Design, synthesis, and investigation of protein kinase C inhibitors: Total syntheses of (+)-calphostin D, (+)-phleichrome, cercosporin, and new photoactive perylenequinones

Article abstract of DOI:10.1021/ja902324j

The total syntheses of the PKC inhibitors (+)-calphostin D, (+)-phleichrome, cercosporin, and 10 novel perylenequinones are detailed. The highly convergent and flexible strategy developed employed an enantioselective oxidative biaryl coupling and a double

Full text of DOI:10.1021/ja902324j

Published on Web 06/02/2009
Design, Synthesis, and Investigation of Protein Kinase C
Inhibitors: Total Syntheses of (+)-Calphostin D,
(+)-Phleichrome, Cercosporin, and New Photoactive
Perylenequinones
Barbara J. Morgan,† Sangeeta Dey,† Steven W. Johnson,‡ and
Marisa C. Kozlowski*,†
Department of Chemistry, Roy and Diana Vagelos Laboratories, UniVersity of PennsylVania,
Philadelphia, PennsylVania 19104, and Department of Hematology/Oncology, UniVersity of
PennsylVania, Philadelphia, PennsylVania 19104
Received March 31, 2009; E-mail: marisa@sas.upenn.edu
Abstract: The total syntheses of the PKC inhibitors (+)-calphostin D, (+)-phleichrome, cercosporin, and
10 novel perylenequinones are detailed. The highly convergent and flexible strategy developed employed
an enantioselective oxidative biaryl coupling and a double cuprate epoxide opening, allowing the selective
syntheses of all the possible stereoisomers in pure form. In addition, this strategy permitted rapid access
to a broad range of analogues, including those not accessible from the natural products. These compounds
provided a powerful means for evaluation of the perylenequinone structural features necessary to PKC
activity. Simpler analogues were discovered with superior PKC inhibitory properties and superior
photopotentiation in cancer cell lines relative to the more complex natural products.
Introduction
cercosporin (3) with a bridging seven-membered ring remained
a challenging synthetic target. Although the structurally related
Several years ago we initiated a program to explore the use
of the Cu-catalyzed enantioselective oxidative biaryl coupling1
in the syntheses of enantiopure perylenequinones and demon-
strated that systems possessing only helical stereochemistry can
be configurationally stable.2 With the development of a key aldol
cycloaddition utilizing a dynamic stereochemistry transfer, we
further completed the first total synthesis of hypocrellin A (4;
Figure 1).3 Prior to our efforts the total syntheses of the (-)-
calphostins A-D (1a-d) and (+)- and (-)-phleichrome (2)
were reported involving diastereoselective biaryl couplings.4
Unfortunately, these couplings afforded mixtures with the wrong
diastereomer usually predominating; additional steps were
required to establish the correct stereochemistry. Furthermore,
1 and 2 are atropisomerically stable, the additional seven-
membered ring in cercosporin lowers the atropisomerization
barrier, allowing 3 to readily atropisomerize at 37 °C (eq 1).5
Herein, we report the first total syntheses of (+)-1d and 3
exploiting a novel double cuprate epoxide opening. The
combination of enantioselective oxidative biaryl coupling,
double cuprate epoxide opening, and decarboxylative function-
alization provides a potentially general means for constructing
a diverse array of perylenequinone analogues with complete
control of the helical and centrochiral stereochemical elements.
As a result, we describe the synthesis of 10 new perylenequinone
analogues as well as (+)-1d, (+)-2, and 3 from a common chiral
binaphthyl precursor that can be generated readily in multigram
batches. The routes to these new compounds are discussed with
respect to chemical efficiency and stereochemistry. Among these
new compounds, we identified several with longer wavelengths
of absorption, potentially leading to superior photosensitizers.
We also report IC50 values for all of the analogues against
protein kinase C (PKC) establishing which elements are the most
† Department of Chemistry.
‡ Department of Hematology/Oncology.
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J. AM. CHEM. SOC. 2009, 131, 9413–9425 9413

A R T I C L E S
Morgan et al.
Figure 1. Perylenequinone natural products.
crucial to inhibition of the regulatory domain. Finally, we report
CC50 values for selected analogues against cancer cell lines.
Background. The perylenequinone family of natural products
(Figure 1) is characterized by a helical chiral extended oxidized
pentacyclic core combined with C7,C7′-substitution containing
centrochiral stereocenters.6 The perylenequinone portion confers
several novel features to these compounds including tautomeric
forms that rapidly interconvert, low barriers to atropisomeriza-
tion, and low barriers to photoexcitation.
Calphostin D, (-)-1d, and phleichrome, (-)-2 are isolates
of the Cladosporium fungi Cladosporium cladosporioides and
Cladosporium phlei, respectively.7,8 Cercosporin, 3, was first
isolated by Kuyama and Tamura from Cercospora kikuchii,9 a
fungus responsible for the “purple speck disease” of soy beans
as well as damage to a host of plant species worldwide.10 The
structures were elucidated through a series of chemical trans-
formations and subsequent spectroscopic analysis.7,8,11,12 The
X-ray structure of 3 was used to assign the absolute and relative
configuration.5 Correlation of the CD and NMR spectra allowed
determination of the helical configurations and relative stere-
ochemistries of the remaining natural products. Related natural
products, hypocrellin (ent-4),13 hypocrellin A (4),14 elsi-
nochromes (5),15 and scutiaquinone (6)16 have also been isolated
from several different fungi.
Figure 2. Perylenequinone 3-D helical chirality and atropisomerization.
From a synthetic perspective, 1-3 contain the same stereo-
chemical elements: helical chirality and stereogenic C7,C7′-2-
hydroxypropyls. Beyond these elements, the perylenequinones
exhibit intriguing architectural aspects, namely keto-enol
tautomerism17,18 and potential atropisomerization of the helical
perylene core (Figure 2). The barrier to atropisomerization of
the helical configuration, which entails rotation of the C2,C2′-
and C7,C7′-groups past one another (Figure 2), varies substan-
tially for the compounds in this series. Whereas the calphostins
and phleichrome are atropisomerically stable, the additional
seven-membered ring in cercosporin lowers the barrier, making
it a particularly challenging synthetic target (see Table 1 and
discussion below).5,19 On the other hand, hypocrellin and
hypocrellin A atropisomerize rapidly at ambient temperature
presenting two sets of sharp peaks in the NMR spectrum.18
The perylenequinones exhibit light-induced activity in bio-
logical systems making them photodynamic therapeutic candi-
dates.20 Photodynamic therapy is a treatment that uses a small
molecule, called a photosensitizer or a photosensitizing agent,
oxygen, and a light source.21 After the photosensitizers are
delivered to target cells/tissues, they are exposed to a specific
wavelength of light forming excited states. In turn, these species
transfer the energy to molecular oxygen to produce singlet
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9414 J. AM. CHEM. SOC. VOL. 131, NO. 26, 2009

Investigation of Protein Kinase C Inhibitors
A R T I C L E S
oxygen as well as other reactive oxygen species that kill cells.
