Structure-activity relationships of the truncated norzoanthamines exhibiting collagen protection toward anti-osteoporotic activity

Article abstract of DOI:10.1016/j.bmc.2014.04.040

The marine alkaloid norzoanthamine is a candidate drug for osteoporosis treatment. Due to its structural complexity, simplified analogues possessing similar biological activities are needed for further research. Recently, we found that the bisaminal unit, representing two-thirds of the original structure, is a bioactive equivalent. We synthesized three kinds of further truncated norzoanthamines and evaluated their collagen protection activities. No analog with collagen protection activity comparable to that of the bisaminal unit was found. Thus, we confirmed the importance of the bisaminal unit for the collagen protection activity. Furthermore, we found that the recognition tolerance of the substrate collagen is relatively large by comparing both enantiomers.

Full text of DOI:10.1016/j.bmc.2014.04.040

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships of the truncated norzoanthamines
exhibiting collagen protection toward anti-osteoporotic activity
⇑
⇑
Hironori Inoue, Kazuho Tokita, Seketsu Fukuzawa , Kazuo Tachibana
Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The marine alkaloid norzoanthamine is a candidate drug for osteoporosis treatment. Due to its structural
complexity, simplified analogues possessing similar biological activities are needed for further research.
Recently, we found that the bisaminal unit, representing two-thirds of the original structure, is a
bioactive equivalent. We synthesized three kinds of further truncated norzoanthamines and evaluated
their collagen protection activities. No analog with collagen protection activity comparable to that of
the bisaminal unit was found. Thus, we confirmed the importance of the bisaminal unit for the collagen
protection activity. Furthermore, we found that the recognition tolerance of the substrate collagen is
relatively large by comparing both enantiomers.
Received 28 February 2014
Revised 17 April 2014
Accepted 18 April 2014
Available online xxxx
Keywords:
Alkaloid
Norzoanthamine
Osteoporosis
Collagen
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
which is a monoaminal unit including the lactone; and southern-
truncated norzoanthamine (s-TZ, 7), which is a monoaminal unit
The members of the zoanthamine1 (1) class of alkaloids possess
unique structures and biological activities.2 Among them, norzo-
anthamine3 (2), isolated from the colonial zoanthid Zoanthus sp.,
suppresses interleukin-6 production and increases bone weight
and density in osteoporosis model mice, making it a candidate
drug for osteoporosis treatment.4,5 In the course of our search for
its mode of action, we found that it accelerates collagen-hydroxy-
apatite composite formation, due to its collagen protection
activity.6 Marine natural products often possess curious or compli-
cated structures, and thus they are difficult to produce by chemical
synthesis for further research. Although the total synthesis of
norzoanthamine has already been achieved,7–9 the design of a sim-
plified structure with similar biological activity represents an
important and promising way to supply natural product-oriented
drugs. According to the structure–activity relationship study of
norzoanthamine, its seco-norzoanthamine methylester (3) exhib-
ited three-fold weaker inhibition of interleukin-6 induction than
that of norzoanthamine.4 Recently, we found a truncated norzo-
anthamine (TZ, 4), which includes two-thirds of the original struc-
ture and exhibits similar collagen protection activity.10 To acquire
more detailed structure–activity relationship information about
norzoanthamine, we divided the bisaminal unit of TZ into three
parts: pseudo-truncated norzoanthamine (p-TZ, 5), which is a lac-
tone-deficient TZ; northern-truncated norzoanthamine (n-TZ, 6),
without the lactone (Scheme 1). Both TZ and p-TZ were previously
synthesized by Kobayashi’s11,12 and Williams’13 groups, respec-
tively, in 1998. In this report, we synthesized more simplified nor-
zoanthamines (p-TZ, n-TZ, and s-TZ) based on Kobayashi’s scheme
and discussed their structure–activity relationships.
2. Results
2.1. Synthesis of p-TZ
The optically pure (99% ee) methyl ester 9 was prepared from
the commercially available 2-methylcyclohexan-1-one (8).14,15
The carbonyl group of the methyl ester 9 was protected by the
Noyori method, to generate the acetal 10. The methyl ester of the
acetal 10 was reduced by DIBAL-H with careful equivalent control,
and the simple aldehyde fragment 11 was obtained. Using the
aldehyde fragment 11 and the sulfone fragment 12,8 the coupling
reaction was conducted to produce the hydroxyl sulfone 13.
Continuously, the hydroxyl group was oxidized by TPAP, and the
sulfone group was removed by sodium mercury amalgam to afford
the ketone 15. One pot deprotection and cyclization were initially
conducted. After boiling in acidic medium and drying over Na2SO4,
the protonated molecular ion peak of the p-TZ 5 (M+H+) was
observed in FAB-MS. Considering the Rf value (0, CH3Cl/MeOH =
3:1) of the obtained product, it should be the acetic acid salt, and
therefore this compound could not be purified by silica gel column
chromatography without a triethylamine-containing eluant. The
⇑
Corresponding authors. Tel.: +81 3 5841 4358; fax: +81 3 5841 4357.
E-mail address: seketsuf@chem.s.u-tokyo.ac.jp (S. Fukuzawa).
http://dx.doi.org/10.1016/j.bmc.2014.04.040
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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2
H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Scheme 1. Simplification of norzoanthamine.
residual triethylammonium acetate was removed by activated
alumina flash chromatography. However, only decomposition
products were observed on TLC. Instead of using silica gel or alu-
mina, the newly prepared product was purified by ODS column
chromatography. After purification, it was quickly subjected to
an NMR measurement, and the characteristic hydrogen peaks of
the aminal and enamine structures were detected. However, this
compound slowly degraded during the NMR measurement, and
its structural elucidation was not completed. Therefore, we could
not be certain whether this isolated compound was the desired
one. In turn, we considered that this enamine structure is not sta-
ble under acidic conditions at high temperature, and thus used
Lewis acid-promoted cyclization. First, the deprotection and the
seven membered ring formation were conducted under acidic con-
ditions with moderate heat.9 Under these conditions, the Boc group
was not deprotected. In the final step, the remaining Boc group was
removed by the Lewis acid.16 The generated secondary amine
formed the enamine structure quickly, and p-TZ 5 was unambigu-
ously obtained (Scheme 2).
2.2. Synthesis of n-TZ
The key intermediate 18 was prepared from the commercially
available diketone 17 according to the Kobayashi’s scheme.11 An
alkyl or an aryl substituent was introduced to the aldehyde frag-
ment 18 by a reductive amination reaction to generate the amines
19. The resultant secondary amino groups were then protected by
Boc groups to obtain the carbamates 20.17 Deprotection of the TBS
groups by TBAF gave the primary alcohols 21. The obtained alco-
hols 21 were oxidized by TPAP to generate the aldehydes 22, and
continuously the Pinnick oxidation reaction was performed to
afford the carboxylic acid 23.7 As the final step, an acid-mediated
cyclization reaction gave n-TZ 6 (Scheme 3).18
2.3. Synthesis of southern truncated norzoanthamine (s-TZ)
The diol 25 was prepared from D-glutamic acid 24 according to
the Kobayashi’s scheme.11 The carbamate and hydroxyl groups of
this intermediate were protected by acetonide groups, to generate
Scheme 2. Reagents and conditions: (a) TMSO(CH2)2OTMS, TMSOTf, CH2Cl2, ꢀ78 °C to rt; (b) DIBAL-H, CH2Cl2, ꢀ78 °C; (c) n-BuLi, THF, ꢀ78 °C; (d) TPAP, NMO, MS4A, CH2Cl2,
rt; (e) 5% Na-Hg, Na2HPO4, MeOH, 0 °C to rt, 63%; (f) AcOH–H2O (96:4), Na2SO4, rt to 100 °C; (g) AcOH–H2O (96:4), rt to 60 °C; (h) TMSI, CH3CN, 0 °C.
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H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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Scheme 3. Reagents and conditions: (a) R-NH2, NaBH3CN, CH3CN, rt; (b) Boc2O, Et3N, DMAP, CH3CN, 0 °C to rt; (c) TBAF, THF, rt; (d) TPAP, NMO, MS4A, CH2Cl2, rt; (e) NaClO2,
NaH2PO4, 2-methyl-2-butene, t-BuOH–H2O, 0 °C to rt; (f) AcOH–H2O (96:4), Na2SO4, rt to 100 °C.
the protected aminal 26. The aminal 26 was oxidized by SO3ꢁPy,
3. Discussion
and then the generated aldehyde 27 was converted to the terminal
olefin 28 by a Wittig reaction. The hydroxyl group was introduced
to the vinyl 28 by hydroboration. The primary alcohol 29 was oxi-
dized to give the aldehyde 30. Surprisingly, in comparison to the
alcohol 26, the alcohol 29 was very unstable. It occasionally
decomposed during NMR measurements, and even during preser-
vation in benzene below ꢀ20 °C. The difference between these
two alcohols is only the length of the carbon skeleton. We could
not find the reason why the additional C1 unit destabilizes the
whole molecule. Furthermore, the aldehyde 30 was also unstable.
