New amide-bearing benzolactam-based protein kinase C modulators induce enhanced secretion of the amyloid precursor protein metabolite sAPPα

Article abstract of DOI:10.1021/jm020350r

Protein kinase C (PKC) is known to participate in the processing of the amyloid precursor protein (APP). Abnormal processing of APP through the action of the β- and γ-secretases leads to the production of the 39-43 amino acid Aβ fragment, which is neuroto

Full text of DOI:10.1021/jm020350r

364
J . Med. Chem. 2003, 46, 364-373
New Am id e-Bea r in g Ben zola cta m -Ba sed P r otein Kin a se C Mod u la tor s In d u ce
En h a n ced Secr etion of th e Am yloid P r ecu r sor P r otein Meta bolite sAP P r
Alan P. Kozikowski,*,† Ireneusz Nowak,† Pavel A. Petukhov,† Rene Etcheberrigaray,‡ Ali Mohamed,‡
Mathew Tan,‡ Nancy Lewin,§ Henry Hennings,§ Larry L. Pearce,§ and Peter M. Blumberg§
Drug Discovery Program, Department of Neurology, Georgetown University Medical Center, 3970 Reservoir Road, NW,
Washington, D.C. 20007-2197, NeuroLogic, Inc., 15010 Broschart Road, Suite 200, Rockville, Maryland 20850, and
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Institutes of Health, Bethesda, Maryland 20892
Received August 14, 2002
Protein kinase C (PKC) is known to participate in the processing of the amyloid precursor
protein (APP). Abnormal processing of APP through the action of the â- and γ-secretases leads
to the production of the 39-43 amino acid Aâ fragment, which is neurotoxic and which is
believed to play an important role in the etiology of Alzheimer’s disease. PKC activation
enhances R-secretase activity, which results in a decrease of the amyloidogenic products of
â-secretase. In this article, we describe the synthesis of 10 new benzolactam V8 based PKC
activators having side chains of varied saturation and lipophilicity linked to the aromatic ring
through an amide group. The Ki values measured for the inhibition of phorbol ester binding to
PKCR are in the nanomolar range and show some correlation with their lipophilicity.
Compounds 5g and 5h show the best binding affinity among the 10 benzolactams that were
synthesized. By use of a cell line derived from an AD patient, significant enhancement of sAPPR
secretion was achieved at 1 µM concentration for most of the compounds studied and at 0.1
µM for compounds 5e and 5f. At 1 µM the enhancement of sAPPR secretion for compounds
5c-h is higher than that observed for the control compound 8-(1-decynyl)benzolactam (BL).
Of interest is the absence of activity found for the highly lipophilic ligand 5i, which has a Ki
of 11 nM. On the other hand, its saturated counterpart 5j, which possesses a comparable Ki
and ClogP, retains activity in the secretase assay. In the hyperplasia studies, 5f showed a
modest response at 100 µg and 5e at 300 µg, suggesting that 5f was approximately 30-fold less
potent than the PKC activator mezerein and 100-fold less potent than TPA. 5e was
approximately 3-fold less active than 5f. On the basis of the effect of unsaturation for other
potent PKC ligands, we would predict that 5e would retain biological activity in most assays
but would show a marked loss of tumor-promoting activity. Compound 5e thus becomes a viable
candidate compound in the search for Alzheimer’s therapeutics capable of modulating amyloid
processing.
In tr od u ction
diabetes, heart disease, cancer, and Alzheimer’s disease,
and thus, both isoform-selective activators and inhibi-
tors may provide novel leads in the development of
therapeutic agents. Bryostatin, a macrocyclic lactone
from the bryozoan Bugula neritina, binds to the DAG
regulatory site of PKC, yet it is not a tumor promoter
but acts as an antineoplastic agent that has been used
to treat murine melanoma.7 In view of the fact that
bryostatin-1 is in clinical evaluation as an anticancer
agent, it is likely that other activators of PKC that are
not tumor promoters will find use in treating neoplastic
conditions.
Protein kinase C (PKC) represents a family of at least
12 serine/threonine kinases that are involved in signal
transduction in response to a host of hormonal, neu-
ronal, and growth factor stimuli.1-4 Differences in
structure as well as substrate requirements have led
to the general classification of the isoforms into three
groups, termed the classical, novel, and atypical PKCs.