Generation of photosensitizers that can be activated by longer
wavelength light, which can penetrate deeper into tissue, is
crucial. The nine photosensitizer compounds (none perylene-
quinones) currently in clinical use as photoactivated anticancer
agents encompass three related structural classes: porphyrins,
chlorins, and phthalocyanins.21 While optimization of current
photosensitizers is warranted, the multiple criteria of desirable
photosensitizers makes discovery of new classes critical to
furthering the practice of photodynamic therapy.
The perylenequinone core is a potent chromophore that
permits the use of these compounds as photosensitizers. Upon
exposure to light, perylenequinones initiate the generation of
reactive oxygen species with high quantum efficiency.20 Fur-
thermore, these compounds selectively bind the C1 regulatory
domain of protein kinase C (PKC),22,23 some forms of which
are upregulated in cancer cells and associated with growth.24
For these reasons, the perylenequinones are candidates for
photodynamic therapy of cancer;20 light-induced activity has
been seen with the natural products against selected tumor cell
lines.25 In addition, they have displayed antiviral26 and immu-
notherapeutic27 properties. The development of new classes of
PDT agents of defined molecular structure, with lower aggrega-
tion tendency, longer absorption wavelengths, high quantum
yields, greater stability, and greater selectivity against cancer
cells is highly desirable.
The perylenequinones are thought to cause cell apoptosis by
two pathways: (1) light-induced production of singlet oxygen
and (2) PKC inhibition. Considerable attention has been devoted
to the development of potent and specific PKC inhibitors.24 The
catalytic domain-acting PKC inhibitors, such as staurosporin
(PKC, IC50 ) 9 nM) and other indolocarbazoles, are highly
potent but not specific for PKC, since they also inhibit other
protein kinases.28 Since the discovery of staurosporin, the
structurally related catalytic site inhibitors ruboxistaurin (PKCꢀΙ,
IC50 ) 4.7 nM; PKCꢀΙΙ, IC50 ) 5.9 nM)29 and enzastaurin
(PKCꢀΙ, IC50 ) 30 nM; PKCꢀΙΙ, IC50 ) 30 nM)30 have been
found to be more selective for the PKCꢀ isozyme relative to
other PKC isozymes and kinases leading to important new
diabetes treatments.31 Significantly, the perylenequinones have
demonstrated to be both potent and specific for PKC [Calphostin
C (1c): PKC, IC50 ) 0.05-0.46 µM; PKA, IC50 ) >100 µM;
PPK, IC50 ) >100 µM; Cercosporin (3): PKC, IC50 ) 0.6-1.3
µM; PKA, IC50 ) >500 µM; PPK, IC50 ) >180 µM] most likely
because they act on a regulatory domain which is unique to
PKC.22,23 To date, evaluation of the perylenequinones as PKC
inhibitors has been confined to natural product derivatives or
simple analogues due to limitations of available synthetic
methods.20,23,32,33 Novel perylenequinones will allow determi-
nation of the structural features necessary for PKC inhibition
as well as generation of structures with improved photodynamic
properties.
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Downie, G. H.; Cuenca, R.; Hu, X.-H.; Childs, C. J. H.; Sibata, C. H.
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Results and Discussion
Retrosynthetic Considerations. We desired a synthetic strat-
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natural products 1-6 (Figure 1) as well as several derivatives.
Examination of prior approaches to the calphostins4 revealed a
common theme in that the stereochemistry of the C7-subsitution
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A R T I C L E S
Morgan et al.
Scheme 1. Retrosynthetic Analysis of the Calphostins
Table 1. Representative Atropisomerization Barriers and Half
Lives
Scheme 2. Common Intermediate to the Perylenequinone Natural
Products
was generated first and then diastereoselective dimerizations
were undertaken (Scheme 1, path b). The moderate diastereo-
selection (1:1-1:8) afforded by the newly formed C1-C1′
biaryl bond presumably arises from the distance between this
center and the C7,C7′-substituents. Furthermore, the predomi-
nant diastereomer did not usually correspond to the calphostin
array although it does match that needed for phleichrome.
In devising a plan to selectively synthesize all of the possible
stereoisomers in pure form, we elected to pursue a different
strategy that mimics the likely biosynthesis.11c,34 By establishing
the axial stereochemistry first (path a in Scheme 1), the
corresponding helical stereochemistry can be generated with
complete stereocontrol. The helical stereochemistry can in turn
be utilized to control the C7,C7′-stereochemistry with good
fidelity as demonstrated in our synthesis of hypocrellin A
(Scheme 2).3 Notably, a synthesis of hypocrellin relying on path
b in Scheme 1 would require oxidation of the initially formed
alcohols resulting in loss of this stereochemical information.
Alternately, the C7,C7′-stereochemistry can be introduced
from an external source, a gambit that permits selective synthesis
a Values are given at 65 °C except for entries 1 and 2 for which
temperatures could not be located. b Ea value.
of all the possible stereoisomers of calphostin D, phleichrome,
and cercosporin. We selected an epoxide opening reaction to
achieve this goal (Scheme 2). The net result is that all of the
target structures (1-5) devolve onto a common synthetic
intermediate, chiral biaryl 13, or its enantiomer, ent-13 (Scheme
2). Furthermore, bisiodide 13 provides a potential avenue for
the synthesis of diverse C7,C7′-analogues by coupling using
Heck, Suzuki, and related methods.
In applying such an approach to cercosporin, the sequencing
of the transformations is critical due to the relatively low
atropisomerization barrier. From measurements that we per-
formed, simple perylenequinones such as 16 enjoy only a
moderatedegreeofatropisomericstability.SincetheC2,C2′,C7,C7′-
groups cannot be coplanar due to steric interactions, a helical
twist is imposed on the central perylenequinone unit (Figure
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Chem. 1975, 39, 1173–1175. (c) Kurobane, I.; Vining, L. C.; McInnes,
A. G.; Smith, D. G.; Walter, J. A. Can. J. Chem. 1981, 59, 422–430.
(d) Chen, H.; Lee, M.-H.; Daub, M. E.; Chung, K.-R. Mol. Microbiol.
2007, 64, 755–770.
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Investigation of Protein Kinase C Inhibitors
A R T I C L E S
2). The strain results in a barrier to atropisomerization for 16
of 28.3 kcal/mol (Table 1, entry 3), substantially lower than
that seen with BINOL (14) (37.1 kcal/mol, Table 1, entry 1).35
Larger C7,C7′-substituents would be expected to sterically
hinder atropisomerization and to confer a higher degree of
atropisomeric stability to the calphostins and phleichromes.
