(S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21- dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene- 1,3(2H)-dione (LY333531) and related analogues: Isozyme selective inhibitors of protein kinase Cβ

Article abstract of DOI:10.1021/jm950588y

Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overactivation of some PKC isozymes has been postulated to occur in several disease states, including diabetic complications. Selective inhibition of overactivated PKC iso

Full text of DOI:10.1021/jm950588y

2664
J . Med. Chem. 1996, 39, 2664-2671
Articles
(S)-13-[(Dim eth yla m in o)m eth yl]-10,11,14,15-tetr a h yd r o-4,9:16,21-d im eth en o-
1H,13H-d iben zo[e,k]p yr r olo[3,4-h ][1,4,13]oxa d ia za cycloh exa d ecen e-1,3(2H)-d ion e
(LY333531) a n d Rela ted An a logu es: Isozym e Selective In h ibitor s of P r otein
Kin a se Câ
Michael R. J irousek,*,† J ames R. Gillig,† Cecile M. Gonzalez,† William F. Heath,† J ohn H. McDonald III,†
David A. Neel,† Christopher J . Rito,† Upinder Singh,† Lawrence E. Stramm,† Anita Melikian-Badalian,‡
Matthew Baevsky,‡ Lawrence M. Ballas,‡ Steven E. Hall,‡ Leonard L. Winneroski,§ and Margaret M. Faul§
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, Sphinx Pharmaceutical Corporation,
Durham, North Carolina 27712, and Lilly Chemical Process Research and Development, Eli Lilly and Company,
Indianapolis, Indiana 46221
Received March 7, 1996X
Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overac-
tivation of some PKC isozymes has been postulated to occur in several disease states, including
diabetic complications. Selective inhibition of overactivated PKC isozymes may offer a unique
therapeutic approach to disease states such as diabetic retinopathy. A novel series of 14-
membered macrocycles containing a N-N′-bridged bisindolylmaleimide moiety is described.
A panel of eight cloned human PKC isozymes (R, âI, âII, γ, δ, ꢀ, ú, η) was used to identify the
series and optimize the structure and associated activity relationship. The dimethylamine
analogue LY333531 (1), (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16,21-dime-
theno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione, inhibits
the PKCâI (IC50 ) 4.7 nM) and PKCâII (IC50 ) 5.9 nM) isozymes and was 76- and 61-fold
selective for inhibition of PKCâI and PKCâII in comparison to PKCR, respectively. The
additional analogues described in the series are also selective inhibitors of PKCâ. LY333531
(1) exhibits ATP dependent competitive inhibition of PKCâI and is selective for PKC in
comparison to other ATP dependent kinases (protein kinase A, calcium calmodulin, caesin
kinase, src tyrosine kinase). The cellular activity of the series was assessed using bovine retinal
capillary endothelial cells. Retinal endothelial cell dysfunction has been implicated in the
development of diabetic retinopathy. Plasminogen activator activity stimulated by a phorbol
ester (4â-phorbol 12,13-dibutyrate) in endothelial cells was inhibited by the compounds in the
series with ED50 values ranging from 7.5 to 0.21 µM. A comparison of the PKC isozyme and
related ATP dependent kinase inhibition profiles is provided for the series and compared to
the profile for staurosporine, a nonselective PKC inhibitor. The cellular activity of the series
is compared with that of the kinase inhibitor staurosporine.
In tr od u ction
peractivation of PKCâ relative to other isozymes (PKCR,
γ, δ, ꢀ, ú, η) in tissue from diabetic animal models, such
as retina and kidney glomeruli, in addition to platelets
from human focused our effort on developing an isozyme
selective antagonist.6 Therapeutically, a PKCâ selective
antagonist could offer a new approach for intervention
in the progression or development of diabetic retinopa-
thy.
The association between the development of diabetic
complications and the level of glycemic control in
diabetic patients has been demonstrated in a number
of studies, including the diabetes control and complica-
tions trial (DCCT).1 Hyperglycemia causes an elevation
of diacylglycerol (DAG), an endogenous second mes-
senger that activates protein kinase C (PKC).2 Tissues
subject to developing diabetic complications contain
elevated levels of DAG.2,3 The elevation in tissue levels
of DAG takes several days to manifest itself and
remains elevated several days after the tissue has been
removed from a hyperglycemic environment. The hy-
pothesis that PKC is overactivated in a diabetic state
and contributes significantly to the development of
diabetic complications encouraged us to initiate a
program to identify a PKC selective antagonist.4,5 Hy-
PKC consists of a family of closely related enzymes
that function as serine and threonine kinases.7 These
enzymes were originally identified as a histone protein
kinase from rat brain and activated by limited proteoly-
sis or by calcium and lipids. Further work revealed
PKC to be the molecular target for a class of tumor
promoters, the phorbol esters.8 Subsequent to the
initial observations that rat brain PKC was composed
of at least four isozymes (R, âI, âII, γ), further screening
of cDNA libraries has identified an additional eight
related proteins to bring the membership of the family
to 12 isozymes or subtypes.9
† Lilly Research Laboratories.
‡ Sphinx Pharmaceutical Corp.
§ Lilly Chemical Process Research and Development.
X Abstract published in Advance ACS Abstracts, J une 15, 1996.
A panel of eight cloned human PKC isozymes (R, âI,
S0022-2623(95)00588-7 CCC: $12.00 © 1996 American Chemical Society

Isozyme Selective Inhibitors of PKCâ
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2665
Sch em e 1a
âII, γ, δ, ꢀ, ú, η) was used to identify and optimize a
series of PKCâ selective antagonists. The panel is
representative of the calcium dependent PKC isoforms
(R, â, γ), the calcium independent isoforms (δ, ꢀ, η), and
the atypical isoforms (ú). Until recently the isozyme
selectivity of the related acyclic bisindolylmaleimide
PKC inhibitors had not been realized.10 Subsequently,
some of the acyclic bisindolylmaleimides have been
reported to preferentially inhibit PKCR.11 Using assay
conditions that simulate the endogenous activation of
PKC with DAG and phosphatidylserine, the macrocyclic
bisindolylmaleimides reported here are PKCâ selective
in comparsion to the other isozymes in the panel.12 The
acyclic bisindolylmaleimides have also been reported to
be competitive inhibitors of ATP binding presumably
by interacting at the ATP binding site.13 An additional
criterion for selectivity is preferential inhibition of the
PKCâ isozymes versus other ATP dependent kinases.
An ATP dependent kinase panel consisting of four
kinases (PKA, calcium calmodulin, casein kinase, src
tyrosine kinase) was used in tandem with the PKC
isozyme panel to demonstrate the kinase selectivity of
the macrocyclic bisindolylmaleimides.
a
(a) Methanesulfonic anhydride, pyridine, CH2Cl2, 52%; (b)
THF, amine for 1 and 3-8, for 2 THF, trifluoromethanesulfonic
anhydride, -78 °C, 33% aqueous methylamine in ethanol, -40
°C, 38%.