Presumably, the moderate yield of this oxidation reaction was
due to this instability. Therefore, all reactions were conducted as
quickly as possible. The aldehyde 30 was cyclized under acidic con-
ditions. When the reaction was performed with 2 N HCl, the yield
was poor and a long reaction time was required. This was probably
because the second cyclization reaction did not proceed quickly,
and became the rate-determining step. Actually, the TLC spot of
the intermediate was observed, and with time this spot disap-
peared and the spot of the carbamate 7-1 appeared. Therefore,
AcOH–H2O (96:4) was selected for the acidic conditions, and the
yield was improved.9 The Boc group of the carbamate 7-1 was then
removed with a 4 N solution of HCl. The resultant amine 7-2 was
too hydrophilic to purify by normal silica gel column chromatogra-
phy. Therefore, the final reaction was performed without further
purification. Aryl substituents were introduced to the secondary
amine by an SN2 reaction, to afford s-TZ 7 (Scheme 4).
For technical reasons, we could not examine all of the samples
at once. Five samples for each collagen protection test are the
maximum. To discuss the structure–activity relationships of the
truncated norzoanthamines, we normalized their collagen protec-
tion activities after an incubation for 180 min. Their relative colla-
gen contents against norzoanthamine 2 were calculated and are
summarized in Table 1. The activity of TZ 4 was equal to that of
norzoanthamine 2, which was previously reported.10 The collagen
protecting activity of p-TZ 5 was weaker than those of norzoanth-
amine 2 and TZ 4. The residual collagen content of p-TZ 5 was 31%
of those of norzoanthamine 2 and TZ 4, which indicated that the
absence of the lactone moiety essentially decreased the activity.
The lack of the hydrophobic space formed by the lactone or the
planarity of the enamine might cause the reduced activity. Further-
more, we divided the bisaminal unit of the active analog TZ 4 into
two monoaminals, n-TZ 6 and s-TZ 7. Both further truncated ana-
logs exhibited slightly different decreasing profiles. The significant
decrease was observed in n-TZ 6, and neither an alkyl nor aryl sub-
stituent on the nitrogen atom affected the activity. On the other
hand, the simplest truncated analog, s-TZ 7, showed interesting
profiles. The simplest unit, the bicyclic monoaminal 7-2, exhibited
31% activity as compared with the natural product 2. The activity
was increased by the addition of hydrophobic aryl groups (7-3,
4) on the nitrogen atom. The activity was dependent on the aro-
matic ring size. Especially, the naphthylmethyl analog 7-5 showed
the highest activity among the truncated norzoanthamines, except
for TZ 4. However, a biphenyl (7-6, 7-8) or carbonyl (carbamate 7-1
and ester 7-7) substituent exerted a negative effect. An ester group
was also present in the seco-norzoanthamine methylester 3, and it
might cause a significant decrease in the collagen protection activ-
ity. The difference between a lactone and an ester is the structural
rigidity. The enhanced fluctuation of the ester group might sup-
press interactions with target molecules. In addition, the enamine
structure without a lactone in the seco-norzoanthamine methyles-
ter 3 is a common characteristic in p-TZ 5. Thus, the lactone
structure is considered to be the key substructure exhibiting the
activity. However, n-TZ 6, the minimum monoaminal-containing
2.4. Collagen protection assay
The collagen protecting activities of all TZ analogs are shown in
Figures 1–5. After an incubation for 180 min, the residual collagen
content of p-TZ 5 was about one-third of that of norzoanthamine 2
(Fig. 1). The monoaminal unit of norzoanthamine 2 including the
lactone (n-TZ 6) showed a significant decrease in collagen protec-
tion activity (Fig. 2). Although the collagen protection activity of
the other monoaminal unit without the lactone (s-TZ 7) was also
weak, its degradation curve profiles were more gradual, as com-
pared with those of n-TZ 6 (Figs. 3–5).
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H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Scheme 4. Reagents and conditions: (a) 2,2-dimethoxypropane, p-TsOHꢁH2O, acetone, rt; (b) SO3ꢁPy, Et3N, DMSO–CH2Cl2, 0 °C to rt; (c) Ph+3CH3Brꢀ, NaHMDS, THF, 0 °C; (d) 9-
BBN, NaHCO3, H2O2, THF, 0 °C to rt; (e) TPAP, NMO, MS4A, CH2Cl2, rt; (f) AcOH–H2O (96:4), 60 °C; (g) 4 N HCl, rt; (h) R-Br, Et3N, THF, rt.
lactone, showed a significant decrease in the collagen protection
activity. Comparing the collagen protection activities of both
monoaminals 6 and 7, the seven-membered bicyclic aminal s-TZ
7 is considered to be the more essential substructure than the lac-
tone-containing bicyclic aminal n-TZ 6. The smallest monoaminal,
7-2, was about three-fold less active than the natural product 2
(Table 1), and its molecular weight (MW 127.2) is nearly one-third
of that of the natural product 2 (MW 481.6).
We previously reported that norzoanthamine 2 functions by
protecting the substrate protein, rather than competitively inhibit-
ing the enzymatic activity. Generally, enzymes and substrate
proteins are chiral molecules. To investigate the mode of action
of bioactive molecules in more detail, comparing both enantiomers
is effective.19 Therefore, we synthesized the enantiomer, (ꢀ)-TZ 31,
from L
-glutamic acid in the same manner as (+)-TZ 4 (Fig. 6)11,12
Figure 1. Collagen protection activities of p-TZ (10 lM sample concentration).
and compared their collagen protection activities (Fig. 7). The
activity was slightly decreased (76%, Table 1). The substrate
type-I collagen is the chiral biopolymer, and is composed primarily
of Gly-Pro-HyPro repeats. The fact that the enantiomer 31 showed
76% activity, as compared with the natural form 4, implied that the
recognition fidelity of the substrate collagen is relatively loose.
The hydrophobic shape fitting might be the driving force for
recognition.
4. Conclusion
As described above, we synthesized three kinds of further
truncated norzoanthamines (p-, n-, and s-TZs), and evaluated their
collagen protection activities. They possessed either the monoami-
nal, lactone-containing aminal (red square in Fig. 8) or the
seven-membered bicyclic aminal (black square in Fig. 8). No analog
with collagen protection activity comparable to the bisaminal
unit (CDEFG ring in Scheme 1) 4 was found (Fig. 8). Thus, we
Figure 2. Collagen protection activities of n-TZs (10 lM sample concentration).
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H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 1
Collagen protection activities of the truncated analogs relative to norzoanthamine 2
Sample
Relative activity (%)
2
100
5
3a
4
5
100
31
17
11
15
23
31
47
45
65
20
25
25
76
6-1
6-2
6-3
7-1
7-2
7-3
7-4
7-5
7-6
7-7
7-8
31
Figure 3. Collagen protection activities of s-TZs (10 lM sample concentration).
a
This data was not shown in this paper.10
Figure 6. Structures of (+)-TZ 4 and (ꢀ)-TZ 31.
units and are referenced to the solvent, that is, 7.24 for CDCl3.
Multiplicities are indicated as: br (broadened), s (singlet), d
(doublet), t (triplet), q (quartet), quint (quintet), sept (septet) or
m (multiplet). Coupling constants (J) are reported in Hertz (Hz).
Low and high resolution mass spectra were recorded on a JEOL
JMS-MS700P mass spectrometer under fast atom bombardment
(FAB) conditions using m-nitrobenzyl alcohol (NBA) as a matrix
or a JEOL JMS-T100LP mass spectrometer with Direct Analysis in
Real TimeÒ unit (DART). IR spectra were recorded on a JASCO FT/
IR-420 spectrometer. Optical rotations were recorded on a JASCO
P-220 digital polarimeter. Analytical thin layer chromatography
(TLC) was performed using E. Merck silica gel 60 F254 plates
(0.25 mm thickness). Column chromatography was performed
using Kanto Chemical silica gel 60 N (40–100 mesh, spherical,
neutral). Anhydrous solvents: acetone, benzene, dichloromethane
(CH2Cl2), diethylether (Et2O), N,N0-dimethylformamide (DMF),
methanol (MeOH), pyridine and tetrahydrofuran (THF) were pur-
chased from Kanto Chemical Co., Inc. or Aldrich, and used without
Figure 4. Collagen protection activities of s-TZs (10 lM sample concentration).
re-confirmed the importance of the bisaminal unit for exhibiting
collagen protection toward an anti-osteoporotic activity. However,
this investigation of further truncated analogs provided more
detailed structure–activity relationship information. We found
that the recognition tolerance of the substrate collagen is relatively
large by comparing both enantiomers. Bottom-up design and syn-
thesis based on our findings will enable us to provide more ratio-
nalized anti-osteoporotic drug candidates in the future.
