduction
Mibefradil 1 (Posicor, Roche®), the first selective T-type
calcium channel blocker, was approved by the FDA in 1997 for
the treatment of hypertension and chronic angina pectoris
(Fig. 1).1–3 Because of its high oral bioavailability and long
half-life,4 it was initially administered to patients as a suitable
as well as convenient once-a-day medication. However, despite
the excellent pharmacokinetic features, this drug was with-
drawn from the market due to unfavorable cardiovascular side
effects, which were derived from a drug–drug interaction likely
resulting from an L-type calcium channel blockade.5
aCenter for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and
Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea
bDepartment of Chemistry, Sogang University, Seoul, 121-742, South Korea
cDepartment of Chemistry, College of Natural Sciences, Seoul National University,
Seoul 151-747, South Korea
dDepartment of Chemistry, Kyung Hee University, 26 Kyungheedae-ro,
Dongdaemun-gu, Seoul, 130-701, South Korea
eDepartment of Biological Chemistry, Korea University of Science and Technology
Fig. 1 Structures of mibefradil 1 and new analogues 2.
(UST), 176 Gajung-dong, 217 Gajungro, Yuseong-gu, Daejeon, 305-333, South Korea.
E-mail: ys4049@kist.re.kr, sjmin@kist.re.kr
†Electronic supplementary information (ESI) available. CCDC 980275. For
A metabolic examination of mibefradil 1 in mammalian
microsomes revealed that most metabolites are formed by a
combination of enzymatic processes, such as cytochrome P450
ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4ob00504j
‡These authors equally contributed to this work.
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(CYP450)-mediated oxidation at saturated and unsaturated compound 2 would be a crucial element for the preparation of
carbons, CYP450-catalyzed dealkylation, and hydrolysis of the the chemical library because there are two consecutive stereo-
ester side-chain.6 In particular, one of the most important genic centers in 2, of which one is quaternary. Once a viable
metabolic processes is oxidation at the benzylic carbon of the synthesis of the left hand side of 2 is established, we were
tetrahydronaphthalene ring. Based on this study, we specu- assured that a set of the mibefradil analogues could be rapidly
lated that such a biotransformation might occur in CYP developed in a convergent way by combining it with various
enzymes to generate some reactive metabolites, which, prior to substituted benzimidazoles.
release from the active site, could cause CYP inactivation.7–9
Herein, we report an efficient and stereoselective synthesis
In order to reduce CYP inhibition without affecting channel of mibefradil derivatives 2 and evaluation of their inhibitory
blocking activity, we considered modifying the structure of activity against the CYP enzymes as well as their calcium
mibefradil 1. Several strategies can be used to possibly mini- channel blocking activities.
mize the impact of CYP inhibition, such as changing the
length of the chain linker, replacing the aromatic groups, or
removing the labile functional groups.10 Considering these
methods, we have designed new mibefradil analogues 2 as
Results and discussion
shown in Fig. 1. First, we imagined that addition of oxygen to Our synthetic plan to achieve diastereoselective synthesis of 2
the site of hydroxylation could improve the stability of 1 by pre- is shown in Scheme 1. Coupling reactions of two fragments 3
venting further metabolic degradation. Second, alteration of and 4 or 5 through reductive amination or oxime formation
the tertiary amine moiety to other preventive linkers such as would be performed at a late stage to afford our target mole-
bulkier tertiary amines or oximes would provide a reasonable cules 2 (Scheme 2). We designed two synthetic approaches
approach to avoid the metabolic dealkylation process. In to construct the important structural framework of dihydro-
addition, the phenolic oxidation on the benzimidazole unit benzopyran 3. In route A, a metal-mediated Reformatsky
could be inhibited by introduction of R1 substituents at the C5 reaction11–14 would be the key strategy for installation of the
position of benzimidazole. Thus, we anticipated that these quaternary center in the functionalized dihydrobenzopyran 3.
structural changes could in turn diminish the formation of In this event, we proposed that the isopropyl group would
metabolic CYP inhibitors.
thereby serve as a steric obstacle to direct stereoselective
To investigate the correlation between structural modifi- addition of an acetate unit to ketone 6. The required chroman-
cation and CYP inhibition, it is necessary to develop a practical 3-one 6 would then be synthesized through an intramolecular
synthetic method to obtain a focused library of mibefradil carbonyl–ene reaction15–19 of 7, followed by oxidation. Alterna-
derivatives. In particular, an efficient and stereoselective syn- tively, an intramolecular carbonyl–ene reaction of 9 in the
thesis of a dihydrobenzopyran ring system on the left side of second approach would result in ring formation and diastereo-
Scheme 1 Our synthetic plan to obtain mibefradil derivatives 2.
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With the ketone 6 in hand, we next investigated metal-
induced Reformatsky reactions to synthesize the β-hydroxy
ester 14. In general, it is difficult to add carbon nucleophiles
to ketones in the Reformatsky reaction due to their low reactiv-
ity. Initially, we attempted a typical Reformatsky reaction of
ketone 6 with bromoacetate using zinc as a metal source, but
we were not able to obtain any desired product. Instead, the
indium-mediated reaction proceeded to give the desired
β-hydroxy ester 14 exclusively in moderate yield.25 The struc-
ture of ester 15a was preliminarily elucidated by NOE experi-
ments. In fact, the NOE correlation between the isopropyl
group and the axial C2 proton indicated that the stereochemi-
cal relationship of the isopropyl group and the hydroxyl group
was cis. However, the formation of 14 under these reaction
conditions was not consistently reproducible on a large scale.
While exploring other reaction conditions, we found that slow
addition of ketone 6 to the solution of Et2Zn and iodoacetate
in Et2O at ambient temperature26,27 afforded the corres-
ponding ester 14 as a single diastereomer in 72% yield. Thus,
by following this protocol, we could efficiently obtain ester 14
on a large scale. The base-catalyzed hydrolysis of ester 14
yielded acid 15, the structure of which was confirmed by X-ray
crystallographic analysis (Fig. 2).28 Finally, ester 14 was easily
transformed to aldehyde 3 by sequential reduction and
oxidation.
Scheme 2 The first synthesis of aldehyde 3: (a) K2CO3, BrCH2CH2OTBS,
DMF, 80 °C, 64%; (b) Ph3PCH(CH3)2I, nBuLi, THF; (c) TBAF, THF, 83%
(2 steps); (d) SO3·Pyr, iPr2NEt, DMSO, CH2Cl2; (e) Me2AlCl, CH2Cl2,
−20 °C, 71% (2 steps); (f) 20% Pd/C, H2, MeOH, 90%; (g) PCC, CH2Cl2,
81%; (h) ICH2CO2Et, Et2Zn, O2, Et2O, rt, 72%; (i) LiOH, MeOH–H2O (3 : 1),
quant.; ( j) LiAlH4, THF, 0 °C, 96%; (k) Dess–Martin periodinane, CH2Cl2, 81%.
selection of 3 simultaneously, which might be of benefit to
reduce the number of reaction steps. Ketone 9 would be pre-
pared from nucleophilic addition of 11 to epoxide 10 and sub-
sequent oxidation. Both precursors 7 and 11 would be readily
accessible starting from 3-fluorosalicylic aldehyde 8.
Alternative synthesis of aldehyde 3
In light of the efficiency for obtaining aldehyde 3 in the first
approach, we started to explore a different approach to alde-
hyde 3 that would be amenable to reasonable scale-up as we
proposed in Scheme 1. The second synthesis of aldehyde 3 is
described in Scheme 3. Alkenylphenol 11 was first prepared in
two steps and in 80% overall yields according to the modified
literature procedure.29 We attempted the reactions of 11 with
epoxide 1030,31 to obtain the corresponding alcohol under
different reaction conditions by varying the bases, solvents
and temperature. Indeed, the best yield was achieved when we
performed the reaction with two equivalents of epoxide 10 in
CH3CN at 90 °C using Cs2CO3 as a base. The ensuing second-
ary alcohol was then converted to the desired ketone 9 in 81%
Synthesis of aldehyde 3
The synthesis of compound 3 via the first route is demon-
strated in Scheme 2. 3-Fluorosalicylic aldehyde20–22 8 was
treated with bromoethyl silyl ether23 and K2CO3 in DMF to give
O-alkylated product, which underwent the Wittig olefination
with isopropylidene ylide and the silyl deprotection to afford
the resulting primary alcohol 12 in good yields. The Swern oxi-
dation of 12 was attempted to obtain aldehyde 7, but it was
susceptible to decomposition during flash column chromato-
graphy. To avoid such decomposition, we decided to conduct
an oxidation reaction under mild conditions, followed by rapid
cyclization without further purification. After screening several
oxidation conditions, we discovered that the Parikh–Doering
oxidation24 of 12 gave the crude aldehyde 7 in a considerably
high yield. Thus, the subsequent carbonyl–ene reaction
immediately proceeded in the presence of dimethylaluminum
chloride, which allowed for conversion of 7 to an inseparable
1 : 1.5 mixture of dihydrobenzopyranols 13. Despite the low
selectivity, it is noteworthy that the desired chromanol ring
was successfully constructed through this ene reaction without
the formation of any side products caused by a Friedel–Craft
type acylation. Hydrogenation of alkene 13 followed by PCC
oxidation produced the chroman-3-one derivative 6 in excel-
lent yields.
Fig. 2 X-ray crystal structure of the acid 15.
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yield by DMP oxidation. The stereoselective intramolecular ene
cyclization of 9 proved to be a significant challenge because
there are few examples of intramolecular carbonyl–ene reac-
tions using isolated ketones as the substrates for cycliza-
tion.32,33 A preliminary evaluation of the carbonyl–ene reaction
of 9 using TiCl4 led to the formation of 16; however, low yield
and low selectivity were observed. After performing reaction
optimization, we obtained the desired alcohol 16 with a high
diastereoselectivity when a solution of 9 in CH2Cl2 was treated
with SnCl4 at −78 to −40 °C for 5.5 h. At last, concurrent
debenzylation and hydrogenation followed by DMP oxidation
proceeded to afford aldehyde 3, which was identical to the
compound synthesized from the first approach. In comparison
to route A, this approach to 9 involved only 7 steps and pro-
vided the target aldehyde 3 in 14% overall yield. Thus, material
for the construction of the mibefradil library was generally pro-
duced via this second route.