Thus, we measured the atropisomerization rate of ent-1d, which
has the same (M)-helicity as 16, in the same solvent (benzene),
and as expected, a higher barrier of 30.2 kcal/mol (Table 1,
entry 4) was found relative to 16. As a result, the atropisomer-
ization barriers for ent-calphostin D (ent-1d) and phleichrome
(2) allow retention of the helical stereochemistry up to 60 °C
(Table 1, entries 4-5).19 The addition of the methylidene bridge
to the perylenequinones as seen in cercosporin (3) is expected
to impose additional strain and lower the atropisomerization
barrier further. For example, the addition of a similar bridge to
BINOL (14) to provide 15 reduces this barrier from 37.235 to
3336 kcal/mol (Table 1, entries 1-2).37
Reports in the literature for cercosporin indicated that rapid
atropisomerization occurs in boiling ether (37 °C),5 whereas a
slower isomerization was observed in DMSO (t1/2 ) 2 d at 60
°C).19 To determine if there is a solvent dependence, we
measured the atropisomerization rate in DMSO and benzene at
65 °C. Indeed, the rate was substantially faster in benzene
corresponding to a barrier of 27.4 kcal/mol vs 28.2 kcal/mol in
DMSO (Table 1, entry 6). These experiments revealed that
temperatures at 60 °C or higher would result in unacceptable
levels of atropisomerization. From a synthetic standpoint, these
results mean that the combination of the perylenequinone and
the methylidene bridge must be reserved to as late as possible.
Furthermore, all subsequent steps must avoid even moderate
temperatures in both the reaction media and purification.
Two distinct approaches fulfill the above requirements. In
the first (bottom of Scheme 3), the perylenequinone (i.e., 7) is
constructed prior to formation of the methylidene bridge in 3.
For this strategy to succeed, the conditions for installation of
the methylidene bridge need to be relatively mild since the
product 3 can undergo significant atropisomerization at 60 °C
(Table 1, entry 6). Since model studies indicated temperatures
of 65 °C were needed to form the methylidene bridge, we turned
to the reverse sequence (top of Scheme 3). Here the methylidene
bridge would be installed early (i.e., 19), also obviating the need
for three sets of orthogonal protecting groups in intermediate 8
(bottom of Scheme 3). For this strategy to succeed, the bridged
binaphthyl compound needs to be configurationally stable, which
is supported by the results from 15 (Table 1, entry 2) which
can readily withstand temperatures up to 90 °C. Furthermore,
the reaction conditions for formation of the perylenequinone
(17 to 3) are very mild (room temperature and lower),3,4c,d which
should prevent any atropisomerization of the sensitive 3 (Table
1, entry 6).
reactions to suppress the ready atropisomerization of 3 (Figure
2). Retrosynthetic installation of the C3,C3′-esters in 18 provides
a means of protecting the C3,C3′-positions for the next operation
and provides functionality needed for highly selective naphthol
coupling and rapid naphthalene synthesis. Deoxygenation to 19
and subsequent removal of the methylidene bridge furnishes
20 with a substitution pattern readily generated via biaryl
coupling.2,38 The epoxide opening disconnection was applied
at this stage to yield an even simpler biaryl 21. This stratagem
reserved diversification of the synthetic sequence to the latest
stage possible providing access to the calphostins, phleichrome,
and cercosporin. However, the retron to 21 requires an
unprecedented epoxide opening; namely, the combination of a
complex biscuprate with the requirement for two epoxide
alkylations. Biaryl 21, in turn, arises from C2,C2′,C4,C4′-
protection of 13 (Scheme 2). Thus, common intermediate 13,
available from our previous studies,3 could be used for the
construction of all the perylenequinone natural products reducing
the total number of steps that would need to be optimized. Due
to the ability to independently generate the two stereochemical
elements, the 2-hydroxypropyl group and the helical chirality,
this synthetic approach would permit the syntheses of the
complete diastereomeric series of 1, 2, and 3, including
compounds not isolated from natural sources. In addition, novel
perylenequinone structures would be easily accessible from
advanced intermediates.
Stereoselective Generation of the C7,C7′ 2-Hydroxypropyls.
In prior syntheses of the calphostins and phleichrome, the
formation of the C7,C7′-stereochemical array has been a central
element. In all cases, incorporation of this element was
intimately coupled to formation of the naphthalene skeleton prior
to dimerization. Our strategy (Scheme 2) reserves introduction
of these substituents until after naphthalene formation and
dimerization, a feature which greatly streamlines production of
the naphthalene portion and allows for maximum diversity in
the synthesis of the natural products and analogues.
To install the stereogenic 2-hydroxypropyl groups we inves-
tigated three routes: (1) diastereoselective reduction of diketone
24; (2) asymmetric methyl-addition to dialdehyde 25; (3)
organocopper epoxide opening with 21 (Scheme 4). Unfortu-
nately, both reduction of the diketone and methyl addition to
the dialdehyde proved to be unproductive, proceeding with low
yield or diastereoselectivity. Apparently, the biaryl axis is too
distant to exercise a high degree of stereocontrol over the
additions to the carbonyls of the C7,C7′-substitutents, an
outcome reminiscent of the poor stereocontrol observed in the
opposite direction (path b, Scheme 1). The last pathway, the
organocopper epoxide openings (Scheme 4) was particularly
attractive in that it allowed access to all of the diastereomers
with full stereocontrol without having to rely on the restrictions
of relative diastereoselection. Furthermore, such a pathway
permits greater flexibility in analogue synthesis. For example,
other epoxides or aziridines could lead to a host of analogues
not available from the natural products. However, the unprec-
edented double alkylation of a highly complex biscuprate with
two epoxides made this venture risky.
With the general strategy defined, the retrosynthesis evolved
to that in the top of Scheme 3. Specifically, helical chiral 3
would arise from axial chiral 17 by means of low temperature
(35) Meka, L.; Reha, D.; Havlas, Z. J. Org. Chem. 2003, 68, 5677–5680.
(36) (a) Yi, R.; Hoz, S. J. Phys. Org. Chem. 2002, 15, 782–786. (b) Park,
J.-W.; Ediger, M. D.; Green, M. M. J. Am. Chem. Soc. 2001, 123,
49–56.
The use of Grignard-derived cuprates in epoxide alkylation
has enjoyed a rich history.39 While complex cuprates have been
used successfully40 and simple biscuprates have been employed
(37) Related compounds with the same dioxapine ring are atropisomerically
stable up to 85-120 °C but are not stable at 220 °C. Papers on the
stability of seven-membered bridged biaryls: (a) Mislow, K.; Hyden,
S.; Schaefer, H. J. Am. Chem. Soc. 1962, 84, 1449–1455. (b) Kurland,
R. J.; Rubin, M. B.; Wise, W. B. J. Phys. Chem. 1964, 2426–2427.
(c) Zhang, M.; Schuster, G. B. J. Phys. Chem. 1992, 96, 3063–3067.