The N-terminal regulatory domain of the classical and
novel PKCs contains a conserved C1 domain comprising
two cysteine-rich zinc fingers that bind to the natural
PKC activator diacylglycerol (DAG) as well as to certain
natural products such as the phorbol esters and the
indolactams. Normally, the classical and novel PKC
isozymes are cytosolic and are translocated to the
membrane upon interaction with Ca2+, whereby they
are activated through interaction with membrane phos-
pholipids and DAG.5,6 PKC has been shown to play a
major role in a variety of disease states including
There has been a growing interest in studying the
relationship between Alzheimer’s disease (AD) and
PKC. Numerous researchers have found defective PKC
in brains8-13 and peripheral tissues of AD patients.14-17
PKC is known to participate in the processing of the
amyloid precursor protein (APP),18 including the amyl-
oidogenic fragments Aâ 1-40 and 1-42. There are three
well-characterized proteolytic routes of amyloid process-
ing, all of which seem to occur in normal and pathologi-
cal states. R-Secretase,19 a metalloprotease neither
completely identified nor characterized, cleaves APP
within the Aâ sequence (between residues K and L),
* To whom correspondence should be addressed. Phone: 202-687-
0686. Fax: 202-687-5065. E-mail: kozikowa@georgetown.edu.
† Georgetown University Medical Center.
‡ NeuroLogic, Inc.
§ National Institutes of Health.
10.1021/jm020350r CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/13/2002

Benzolactam-Based PKC Modulators
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 365
Sch em e 1a
a
Reagents and conditions: (a) HNO3, Ac2O, 5 min, 0 °C, 92%; (b) Pd/C, H2, 3 h, room temp; (c) RCOCl, Et3N, 4 h, room temp; (d)
Na2CO3, MeOH, 1.5 h, room temp; (e) Pd/C, H2, 3 h, room temp, 90%.
generating a large secreted fragment termed sAPPR and
a smaller intracellular fragment P3. Both fragments are
of no pathological significance. The process leading to
Aâ formation or “amyloidogenic” processing and ulti-
mately Aâ deposition and plaque formation in AD
involves the participation of another enzyme, â-secre-
tase.20,21 This enzyme cleaves just outside the Aâ
sequence, leaving a large membrane-bound fragment
that is later cleaved by a yet to be identified γ-secre-
tase22 at position 711 or 713 and thereby generating Aâ
1-40 or 1-42, respectively. In-depth description and
discussion of APP processing can be found elsewhere.23-26
PKC activators belonging to the phorbol family have
been shown to dramatically enhance R-secretase activ-
ity, thus leading to enhanced secretion of sAPPR.18,27-32
Because the secretases seem to compete for a single pool
of APP,33,34 enhancing R-secretase activity would result
in a decrease of amyloidogenic products of â-secretase.
In fact, a direct decrease of Aâ 1-40 has been docu-
mented after PKC activation.27,28 A few other studies
fail to show the direct relationship between enhanced
sAPPR and decreased Aâ 1-40. However, they still show
that PKC activation has “positive” effects as reflected
in an increase in the nonpathogenic sAPPR or a decrease
in â-amyloid.35-37 We have shown that 8-(1-decynyl)-
benzolactam38 restores an otherwise abnormal K chan-
nel activity in fibroblasts from AD patients, linked to
enhanced translocation of PKCR.39,40 Furthermore, the
same compound was later shown to significantly en-
hance sAPPR secretion.41
In continuation of these studies, we have now exam-
ined the effect of altering the side chain appendage of
the core benzolactam through introduction of both
saturated and unsaturated amide residues. Interest-
ingly, as described herein, we find that the majority of
these ligands are more active than the 8-(1-decynyl)-
benzolactam in promoting the non-amyloidogenic R-
processing of APP.
Design a n d Syn th esis. An important role has been
ascribed to the nature of the side chains present in
certain PKC activators, since unsaturation in these side
chains tends to increase inflammatory activity while
decreasing tumor-promoting activity. For example, octa-
hydromezerein is a tumor promoter while mezerein
itself acts as a nonpromoting inflammatory agent.42
Likewise, 12-O-acetylphorbol 13-(2,4-decadienoate) has
been shown to have practically no effect as a tumor
promoter in contrast to its saturated counterpart, 12-
O-acetylphorbol 13-decanoate.43 Guided by these obser-
vations, we chose to compare the properties of benzo-

366 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Kozikowski et al.