Scheme 3 The second approach to aldehyde 3: (a) iPrMgBr, Et2O, 86%; Synthesis of benzimidazole fragments
(b) μW (120W), hexane, 93%; (c) 10, Cs2CO3, CH3CN, 90 °C, 52%; (d)
In order to use benzimidazoles containing various substitu-
ents for assembly of two fragments as we planned in
Scheme 1, we prepared a set of amines 4, aldehydes 21 and
Dess–Martin periodinane, CH2Cl2, 94%; (e) SnCl4, CH2Cl2, −78 °C to
−40 °C, 53% (dr = 10 : 1); (f) H2, Pd/C, MeOH, 86%; (g) Dess–Martin peri-
odinane, CH2Cl2, 81%.
Scheme 4 Synthesis of benzimidazole fragments 4, 21 and 5: (a) ClCO2iBu, Et3N, diamines 18, THF; (b) pTsOH·H2O, toluene, Deans–Stark; (c) H2,
10% Pd/C, MeOH (for 4a–d and 4f); (d) 6 N aq. HCl, 110 °C (for 4e and 4g); (e) pTsCl, DMAP, Et3N, CH2Cl2–THF (1 : 1); (f) Dess–Martin periodinane,
CH2Cl2; (g) diamines 18, conc. HCl, reflux; (h) N-hydroxyphthalimide, DIAD, PPh3, THF; (i) H2NNH2, EtOH, reflux, then 1 M HCl in Et2O. aIsolated
yields over two steps.
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O-alkylated hydroxyl amines 5 as shown in Scheme 4. N-CbZ pro- whole process provided reasonable amounts of materials for
tected amino acid 17, prepared from the treatment of γ-amino- the next steps.
butyric acid with CbzCl and 4 N NaOH,34 was transformed to
amides 19 via mixed anhydride intermediates. Condensation
of 19 with azeotrophic removal of water produced the corres-
Complete synthesis of mibefradil analogues
ponding benzimidazoles, which was subjected to Cbz depro-
tection by either hydrogenolysis or acid-catalyzed hydrolysis to
afford free amines 4. Considering a role reversal of two frag-
ments for coupling reactions (see below), we synthesized alde-
hydes 21 as alternative components by conducting selective
tosylation of readily available benzimdazoles 2035 and consecu-
tive DMP oxidation. At this time, we should point out that all
attempted direct oxidations of 20 using different reagents such
as SO3·pyr, PCC, DMP, TPAP/NMO or Swern failed without
blocking nitrogen of the benzimidazole moiety in 20. On the
other hand, O-alkylated hydroxyl amines 5 were also prepared
in three steps: the elaboration of 3-hydroxypropionitrile 22 to
hydroxyethyl benzimidazoles 23 through condensation with
phenylenediamines 18 under acidic conditions, Mitsunobu
reaction and hydrazinolysis followed by in situ salt formation.
Although the yields of several reactions were relatively low, the
The complete synthesis of mibefradil derivatives 2 is illustrated
in Scheme 5. Reductive amination of 3 with amines 4 and 25
produced the first series of analogues 2a and 2b. The second
series of compounds 2c (R2 = iPr) were obtained by subsequent
reductive amination of 2a with acetone in the presence of
acetic acid. However, conversion of 2a to tertiary amines 2d
(R2 = cyclopropyl) or 2e (R2 = CF3CH2) using reductive amin-
ation, metal-catalyzed coupling reactions or direct N-alky-
lations36,37 proved to be problematic. As indicated in the
previous section, aldehydes 21 were regarded as surrogate frag-
ments for coupling reactions. Thus, reductive aminations of 3
with cyclopropylamine and trifluoroethylamine gave secondary
amines 24, which could be transformed into the desired 2d
and 2e by second reductive amination with aldehydes 21 and
subsequent removal of the tosyl group.38 Towards the last ana-
logues of mibefradil, condensation reactions of aldehydes 3
Scheme 5 Reagents and conditions: (a) 4 or 25, NaBH3CN, AcOH, MeOH; (b) acetone, NaBH3CN, AcOH, MeOH (from 2a only); (c) R2NH2, AcOH,
NaBH3CN, MeOH; (d) 21, AcOH, NaBH3CN, MeOH; (e) TBAF, THF; (f) 5, K2CO3, MeOH. aIsolated yields over two steps.
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Table 2 CYP inhibitiona and microsomal stabilityb of the selected com-
pounds 2
with 5 in the presence of K2CO3 provided a series of oximes 2f
in moderate yields.
% CYP isozyme remaining activityc
HLMd
Channel blocking activity and in vitro stability
% Remaining
@ 30 min
In vitro screening of mibefradil analogues 2 was performed
with HEK293 cells which stably expressed T-type calcium
channel α1G and α1H subtypes using an FDSS6000 instru-
ment.39 As summarized in Table 1, 19 and 18 compounds out
of the total 22 new analogues respectively displayed higher
than 50% inhibitory activities against each T-type calcium
channel (α1G and α1H) at 10 μM, which indicated that the
overall structural modification of mibefradil 1 in this study did
not significantly reduce the T-type calcium channel blockade
as we expected. In particular, the structural change of the ter-
tiary amine moiety to other functional groups such as a sec-
ondary amine (2a), a larger tertiary amine (2b–e), or oxime (2f)
exerted a great influence on the channel blocking activities,
whereas the installation of various substituents to the benzimi-
dazole moiety in 2 did not induce any critical change of inhibi-
tory activities against T-type calcium channels.
After we confirmed the in vitro potencies of our compounds
2, we evaluated their inhibitory activities against five CYP450
isozymes including CYP2D6 and CYP3A4, two of the most
abundant CYP enzymes in human liver. As shown in Table 2,
we measured the remaining activity of the CYP enzymes after
treatment with the selected compounds 2 using CYP450
screening kits.40 In general, the blocking activities of several
compounds against the CYP enzymes were considerably
improved in comparison with those of mibefradil 1. In particu-
lar, compound 2aa containing dihydrobenzopyran and a sec-
ondary amine linker exhibited significantly increased CYP
remaining activities. Although the inhibitory values of the
selected 2c (R2 = iPr) against CYP3A4 isozyme were highly
reduced relative to that of mibefradil 1, we could not observe
any enhancement of those activities against CYP2D6. In con-
trast, the CYP inhibitory activities of the selected oximes 2f
were increased over that of 1, which might be a consequence
of the hydrolysis of oxime due to its liability to acid, but this is
Compds 1A2
2D6
2C9
3A4
2C19
2aa
2ab
2ac
2ad
2ae
2af
2ag
2ba
2ca
2cc
2da
2db
2fa
2fb
2fc
2fd
1
59.46 84.25 109.58
39.91 55.00 75.32
25.71 19.94 37.23
26.20 40.46 83.26
16.01
59.86
19.43
43.07
26.36
58.75
49.06
2.75
3.27
1.86
3.95
1.81
5.15
8.68
33.48
85.69
5.91
21.15
14.69
42.97
54.99 17.44
47.45 4.57
5.52
—
—
—
—
51.54 121.08 28.64
90.38
31.65 44.26 12.89
77.56 10.56 110.18
46.94 56.71
45.55 49.66
19.66
12.05 12.76
30.26 9.92
10.86 11.22
—
5.72
—
1.19
—
0.11
0.38
0.30
111.44
56.02
56.71
52.27
19.42
39.74
16.84
99.00
7.79
4.73
8.07
9.54
5.61
8.07
3.38
3.79
55.57
26.39
54.52
11.45
5.72
8.09
3.05
9.45
10.81
8.52
8.75
2.93
24.70
4.82 39.70 56.66
a Each data represent the mean of triplicate experiments. b Each data
represent the mean of duplicated experiments. c The remaining activity
of each isozyme was obtained at a concentration of 10 μM using
fluorogenic Vivid® CYP450 screening kits. d HLM, human liver
microsomes.
not clearly conclusive at this point. The overall result implied
that our approach to structural modification of mibefradil 1
was significantly effective in reducing its CYP inactivation.
In addition to the CYP inhibition experiments, we exam-
ined the metabolic stability of the representative compounds 2
of each series in human hepatic microsome. The result
showed that the metabolic stabilities of the tested compounds
2 were also dependent on the structural modification of the
tertiary amine moiety of 1 (Table 2). In fact, compound 2aa
turned out to be highly stable, remaining up to 75% at 30 min
after treatment of 2aa in pooled human hepatic microsomes,
while other compounds having piperidine (2ba), isopropyl
amine (2cc), cyclopropyl amine (2db), and oxime (2fc)
appeared to be rapidly decomposed. Given the comprehensive
data of 2aa with regard to calcium channel blocking activities,
CYP inhibition, and metabolic stability, we therefore con-
cluded that the structural revision of 1 into 2aa by insertion of
oxygen at the benzylic position of the tetrahydronaphthalene
ring and replacement of the tertiary amine by a secondary
amine could effectively improve the metabolic stability of
mibefradil 1 in both the CYP enzymes and the hepatic micro-
somes, maintaining the in vitro efficacy against T-type calcium
channels.