(38) Kozlowski, M. C.; Dugan, E. C.; DiVirgilio, E. S.; Maksimenka, K.;
Bringmann, G. AdV. Synth. Catal. 2007, 349, 583–594.
9
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A R T I C L E S
Morgan et al.
Scheme 3. Retrosynthesis of Cercosporin
in epoxide alkylation,41,42 we could locate no reports of a highly
functionalized dianion effecting two ring openings. Our primary
concerns were (1) the metal-halogen exchange on an electron-
rich system and in the presence of the C3-methyl esters; (2)
the stability of the electron-rich bisarylcuprate, and (3) the
requirement that the biscuprate be the limiting reagent rather
than in excess as is typical.
the opportunity for convergence as late as possible in the
synthesis was appealing allowing analogues to be produced from
one late intermediate. To examine epoxide opening as late as
possible, 28, in which the oxidation of the C5,C5′-positions had
already been accomplished, was utilized (Table 2, entry 2). An
arene with acetate groups intact was the major product of this
reaction arising from protodemetalation, which indicates that
either cuprate formation or epoxide opening was sluggish.
Presumably, steric gearing from the C5,C5′-positions hinders
the C7,C7′-Grignards slowing subsequent reactions at these
positions. Moving the epoxide alkylation prior to C5,C5′-
oxidation but after formation of the methylidene bridge was
examined next (Table 2, entry 3). While the desired product 31
did form in 40% yield, the methylidene bridge caused the
C3,C3′-esters to be less hindered resulting in a competing
halodecarboxylation. To alleviate this problem, the double
epoxide alkylation was investigated with different C2,C2′-
substitution (Table 2, entry 4). The C2,C2′-bismethoxy, bis-
benzyloxy, and bisisopropoxy analogues all reacted well
providing the corresponding products with two new stereocenters
in 65-75% yield, corresponding to the 81-87% yield for each
of the two individual coupling reactions. Furthermore, reaction
of the same M-chiral biaryl 32a with the enantiomeric epoxide
provided 34, the diastereomer of 33a in 74% yield (Table 2,
entry 5). While different rates might be expected via matched
and mismatched reactions of the enantiomeric epoxides (R)- and
(S)-propylene oxide with (M)-32a, no difference in the reaction
rate was seen here. Apparently, the stereochemistry of the biaryl
is distant from the reacting centers and exerts little control, in
line with previous observations (see Scheme 4). Together, these
results validated the use of the epoxide dialkylation as a key
bond construction providing a highly flexible synthetic route.
For example, biaryl 32 can be used to furnish 33a corresponding
to ent-phleichrome (ent-2) and 34 corresponding to ent-
calphostin D (ent-1d) as well as 33b and 33c which can either
provide analogues with different C2,C2′-subsititution or serve
as precursors toward cercosporin (3).
To fully evaluate the proposed epoxide-opening reaction,
several naphthalenes were investigated revealing that a C7-
iodide permitted the best coupling and that robust alkyl
protecting groups needed to be employed instead of acetates.
Hindered bases (t-BuLi, i-PrMgBr) were found to attenuate
addition to the C3-methyl ester. Encouragingly, the initial
epoxide opening with 26 yielded modest amounts of the desired
product (40%) with complete regioselectivity (Table 2). Rigor-
ously maintained air- and water-free conditions drastically
decreased the amount of protodemetalation, improving the yield
of 27 to 77% (entry 1a). An examination of the cuprate addition
revealed that additives such as TMSCl or HMPA or lower
temperatures (-78 °C) provided no improvement. Upon screen-
ing the copper reagent and solvents (Table 2, entries 1a-d),
CuI and THF were found to be optimal. Careful purification of
the CuI by recrystallization proved to be the most critical
element providing product 27 in 87% yield (Table 2, entry 1e).
Due to the requirement for a C4-acetate during biaryl
coupling2 (see below) which is not tolerated in the epoxide
opening, the epoxide-opening reaction was reserved until after
biaryl coupling to reduce the number of protecting group steps.
While the task of forming a biscuprate and undertaking two
epoxide alkylations on such a complex dianion was daunting,
(39) (a) Lipshutz, B. H.; Kozlowski, J.; Wilhelm, R. S. J. Am. Chem. Soc.
1982, 104, 2305–2307. (b) Smith, J. G. Synthesis 1984, 629–656. (c)
Sheperd, T.; Aikins, J.; Bleakman, D.; Cantrell, B.; Rearick, J.; Simon,
R.; Smith, E.; Stephenson, G.; Zimmerman, D. J. Med. Chem. 2002,
45, 2101–2111. (d) Taber, D. F.; He, Y. J. Org. Chem. 2005, 70, 7711–
7714. (e) Nakamura, E.; Mori, S. Angew. Chem., Int. Ed. 2000, 39,
3751–3771. (f) Review of organocopper reagents: Lipshutz, B. H.;
Sengupta, S. Org. React. 1992, 41, 135.
Total Synthesis of (+)-Phleichrome and (+)-Calphostin D.
Our total synthesis efforts commenced with the simpler peryle-
nequinone compounds lacking the methylidene bridge. As
outlined in Scheme 5, the bisiodide 13 was generated in a
straightforward manner using the protocol that we reported for
its enantiomer.3 Thus, the M-biaryl was produced in good yield
and enantioselectivity (81% ee) utilizing the (S,S)-diaza-cis-
decalin copper catalyst.1 The high crystallinity of bisiodide 13
(40) (a) Ireland, R. E.; Gleason, J. L.; Gegnas, L. D.; Highsmith, T. K. J.
Org. Chem. 1996, 61, 6856–6872. (b) Ireland, R. E.; Liu, L.; Roper,
T. D. Tetrahedron 1997, 53, 13221–13256.
(41) Cahill, S.; Evans, L. A.; O’Brien, M. Tetrahedron Lett. 2007, 48, 5683–
5686.
(42) For simple bis-Grignard and bis-lithium reagents in epoxide opening: (a)
Bianchetti, G. Farmaco, Ed. Sci. 1957, 12, 441–448. (b) Ghorai, P.;
Dussault, P. H.; Hu, C. Org. Lett. 2008, 10, 2401–2404. (c) Gordon,
B., III.; Blumenthal, M.; Mera, A. E.; Kumpf, R. J. J. Org. Chem.
1985, 50 (9), 1540–1542.
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Investigation of Protein Kinase C Inhibitors
A R T I C L E S
Scheme 4. Pathways to Installation of the C7,C7′-Stereoarray
The C3-ester is integral to the synthesis of the naphthalene and
also provides a necessary coordination site for the biaryl
coupling catalyst. Large quantities of common intermediate 13
could be generated, which was used in the synthesis of three
natural products and 10 analogues.
