Total synthesis of amino-functionalized calphostin analogs as potent and selective inhibitors of protein kinase C (PKC)

Article abstract of DOI:10.1002/bkcs.10908

As potential protein kinase C (PKC) inhibitors and photodynamic agents, the novel amino-functionalized calphostin analog 1,12-bis((benzoyl-amino)methyl)-3,10-perylenequinone was successfully prepared by dimerization of the key intermediate 3-(benzoylamino

Full text of DOI:10.1002/bkcs.10908

Article
DOI: 10.1002/bkcs.10908
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H. Kim et al.
KOREAN CHEMICAL SOCIETY
Total Synthesis of Amino-Functionalized Calphostin Analogs as Potent
and Selective Inhibitors of Protein Kinase C (PKC)
Hyoyeon Kim,† Choon-Ho Song,† Dae-Hyuk Kim,‡ Nam-Gin Jung,§ Seung-Jae Lee,¶, and
*
Beom-Tae Kim†,‡,k,
*
†Department of Bioactive Material Sciences, Center of Bioactive Materials, Jeonju 561-756, Korea.
*E-mail: bkim002@jbnu.ac.kr
‡Department of Molecular Biology, College of Natural Science, Jeonju 561-756, Korea
§Department of Crop Science and Biotechnology, College of Agriculture and Life Science, Jeonju 561-
756, Korea
¶Department of Chemistry, College of Natural Science, Jeonju 561-756, Korea
kKeunsaram Educational Development Institute, Chonbuk National University, Jeonju 561-756, Korea
*E-mail: slee026@jbnu.ac.kr
Received June 2, 2016, Accepted July 28, 2016
As potential protein kinase C (PKC) inhibitors and photodynamic agents, the novel amino-functionalized
calphostin analog 1,12-bis((benzoyl-amino)methyl)-3,10-perylenequinone was successfully prepared by
dimerization of the key intermediate 3-(benzoylamino)methyl-1,2-naphthoquinone (9), which was synthe-
sized by an efficient and relatively short synthetic sequence (eight steps) with satisfactory overall yield.
The naturally occurring form of perylenequinone 12 was prepared by consecutive methylation and
demethylation reactions. In our synthetic strategy, it was beneficial that the amino functionality of 1,2-
naphthoquinone 9 could be easily introduced at an early synthetic stage and subsequently dimerized to
prepare various potentially bioactive perylenequinones.
Keywords: Amino-functionalized calphostin, Naphthoquinone, Perylenequinone, Protein kinase C
Introduction
Another crucial structural factor that affects photodynamic activ-
ity is substituents at the 1- and 12-positions of the perylenequi-
none core.7 In fact, a previous analysis of the structural features
of perylenequinones showed that the functional diversity of sub-
stituents at the 1- and 12-positions of perylenequinones is likely
to play a crucial role in such activity.
Calphostins A–D are biologically significant perylenequi-
nones that were isolated in 1989 from the fungi Cladospor-
ium cladosporioides (Figure 1).1 The calphostins were
found to be potent and selective inhibitors of protein kinase
C (PKC),2 a regulatory enzyme crucial to cell division and
differentiation.3 Thus, because of their unique inhibitory
activity against PKC, considerable attention has been paid
to the application of calphostins as potential anticancer
therapy agents for a variety of cancers.4
The perylenequinone core of the calphostins is a crucial chro-
mophore to their photodynamic activity, which upon exposure
to light generates reactive oxygen species with high quantum
efficiencies.5 Thus, the inhibitory potential of calphostins to
PKC has been suggested as due to their light-dependent free-
radical formation and the resultant local damage. However,
despite the photodynamic properties of perylenequinones,
including calphostins, their practical application requires over-
coming several restrictions, i.e., poor aggregation tendency, rela-
tively short absorption wavelengths, low quantum yields, low
solubility, and poor stability in an aqueous environment. One of
the notable endeavors dedicated to improving the properties
mentioned above was the introduction of an amino functionality
to the perylenequinone core to prepare various amino-substituted
derivatives.6 Representative work was performed with
hypocrellin B, a similar perylenequinone natural product.
Taking the factors mentioned above into consideration, we
designed a calphostin derivative substituted with amino func-
tionality at the 1- and 12-positions of the perylenequinone core
as a target compound (Scheme 1, compound 12). A literature
survey of the synthesis of the perylenequinone core7,8 showed
that a synthetic strategy involving 1,2-naphthoquinone as a
key intermediate, which could be converted to the dimerized
3,10-perylenequinone, would be the most promising (Scheme
1). In our synthetic approach to the target molecule, initial
work focused on the preparation of 3-(benzoylamino)methyl-
1,2-naphthoquinone (9) as the key intermediate, which was
prepared starting from the readily available naphthoate9 (3).