Table 1 In vitro T-type calcium channel blocking activitiesa of mibefra-
dil analogues 2
α1G
(10 μM)
α1H
(10 μM)
α1G
(10 μM)
α1H
(10 μM)
Compds
Compds
2aa
2ab
2ac
2ad
2ae
2af
2ag
2ba
2ca
2cb
2cc
2cd
71.20
66.04
65.19
60.02
62.59
59.76
59.69
68.37
64.57
63.63
61.74
63.49
66.55
47.90
60.54
55.32
56.20
58.37
66.73
70.76
59.16
54.58
64.41
59.10
2ce
2cf
2cg
2da
2db
2ea
2fa
2fb
2fc
2fd
1
67.42
53.67
64.35
46.85
47.56
11.76
53.29
59.28
58.65
60.37
79.19
65.61
62.28
69.56
44.56
44.16
38.10
55.33
52.70
54.53
52.11
80.21
Conclusion
In this article, we have established a synthesis of the chemical
library of mibefradil analogues 2 which is based on the con-
struction of the dihydrobenzopyran structure by means of the
a Ca2+ flux assay; % Inhibition value was obtained at 10 μM (see the
detail assay procedure in the Experimental section).
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diastereoselective intermolecular Reformatsky reaction and the 55.2 mmol) in anhydrous DMF (60 mL) was heated at 80 °C for
intramolecular carbonyl–ene cyclization. These protocols pro- 30 min. 2-Bromoethoxy tert-butyldimethylsilane (16.94 g,
vided efficient access to the fragment 3 at the left hand side of 70.8 mmol) was added and the reaction mixture was stirred at
2, which served as the immediate precursor for coupling reac- the same temperature for an additional 3 h. The solvent was
tions with amines or aldehydes. In particular, the second strat- removed under reduced pressure to give the crude material,
egy through the carbonyl–ene reaction led to the formation of which was dissolved in ethyl acetate (100 mL). The resulting
aldehyde 3 within only 7 steps in a highly stereoselective solution was treated with aqueous NH4Cl (300 mL) and was
manner, which has allowed for practical and convenient prepa- extracted with ethyl acetate (3 × 200 mL). The combined
ration of mibefradil analogues 2. Accordingly, the synthesis of organic layers were washed with brine, dried over anhydrous
2 has been successfully achieved by simple or dual reductive MgSO4 and concentrated in vacuo. The crude residue was puri-
amination strategies using aldehyde
substrate.
3
as
a
switchable fied by flash column chromatography on silica gel (EtOAc–
n-hexane = 1 : 20) to afford the corresponding ether (10.00 g,
A total of 22 mibefradil derivatives were synthesized and 64%) as a colorless oil; 1H-NMR (400 MHz, CDCl3) δ 10.40
evaluated for in vitro activities. Most compounds 2 were con- (s, 1H), 7.84–7.85 (m, 1H), 6.70–6.74 (m, 2H), 4.14 (t, J = 4.6 Hz,
firmed to be potent T-type calcium channel blockers compar- 2H), 4.01 (t, J = 4.6 Hz, 2H), 0.88 (s, 9H), 0.07 (s, 6H). 13C-NMR
able to mibefradil 1. Moreover, in vitro stabilities of the selected (100 MHz, CDCl3) δ 188.2, 167.6 (d, J = 254.4 Hz), 163.2 (d, J =
compounds 2 in the CYP enzymes and the human hepatic 11.0 Hz), 130.6 (d, J = 11.5 Hz), 121.9, 108.3 (d, J = 22.1 Hz),
microsomes were also significantly improved. In particular, we 100.9 (d, J = 25.5 Hz), 70.5, 61.6, 25.8, 18.29, −5.4. HRMS-CI
found that analogue 2aa, which has a dihydrobenzopyran ring (m/z): [M + H]+ calcd for C15H24FO3Si 299.1479, found 299.1479.
and a secondary amine linker, showed high % remaining activi-
To a solution of isopropyltriphenylphosphonium iodide
ties of the tested CYP enzymes, retaining the high inhibitory (19.7 g, 45.6 mmol) in THF (80 mL) was slowly added a solu-
activity against T-type calcium channels. These findings high- tion of n-BuLi (31.7 mL, a 1.6 M solution in n-hexane,
light the importance of the structural modification of 1 for redu- 50.6 mmol) at 0 °C. After the reaction was stirred for 30 min, a
cing CYP inhibition and metabolic degradation.
solution of the above aldehyde (7.56 g, 25.3 mmol) in THF
(20 mL) was added. The reaction mixture was stirred for 2 h at
room temperature and quenched with NH4Cl (150 mL). The
resulting solution was extracted with ethyl acetate (3 ×
150 mL), washed with brine, and dried over anhydrous MgSO4.
The solvent was removed under reduced pressure to give the
Experimental section
General
All reactions were carried out under dry nitrogen unless other- crude alkene, which was used for the next step without further
wise indicated. Commercially available reagents were used purification; 1H-NMR (300 MHz, CDCl3 δ 7.09–7.15 (m, 1H),
without further purification. Solvents and gases were dried 6.60–6.65 (m, 2H), 6.26 (s, 1H), 4.00 (m, 4H), 1.91 (s, 3H), 1.79
according to standard procedures. Organic solvents were evap- (s, 3H), 0.93 (s, 9H), 0.12 (s, 6H). 13C-NMR (75 MHz, CDCl3)
orated with reduced pressure using a rotary evaporator. δ 162.3 (d, J = 324.5 Hz), 157.7 (d, J = 127.0 Hz), 135.2, 131.4
Analytical thin layer chromatography (TLC) was performed (d, J = 127.0 Hz), 123.9 (d, J = 47.0 Hz), 120.3, 106.5 (d, J =
using glass plates precoated with silica gel (0.25 mm). TLC 27.4 Hz), 100.0 (d, J = 34.0 Hz), 70.1, 62.2, 26.8, 26.2, 19.7,
plates were visualized by exposure to UV light (UV), and then 18.7, −5.1. HRMS-EI (m/z): [M]+ calcd for C18H29FO2Si
visualized with a p-anisaldehyde stain followed by brief 324.1921, found 324.1926.
heating on a hot plate. Flash column chromatography was per-
To a solution of the above alkene (8.22 g, 25.3 mmol) in dis-
formed using silica gel 60 (230–400 mesh, Merck) with the tilled THF (60 mL) was added a solution of tetrabutyl-
indicated solvents. 1H and 13C spectra were recorded on ammonium fluoride (30.6 mL, 1.0 M solution in THF,
1
Bruker 300, Bruker 400 or Varian 300 NMR spectrometers. H 30.6 mmol) at 0 °C. After stirring for 2 h, the reaction mixture
NMR spectra are represented as follows: chemical shift, multi- was quenched with NH4Cl (200 mL). The solvent was partially
plicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multi- removed under reduced pressure. The resulting mixture was
1
plet), integration, and coupling constant (J) in hertz (Hz). H extracted with CH2Cl2 (3 × 150 mL). The organic layers were
NMR chemical shifts are reported relative to CDCl3 (7.26 ppm). washed with brine, dried over anhydrous MgSO4 and concen-
13C NMR was recorded relative to the central line of CDCl3 trated in vacuo. The resulting residue was purified by flash
(77.0 ppm). HPLC data were acquired from a Waters Alliance column chromatography on silica gel (EtOAc–n-hexane = 1 : 4)
System with a UV detector set to 254 and 280 nm. Samples to afford alcohol 12 (4.44 g, 83%, 2 steps) as a pale yellow oil;
were injected (10 μL) onto a Waters Sunfire 4.6 × 150 mm, 1H-NMR (400 MHz, CDCl3) δ 7.10–7.13 (m, 1H), 6.58–6.67 (m,
5.0 μM, C18 column maintained at 25.8 °C. A linear gradient 2H), 6.21 (s, 1H) 4.04 (t, J = 3.7 Hz, 2H), 3.95 (t, J = 3.7 Hz, 2H),
from 30% to 100% B (MeCN) in 20 min was followed by 2.24 (s, 1H), 1.91 (s, 3H), 1.77 (s, 3H). 13C-NMR (100 MHz,
pumping 100% B for another 10 minutes with A being H2O + CDCl3) δ 162.1 (d, J = 243.5 Hz), 156.9 (d, J = 9.3 Hz), 135.8,
0.1 M NH4OAc (or NH4HCO2). The flow rate was 1.0 mL min−1
.
131.1 (d, J = 9.3 Hz), 123.787, 119.5, 107.0 (d, J = 20.8 Hz),
2-(5-Fluoro-2-(2-methylprop-1-enyl)phenoxy)ethanol (12). A 100.2 (d, J = 25.2 Hz), 70.0, 61.3, 26.5, 19.5. HRMS-EI (m/z):
solution of aldehyde 8 (7.74 g, 55.2 mmol) and K2CO3 (7.63 g, [M]+ calcd for C12H15FO2 210.1056, found 210.1054.
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2-(5-Fluoro-2-(2-methylprop-1-enyl)phenoxy)acetaldehyde J = 21.2 Hz), 103.8 (d, J = 24.1 Hz), 71.0, 64.8, 44.2, 27.7, 21.0,
(7). To a solution of the alcohol 12 (628 mg, 2.99 mmol) in 19.4. HRMS-EI (m/z): [M]+ calcd for [C12H15FO2]+ 210.1056,
dry CH2Cl2 (15 mL) were sequentially added DMSO (4 mL), found, 210.1055; minor isomer: 1H-NMR (300 MHz, CDCl3)
iPr2NEt (8 mL) and SO3·pyridine (2.38 g, 14.9 mmol) at 0 °C. δ 6.98–7.03 (m, 1H), 6.53–6.63 (m, 2H), 4.12–4.14 (m, 3H),
The reaction mixture was allowed to stir at the same tempera- 3.93–4.02 (m, 1H), 2.43 (d, J = 7.8 Hz, 1H), 2.10 (s, 1H),
ture for 1 h. It was the quenched with NH4Cl (10 mL), acidified 1.63–1.70 (m, 1H), 0.96 (d, J = 6.6 Hz, 6H). 13C-NMR (75 MHz,
with 3 N HCl until pH 2 was reached, and finally extracted CDCl3) δ 162.5 (d, J = 242.3 Hz), 154.5 (d, J = 12.0 Hz), 132.9
with diethyl ether (3 × 45 mL) and washed with brine. The (d, J = 9.5 Hz), 118.0, 107.9 (d, J = 21.0 Hz), 103.9 (d, J =
organic layers were dried over anhydrous MgSO4 and concen- 24.1 Hz), 67.7, 65.2, 48.6, 33.1, 21.5, 20.3.
trated in vacuo to afford the crude aldehyde 7, which was used
To a solution of a mixture of the above alcohols (200 mg,
for the next step without purification; 1H-NMR (400 MHz, 0.95 mmol) in dry CH2Cl2 (50 mL) was added pyridinium
CDCl3) δ 9.83 (s, 1H), 7.13–7.16 (m, 1H), 6.66–6.70 (m, 1H), chlorochromate (615 mg, 2.85 mmol) at 0 °C. The reaction
6.43–6.46 (m, 1H), 6.26 (s, 1H), 4.52 (s, 2H), 1.91 (s, 3H), 1.77 mixture was allowed to stir at room temperature for 2 h. The
(s, 3H). 13C-NMR (75 MHz, CDCl3) δ 199.2, 162.2 (d, J = resulting solution was filtered through a pad of Celite® and
326.5 Hz), 156.2, 136.6, 131.8 (d, J = 12.0 Hz), 124.2, 119.5, silica gel and the solvent was removed under reduced pressure.