The C2,C2′,C4,C4′-phenols of 13 were methylated by means
of excess NaH in undistilled DMF with excess MeI (Scheme
5). The small amount of water in DMF was converted to NaOH,
which cleaved the acetates. Subsequently, MeI alkylated all four
positions with high efficiency (94% yield). At this stage,
formation of the biscuprate and double epoxide alkylation were
undertaken following the method described above (see Table 2
and accompanying discussion). Both (R)- and (S)-propylene
oxide were employed to provide the diastereomers (M,R,R)-
33a and (M,S,S)-34 in good yield. After benzylation of the newly
formed alcohols, the C5,C5′-oxidation was undertaken. We
found that our previously reported protocol3 for this transforma-
tion using Kita’s method [(PhI(OCOCF3)2, (CF3)2CHOH,
provided a simple means of enantioenrichment with one
trituration giving >99% ee material and excellent mass recovery.
Table 2. Screening Substrates and Conditions for Epoxide Opening
a 2.5 equiv of i-PrMgBr; 1 equiv of Cux; epoxide source for entries 1-4 is (R)-propylene oxide and for entry 5 is (S)-propylene oxide. b Conversion.
c Isolated yield in parentheses. d Inseparable byproducts.
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A R T I C L E S
Morgan et al.
Scheme 5. Total Synthesis of (+)-Phleichrome and (+)-Calphostin D
NaOAc]43 was very sensitive to any changes in the oxidation
potential of the substrate requiring reoptimization for each new
precursor. As a result, we devised a more robust C5,C5′-
oxidation utilizing a palladium-catalyzed O-arylation. While
these transformations have been reported for a host of systems,44
we aimed to determine their applicability to highly functional-
ized, hindered, electron-rich systems. Thus, chlorination was
effected with high efficiency (>90% yield) using sulfuryl
chloride to provide (M,R,R)-36 and (M,S,S)-36. Here, the
C3,C3′-esters again proved critical; while not present in the
natural products, they serve to protect the C3,C3′-positions from
halogenation. Disappointingly, direct O-arylation of the bis-
chloride with benzyl alcohol and other alcohols failed, likely
due to the hindered nature of the C5,C5′-position. Gratifyingly,
hydroxylation using KOH and a palladium catalyst45 proceeded
extremely well and was found to be remarkably general for a
range of substrates. While the product bisphenol was not stable,
immediate protection with benzyl bromide furnished (M,R,R)-
37 and (M,S,S)-37. A control reaction using KOH without
palladium catalyst did not produce any of the bisphenol
indicating that an addition/elimination pathway of the conjugate
ester was not responsible for the results.
The next task was removal of the C3,C3′-esters which were
immune to aqueous basic hydrolysis even at elevated temper-
atures, due to their steric hindrance. We discovered that an SN2
displacement of the methyl esters could be accomplished with
NaCN in DMSO to provide the bisacid.3 However, conventional
aromatic decarboxylation protocols46 required temperatures that
would atropisomerize the biaryl. As a consequence we devel-
oped a palladium-catalyzed decarboxylation protocol,47 which
worked well in these systems.3 Unfortunately, we discovered a
competing C-H insertion reaction occurred with the meth-
ylidene bridge series en route to cercosporin (see below). Thus,
we undertook development of a new strategy with 37. Reex-
amination of other decarboxylation methods48 yielded no success
so our attention turned to decarbonylation.49 Formation of the
requisite dialdehyde proved to be more efficient by overreduc-
tion using DIBAL and then oxidation with ortho-iodoxybenzoic
acid (IBX) (Scheme 5). Decarbonylation with 2 equiv of
Wilkinson’s catalyst to (M,R,R)-38 and (M,S,S)-38 then pro-
ceeded smoothly provided that rigorously oxygen-free conditions
were employed.
The remaining steps of the synthesis could all be conducted
under very mild conditions. Removal of the four benzyl ethers
occurred readily and was followed by oxidative cyclization with
MnO2 to reveal the central perylenequinone core.4c,d Directed
4b,e,f
deprotection of the C4,C4′-methyl ethers with MgI2
fur-
nished the enantiomers of the natural products phleichrome (ent-
2) and calphostin D (ent-1d), respectively (Scheme 5). Each
synthesis proceeded in a total of 17 steps with ent-2 being
formed in an overall yield of 5.3% (average of 87% per step)
and ent-1d being formed in an overall yield of 5.2% (average
of 87% per step). With the enantiomers of the natural products
available, it was now possible to assess the impact of the helical
and centrochiral centers on the biological activity.
First Total Synthesis of Cercosporin. Starting from common
intermediate 13 (see Scheme 5), orthogonal C2,C2′-benzyl ethers
were installed via a Mitsunobu reaction. A subsequent one-pot
deacylation/methylation of the C4,C4′-positions afforded 32b
with high yield and scalability (Scheme 6). The double
alkylation of the biscuprate of 32b with (R)-propylene oxide
furnished 33b in high yield as a single diastereomer. The newly
formed 2-hydroxypropyl groups were then masked as benzyl
ethers furnishing 39. Subsequent chemoselective debenzylation
of the more labile C2,C2′-benzyl ethers of 39 was efficiently
accomplished using Pd/C poisoned with pyridine. Installation
(43) Kita, Y.; Tohma, H.; Hatanaka, K.; Takada, T.; Fujita, S.; Mitoh, S.;
Sajurai, H.; Oka, S. J. Am. Chem. Soc. 1994, 116, 3684–3691.
(44) Reviews: (a) Muci, A. R.; Buchwald, S. L. Top. Curr. Chem. 2002,
219, 131–209. (b) Hartwig, J. F. Nature 2008, 455, 314–322. (c) Carril,
M.; SanMartin, R.; Dominguez, E. Chem. Soc. ReV. 2008, 37, 639–
647.
(45) (a) Torraca, K. E.; Huang, X.; Parrish, C. A.; Buchwald, S. L. J. Am.
Chem. Soc. 2001, 123, 10770–10771. (b) Vorogushi, A. V.; Huang,
X.; Buchwald, S. L. J. Am. Chem. Soc. 2005, 127, 8146–8149. (c)
Anderson, K. W.; Ikawa, T.; Tundel, R. E.; Buchwald, S. L. J. Am.
Chem. Soc. 2006, 128, 10694–10695.
(47) Dickstein, J. S.; Mulrooney, C. A.; O’Brien, E. M.; Morgan, B. J.;
Kozlowski, M. C. Org. Lett. 2007, 9, 2441–2444.
(48) (a) Garner, P.; Anderson, J. T.; Dey, S. J. Org. Chem. 1998, 63, 5732–
5733. (b) Chimiak, A.; Przychodzen, W.; Rachon, J. Heteroatom.
Chem. 2002, 13, 169–194.
(46) (a) Olah, G. A.; Laali, K.; Mehrotra, A. K. J. Org. Chem. 1983, 48,
3360–3362. (b) Cohen, T.; Schambach, R. A. J. Am. Chem. Soc. 1970,
92, 3189–3190. (c) Barton, D. H. R.; Lacher, B.; Zard, S. Z.