The selective low nanomolar inhibition of PKCâI and
PKCâII with 1 versus other calcium dependent isozymes
(PKCR and PKCγ) is remarkable given the high degree
of shared homology in the ATP binding region.18 The
IC50 values for inhibition of the calcium independent
isozymes (PKCδ and PKCꢀ) and calcium dependent
isozymes (PKCR and PKCγ) by 1 are comparable. The
selectivity ratio (PKCx/PKCâII IC50 value) of 1 for the
PKCâ isozymes versus PKCR, PKCγ, PKCδ, and PKCꢀ
is also comparable. PKCú, an example of an atypical
isoform, was least sensitive to inhibition by either the
compounds in this series or staurosporine. The activity
profile of methylamine 2 was similar to that of di-
methylamine 1 in both activity (IC50 ) 5 nM) and
selectivity (94-fold versus PKCR) for PKCâ. Primary
amine 3, pyrrolidine 4, morpholine 7, and piperazine 8
were weaker inhibitors of PKCâ than either 1 or 2. The
benzylamine analogue 5 was one of the least active
compounds and exhibited lower selectivity for PKCâ in
comparison to the other isozymes in the panel. When
the basic amine site is altered such as in sulfonamide
6, the selectivity ratio of PKCâII versus PKCR is
increased (>114-fold selectivity) relative to benzylamine
5 (20-fold selectivity). Alcohol 9 exhibited moderate
isozyme selectivity. Staurosporine, an indolocarbazole,
did not exhibit significant selectivity toward any of the
calcium dependent isozymes (PKCR, âI, âII, γ).19 Stau-
rosporine also had minimal selectivity for the calcium
dependent isozymes as a class versus the calcium
independent (PKCδ, ꢀ, η) isozymes.20
A number of compounds containing a bisindolyl-
maleimide structural component have been implicated
as competitive inhibitors of ATP binding.10,11 LY333531
(1) displays kinetics consistent with direct competition
for ATP binding (Figure 1) to PKCâI. The ATP con-
centration was varied in a series of inhibition experi-
ments of PKCâI activity with 1. A double-reciprocal
dose-response plot (Figure 1A) of data from the inhibi-
tion experiments was consistent with 1 acting as a direct
competitive inhibitor with ATP for binding to PKCâI.
The apparent ki (2 nM) of 1 obtained from a Dixon plot
(Figure 1B) is consistent with the observed IC50 value
for inhibition of PKCâI (4.7 nM) and PKCâII (5.9 nM).
The cellular activity of the series was assessed in an
endothelial cell culture assay measuring the inhibition
of plasminogen activator (PA) activity.14 Endothelial
cell dysfunction is a key consideration in the develop-
ment of diabetic complications and the progression of
diabetic retinopathy.15 Retinal capillary endothelial
cells were treated with 4â-phorbol 12,13-dibutyrate (4â-
PDBu) that stimulated PA activity. Plasminogen acti-
vator activity was determined in the cell lysate with the
synthetic substrate H-D-Val-L-Leu-L-Lys-p-nitroaniline
dihydrochloride and plasminogen. In the presence of
the PKC inactive diastereomer 4R-phorbol 12,13-di-
butyrate (4R-PDBu), no PA activity above the basal level
was detected. Cellular toxicity of the series was deter-
mined in a parallel series of cultures using a neutral
red viability assay to monitor lysosomal integrity.16
Ch em istr y
We recently reported the synthesis of a new structural
class of protein kinase C inhibitors that are macrocyclic
bisindolylmaleimides.17 The series represented by com-
pounds 1-9 are members of this structural class and
possess an activity profile that could be of utility in
several disease states. LY333531 (1), the primary
amine 3, pyrrolidine 4, benzylamine 5, sulfonamide 6,
morpholine 7, and piperazine 8 were prepared from
alcohol 9 (Scheme 1). Alcohol 9 was reacted with
methanesulfonic anhydride to produce a mesylate that
underwent displacement with dimethylamine, ammo-
nia, pyrrolidine, the sodium salt of benzenesulfonamide,
morpholine, or piperazine to yield the desired com-
pounds 1 and 3-8, respectively. The secondary amine
2 was prepared from alcohol 9 by displacement of the
corresponding triflate with methylamine as formation
of the N-methylmaleimide of 2 was observed when the
corresponding mesylate of 9 was reacted with methyl-
amine.
Biologica l Activity
P KC Isozym e Selectivity. LY333531 (1), and the
additional analogues 2-9, produced selective inhibition
of PKCâ when assayed in the isozyme panel (Table 1).

2666 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Ta ble 1. PKC Isozyme IC50 Valuesa
J irousek et al.
PKC isozyme IC50 (µM)
compd
R
âI
âII
γ
δ
ꢀ
ú
η
1
2
3
4
5
6
7
8
9
0.36
0.24
1.1
3.5
6.3
>5.0
3.5
1.8
0.72
0.045
0.0047
0.005
0.048
0.12
0.33
0.17
0.049
0.030
0.073
0.023
0.0059
0.005
0.033
0.044
0.31
0.044
0.038
0.024
0.028
0.019
0.30
0.27
2.1
2.2
6.5
>5.0
2.5
3.2
2.0
0.25
0.17
2.6
3.3
7.8
0.88
2.0
2.8
0.69
0.028
0.60
1.1
>5.0
3.8
57
>5.0
>5.0
>5.0
79
>100
22
44
55
54
>5.0
>5.0
>5.0
8.7
0.052
0.031
0.24
0.35
2.5
1.9
1.7
2.00
0.30
0.005
staurosporine
0.11
0.018
>1.5
a
Measurements are the average of at least three independent determinations from eight-point titration curves. The typical standard
deviation was 30% of the IC50 value. Cloned human isozymes were used in the assay that were activated by DAG using phosphatidylserine
vesicles incubated with [32P]ATP and histone or myelin basic protein as a substrate.
F igu r e 1. Lineweaver-Burk (A) and Dixon (B) plots for inhibition of PKCâI isozyme with LY333531 (1) (average of two
independent determinations).
Ta ble 2. ATP Dependent Kinase IC50 Values and IC50 Values
selective inhibitor against src tyrosine kinase. A com-
parison of the data from Tables 1 and 2 indicates that
for Inhibition of Rat Brain PKCa
kinase IC50 (µM)
staurosporine is a more potent inhibitor of both calcium
calmodulin kinase and src tyrosine kinase than either
PKCâII or most of the other PKC isozymes tested. A
high degree of species homology exists between the PKC
isozymes.21 The activity profile of the LY333531 series
was optimized against the individual human PKC
isozymes; however, 1-3 and 9 are also nanomolar
potent inhibitors of rat brain PKC (RB-PKC) which
consists of a mixture of isozymes from this species.