108.1 (d, J = 28.0 Hz), 100.5 (d, J = 34.1 Hz), 73.4, 26.8, 19.8. The resulting residue was purified by flash column chromato-
HRMS-EI (m/z): [M]+ calcd for C12H13FO2 208.0899, found graphy on silica gel (EtOAc–n-hexane = 1 : 10) to afford ketone
208.0898.
6 (160 mg, 81%) as an oil; 1H-NMR (300 MHz, CDCl3)
7-Fluoro-4-(prop-1-en-2-yl)chroman-3-ol (13). To a solution δ 7.00–7.02 (m, 1H), 6.74–6.80 (m, 2H), 4.58 (d, J = 18.1 Hz,
of the above aldehyde 7 (622 mg, 2.99 mmol) in dry CH2Cl2 1H), 4.27 (dd, J = 1.3, 18.1 Hz, 1H), 3.13 (d, J = 7.4 Hz, 1H),
(15 mL) cooled to −20 °C was added a solution of Me2AlCl 2.20 (m, 1H), 0.98 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H).
(2.99 mL, 1.0 M in n-hexane). The reaction mixture was stirred 13C-NMR (75 MHz, CDCl3) δ 209.4, 162.7 (d, J = 326 Hz), 155.3
at the same temperature for 30 min, quenched with NaHCO3 (d, J = 15.0 Hz), 131.0 (d, J = 13.0 Hz), 120.6 (d, J = 4.0 Hz),
(20 mL) at 0 °C and filtered through a pad of Celite®. The fil- 110.1 (d, J = 29.0 Hz), 105.6 (d, J = 32.0 Hz), 72.72, 58.0, 32.4,
trate was extracted with CH2Cl2 (3 × 30 mL). The organic layers 20.5, 20.0. HRMS-EI (m/z): [M]+ calcd for C12H13FO2 208.0899,
were washed with brine, dried over anhydrous MgSO4 and con- found 208.0899.
centrated in vacuo. The resulting residue was purified by flash
( )-(3S,4S)-Ethyl 2-(7-fluoro-3-hydroxy-4-isopropylchroman-3-
column chromatography on silica gel (EtOAc–n-hexane = 1 : 5) yl)acetate (14). In a two neck round bottom flask equipped
to afford an inseparable mixture of alcohols 13 (440 mg, 71%, with a CaCl2 tube, diethyl ether (8 mL) and ethyl iodoacetate
1
2 steps, dr = 1.5 : 1) as a pale yellow oil; major isomer: H-NMR (0.26 mL, 2.16 mmol) were added at room temperature. Et2Zn
(300 MHz, CDCl3) δ 7.01–7.06 (m, 1H), 6.55–6.63 (m, 2H), 5.22 (4.32 mL of a 1 M solution in n-hexane) was added and
(d, J = 5.0 Hz, 1H), 4.83 (d, J = 0.7 Hz, 1H), 4.03–4.28 (m, 3H), immediately the addition of a solution of ketone 7 (225 mg,
3.70 (d, J = 4.3 Hz, 1H), 1.87 (s, 3H). 13C-NMR (100 MHz, 1.08 mmol) in diethyl ether (3 mL) was started using a syringe
CDCl3) δ 162.1 (d, J = 243.0 Hz), 155.3 (d, J = 12.2 Hz), 144.4, pump over a 50 min period. Then, a new portion of Et2Zn
131.6 (d, J = 9.5 Hz), 117.2, 116.5, 108.2 (d, J = 21.4 Hz), 103.6 (4.32 mL of a 1.0 M solution in n-hexane) was added. The
(d, J = 21.5 Hz), 68.5, 63.7, 47.7, 23.0. HRMS-EI (m/z): [M]+ resulting solution was stirred for 1.5 h and quenched with
calcd for C12H13FO2 208.0899, found 208.0898; minor isomer: NH4Cl (20 mL). It was extracted with diethyl ether (3 × 20 mL).
1H-NMR (300 MHz, CDCl3) δ 6.93–6.98 (m, 1H), 6.54–6.65 (m, The organic layers were washed with brine, dried over anhy-
2H), 5.12 (d, J = 13.2 Hz, 1H), 4.79 (d, 0.7 Hz, 1H), 3.90–4.25 drous MgSO4 and concentrated in vacuo. The resulting residue
(m, 3H), 3.41 (d, J = 6.1 Hz, 1H), 1.70 (s, 3H). 13C-NMR was purified by flash column chromatography on silica gel
(100 MHz, CDCl3) δ 162.3 (d, J = 243.2 Hz), 155.0 (d, J = (EtOAc–n-hexane = 1 : 10) to afford β-hydroxy ester 14 (230 mg,
11.8 Hz), 144.5, 131.2 (d, J = 9.6 Hz), 117.6, 116.9, 108.5 (d, J = 72%); 1H-NMR (400 MHz, CDCl3) δ 6.92–6.96 (m, 1H),
21.5 Hz), 103.6 (d, J = 21.9 Hz), 67.7, 64.8, 52.3, 19.3.
7-Fluoro-4-isopropylchroman-3-one (6). To
6.51–6.61 (m, 2H), 4.33 (s, 1H), 4.17 (q, J = 5.9 Hz, 2H) 4.06 (d,
solution of J = 11.0 Hz, 1H), 3.91 (dd, J = 2.1, 11.0 Hz, 1H), 2.56 (m, 1H),
a
alkene 13 (730 mg, 3.54 mmol) in MeOH (10 mL) was added 2.55 (d, J = 16.8 Hz, 1H), 2.45 (d, J = 16.8 Hz, 1H), 2.32–2.36
Pd/C (146 mg, 10% (w/w)) at room temperature. The reaction (m, 1H),), 1.25 (t, J = 7.1 Hz, 3H) 1.13 (d, J = 6.9 Hz, 3H), 0.64
mixture was stirred under a hydrogen atmosphere (with the (d, J = 7.0 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 173.2, 162.4
aid of a hydrogen balloon) for 3 h. It was filtered through a (d, J = 242.3 Hz), 153.3 (d, J = 11.8 Hz), 131.8 (d, J = 9.4 Hz),
pad of Celite® and the solvent was removed under reduced 118.2, 107.4 (d, J = 21.5 Hz), 103.3 (d, J = 24.3 Hz), 69.0, 68.1,
pressure to afford isopropylchroman-3-ol (663 mg, 90%) as a 61.1, 50.8, 41.8, 28.3, 25.0, 21.1, 14.1. HRMS-CI (m/z): [M + H]+
pale yellow liquid; major isomer: 1H-NMR (400 MHz, CDCl3) calcd for C16H22FO4 297.1502, found 297.1510.
δ 7.13–7.16 (m, 1H), 6.56–6.68 (m, 2H), 4.31–4.32 (m, 1H), 4.22
( )-(3S,4S)-2-(7-Fluoro-3-hydroxy-4-isopropylchroman-3-yl)-
(dd, J = 4.2, 11.2 Hz, 1H), 4.09 (dt, J = 1.0, 11.2 Hz, 1H), 2.82 acetic acid (15). To a solution of β-hydroxy ester 14 (230 mg,
(m, 1H), 2.41–2.46 (m, 1H), 1.16 (d, J = 6.8 Hz, 3H), 1.05 (d, J = 0.78 mmol) in MeOH (3 mL) and H2O (1 mL) was added
7.0 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.0 (d, J = 242.7 LiOH·H2O (98 mg) at room temperature. The reaction mixture
Hz), 155.1 (d, J = 11.7 Hz), 130.0 (d, J = 9.5 Hz), 118.3, 108.1 (d, was stirred at the same temperature for 15 h and quenched
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with 3 N HCl until pH 2 was reached. It was extracted with isobutyl alcohol (2.22 g, 86%) as a pale brown oil; 1H-NMR
ethyl acetate (3 × 30 mL), washed with brine, and dried over (400 MHz, CDCl3) δ 8.45 (s, 1H), 6.82 (dd, J = 6.5, 8.3 Hz, 1H),
anhydrous MgSO4. The solvent was completely removed under 6.54 (td, J = 1.8, 5.4 Hz, 1H), 6.50 (dd, J = 2.8, 8.3 Hz, 1H), 4.48
reduced pressure to give carboxylic acid 15 (208 mg, quant.) as (d, J = 7.0 Hz, 1H), 3.08 (s, 1H), 2.10–1.98 (m, 1H), 1.02 (d, J =
a solid; 1H-NMR (400 MHz, CDCl3) δ 6.96–6.98 (m, 1H), 6.7 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz,
6.53–6.63 (m, 2H), 4.08 (d, J = 11.1 Hz, 1H), 3.97 (dd, J = 2.1, CDCl3) δ 162.9 (d, 1J = 243 Hz), 157.0 (d, 3J = 12 Hz), 129.1
3
4
2
11.1 Hz, 1H), 2.65 (d, J = 17.0 Hz, 1H), 2.61–2.62 (m, 1H), 2.57 (d, J = 10 Hz), 122.0 (d, J = 3 Hz), 106.2 (d, J = 21 Hz), 104.4
2
(d, J = 17.0 Hz, 1H), 1.14 (d, J = 7.0 Hz, 3H), 0.66 (d, J = 7.0 Hz, (d, J = 24 Hz), 81.5, 34.5, 19.1, 18.1. LRMS-EI (m/z): [M]+ calcd
1H). 13C-NMR (100 MHz, CDCl3) δ 178.1, 162.5 (d, J = 243.0 for C10H13FO2 184.09, found 184.