5. Experimental section
5.1. General
1H NMR spectra were recorded on a JEOL JNM-AL400 spectrom-
eter operating at 400 MHz (1H). Chemical shifts are reported in d
Figure 5. Collagen protection activities of s-TZs (10
lM sample concentration).
Figure 7. Collagen protection activities of TZs (10 lM sample concentration).
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H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 8. Structure–activity relationships of the truncated norzoanthamines.
further drying. All other reagents and solvents were purchased at
highest commercial grade and used as supplied unless otherwise
noted. All moisture sensitive reactions were performed under a
static argon atmosphere in oven-dried glassware, otherwise noted.
5.4. tert-Butyl (R)-2,2-dimethyl-5-((S)-2-methyl-6-((R)-6-
methyl-1,4-dioxaspiro[4.5]decan-6-yl)-4-oxohexyl)oxazolidine-
3-carboxylate (15)
To a solution of sulfone 12 (131 mg, 0.330 mmol) in THF (3 mL)
were added t-BuLi (0.209 mL, 0.330 mmol) at ꢀ78 °C, and then the
resulting mixture was stirred at that temperature for 30 min. To
this mixture was added a solution of aldehyde 11 (75.0 mg,
0.353 mL) in THF (4 mL) at ꢀ78 °C, and then the resulting mixture
was stirred at that temperature for 30 min. The reaction was
quenched with a saturated aqueous solution of NH4Cl. The aqueous
layer was extracted three times with EtOAc. The combined organic
layer was washed with water and brine, dried over Na2SO4, fil-
trated and concentrated under reduced pressure. Purification by
silica gel column chromatography (hexane/EtOAc = 9:1) gave
197 mg (92% yield) of diastereomeric mixture of the hydroxyl sul-
fone 13 as yellow oil. To a solution of the diastereomeric mixture of
hydroxyl sulfone 13 in CH2Cl2 (4 mL) was added MS4A (200 mg),
NMO (114 mg, 0.973 mmol) and TPAP (1 portion) at room temper-
ature. The resulting suspension was stirred at room temperature
for 30 min. Purification by silica gel column chromatography
(hexane/EtOAc = 4:1) gave 167 mg (85% yield) of diastereomeric
mixture of the sulfone 14 as yellow oil. To a solution of diastereo-
meric mixture of the sulfone 14 in MeOH (4 mL) was added
Na2HPO4 (1.17 g, 8.24 mmol) and 5% Na-Hg (1.17 g) at 0 °C. The
resulting suspension was stirred at that temperature for 2 h. The
reaction mixture was diluted with a saturated aqueous solution
of NH4Cl and EtOAc at room temperature, and the mixture was
stirred at room temperature 30 min. The aqueous layer was
extracted three times with EtOAc. The combined organic layer
was washed with brine, dried over Na2SO4, filtrated and concen-
trated under reduced pressure. Purification by silica gel column
5.2. Methyl (R)-3-(6-methyl-1,4-dioxaspiro[4.5]decan-6-
yl)propanoate (10)
To a solution of the ketone 9 (302 mg, 1.52 mmol) in CH2Cl2
(5.3 mL) was added TMSO(CH2)2OTMS (1.12 mL, 5.43 mmol) at
room temperature. After the resulting mixture was cooled to
ꢀ78 °C, to this mixture was added a solution of TMSOTf (55
lL,
0.304 mmol) and the mixture was stirred at same temperature.
After 30 min, the mixture was allowed to warm to room tempera-
ture and kept at this temperature for 12 h. The reaction was
quenched with a saturated aqueous solution of NaHCO3. The aque-
ous layer was extracted with CH2Cl2 three times. The combined
organic layer was dried over Na2SO4, filtrated and concentrated
under reduced pressure. Purification by silica gel column chroma-
tography (hexane/EtOAc = 9:1) gave 286 mg (78% yield) of the ace-
26
tal 10 as colorless oil: [
a]
+0.23 (c 0.44, CHCl3); 1H NMR
D
(400 MHz, CDCl3) d 3.85–3.76 (m, 4H), 3.55 (s, 3H), 2.24–2.11 (m,
2H), 1.75–1.60 (m, 2H), 1.53–1.40 (m, 4H), 1.31 (br s, 4H), 0.81
(s, 3H); 13C NMR (100 MHz, CDCl3) d 175.1, 112.7, 64.9, 64.6,
51.5, 40.8, 34.7, 30.5, 30.1, 29.2, 23.5, 20.7, 19.3; IR (film, cmꢀ1
)
2944, 2878, 1738, 1436, 1174, 1090; HRMS (FAB) calcd for
C
13H23O4 [(M+H)+] 243.1518, found 243.1552.
5.3. (R)-3-(6-Methyl-1,4-dioxaspiro[4.5]decan-6-yl)propanal
(11)
To a solution of the acetal 10 (270 mg, 1.11 mmol) in CH2Cl2
(1 mL) was added 0.99 M DIBAL-H (1.24 mL, 1.36 mmol) in hexane
at ꢀ78 °C, and the mixture was stirred at that temperature for
30 min. The reaction mixture was quenched with MeOH and the
mixture was allowed to warm to 0 °C. To this mixture was added
a saturated aqueous solution of potassium sodium tartrate at that
temperature and the mixture was stirred at room temperature
overnight. The aqueous layer was extracted three times with CH2-
Cl2. The combined organic layer was washed with brine, dried over
Na2SO4, filtrated and concentrated under reduced pressure. Purifi-
cation by silica gel column chromatography (hexane/EtOAc = 9:1)
chromatography (hexane/EtOAc = 5:1) gave 102 mg (80% yield) of
24
the ketone 15 as yellow oil : [
a]
D
ꢀ11.6 (c 0.25, CHCl3); 1H
NMR (400 MHz, CDCl3) d 4.10–4.03 (m, 1H), 3.94–3.85 (m, 4H),
3.71–3.58 (m, 1H), 3.03–2.96 (m, 1H), 2.48–2.24 (m, 4H), 2.11 (br
s, 1H), 1.74–1.39 (m, 27H), 0.93 (d, J = 6.6 Hz, 3H), 0.88 (s, 3H);
13C NMR (100 MHz, CDCl3) d 211.2, 152.0, 112.7, 93.0, 79.7, 71.8,
67.0, 64.9, 64.6, 51.2, 49.9, 40.6, 39.8, 38.3, 34.8, 30.4, 28.7, 28.5,
26.8, 24.7, 23.5, 20.7, 20.1, 19.4; IR (film, cmꢀ1) 2932, 2875,
1698, 1393, 1176, 1092; HRMS (FAB) calcd for
C26H46O6N
[(M+H)+] 468.3247, found 468.3353.
gave 197 mg (83% yield) of the aldehyde 11 as colorless oil:
+30.1 (c 2.5, CHCl3); 1H NMR (400 MHz, CDCl3) d 9.75 (s
5.5. tert-Butyl (1R,3S,5S)-3-methyl-5-(2-((R)-1-methyl-2-
oxocyclohexyl)ethyl)-8-oxa-6-azabicyclo[3.2.1]octane-6-
carboxylate (16)
26
[a
]
D
1H), 3.95–3.86 (m, 4H), 2.45–2.30 (m, 2H), 1.82–1.66 (m, 2H),
1.61–1.51 (m, 4H), 1.41 (br s, 4H), 0.90 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 203.4, 112.7, 64.9, 64.6, 40.6, 39.2, 34.8, 30.4,
27.0, 23.5, 20.7, 19.5; IR (film, cmꢀ1) 2933, 2865, 2716, 2716,
1725, 1176, 1091; HRMS (FAB) calcd for C12H21O3 [(M+H)+]
213.1412, found 213.1483.