Ta ble 1. ClogP and Ki Values of BL and Amides 5a -j
lactam derivatives having unsaturated and saturated
side chains of the same length. We thus attached
aliphatic side chains, both trans,trans dienic and satu-
rated, linked to the benzolactam scaffold by means of
an amide bond and terminating with a methyl, phenyl,
p-trifluorophenyl, bis(3,5-trifluoromethyl)phenyl, and p-
(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooct-1-enyl)phenyl
or p-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)phenyl
groups. The terminal aryl and fluoroalkylaryl groups
will serve to increase the ClogP, as well as to modify
the association of the protein-ligand complex with the
membrane.44 Linkage of the side chain to the benzo-
lactam core by an amide bond was chosen for its
synthetic convenience. It was also anticipated that this
amido group might be able to form additional hydrogen
bonds with proximal amino acid residues comprising the
binding loops of PKCR. This possibility finds support
in our molecular modeling studies as described below.
The synthesis of these 8-substituted benzolactam
derivatives containing an amide moiety commences
from the unsubstituted benzolactam, the synthesis of
which has previously been published.38 After O-acetyl-
ation, the benzolactam 1 can be nitrated under mild
conditions with a solution of nitric acid in acetic
anhydride (Scheme 1). The reaction produces exclusively
the 8-nitro derivative 2 in an excellent yield of 92%,
provided that the reaction is promptly terminated after
completion. Prolonged exposure to the nitration reagent
leads to a mixture of incompletely characterized prod-
ucts, with overnitration probably being the main proc-
ess. Catalytic reduction of the nitro group over Pd/C
afforded the desired amino derivative 3, which was then
reacted without purification with acyl chlorides 4a -f.
The resulting amides were subjected to deacetylation
under basic conditions to obtain the final products 5a -
c, 5e, 5g, and 5i in 31-42% yield over the three steps.
Catalytic reduction of the unsaturated side chain ap-
pendages of 5c, 5e, 5g, and 5i over Pd/C provided the
saturated analogues 5d , 5f, 5h , and 5j. The synthesis
of the required trifluoromethylated acid chlorides was
carried out using a modification of Huang’s method,45
involving the reaction between methoxycarbonylallyl-
idenetriphenylarsorane generated in situ and the req-
uisite benzaldehyde. Subsequent base hydrolysis of the
ester and reaction with thionyl chloride provided the
desired acylating agent. For the synthesis of 5i, the
required acid chloride was prepared by coupling of the
E,E-isomer of methyl 5-(4-iodophenyl)-2,4-pentadienoate
and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-1-octene in the
presence of Pd(OAc)2, NaHCO3, and n-Bu4NHSO4 in
DMF under nitrogen,46 followed by base hydrolysis and
acid chloride formation.
sAP P r Secr etion . The ability of our compounds to
enhance sAPPR secretion using a cell line derived from
an AD patient is summarized as bar graphs in Figure
1. All values are expressed relative to DMSO alone.
Four to six independent experiments were conducted
for each compound at a concentration of 1 µM, and
duplicate experiments for all the compounds were
carried out at a concentration of 0.1 µM. A substantial
effect was observed at 1 µM for all compounds tested
with the exception of compound 5i. The effect on sAPPR
secretion correlates reasonably well with the Ki for
inhibition of PDBU. Thus, compounds 5a and 5b exhibit
only modest effects in this assay, since their Ki values
are between 200 and 2000 nM. Of interest is the absence
of activity found for the highly lipophilic ligand 5i, which
has a Ki of 11 nM. On the other hand, its saturated
counterpart 5j, which possesses a comparable Ki and
ClogP, retains activity in the secretase activity. The
Bin d in g St u d ies. The interaction of the benzo-
lactams 5a -j with PKC was assessed by determining
their ability to displace bound [20-3H]phorbol 12,13-
dibutyrate (PDBU) from recombinant PKCR in the
presence of phosphatidylserine. The partition coef-
ficients (ClogP) were calculated according to the frag-
ment-based program KOWWIN 1.63.47 The results are
presented in Table 1. The Ki values of all compounds
are in the nanomolar range, and the calculated log P
values are in the range 2-11. Dienic ligands and their
saturated counterparts generally display comparable
binding affinities except in the cases of 5a and 5b.