Tetrahedron Lett. 1985, 26, 5939–5942.
(49) (a) Ohno, K.; Tsuji, J. J. Am. Chem. Soc. 1968, 90, 99–107. (b) Abu-
Hasanayn, F.; Coldman, M. E.; Goldman, A. S. J. Am. Chem. Soc.
1992, 114, 2520. (c) Kreis, M.; Palmelund, A.; Bunch, L.; Madsena,
R. AdV. Synth. Catal. 2006, 348, 2148–2154.
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Investigation of Protein Kinase C Inhibitors
A R T I C L E S
Scheme 6. First Total Synthesis of Cercosporin
of the methylene bridge was not trivial, presumably due to
disfavorable entropy in forming the seven-membered ring
combined with ring strain. Of all the potential methylidene
equivalents, ring formation was most facile with BrCH2Cl vs
BrCH2I or BrCH2Br. Apparently, there is a balance between
the leaving group ability of the second halide and its size with
larger halides greatly decelerating the first alkylation event. Even
so, formation of the seven-membered ring of 40 required heating
to 65 °C, which was well below the atropisomerization threshold
(see Table 1, entry 2).
As described above (see Scheme 5, 33a/34 to 37 and
accompanying text), we elected to use a halogenation/O-
arylation reaction to install the C5,C5′-oxygenation. The C5,C5′-
chlorination proceeded uneventfully, but the chloro to alkoxy
interchange was difficult. After optimization of the reaction
conditions, we found that the catalyst system derived from
Pd2dba3 and the X-phos(t-Bu) ligand proved to be effective in
the coupling of 40 with KOH to provide the desired bisphenol.
Unfortunately, the resulting product was highly unstable and
could not withstand a one-pot alkylation protocol. Isolation of
the bisphenol under carefully controlled conditions, however,
proceeded well. Immediate exposure to standard benzylation
procedures (BnBr, NaH, DMF) furnished key intermediate 41
in 70% yield.
As alluded to above (see Scheme 5, 37 to 38 and accompany-
ing text), decarboxylation of 41 proved difficult. We had
developed a palladium-catalyzed protodecarboxylation for this
application which succeeded in related systems.47 With 41,
however, this procedure failed due to a competing C-H
insertion reaction of the methylidene in the seven-membered
ring. Fortunately, decarbonylation of the corresponding dialde-
hyde with excess Wilkinson’s catalyst proceeded well, provided
that rigorously air-free conditions were employed. Furthermore,
the relatively high temperatures (85 °C in diglyme) required
for this process were well-tolerated in accord with our expecta-
tions (Table 1, entry 2) and no atropisomerization was observed.
After deprotection of 42, the bisphenol was oxidized by MnO2
to afford perylenequinone (Scheme 6).4c,d As discussed in the
retrosynthetic analysis, this final cyclization step was held until
the end of the synthesis because formation of the perylene-
quinone reduces the dihedral angle between the upper and lower
portions from 50° in axial chiral 42 to 20° in helical chiral 3,
further facilitating atropisomerization. Directed deprotection of
the C4,C4′-methyl ethers was accomplished selectively with
MgI2,4b,e,f completing the first total synthesis of cercosporin (3)
in 20 steps and 2.8% overall yield (86% average yield per step).
Notably, none of the diastereomer of 3 arising from the facile
atropisomerization was observed in this sequence. The synthetic
material possessed NMR and CD spectra identical to those from
a sample of the natural product.
Design of Novel Perylenequinone Structures. The two peryle-
nequinone apoptosis pathways allow for the therapeutic effects
to be localized in cancer therapy two distinct ways: (1) via
direction of a specific wavelength of light to the region of the
tumor and (2) via sequestration in cells with high levels of PKC.
Since most tumor cells appear to have multiple growth and
survival mechanisms, the two apoptosis pathways and means
of therapeutic localization render the perylenequinones espe-
cially promising targets. As a result, we targeted derivatives to
both increase the photodynamic activity and also probe PKC
binding. To increase the photoresponse in the photodynamic
window (600-900 nm), the perylenequinone chromophore was
extended through C3,C3′-substitution. Additionally, we designed
and synthesized perylenequinones in a systematic manner to
examine the effects of the helical chirality, C2,C2′-substitution,
C3,C3′-substitution, and C7,C7′-substitution, which have thus
far seemed to be the most integral to PKC inhibition.22 Most
perylenequinone analogues examined to date are derived from
the natural product themselves;23 generation of the above
variants from the natural products is difficult or not feasible.
The lack of information has limited the conclusions drawn
between structure and PKC inhibition. On the other hand, our
synthetic strategy allows rapid and convergent synthesis of a
large number of novel analogues for biological and photophysi-
cal evaluation.
The versatility of our perylenequinone strategy (Scheme 2)
enabled the syntheses of the stereoisomers of 1-4, including
the natural products themselves (Figure 3). Utilizing the
enantioselective biaryl coupling we can access both enantiomers
of the common intermediate (P-13 and M-13). In addition, the
copper-mediated epoxide openings are integral to this evaluation,
allowing entry to the unnatural isomers of 1, 2, and 3, which
would not be possible if a stereochemistry transfer reaction was
used in the construction of the stereogenic 2-hydroxypropyl
groups. Thus, our approach permits a systematic assessment of
the helical stereochemistry and the C7,C7′-stereochemistry on
PKC inhibition.
Docking studies were performed to determine what struc-
tural modifications might lead to higher activity. Starting with
the PKCδ C1 regulatory domain obtained from a cocrystal
structure with a phorbol ester,50 ent-phleichrome (ent-2),
which contains the helical and centrochiral stereochemistries
that led to the best binding (see below), was docked into the
phorbol binding domain utilizing Autodock3.51 The lowest
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A R T I C L E S
Morgan et al.
Scheme 7. Synthesis of C2,C2′-Analogues
energy binding mode places the C2,C2′-methoxy groups into
a hydrophobic pocket of the binding site. Since there was
unfilled space in this pocket, we proposed to modify the
C2,C2′-substitution to include the more hydrophobic isopro-
poxy and n-propoxy groups (48a and 48b). Docking of these
two analogues of (ent-2) indicated superior binding and is
expected to lead to higher affinity.52 As seen in Scheme 7,
these novel perylenequinones could be synthesized by
selective alkylation of an advanced intermediate (44) avail-
able from the cercosporin synthesis (step 11, Scheme 6). The
remaining steps followed the protocol developed above for
calphostin/phleichrome (Scheme 5) and cercosporin (Scheme
6) providing unnatural congeners 48a and 48b in a straight-
forward manner (Scheme 7). The yield for the last three steps
was lower than those obtained above for cercosporin due to
the larger C2,C2′-n-propoxy and -iso-propoxy groups which
slow the perylenequinone forming reaction.