The ketone 15 (13.7 mg, 0.0293 mmol) was dissolved in AcOH–
H2O (96:4, 2 mL) at room temperature, and then the resulting
mixture was stirred at 60 °C for 14 h. After the reaction mixture
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H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
was cooled to room temperature, the solution was concentrated
under reduced pressure. Purification by silica gel column chroma-
3.81–3.75 (m, 4H), 3.60 (t, J = 7.3 Hz, 2H), 2.52 (t, J = 7.3 Hz, 2H),
2.26 (s, 2H), 2.13 (s, 2H), 1.71–1.23 (m, 13H), 0.97 (s, 3H), 0.87
(s, 9H), 0.85 (s, 3s), 0.03 (s, 6H); 13C NMR (100 MHz, CDCl3) d
140.4, 129.3, 128.4, 128.3, 126.8, 126.6, 114.2, 64.8, 63.0, 60.8,
54.0, 51.1, 47.3, 46.6, 40.2, 32.2, 31.0, 28.2, 26.8, 26.0, 22.3, 19.5,
19.3, 18.4, ꢀ5.2; IR (film, cmꢀ1) 3297, 2929, 2881, 1643, 1111,
1078; HRMS (FAB) calcd for C28H50O3NSi [(M+H)+] 476.3482, found
476.3580.
tography (hexane/EtOAc = 9:1) gave 7.5 mg (70% yield) of the car-
21
bamate 16 as colorless oil: [
a]
+86.6 (c 0.52, CHCl3); 1H NMR
D
(400 MHz, CDCl3) d 4.36–4.34 (m, 1H), 3.52–3.33 (m, 2H), 2.47–
2.38 (m, 1H), 2.28–2.13 (m, 2H), 2.08–1.88 (m, 3H), 1.84–1.69
(m, 3H), 1.66–1.50 (m, 5H), 1.44 (s, 9H), 1.33–1.22 (m, 2H), 1.14
(dd, J = 11.9, 12.8 Hz, 1H), 1.00 (s, 3H), 0.86 (d, J = 6.4 Hz, 3H); 13C
NMR (100 MHz, CDCl3) d 216.3, 152.4, 94.0, 79.7, 72.5, 51.1, 48.0,
41.5, 39.7, 38.7, 37.7, 30.1, 29.7, 28.4, 27.6, 24.1, 22.2, 21.5, 21.0;
IR (film, cmꢀ1) 2955, 2928, 1694, 1391, 1162, 1037; HRMS (FAB)
calcd for C21H36O4N [(M+H)+] 366.2566, found 366.2625.
5.9. 3-((6S,7R)-7-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-6,
7-dimethyl-1,4-dioxaspiro[4.5]decan-6-yl)-N-(2-
methoxyethyl)propan-1-amine (19-3)
5.6. Pseudo-truncated norzoanthamine (p-TZ, 5)
92% as yellow oil: [a]
16 +7.5 (c 0.17, CHCl3); 1H NMR (400 MHz,
D
CDCl3) d 3.95–3.86 (m, 2H), 3.79 (dd, J = 6.1, 6.1 Hz, 2H), 3.60 (t,
J = 7.3 Hz, 2H), 3.50 (t, J = 5.1 Hz, 2H), 3.34 (s, 3H), 2.80 (t,
J = 5.2 Hz, 2H), 2.60–2.49 (m, 2H), 2.25 (br s, 1H), 2.06 (br s, 2H),
1.63–1.21 (m, 10H), 0.98 (s, 3H), 0.88 (s, 9H), 0.87 (s, 3H), 0.03 (s,
3H); 13C NMR (100 MHz, CDCl3) d 114.2, 71.7, 64.9, 62.9, 60.8, 59.2,
58.8, 51.7, 49.2, 46.5, 43.7, 40.2, 32.1, 30.9, 28.2, 26.6, 26.0, 22.3,
19.3, 18.4, –5.2; IR (film, cmꢀ1) 3326, 2928, 2880, 1253, 1078; HRMS
(FAB) calcd for C24H50O4NSi [(M+H)+] 444.3431, found 444.3523.
To a solution of the carbamate 16 (7.5 mg, 0.0205 mmol) in CH3-
CN (1 mL) was added TMSI (5.6 lL, 0.0409 mmol) at 0 °C, and the
mixture was stirred at that temperature for 15 min. The reaction
mixture was quenched with a saturated aqueous solution of
NaHCO3 and the mixture was stirred at that temperature for
5 min. The aqueous layer was extracted three times with CHCl3.
The combined organic layer was dried over Na2SO4, filtrated and
concentrated under reducing pressure. Purification by silica gel
column chromatography (CHCl3/MeOH = 19:1) gave 2.3 mg (45%
5.10. General procedure of preparation of carbamate (20)
+78.6 (c 0.14, CHCl3); 1H
21
yield) of the p-TZ 5 as yellow oil: [
a]
D
NMR (400 MHz, CDCl3) d 5.34 (t, J = 4.1 Hz, 1H), 4.08–4.04 (m,
1H), 2.88–2.78 (m, 2H), 2.38–2.25 (m, 2H), 2.14–2.11 (m, 2H),
2.03–1.99 (m, 3H), 1.80–1.58 (m, 3H), 1.50–1.30 (m, 5H), 1.19 (d,
J = 6.9 Hz, 3H), 0.96 (s, 3H); 13C NMR (100 MHz, CDCl3) d 147.7,
140.4, 114.4, 109.8, 71.5, 70.5, 47.0, 37.8, 36.3, 32.4, 32.1, 29.7,
25.6, 25.2, 23.7, 22.9, 18.1 (involving peaks due to imine); IR (film,
cmꢀ1) 2924, 2847, 1636, 1456, ; HRMS (FAB) calcd for C16H26ON
[(M+H)+] 248.1936, found 248.1994.
To a solution of the amine 19-1 (24.0 mg, 0.0543 mmol) in CH3CN
(1 mL) was added Et3N (19.0 lL, 0.136 mmol), Boc2O (19.0 lL,
0.0827 mmol) and DMAP (1 portion) at 0 °C, and the mixture was
stirred at room temperature for 12 h. The reaction mixture was
quenched with a saturated aqueous solution of NH4Cl. The aqueous
layer was extracted three times with EtOAc. The combined organic
layer was washed with brine, dried over Na2SO4, filtrated and
concentrated under reduced pressure. Purification by silica gel col-
umn chromatography (hexane/EtOAc = 14:1) gave 27.5 mg (95%
yield) of tert-butyl butyl(3-((6S,7R)-7-(2-((tert-butyldimethylsi-
5.7. General procedure of preparation of the amine (19)
lyl)oxy)ethyl)-6,7-dimethyl-1,4-dioxaspiro[4.5]decan-6-yl)propyl)
+15.4 (c 0.057, CHCl3); 1H
18
To
a
solution of 3-((6S,7R)-7-(2-((tert-butyldimethylsi-
carbamate (20-1) as yellow oil: [
a]
D
lyl)oxy)ethyl)-6,7-dimethyl-1,4-dioxaspiro[4.5]decan-6-yl)propanal
(18, 32.0 mg, 0.0832 mmol) in CH3CN (1 mL) was added n-butyl-
amine (25.0 lL, 0.253 mmol) and NaBH3CN (10.0 mg, 0.159 mmol)
NMR (400 MHz, CDCl3) d 3.96–3.86 (m, 2H), 3.83–3.75 (m, 2H),
3.60 (t, J = 7.3 Hz, 2H), 3.07 (br, 4H), 1.56–1.17 (m, 31H), 0.97 (s,
3H), 0.89 (t, J = 7.3 Hz, 3H), 0.87 (s, 12H), 0.03 (s, 6H); 13C NMR
(100 MHz, CDCl3) d 155.6, 114.2, 78.7, 64.9, 62.9, 60.7, 48.3, 46.8,
46.5, 40.2, 36.3, 32.1, 31.0, 30.4, 28.5, 27.6, 26.0, 25.5, 22.4, 20.1,
19.3, 18.4, 15.9, 13.9, –5.2; IR (film, cmꢀ1) 2929, 2876, 1693, 1251,
1078; HRMS (FAB) calcd for C30H60O5NSi [(M+H)+] 542.4163, found
542.4217.
at room temperature, and the mixture was acidified (pH = 6) by
adding AcOH, and stirred at that temperature for 12 h. The reaction
mixture was quenched with a saturated aqueous solution of
NaHCO3. The aqueous layer was extracted three times with CHCl3.
