Total synthesis of natural derivatives and artificial analogs of 13-oxyingenol and their biological evaluation

Article abstract of DOI:10.1039/c6ob02268e

We have established an efficient synthetic methodology for the 13-oxyingenol natural derivative (13-oxyingenol-13-dodecanoate-20-hexanoate), featuring a ring-closing olefin metathesis reaction for the “direct” construction of a highly strained inside-outside framework and a Mislow-Evans-type [2,3]-sigmatropic rearrangement for the stereoselective introduction of the hydroxy group at C5. We also synthesized artificial analogs of 13-oxyingenol and ingenol by using our synthetic strategy. In vitro activation assays of protein kinase C (PKC) α and δ revealed that the dodecanoyl group at O13 on 13-oxyingenol analogs had a significant role in PKCδ activation. The PKCα- or PKCδ-activating 13-oxyingenol and ingenol analogs induced both distinct morphological changes and increases of CD11b expression in HL-60 cells, which would be typical signs of HL-60 cell differentiation to macrophage-like cells, as expected by previous reports. Intriguingly, however, similar differentiation phenotypes were observed with the use of 13-oxyingenol natural derivatives and 13-oxyingenol-13-dodecanoate showing a remarkably less potent PKCα or PKCδ activation ability, which the PKC inhibitor G?6983 diminished. This indicated the involvement of other PKC isozymes or related kinase activities. 13-Oxyingenol analogs, which induced HL-60 cell differentiation, also induced HL-60 cell death, similar to the action of a phorbol ester, a strong PKC activator.

Full text of DOI:10.1039/c6ob02268e

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Takeharu Haino et al.
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Total synthesis of natural derivative and artificial analogs of 13-
oxyingenol and their biological evaluation
Takayuki Ohyoshi,a Yuki Tamura,b Ichiro Hayakawa,*c Go Hirai,*b,d,e,f Yamato Miyazawa,a Shota
Funakubo,a Mikiko Sodeoka*,b,d,e and Hideo Kigoshi*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We have established an efficient synthetic methodology for 13-oxyingenol natural derivative (13-oxyingenol 13-
dodecanoate 20-hexanoate), featuring a ring-closing olefin metathesis reaction for the "direct" construction of a highly
strained inside–outside framework and a Mislow–Evans-type [2,3]-sigmatropic rearrangement for the stereoselective
introduction of the hydroxy group at C5. We also synthesized artificial analogs of 13-oxyingenol and ingenol by using our
synthetic strategy. In vitro activation assays of protein kinase C (PKC) α and δ revealed that the dodecanoyl group at O13
on 13-oxyingenol analogs had a significant role in PKCδ activation. The PKCα- or PKCδ-activating 13-oxyingenol and ingenol
analogs induced both distinct morphological changes and increases CD11b expression in HL-60 cells, which would be
typical signs of HL-60 cell differentiation to macrophage-like cells, as expected by previous reports. Intriguingly, however,
similar differentiation phenotypes were observed with the use of 13-oxyingenol natural derivatives and 13-oxyingenol 13-
dodecanoate showing a remarkably less potent PKCα or PKCδ activation ability, which PKC inhibitor Gö6983 diminished.
This indicated the involvement of other PKC isozymes or related kinase activities. 13-Oxyingenol analogs, which induced
HL-60 cell differentiation, also induced HL-60 cell death, similar to the action of a phorbol ester, a strong PKC activator.
www.rsc.org/
effect of 3 (5,200,000 pM) or of anti-HIV drug zidovudine
(Retrovir®) (4,100 pM) under the same assay conditions.
Introduction
13-oxyingenol 13-dodecanoate 20-hexanoate (13-oxyingenol
natural derivative; 13-oxyingenol derivative) (
from Euphorbia kansui plants by Uemura et al. (Figure 1).1 The
structural features of is a high degree of oxygenation and a
1) was isolated
1
highly strained unique bicyclo[4.4.1]undecane skeleton with
trans intrabridgehead stereochemistry at the BC rings, which is
called an inside–outside framework. Ingenol (
3
) has the same
framework of 1 2 and ingenol 3-angelate (Picato®) (
,
4
) was
approved by the FDA as a topical treatment for solar
keratosis.3 In 1996, Fujiwara et al. reported the structure–
activity relationship study of 1.4 As a results, a 13-oxyingenol
analog, RD4-2227 (2), showed a potent inhibitory effect at
picomolar concentrations on HIV-1 (HTLV-IIIB) replication in
MT-4 cells (EC50 = 140 pM). This is stronger than the inhibitory
Fig. 1 Structures of 13-oxyingenols and ingenols.
a.Department of Chemistry, Graduate School of Pure and Applied Sciences,
University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8571, Japan. E-mail:
kigoshi@chem.tsukuba.ac.jp
Ingenol analogs are reported to be involved in the modulation
of protein kinase C (PKC) enzyme activity.5 PKCs are a family of
serine/threonine kinases and are key mediators of many
intracellular signal transduction pathways involved in cell
growth, differentiation, carcinogenesis and cell death.6 PKCs
are categorized into three classes: conventional (cPKCs), novel
(nPKCs), and atypical (aPKCs). The binding of ligands such as
endogenous second messenger diacylglycerol (DAG) and
tumor promoter phorbol esters (e.g., phorbol 12-myristate 13-
acetate; PMA) to the C1 domain in cPKCs and nPKCs, but not in
b.Synthetic Organic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama
351-0198, Japan. E-mail: gohirai@riken.jp; sodeoka@riken.jp
c. Division of Applied Chemistry, Graduate School of Natural Science and
Technology, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-
8530, Japan. E-mail: ichiro.hayakawa@okayama-u.ac.jp
d.RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama
351-0198, Japan
e. AMED-CREST, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
f. Present address: Graduate School of Pharmaceutical Sciences, Kyushu University,
Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
† Electronic Supplementary Informaꢀon (ESI) available: Experimental protocols,
characterization of data and NMR spectra of all new compounds. See
DOI: 10.1039/x0xx00000x
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aPKCs, triggered the enhancement of enzymatic activity. The portion by using ring-opening reaction of the epoxy group in 5.