Several dimerization conditions that could lead to successful
preparation of 3,10-perylenequinone 10 were investigated. In
an acid-catalyzed dimerization, along with the desired pery-
lenequinone 10, two compounds known to be key intermedi-
ates to 3,10-perylenequinone were isolated, and their
structures were elucidated on the basis of spectroscopic anal-
ysis. Here, we provide the details of the synthesis of the key
intermediate 3-(benzoylamino)methyl-1,2-naphthoquinone
(9), its dimerization compound 1,12-bis((benzoyl-amino)
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(5.0 g, 30.09 mmol) and diethyl succinate (14.13 mL,
84.25 mmol) in toluene (15 mL) was added dropwise. After
stirring for 2.5 h, the reaction mixture was quenched by
cautiously adding 1 M aqueous HCl solution (120 mL).
The organic layer was separated, and the pH was adjusted
to ~5 with 1 M aqueous K2CO3. The aqueous layer was
separated and re-acidified with 10% aqueous HCl. The
resulting precipitate was partitioned into diethyl ether
(150 mL). The organic layer was washed with water
(150 × 4 mL) and brine (150 mL), dried over Na2CO3, fil-
tered, and evaporated in vacuo to give a yellowish solid res-
idue. The residue was subjected to flash column
chromatography (silica gel) to afford butenoic acid
2 (8.55 g, 97%) as a yellowish solid: mp 88–90ꢀC; 1H
NMR (400 MHz, CDCl3): δ 7.83 (s, 1H, H-4), 6.49 (d, 2H,
J = 1.96 Hz, H-200’, 600’), 6.44 (t, 1H, J = 2.44 Hz, H-400’),
4.28 (q, 2H, J = 7.32 Hz, −CH2–), 3.77 (s, 6H, −OCH3),
3.69 (d, H, J = 1.48 Hz, H-20), 3.57 (d, H, J = 2.44 Hz, H-
20), 1.32 (t, 3H, J = 6.84 Hz, −CH3); 13C NMR (100 MHz,
CDCl3): δ 175.80, 167.63, 160.87, 142.52, 136.52, 125.76,
106.85, 101.42, 61.52, 55.42, 33.87, 14.16.
Figure 1. Structure of the calphostins A–D.
methyl)-3,10-perylenequinone (10), and the final naturally
occurring form, 4,9-dihydroxy-2,6,7,11-tetramethoxy-3,10-
perylenequinone (12).
Compound 12 is a new type of nitrogen-containing pery-
lenequninone prepared via total synthesis, which to the best
of our knowledge has not been found in natural sources to
date. It is notable that compound 12 could be a platform
compound to develop structurally diverse and novel amino-
perylenequinones.
Ethyl 4-acetoxy-5,7-dimethoxy-2-naphthoate (3). The
neat mixture of butenoic acid 2 (6.98 g, 23.71 mmol), acetic
anhydride (11.17 mL, 118.55 mmol), and sodium acetate
(3.69 g, 45.05 mmol) was heated for 1 h at 140ꢀC under an
Ar atmosphere. After checking the completeness of the reac-
tion by the disappearance of compound 2 in thin-layer chro-
matography, the reaction mixture was cooled in an ice bath
and quenched by adding water (15 mL). The resulting precip-
itate was collected, washed thoroughly with water (50 mL ×
5), and dissolved in EtOAc (100 mL). The organic layer was
dried over Na2CO3, filtered, and evaporated in vacuo to give
a crude product. The residue was subjected to flash column
chromatography (silica gel) to afford naphthoate 3 (5.87 g,
78%) as a yellowish solid: mp 129 ~130ꢀC; APCIMS m/z
[M + H]+ Calcd 319.1182 for C15H19O6: Found: 318.9972;
1H NMR (400 MHz, CDCl3): δ 8.30 (d, 1H, J = 1.9 Hz, H-
1), 7.48 (d, 1H, J = 1.9 Hz, H-3), 6.85 (d, 1H, J = 2.4 Hz,
H-8), 6.58 (d, 1H, J = 2.4 Hz, H-6), 4.39 (q, 2H, J = 7.3 Hz,
−CH2-), 3.89 (d, 6H, J = 1 Hz -OCH3), 2.35 (s, 3H, −CH3),
1.39 (t, 3H, J = 7.3 Hz, −CH3); 13C NMR (100 MHz,
CDCl3): δ 170.04, 165.87, 158.74, 156.19, 146.82, 137.00,
128.74, 127.87, 117.17, 116.60, 101.55, 100.14, 61.20,
56.17, 55.43, 20.88, 14.33.
Experimental Section
Chemicals and Instrumentation. Melting points were
recorded on an MEL-TEMP melting point apparatus
(Laboratory Devices Inc., USA) and were uncorrected.