The combined organic layer was dried over Na2SO4, filtrated and
concentrated under reduced pressure. Purification by silica gel col-
umn chromatography (CHCl3/MeOH = 19:1) gave 24.3 mg (58%
yield) of N-(3-((6S,7R)-7-(2-((tert-butyldimethylsilyl)oxy)ethyl)-
5.11. tert-Butyl benzyl(3-((6S,7R)-7-(2-((tert-butyldimethylsilyl)
oxy)ethyl)-6,7-dimethyl-1,4-dioxaspiro[4.5]decan-6-yl)
propyl)carbamate (20-2)
6,7-dimethyl-1,4-dioxaspiro[4.5]decan-6-yl)propyl)butan-1-amine
17
(19-1) as colorless oil: [
a]
D
+38.7 (c 0.038, CHCl3); 1H NMR
+12.2 (c 0.13, CHCl3); 1H NMR
18
(400 MHz, CDCl3) d 3.91 (ddd, J = 15.4, 11.0, 7.8 Hz, 2H), 3.81–
3.78 (m, 2H), 3.60 (t, J = 7.3 Hz, 2H), 2.61 (t, J = 7.6 Hz, 2H), 2.54
(m, 2H), 1.73–1.20 (m, 16H), 0.98 (s, 3H), 0.90 (t, J = 7.3 Hz, 3H),
0.87 (s, 12H), 0.03 (s, H); 13C NMR (100 MHz, CDCl3) d 114.2,
67.1, 64.9, 63.0, 60.8, 51.7, 49.7, 46.6, 40.2, 32.2, 32.0, 30.9, 28.3,
73% as yellow oil: [
a]
D
(400 MHz, CDCl3) d 7.31–7.10 (m, 5H), 4.47–4.32 (m, 2H), 3.92–
3.67 (m, 4H), 3.59 (t, J = 7.3 Hz, 2H), 3.04–3.00 (br, 2H), 2.42 (br
s, 1H), 1.59–1.23 (m, 20H), 0.94 (s, 3H), 0.87 (s, 9H), 0.83 (s, 3H),
0.03 (s, 6H); 13C NMR (100 MHz, CDCl3) d 138.7, 128.4, 127.7,
127.1, 127.0, 114.2, 79.3, 64.9, 62.8, 60.7, 49.8, 48.0, 46.5, 40.2,
32.0, 32.0, 30.9, 28.5, 27.6, 26.0, 24.9, 22.4, 19.3, 18.4, 16.0, ꢀ5.2;
IR (film, cmꢀ1) 2928, 2882, 1694, 1139, 1077; HRMS (FAB) calcd
for C33H58O5NSi [(M+H)+] 576.4006, found 576.4053.
26.7, 26.1, 22.3, 20.5, 19.3, 18.4, 15.8, 14.0, ꢀ5.2; IR (film, cmꢀ1
)
3309, 2927, 2874, 1253, 1078; HRMS (FAB) calcd for C25H52O3NSi
[(M+H)+] 442.3638, found 442.3721.
5.8. N-Benzyl-3-((6S,7R)-7-(2-((tert-butyldimethylsilyl)oxy)
ethyl)-6,7-dimethyl-1,4-dioxaspiro[4.5]decan-6-yl)propan-1-
amine (19-2)
5.12. tert-Butyl (3-((6S,7R)-7-(2-((tert-butyldimethylsilyl)
oxy)ethyl)-6,7-dimethyl-1,4-dioxaspiro[4.5]decan-6-yl)
propyl)(2-methoxyethyl)carbamate (20-3)
17
Quant as colorless oil: [
a]
+1.3 (c 0.68, CHCl3); 1H NMR
D
(400 MHz, CDCl3) d 7.40–7.34 (m, 2H), 7.32–7.29 (m, 3H), 4.48
89% as colorless oil: [
a]
+8.0 (c 0.16, CHCl3); 1H NMR
17
D
(d, J = 6.1 Hz, 1H), 4.27 (d, J = 5.1 Hz, 1H), 3.94–3.83 (m, 2H),
(400 MHz, CDCl3) d 3.96–3.86 (m, 2H), 3.83–3.73 (m, 2H), 3.60 (t,
Please cite this article in press as: Inoue, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.040

8
H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
J = 7.1 Hz, 2H), 3.46 (br, 2H), 3.32 (br s, 5H), 3.11 (br, 2H), 2.31 (br,
1H), 1.53–1.16 (m, 20H), 0.97 (s, 3H), 0.87 (s, 9H), 0.86 (s, 3H), 0.03
(s, 6H); 13C NMR (100 MHz, CDCl3) d 155.4, 114.2, 79.1, 71.2, 65.0,
62.9, 60.7, 58.8, 49.8, 46.7, 46.5, 40.2, 37.0, 32.1, 31.0, 28.5, 27.5,
26.0, 25.3, 22.4, 19.3, 18.4, 16.1, –5.2; IR (film, cmꢀ1) 2929, 2882,
1695, 1251, 1078; HRMS (FAB) calcd for C29H58O6NSi [(M+H)+]
544.3955, found 544.4011.
5.16. General procedure of preparation of northern-truncated
norzoanthamine (n-TZ, 6)
The carboxylic acid 23-1 (6.8 mg, 0.0154 mmol) was dissolved
in AcOH–H2O (96:4, 2 mL) at room temperature, and then the
resulting mixture was stirred at 100 °C for 12 h. After the reaction
mixture was cooled to room temperature, anhydrous Na2SO4 was
added to the mixture. The mixture was stirred for 1 h at that tem-
perature, and filtered and washed with MeOH. The filtrate was
concentrated under reduced pressure. Purification by silica gel col-
5.13. General procedure of preparation of carboxylic acid (23)
To a solution of carbamate 20-1 (27.5 mg, 0.0507 mmol) in THF
umn chromatography (CHCl3/MeOH = 9:1) gave 2.2 mg (51% yield)
–21.8 (c 0.022, CHCl3); 1H
23
(1 mL) was added TBAF (1 M; 60.0
l
L, 0.0600 mmol) at room tem-
of the n-TZ 6-1 as colorless oil : [
a]
D
perature, and then resulting mixture was stirred at that tempera-
ture for 16 h. The reaction was quenched with water. The
aqueous layer was extracted three times with EtOAc. The com-
bined organic layer was dried over Na2SO4, filtrated and concen-
trated under reduced pressure to afford the crude tert-butyl
butyl(3-((6S,7R)-7-(2-hydroxyethyl)-6,7-dimethyl-1,4-dioxaspiro
[4.5]decan-6-yl)propyl)carbamate(21-1) as yellow oil.
To a solution of the crude alcohol 21-1 in CH2Cl2 (1 mL) was
added MS4A (36.0 mg), NMO (17.8 mg, 0.152 mmol) and TPAP (1
portion) at room temperature. The resulting suspension was stir-
red at room temperature for 10 min. Purification by silica gel col-
umn chromatography (hexane/EtOAc = 4:1) gave tert-butyl butyl
(3-((6S,7R)-6,7-dimethyl-7-(2-oxoethyl)-1,4-dioxaspiro[4.5]decan-6-
yl)propyl)carbamate (22-1) as yellow oil.
To a solution of aldehyde 22-1 in t-BuOH (1 mL) and water
(0.2 mL) was added 2-methyl-2-butene (0.27 ml, 2.54 mmol) and
aqueous solution of NaClO2 (27.5 mg, 0.304 mmol) and NaH2PO4
(21.9 mg, 0.183 mmol) at 0 °C dropwise. The resulting mixture
was stirred at room temperature for 3 h. The reaction was
quenched with a saturated aqueous solution of NH4Cl. The aqueous
layer was extracted three times with EtOAc. The combined organic
layer was dried over Na2SO4, filtrated and concentrated under
reduced pressure. Purification by silica gel column chromatogra-
phy (hexane/EtOAc = 4:1) gave 15.8 mg (71% yield) of 2-((6S,7R)-
NMR (400 MHz, CDCl3) d 3.00–2.88 (m, 2H), 2.65 (dd, J = 11.5,
4.6 Hz, 1H), 2.52 (dd, J = 19.0, 1.7 Hz, 1H), 2.40 (ddd, J = 14.4, 7.1,
7.1 Hz, 1H), 2.22 (d, J = 19.0 Hz, 1H), 2.14–2.09 (m, 1H), 1.90–1.79
(m, 2H), 1.70–1.37 (m, 8H), 1.26–1.14 (m, 6H), 0.87 (t, J = 7.3 Hz,
3H), 0.84 (s, 3H); 13C NMR (100 MHz, CDCl3) d 174.1, 100.9, 49.5,
47.1, 41.2, 38.6, 35.9, 35.2, 31.1, 29.1, 27.2, 23.1, 20.6, 20.4, 18.5,
17.8, 14.0; IR (film, cmꢀ1) 2918, 2871, 1716; HRMS (FAB) calcd
for C17H30O2N [(M+H)+] 280.2198, found 280.2266.