Ca
casein
Kd
src-
TKe
RB-
compd
PKAb
calmodulinc
PKCf
1
2
3
4
5
6
9
>100
6.2
2.2
2.7
5.3
4.4
56
6.0
0.004
>100 >100
>100 NT
>100 89
0.32
0.16
0.79
1.9
49
83
>100
>100
>100
>100
>100 11
>100 >100
4.2
>100 >100 10
>100 >100
0.63
0.19
staurosporine
0.10
14 0.001
Cellu la r Activity. An endothelial cell-based plas-
minogen activator assay, coupled with a neutral red
viability assay, was used to determine the cellular
activity of the series. The activity of the series was
measured as inhibition of the induced elevated plasmi-
nogen activator activity stimulated by â-PDBu. The
dose-response curve (Figure 2) obtained for 1 indicates
that these compounds effectively block PKC-mediated
events such as the release of plasminogen activator with
minimal cellular toxicity (left axis verses right axis).
Cellular activity is maintained through the series with
1 having an EC50 in the 200 nM range (Table 3).
Secondary amine 2, pyrrolidine 4, and piperazine 8 also
have submicromolar EC50 values in this assay. Primary
amine 3, benzylamine 5, sulfonamide 6, morpholine 7,
and alcohol 9 are active in the micromolar range. None
of the compounds were found to be toxic in the neutral
red viability assay. Comparatively, staurosporine was
toxic in the endothelial cell culture assay at the ED50
dose.
a
The values are the average of at least three independent
b
determinations from eight-point titration curves. Bovine heart
cAMP dependent protein kinase catalytic subunit kinase assay.
c Purified mammalian brain calcium calmodulin dependent protein
d
kinase assay. Purified rat brain casein protein kinase II assay.
e src protein tyrosine kinase assay. f Purified rat brain protein
kinase C assay.
Kin a se Selectivity. The compounds 1-9 in the
series are also PKC selective relative to other ATP
dependent kinases. Comparison of IC50 values for
inhibition of the catalytic subunit of PKA, calcium
calmodulin kinase, casein type II kinase, or src tyrosine
kinase indicates 1 has 3 orders of magnitude selectivity
for PKCâII versus calcium calmodulin (Table 2) and was
inactive against the other ATP dependent kinases
tested. Secondary amine 2, primary amine 3, pyrroli-
dine 4, benzylamine 5, and alcohol 9 also have a similar
selectivity pattern for PKC versus these kinases. The
sulfonamide 6 was 1 order of magnitude less active in
inhibition of calcium calmodulin kinase than the other
compounds in the series. Pyrrolidine 4 was the least

Isozyme Selective Inhibitors of PKCâ
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2667
determined using a Nicolet DX10 FT-IR spectrometer. Silica
gel flash chromatography was performed using Mallinckrodt
SilicAR 230-400 mesh. Reverse phase chromatography was
performed on a Waters HPLC system and a LC-908-G30
recycling preparative HPLC from J apan Analytical Instru-
ment. In general, commercially available dry solvents were
used. Acetone was distilled from anhydrous calcium sulfate
prior to use.
(S)-10,11,14,15-Tetr a h yd r o-13-(h yd r oxym eth yl)-4,9:16,-
21-d im eth en o-1H,13H-d iben zo[e,k]p yr r olo[3,4-h ][1,4,13]-
oxa d ia za cycloh exa d ecen e-1,3(2H)-d ion e (9). To a stirred
solution of (S)-methyl 4-[(tert-butyldiphenylsilyl)oxy]-3-hy-
droxybutyrate (21.5 g, 57.7 mmol) in cyclohexane (400 mL) at
ambient temperature under a nitrogen atmosphere was added
allyl trichloroacetamidate (23.4 g, 115 mmol) followed by
trifluoromethanesulfonic acid (1 mL, 50 µL/g alcohol) in five
portions over 30 min. After stirring for 70 h, the reaction
mixture was filtered to remove the precipitate followed by
concentration of the filtrate in vacuo. The concentrate was
eluted through a pad of silica gel using an ethyl acetate in
hexane gradient (0-10%). The eluant was concentrated to
give a mixture containing (S)-methyl 4-[(tert-butyldiphenyl-
silyl)oxy]-3-(allyloxy)butyrate (27 g, expected mass 24 g). NMR
analysis of this mixture indicated the presence of residual
imidate (ca. 10%). This mixture was used in the next reaction
without further purification.
To a stirred solution of the above ester (23.8 g, 57 mmol) in
anhydrous THF (1.0 L) at -75 °C under a nitrogen atmosphere
was added a 1 M solution of DIBAL-H in toluene (231 mL)
dropwise over 40 min. After 1.5 h, the mixture was warmed
up to -10 °C and the reaction quenched with 5% water in
MeOH and Celite. The suspended solids were filtered and
rinsed with THF. The filtrate was concentrated and parti-
tioned between ether and 20% citric acid. The ether layer was
separated, dried, filtered, and concentrated in vacuo to afford
the crude alcohol. The crude alcohol was eluted through a pad
of silica gel with chloroform to obtain the purified alcohol (20.6
g, 93%).
F igu r e 2. Dose-response of LY333531 (1) inhibition of
â-PDBu-stimulated plasminogen activator activity (left axis).
The compound blocks PA activity with minimal cellular
toxicity (right axis) (average values of two determinations, see
Table 3).
Ta ble 3. Endothelial Cell-Based Plasminogen Activator Assay
Activity
compd
EC50 (µM)a
Tox (µM)b
nc
50
1
2
3
4
5
6
7
8
9
0.21 ((0.08)d
0.24 ((0.07)d
7.5 ((1.5)e
>20
>20
>20
>10
>20
>2
>20
>20
>20
<0.02
5
7
2
2
2
2
2
2
2
1
0.55 ((0.15)e
4.3 ((0.81)e
>2
3.5 (+0.52)e
0.55 ((0.23)e
3.0 ((0.53)e
0.02
staurosporine
a
EC50 is the effective concentration for 50% inhibition of PA
b
activity relative to control by â-PDBu stimulation. Tox is the
50
concentration for 50% cell death determined by lysosomal staining
in the neutral red viability assay. c n is the number of independent
determinations. σ -1 standard deviation. e Average value of two
d
The alcohol (20.6 g, 53.6 mmol) was dissolved in MeOH (500
mL) under a nitrogen atmosphere at -78 °C. Ozone was
bubbled through the solution for 12 min, and excess ozone was
apparent from the blue color of the solution. Nitrogen was
passed through the reaction mixture displacing the excess
independent determinations assayed in duplicate.
Con clu sion s
In summary a novel class of macrocyclic bisindolyl-
maleimides has been synthesized. LY333531 and re-
lated compounds exhibit nanomolar inhibition of PKCâ
and at least 60-fold selectivity for PKCâ versus PKCR.