crystal structure of the PKCδ C1 domain with phorbol ester To introduce the requisite functional groups at the A-ring
revealed crucial hydrogen bonding interactions between the portion, we decided to use the introduced hydroxy group at
hydrophilic cleft of the C1 domain and phorbol esters.7 Some C2. The secondary hydroxy group at the C5 in the B-ring
ingenol derivatives, especially those that have an acyl group on portion would be introduced by using regio- and
the hydroxyl group at C3, were reported to be C1 domain stereoselective epoxidation of allylic alcohol
ligands and to activate PKC, which was explained by their subsequent ring-opening reaction of the epoxy group in
structural similarities with phorbol esters. For 13-oxyingenol Allylic alcohol
analogs, RD4-2227 (
) was identified as a potent nontoxic PKC dihydroxylation at the most strained ∆3,4 olefin in triene
activator (no toxicity was observed in mice up to 0.1 mg kg– which might be obtained from tetracyclic ketone
1day–1 by intraperitoneal injection).4a Non-tumor-promoting outside framework of
was prepared by using an RCM
PKC activators, such as bryostatin 1,8 prostratin,9 and reaction of diene
. Diene
6 and the
5.
6
was prepared by regio- and stereoselective
2
7,
8. The inside–
8
9
9
can be synthesized from spiro-
aplysiatoxin analogs,10 have been attracting attention as ketone 10
potential therapeutic agents for acquired immune deficiency
syndrome (AIDS), tumors, and Alzheimer's disease. Thus, the
activities of 13-oxyingenol analogs, which have an additional
functionalization site at the C13 position in 13-oxyingenol as
compared to ingenol analogs, have been of great interest.
.
TBSO
O
epoxide
opening
regioꢀ and stereoꢀ
selective epoxidation
H
13ꢀoxyingenol
However, the structure‒activity relationships of 13-oxyingenol
B
derivative (1)
5
derivatives are still largely unknown, which hampers further
rational molecular design and obscures the merit of 13-
oxyingenols for use as parent compounds. We anticipated that
BzO
HO
O
OTBS
5
TBSO
O
TBSO
the development of an efficient synthetic methodology for
1
would enable us to provide a practical synthetic route for
research supply of its analogs and to conduct further biological
O
regioꢀ and stereoꢀ
selective dihydroxylation
H
H
studies in order to uncover the characteristic features of 1.
4
3
The unique stereostructures of the ingenol family have made
them attractive targets for total synthesis.11 Four groups have
BzO
HO
5
OTBS
OTBS
TBSO
achieved the total synthesis of ingenol (3
): Winkler,12 Tanino–
6
7
Kuwajima,13 Wood,14 and Baran15. Also, several groups have
reported the synthesis of the ingenol skeleton.16 Whereas
many groups have constructed the highly strained inside–
TBSO
O
MPMO
MPMO
H
H
RCM
2
outside framework of
3 by using rearrangement or
fragmentation reactions, we developed a "direct" construction
method using the ring-closing olefin metathesis (RCM)
reaction.17 We then achieved the formal synthesis of optically
O
H
H
OTBS
OTBS
8
9
active
3
using this methodology as a key step.18 Wood et al.
TBSO
accomplished the total synthesis of ingenol by using similar
approach in 2004.14 We also have investigated the synthesis of
13-oxyingenol derivative
(
1
).19 In 2012, we preliminarily
The highlights of this
MPMO
A
reported the total synthesis of 1 20
.
O
approach are the use of an RCM reaction for the direct
construction of an inside–outside framework and a Mislow–
Evans-type [2,3]-sigmatropic rearrangement21 for the
stereoselective introduction of a hydroxy group at C5. We
describe herein the history of the total synthesis of 13-
H
10
Scheme 1 Retrosynthetic pathway of 13-oxyingenol derivative (1).
oxyingenol derivative
(1). Furthermore, we discuss the
The synthesis of epoxide 5 started from the spiro-ketone 10,
which is our previous intermediate (Scheme 2).19c Thus, the
introduction of an allyl group to the C8 position of spiro-
synthesis and activities against PKC of the C3-acyloxy 13-
oxyingenol and ingenol analogs.
ketone 10 with iodide 11 gave diene
9. Diene 9 was treated
with the second-generation Hoveyda–Grubbs (HG-II) catalyst
22
(
37)
to afford tetracyclic ketone 8, which possessed the
Results and disscussion
inside–outside framework. We attempted the stereoselective
Construction of the A-ring portion of 13-oxyingenol derivative (1)
introduction of the functional groups at the A- and B-rings
from
8. The removal of the MPM group of 8 followed by
The retrosynthetic pathway of 13-oxyingenol derivative (1) is
oxidation of the resulting secondary hydroxy group with Dess–
shown in Scheme 1. We planned to construct the B-ring
2 | J. Name., 2012, 00, 1-3
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TBSO
TBSO
Martin periodinane23 gave diketone 12. We next tried to
synthesize enone 13 from 12. Regioselective deprotonation at
the H3 in 12 and silylation of the resultant enolate with TMSCl
gave an enol silyl ether as the sole product, which was oxidized
by using Ito–Saegusa oxidation24 to afford 13 (95% yield in two
steps). In this deprotonation of 12, deprotonation at the H1
did not proceed, presumably due to the steric hindrance of the
methyl group at the C18. Enone 13 was converted into an enol
triflate, which was subjected to the Negishi coupling25 reaction
8
8
MPMO
H
MPMO
a
O
H
O
OTBS
I
H
OTBS
11
10
9
TBSO
TBSO
O
18
O
with Me2Zn to afford triene
7. Regio- and stereoselective
b
c, d
1
MPMO
O
H
H
dihydroxylation of 7 with a stoichiometric amount of OsO4
3
gave diol 14. The high regio- and stereoselectivity could be
explained by the shielding of the ∆1,2 olefin by the steric
hindrance of the C18 methyl group and by the enhancement of
the reactivity by the pyramidal distortion of ∆3,4 olefin. We
succeeded in synthesizing the A-ring portion of 13-oxyingenol
H
H
OTBS
OTBS
8
12
TBSO
O
TBSO
O
derivative (
attempted to functionalize the B-ring portion of
of the secondary hydroxy group of 14 with a benzoyl group
gave allylic alcohol , which was converted into epoxide as a
1) by using the C2 hydroxy group. Next, we
1
2
O
H
H
1. Protection
e, f
g, h
4
3
6
5
OTBS
OTBS
single isomer. In this epoxidation, stereoselectivity was
controlled by the tertiary hydroxy group at the C4 and the cage
13
7
TBSO
TBSO
13
15
structure of 6. The stereochemistry of 5 was established by X-
18
O
O
ray crystallographic analysis of crystalline p-bromobenzoate 15
11
i
k
(Figure 2).