5.17. n-TZ 6-2
24
92% As colorless oil: [
a]
ꢀ8.54 (c 0.073, CHCl3); 1H NMR
D
(400 MHz, CDCl3) d 7.27–7.24 (m, 2H), 7.20–7.15 (m, 3H), 4.39
(d, J = 15.6 Hz, 1H), 4.31 (br s, 1H), 4.15–3.78(m, 1H), 3.35 (d,
J = 15.6 Hz, 1H), 4.15–4.14 (br d, 1H), 2.88–2.81 (m, 1H), 2.62–
2.59 (m, 1H), 2.52 (dd, J = 19.0, 1.7 Hz, 1H), 2.23 (d, J = 19.0 Hz,
1H), 2.24 (s, 1H), 2.12–2.03 (m, 2H), 1.88–1.76 (m, 3H), 1.62–
1.41 (m, 5H), 1.29 (s, 3H), 1.19–1.12 (m, 1H), 0.84 (s 3H); 13C
NMR (100 MHz, CDCl3) d 173.8, 141.1, 128.5, 128.3, 127.3, 126.6,
104.5, 52.9, 47.6, 41.2, 38.8, 36.0, 35.1, 29.5, 27.1, 23.1, 20.5,
18.4, 17.7; IR (film, cmꢀ1) 2948, 1715, 1714, 1697; HRMS (FAB)
calcd for C20H28O2N [(M+H)+] 314.2042, found 314.2142.
5.18. n-TZ 6-3
6-(3-((tert-butoxycarbonyl)(butyl)amino)propyl)-6,7-dimethyl-1,
ꢀ15.2 (c 0.053, CHCl3); 1H NMR
18
22
4-dioxaspiro[4.5]decan-7-yl)acetic acid (23-1) as yellow oil: [
a
]
59% as colorless oil: [
a]
D
D
+18.2 (c 0.042, CHCl3); 1H NMR (400 MHz, CDCl3) d 3.99–3.90 (m,
2H), 3.83–3.75 (m, 2H), 3.16–3.02 (m, 5H), 2.21 (d, J = 13.1 Hz,
1H), 1.64–1.42 (m, 21H), 1.31–1.21 (m, 3H), 1.04 (s, 3H), 1.00 (s,
3H), 0.89 (t, J = 7.3 Hz, 3H); IR (film, cmꢀ1) 3127, 2929, 2873,
1697, 1152; HRMS (FAB) calcd for C24H44O6N [(M+H)+] 442.3090,
found 442.3168.
(400 MHz, CDCl3) d 3.44–3.39 (m, 2H), 3.32 (s, 3H), 3.13 (ddd,
J = 15.1, 7.1, 7.1 Hz, 1H), 3.06–2.99 (m, 1H), 2.72–2.65 (m, 2H),
2.53 (dd, J = 19.0, 1.7 Hz, 1H), 2.22 (d, J = 19.0 Hz, 1H), 2.08–2.05
(m, 1H), 1.89–1.79 (m, 2H), 1.72–1.63 (m, 2H), 1.56–1.45 (m, 4H),
1.22–1.14 (m, 4H), 0.84 (s, 3H); 13C NMR (100 MHz, CDCl3) d 173.8,
104.7, 71.3, 58.9, 49.5, 48.1, 41.2, 38.6, 35.9, 35.1, 29.2, 27.1, 23.1,
20.6, 18.4, 17.7; IR (film, cmꢀ1) 2947, 2874, 1716, 1116; HRMS
(FAB) calcd for C16H28O3N [(M+H)+] 282.1991, found 282.2086.
5.14. 2-((6S,7R)-6-(3-(Benzyl(tert-butoxycarbonyl)
amino)propyl)-6,7-dimethyl-1,4-dioxaspiro[4.5]decan-7-yl)
acetic acid (23-2)
5.19. tert-Butyl (R)-5-((S)-3-hydroxy-2-methylpropyl)-2,
2-dimethyloxazolidine-3-carboxylate (26)
73% Yield as yellow oil: [
a]
+1.7 (c 0.58, CHCl3); 1H NMR
18
D
(400 MHz, CDCl3) d 7.31–7.20 (m, 5H), 4.48–4.32 (m, 2H), 3.96–
3.68 (m, 4H), 3.08 (br d, 3H), 2.17 (d, J = 12.9 Hz, 1H), 1.60–1.22
(m, 20H), 1.00 (s, 3H), 0.97 (s, 3H); IR (film, cmꢀ1) 3164, 2933,
To a solution of tert-butyl ((2R,4S)-2,5-dihydroxy-4-methylpen-
tyl)carbamate (25, 656 mg, 2.81 mmol) in dry acetone (56 mL)
was added 2,2-dimethoxypropane (1.04 mL, 8.46 mmol) and
p-TsOHꢁH2O (26.7 mg, 0.140 mmol) at room temperature, and then
the resulting mixture was stirred at that temperature for 4 h. The
reaction was quenched with a saturated aqueous solution of
NaHCO3. The aqueous layer was extracted three times with EtOAc.
The combined organic layer was washed with brine, dried over
Na2SO4, filtrated and concentrated under reduced pressure. Purifi-
cation by silica gel column chromatography (hexane/EtOAc = 3:1)
gave 561 mg (73% yield) of the acetal 26 as colorless oil: 1H NMR
(400 MHz, CDCl3) d 4.16 (ddd, J = 10.6, 6.4, 5.6 Hz, 1H), 3.68–3.60
(br d, 2H), 3.55–3.43 (m, 2H), 3.04 (br dd, 1H), 2.04 (br s, 1H),
1.83 (dt, J = 12.5, 6.3 Hz, 1H), 1.63–1.40 (m, 17H), 0.95 (d,
2879, 1692, 1247, 1167; HRMS (FAB) calcd for
C27H42O6N
[(M+H)+] 476.2934, found 476.2969.
5.15. 2-((6S,7R)-6-(3-((tert-Butoxycarbonyl)(2-methoxyethyl)
amino)propyl)-6,7-dimethyl-1,4-dioxaspiro[4.5]decan-7-yl)
acetic acid (23-3)
1.7
82% As yellow oil: [
a
]
D
+10.2 (c 0.083, CHCl3); 1H NMR
(400 MHz, CDCl3) d 4.00–3.92 (m, 2H), 3.84–3.74 (m, 2H), 3.46
(br s, 2H), 3.32 (br s, 5H), 3.15–3.13 (m, 3H), 2.23 (d, J = 13.3 Hz,
1H), 1.63–1.43 (m, 20H), 1.05 (s, 3H), 1.01 (s, 3H); IR (film, cmꢀ1
)
3154, 2929, 2878, 1728, 1693, 1154, 1118; HRMS (FAB) calcd for
J = 7.1 Hz, 3H); HRMS (FAB) calcd for C
14H28O4N [(M+H)+]
C
23H42O7N [(M+H)+] 444.2883, found 444.3004.
274.1940, found 274.2021.
Please cite this article in press as: Inoue, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.040

H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
5.20. tert-Butyl (R)-2,2-dimethyl-5-((S)-2-methylbut-3-en-1-
yl)oxazolidine-3-carboxylate (28)
(film, cmꢀ1) 2977, 2877, 2719, 1725, 1698, 1394; HRMS (FAB) calcd
for C15H28O4N [(M+H)+] 286.1940, found 286.2027.
To a solution of the acetal 26 (146 mg, 0.534 mmol) in CH2Cl2
(5 mL) and DMSO (1.5 mL) was added Et3N (0.595 mL, 4.27 mmol)
and SO3ꢁPy (425 mg, 2.67 mmol) at 0 °C, and then the resulting
mixture was stirred at room temperature for 1 h. The reaction
was quenched with a saturated aqueous solution of NH4Cl. The
aqueous layer was extracted three times with CHCl3. The combined
organic layer was washed with brine, dried over Na2SO4, filtrated
and concentrated under reduced pressure to afford the crude alde-
hyde 27 as colorless oil.