Hz), 153.2 (d, J = 11.2 Hz), 131.8 (d, J = 9.5 Hz), 118.0, 107.7 (d,
In a sealed tube, a solution of the above isobutyl alcohol
J = 21.5 Hz), 103.5 (d, J = 24.2 Hz), 69.1, 67.9, 50.7, 41.7, 28.4, (952 mg, 5.17 mmol) in n-hexane (2 mL) was stirred using a
25.0, 21.1. HRMS-CI (m/z): [M + H]+ calcd for C14H18FO4 microwave (170 °C, 120 W) for 15 min. The reaction mixture
269.1189, found 269.1187.
was treated with water (30 mL) and extracted with CH2Cl2 (3 ×
( )-(3S,4S)-2-(7-Fluoro-3-hydroxy-4-isopropylchroman-3-yl)- 30 mL). The organic layers were washed with brine, dried over
acetaldehyde (3). To a suspension of the lithium aluminium anhydrous MgSO4 and concentrated in vacuo. The resulting
hydride (184 mg, 4.92 mmol) in dry THF (16.4 mL) was added residue was purified by flash column chromatography on silica
β-hydroxy ester 14 (487 mg, 1.64 mmol) at 0 °C. The reaction gel (EtOAc–n-hexane = 1 : 6) to afford alkene 11 (800 mg, 93%)
1
mixture was allowed to stir at room temperature for 2 h. as a red brown oil; H-NMR (400 MHz, CDCl3) δ 6.97 (dd, J =
The reaction mixture was quenched with 30% NaOH (30 mL) 7.0, 7.8 Hz, 1H), 6.65–6.57 (m, 2H), 6.04 (s, 1H), 5.27 (d, J =
for 20 min. The resulting solution was extracted with ethyl 1.4 Hz, 1H), 1.93 (d, J = 1.2 Hz, 3H), 1.66 (d, J = 0.9 Hz, 3H).
1
acetate (3 × 30 mL), washed with brine, and dried over 13C-NMR (100 MHz, CDCl3) δ 165.5 (d, J = 240 Hz), 153.9 (d,
anhydrous MgSO4. The solvent was completely removed under 3J = 12 Hz), 114.4, 130.6 (d, 3J = 10 Hz), 120.6 (d, 4J = 3 Hz),
reduced pressure. The resulting residue was purified by flash 117.8, 107.1 (d, 2J = 21 Hz), 102.4 (d, 2J = 25 Hz), 25.7, 19.3.
column chromatography on silica gel (EtOAc–n-hexane = 1 : 4) LRMS-EI (m/z): [M]+calcd for C10H11FO 166.08, found 166.
to afford the corresponding diol (400 mg, 96%) as a colorless
oil. The spectroscopic data is identical to that of 16a (see butan-2-one (9). To
below). 1.32 mmol) in anhydrous CH3CN (13 mL) were added epoxide
4-(Benzyloxy)-1-(5-fluoro-2-(2-methylprop-1-en-1-yl)phenoxy)-
a
solution of alkene 11 (220 mg,
Dess–Martin periodinane (226 mg, 533 μmol) was added to 10 (472 mg, 2.65 mmol) and Cs2CO3 (647 mg, 1.99 mmol) at
a solution of the above diol (90.3 mg, 355 μmol) in CH2Cl2 room temperature. The reaction mixture was allowed to warm
(4 mL). After stirring for 2 h, the reaction mixture was to reflux for 21 h. The reaction solution was extracted with
quenched with Na2S2O3 (10 mL) and sat. NaHCO3 (10 mL). CH2Cl2 and washed with brine. The resulting residue was puri-
The resulting mixture was extracted with Et2O (3 × 15 mL). The fied by flash column chromatography on silica gel (EtOAc–
organic layers were washed with brine, dried over anhydrous n-hexane = 1 : 4) to afford the corresponding alcohol (0.24 g,
MgSO4 and concentrated in vacuo. The resulting residue was 52%) as a brown oil; 1H-NMR (400 MHz, CDCl3) δ 7.38–7.26
purified by flash column chromatography on silica gel (EtOAc– (m, 5H), 7.11 (t, J = 7.6 Hz, 1H), 6.64 (td, J = 2.3, 8.3 Hz, 1H),
n-hexane = 1 : 4) to afford aldehyde 3 (72.4 mg, 81%) as a pale 6.58 (dd, J = 2.2, 10.8 Hz, 1H), 6.19 (s, 1H), 4.55 (s, 1H), 4.22 (s,
1
brown oil; H-NMR (300 MHz, CDCl3)δ 9.77 (s, 1H), 6.95 (dd, 1H), 3.94–3.87 (m, 2H), 3.78–3.67 (m, 2H), 2.96 (s, 1H)
J = 6.4, 8.4 Hz, 1H), 6.60 (td, J = 2.5, 8.3 Hz, 1H), 6.53 (dd, J = 1.97–1.88 (m, 5H), 1.76 (s, 1H).13C-NMR (100 MHz, CDCl3)
1
2.6, 10.5 Hz, 1H), 4.02 (d, J = 11.0 Hz, 1H), 3.93 (dd, J = 2.0, δ 162.1 (d, J = 243 Hz), 156.9 (d, 3J = 9 Hz), 138.0, 135.6, 131.0
3
4
11.0 Hz, 2H), 3.61 (s, 1H), 2.72 (dd, J = 18.7, 39.9 Hz, 2H), (d, J = 9 Hz), 128.5, 127.8, 127.7, 123.7 (d, J = 4 Hz), 119.5,
2.60–2.58 (m, 1H), 2.35–2.24 (m, 1H), 1.12 (d, J = 7.0 Hz, 3H), 106.8 (d, 2J = 20 Hz), 100.2 (d, 2J = 26 Hz), 73.3, 72.5, 68.9, 67.8,
0.62 (d, J = 7.0 Hz, 3H).13C-NMR (100 MHz, CDCl3) δ 203.5, 33.1, 26.5, 19.5. HRMS-ESI (m/z): [M + H]+ calcd for C21H26FO3
162.4 (d, 1J = 243 Hz), 153.3 (d, 3J = 12 Hz), 131.8 (d, 3J = 9 Hz), 345.1861, found 345.1861.
4
2
2
118.2 (d, J = 3 Hz), 107.6 (d, J = 21 Hz), 103.4 (d, J = 24 Hz),
70.1, 68.1, 50.8, 28.1, 25.1, 21.1. HRMS-ESI (m/z): [M + H]+ a solution of the above alcohol (600 mg, 1.74 mmol) in CH2Cl2
calcd for C14H18FO3 253.1235, found 253.1201. (18 mL). After stirring for 1 h, the reaction mixture was
Dess–Martin periodinane (1.11 g, 2.62 mmol) was added to
5-Fluoro-2-(2-methylprop-1-en-1-yl)phenol (11). To a solu- quenched with Na2S2O3 (20 mL) and sat. NaHCO3 (20 mL).
tion of aldehyde 8 (1.97 g, 14.0 mmol) in Et2O (12 mL) cooled The resulting mixture was extracted with Et2O (3 × 60 mL). The
to 0 °C was slowly added isopropylmagnesium bromide organic layers were washed with brine, dried over anhydrous
(56 mL, 1.0 M in THF) for 30 min. The reaction was monitored MgSO4 and concentrated in vacuo. The resulting residue was
by TLC, quenched with saturated aqueous NH4Cl (50 mL) after purified by flash column chromatography on silica gel (EtOAc–
completion of the reaction, and extracted with Et2O (3 × n-hexane = 1 : 10) to afford the ketone 9 (565 mg, 95%) as a col-
75 mL). The organic extracts were washed with water and orless oil; 1H-NMR (400 MHz, CDCl3) δ 7.37–7.27 (m, 5H), 7.16
brine, dried over MgSO4, and concentrated under reduced (dd, J = 7.2, 8.0 Hz, 1H), 6.68 (td, J = 2.4, 8.3 Hz, 1H), 6.45 (dd,
pressure. The resulting residue was purified by flash column J = 2.4, 10.6 Hz, 1H), 6.30 (s, 1H), 4.59 (s, 2H), 4.54 (s, 2H),
chromatography on silica gel (EtOAc–n-hexane = 1 : 4) to afford 3.81 (t, J = 6.1 Hz, 2H), 2.85 (t, J = 6.1 Hz, 2H), 1.94 (d, J =
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1.1 Hz, 3H), 1.80 (d, J = 1.0 Hz, 3H). 13C-NMR (100 MHz, 2J = 24 Hz), 71.2, 68.0, 59.6, 50.8, 38.5, 27.8, 25.4, 21.1.
CDCl3) δ 205.2, 162.0 (d, 1J = 244 Hz), 156.1 (d, 3J = 9 Hz), HRMS-ESI (m/z): [M + H]+ calcd for C14H20FO3 255.1391, found
3
137.9, 135.9, 131.3 (d, J = 9 Hz), 128.5, 127.8, 127.7, 123.8 (d, 255.1379.