Mass spectra were recorded on a Synapt HDMS mass spec-
1
trometer (Waters, Milford, MA, USA). H and 13C NMR
spectra were recorded on a JEOL 400 MHz or 600 MHz
spectrometer (Akishima, Japan). Chemical shifts are shown
in δ values (ppm), with tetramethylsilane (TMS) as the
internal standard. UV spectra were recorded on an HP UV–
visible Chemstation spectrophotometer (Hewlett-Packar
Company, Palo Alto, CA, USA). All chemicals were pur-
chased from Sigma-Aldrich Co. (St. Louis, MO, USA), and
all solvents for column chromatography were of reagent
grade and were purchased from commercial sources.
(E)-4-(3,5-Dimethoxyphenyl)-3-(ethoxycarbonyl)but-
3-enoic acid (2). A suspension of sodium hydride (3.01 g,
75.23 mmol, 60% dispersion in mineral oil, washed three
times with toluene) in dry toluene (40 mL) was precooled
in an ice bath under an Ar atmosphere and was activated
with a catalytic amount of EtOH (0.5 equiv). To this sus-
pension, a solution of 3,5-dimethoxybenzaldehyde (1)
Scheme 1. Retrosynthetic analysis of amino-functionalized calphostin derivative (12) from 1,2-naphthoquinone (9).
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Total Synthesis of Amino-Functionalized Calphostin Analogs
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3-Hydroxymethyl-6,8-dimethoxynaphthalen-1-ol (4).
(400 MHz, CDCl3): δ 9.07 (s, 1H, −OH), 7.83 (dd, 2H,
J = 5.2; 2.8 Hz), 7.69 (dd, 2H, J = 5.6; 2.4 Hz), 7.18 (d,
1H, J = 1.0 Hz, H-1), 6.73 (d, 1H, J = 1.4 Hz, H-8), 6.66
(d, 1H, J = 2.0 Hz H-3), 6.39 (d, 1H, J = 2.0 Hz, H-6),
4.87 (s, 2H, −CH2-), 3.97 (s, 3H, −OCH3), 3.84 (s, 3H,
−OCH3); 13C NMR (100 MHz, CDCl3): δ 171.25, 170.23,
160.88, 159.88, 157.88, 142.56, 137.54, 134.41, 134.41,
125.98, 125.94, 124.30, 123.28, 120.18, 111.06, 111.03,
103.86, 96.74, 57.04, 56.08, 45.89.
Naphthoate 3 (3.11 g, 9.76 mmol) in dry THF (25 mL)
was slowly added over 30 min to a precooled suspension
of lithium aluminum hydride (1.85 g, 48.81 mmol) in dry
THF (20 mL). After stirring for 1.5 h at room temperature,
the reaction was quenched by slowly adding sat. NH4Cl
(10 mL). The resulting sludge was removed by filtration
and washed with ethyl acetate (100 mL × 3). The com-
bined organic layers were dried over Na2CO3, filtered, and
evaporated in vacuo to give naphthalenol 4 as a pale yel-
lowish solid (2.23 g, 98%). Thin-layer chromatography
(EtOAc:Hexane (3/7, v/v), Rf = 0.35) showed a single
clean spot corresponding to compound 4, which was used
in the next reaction without further purification: mp
129 ~130ꢀC; APCIMS m/z [M + H]+ Calcd 235.0970 for
3-Aminomethyl-6,8-dimethoxynaphthalen-1-ol
(7).
Compound 6 (430 mg, 1.18 mmol) was dissolved in a
mixed solution of ethanol/toluene (2:1, v/v), and hydrazine
monohydrate (0.92 ml, 18.94 mmol) was added at room
temperature under an Ar atmosphere. After stirring for 2 h
at 90ꢀC, the reaction mixture was cooled in an ice bath,
and the pH was adjusted to two with 2 N aqueous HCl.
The mixture was stirred for another 30 min, then neutra-
lized to pH 7 with 1 M aqueous NaOH, and partitioned
with CHCl3 (20 mL × 3). The combined organic layers
were washed with water (50 × 2 mL) and brine (50 mL),
dried over MgSO4, filtered, and evaporated in vacuo to give
an oily residue. The residue was subjected to flash column
chromatography (silica gel) to afford aminomethyl com-
pound 7 (261 mg, yellow oil, 95%) as an oil: mp:
76–77ꢀC; APCIMS m/z [M + H]+ Calcd 234.1130 for
C13H16NO3: Found: 234.0664; 1H NMR (400 MHz,
CDCl3): δ 7.05 (s, 1H, H-4), 6.63 (S, 2H, H-5,7), 6.35 (d,
1H, J = 2.0 Hz, H-2), 3.92 (s, 3H, −OCH3), 3.83 (s, 2H,
−CH2-), 3.82 (s, 3H, −OCH3) 13C NMR (100 MHz,
CDCl3): δ 157.87, 157.11, 154.72, 143.52, 137.56, 115.35,
109.74, 107.79, 99.34, 97.29, 56.98, 55.32, 36.39.