Benzolactam-Based PKC Modulators
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 367
F igu r e 1. Secretion of sAPPR. Secretion was determined by densitometry analyses of immunoblots. Each bar represents a different
treatment (all 1 µM). Results are from 4 to 6 independent experiments, except for 5f,i,j (triplicate), and 12 repeats for BL (BL is
the “+” reference or control). The units are relative to the signal obtained from cells treated with solvent (DMSO) alone, which
reflects nonstimulated or basal secretion of sAPPR. The normalization was applied to each blot. Increased sAPPR was particularly
marked after stimulation with 5e-g (Kruskal-Wallis and Dunn’s post-test). Noticeable increases were also achieved with 5c,d ,h
and, to a lesser extent, BL. The inset shows that 5e and 5f also have a significant effect at 0.1 µM. Compounds 5c,g,h also induce
noticeable secretion. Most experiments for 0.1 µM are duplicates. Six repeats were carried out for 5g and 5h and single experiments
for 5a and 5b.
enhancement of sAPPR secretion by compounds 5c-h
is higher than that observed for 8-(1-decynyl)benzo-
lactam. Compounds 5c,e-h enhanced sAPPR secretion
even at 0.1 µM, although to a lesser degree than at the
higher concentration. A noticeable difference is observed
between most of the compounds and the ligands 5e and
5f, which are both lipophilic and bind potently to PKCR.
Their activity in the sAPPR assay dropped by merely
one-half when the concentration was decreased 10-fold.
A representative Western blot illustrates the sAPPR
signal analyzed for all of the compounds (Figure 2).
Hyp er p la sia Assa ys. Compounds 5e and 5f were
evaluated for induction of hyperplasia after topical
application to the shaved backs of outbred Sencar mice
(NCIsFrederick, Frederick, MD). The extent of hyper-
plasia was estimated in terms of the number of cell
layers in the epidermis (Table 2). The potencies of 5e
and 5f were compared with those of TPA and of
mezerein. Detectable hyperplasia was observed after a
single application of 1 µg of TPA or after a single
application of 3 µg of mezerein. 5f showed a modest
response at 100 µg and 5e at 300 µg, suggesting that 5f
was approximately 30-fold less potent than mezerein
and 100-fold less potent than TPA. 5e was approxi-
mately 3-fold less active than 5f. Similar relationships
were observed after four applications, although the
extent of hyperplasia was somewhat more marked.
F igu r e 2. sAPPR detection. Conventional immunoblotting
Dock in g a n d MD Sim u la tion . The truncated ver-
sion of 8-(1-decynyl)benzolactam (BL) bearing only an
acetylene group at position 8 and generic ligand 5
containing -NH(CO)-CHdCHMe (E double bond) as
a side chain appendage were docked to the binding
domain of PKCR C1b, and the region within an 8 Å
sphere around the ligand was subjected to molecular
dynamics simulations. Molecular modeling revealed
that truncated BL forms four highly populated (close
to 100%) hydrogen bonds with amino acid residues Ser
111, Thr113, Leu 122, and Gly124. It also forms a less
techniques were used to detect secreted sAPPR. Representative
blots show the signal obtained with various compounds used
at (A) 1 and (B) 0.1 µM. The two panels represent distinct
experiments and blots.
populated hydrogen bond with Tyr 109 (21%) (Figure
3, Table 3). Generic ligand 5 also forms four hydrogen
bonds with an occupancy close to 100% with Ser 111,
Thr 113, Leu 122, and Gly 124. Like BL, ligand 5 forms
a low occupancy bond with Tyr 109 (16%). However, in
comparison to BL, the hydrogen atom from the amido

368 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Kozikowski et al.
Ta ble 2. Hyperplasia Induced by Compounds 5e and 5f in
Comparison to TPA and Mezerein Estimated by the Number of
Cell Layers in the Epidermis
epidermal cell layers
dose
single
four
compound
(µg/application) application
applications
acetone control
1-2
1-2
5-7
3-7
1-4
3-7
2-6
1-3
2-4
2-5
1-4
1-2
1-2
3-7
2-4
1-3
1-3
1-2
1-2
2
1
0.3
10
3
1
300
100
30
10
3
300
100
30
10
3
2-5
2-4
1-3
3-6
2-6
1-2
1-4
1-2
1-2
1-2
1-2
2-6
1-5
1-2
1-2
1-2
1-2
TPA
mezerein
5e
5f
1
group located at position 8 of 5 is able to form one
additional hydrogen bond with Ser 110, which shows
an occupancy of 67%.