H
H
8
10
3
4
4
6
5
HO
HO
RO
RO
OTBS
O
OTBS
l, m
14 R = H
6 R = Bz
5 R = Bz
15 R = ꢀBrBz
j
p
Scheme 2 Synthesis of epoxide
HMPA, –10 °C, 90%; (b) HG-II
5
(
. Reagents and conditions: (a) 11, LDA, THF,
37), toluene, reflux, 98%; (c) DDQ, tBuOH,
phosphate buffer (pH = 6.6), CH2Cl2, rt, 61%; (d) Dess–Martin periodinane,
CH2Cl2, rt, 87%; (e) TMSCl, LHMDS, Et3N, THF, –78 °C; (f) Pd(OAc)2, MeCN, rt, 95%
in two steps; (g) Tf2NPh, LHMDS, THF, 0 °C; (h) Pd(PPh3)4, Me2Zn, THF, rt, quant
in two steps; (i) OsO4, py, THF, 0 °C, then NaHSO3 aq, rt, 99%; (j) BzCl, Et3N,
DMAP, CH2Cl2, 0 °C, 94%; (k) tBuOOH, VO(acac)2, benzene, rt, quant; (l) K2CO3,
MeOH, rt; (m) p-BrBzCl, Et3N, DMAP, CH2Cl2, 83% in two steps. TBS = tert-
butyldimethylsilyl, LDA = lithium diisopropylamide, THF = tetrahydrofuran, HMPA
= hexamethylphosphoramide, DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone,
TMS
=
trimethylsilyl, Ac
=
acetyl, Ph
=
phenyl, LHMDS
=
lithium
= N,N-
bis(trimethylsilyl)amide, Tf
dimethylaminopyridine, Bz = benzoyl, acac = acetylacetonyl.
=
trifluoromethanesulfonyl, DMAP
Fig. 2 X-ray crystallographic structure of 15 (CCDC 1508569)
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With a fully substituted tetracyclic compound
5 in hand, we
Table 1 Ring-opening reaction of epoxide 5
next tried the ring-opening reaction of the epoxy group in
5
(Table 1). In entries 1 and 2, the ring-opening reaction of the
epoxy group was investigated by using TfOH as a Brønsted acid
or Al(OiPr)3 as a Lewis acid.26 However, the desired compound
16 was not obtained. Next, the ring-opening reaction was
examined by using TMSOTf and DBU (entry 3).27 Again,
however, the reaction did not give 16, but rather undesired
compounds 17 and 18. The production of these undesired
compounds can be explained as follows (Scheme 3). First, the
TBSO
TBSO
O
O
19
H
9
H
conditions
6
HO
BzO
HO
BzO
O
OTBS
HO
OTBS
20
5
16
carbonyl group at the C9 in
5 was activated by TMSOTf as 20,
entry
conditions
result
and allylic cations 21 and 22 were generated by the migration
of blue or green bonds, respectively. The methyl proton at the
C19 in the resulting cations 21 and 22 were removed by DBU
to give compounds 17 and 18, respectively. Because the
desired epoxide-opening reaction under acidic conditions did
not afford 16, we next investigated the reaction under basic
1
2
3
4
TfOH, CH2Cl2, 0 °C
decomposition
decomposition
Al(OiPr)3, toluene, reflux
TMSOTf, 2,6ꢀlutidine, DBU, rt
LDA, THF, 0 °C
mixture of compounds 17 and 18
complex mixture
conditions (entries 4 and 5). The ring-opening reaction of
with LDA was attempted, but 16 was not obtained (entry 4).
This reaction afforded complex mixture containing
byproducts, such as those that seemed to form via the allylic
5
5
6
N
Al
, toluene, 0 °C decomposition
a
Cp2TiCl, THF, rt
compound 19
anion at the C19 in 5. In entry 5, the treatment of 5 by an
aluminum amide28 did not give the desired ring-opening
O
OTBS
compound 16. We next attempted the ring-opening reaction
TMSO
under radical conditions (entry 6). Treatment of
5 with
O
Cp2TiCl29 gave only the undesired compound 19 and not the
desired 16. A plausible reaction pathway for the formation of
19 is shown in Scheme 4. The generated tertiary radical at the
BzO
TMS
O
HO
BzO
O
OTBS
OH
OTBS
C6 in 23 attacked to the carbonyl group to give oxy-radical 24
.
TBSO
OR
19
The fragmentation of 24 and the subsequent ring-opening
reaction of the cyclopropane ring formed compound 19. The
structure of 19 was determined by X-ray crystallographic
analysis.30 Despite these attempts, we could not establish
reaction conditions for the ring-opening reaction of the epoxy
17
H
O
OR
group in
constructing the A- and B-ring portions of 13-oxyingenol
derivative ( ).
5. Therefore, we reexamined the strategy of
BzO
OTBS
18
R = TMS
1
DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, Cp = cyclopentadienyl.
4 | J. Name., 2012, 00, 1-3
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using Mislow–Evans-type [2,3]-sigmatropic rearrangement.21,31
This strategy has the benefit of constructing the ∆6,7 olefin and
the C5 stereocenter at once. This precursor 27 might be
obtained from alcohol 28 by SN2 reaction with a selenide anion
at the C7. Alcohol 28 was synthesized by an aldol reaction
between 10 and the unsaturated aldehyde 30 for the
introduction of the hydroxy group at the C7 and the ring-
TBSO
O
TMS
H
BzO
O
O
OTBS
TMS
20
closing olefin metathesis reaction of the resultant 29
.
OTBS
TBSO
OR
TBSO
O
TMSO
19
functionalization
of Aꢀring portion
H
O
H
H
MPMO
13ꢀoxyingenol
derivative (1)
A
H
OTBS
7
BzO
TMS
O
5
19
O
OTBS
6
OR
BzO
HO
OMPM
22
21
26
R = TMS
TBSO
O
DBU
DBU
Mislow–Evans type
[2,3]ꢀsigmatropic
rearrangement
OTBS
TBSO
OR
MPMO
H
TMSO
O
SeAr
H
O
H
H
OMPM
27
OTBS
TBSO
TBSO
BzO
O
OTBS
OR
TMS
BzO
O
17
18
R = TMS
MPMO
H
MPMO
H
RCM
Scheme 3 Proposed reaction mechanism for the formation of compounds 17 and
18.