Kinase selectivity is also maintained in the series;
LY333531 is several orders of magnitude more selective
for inhibition of PKCâ in comparsion to other ATP
dependent kinases such as calcium calmodulin (1060-
fold selective) or src tyrosine kinase (2200-fold selective).
In comparison, staurosporine is a more potent inhibitor
of both calcium calmodulin and src tyrosine kinase than
PKC.
(12.2 g, 321 mmol) was added to the reaction
ozone, and NaBH4
mixture. The reaction mixture was warmed to ambient
temperature followed by concentration in vacuo to give a
residue that was eluted through a pad of silica gel with ethyl
acetate. Concentration of the eluant gave the diol (16.4 g, 79%)
as a colorless oil.
The diol (15.7 g, 40.4 mmol) was dissolved in ether (600 mL)
containing triethylamine (16.8 mL, 121 mmol) at 0 °C under
a nitrogen atmosphere. To this mixture was added methane-
sulfonyl chloride (9.38 mL, 121 mmol). After stirring for 3 h,
the reaction mixture was filtered; the filtrate was washed with
water and brine, dried over MgSO4, and concentrated to give
the bismesylate (21.9 g, 99% yield).
The retinal capillary endothelial cell activity is en-
couraging in light of the importance of endothelial cell
dysfunction in the development of diabetic retinopathy.
The importance of PKC and the implications of specific
isozymes in the development of diabetic complications
and other disease states suggest these agents may offer
a novel therapeutic approach.
To a stirred solution of the bismesylate (21.9 g) in freshly
distilled dry acetone (1.4 L) were added NaI (90.4 g, 603 mmol)
and NaHCO3 (170 mg, 2 mmol). After 24 h at 56 °C the
reaction mixture was filtered, concentrated, and partitioned
between ether and 10% Na2SO3. The ether layer was sepa-
rated, washed with brine, dried over MgSO4, and concentrated
in vacuo to give the bisiodide (S)-1-[(tert-butyldiphenylsilyl)-
oxy]-2-[(2-iodoethyl)oxy]-4-iodobutane (17.9 g, 73.2%).
The above bisiodide (17.9 g, 29.4 mmol) and N-methyl-
bisindolylmaleimide (10.0 g, 29.4 mmol) were dissolved in
anhydrous DMF (80 mL). This solution was added over 72 h
to a stirred suspension of cesium carbonate (38.3 g, 118 mmol)
in anhydrous DMF (1.7 L) at 50 °C under a nitrogen atmo-
sphere. The reaction mixture was concentrated and parti-
tioned between CHCl3 and 1 N HCl, and the aqueous layer
was extracted with chloroform and ethyl acetate. The com-
bined organic layers were washed with 1 N HCl, water, and
brine, dried over MgSO4, and concentrated in vacuo. The
crude product was used without further purification.
Exp er im en ta l Section
Melting points were determined on a MEL-TEMP II ap-
paratus and are uncorrected. NMR spectra were recorded on
a GE QE-300 spectrometer. All chemical shifts are reported
in parts per million (δ) relative to tetramethylsilane. The
following abbreviations are used to denote signal patterns: s
) singlet, d ) doublet, t ) triplet, b ) broad, m ) multiplet.
The mass spectral data were determined on VG ZAB-2SE (fast
atom bombardment conditions) and VG ZAB-3F (field desorp-
tion conditions) spectrometers. Infrared spectral data were

2668 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
J irousek et al.
The above product was suspended in ethanol (700 mL)
containing 5 N KOH (800 mL). The stirred suspension was
heated (80 °C), and after 72 h the reaction mixture was
concentrated, cooled to 0 °C, and acidified with 5 N HCl. A
violet precipitate formed that was collected. The precipitate
was eluted through a pad of silica gel with ethyl acetate to
obtain the cyclized product (8.7 g).
h yd r och lor id e (2). To a stirred solution of (S)-10,11,14,15-
tetrahydro-13-(hydroxymethyl)-4,9:16,21-dimetheno-1H,13H-
dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-
1,3(2H)-dione (1.32 g, 3.0 mmol) in anhydrous tetrahydrofuran
(120 mL) under nitrogen was added 2,4,6-collidine (1.19 mL,
9 mmol) followed by trifluoromethanesulfonic anhydride (1.52
mL, 9 mmol) at -78 °C. The reaction mixture was stirred for
40 min, at which time a solution of 33% methylamine in
ethanol (19 mL, 150 mmol) was added. The reaction mixture
was stirred at -78 °C for 10 min, at which time the dry ice/
acetone bath was replaced with a dry ice/acetonitrile bath and
the reaction mixture allowed to warm to room temperature
over 18 h. The reaction mixture was concentrated in vacuo
to yield a precipitate that was collected on a filter plate
washing with ethyl acetate. The filtrate was washed with
water, and the aqueous layer was back-extracted with ethyl
acetate. The combined organic phases were dried over MgSO4,
filtered, and concentrated in vacuo to give a residue. The
residue and the precipitate were combined and eluted through
a silica gel column with a 50% ethyl acetate/hexane to ethyl
acetate and then 2% isopropylamine/ethyl acetate gradient.
Removal of the eluting solvent and conversion of the free base
to the hydrochloride salt was the same as described previously
to give (S)-13-[(monomethylamino)methyl]-10,11,14,15-tet-
rahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-
h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione monohydro-
chloride, 2 (520 mg, 38%), as a red powder: 1H NMR (300 MHz,
DMSO-d6) δ 1.19-2.04 (m, H), 2.14-2.22 (m, 1H), 2.45-2.55
(m, 3H), 2.91-2.97 (m, 1H), 3.17-3.26 (m, 1H), 3.53-3.61 (m,
1H), 3.63-3.78 (m, 1H), 3.78-3.82 (m, 1H), 4.05-4.43 (m, 4H),
7.08 (t, J ) 7.51 Hz, 2H), 7.17 (t, J ) 7.48 Hz, 2H), 7.41-7.50
(m, 3H), 7.53 (d, J ) 8.00 Hz, 1H), 7.77 (t, J ) 7.26 Hz, 2H),
8.61 (bs, 2H), 10.91 (s, 1H); IR (KBr) cm-1 2995, 2997, 2712,
1699, 1524, 1507, 1395, 1355, 748, 741; HRMS (FAB) calcd
for C27H27N4O3 455.2083, found 455.2086; OR [R]D 80.8° (c 0.5,
MeOH) at 25 °C. Anal. (C27H26N4O3‚HCl) C, H, N.