1
C13H15O4: Found: 235.0227; H NMR (400 MHz, CDCl3):
δ 9.09 (s, 1H, −OH), 7.14 (s, 1H, H-4), 6.69 (d, 1H,
J = 1.4 Hz, H-5), 6.67 (d, 1H, J = 2.4 Hz, H-2), 6.42 (d,
1H, J = 2.4 Hz, H-7), 4.70 (s, 2H, −CH2-), 3.99 (s, 3H,
−OCH3), 3.86 (s, 3H, −OCH3); 13C NMR (100 MHz,
CDCl3): δ 157.88, 157.09, 154.75, 141.26, 137.45, 115.37,
110.12, 107.17, 99.50, 97.53, 65.17, 56.09, 55.30.
3-Chloromethyl-6,8-dimethoxynaphthalen-1-ol (5). To
a mixture of triphenylphosphine (4.87 g, 18.58 mmol) and
carbon tetrachloride (3.59 mL, 37.15 mmol) in dry THF
(5 mL) under an Ar atmosphere was added compound
4 (1.09 g, 4.64 mmol). After stirring for 1.5 h at 70ꢀC, the
reaction was quenched by adding water (150 mL), and the
mixture was partitioned with EtOAc (50 mL × 4). The
combined organic layers were washed with water
(50 × 4 mL) and brine (100 mL), dried over Na2CO3, fil-
tered, and evaporated in vacuo to give a yellowish solid
residue. The residue was subjected to flash column chroma-
tography (silica gel) to afford product 5 (0.91 g, 78%,) as a
white solid: mp 130–131ꢀC; APCIMS m/z [M + H]+ Calcd
253.0631 for C13H14ClO3: Found: 252.9911; 1H NMR
(400 MHz, CDCl3): δ 9.12 (s, 1H, −OH), 7.17 (d, 1H,
J = 1.4 Hz, H-4), 6.73 (d, 1H, J = 1.4 Hz, H-5), 6.68 (d,
1H, J = 2.4 Hz, H-2), 6.45 (d, 1H, J = 2.0 Hz, H-7), 4.60
(s, 2H, −CH2-), 4.00 (s, 3H, −OCH3), 3.87 (s, 3H,
−OCH3); 13C NMR (100 MHz, CDCl3): δ 158.10, 157.07,
154.98, 137.42, 137.27, 117.53, 110.47, 108.52, 99.58,
98.09, 56.16, 55.34, 46.32.
2-((4-Hydroxy-5,7-dimethoxynaphthalen-2-yl) methyl)
isoindoline-1,3-dione (6). To a solution of compound
5 (0.84 g, 3.33 mmol) in DMF (5 mL) under an Ar atmos-
phere, potassium phthalimide was added (1.24 g,
6.67 mmol) at room temperature. After stirring for 2 h at
50ꢀC, the reaction mixture was cooled in an ice bath and
diluted with EtOAc (100 mL). The solution was washed
with water (50 × 4 mL) and brine (50 mL), dried over
MgSO4, filtered, and evaporated in vacuo to give a solid.
The residue was subjected to flash column chromatography
(silica gel) to afford product 6 (0.98 g, 81%) as a white
solid: mp: 231–232ꢀC; APCIMS m/z [M + H]+ Calcd
364.1185 for C21H18NO5: Found: 363.0417; 1H NMR
3-(Benzoylamino)methyl-6,8-dimethoxy-naphthalene-
1-ol (8). To
a solution of compound 7 (203 mg,
0.87 mmol) in CH3CN (5 mL) was added benzoyl chloride
(303 μL, 2.61 mmol) at room temperature under an Ar
atmosphere. After stirring for 1 h at 50ꢀC, K2CO3
(120 mg, 0.87 mmol) was added to the reaction, and the
mixture was stirred for another 1 h at room temperature.
The mixture was diluted with water (10 mL) and parti-
tioned with EtOAc-CHCl3 (20 mL × 3). The combined
organic layers were washed with water (50 × 2 mL) and
brine (50 mL), dried over MgSO4, filtered, and evaporated
in vacuo to give a solid residue. The residue was subjected
to flash column chromatography (silica gel) to afford ben-
zoylamino compound 8 (285 mg, 97%) as a colorless solid:
mp 183–184ꢀC; APCIMS m/z [M + H]+ Calcd 338.1392
for C20H20NO4: Found: 338.0942; 1H NMR (400 MHz,
CDCl3): δ 9.13 (s, 1H, −OH), 7.81 (m, 2H), 7.49 (m, 1H),
7.42 (m, 2H), 7.14 (s, 1H, H-4), 6.69 (d, 1H, J = 1.5 Hz,
H-5), 6.67 (d, 1H, J = 2.0 Hz, H-7), 6.50 (s, 1H, −NH),
6.43 (d, 1H, J = 2.5 Hz, H-2), 4.67 (d, 2H, J = 5.4 Hz,
−CH2-), 4.01 (s, 3H, −OCH3), 3.87 (s, 3H, −OCH3); 13C
NMR (100 MHz, CDCl3): δ 168.73, 159.63, 158.62,
156.64, 141.43, 136.45, 135.37, 133.35, 124.31, 124.02,
123.32, 121.30, 119.23, 110.18, 110.15, 103.04, 95.98,
56.59, 55.64, 45.53.