Whereas the docking studies described above indicated a
possible interaction of the C2,C2′-substituents with a hydro-
phobic pocket in the regulatory domain site, the much higher
affinity of calphostin A (Figure 1, 1a, R1 ) R2 ) COPh, IC50
) 0.25 µM) compared to calphostin D (1d, R1 ) R2 ) H, IC50
) 6.4 µM) points to interaction of the C7,C7′-portion with the
binding site.22b While it may be possible that the perylene-
quinone lies in a sufficiently deep pocket to undergo interactions
with both the western and eastern portions of the structure, we
expect that C2,C2′- and C7,C7′-analogues will be invaluable
in elucidating the relative importance of each of these interac-
tions. Our first step was to determine whether the stereogenic
hydroxyl groups on the C7,C7′-substituents of the natural
products were critical to binding. Thus, the simpler C7,C7′-
propyl substitution was incorporated into analogue 16 (Scheme
8). Based upon the improVed activity of this compound relative
to the parent natural products (ent-1d and ent-2) with the C7,C7′-
2-hydroxypropyl substitution (see below), a series of derivatives
(53-57) incorporating the C7,C7′-propyl groups were designed
to probe the effect of substitution at the C3,C3′-positions.
Specifically, bromo (57), ester or acid (53, 55, 56), and vinyl
(54) C3,C3′-substitutions were proposed to increase the absorp-
tion wavelength of the perylenequinone chromophore (Scheme
8). The use of the C3,C3′-methyl ester in our synthetic strategy
proved very useful at this juncture allowing all of these structures
to be quickly accessed either directly or by decarboxylative
functionalization (Scheme 8).
The synthesis of 16 and 53-57 (Scheme 8) commenced with
racemic bisiodide 13, a byproduct of the trituration to provide
M-13 (see Scheme 5). Cross coupling with the iodides of 13 is
a versatile and simple means to introduce any desirable C7,C7′-
substitution. In this case, Suzuki coupling with pinacol allyl-
boronate provided bisallyl 49.3 Subsequent hydrogenation
yielded the bis-n-propyl compound. The lack of functionalization
of the C7,C7′-groups allowed facile C5,C5′-hydroxylation with
Kita’s reagent43 to provide 50 following our first generation
protocol.3
Compound 50 represents the first branchpoint intermediate
in the analogue syntheses; a small amount was subjected to
MnO2 followed by MgI2 to effect oxidative cyclization2 and
deprotection furnishing analogue 53. The remainder of branch-
point intermediate 50 was subjected to benzyl bromide and NaH
to protect the C5,C5′-naphthols in preparation for ester hy-
drolysis to provide the next key branchpoint intermediate,
biascid 51. Ester hydrolysis here was surprisingly facile relative
to congeners 37 (Scheme 5) and 41 (Scheme 6). Presumably,
the smaller C7,C7′-groups alleviate gearing interactions that
sterically hinder the C3,C3′-esters.
Bisacid 51 was subject to three different fates. In the first
pathway, a palladium-catalyzed decarboxylative Heck reaction
of 51 followed by perylenequinone formation provided bisstyryl
Figure 3. Common enantiopure intermediates to allow evaluation of helical
chirality.
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9422 J. AM. CHEM. SOC. VOL. 131, NO. 26, 2009

Investigation of Protein Kinase C Inhibitors
A R T I C L E S
Scheme 8. Use of Common Intermediates in the Syntheses of C3,C3′,C7,C7′-Analogues
derivative 54.53 In the second pathway, the C5,C5′-benzyl ethers
were cleaved and the more acidic carboxylic acids were then
selectively benzylated using BnBr and K2CO3. This tactic was
necessary because the carboxylic acids did not survive the
MnO2-mediated oxidative cyclization, nor was selective hydro-
genation of the bisbenzyl ester of 51 successful. Subsequent
perylenequinone formation yielded 55, which could then be
further hydrogenated to yield the perylenequinone bisacid 56.
In the third pathway, protodecarboxylation using a palladium
source furnished 52,47 which was then transformed to peryle-
nequinone 1654 following protocols described above. Peryle-
nequinone 16 could, in turn, be treated with Br2 to provide
bisbromide 57. Subsequent enzyme assays with this analogue
series revealed high activity for 16, stimulating us to also
undertake a synthesis of M-16 since we had established that
the M-isomers are more potent (see below).
inhibit PKC phosporylation, providing a benchmark comparison
of a broad range of the natural products and analogues to
establish the most critical elements for PKC inhibition.
As seen in Table 3, there are clear trends between the
perylenequinone architecture and PKC-inhibitory activity. No-
tably, these results represent the first systematic evaluation of
helical chirality on PKC inhibition. We found a direct correlation
between helical chirality and inhibition; the M-perylenequinones
were 1.8-20 times more potent than the corresponding P-
isomers with the same C7,C7′-stereochemistry [(+)-1 vs (-)-
2, (+)-2 vs (-)-1, and 3 vs epi-3]. For the hypocrellins (4 and
ent-4), the helical stereochemistry atropisomerizes rapidly at
room temperature and the compounds are 4:1 equilibrium
mixtures of the two atropisomers.18 In line with the above
results, the greatest PKC inhibition was observed for the
compound where the M-helicity predominates (ent-4). Particu-
larly noteworthy is that the unnatural isomers (+)-1 and (+)-2
were more potent than the natural products (-)-1 and (-)-2.
With respect to the stereochemistry of the C7,C7′-substitution,
the R,R-array was found to offer slightly better (1.7-fold) activity
than the S,S-array [(+)-1 vs (+)-2, (-)-1 vs (-)-2].
Docking models of the perylenequinones indicated that larger
hydrophobic groups at the C2,C2′-positions better fill a pocket
in the binding site (see above).52 Analogues of the most active
stereochemical array (M,R,R) were thus constructed to probe
the role of the C2,C2′-substitution (Scheme 7). Pleasingly, the
bisisopropyl 48a (0.8 µM) and bis-n-propyl 48b (1.5 µM) were
more potent than the parent compound (+)-2 (3.5 µM) providing
support for the docking model.
Protein Kinase C Inhibition. The activity of the calphostins
and hypocrellin A are thought to arise in part by modulation of
protein kinase C (PKC), a family of C1 domain containing
kinases. The PKCs are membrane bound proteins that play
important roles in intracellular signal transduction effecting
proliferation, differentiation, and apoptosis and have been
implicated in a variety of disease states including diabetes,
cancer, heart disease, and cognitive disfunction.24,55 Because
the C1 domain is found in only a small subset of the human
kinome,56 selectivity relative to the many other biologically
important kinases may be achievable. In light of this information,
the newly synthesized analogues were tested for their ability to
(50) Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J. H. Cell
1995, 81, 917–924.