OH
7
OH
7
O
H
H
OMPM
OHC
OMPM
28
29
TBSO
O
TBSO
O
OMPM
aldol reaction
10
+
H
H
30
Scheme 5 Modified retrosynthetic pathway of 13-oxyingenol derivative (
H
6
1).
BzO HO
TiO
BzO HO
TiO
OTBS
OTBS
23
24
We prepared precursors for the RCM reaction, such as 29
,
O
TBSO
O
from spiro-ketone 10 (Scheme 6). The aldol reaction between
10 and unsaturated aldehyde 3032 gave allylic alcohol 29 as a
single diastereomer. The stereochemistry of C7 in 29 was
determined after the RCM reaction. Aldol 29 was converted
into acetate 31, TMS ether 32, and unsaturated ketone 33 as
the precursors for the RCM reaction.
ringꢀopening
reaction of
cyclopropane
O
OTBS
OTBS
HO
BzO
HO
BzO
OH
OH
19
25
Scheme 4 Proposed reaction mechanism of formation of compound 19
.
Construction of the B-ring portion of 13-oxyingenol derivative (1)
The modified retrosynthetic pathway of 13-oxyingenol
derivative (1) is shown in Scheme 5. As mentioned above, we
established the functionalization of the A-ring portion. In this
section, we mainly describe the construction of the B-ring
portion of 1
. We planned to introduce the ∆6,7 olefin and chiral
secondary hydroxy group at the C5 in the B-ring portion by
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Table 2 Optimization of RCM reaction
TBSO
TBSO
TBSO
O
TBSO
MPMO
MPMO
H
a
OH
7
OMPM
TBSO
MPMO
conditions
reflux
O
O
MPMO
H
H
R1
R2
R1
OHC
H
H
R2
OMPM
O
H
10
29
H
30
OMPM
OMPM
29 R1 = H, R2 = OH
31 R1 = H, R2 = OAc
32 R1 = H, R2 = OTMS
33 R1 = R2 = O
28 R1 = H, R2 = OH
34 R1 = H, R2 = OAc
35 R1 = H, R2 = OTMS
36 R1 = R2 = O
MPMO
H
R1
b, c, or d
7
R2
O
H
precursor
entry
catalyst
of RCM
solvent
yield
OMPM
31 R1 = H, R2 = OAc
32 R1 = H, R2 = OTMS
33 R1 = R2 = O
toluene
toluene
toluene
benzene
1
2
29
31
32
31
37
37
37
37
54%
64%
Scheme
6 Syntheses of precursors for the RCM reaction. Reagents and
3
4
49%
0%
conditions: (a) 30, NaHMDS, THF, –78 °C; (b) Ac2O, DMAP, py, 50 °C, 83% in two
steps for 31; (c) TMSCl, imidazole, DMF, rt, 47% in two steps for 32; (d) Dess–
Martin periodinane, CH2Cl2, rt, 81% in two steps for 33. MPM = p-methoxybenzyl,
NaHMDS = sodium bis(trimethylsilyl)amide, DMF = N,N-dimethylformamide.
5a)
6
31
31
31
31
33
1,2ꢀdichlorobenzene
trifluorobenzene
37
37
25%
0%
Next, the RCM reactions of the precursors were optimized
toluene
toluene
toluene
7
38
39
37
51%
0%
(Table 2). We examined the RCM reactions of precursors 29
and 31–
33 with HG-II catalyst (37).22 First, the RCM reaction of
8
9
86%
allylic alcohol 29 was attempted, but the yield was moderate
(54%) (entry 1). Because this reaction gave spiro-ketone 10, we
supposed that a retro-aldol reaction occurred under this RCM
condition. We next investigated the RCM reactions with
acetate 31 and the TMS ether 32. However, the yields were
still moderate (entry 2: 64%, entry 3: 49%). These results
indicated that the RCM reaction was most efficiently effected
by using 31. Therefore, we next screened solvents by using 31
as a precursor. In entry 4, we attempted the RCM reaction in
boiling benzene, but the yield was low. Halogenated aromatic
solvents, such as 1,2-dichlorobenzene (reaction at 120 °C) and
trifluorobenzene (reaction at reflux) (entries 5 and 6) did not
improve the yields. We also screened an assortment of Ru
catalysts. The RCM reaction with highly active Ru catalyst 3833
afforded tetracyclic ketone 34 in 51% yield (entry 7), whereas
the RCM reaction with the less-hindered Ru catalyst (Stewart–
Grubbs catalyst) 3934 did not yield the desired compound 34
(entry 8). These results indicated that the use of acetate 31
cannot improve the yield of the RCM reaction. Next, we tried
the RCM reaction with α,β-unsaturated ketone 33 as a
precursor, because the steric hindrance around the C7 of the
α,β-unsaturated ketone group at C7 is smaller in 33 than in
acetate 31 and because the reactivity of the enone is suitable
for the RCM reaction. The reaction of α,β-unsaturated ketone
33 with HG-II catalyst (37) in boiling toluene proceeded
smoothly to afford the desired tetracyclic ketone 36 in 86%
yield (entry 9). On the basis of these results, we established an
optimized precursor and reaction conditions for the RCM
reaction.
a) This reaction was carried out at 120 °C.
N
N
N
N
N
N
Mes
Mes
Mes
Mes
oꢀTol
oꢀTol
Cl
Cl
Cl
Cl
Cl
Cl
Ru
O
Ru
O
Ru
NO2
O
37
38
39
Mes = mesityl, Tol = p-tolyl.
With tetracyclic ketone 36, which has an oxygen functional
group at the C7, in hand, we examined the construction of the
B-ring portion. Reduction of the carbonyl group at the C7 in 36
proceeded from the convex face in a highly stereoselective
manner to give allylic alcohol 28 (Scheme 7). Mesylation of the
resultant hydroxy group of 28 by the Tanabe method35
afforded mesylate 40, and the introduction of a phenylselenyl
group by the selenide anion prepared from (PhSe)2 and NaBH4
gave selenide 41. Oxidation of 41 by m-CPBA gave a
selenoxide, which was treated with P(OMe)3 to afford [2,3]-
sigmatropic rearrangement product 26. Thus, we established
the construction of the B-ring portion by using [2,3]-
sigmatropic rearrangement as a key step.