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Total Synthesis of Amino-Functionalized Calphostin Analogs
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3-(Benzoylamino)methyl-6,8-dimethoxy-1,2-naphtho-
N,N0-(50,60-dihydroxy-2,20,4,40-tetramethoxy-5,6-dioxo-
5,6-dihydro-1,10-binaphthyl-7,70-diyl)bis(methylene)
dibenzamide (13). mp: 158–159ꢀC APCIMS m/z [M + H]+
Calcd 703.2292 for C40H33N2O10: Found: 701.1942; UV
quinone (9). To a solution of compound 8 (285 mg,
0.84 mmol) in DMF (5 mL) was added iodoxybenzoic
acid (IBX) (498 mg, 45 wt%, 0.84 mmol) at room temper-
ature under an Ar atmosphere. After stirring for 1 h, the
mixture was diluted with water (10 mL) and partitioned
with EtOAc (20 mL × 3). The combined organic layers
were washed with water (100 mL) and brine (100 mL),
dried over Na2SO4, filtered, and evaporated in vacuo to
give a dark brownish solid. The residue was subjected to
flash column chromatography (silica gel) to afford 1,2-
naphthoquinone 9 (260 mg, 88%) as a dark red solid: mp:
175–176ꢀC; APCIMS m/z [M + H]+ Calcd 352.1185 for
C20H18NO5: Found: 352.1186; IR 3337, 1674, 1651,
1594, 1327 cm−1; UV λmax (CHCl3) 268 (log ε = 4.45),
1
λmax (CHCl3) 266 (log ε = 4.57), 413 (log ε = 4.10); H
NMR (400 MHz, CDCl3): δ 7.63 (d, 2H, J = 7.32 Hz), 7.47
(d, 2H, J = 7.8 Hz), 7.40 (m, 1H), 7.35 (m, 1H), 7.32 (s, 1H,
H-80), 7.28 (m, 2H), 7.21 (t, 2H, J = 7.8 Hz), 7.10 (m, 1H,
J = 5.8 Hz, −NH), 4.19 (m, 4H, −CH2–), 4.09 (s, 3H,
−OCH3), 3.89 (s, 3H, −OCH3), 3.85 (s, 3H, −OCH3), 3.75
(s, 3H, −OCH3); 13C NMR (100 MHz, CDCl3): δ 181.45,
180.46, 175.90, 175.15, 167.74, 166.49, 166.12, 165.83,
163.08, 147.32, 139.20, 138.11, 137.39, 136.52, 135.60,
133.90, 133.49, 131.67, 131.51, 128.50, 128.40, 126.89,
126.69, 117.34, 112.90, 112.79, 110.68, 98.16, 96.57, 56.52,
56.43, 56.36, 55.77, 38.54, 36.68.
1
414 (log ε = 4.18); H NMR (400 MHz, CDCl3): δ 7.78
(m, 2H), 7.45 (m, 1H), 7.37 (t, 2H, J = 7.0 Hz), 7.30
(s, 1H, H-4), 7.09 (t, 1H, −NH, J = 5.8 Hz), 6.40 (d, 1H,
J = 2.0 Hz, H-7), 6.36 (d, 1H, J = 2.0 Hz, H-5), 4.33
(d, 2H, J = 5.8 Hz -CH2-), 3.89 (s, 3H, −OCH3), 3.86
(s, 3H, −OCH3); 13C NMR (100 MHz, CDCl3): δ 181.74,
175.47, 167.53, 166.56, 165.58, 143.12, 138.09, 136.06,
133.88, 131.65, 128.53, 127.05, 112.53, 110.09, 99.29,
56.27, 55.90, 38.74.
N,N0-(2,20,4,40-tetramethoxy-5,50,6,60-tetraoxo-5,50,6,60-
tetrahydro-1,10-binaphthyl-7,70-diyl)bis(methylene)
dibenzamide (14). mp: 180–181ꢀC APCIMS m/z [M + H]+
Calcd 701.2135 for C40H33N2O10: Found: 701.2132; UV
λmax (CHCl3) 261 (log ε = 4.56), 430 (log ε = 4.10);
1H NMR (400 MHz, CDCl3): δ 7.54 (m, 6H, −NH,
Hb-1,10,5,50), 7.39 (t, 2H, Hb-3,30 , J = 7.8 Hz), 7.23 (t, 4H,
Hb-2,204,40, J = 7.8 Hz), 6.84 (s, 2H, Ha-8,80), 6.39 (s, 2H,
Ha −3,3’), 4.16 (m, 4H, −CH2-), 3.90 (s, 6H, −OCH3),
3.76 (s, 6H, −OCH3); 13C NMR (100 MHz, CDCl3):
δ179.92, 175.44, 167.97, 165.42, 164.71, 136.77, 136.32,
135.67, 133.47, 132.05, 128.63, 126.37, 116.95, 112,13,
96.09, 56.25, 56.19, 38.08. Method B. A solution of 1,2-
naphthoquinone 8 (56 mg, 0.16 mmol) and anhydrous ferric
chloride (65 mg, 0.40 mmol) in 2 mL of anhydrous acetoni-
trile was stirred at room temperature for 3 h. The reaction
mixture was poured into 3% aqueous HCl, and then
extracted with chloroform (50 mL × 3). The combined
organic layers were washed with water (30 mL × 3) and
brine (50 mL), dried over Na2SO4, filtered, and evaporated
in vacuo to afford a dark red solid. This solid was subjected
to flash column chromatography (silica gel) to afford peryle-
nequinone 10 (8 mg, 14%).