4J = 3 Hz), 119.5, 107.4 (d, 2J = 21 Hz), 100.4 (d, 2J = 25 Hz),
( )-(3S,4S)-3-(2-((3-(1H-Benzo[d]imidazol-2-yl)propyl)amino)-
73.6, 73.3, 64.9, 39.5, 26.5, 19.5. LRMS-EI (m/z): [M]+ calcd for ethyl)-7-fluoro-4-isopropylchroman-3-ol (2aa). Acetic acid was
C21H23FO3 342.16, found 342. added to a solution of amine 4a (98.2 mg, 561 μmol) in anhy-
( )-(3S,4S)-3-(2-(Benzyloxy)ethyl)-7-fluoro-4-(prop-1-en-2-yl)- drous MeOH (4.5 mL) until pH 6 was reached. To the resulting
chroman-3-ol (16). To a solution of ketone 9 (32.4 mg, solution was added sodium cyanoborohydride (26.4 mg,
94.6 μmol) in dry CH2Cl2 (1 mL) was added SnCl4 (27.1 mg, 420 μmol) and aldehyde 3 (72.4 mg, 280 μmol). The reaction
104 μmol) at −78 °C. The reaction mixture was allowed to stir mixture was stirred at room temperature for 18 h. The reaction
at the same temperature for 2.5 h. The mixture was allowed to was monitored by TLC, quenched with saturated aqueous
warm up to −40 °C over 3 h. After completion of the reaction NaHCO3 (10 mL) after completion of the reaction, and
(monitored by TLC), it was quenched with saturated aqueous extracted with CH2Cl2 (3 × 10 mL). The organic layers were
NaHCO3 (10 mL), extracted with CH2Cl2 (3 × 10 mL) and washed with brine, dried over anhydrous MgSO4 and concen-
washed with brine. The organic layers were dried over anhy- trated in vacuo. The resulting residue was purified by flash
drous MgSO4 and concentrated in vacuo. The resulting residue column chromatography on silica gel (CH2Cl2–MeOH–H2O–
was purified by flash column chromatography on silica gel NH4OH = 80 : 20 : 1 : 1) to afford amine 2aa (104 mg, 91%) as a
(EtOAc–n-hexane = 1 : 4) to afford alcohol 16 (17.1 mg, 53%) as brown oil; 1H-NMR (400 MHz, CDCl3) δ 7.48–7.46 (m, 2H),
a colorless oil; major isomer: 1H-NMR (400 MHz, CDCl3) 7.19–7.16 (m, 2H), 6.91 (dd, J = 6.6, 8.4 Hz, 1H), 6.55 (td, J =
δ 7.39–7.26 (m, 5H), 6.96–6.92 (m, 1H), 6.60 (td, J = 2.6, 8.4 Hz, 2.6, 8.3 Hz, 1H), 6.47 (dd, J = 2.5, 10.2 Hz, 1H), 4.06 (d, J =
1H), 6.55 (dd, J = 2.6, 8.4 Hz, 1H), 5.13 (t, J = 1.6 Hz, 1H), 4.66 11.0 Hz, 1H), 3.90 (dd, J = 1.8, 11.0 Hz, 1H), 2.94–2.88 (m, 4H),
(t, J = 0.8 Hz, 1H), 4.54 (d, J = 2.2 Hz, 2H), 4.11 (d, J = 11.0 Hz, 2.71–2.58 (m, 2H), 2.53 (s, 1H), 2.37–2.29 (m, 1H), 1.96 (m, J =
1H), 3.90 (dd, J = 1.4, 11.0 Hz, 1H), 3.83–3.72 (m, 2H), 3.61 (s, 6.3 Hz, 2H), 1.69–1.54 (m, 2H), 1.13 (d, J = 7.0 Hz, 3H), 0.61 (d,
1H), 3.43 (s, 1H), 2.09–2.02 (m, 1H), 1.84–1.77 (m, 4H). J = 6.9 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 162.3 (d, 1J =
1
13C-NMR (100 MHz, CDCl3) δ 162.1 (d, J = 243 Hz), 154.3 (d, 242 Hz), 154.4, 153.5 (d, 3J = 12 Hz), 138.2, 131.7 (d, 3J = 10 Hz),
4
2
3J = 12 Hz), 146.4, 137.4, 131.5 (d, 3J = 10 Hz), 128.6, 128.0, 122.4, 118.9 (d, J = 3 Hz), 114.6, 107.1 (d, J = 21 Hz), 103.1 (d,
127.9, 119.11 (d, J = 3 Hz), 117.4, 108.3 (d, J = 21 Hz), 103.4 2J = 2.5 Hz), 71.1, 67.8, 51.6, 47.7, 44.9, 34.8, 27.8, 27.1, 26.5,
(d, 2J = 25 Hz), 73.7, 69.7, 69.0, 66.8, 53.2, 36.3, 23.4. 25.5, 21.0. HPLC: 99.5%, RT 12.02 min. HRMS-ESI (m/z): [M +
HRMS-ESI (m/z): [M + H]+ calcd for C21H24FO3 343.1704, found H]+ calcd for C24H31FN3O2 412.2395, found 412.2398.
4
2
343.1705; minor isomer: 1H-NMR (400 MHz, CDCl3) δ 7.38–7.28
( )-(3S,4S)-3-(2-(3-((1H-Benzo[d]imidazol-2-yl)methyl)piperi-
(m, 5H), 6.98 (dd, J = 6.8, 8.2 Hz, 1H), 6.62–6.55 (m, 2H), 5.02 din-1-yl)ethyl)-7-fluoro-4-isopropylchroman-3-ol (2ba). By fol-
(t, J = 1.5 Hz, 1H), 4.81 (s, 1H), 4.5 (s, 2H), 4.03 (d, J = 0.6 Hz, lowing the same procedure as that used for the synthesis of
2H), 3.86–3.74 (m, 2H), 3.72 (s, 1H), 3.49 (s, 1H), 2.00–1.93 2aa, the reaction of amine 25 (56 mg, 261 μmol), sodium
(m, 1H), 1.76–1.70 (m, 1H), 1.67 (d, J = 0.5 Hz, 3H). 13C-NMR cyanoborohydride (16 mg, 261 μmol), and aldehyde 3 (44 mg,
1
3
(100 MHz, CDCl3)δ 162.1 (d, J = 243 Hz), 154.3 (d, J = 12 Hz), 174 μmol) in MeOH (2 mL) gave an inseparable 1 : 1 mixture of
144.5, 137.4, 131.6 (d, 3J = 10 Hz), 128.6, 128.0, 127.9, 118.5 amines 2ba (37 mg, 47%) after purification by column
4
2
2
(d, J = 3 Hz), 117.2, 108.3 (d, J = 21 Hz), 103.4 (d, J = 25 Hz), chromatography on silica gel (CH2Cl2–MeOH = 10 : 1) as an oil;
73.6, 70.3, 69.8, 66.5, 54.6, 33.5, 22.3. LRMS-EI (m/z): [M]+ calcd 1H-NMR (400 MHz, CDCl3) δ 7.57 (m, 2H + 2H′), 7.26–7.19 (m,
for C21H23FO3 342.16, found 342.
2H + 2H′), 6.96 (dd, J = 6.8, 8.4 Hz, 1H), 6.94 (dd, J = 6.8,
( )-(3S,4S)-7-Fluoro-3-(2-hydroxyethyl)-4-isopropylchroman- 8.4 Hz, 1H′), 6.57 (td, J = 2.4, 8.4 Hz, 1H), 6.55 (td, J = 2.4,
3-ol (16a). To a solution of the alkene 16 (17.1 mg, 50.0 μmol, 8.4 Hz, 1H′), 6.51–6.47 (m, 1H + 1H′) 4.16 (d, J = 10.7 Hz, 1H),
only major) in MeOH (1 mL) was added Pd/C (5 mg, 10% 4.10 (d, J = 11.2 Hz, 1H′), 3.94–3.86 (m, 1H + 1H′), 3.05–2.73
(w/w)) at room temperature. The reaction mixture was stirred (m, 3H + 3H′), 2.62–2.21 (m, 8H + 8H′) 1.82–1.40 (m, 4H +
under a hydrogen atmosphere (with the aid of a hydrogen 4H′), 1.25 (d, J = 6.4 Hz, 3H), 1.15 (d, J = 6.8 Hz, 3H′), 0.69 (d,
balloon) for 7 h. It was filtered through a pad of Celite® and J = 7.2 Hz, 3H), 0.67 (d, J = 8.0 Hz, 3H′). 13C-NMR (100 MHz,
the solvent was removed under reduce pressure. The resulting CDCl3) δ 162.4 (d, 1J = 242 Hz, C), 162.3 (d, 1J = 242 Hz, C′),
3
3
3
residue was purified by flash column chromatography on silica 153.6 (d, J = 11 Hz, C), 153.5 (d, J = 11 Hz, C′), 131.8 (d, J =
gel (EtOAc–n-hexane = 1 : 2) to afford the alcohol 16a (10.9 mg, 9 Hz, C), 131.7 (d, 3J = 10 Hz, C′), 122.2 (d, 4J = 2 Hz, C), 119.0 (d,
86%) as a colorless oil; H-NMR (300 MHz, CDCl3) δ 6.94 (dd, 4J = 2 Hz, C′), 118.8 (C + C′), 114.5 (C + C′), 107.1 (d, 2J = 22 Hz,
1
2
2
J = 8.9, 11.1 Hz, 1H), 6.55 (td, J = 3.4, 11.1 Hz, 1H), 6.49 (dd, J = C), 107.0 (d, J = 22 Hz, C′), 103.1 (d, J = 24 Hz, C), 103.1 (d,
3.3, 13.6 Hz, 1H), 4.07 (d, J = 11.0 Hz, 1H), 3.97 (dd, J = 2.4, 2J = 25 Hz, C′), 71.6 (C + C′), 68.7 (C + C′), 67.2 (C + C′), 54.8
8.2 Hz, 1H), 4.03–3.95 (m, 2H), 2.59 (s, 1H), 2.38–2.27 (m, 1H), (C), 54.7 (C′), 53.4 (C + C′), 52.4 (C + C′), 51.0 (C + C′), 35.5 (C),
1.84–1.68 (m, 2H), 1.15 (d, J = 7.0 Hz, 3H), 1.14 (d, J = 7.0 Hz, 35.3 (C′), 32.1 (C), 31.7 (C′), 29.8 (C), 29.4 (C′), 28.1 (C), 27.8
3H), 0.64 (d, J = 7.0 Hz, 3H). 13C-NMR (100 MHz, CDCl3) (C′), 25.7 (C), 25.5 (C′), 21.2 (C), 21.1 (C′). HPLC: 97.6%, RT
δ 162.3 (d, 1J = 242 Hz), 153.6 (d, 3J = 12 Hz), 131.6 (d, 8.67 min. HRMS-ESI (m/z): [M + H]+ calcd for C27H35FN3O2
4
2
3J = 10 Hz), 119.0 (d, J = 3 Hz), 107.1 (d, J = 21 Hz), 103.2 (d, 452.2708, found 452.2704.