(S)-13-(Am in om eth yl)-10,11,14,15-tetr a h yd r o4,9:16,21-
d im et h en o-1H ,13H -d ib en zo[e,k ]p yr r olo[3,4-h ][1,4,13]-
oxa d ia za cycloh exa d ecen e-1,3(2H )-d ion e Mon oh yd r o-
ch lor id e (3). The mesylate (S)-10,11,14,15-tetrahydro-13-
[[(methylsulfonyl)oxy]methyl]-4,9:16,21-dimetheno-1H,13H-
dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-
1,3(2H)-dione (85 mg, 0.164 mmol) was dissolved in dioxane
(17 mL) containing ammonium hydroxide (6 mL, concentrated)
in a sealed tube. After 24 h at 60 °C, the reaction mixture
was cooled to 0 °C and concentrated producing a red residue
that was purified using reverse phase gel filtration HPLC (85%
MeCN/H2O, 0.01% TFA). Evaporation of the eluting solvent
produced the TFA salt of (S)-13-(aminomethyl)-10,11,14,15-
tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo-
[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione. The TFA
salt was partitioned between ethyl acetate and 0.1 N NaOH,
the ethyl acetate layer was concentrated, and the residue was
dissolved in MeOH (2 mL). To this solution was added 4 N
HCl/dioxane (1 mL, 1:1 by volume), and the reaction mixture
was stirred for 1 h, at which time the reaction mixture was
concentrated to produce (S)-13-(aminomethyl)-10,11,14,15-
tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo-
[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione mono-
hydrochloride, 3 (12 mg, 15%), as a violet solid: 1H NMR (300
MHz, DMSO-d6) δ 1.90-2.06 (m, 1H), 2.06-2.15 (m, 1H),
2.70-2.86 (m, 1H), 2.96-3.06 (m, 1H), 3.55-3.66 (m, 1H),
3.73-3.80 (m, 1H), 4.00-4.42 (m, 5H), 7.06 (t, J ) 7.23 Hz,
2H), 7.15 (t, J ) 7.32 Hz, 2H), 7.72 (d, J ) 6.03 Hz, 1H), 7.76
(d, J ) 6.34 Hz, 1H), 8.08 (bs, 3H), 10.91 (s, H); HRMS (FAB)
calcd for C26H25N4O3 441.1927, found 441.1918; OR [R]D 65.5°
(c 0.5, MeOH) at 25 °C.
The precipitate (8.7 g, 19.7 mmol) was dissolved in anhy-
drous DMF (1 L) followed by addition of a mixture of
1,1,1,3,3,3-hexamethyldisilazane (41.6 mL, 197 mmol) and
methanol (4 mL, 98.5 mmol) under a nitrogen atmosphere.
After stirring for 40 h, the DMF was removed in vacuo, and
MeCN/1 N HCl (2:1, v/v) was added to the residue. After
stirring for 1 h, the reaction mixture was concentrated and
extracted with ethyl acetate, dried over MgSO4, filtered, and
concentrated to give a solid that was eluted through a pad of
silica gel with CH2Cl2/MeCN (9:1) to obtaine pure (S)-10,11,-
14,15-tetrahydro-13-(hydroxymethyl)-4,9:16,21-dimetheno-1H,-
13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-
1,3(2H)-dione, 9 (6.5 g, 75%), as a magenta solid: 1H NMR
(300 MHz, DMSO-d6) δ 1.92-2.14 (m, 2H), 3.64-3.30 (m, 4H),
3.91 (m, 1H), 4.16-4.37 (m, 4H), 7.12 (t, J ) 7.17 Hz, 2H),
7.18 (t, J ) 8.12 Hz, 2H), 7.49 (dd, J ) 19.12, 19.85 Hz, 4H),
7.80 (d, J ) 8.09 Hz, 1H), 7.84 (d, J ) 8.09 Hz, 1H), 10.96 (bs,
1H); IR (KBr) cm-1 3446, 2931, 1703, 1470, 1288, 743; HRMS
(FAB) calcd for C26H23N3O4 441.1767, found 442.1771 (M+
1); OR [R]D -11.26° (c 1.0, MeOH) at 25 °C. Anal. C26H23N3O4-
(H2O)1.5 C, H, N.
+
(S)-13-[(Dim eth ylam in o)m eth yl]-10,11,14,15-tetr ah ydr o-
4,9:16,21-d im eth en o-1H,13H-d iben zo[e,k]p yr r olo[3,4-h ]-
[1,4,13]oxa d ia za cycloh exa d ecen e-1,3(2H)-d ion e Mon o-
h yd r och lor id e (1). The title compound was prepared by the
following sequence of reactions. To a stirred suspension of 9,
(S)-10,11,14,15-tetrahydro-13-(hydroxymethyl)-4,9:16,21-dime-
theno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiaza-
cyclohexadecene-1,3-(2H)-dione (8.9 g, 20 mmol), in anhydrous
CH2Cl2 (800 mL) under nitrogen at ambient temperature was
added pyridine (4.85 mL, 60 mmol) followed by methane-
sulfonic anhydride (4.21 g, 24 mmol). After 16 h, the reaction
mixture was washed with 0.1 N HCl and brine, dried over
MgSO4, filtered, and concentrated in vacuo to afford a magenta
solid that was purified by gradient elution through a column
of silica gel (0-10% MeCN in CH2Cl2) to obtain (S)-10,11,14,-
15-tetrahydro-13-[[(methylsulfonyl)oxy]methyl]-4,9:16,21-dime-
theno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiaza-
cyclohexadecene-1,3(2H)-dione (9.4 g, 90%).
To a stirred solution of (S)-10,11,14,15-tetrahydro-13-[[(me-
thylsulfonyl)oxy]methyl]-4,9:16,21-dimetheno-1H,13H-diben-
zo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-
dione (2.8 g, 5.39 mmol) in THF (300 mL) was added
dimethylamine (100 mL, 40% in water). The reaction vessel
was sealed and heated to 50 °C. After 24 h the reaction vessel
was cooled to 0 °C and the mixture concentrated in vacuo. The
residue was eluted through a column of silica gel with a
gradient of ethyl acetate to 10% triethylamine in ethyl acetate
to give the free base as a violet solid (1.7 g, 67%).
The free base (1.7 g, 3.6 mmol) was suspended in methanol
(300 mL) and treated with 1.0 N anhydrous HCl in ether (10
mL, 10 mmol). After stirring for 30 min, a bright orange
precipitate formed and was collected. The orange cake was
washed with ether and dried under vacuum to give the
hydrochloride salt of (S)-13-[(dimethylamino)methyl]-10,11,-
14,15-tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyr-
rolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione mono-
hydrochloride, 1 (1.6 g, 88% yield): 1H NMR (300 MHz, DMSO-
d6) δ 2.01-2.16 (m, 1H), 2.26-2.40 (m, 1H), 2.68 (s, 6H), 3.10-
3.24 (m, 1H), 3.24-3.34 (m, 1H), 3.60-3.74 (m, 1H), 3.74-
3.92 (m, 2H), 4.08-4.22 (m, 1H), 4.22-4.44 (m, 3H), 7.04-
7.26 (m, 4H), 7.40-7.60 (m, 4H), 7.76-7.86 (m, 2H), 10.59 (bs,
1H), 10.96 (s, 1H); IR (KBr) cm-1 3411.55, 3163.66, 1691.79,
1623.32, 1512.38, 1474.77, 1314.88, 1206.63; MS (FD) calcd
for C28H29N4O3Cl 504.5, found 468 (M+ - HCl); OR [R]D -28.7°
(c 1.0, EtOH) at 25 °C. Anal. (C28H29N4O3Cl) C, H, N.