1,12-Bis((benzoylamino)methyl)-2,4,6,7,9,11-hexam-
ethoxy-3,10-perylenequinone (11). To a solution of peryle-
nequinone 10 (7 mg, 0.01 mmol) in THF (2 mL) was added
tetrabutylammonium fluoride (TBAF; 30 μL, 0.03 mmol,
1.0 M in THF) followed by iodomethane (1 mL). After stir-
ring at room temperature for 2 h, the mixture was diluted
with CHCl3 (20 mL) and washed with sat. NaHCO3 (20 mL)
and water, dried over Na2SO4, filtered, and evaporated in
vacuo to afford a dark red solid. This solid was subjected to
flash column chromatography (silica gel) to afford hexam-
ethoxyperylenequinone 11 (6 mg, 83%) as a dark red solid:
mp: 153–154ꢀC; APCIMS m/z [M + H]+ Calcd 729.2448 for
C42H37N2O10: Found: 729.2450; IR 3440, 1606, 1461 cm-1;
UV λmax (CHCl3) 266 (log ε = 3.90), 468 (log ε = 3.68);
1H NMR (400 MHz, CDCl3): δ 7.58 (d, 4H, J = 8.0 Hz,
Hb-1,1’,5,5’), 7.4 (t, 2H, J = 7.3 Hz, Hb-3,3’), 7.30 (t, 4H,
J = 7.3 Hz, Hb-2,2’,4,4’), 6.91 (t, 2H, −NH), 6.75(s, 2H,
1,12-Bis((benzoylamino)methyl)-2,11-dihydroxy-4,6,7,9,-
tetramethoxy-3,10-perylenequinone (10). Method A. A neat
solution of 1,2-naphthoquinone 9 (80 mg, 0.23 mmol) in
trifluoroacetic acid (1 mL) was stirred at 0ꢀC for 2 h. The
solution instantly turned dark green and became dark blue
over 30 min. The reaction mixture was poured into ice-cold
water and was extracted with chloroform (20 mL × 3).
The combined organic layers were washed with water
(30 mL × 3) and brine (50 mL), dried over Na2SO4, fil-
tered, and evaporated in vacuo to afford a dark red solid.
This solid was subjected to flash column chromatography
(silica gel) to afford perylenequinone 10 (16 mg, 42%) as a
dark red solid (CHCl3:MeOH (9/1, v/v), Rf = 0.41). In
addition to 10, a mixture of two by-products was eluted,
and the corresponding fractions were evaporated in vacuo
to give a dark yellow solid (48 mg). This mixture was
subjected again to careful flash column chromatography
to afford compounds 13 (7 mg, Rf = 0.46, 5%) and
binaphthoquinone 14 (4 mg, Rf = 0.48, 8%).
Compound 10: mp: 171–172ꢀC APCIMS m/z [M + H]+
Calcd 701.2135 for C40H33N2O10: Found: 701.2138; IR
3467, 3304, 1594, 1464 cm-1; UV λmax (CHCl3) 269 (log
1
ε = 5.13), 506 (log ε = 4.90), 564 (log ε = 4.58); H NMR
(400 MHz, CDCl3): δ 8.41 (s, 2H, –OH), 7.55 (d, 4H,
J = 7.3 Hz, Hb-1,10,5,50), 7.37 (t, 2H, J = 7.3 Hz, Hb-3,30),
7.28 (t, 4H, J = 7.3 Hz, Hb-2,20,4,40), 7.18 (t, 2H, −NH),
6.71 (s, 2H, Ha-5,50), 5.0 (dd, 2H, J = 2.0 Hz, −CH2–),
4.38 (dd, 2H, J = 5.9 Hz –CH2–), 3.95 (s, 3H, −OCH3),
3.90 (s, 3H, −OCH3), 13C NMR (100 MHz, CDCl3): δ
175.66, 166.75, 165.36, 164.87, 150.21, 134.28, 131.30,
130.74, 129.43, 128.40, 126.86, 118.88, 111.94, 106.27,
94.07, 56.62, 56.21, 39.90.