5.23. tert-Butyl (1R,3S,5S)-3-methyl-8-oxa-6-
azabicyclo[3.2.1]octane-6-carboxylate (7-1)
The aldehyde 30 (38.1 mg, 0.134 mmol) was dissolved in
AcOH–H2O (96:4, 8 mL) at room temperature, and then the result-
ing mixture was stirred at 60 °C for 18 h. After the reaction mixture
was cooled to room temperature, the solution was concentrated
under reduced pressure. Purification by silica gel column chroma-
tography (hexane/EtOAc = 4:1) gave 24.9 mg (83% yield) of the
To
a
solution of methyltriphenylphosphonium bromide
protected aminal 7-1 as colorless oil: [a]
26 +50.0 (c 0.092, CHCl3);
D
(286 mg, 0.801 mmol) in THF (4 mL) was added NaHMDS
(0.694 mL, 0.749 mmol) at 0 °C, and then the resulting mixture
was stirred at that temperature for 1 h. The solution of the crude
aldehyde 27 in THF (3 mL) was added to this reaction mixture
via cannula at 0 °C, and then the resulting mixture was stirred at
that temperature for 2 h. The reaction mixture was quenched with
a saturated aqueous solution of NH4Cl. The aqueous layer was
extracted three times with EtOAc. Organic layer was washed with
brine, dried over Na2SO4, filtrated and concentrated under reduced
pressure. Purification by silica gel column chromatography (hex-
1H NMR (400 MHz, CDCl3) d 5.44 (br d, 1H), 4.52 (m, 1H), 3.51–3.46
(br dd, 1H), 3.25–3.20 (br dd, 1H), 1.96–1.80 (m, 2H), 1.58–1.20 (m,
12H), 0.90 (d, J = 6.3 Hz, 3H); HRMS (FAB) calcd for C12H22O3N
[(M+H)+] 228.1521, found 228.1610.
5.24. (1R,3S,5S)-3-Methyl-8-oxa-6-azabicyclo[3.2.1]octane (7-2)
The solution of the protected aminal 7-1 (8.16 mg, 0.0359 mmol)
in 4 N HCl–EtOAc (1 mL) was stirred at room temperature for
30 min. The filtrate was concentrated under reduced pressure to
afford the amine 7-2 as yellow oil (quantitative yield): 1H NMR
(400 MHz, CDCl3) d 5.58 (s, 1H), 4.71 (s, 1H), 3.42 (br, 2H), 2.27–
2.17 (m, 2H), 1.70–1.44 (m, 3H), 0.95 (d, J = 6.3 Hz, 3H); 13C NMR
(100 MHz, CDCl3) d 101.6, 87.81, 46.16, 37.53, 36.70, 22.49, 21.93.
ane/EtOAc = 19:1) gave 120 mg (83% yield) of the terminal olefin
28 as colorless oil: [
a
]
D
ꢀ64.9 (c 0.029, CHCl3); 1H NMR
24
(400 MHz, CDCl3) d 5.63 (ddd, J = 18.1, 10.0, 8.1 Hz, 1H), 4.99 (d,
J = 17.1 Hz, 1H), 4.93 (d, J = 10.5 Hz, 1H), 4.02 (ddt, J = 10.4, 6.2,
5.2 Hz, 1H), 3.70–3.50 (br d, 1H), 3.01–2.90 (br dd, 1H), 2.30 (m,
1H), 1.75–1.41 (m, 15H), 1.01 (d, J = 6.6 Hz, 3H); IR (film, cmꢀ1
)
5.25. General procedure of preparation of southern-truncated
norzoanthamine (s-TZ, 7)
2933, 2872, 1702, 1393; HRMS (FAB) calcd for
C15H28O3N
[(M+H)+] 270.1991, found 270.2054.
To a solution of the amine 7-2 (2.64 mg) in CH3CN (0.5 mL) was
5.21. tert-Butyl (R)-5-((R)-4-hydroxy-2-methylbutyl)-2,
added BnBr (20.0 lL, 0.168 mmol) and Et3N (22.5 lL, 0.162 mmol)
2-dimethyloxazolidine-3-carboxylate (29)
at room temperature. The resulting suspension was stirred at room
temperature for 12 h. The reaction mixture was quenched with a
saturated aqueous solution of NH4Cl, and basidified with a satu-
rated aqueous solution of NaHCO3. The aqueous layer was
extracted three times with EtOAc. Organic layer was washed with
brine, dried over Na2SO4, filtrated and concentrated under reduced
pressure. Purification by silica gel column chromatography (hex-
ane/EtOAc = 9:1) gave 1.58 mg (58% yield for 2 steps) of s-TZ 7-3
as colorless oil: 1H NMR (400 MHz, CDCl3) d 7.37–7.20 (m, 5H),
4.70 (s, 1H), 4.48 (m, 1H), 3.90 (d, J = 13.4 Hz, 1H), 3.76 (d,
J = 13.4 Hz, 1H), 3.03 (d, J = 9.8 Hz, 1H), 2.64 (dd, J = 9.8, 6.6 Hz,
1H), 2.14–2.02 (m, 1H), 1.40–1.34 (m, 2H), 1.25–1.17 (m, 2H),
0.87 (d, J = 6.6 Hz, 3H); IR (film, cmꢀ1) 2916, 2843, 1635, 1507;
To a solution of the terminal olefin 28 (120 mg, 0.445 mmol) in
THF (5 mL) was added 9-BBN-H (1.07 mL, 0.535 mmol) at 0 °C, and
the mixture was stirred at room temperature for 2 h. To this mix-
ture was added a saturated aqueous solution of NaHCO3 (15 mL)
and 30% H2O2 (7.5 mL) at 0 °C and the mixture was stirred at room
temperature for 1 h. The reaction mixture was quenched with a
saturated aqueous solution of Na2S2O3. The aqueous layer was
extracted three times with EtOAc. Organic layer was washed with
brine, dried over Na2SO4, filtrated and concentrated under reduced
pressure. Purification by silica gel column chromatography (hex-
ane/EtOAc = 3:2) gave 104 mg (81% yield) of the alcohol 29 as col-
24
orless oil: [
a]
D
ꢀ49.2 (c 0.094, CHCl3); 1H NMR (400 MHz, CDCl3)
HRMS (DART) calcd for
218.1560.
C
14H20ON [(M+H)+] 218.1467, found
d 4.12 (ddt, J = 10.2, 6.4, 5.6 Hz, 1H), 3.68 (br t, 2H), 3.62 (br dd, 1H),
2.98 (br dd, 1H), 1.73–1.36 (m, 20H), 0.95 (d, J = 6.3 Hz, 3H); IR
(film, cmꢀ1) 3431, 2932, 2875, 1701, 1393; HRMS (FAB) calcd for
5.26. s-TZ (7-4)
C
15H30O4N [(M+H)+] 288.2097, found 288.2190.
72% as colorless oil: 1H NMR (400 MHz, CDCl3) d 7.25 (d,
J = 8.0 Hz, 2H), 7.10 (d, J = 8.1 Hz, 2H), 4.70 (s, 1H), 4.48 (m, 1H),
3.86 (d, J = 13.4 Hz, 1H), 3.72 (d, J = 13.4 Hz, 1H), 3.03 (d,
J = 9.8 Hz, 1H), 2.64 (dd, J = 9.7, 6.4 Hz, 1H), 2.57 (t, J = 7.8 Hz,
2H), 2.14–2.02 (m, 1H), 1.62–1.54 (m, 9H), 1.25–1.17 (m, 6H),
0.92–0.87 (m, 6H); 13C NMR (100 MHz, CDCl3) d 128.24, 128.21,
93.2, 74.6, 61.9, 57.5, 41.2, 38.7, 35.3, 33.6, 23.0, 22.4, 21.6, 13.9;
IR (film, cmꢀ1) 2916, 2843, 1635, 1507; HRMS (DART) calcd for
C18H28ON [(M+H)+] 274.2093, found 274.2159.