5678 | Org. Biomol. Chem., 2014, 12, 5669–5681
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( )-(3S,4S)-3-(2-((3-(1H-Benzo[d]imidazol-2-yl)propyl)(isopro-
(3 × 10 mL). The organic layers were washed with brine, dried
pyl)amino)ethyl)-7-fluoro-4-isopropylchroman-3-ol (2ca). To a over anhydrous MgSO4 and concentrated in vacuo. The result-
solution of amine 2aa (33.8 mg, 82.1 μmol) in anhydrous ing residue was purified by flash column chromatography on
MeOH (1 mL) were added acetic acid (9.40 μL, 164 μmol), silica gel (EtOAc–n-hexane = 1 : 1) to afford tertiary amine
1
sodium cyanoborohydride (10.3 mg, 164 μmol) and acetone (33.8 mg, 66%) as a colorless oil; H-NMR (400 MHz, CDCl3) δ
(24.1 μL, 329 μmol). The reaction mixture was stirred for 19.5 h 8.04–8.02 (m, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 6.4 Hz,
at room temperature. The reaction was monitored by TLC, 1H), 7.37–7.30 (m, 2H), 7.29–7.26 (m, 2H), 6.92 (dd, J = 6.8, 8.4
quenched with saturated aqueous NaHCO3 (10 mL) after com- Hz, 1H), 6.75 (s, 1H), 6.55 (td, J = 2.4, 8.4 Hz, 1H), 6.49 (dd, J =
pletion of the reaction, and extracted with CH2Cl2 (3 × 15 mL). 2.4, 10.4 Hz, 1H), 4.00 (d, J = 11.2 Hz, 1H), 3.90 (dd, J = 1.8,
The organic layers were washed with brine, dried over anhy- 11.0 Hz, 1H), 3.15 (t, J = 7.4 Hz, 1H), 2.98–2.84 (m, 4H), 2.52 (s,
drous MgSO4 and concentrated under reduced pressure. The 1H), 2.38 (s, 3H), 2.35–2.21 (m, 3H), 1.79 (s, 1H), 1.64 (s, 2H),
resulting residue was purified by flash column chromato- 1.11 (d, J = 6.8 Hz, 3H), 0.59 (d, J = 7.2 Hz, 7H). 13C-NMR
1
3
graphy on silica gel (CH2Cl2–MeOH = 10 : 1) to afford amine (100 MHz, CDCl3) δ 162.3 (d, J = 242 Hz), 154.4, 153.7 (d, J =
2ca (17.3 mg, 46%) as a colorless oil; 1H-NMR (400 MHz, 12 Hz), 146.0, 141.9, 135.5, 133.2, 131.7 (d, J = 10 Hz), 130.3,
3
4
CDCl3) δ 7.52–7.48 (m, 2H), 7.23–7.19 (m, 2H), 6.92 (dd, J = 126.8, 124.8, 124.7, 119.8, 119.3 (d, J = 3 Hz), 113.6, 106.7 (d,
6.6, 8.6 Hz, 1H), 6.55 (td, J = 2.5, 8.3 Hz, 1H), 6.48 (dd, J = 2.4, 2J = 21 Hz), 103.0 (d, J = 24 Hz), 70.6, 68.4, 54.5, 51.8, 51.7,
2
10.4 Hz, 1H), 4.11 (d, J = 10.8 Hz, 1H), 3.93 (d, J = 2.2, 11.0 Hz, 37.4, 32.2, 28.0, 27.7, 25.5, 23.7, 25.5, 23.7, 21.7, 21.0, 6.9, 6.2.
1H), 3.22 (m, J = 6.8 Hz, 1H), 3.03–2.90 (m, 2H), 2.77 (t, J =
6.0 Hz, 2H), 2.63–2.58 (m, 3H), 2.45–2.38 (m, 1H), 2.10–2.04 distilled THF (0.6 mL) was added a solution of tetrabutyl-
solution in THF,
To a solution of the above product (18.2 mg, 30.0 μmol) in
(m, 2H), 1.15 (d, J = 6.8 Hz, 3H), 1.08 (d, J = 6.4 Hz, 6H), 0.64 ammonium fluoride (300 μL, 1.0
M
(d, J = 6.8 Hz, 3H). 13C-NMR (100 MHz, CDCl3)δ 162.4 (d, J = 30.6 mmol) at room temperature. The reaction mixture was
242 Hz), 154.5, 153.6 (d, 3J = 11 Hz), 138.3, 131.6 (d, 3J = 9 Hz), allowed to warm to reflux for 4 h. The resulting mixture was
122.3, 118.7 (d, 4J = 3 Hz), 114.6, 107.0 (d, 2J = 22 Hz), 103.1 (d, extracted with Et2O. The organic layers were washed with
2J = 24 Hz), 71.3, 68.4, 51.4, 49.5, 48.6, 45.5, 32.2, 27.7, 26.5, brine, dried over anhydrous MgSO4 and concentrated in vacuo.
28.8, 25.5, 20.9, 17.3, 16.6. HPLC: 98.7%, RT 15.47 min. The resulting residue was purified by flash column chromato-
1
HRMS-ESI (m/z): [M + H]+ calcd for C27H37FN3O2 454.2864, graphy on silica gel (CH2Cl2–MeOH = 10 : 1) to afford amine
1
found 454.2867.
2da (7.40 mg, 55%) as a pale colorless oil; H-NMR (400 MHz,
( )-(3S,4S)-3-(2-(Cyclopropylamino)ethyl)-7-fluoro-4-isopropyl- CDCl3) δ 7.57–7.55 (m, 2H), 7.26–7.21 (m, 2H), 6.93 (dd, J =
chroman-3-ol (24a). To a solution of the aldehyde 3 (31.8 mg, 6.6, 8.5 Hz, 1H), 6.56 (td, J = 2.6, 8.3 Hz, 1H), 6.49 (dd, J = 2.6,
126 μmol) in dry MeOH (1.5 mL) were sequentially added 10.2 Hz, 1H), 4.10 (d, J = 10.9 Hz, 1H), 3.93 (dd, J = 2.1, 10.8
cyclopropylamine (14.4 mg, 252 μmol) and sodium cyanoboro- Hz, 1H), 2.99–2.87 (m, 4H), 2.70–2.66 (m, 2H), 2.57 (s, 1H),
hydride (11.9 mg, 189 μmol) at room temperature. The reac- 2.42–2.34 (m, 1H), 2.89–2.18 (m, 2H), 1.78–1.72 (m, 1H), 1.62
tion was allowed to stir for 18 h at the same temperature. The (t, J = 5.9 Hz, 2H), 1.18 (d, J = 7.0 Hz, 3H), 0.70–0.59 (m, 7H).
reaction mixture was quenched with NaHCO3 (10 mL), 13C-NMR (75 MHz, CDCl3) δ 162.4 (d, 1J = 241 Hz), 154.4, 153.5
extracted with CH2Cl2 (3 × 10 mL) and washed with brine. The (d, 3J = 12 Hz), 138.6, 131.6 (d, 3J = 9.8 Hz), 122.3, 118.8 (d, 4J =
2
2
organic layers were dried over anhydrous MgSO4 and concen- 3 Hz), 114.7, 107.0 (d, J = 22 Hz), 103.1 (d, J = 24 Hz), 75.6,
trated in vacuo. The resulting residue was purified by flash 68.3, 54.3, 53.0, 51.4, 37.9, 32.2, 27.8, 26.6, 25.6, 25.2, 21.0, 6.7,
column chromatography on silica gel (CH2Cl2–MeOH = 10 : 1) 6.2. HPLC: 96.1%, RT 19.06 min. HRMS-ESI (m/z): [M + H]+
1
to afford amine 24a (24.8 mg, 67%) as a colorless oil; H-NMR calcd for C27H35FN3O2 452.2708, found 452.2710.
(400 MHz, CDCl3) δ 6.92 (dd, J = 6.7, 8.4 Hz, 1H), 6.54 (td, J =
( )-(3S,4S)-3-(2-((3-(1H-Benzo[d]imidazol-2-yl)propyl)(2,2,2-tri-
2.6, 8.4 3.03–2.96 (m, 2H), 2.49 (m, 1H), 2.33–2.25 (m, 1H), fluoroethyl)amino)ethyl)-7-fluoro-4-isopropylchroman-3-ol (2ea).