Prior reports indicated that acylation of the C7,C7′-hydroxy-
propyl group provided superior activity (i.e., calphostin A, 1a,
R1 ) R2 ) COPh, 0.25 µM vs calphostin D, 1d, R1 ) R2 ) H,
6.4 µM; Figure 1).22b To determine if this result arises from a
simple increase in hydrophobicity and removal of the two
hydroxyl H-bond donors, the C7,C7′-n-propyl analogues were
synthesized (Scheme 8). Notably, this compound possessed
significant potency with racemic 16 exhibiting an IC50 of 1.2
µM indicating that hydrophobic C7,C7′-groups are indeed
optimal (Table 3). Based upon our discovery that the M-helicity
(51) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.;
Belew, R. K.; Olson, A. J. J. Comput. Chem. 1998, 19, 1639–1662.
(52) See Supporting Information.
(53) (a) Myers, A. G.; Tanaka, D.; Mannion, M. R. J. Am. Chem. Soc.
2002, 124, 11250–11251. (b) Tanaka, D.; Romeril, S. P.; Myers, A. G.
J. Am. Chem. Soc. 2005, 127, 10323–10333.
(54) Racemic-24 has been reported previously. See ref 4f.
(55) Reviews on Protein Kinase C: (a) Nishizuka, Y. Nature 1984, 308,
693–698. (b) Nishizuka, Y. Science 1986, 233, 305–312.
(56) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,
S. Science 2002, 298, 1912–1934.
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A R T I C L E S
Morgan et al.
Table 3. Perylenequinone Inhibition of PKC
a PKC evaluation obtained from ref 23.
provided superior inhibition, enantiopure M-16 was generated
(see Scheme 8) and was found to exhibit an IC50 of 0.4 µM.
Based on these data, P-16 is indeed a poorer inhibitor (2.0 µM)
supporting our prior conclusions. Enantiopure M-16 is a 9-25
times better inhibitor than the nearest congeners, (+)-1 and (+)-
2, and 16-28 times better than the corresponding natural
products, (-)-1 and (-)-2.
The finding that the stereogenic C7,C7′-substitution is not
necessary for potency and that architecturally simple structures
such as 16 can be employed motivated us to examine C3,C3′-
analogues of the C7,C7′-n-propyl series (Scheme 8). In doing
so, compounds (53-57) with esters, acids, styryls, or bromo
groups at the C3,C3′-positions were designed to alter the
chromophore to improve the absorption profile. Analogues
53-57 displayed increased absorption in the 600-800 nm range
compared to the natural products. Assessing the effects on PKC
inhibition, the bisacid 56 was found to decompose rapidly. On
the other hand, 53-54 and 57 were found to be 2-4 times less
potent than the parent analogue 16, having the same architecture
but no C3-substitution.
The assays described above were performed with PKC
containing regulatory and catalytic domains. To determine
whether these perylenequinones were indeed inhibiting phos-
porylation by acting on the regulatory domain, assays were
performed with PKC containing only the catalytic subunit. Over
85% of the catalytic subunit activity was retained even when
50 µM 16 was employed which is well above the IC50 (1.2 µM)
seen with the whole enzyme.
Cancer Cell Lines. To determine whether the structural effects
on PKC inhibition are related to anticancer activity, eight cancer
cell lines were screened against the most potent M-isomers: ent-
phleichrome [(+)-2], cercosporin (3), and hypocrellin (ent-4).
Finally, the most potent analogue, 16, was included. All of the
compounds exhibited micromolar or submicromolar CC50 values
for growth inhibition in line with their light activated mechanism
of action. Overall, cercosporin (3) was the most active com-
pound in every cell line exhibiting CC50 values of 0.12-0.27
µM across the series. The remaining compounds exhibited
similar activities across all of the cell lines with the exception
of simple analogue 16, which was a poorer inhibitor of HT29
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9424 J. AM. CHEM. SOC. VOL. 131, NO. 26, 2009

Investigation of Protein Kinase C Inhibitors
A R T I C L E S
Table 4. CC50 (µM) Values of the Perylenequinone against Cancer
photoactive compounds. The analogues provided an assessment
of the structural features important to PKC inhibition with
M-helicity and hydrophobic C7,C7′-substitution being identified
as advantageous. Substitution at the C2,C2′- and C3,C3′-
positions was well tolerated and led to compounds simpler than
the natural products, but possessing superior absorption profiles
which may provide an avenue to superior photosensitizers.
Finally, cercosporin (3) and simple analogue 16 provide superior
inhibition of growth activity in cancer cell lines, and the latter
displayed higher levels of photopotentiation than any of the
natural products. Further biological evaluation of these and
additional compounds, including selective inhibition among the
PKC isoforms, is underway and will be reported in due course.
Cell Lines
NCI- SK-
H460 PC3 MEL-5 SN12C U251 A2780 MCF7
JHU-012 Head
and Neck
photo-
potentiation
cmpd lung prostate skin
colon brain ovary breast light darkb
(+)-2 0.52 0.71 0.86 0.66 0.49 0.28 1.1 0.81 2.8
0.13 0.26 0.15 0.17 0.15 0.12 0.20 0.16 0.78
ent-4 0.49 0.51 0.57 1.0 0.57 0.41 0.84 1.0 1.9
16 0.51 0.71 0.55 0.58 0.50 0.22 0.84 0.23 1.3
3.5
4.9
1.9
5.8
3
a All assays conducted with a 30 min exposure to a 32 W light at 5
cm. b Assays conducted without 30 min light exposure.
and a superior inhibitor of A2780 and JHU-012 relative to all
the compounds except cercosporin (3). Given the selectivity of
cercosporin (3) and simple analogue 16 for the head and neck
cancer line JHU-012, the compounds were further examined
without light exposure in this line. The photopotentiation was
strongest with cercosporin (4.9-fold) among the natural products.
Remarkably, the photopotentiation was even more pronounced
for simple analogue 16 (5.8-fold).
Acknowledgment. We are grateful to the NIH CA-109164 for
financial support and to Novartis and the American Chemical
Society for graduate fellowships (B.J.M.). Special thanks to E. M.
Daub and Quest Pharmaceuticals for providing samples of cer-
cosporin and hypocrellin, respectively. We thank James Ianni for
assistance with the docking studies, Feng Gai and Virgil Percec
for assistance with CD measurements, and Barry Cooperman for
assistance with the enzyme assays.
Concluding Remarks
In summary, the first total syntheses of (+)-1 and 3 and 10
perylenequinone analogues have been accomplished in an
efficient manner beginning with a common chiral binaphthalene.
The approach employing enantioselective oxidative biaryl
coupling, double cuprate epoxide opening, and decarboxylative
functionalization permits access with complete control of the
helical and centrochiral stereochemical elements of these
Supporting Information Available: Full experimental details
including synthetic procedures, atropisomerization data, biologi-
cal assays, and spectral data. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA902324J
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J. AM. CHEM. SOC. VOL. 131, NO. 26, 2009 9425