Discu ssion a n d Con clu sion s
The nature of the benzolactam side chain has a
substantial effect on the ability of the benzolactams to
increase the formation of sAPPR. This is most clearly
seen in comparing the decynyl-substituted benzolactam
with the benzolactams 5c,e-g. Of particular note is the
improved activity of 5f in comparison to 8-(1-decynyl)-
benzolactam, although the ClogP of the former is one
log unit lower while their Ki values are very similar.
On the basis of our modeling studies, we find that the
side chain amide group of these new benzolactams is
able to form an additional H-bonding interaction with
serine residue 110 present in loop A. We suggest that
the effect of this extra H bond on the orientation of the
side chain coupled with the possible interaction of the
side chain amide carbonyl with complementary groups
present in the membrane phospholipids may better
anchor the PKC-ligand complex to the membrane,
allowing it to be a more effective mediator of the events
resulting in R-secretase activation. Compounds 5e and
5f are relatively potent PKC ligands49 exhibiting binding
affinities that are only 40 times poorer than that of
PDBU (phorbol 12,13-dibutyrate). The in vitro assays
show the most dramatic increase in the secretion of
nonpathogenic sAPPR in the cases of 5e and 5f.
Concerning the hyperplasia results, our findings with
5e and 5f are consistent with the structure-activity
relations that have been shown for other classes of DAG
mimetics derived from phorbol and related diterpenes.
Here, the concept that has emerged is that interaction
with the binding site on protein kinase C is relatively
independent of the pattern of side chain substitution.
This independence can be understood from the X-ray
crystallographic analysis of the complex between the
C1b binding domain of protein kinase C and phorbol
13-acetate,48 in which the primary hydroxyl group (C20)
and several of the secondary hydroxy groups of the
constrained diterpene ring system insert into a hydro-
F igu r e 3. Hydrogen bonding interactions between PKCR C1b
domain and ligands BL (A) and generic 5 (B).
philic pocket in an otherwise hydrophobic face of the
C1 domain, whereas the ester side chains project away
from the C1 domain and presumably interact with the
phospholipid bilayer. Computer modeling and site-
directed mutagenesis have permitted extrapolation of
this model to other classes of constrained ligands,
including the resiniferonol derivative thymeleatoxin,53
the ingenol 3-monoesters,53 constrained diacylglycerol
lactones,54 and the indole alkaloids.55 In all of these
cases, the side chains project away from the C1 domain.
Although the side chains do not directly interact with
the C1 domain, studies at the cellular level clearly
indicate that the side chains can influence into which
membranes PKC can insert. This is most evident in the
case of PKCδ.56 PKCδ inserts into the plasma and

Benzolactam-Based PKC Modulators
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 369
Ta ble 3. Hydrogen Bond Interactions between PKCR C1b Domain and the Modeled Ligands
parameters of hydrogen bonds
occupancya
(%)
average
lifetime (ps)
b
hydrogen bond
rDA (Å)
rAH (Å)
R
D-H-A (deg)
ω
H-A-AA (deg)
BL
O3-CO‚‚‚HNGly124
H4-NH‚‚‚COLeu122
O11-OH‚‚‚HNThr113
H11-OH‚‚‚COSer111
H11-OH‚‚‚COTyr109
100
85
90
99
21
50.0
42.4
44.8
49.6
10.3
2.65 ( 0.17
3.06 ( 0.31
2.85 ( 0.30
2.49 ( 0.12
3.67 ( 0.47
1.76 ( 0.17
2.15 ( 0.34
2.05 ( 0.30
1.77 ( 0.20
2.92 ( 0.49
148.0 ( 13.6
152.7 ( 9.2
138.7 ( 17.8
132.7 ( 16.7
137.7 ( 12.8
35.4 ( 20.8c
-57.0 ( 10.6d
26.2 ( 29.1e
-53.3 ( 57.7f
132.7 ( 28.1g
Generic Ligand 5
2.57 ( 0.12
2.94 ( 0.23
2.71 ( 0.18
2.59 ( 0.16
3.76 ( 0.46
3.19 ( 0.59
O3-CO‚‚‚NHGly124
H4-NH‚‚‚COLeu122
O11-OH‚‚‚NHThr113
H11-OH‚‚‚COSer111
H11-OH‚‚‚COTyr109
NH8-NHCO‚‚‚COSer110
100
94
98
96
16
67
50.0
47.2
49.1
47.9
7.9
1.74 ( 0.13
2.03 ( 0.26
1.93 ( 0.20
1.99 ( 0.26
3.03 ( 0.53
2.45 ( 0.91
138.9 ( 12.5
152.4 ( 8.4
135.3 ( 16.0
121.7 ( 16.1
136.2 ( 12.9
139.5 ( 31.5
33.4 ( 23.0c
-52.7 ( 11.1d
13.7 ( 14.7e
-78.2 ( 13.0f
115.3 ( 29.7g
79.8 ( 13.1h
33.5
a
b
Resolution of 50 fs, counted as hydrogen bond only if rAH < 2.5 Å. Average distance during 50 ps production phase. “D” refers to a
donor atom. “A” refers to an acceptor atom. c Torsion angle HGly124-O-C-Nligand
HThr113-O-C-Hligand
.