2.12–2.07 (m, 1H), 1.64–1.58 (m, 1H), 1.53–1.47 (m, 1H), 1.13 By following the same procedure as that used for the synthesis
(d, J = 6.9 Hz, 3H), 0.60 (d, J = 7.0 Hz, 3H), 0.52–0.40 (m, 4H). of 2da, the reaction of amine 24b (38.8 mg, 116 μmol), acetic
1
13C-NMR (100 MHz, CDCl3) δ 162.2 (d, J = 242 Hz), 153.7 (d, acid (13.3 μL, 231 μmol), sodium cyanoborohydride (10.9 mg,
3
4
3J = 11 Hz), 131.7 (d, J = 10 Hz), 119.3 (d, J = 3 Hz), 106.8 (d, 174 μmol) and aldehyde 21a (44.8 mg, 136 μmol) in MeOH
2J = 22 Hz), 102.5 (d, J = 24 Hz), 70.6, 68.1, 51.8, 45.4, 35.6, (1 mL) gave a tertiary amine intermediate (57.2 mg, 76%) after
2
30.6, 27.8, 25.5, 21.1, 6.3, 6.1.
purification by column chromatography on silica gel (CH2Cl2–
1
( )-(3S,4S)-3-(2-((3-(1H-Benzo[d]imidazol-2-yl)propyl)(cyclo- n-hexane = 1 : 2) as a colorless oil; H-NMR (400 MHz, CDCl3)
propyl)amino)ethyl)-7-fluoro-4-isopropylchroman-3-ol (2da). To δ 8.05–8.03 (m, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.66–7.64 (m, 1H),
a solution of amine 24a (24.8 mg, 85.0 μmol) in anhydrous 7.38–7.26 (m, 4H), 6.93 (dd, J = 6.7, 8.4 Hz, 1H), 6.55 (td, J =
MeOH (1 mL) were added acetic acid (9.68 μL, 169 μmol), 2.6, 8.3 Hz, 1H), 6.49 (dd, J = 2.5, 10.2 Hz, 1H), 4.04 (d, J = 11.1
sodium cyanoborohydride (10.6 mg, 169 μmol) and aldehyde Hz, 1H), 3.91 (dd, J = 1.9, 11.1 Hz, 1H), 3.20–3.11 (m, 1H),
21a (38.4 mg, 117 μmol). The reaction was stirred for 17.5 h at 2.94–2.89 (m, 2H), 2.88–2.83 (m, 2H), 2.55 (s, 1H), 2.38–2.32 (m,
room temperature. The reaction mixture was monitored by 4H), 2.18–2.11 (m, 2H), 1.71–1.56 (m, 2H), 1.10 (d, J = 7.0 Hz,
TLC, quenched with saturated aqueous NaHCO3 (10 mL) after 3H), 0.60 (d, J = 6.9 Hz, 3H).13C-NMR (100 MHz, CDCl3) δ 162.3
1
3
completion of the reaction, and extracted with CH2Cl2 (d, J = 242 Hz), 153.9, 153.6 (d, J = 12 Hz), 146.1, 141.8, 135.4,
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3
1
133.2, 131.7 (d, J = 9 Hz), 130.3, 126.8, 125.4 (d, J = 279 Hz), fluo3/AM and 0.001% Pluronic F-127 in a Hepes-buffered solu-
4
2
124.9, 124.7, 119.8, 119.1 (d, J = 3 Hz), 113.6, 106.9 (d, J = 21 tion composed of (in mM): 115 NaCl, 5.4 KCl, 0.8 MgCl2, 1.8
Hz), 103.1 (d, J = 24 Hz), 70.5, 68.3, 54.7 (q, J = 30 Hz), 53.3, CaCl2, 20 Hepes, and 13.8 glucose (pH 7.4). During the fluo-
51.4, 51.3, 33.2, 27.7, 27.2, 25.3, 23.8, 21.6, 21.0.
rescence-based FDSS6000 assay, a1G T-type Ca2+ channels were
2
2
The reaction of the above tertiary amine (57.2 mg, activated using a high concentration of KCl (70 mM) in 10 mM
88.3 μmol) and tetrabutylammonium fluoride (883 μmol, CaCl2 contained Hepes-buffered solution and the increase in
1.0 M solution in THF) in THF (1 mL) gave amine 2ea [Ca2+]i by KCl-induced depolarization was detected. During the
(16.0 mg, 57%) after purification by column chromatography whole procedure, cells were washed using a BIO-TEK 96-well
on silica gel (33.0 mg, 76%) as a yellow oil; 1H-NMR (400 MHz, washer. All data were collected and analyzed using FDSS6000
CDCl3) δ 7.56 (s, 2H), 7.26–7.22 (m, 2H), 6.93 (dd, J = 6.6, and related software (Hamamatsu, Japan).
8.4 Hz, 1H), 6.56 (td, J = 2.6, 8.3 Hz, 1H), 6.49 (dd, J = 2.6,
CYP inhibition assay
10.2 Hz, 1H), 4.12 (d, J = 11.0 Hz, 1H), 3.90 (dd, J = 2.1, 11.0
Hz, 1H), 3.05 (q, J = 9.4 Hz, 2H), 2.94 (t, J = 7.1 Hz, 2H), 2.87 (t, The test compound (40 μL of a 25 μM solution of the com-
J = 5.7 Hz, 2H), 2.67 (t, J = 6.6 Hz, 2H), 2.58 (s, 1H), 2.44–2.37 pound in distilled water) was seeded onto a 96-well plate and
(m, 1H), 2.11–2.04 (m, 2H), 1.65–1.53 (m, 2H), 1.15 (d, J = then 50 μl of the Master Pre-Mix (CYP450 BACULOSOMES®
7.0 Hz, 3H), 0.64 (d, J = 6.9 Hz, 3H). 13C-NMR (100 MHz, Reagent and Regeneration System) was added. The plate was
CDCl3) δ 162.4 (d, 1J = 242 Hz), 154.0, 153.5 (d, 3J = 12 Hz), incubated for 20 min at 37 °C to allow the compound to inter-
3
1
131.6 (d, J = 9 Hz), 130.0, 125.3 (q, J = 277 Hz), 122.4, 118.7 act with CYP450. The reaction was initiated by adding Vivid®
(d, J = 3 Hz), 115.7, 107.1 (d, J = 22 Hz), 103.1(d, J = 24 Hz), CYP450 substrate/NADP+ buffer (10 μL). The remaining
71.3, 68.2, 55.0 (q, 2J = 31 Hz), 53.2, 52.4, 51.2, 33.1, 27.8, 26.0, enzyme activity was measured by reading the amount of fluo-
25.5, 25.3, 20.9. HPLC: 96.6%, RT 17.68 min. HRMS-ESI (m/z): rescent product using a fluorescent plate reader. The reference
4
2
2
[M + H]+ calcd for C26H32F4N3O2 494.2425, found 494.2426.
( )-(3S,4S)-2-(7-Fluoro-3-hydroxy-4-isopropylchroman-3-yl)acet-
aldehyde O-(2-(1H-benzo[d]imidazol-2-yl)ethyl) oxime (2fa). To
inhibitor for each CYP isozyme is indicated in Table 2.
Microsomal stability assay
a solution of aldehyde 3 (68 mg, 0.27 mmol) in MeOH (1 mL) Human liver microsomes (0.5 mg mL−1) and the test com-
were added K2CO3 and N-alkoxyamine 5a (38 mg, 0.18 mmol) pound (1 μM) in potassium phosphate buffer (0.1 M, pH 7.4)
at 0 °C. The reaction mixture was stirred for 30 min at room were incubated at 37 °C. Reactions were initiated by the
temperature. The solvent of the reaction mixture was removed addition of β-NADPH (1.2 mM) and continued at 37 °C. After
under reduced pressure. The resulting mixture was extracted incubation for 30 min, the reaction was stopped by addition of
with EA and washed with brine. The organic layer was dried acetonitrile with the internal standard. The samples were ana-
over anhydrous MgSO4 and concentrated in vacuo. The result- lyzed using LC–MS/MS. Mibefradil was used as a reference
ing residue was purified by flash column chromatography on standard. The results are expressed as the percentage of drug
silica-gel (EtOAc–n-hexane = 1 : 1) to give oxime 2fa (21 mg, remaining after incubation.
1
28%); H-NMR (300 MHz, CDCl3) δ 7.53–7.54 (m, 2H), 7.48 (t,
J = 6.0 Hz, 1H), 7.19–7.25 (m, 2H) 6.95 (dd, J = 6.6, 8.3 Hz, 1H),
6.51–6.62 (m, 2H), 4.40–4.47 (m, 2H), 4.06 (d, J = 11.0 Hz, 1H),
Acknowledgements
3.87 (dd, J = 1.8, 11.0 Hz, 1H), 3.21–3.36 (m, 2H), 2.59 (s, 1H),
This research was supported by the Korea Institute of Science
2.31–2.46 (m, 3H), 1.13 (d, J = 7.0 Hz, 3H), 0.66 (d, J = 6.9 Hz,
and Technology (KIST-2E24670, 2E25023, 2E24510, 2V03370)
3H). 13C-NMR (100 MHz, CDCl3) δ 162.4 (d, J = 242.0 Hz),
and the National Research Foundation of Korea (NRF-
153.5 (d, J = 12.0 Hz), 152.2, 149.1, 137.5, 131.7 (d, J = 9.0 Hz),
2013R1A1A2005550) funded by the Ministry of Education,
122.8, 118.5 (d, J = 3.0 Hz), 114.7, 107.5 (d, J = 21.0 Hz), 103.4
Science and Technology.
(d, J = 24.0 Hz),70.9, 70.4, 68.2, 50.8, 38.5, 29.1, 28.3, 25.1,
21.0. HPLC: 95.1%, RT 17.34 min. HRMS-ESI (m/z): [M + H]+
calcd for C23H27FN3O3 412.2031, found 412.2030.
Notes and references
In vitro assay for T-type calcium channels blockade
1 W. Osterrieder and M. Holck, J. Cardiovasc. Pharmacol.,
HEK293 cells which stably express both α1G (or α1H) and Kir2.1
subunits were grown in Dulbecco’s modified Eagle’s medium
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(100 U mL−1), streptomycin (100 μg mL−1), geneticin (500 μg
mL−1), and puromycin (1 μg mL−1) at 37 °C in a humid atmos-
phere of 5% CO2 and 95% air. Cells were seeded onto 96-well
black wall clear bottom plates at a density of 4 × 104 cells per
well and were used the next day for the high-throughput
screening (HTS) FDSS6000 assay. For the FDSS6000 assay, cells
were incubated for 60 min at room temperature with 5 μM
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