(S)-13-[(Mon om eth ylam in o)m eth yl]-10,11,14,15-tetr ah y-
d r o-4,9:16,21-d im eth en o-1H,13H-d iben zo[e,k]p yr r olo[3,4-
h ][1,4,13]oxa d ia za cycloh exa d ecen e-1,3(2H)-d ion e Mon o-
(S)-13-(P yr r olid in om eth yl)-10,11,14,15-tetr a h yd r o-4,9:
16,21-d im eth en o-1H,13H-d iben zo[e,k]p yr r olo[3,4-h ][1,4,-
13]oxa d ia za cycloh exa d ecen e-1,3(2H)-d ion e Mon oh yd r o-
ch lor id e (4). Compound
4 was prepared as described
previously for compound 3 using pyrrolidine in THF (28.8 mg,
43%): 1H NMR (300 MHz, DMSO-d6) δ 1.72-2.10 (m, 5H),
2.20-2.34 (m, 1H), 2.86-3.02 (m, 2H), 3.04-3.18 (m, 4H),
3.52-3.64 (m, 1H), 3.66-3.74 (m, 1H), 3.75-3.82 (m, 1H),
4.04-4.48 (m, 4H), 7.10 (t, J ) 7.50 Hz, 2H), 7.18 (t, J ) 7.50

Isozyme Selective Inhibitors of PKCâ
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2669
Hz, 2H), 7.44 -7.53 (m, 4H), 7.81 (t, J ) 7.50 Hz, 2H), 9.77
(bs, 1H), 10.96 (s, 1H); IR (KBr) cm-1 3399.01, 1710.11,
1621.38, 1469.94, 1335.88, 1196.98; HRMS (FAB) calcd for
C30H31N4O3 495.2396, found 495.2404; OR [R]D 20.3° (c 1.0,
MeOH) at 25 °C.
[3,4-h ][1,4,13]oxa d ia za cycloh exa d ecen e-1,3(2H )-d ion e
Mon oh yd r och lor id e (8). As described for compound 3, the
mesylate (S)-10,11,14,15-tetrahydro-13-[[(methylsulfonyl)oxy]-
methyl]-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-
h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione (150 mg, 0.29
mmol) was reacted with N-methylpiperazine (0.64 mL, 20
equiv) to give a dark red solid that was converted to its
hydrochloride salt using 1 N HCl in ether to provide (S)-13-
[(N-methylpiperazino)methyl]-10,11,14,15-tetrahydro-4,9:16,-
21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]-
oxadiazacyclohexadecene-1,3(2H)-dione monohydrochloride, 8
(S)-13-[(Ben zyla m in o)m eth yl]-10,11,14,15-tetr a h yd r o-
4,9:16,21-d im eth en o-1H,13H-d iben zo[e,k]p yr r olo[3,4-h ]-
[1,4,13]oxa d ia za cycloh exa d ecen e-1,3(2H)-d ion e Mon o-
h yd r och lor id e (5). Compound 5 was prepared as described
previously for compound 3 reacting the mesylate with benzyl-
amine (45-fold excess) to yield 5 (10.2 mg, 9.3%) as a dark
violet solid: 1H NMR (300 MHz, DMSO-d6) δ 2.00-2.07 (m,
1H), 2.19-2.22 (m, 1H), 2.80-2.91 (m, 1H), 3.11-3.15 (m, 1H),
3.50-3.64 (m, 1H), 3.70-3.77 (m, 1H), 3.81-3.88 (m, 1H),
3.99-4.42 (m, 6H), 7.11 (t, J ) 9 Hz, 1H), 7.19 (t, J ) 9 Hz,
1
(96 mg, 59%), as a purple solid: H NMR (300 MHz, DMSO-
d6) δ 2.01-2.03 (m, 1H), 2.22-2.25 (m, 1H), 2.73 (s, 3H), 3.32-
3.62 (m, 12H), 3.81-3.89 (m, 1H), 4.08-4.35 (m, 4H), 7.05-
7.10 (m, 2H), 7.14-7.19 (m, 2H), 7.43-7.52 (m, 4H), 7.73-
7.77 (m, 2H), 10.92 (s, 1H), 11.7 (bs, 1H); IR (KBr) cm-1 1707,
2666, 3046, 3408; Anal. (C31H33ClN5O3‚HCl‚2H2O) C, H, N.
MS (FD) calcd for C31H33ClN5O3 523.64 (free base), found 523.
Biologica l Meth od s. Cell Cu ltu r e. Retinal capillary
endothelial cell cultures were initiated from bovine eyes using
a modification of the procedure of Buzney.14 Bovine eyes were
transported on ice from a local abattoir. Extraocular muscle
was trimmed from the eye, and the eye was bisected posterior
to the ora serrata. The vitreous and anterior portion of the
eye were discarded, and the neuroretina was gently dissected
from the posterior eyecup. The retinas from 20 cattle were
pooled and homogenized with 5 strokes of a Teflon/glass
homogenizer in Hank’s saline. The homogenate was passed
through a 350 µm filter to remove large debris and a 210 µm
filter to remove large vessels, and the microvessels were
trapped on a 85 µm filter. The microvessels were resuspended
in Hank’s saline and digested with 7.5 mg/mL bacterial
collagenase type D (Boehringer Mannheim, Indianapolis, IN)
for 1 h at 37 °C. The cells were pelleted by centrifugation
(100g, 10 min), resuspended in 5 mL of endothelial growth
media (EGM; Clonetics, San Diego, CA), and seeded in a
gelatin-coated T25 flask. After 24 h the cells were trypsinized
and replated in a gelatin-coated T225 flask. At 7 days and
again at 14 days, the cultures were labeled with Dil-AcLDL
(Stoughton, MA) and endothelial cells were separated from
contaminating cell types using a fluorescent cell sorter. Cells
were used between the 3rd and 10th passages. As a compari-
son, human dermal fibroblasts (Clonetics), between the 5th
and 10th passages, also were studied.