Torsion angle Hligand-OLeu122-C-N. e Torsion angle
d
g
h
.
f Torsion angle Hligand-OSer111-C-N. Torsion angle Hligand-OTyr109-C-N. Torsion angle Hligand-OSer110-C-N.
nuclear membranes in response to the tumor-promoting
derivative 12-deoxyphorbol 13-tetradecanoate, whereas
it goes to the nuclear membrane and punctate ag-
gregates, but not the plasma membrane, after treatment
with 12-deoxyphorbol 13-acetate, a derivative that is
inflammatory but that acts to inhibit tumor promotion.
The important influence of hydrophobicity in the trans-
location of PKCδ was similarly shown for a series of
homologous, symmetrically substituted phorbol 12,13-
diesters, where both the kinetics and ultimate position
of translocation of PKCδ depended on the hydrophobic-
ity.57 Since subcellular location should drive the avail-
ability of substrates for PKC, the ability of side chains
on PKC ligands to control localization could plausibly
contribute to differences in their biological activities.
In the case of derivatives that differ by being either
unsaturated or saturated in their side chains, the
biological effect has been consistent but the mechanism
remains unresolved. In early studies, Hecker and co-
workers described several series of diterpene esters in
which unsaturation was associated with retention of
inflammatory activity but loss of tumor-promoting
activity. Examples include phorbol 12,13-dihexanoate
versus phorbol 12,13-dihexa-2,4-dienoate,43 phorbol 12-
tetradecanoate 13-acetate versus phorbol 12-tetradeca-
2,4,6,8-tetraenoate 13-acetate,58 and 12-deoxyphorbol
13-decanoate versus 12-deoxyphorbol 13-deca-2,4-di-
enoate.59 Some of us demonstrated that mezerein, a
phorbol-related diterpene with a 5-phenylpenta-2,4-
dienoate side chain at C12 that is only weak as a
complete tumor promoter, regained tumor-promoting
activity upon hydrogenation to generate octahydro-
mezerein.60 However, Mezerein did not induce any
striking difference in the pattern of PKC translocation,
suggesting that unsaturation may have a more subtle
effect on PKC function, such as positioning PKC relative
to cholesterol-rich rafts in the plasma membrane.
For 5e and 5f, we find similar affinities for PKCR and
only a modest, 3-fold decrease in potency for skin
hyperplasia for the unsaturated derivative. On the basis
of the effect of unsaturation for other potent PKC
ligands, we would predict that 5e would retain biological
activity in most assays but would show a marked loss
of tumor-promoting activity.
absence of activity observed in the sAPPR assay for the
highly fluorinated analogue 5i. Its binding affinity for
PKCR is good, and its calculated lipophilicity is ex-
tremely high. It is unclear as to what is happening in
this case, since its saturated counterpart is still effec-
tive, and thus solubility issues may be eliminated from
consideration. However, we hypothesize that the some-
what more rigid side chain of the ligand 5i is responsible
for the lack of activity observed in the sAPPR assay.
Particularly, since all double bonds in 5i are trans and
the orientation of the amido group in 5i is fixed because
of the presence of a hydrogen bond interaction with Ser
110, the overall rigidity of the long lipophilic side chain
of 5i is greater than that of 5j. Although, the amido
group in position 8 of 5j also forms a hydrogen bond
with Ser 110, unlike 5i, the side chain of 5j is much
more flexible because of its saturated nature. These
differences in side chain rigidity may accordingly alter
the subcellular localization of PKC and contribute to the
observed differences in their effects on secretase activity.
In light of the present findings, we believe that
compounds such as 5e that are capable of enhancing
sAPPR secretion are worth exploring further as possible
therapeutics in the treatment of Alzheimer’s disease.