5.22. tert-Butyl (R)-2,2-dimethyl-5-((S)-2-methyl-4-
oxobutyl)oxazolidine-3-carboxylate (30)
To a solution of the alcohol 29 (104 mg, 0.362 mmol) in CH2Cl2
(3.5 mL) was added MS4A (104 mg), NMO (127 mg, 1.08 mmol)
and TPAP (1 portion) at room temperature. The resulting suspen-
sion was stirred at room temperature for 30 min. Purification by
silica gel column chromatography (hexane/EtOAc = 7:1) gave
25
71.0 mg (69% yield) of the aldehyde 30 as colorless oil: [a]
D
ꢀ39.6 (c 0.12, CHCl3); 1H NMR (400 MHz, CDCl3) d 9.74 (s, 1H),
4.07 (ddd, J = 10.4, 6.4, 5.6 Hz, 1H), 3.71–3.58 (br dd, 1H), 3.05–
2.96 (br dd, 1H), 2.47 (d, J = 10.7 Hz, 1H), 2.27 (d, J = 10.6 Hz, 1H),
2.26–2.18 (m, 1H), 1.64–1.43 (m, 17H), 1.01 (d, J = 6.6 Hz, 3H); IR
5.27. s-TZ (7-5)
65% as colorless oil: 1H NMR (400 MHz, CDCl3) d 7.78 (dd,
J = 8.2, 5.8 Hz, 4H), 7.53 (d, J = 7.3 Hz, 1H), 7.46–7.40 (m, 2H),
Please cite this article in press as: Inoue, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.040

10
H. Inoue et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4.75 (s, 1H), 4.51 (m, 1H), 4.06 (d, J = 13.7 Hz, 1H), 3.91 (d,
J = 13.4 Hz, 1H), 3.03 (d, J = 9.8 Hz, 1H), 2.71 (dd, J = 9.7, 6.4 Hz,
1H), 2.16–2.08 (m, 1H), 1.25–1.17 (m, 4H), 0.92–0.87 (d,
J = 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 137.8, 133.3, 132.5,
127.9, 127.7, 127.6, 126.9, 126.7, 125.9, 125.5, 93.3, 77.2, 62.3,
57.5, 38.7, 30.9, 23.0, 21.8; HRMS (DART) calcd for C18H22ON
[(M+H)+] 268.1623, found 268.1713.
San Jose, CA, USA) was diluted with 50 mM Tris buffer (pH 7.4),
containing 100 mM NaCl and 5 mM CaCl2, to make a 1.11 mg/mL
solution. A 450
lL portion of the substrate collagen solution was
placed in a 1.5-mL microtube. A 50
l
L aliquot of each compound,
in 50 mM Tris buffer (pH 7.4) containing 100 mM NaCl and 5 mM
CaCl2, was added, and the solution was gently mixed by vortexing.
The resultant mixture was then incubated at 37 °C for 5 min. When
the sample solution became cloudy, the sample preparation was
performed again. The enzymatic reaction was started by adding
5.28. s-TZ (7-6)
5 lL of 2 mg/mL collagenase (from Clostridium histlyticum, 1000
Mandle units/mg, EC 3. 4. 24. 3, Lot. LTK2485, Wako, Osaka, Japan)
at 37 °C. After various time intervals (30, 60, 120, 180 min), an
80 lL aliquot was collected in a 1.5-mL microtube, and was stained
with 1.0 mL Sirius red dye solution (Biocolor, Carrickfergus, County
Antrim, UK). After gentle mixing for 30 min at room temperature,
the collagen-dye complex was centrifuged at 15,000 rpm and
4 °C for 10 min. The supernatant was then drained to remove the
unbound dye solution. The pellet was dissolved in 1.0 mL of
77% As colorless oil: 1H NMR (400 MHz, CDCl3) d 7.57 (dd,
J = 11.3, 8.5 Hz, 4H), 7.43–7.39 (m, J = 7.3 Hz, 5H), 7.46–7.40 (m,
2H), 4.75 (s, 1H), 4.51 (m, 1H), 4.06 (d, J = 13.7 Hz, 1H), 3.91 (d,
J = 13.4 Hz, 1H), 3.03 (d, J = 9.8 Hz, 1H), 2.71 (dd, J = 9.7, 6.4 Hz,
1H), 2.16–2.08 (m, 1H), 1.25–1.17 (m, 4H), 0.89 (d, J = 6.5 Hz,
3H); 13C NMR (100 MHz, CDCl3) d 207.0, 128.7, 127.0, 93.3, 87.7,
61.8, 58.2, 57.6, 54.3, 41.2, 38.7, 30.9, 29.7; HRMS (DART) calcd
for C20H24ON [(M+H)+] 294.1780, found 294.1860.
0.1 N NaOH with vigorous mixing, and then 200 lL of the mixture
5.29. s-TZ (7-7)
was transferred to a 96-well microtiter plate to measure the absor-
bance of the released dye at 540 nm, using a VARIOSKAN FLASH
microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
All measurements were performed more than 3 times, and average
values and errors were computed.
62% As colorless oil: 1H NMR (400 MHz, CDCl3) d 8.02 (s, 1H),
7.89 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.37 (t, J = 7.8 Hz,
1H), 4.69 (s, 1H), 4.50–4.48 (m, 1H), 3.95 (d, J = 13.4 Hz, 1H), 3.90
(s, 3H), 3.78 (d, J = 13.6 Hz, 1H), 3.03 (d, J = 9.8 Hz, 1H), 2.63 (dd,
J = 9.7, 6.4 Hz, 1H), 2.12–2.05 (m, 1H), 1.49–1.31 (m, 3H), 1.25–
1.18 (dt, J = 6.3, 2.4 Hz, 2H), 0.88 (d, J = 6.5 Hz, 3H); 13C NMR
(100 MHz, CDCl3) d 141.1, 133.6, 130.8, 130.1, 129.0, 128.8, 94.1,
75.3, 62.4, 58.2, 56.1, 53.8, 52.8, 41.4, 39.3, 23.7, 22.4; HRMS
(DART) calcd for C16H22O3N [(M+H)+] 276.1521, found 276.1589.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research on Priority Areas: Creation of Biologically Functional
Molecules (17035021 and 18032025), and The Global COE Program
for Chemistry Innovation through Cooperation of Science and
Engineering, from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, and SEKISUI CHEMICAL CO., LTD.
5.30. s-TZ (7-8)
77% As colorless oil: 1H NMR (400 MHz, CDCl3) d 7.78 (d,
J = 7.8 Hz, 1H), 7.50 (dt, J = 7.6, 1.5 Hz, 1H), 7.25 (m, 3H), 4.73 (s,
1H), 4.51–4.49 (m, 1H), 3.95 (d, J = 13.4 Hz, 1H), 3.80 (d,
J = 13.6 Hz, 1H), 3.61 (s, 3H), 3.07 (d, J = 9.8 Hz, 1H), 2.67 (dd,
J = 9.7, 6.4 Hz, 1H), 2.12–2.07 (m, 1H), 1.27–1.20 (dt, J = 6.3,
2.4 Hz, 2H), 0.88 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
175.9, 139.8, 139.2, 131.2, 130.1, 129.7, 128.2, 128.0, 127.1, 96.1,
93.4, 74.6, 61.9, 57.5, 51.9, 41.1, 38.6, 23.0, 21.8; HRMS (DART)
calcd for C22H26O3N [(M+H)+] 352.1834, found 352.1894.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.040.
References and notes
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ꢀ2.4 (c 0.152, CHCl3); 1H
24
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62% Of colorless amorphous: [
a]
D
NMR (400 MHz, CDCl3) d 4.51–4.49 (m, 1H), 3.19 (t, J = 14.6 Hz,
1H), 3.19 (d, J = 14.4 Hz, 1H), 2.52 (dd, J = 19.0, 2.2 Hz, 1H), 2.22
(d, J = 19.0 Hz, 1H), 2.30–2.20 (m, 1H), 2.06 (dd, J = 13.2, 5.1 Hz,
1H), 1.99 (dd, J = 14.2, 4.6 Hz, 1H), 1.91–1.64 (m, 6H), 1.63–1.49
(m, 3H), 1.41 (td, J = 13.2, 2.9 Hz, 1H), 1.25–1.10 (m, 1H), 1.17 (s,
3H), 1.05 (t, J = 12.7 Hz, 1H), 0.87 (s, 3H), 0.87 (t, J = 5.3 Hz, 3H);
13C NMR (100 MHz, CDCl3) d 173.6, 105.6, 90.2, 74.5, 47.7, 44.8,
41.1, 39.2, 35.9, 35.5, 30.5, 30.1, 25.3, 23.6, 23.3, 22.1, 18.8, 18.6;
IR (film, cmꢀ1) 2941, 1705, 1408, 1394, 1258; HRMS (DART) calcd
for C19H30O3N [(M+H)+] 320.2147, found 320.2243.
12. Hikage, H.; Furukawa, H.; Takao, K.; Kobayashi, S. Tetrahedron Lett. 1998, 39,
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5.32. Collagen protection assay
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The TZ analogs were dissolved in 1 N HCl. The resultant solution
was then passed through a syringe filter to remove the insoluble
impurities. After condensation, the weights of the hydrochloride
salts were accurately measured. Norzoanthamine and buffer only
were selected as positive and negative controls, respectively. Inci-
dentally, the NZ preserved in our laboratory was used. Acid solubi-
lized type I collagen from rat tail stock solution (BD Biosciences,
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Please cite this article in press as: Inoue, H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.040