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Total Synthesis of Amino-Functionalized Calphostin Analogs
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Ha-5,5’), 4.79(d, 4H, −CH2–, J = 5.8 Hz), 4.26 (s, 3H,
−OCH3), 4.14 (s, 3H, −OCH3), 4.07 (s, 3H, −OCH3); 13C
NMR (100 MHz, CDCl3): δ 178.08, 166.69, 164.26, 163.24,
154.75, 134.21, 131.27, 130.99, 130.13, 129.98, 128.38,
126.86, 111.35, 108.83, 94.95, 61.16, 56.52, 56.11, 39.84.
1,12-Bis((benzoylamino)methyl)-4,9-dihydroxy-2,6,7,11-
tetramethoxy-3,10-perylenequinone (12). To a solution of
perylenequinone 11 (21 mg, 0.03 mmol) in THF (2 mL)
under an Ar atmosphere, MgI2 in ether was added (16 mg,
0.058 mmol, 0.07 M in Et2O). The solution instantly turned
dark green. After confirming the reaction was complete by
the disappearance of the starting material 13 by thin-layer
chromatography, the mixture was diluted with EtOAc
(25 mL) and washed with sat. NH4Cl (20 mL) and water,
dried over Na2SO4, filtered, and evaporated in vacuo to afford
a dark red solid. This solid was subjected to flash column
chromatography (silica gel) to afford perylenequinone 12
(9 mg, 45%) as a dark red solid: mp: 251–252ꢀC APCIMS
m/z [M + H]+ Calcd 701.2135 for C40H33N2O10: Found:
701.2138; IR 3446, 1617, 1462 cm–1; UV λmax (CHCl3)
269 (log ε = 4.28), 470 (log ε = 4.00), 555 (log ε = 3.54);
1H NMR (400 MHz, CDCl3): δ 15.84 (s, 2H,-OH), 7.48
(m, 4H, Hb-1,1’5,5’), 7.33 (t, 2H, J = 7.3 Hz, Hb-3,3’), 7.24
(t, 4H, J = 7.3 Hz, Hb-2,2’4,4’), 6.90 (m, 2H, −NH), 6.36
(s, 2H, Ha-5,5’), 5.1 (dd, 2H, J = 7.3 Hz, −CH2–), 4.8
(dd, 2H, J = 3.7;4.4 Hz –CH2–), 4.30 (s, 3H, −OCH3), 3.94
(s, 3H, −OCH3); 13C NMR (100 MHz, CDCl3): δ 183.38,
167.81, 167.34, 166.74, 151.46, 134.32, 133.85, 131.51,
128.44, 126.84, 125.32, 124.06, 120.19, 107.68, 102.59,
61.79, 56.45, 40.00.
The synthesis began by condensing 3,5-dimethoxybenzaldehyde
(1) with diethyl succinate to produce butenoic acid 2. Com-
pound 2, on boiling with anhydrous sodium acetate in acetic
anhydride for a short time (1 h), gave the annulated product
naphthoate 3 in acceptable yield (78%).9 The ester group
of naphthoate 3 was sequentially converted to alcohol 4 -
(LAH/THF, 98%)10 and subsequently to chloride 5 (CCl4/
PPH3, 78%).11 Conversion of chloride 5 to aminomethylene
compound 7 via compound 6 was accomplished via Gabriel
reaction12 through two steps (potassium phthalimide, and
then N2H4/H2O) with 77% overall yield. Aminomethylene
compound 7 could be a versatile platform compound to
introduce diverse amino substituents to the 1- and 12-
positions of the perylenequinone core (10) by N-alkylation
or N-acylation, leading to structurally diverse amino-
functionalized calphostin derivatives for investigation of
structure–activity relationships. In the next step, amino-
functionalized 1,2-naphthoquinone (9), to which an appro-
priate amino functionality could be introduced, was prepared
in two steps. The first step involved N-benzoylation (BzCl,
K2CO3) of compound 7 to afford compound 8 in excellent
yield (97%). A benzoyl functionality at the amino group of
compound 7 was preferentially considered because calphos-
tins A–C have O-benzoyl group(s) at the sidechains of the
1- and 12-positions of the perylenequinone, as shown in the
calphostin structures (Figure 1). The next step, namely oxi-
dation of compound 8 to 1,2-naphthoquinone 9, proceeded
smoothly with a good yield (88%) through the agency of
IBX in THF. IBX, a hypervalent iodine compound, is a
promising reagent for regiocontrolled oxidation of polycy-
clic aromatic phenols to their corresponding aromatic
quinones.13 Application of IBX in this oxidation step consid-
erably improved the yield, which was not achieved in our
previous efforts to prepare 1,2-naphthoquinone derivatives
using other oxidation conditions.14
Results and Discussion
Our initial work focusing on the construction of amino-
functionalized 1,2-naphthoquinone (9) is shown Scheme 2.