6 | J. Name., 2012, 00, 1-3
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ARTICLE
TBSO
TBSO
O
TBSO
TBSO
O
O
O
MPMO
HO
H
H
a
a
MPMO
MPMO
H
2
H
b, c
O
OH
H
H
H
H
OH
HO
HO
OMPM
OMPM
TBSO
OMPM
36
28
TBSO
42
26
TBSO
O
TBSO
O
O
O
MPMO
MPMO
H
H
O
H
O
H
d, e
OMs
A
b
c
SePh
H
H
H
OMPM
OMPM
O
40
41
O
O
O
TBSO
O
43
44
desired
MPMO
H
d, e
TBSO
B
H
HO
OMPM
OH
H
26
Scheme 7 Synthesis of allylic alcohol 26. Reagents and conditions: (a) DIBAL,
toluene, –78 °C, 96%; (b) MsCl, Me2N(CH2)3NMe2, toluene, 0 °C; (c) (PhSe)2,
NaBH4, THF, EtOH, rt, 96% in two steps; (d) m-CPBA, THF, –78 °C; (e) P(OMe)3,
O
MeOH,
0 °C, 76% in two steps. Ms = methanesulfonyl, m-CPBA = m-
chloroperbenzoic acid.
O
O
45
undesired
Scheme 8 Attempt to functionalize the A-ring portion. Reagents and conditions:
Next, we tried to construct the A-ring portion of 13-oxyingenol
derivative ( ) using a similar strategy as that shown in Scheme
1
(a) DDQ, tBuOH, phosphate buffer (pH
= 6.6), CH2Cl2, rt, 52%; (b) 2-
2. Removal of two MPM groups in 26 afforded triol 42 (Scheme
8). The 1,3-diol group in 42 was protected as an acetonide, and
the remaining secondary hydroxy group at the C2 was oxidized
methoxypropene, PPTS, benzene, rt; (c) Dess–Martin periodinane, PPTS, CH2Cl2,
rt, 90% in two steps; (d) TMSCl, LHMDS, Et3N, THF, –78 °C; (e) Pd(OAc)2, MeCN,
rt. PPTS = pyridinium p-toluenesulfonate.
by Dess–Martin periodinane to give diketone 43 36
We next
.
attempted the Ito–Saegusa oxidation of 43. However, we did
not obtain the desired enone 44, but rather an undesired
We can interpret the mechanism underlying the undesired
reaction in the following way (Scheme 9): deprotonation of H3
in diketone 43 gave anion 46, the fragmentation of which
afforded enolate 47. Enolate 47 was then transformed into
dienolate 48, which was converted into 45 by an electrocyclic
reaction. We thought that this undesired reaction was due to
the highly strained pentacyclic skeleton of 43. Therefore, we
tried to convert the acetonide group into another protecting
group to decrease the strain.
compound 45
.
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Journal Name
TBSO
O
TBSO
TBSO
O
TBSO
O
O
9
O
O
MPMO
HO
H
H
H
H
deprotonation
a, b
c, d
3
3
B
H
H
O
O
HO
OMPM
TESO
OH
O
O
26
49
TBSO
TBSO
O
43
46
TBSO
TBSO
O
O
e, f
O
g, h
H
H
O
O
O
O
H
H
enolization
H
TESO
OTBS
TESO
OTBS
51
50
O
O
TBSO
TBSO
O
O
O
O
47
48
i
H
H
4
4
7
3
7
3
TBSO
O
TBSO
HO
TESO
HO
6
6
TESO
OTBS
OTBS
52
53
desired
OH
H
H
electrocyclic
reaction
TBSO
O
O
O
O
O
O
O
H
7
OH
45
48
OH
OTBS
Scheme 9 Proposed reaction mechanism of formation of compound 45
.
6
TESO
54
undesired
76%
Protection of the secondary hydroxy group in 26 with a TES
group and removal of two MPM groups afforded diol 49
(Scheme 10). The primary alcohol in 49 was protected as a TBS
group, and the remaining secondary hydroxy group at the C2
Scheme 10 Study of functionalization of the A-ring portion. Reagents and
conditions: (a) TESOTf, 2,6-lutidine, CH2Cl2, 0 °C, 89%; (b) DDQ, tBuOH, phosphate
buffer (pH = 6.6), CH2Cl2, rt, 84%; (c) TBSCl, Et3N, DMAP, CH2Cl2, rt, quant; (d)
Dess–Martin periodinane, CH2Cl2, rt, 91%; (e) TMSCl, LHMDS, Et3N, THF, –78 °C;
(f) Pd(OAc)2, MeCN, rt, 50% in two steps; (g) Tf2NPh, LHMDS, THF, 0 °C; (h)
Pd(PPh3)4, Me2Zn, THF, rt, 77% in two steps; (i) OsO4, THF, py, 0 °C then NaHSO3,
rt. TES = triethylsilyl, py = pyridine.
was oxidized by Dess–Martin periodinane to give diketone 50
.
Diketone 50 was oxidized to enone 51 by using Ito–Saegusa
oxidation. In this case, a rearrangement compound, such as
compound 45, was not afforded. Enone 51 was converted into We found it difficult to introduce functional groups at the A-
an enol triflate, which was transformed into triene 52 by using ring portion after the construction of the B-ring portion. Thus,
Negishi coupling. Next, the regio- and stereoselective we next tried to construct the A-ring portion before
dihydroxylation of 52 with a stoichiometric amount of OsO4 constructing the B-ring portion. Removal of two MPM groups
was carried out. However, the desired diol 53 was not in 34 and subsequent hydrolysis of the acetyl group gave a
obtained, but rather the undesired compound 54, which was triol, which was converted into acetonide 55 (Scheme 11).
oxidized at the ∆6,7 olefin, was obtained. We thought that Acetonide 55 was transformed into diol 56 by using our
steric hindrance of the TES group in 52 interfered with the established synthetic strategy (Scheme 2). Protection of the
electrophilic attack of OsO4 at the ∆3,4 olefin.
diol group in 56 with a cyclic carbonate group and removal of
the TBS group gave a diol, which was transformed into alcohol
57 by selective silylation of the primary hydroxy group. We
next examined the introduction of a phenylselenyl group at
the C7 in 57. The mesylation of 57 afforded mesylate 58, which
was treated with (PhSe)2 and NaBH4. However, selenide 59
was not obtained. This suggested that steric hindrance of the
cyclic carbonate group in 58 interfered with the nucleophilic
attack by a selenide anion.