To study PA activity retinal capillary endothelial cells were
seeded into 96-well plates and grown to confluence (105 cells/
well) in EGM containing 10% fetal bovine serum. Fibroblasts
were maintained in Dulbecco’s modified Eagles media (DME)
supplemented with 10% fetal bovine serum. In both cell types
the media were changed to DME with 10% fetal bovine serum
24 h prior to the assay. Cultures were treated with the phorbol
esters 4â-phorbol 12,13-dibutyrate (4â-PDBu), 4R-phorbol 12,-
13-dibutyrate (4R-PDBu), and 4â-phorbol 12-myristate 13-
acetate (PMA), or the compound was dissolved in DMSO and
incubated at 37 °C for 48 h. Cell toxicity was determined in
a parallel series of cultures using a neutral red assay to
monitor lysosomal integrity.
1H), 7.38-7.46 (m, 6H), 7.53-7.61 (m, 5H), 7.80 (d, J ) 9 Hz,
-1
2H), 9.05 (bs, 1H), 10.96 (s, 1H); IR (KBr) cm
3020, 1711,
1468, 1193; MS (FD) calcd 567.11, found 531 (M+ - HCl). Anal.
(C33H31N4O3‚H2O) C, H; N: calcd, 9.58; found, 9.13.
(S)-13-(Ben zen esu lfon a m id om et h yl)-10,11,14,15-t et -
r ah ydr o-4,9:16,21-dim eth en o-1H,13H-diben zo[e,k]pyr r olo-
[3,4-h ][1,4,13]oxa d ia za cycloh exa d ecen e-1,3(2H )-d ion e
Mon oh yd r och lor id e (6). To a suspension of sodium hydride
(264 mg, 33 mmol) in anhydrous dimethylformamide (15 mL)
under nitrogen at 5 °C was added benzenesulfonamide (786
mg, 5 mmol) dissolved in anhydrous dimethylformamide (10
mL). The reaction mixture was stirred at room temperature
for 2 h. The resulting sodium salt was cooled to 5 °C followed
by slow addition of the mesylate (S)-10,11,14,15-tetrahydro-
13-[[(methylsulfonyl)oxy]methyl]-4,9:16,21-dimetheno-1H,13H-
dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3-
(2H)-dione (520 mg, 1 mmol) dissolved in anhydrous dimethyl-
formamide (10 mL). The reaction mixture was heated (50 °C)
for 20 h and then partitioned between 1 N HCl (200 mL) and
dichloromethane (400 mL). The organic layer was separated,
and the aqueous layer was further extracted with dichlo-
romethane (2 × 100 mL). The combined organic phases were
washed with brine, dried over MgSO4, filtered, and evaporated
in vacuo to give a solid that was purified by silica gel
chromatography eluting the column sequentially with dichlo-
romethane, 5% acetonitrile in dichloromethane, and finally
10% acetonitrile in dichloromethane to provide (S)-13-(benze-
nesulfonamidomethyl)-10,11,14,15-tetrahydro-4,9:16,21-dime-
theno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiaza-
cyclohexadecene-1,3(2H)-dione monohydrochloride, 6 (86 mg,
15%), as a purple solid: 1H NMR (300 MHz, DMSO-d6): δ
1.90-1.95 (m, 1H), 2.00-2.07 (m, 1H), 2.76-2.82 (m, 1H),
2.90-2.96 (m, 1H), 3.25-3.45 (m, 2H), 3.66-3.70 (m, 1H),
3.98-4.09 (m, 2H), 4.15-4.21 (m, 1H), 4.26-4.32 (m, 1H),
7.04-7.18 (4H, m), 7.36-7.39 (m, 3H), 7.47-7.59 (m, 4H), 7.66
(t, J ) 6.00 Hz, 1H), 7.71-7.78 (m, 4H), 10.88 (s, 1H); IR (KBr)
cm-1 1697, 3420; MS (FD) calcd for C32H28N4O5S 580.67, found
580. Anal. (C32H28N4O5S) C, H, N.
(S)-13-(Mor p h olin om eth yl)-10,11,14,15-tetr a h yd r o-4,9:
16,21-d im eth en o-1H,13H-d iben zo[e,k]p yr r olo[3,4-h ][1,4,-
13]oxa d ia za cycloh exa d ecen e-1,3(2H)-d ion e Mon oh yd r o-
ch lor id e (7). As described for compound 3, the mesylate (S)-
10,11,14,15-tetrahydro-13-[[(methylsulfonyl)oxy]methyl]-4,9:
16,21-dimetheno-1H,13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]-
oxadiazacyclohexadecene-1,3(2H)-dione (150 mg, 0.29 mmol)
was reacted with morpholine (0.50 mL, 20 equiv) to give a dark
red solid that was converted to the its hydrochloride salt using
1 N HCl in ether to provide (S)-13-(morpholinomethyl)-10,-
11,14,15-tetrahydro-4,9:16,21-dimetheno-1H,13H-dibenzo[e,k]-
pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione
monohydrochloride, 7 (109 mg, 69%), as a purple solid: 1H
NMR (300 MHz, DMSO-d6) δ 1.99-2.04 (m, 1H), 2..23-2.30
(m, 1H), 2.95-3.03 (m, 1H), 3.18-3.37 (m, 5H), 3.51-3.88 (m,
7H), 4.07-4.39 (m, 4H), 7.06-7.11 (m, 2H), 7.17-7.18 (m, 2H),
7.39 (s, 1H), 7.42 (s, 1H), 7.47 (d, J ) 8.00 Hz, 1H), 7.51 (d, J
) 8.00 Hz, 1H), 7.73-7.79 (m, 2H), 10.73 (bs, 1H), 10.92 (s,
P la sm in ogen Activa tor Assa y. Following treatment, the
cells were lysed with 25 mM NH4OH in 0.5% Triton X-100.
Plasminogen activator activity was determined in the cell
lysate using the synthetic substrate H-D-valyl-L-leucyl-lysine-
p-nitroaniline dihydrochloride (Kabi, Mo¨lndal, Sweden) using
a modification of the procedure of Verheijen et al.16 Briefly, a
plasminogen-substrate solution was prepared which con-
tained 1 mM synthetic substrate and 0.35 unit of plasminogen
(Kabi)/1 mL of buffer (50 mM Tris HCl, pH 8.0, with 0.005%
Tween 80). To a 50 µL aliquot of the cell lysate was added
200 µL of the plasminogen-substrate solution, the mixture
was incubated at 25 °C for 45 min (endothelial cells) or 2 h
(fibroblasts), and the plates were read at 410 nm. A standard
curve was constructed using purified human urokinase or tPA
(American Diagnostic, Greenwich, CT).
1H); IR (KBr) 1704, 2926, 3393, 3596; Anal. (C30H31
-
ClN4O‚0.5CH3OH) C, N; H: calcd, 5.91; found, 4.65. MS (FD)
Ca lciu m Ca lm od u lin Dep en d en t P r otein Kin a se Assa y
(Ca M). The calcium calmodulin dependent protein kinase
assay is described in J . Neurosci. 1983, 3, 818-831. The assay
components were in a total volume of 250 µL: 55 mM HEPES
calcd for C30H31ClN4O 510.60 (free base), found 510.