Scheme 2. The synthetic route to 3-(benzoylamino)methyl-1,2-naphthoquinone (9). (a) diethyl succinate, NaH; toluene, 0ꢀC ! rt;
(b) Ac2O, AcONa, 140ꢀC; (c) LAH, THF, rt; (d) CCl4, PPH3, THF, reflux; (e) Potassium phthalimide, DMF, 50ꢀC; (f ) N2H4/H2O, EtOH,
reflux; (g) Benzoyl chloride, K2CO3, CH3CN, rt; (h) IBX, DMF, rt.
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Total Synthesis of Amino-Functionalized Calphostin Analogs
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Scheme 3. Preparation of target compound, namely the naturally occurring form of amino-functionalized perylenequinone (12), from 1,2-
naphthoquinone (9). (a) TFA, 0ꢀC; (b) 10% FeCl3, CH3CN, rt; (c) TBAF, THF, MeI, rt; (d) MgI2, THF, rt.
Scheme 4. Dimerization mechanism proposed by Merlic et al.7
k,14a
With key intermediate 3-(benzoylamino)methyl-1,2-
naphthoquinone (9) in hand, we continued the preparation
of the perylenequinone skeleton, (1,12-bis((benzoylamino)
methyl)-2,11-dihydroxy-4,6,7,9,-tetramethoxy-3,10-peryle-
nequinone (10). In our previous work on dimerization of
1,2-naphthoquinone to construct the perylenequinone skel-
eton, several reaction conditions were investigated.14a
Thus, we preferentially applied two types of reaction condi-
tions, namely acid-catalysis and oxidative dimerization, to
construct perylenequinone 10 (Scheme 3). Under the acid-
catalyzed reaction conditions (trifluoroacetic acid/0ꢀC),8g
the desired perylenequinone 10 (mp 171–172ꢀC) was
obtained as a dark red solid in modest yield (48%). The
generated the desired product perylenequinone 10,
these conditions were not satisfactory because of the low
(<34%) and large fluctuations in yield.
The final naturally occurring form of perylenequinone 12
was prepared by consecutive methylation and demethyla-
tion reactions (Scheme 3). Thus, the hydroxyl groups at
C-2 and C-11 of compound 10 were methylated by treat-
ment with MeI and TBAF with good yield (83%). Finally,
selective demethylation of the C4, C9-methyl ethers of
compound 11 by treatment with MgI27,8k resulted in peryle-
nequinone 12 with modest yield (45%).
The dimerization of 1,2-naphthoquinone 9 to 3,10-
perylenequinone 10 deserves additional discussion. In this
reaction, along with the dimerized product perylenequinone
10, two other compounds were isolated as reddish solids in
small amounts (Scheme 4). Their chemical structures were
determined to be 13 and binaphthoquinone 14 on the basis
of their spectroscopic data, which showed that these com-
pounds were not perylenequinones but were evidently not
ring-closed structures either. In several previous reports on
the dimerization of 1,2-naphthoquinone under acid-
catalyzed conditions,7,8d,g coupled but not ring-closed
1
structure of compound 10 was elucidated by analysis of H
and 13C NMR, high-resolution mass spectrum, as well as
UV and IR spectra. Three broad absorption bands at
269, 506, and 564 nm in the UV spectrum were reported to
be typical for the quinone system.15 Proton signals for the –
OH (δ 8.41, broad singlet, 2H) and –NHBz (δ 7.18, triplet,
2H) in the 1H NMR spectrum of compound 10 provide
additional convincing evidence of the structure. While
oxidative dimerization conditions (10% FeCl3, CH3CN)7,8d,
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give a perylenequinone skeleton (Scheme 4). Compounds
13 and 14 identified in our experiment were shown to
exactly correspond to the reported intermediates.
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Conclusion
A novel amino-functionalized perylenequinone derivative,
1,12-bis((benzoyl-amino)methyl)-3,10-perylenequinone (12),
was successfully prepared by dimerization of the key
intermediate, 3-(benzoylamino)methyl-1,2-naphthoquinone
(9), which was synthesized by an efficient and relatively
short synthetic route (eight steps) with satisfactory overall
yield. The naturally occurring form of perylenequinone 12
was prepared by consecutive methylation and demethyla-
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cial that the amino functionality of 1,2-naphthoquinone
9 was easily introduced in the early synthetic stages, and
consequently dimerized to prepare various potentially bio-
active perylenequinones as potential PKC inhibitors and
photodynamic agents. However, more elaborate experi-
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9 should be considered to overcome the considerably low
yield in this step. The inhibitory activities of three syn-
thetic perylenequinones 10, 11, and 12 are currently under
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Acknowledgment
This work was carried out with the support of the “Cooper-
ative Research Program for Agricultural Science & Tech-
nology Development (Project No. PJ009998022016),”
Rural Development Administration, Republic of Korea.
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