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group at the C7 (Scheme 12). Two MPM groups in selenide 41
were removed by using DDQ to afford diol 60. Selective
protection of the primary hydroxy group of 60 with a TBS
group and the remaining secondary hydroxy group at the C2
was oxidized by Parikh–Doering oxidation37 to give diketone
62. Other oxidation conditions, such as Dess–Martin oxidation
and Ley–Griffith oxidation,38 gave Mislow–Evans-type
rearrangement byproducts along with desired 62. Diketone 62
was oxidized into enone 63 by using Ito–Saegusa oxidation.
Enone 63 was treated with LHMDS and Tf2NPh to form an enol
triflate, which was subjected to Negishi coupling to afford
triene 64. Regio- and stereoselective dihydroxylation of the
TBSO
TBSO
O
O
a–c
MPMO
HO
H
H
OAc
OMPM
TBSO
O
H
H
O
34
55
TBSO
O
O
8%
in 6 steps
H
H
d–f
4
7
O
OH
∆
3,4 olefin in triene 64 with a stoichiometric a mount of OsO4
HO
HO
O
O
O
OTBS
gave the desired diol 65. During conversion of selenide 41 into
65, the phenylselenyl group was not affected by oxidizing
agents, such as DDQ, SO3·py–DMSO, and OsO4. We next
examined the introduction of the C5 hydroxy group by using a
Mislow–Evans-type rearrangement. The oxidation of the
aromatic selenide group in 65 using m-CPBA and subsequent
[2,3]-sigmatropic rearrangement with P(OMe)3 afforded triol
66. Thus, both the A- and B-ring portions of 13-oxyingenol
56
57
O
TBSO
O
TBSO
O
H
H
g
h
4
7
OMs
SePh
O
O
O
O
OTBS
OTBS
58
59
derivative (1) were successfully constructed. The removal of
O
O
two TBS groups in 66 afforded 13-oxyingenol (67), and four
hydroxy groups at the A- and B-ring portions of 67 were
Scheme 11 Attempt to functionalize the B-ring portion. Reagents and conditions:
(a) DDQ, tBuOH, phosphate buffer (pH = 6.6), CH2Cl2, rt; (b) 1 M LiOH aq, THF,
MeOH, rt; (c) 2-methoxypropene, PPTS, benzene, rt, 87% in three steps; (d)
carbonyl diimidazole, benzene, rt; (e) PPTS, EtOH, 50 °C; (f) TBSCl, Et3N, DMAP,
CH2Cl2, rt, 60% in three steps; (g) MsCl, Me2N(CH2)3NMe2, toluene, 0 °C; (h)
(PhSe)2, NaBH4, THF, EtOH, rt to reflux.
protected as two acetonide groups to afford compound 68
.
The introduction of the dodecanoyl ester group at the
remaining tertiary hydroxy group at C13 in 68 gave 69, which
was hydrolyzed into 13-oxyingenol 13-dodecanoate
Finally, selective acylation of the primary hydroxy group in
tetraol 70 formed 13-oxyingenol derivative ( ). The spectral
data (1H NMR and 13C NMR spectroscopy, HRMS, and optical
rotation) of synthetic were in full agreement with those of
(70).
Total synthesis of 13-oxyingenol derivative (1)
1
As described above, we constructed each of the A- and B-ring
portions of 13-oxyingenol. However, we were unable to
construct both of A- and B-rings in a molecule (Schemes 8 and
10). Therefore, we attempted to construct the A-ring portion
at the next stage of the introduction of an aromatic selenyl
1
the natural product, thus completing the total synthesis (1.4%
overall yield in 41 steps).
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Journal Name
TBSO
O
TBSO
O
TBSO
O
TBSO
O
d, e
H
a
b
c
MPMO
HO
HO
O
H
H
H
SePh
SePh
SePh
SePh
H
H
H
H
OMPM
OH
OTBS
OTBS
41
60
61
62
TBSO
O
TBSO
O
TBSO
TBSO
O
O
O
f, g
h
i, j
k
H
H
H
H
A
B
SePh
SePh
SePh
HO
HO
HO
HO HO
OTBS
OTBS
OTBS
OTBS
63
64
65
66
HO
HO
C11H23COO
O
C11H23COO
O
C11H23COO
13
O
O
O
H
H
H
n
H
H
o
l
m
HO HO
HO
HO HO
HO
HO
HO
HO
O
O
O
O
O
O
OH
OH
OCOC5H11
O
O
67
70
13ꢀoxyingenol derivative (1)
68
Scheme 12 Total synthesis of 13-oxyingenol derivative (
69
t
). Reagents and conditions: (a) DDQ, BuOH, phosphate buffer (pH = 6.6), CH2Cl2, rt, quant; (b) TBSCl, Et3N,
1
DMAP, CH2Cl2, rt, 97%; (c) SO3
THF, –40 °C; (g) Pd(PPh3)4, Me2Zn, THF, rt; (h) OsO4, py, THF, then NaHSO3, rt, 64% in three steps; (i) m-CPBA, THF, –78 °C; (j) P(OMe)3, MeOH, 0 °C, 61% in two steps;
·py, DMSO, Et3N, CH2Cl2, rt, quant; (d) TMSCl, LHMDS, Et3N, THF, –78 °C; (e) Pd(OAc)2, DMSO, rt, 64% in two steps; (f) Tf2NPh, LHMDS,
(k) HF·py, THF, py, rt, quant; (l) 2,2-dimethoxypropane, PPTS, CH2Cl2, rt; (m) C11H23CO2H, EDCl, DMAP, CH2Cl2, rt, 63% in two steps; (n) 1 M HCl, THF, rt, 99%; (o)
(C5H11CO)2O, Et3N, CH2Cl2, –20 °C, 85%. DMSO = dimethylsulfoxide, EDCl = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.