(S)-13-[(N-Met h ylp ip er a zin o)m et h yl]-10,11,14,15-t et -
r ah ydr o-4,9:16,21-dim eth en o-1H,13H-diben zo[e,k]pyr r olo-

2670 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
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1993, 329, 304-309. (c) D’Amico, D. J . Diseases of the Retina.
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328, 1676-1685.
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5,
2.75 mM dithiothreitol, 2.2 mM EGTA (ethylenebis(oxyethyl-
enenitrilo)tetraacetic acid; used in the blank buffer), 1.1 mM
calcium chloride (Sigma, St. Louis, MO; used in the control
buffer), 10 mM magnesium chloride (Sigma, St. Louis, MO),
200 µg/mL histone type HL (Worthington), 10 µL of DMSO or
DMSO/inhibitor, and 30 µM [γ-32P]ATP (DuPont). The reac-
tion was initiated by the addition of calcium calmodulin
dependent protein kinase (isolated from rat brain homogenate),
the mixture was incubated at room temperature for 10 min,
and the reaction was stopped by adding 0.5 mL of ice cold
trichloroacetic acid (Amresco) followed by 100 µL of 1 mg/mL
bovine serum albumin (Sigma, St. Louis, MO). The precipitate
was collected by vacuum filtration on glass fiber filters and
quantified by counting in a beta scintillation counter.
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of protein kinase
C by elevation of glucose concentration:
Proposal for mechanism in the development of diabetic
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W. F.; King, G. L. Preferential elevation of protein kinase C
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of diabetic rats: differential reversibility to glycemic control by
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11059-11063.
(7) (a) Lester, D. S., Epand, R. M., Eds.; Protein Kinase C; Current
concepts and Future Perspectives; Ellis Harwood: New York,
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Y. Protein Kinase C, calcium and phospholipid degradation.
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(8) (a) Nishizuka, Y. Intracellular signaling by hydrolysis of phos-
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Ca sein P r otein Kin a se II Assa y (CK-II). The casein
protein kinase II assay is described in Neurochem. Res. 1988,
13, 829-836. The assay components were in a total volume
of 250 µL: 20 mM Tris HCl, pH 7.5, 5 mM sodium fluoride,
50 mg/mL casein (Sigma, St. Louis, MO), 10 mM magnesium
chloride (Sigma, St. Louis, MO), 10 µL of DMSO or DMSO/
inhibitor, and 30 µm [γ-32P]ATP (DuPont). Initiation of the
reaction was performed by addition of casein protein kinase
II (isolated from rat brain homogenate), the mixture was
incubated at room temperature for 10 min, and the reaction
was stopped by the addition of 0.5 mL of ice cold trichloroacetic
acid (Amresco) followed by 100 µL of 1 mg/mL bovine serum
albumin (Sigma, St. Louis, MO). The precipitate was collected
by vacuum filtration on glass fiber filters and quantified by
counting in a beta scintillation counter.
cAMP Dep en d en t P r otein Kin a se Ca ta lytic Su bu n it
Assa y (P KA). The assay components were in a total volume
of 250 µL: 20 mM HEPES (Sigma, St. Louis, MO) buffer, pH
7.5, 200 µg/mL histone type HL (Worthington), 10 mM
magnesium chloride (Sigma, St. Louis, MO), 10 µL of DMSO
or DMSO/inhibitor, and 30 µM [γ-32P]ATP (DuPont). The
reaction was initiated by addition of bovine heart cAMP
dependent kinase catalytic subunit (Sigma, St. Louis, MO),
the mixture was incubated at 30 °C for 10 min, and the
reaction was stopped by adding 0.5 mL of ice cold trichloro-
acetic acid (Amresco) followed by 100 µL of 1 mg/mL bovine
serum albumin (Sigma). The precipitate was collected by
vacuum filtration on glass fiber filters employing a TOMTEC
system and quantified by counting in a beta scintillation
counter. This assay was done identically to the PKC enzyme
assay except that no phospholipids or diacylglycerol was
employed in the assay and the histone substrate was specific
for the cAMP dependent catalytic subunit enzyme.
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D. R.; Wadsworth, J .; Wilkinson, S. E. Inhibitors of protein
kinase C. 3. Potent and highly selective bisindolylmaleimides
by conformational restriction. J . Med. Chem. 1993, 36, 21-29.
(b) Toullec, D.; Pianetti, P.; Coste, H.; Bellevergue, P.; Grand-
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Loriolle, F.; Duhamel, L.; Charon, D.; Kirilovsky, J . The bisin-
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(11) (a) Wilkinson, S. E.; Parker, P. J .; Nixon, J . S. Isozyme specificity
of bisindolylmaleimides, selective inhibitors of protein kinase C.
Biochem. J . 1993, 294, 335-337. (b) Birchall, A. M.; Bishop, J .;
Bradshaw, D.; Cline, A.; Coffey, J .; Elliot, L. H.; Gibson, V. M.;
Greenham, A.; Hallam, T. J .; Harris, W.; Hill, C. H.; Hutchings,
A.; Lamont, A. G.; Lawton, G.; Lewis, E. J .; Maw, A.; Nixon, J .
S.; Pole, D.; Wadsworth, J .; Wilkinson, S. E. Ro-32-0432, a
selective and orally active inhibitor of protein kinase C prevents
T-cell activation. J . Pharmacol. Exp. Ther. 1994, 268 (2), 922-
929. (c) Davis, P. D.; Hallman, T. J .; Harris, W.; Hill, C. H.;
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(13) (a) Calcium calmodulin dependent protein kinase assay: Kennedy,
M. B.; Mcguinness, T.; Greengard, P. J . Neurosci. 1983, 3, 818-
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vesicles consisting of 120 µg/mL phosphatidylserine (Avanti
Polar Lipids) and sufficient diacylglycerol (Avanti Polar Lipids)
to activate the enzyme to maximum activity in 20 mM HEPES
buffer (Sigma, St. Louis, MO), pH 7.5, 940 µM calcium chloride
(Sigma, St. Louis, MO) for assaying the R, âI, âII, and γ
enzyme only, 1 mM EGTA for all the enzymes, 10 mM
magnesium chloride (Sigma, St. Louis, MO), and 30 µM [γ-32P]-
ATP (DuPont). For all the enzymes either histone type HL
(Worthington) or myelin basic protein was used as substrate.
The assay was started by addition of protein kinase C enzyme
incubated at 30 °C for 10 min and stopped by adding 0.5 mL
of cold trichloroacetic acid (Amresco) followed by 100 µL of 1
mg/mL bovine serum albumin (Sigma, St. Louis, MO). The
precipitate was collected by vacuum filtration on glass fiber
filters employing a TOMTEC filtration system and quantified
by counting in a beta scintillation counter.
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