Synthesis of artificial analogs of the 13-oxyingenols and ingenols
To investigate the structure–activity relationship of 13-
oxyingenol derivative (
1
), we prepared artificial analogs of 13-
) and ingenol 3-angelate (4)
oxyingenol. Because RD4-2227 (
2
show potent biological activity, we focused on the C3-acyloxy
13-oxyingenol analogs. We synthesized the C3-acyloxy analogs
from our synthetic intermediate 70 (Scheme 13). Selective
protection of the 1,3-diol group at C5 and C20 with an
acetonide group gave mono acetonide 71.39 Selective acylation
of the O3-hydroxy group and removal of the acetonide group
afforded various C3-acyloxy 13-oxyingenol analogs, pivalate
2
(RD4-2227), angelate 76, tetradecanoate 77, and benzoate 78
.
On the other hand, to examine the effect of the dodecanoyl
ester group at the C13 in 13-oxyingenol analogs, C3-acyloxy
ingenol analogs
40
4
and 84
–86 were also prepared from ingenol
(3)
by using the same strategy for the C3-acyloxy 13-
oxyingenol analogs.
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we observed in our isobenzofuranone ligands before.41 The
branched pivaloyl derivatives ( and 84) showed stronger
R1
R1
13
O
13
O
2
activation than the straight chain myristoyl analogs (77 and
85). These results indicated that 13-oxyingenol analogs with a
long acyl chain at the C13 position and a short bulky acyl group
at the C3 position have the potential for broad activation
ability toward both cPKCs and nPKCs. On the other hand, the
activation abilities of compound 77 having two long straight
acyl chains similar to DAGs were low, and indeed were lower
than those of compound 85, which lacks a long acyl chain at
C13.
H
H
a
b or c
3
5
HO
HO
HO
HO
HO
OH
O
20
O
70 R1 = OCOC11H23
3 R1 = H (ingenol)
71 R1 = OCOC11H23
79 R1 = H
R1
72, 80 R2 = tꢀBu
73, 81 R2
O
HL-60 cell differentiation and cell death
H
=
O
3
We further examined the biological effects of 13-oxyingenol
analogs at the cellular level. It has been reported that ingenol
O HO
R2
O
74, 82 R2 = C13H27
75, 83 R2 = Ph
3-angelate (4) induced the differentiation and apoptosis of HL-
O
60 cells, and that PKCδ would play a critical role in these
effects.42 Since CD11b was reported as an early marker of HL-
60 cell differentiation, the expression levels of CD11b of HL60
cells treated with PMA and the ingenol and 13-oxyingenol
analogs (1 μM, 24 hr) were measured by flow cytometry. The
relative expression levels (%) compared to PMA are shown in
Table 3. CD11b expression was greatly induced by all of the
72–75 R1 = OCOC11H23
80–83 R1 = H
d
R1
O
78% for 2; 89% for 84
in 2 steps, respectively
2, 84 R2 = tꢀBu
compounds except compounds 67 and
3 (see also Figure S6).
81% for 76; 89% for 4
in 2 steps, respectively
H
76, 4 R2
=
Furthermore, morphological changes in HL-60 cells, cell
aggregation, cell attachment and pseudopodia formation,
induced by these ingenol and 13-oxyingenol analogs were
confirmed by microscopic observations (Figure 3 and Figure
S5).
O
3
5
O
HO
HO
R2
50% for 77; 80% for 85
in 2 steps, respectively
77, 85 R2 = C13H27
78, 86 R2 = Ph
OH
20
2, 76–78 R1 = OCOC11H23
4, 84–86 R1 = H
82% for 78; quant. for 86
in 2 steps, respectively
In addition, the induction of apoptosis by the treatment of
these compounds (1 μM, 48 hr) was also quantified by
measuring the sub-G1 population by flow cytometry. Similar to
Scheme 13 Syntheses of the artificial analogs of 13-oxyingenol and ingenol.
Reagents and conditions: (a) p-TsOH·H2O, acetone, rt, 50% for 71, 57% for 79; (b)
(RCO)2O, Cs2CO3, MeCN, rt, for 72–74, 80–82; (c) BzCl, Et3N, DMAP, CH2Cl2, rt, for
75 and 83; (d) 0.5% HCl aq, MeOH, rt. Ts = p-toluenesulfonyl.
ingenol 3-angelate (
4), these compounds induced apoptosis
(Table 3 and S7).
Compounds 67 and
3, each lacking the ability to active PKCα
PKCα and PKCδ activation abilities of 13-oxyingenol natural
derivative (1) and its artificial analogs
and δ, did not induce morphological changes, expression of
CD11b, or the death of HL-60 cells. These results suggested
that PKCδ activation might be involved in the mode of action
of HL-60 differentiations and apoptosis induced by ingenol and
13-oxyingenol analogs, as reported previously.42 However, 13-
The enhancement of PKC activity by 13-oxyingenol and ingenol
analogs was evaluated in vitro for PKCα (as a representative of
cPKCs) and PKCδ (as a representative of nPKCs). First, the
effects of the compounds (10 nM for PKCα and 100 nM for
PKCδ) on the enzyme activities were examined. As shown in
Table 3, analogs without the acyl group on the O3 position (R2
= H) did not show significant activation of either PKCα or PKCδ,
indicating that the 3-acyloxy group is essential for PKC
activation as suggested previously.4a,15c Other derivatives
showed the dose-dependent activation of PKCα and PKCδ
(Figures S2 and S4). Regardless of the presence or absence of
the 13-acyloxy group (R1), a similar activation level of PKCα
oxyingenol derivatives (
1) and 13-oxyingenol 13-dodecanoate
(
70), each showing less potent PKCδ activation in vitro than
PMA, significantly induced HL-60 cell differentiation and
apoptosis (Table 3, Figures S3, S5, and S6), suggesting that
these compounds induce activations of other PKC isozymes,
although the possibility that migration of acyl groups in
1 and
70 altered these compounds to be stronger PKC activators
δ
could not be excluded.39b Therefore, the effects of broad-
spectrum PKC inhibitor Gö6983 on CD11b expression were
examined. Interestingly, Gö6983 suppressed the up-
was observed for ingenol analogs (
analogs ( 76 78). In contrast, it should be noted that PKCδ
activity was clearly susceptible to the 13-acyloxy group.
Though compounds 76, and 78 exhibited remarkable
activation of PKCδ, the corresponding ingenol analogs ( 84
4, 84–86) and 13-oxyingenol
regulations of CD11b induced by
1 and 70 as well as other
2
,
–
compounds (Figure S8). These results indicated that the
activation of other PKC isozyme (or other kinases that Gö6983
inhibits) might contribute to HL-60 cells differentiation induced
by these 13-oxyingenols.15c
2,
4,
,
and 86) showed much lower activation. The bulkiness of the 3-
acyl group (R2) also seemed to affect PKCα activation ability, as
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Journal Name
Table 3 Biochemical and biological activities of 13-oxyingenol or ingenol analogsa
R1
CH3(CH2)12COO
OAc
O
H
H
H
H
OH
3
5
R2O HO
O
HO
HO
OR3
OH
20
ingenol analogs
PMA
Compounds
R1
R2
R3
PKCα [%]
PKCδ [%]
CD11b [%]
apoptosis [%]
13-oxyingenols
1
OCOC11H23
OH
H
H
H
COC5H11
19.9 (5.2)
16.9 (4.9)
17.0 (1.6)
96.6 (4.1)
98.3 (8.8)
69.7 (10.9)
88.3 (4.2)
14.2 (0.8)
11.5 (1.1)
11.8 (2.0)
124 (1.4)
118 (7.9)
82.2 (8.2)
109 (10.1)
152 (4.4)
13.6 (3.6)
170 (1.1)
131 (2.3)
138 (4.4)
162 (2.1)
130 (4.1)
40.8 (1.9)
6.7 (0.88)
35.6 (1.3)
40.6 (1.5)
40.2 (2.2)
37.4 (0.87)
41.3 (1.2)
67
H
H
H
H
H
H
70
OCOC11H23
OCOC11H23
OCOC11H23
OCOC11H23
OCOC11H23
2
COCMe3
76
COCMe=CHMe
COC13H27
COPh
77
78
ingenols
3
H
H
H
H
H
H
H
H
H
H
H
12.1 (3.1)
119 (3.7)
98.0 (4.5)
88.3 (6.7)
85.7 (12.0)
100
8.0 (2.3)
59.6 (5.8)
37.6 (3.3)
103 (3.8)
42.0 (7.0)
100
14.6 (3.0)
128 (2.7)
141 (4.3)
130 (2.1)
126 (2.0)
100
9.2 (0.80)
42.5 (0.85)
41.2 (1.2)
43.4 (0.58)
36.5 (1.2)
43.7 (2.0)
84
COCMe3
COCMe=CHMe
COC13H27
COPh
4
85
86
PMA
a PKCα and PKCδ; The increased activity by the treatment of the compounds (10 nM for PKCα; 100 nM for PKCδ) are presented as relative acitivity (% of control PMA);
CD11b; Expression levels of CD11b induced by the compounds (1 μM, 24 h) in HL-60 cells are presented as the relative activity (% of control PMA); apoptosis;
Apoptosis-inducing activity of the compounds (1 μM) for HL-60 cells was shown as population of sub G1 cells.
12 | J. Name., 2012, 00, 1-3
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from the Uehara Memorial Foundation. We would like to
thank Profs. Akira Sekiguchi and Masaaki Ichinohe (University
of Tsukuba) for the X-ray crystallographic analysis and helpful
discussions. We thank the Takasago International Corp. for
their gift of (S)-β-hydroxy-γ-butyrolactone. Also, we would like
to thank Professor Daisuke Uemura (Kanagawa University) for
providing a sample of 13-oxyingenol natural derivative and
ingenol and for his helpful discussion.
Notes and references
1
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1
was called "13-oxyingenol derivative" in reference 1a. In this
paper, we call synthetic artificial analogs of C3-acyloxy 13-
oxyingenol (2, 76–78) and ingenol (4, 84–86) as "analogs"
(not derivative).
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,
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Fig. 3 Microscopic observations of HL-60 cells treated with 13-oxyingenol
analogs. HL-60 cells were treated with PMA (1 μM) or 13-oxyingenol analogs (67
or 70, 1 μM) for 24 h.
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Conclusions
We have achieved the total synthesis of 13-oxyingenol
derivative (
of an inside–outside framework and Mislow–Evans-type [2,3]-
sigmatropic rearrangement for the stereoselective
introduction of hydroxy group at C5. The efficient
1), featuring the RCM reaction for the construction
6
7
a
functionalization of the A- and B-ring portions was established
by using C2 and C7 hydroxy groups. Furthermore, we have
investigated the structure–activity relationships (SAR) of 13-
8
9
oxyingenol derivative (1) and ingenol (3). SAR studies of
synthetic 13-oxyingenol analogs for PKCα and PKCδ activation
reinforced the importance of esterification at the C3 hydroxyl
A. Zack and P. A. Wender, Nature Chem., 2012, 4, 705 and
references cited therein.
group for PKC activation, and
2 and 76 proved to be the most
(a) Z. Szállási, W. Kristopher, K. W. Krausz and P. M.
Blumberg, Carcinogenesis, 1992, 13, 2161; (b) E. J. Beans, D.
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powerful PKCδ activators. We also found that there are 13-
oxyingenol analogs having a long hydrophobic alkyl chain that
induced HL-60 cell differentiation and cell death, though they
were poorer PKCα and PKCδ activators than others or PMA.
This showed the potential of 13-oxyingenol as a new scaffold
for the development of therapeutic agents against acute
myeloid leukemia. Various artificial analogs of 13-oxyingenol
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prepared by our synthetic strategy of
these new findings.
1 enabled us to obtain
Acknowledgements
12 J. D. Winkler, M. B. Rouse, M. F. Greaney, S. J. Harrison and
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13 K. Tanino, K. Onuki, K. Asano, M. Miyashita, T. Nakamura, Y.
This work was supported by Grant-in-Aid for Scientific
Research on Innovative Areas ‘Chemical Biology of Natural
Products’ (Grant Numbers JP23102014, JP26102744) and
‘Homeostatic Regulation by Various Types of Cell Death’ (Grant
Number JP26110004) from the Ministry of Education, Culture,
Sports, Science, and Technology (MEXT), Japan; and by a grant
Takahashi and I. Kuwajima, J. Am. Chem. Soc., 2003, 125
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14 A. Nickel, T. Maruyama, H. Tang, P. D. Murphy, B. Greene, N.
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14 | J. Name., 2012, 00, 1-3
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