Such compounds could be used alone or in combination
with â-secretase inhibitors to decrease the formation of
the neurotoxic â-amyloid peptide, thereby slowing the
progression of the disease process.72
Exp er im en ta l Section
An a lysis of In h ibition of [3H]P DBU Bin d in g by Non -
r a d ioa ctive Liga n d s. Enzyme-ligand interactions were
analyzed by competition with [3H]PDBU binding to the single
isozyme PKCR as described previously.50
Cells a n d Cell Cu ltu r e. A well-characterized cell line from
an AD patient (AG06848) was obtained from the Coriell Cell
Repository (Camden, NJ ), and then it was cultured and
maintained as described41 except that the plastic material was
a 6 cm diameter Petri dish. Cells were typically grown to
confluence, which took approximately 4-5 days. Cells were
maintained in a complete medium until 2 h prior to treatment.
At that point, the medium was replaced by DMEM without
serum and left undisturbed for 2 h. Then, the cells were treated
with 0.1 or 1 µM of the compounds to be tested. 8-(1-Decynyl)-
benzolactam V (BL) and DMSO were used as positive and
negative controls, respectively. The concentration of DMSO
was maintained at less than 1% in all cases. The medium was
collected after 3 h to measure the sAPPR secretion.
Of considerable interest in understanding the com-
plexity of these side chain interactions is the complete

370 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
Kozikowski et al.
sAP P r Deter m in a tion s. The concentration of secreted
sAPPR was measured using conventional immunoblotting
techniques,51 following with minor modifications the protocol
described elsewhere.41 Precipitated protein extracts from each
dish/treatment were loaded into freshly prepared 8% acryl-
amide Tris-HCl minigels and separated by SDS-PAGE. The
volume of sample loaded was corrected for total cell protein
per dish. Proteins were then electrophoretically transferred
to PVDF membranes. Membranes were saturated with 5%
nonfat dry milk to block nonspecific binding. Blocked mem-
branes were incubated overnight at 4 °C with the commercially
available antibody 6E10 (1:500), which recognizes sAPPR in
the conditioned medium (SENETEK).41,52 After being washed,
the membranes were incubated at room temperature with
horseradish peroxidase conjugated antimouse IgG secondary
antibody (J ackson’s Laboratories). The signal was then de-
tected using enhanced chemiluminescence followed by expo-
sure of Hyperfilm ECL (Amersham). The band intensities were
quantified by densitometry using a BioRad GS-800 calibrated
scanning densitometer and Multianalyst software (BioRad).
11.2 Hz), 5.97 (s, 1H), 6.90 (d, 1H, J ) 9.0 Hz), 8.01 (d, 1H, J
) 2.4 Hz), 8.08 (dd, 1H, J ) 2.7, 9.0 Hz); 13C NMR (CDCl3) δ
18.9, 20.5, 27.5, 33.5, 37.5, 51.5, 66.8, 66.9, 116.3, 123.8, 128.0,
139.2, 156.3, 170.2, 171.5; MS m/z 349 (M+, 26%), 332, 306,
278, 203, 177, 133, 43 (100%). Anal. (C17H23N3O5) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of (2S,5S)-8-
(Acyla m in o)ben zola cta m s V (5a -d ). A mixture of nitro
compound 2 (100 mg, 287 µmol) and 10% Pd/C (45 mg) in
ethanol (20 mL) was shaken in a Parr apparatus under 4.0
atm of hydrogen at room temperature for 3 h. The mixture
was concentrated, and the residue was dried under vacuum
for 1 h at 60 °C. To the crude amine 3, dry THF (4 mL) was
added under inert atmosphere, followed by dry Et3N (0.5 mL)
and the acyl chloride (1.6 equiv). The mixture was stirred at
room temperature for 4 h and then was filtered and concen-
trated. Preparative TLC (ethyl acetate as developing solvent)
provided an intermediate, which was dissolved in ethanol (30
mL). A solution of sodium carbonate (60 mg) in water (6 mL)
was added with vigorous stirring. After 1.5-2 h at room
temperature, the solution was concentrated and water (20 mL)
was added. Extraction with ethyl acetate (4 × 30 mL) provided
the crude amides 5a -d , which were purified by preparative
TLC as above.
Hyp er p la sia Stu d ies. Mice were 7 weeks old at the
beginning of the treatments and were in the resting phase of
the hair cycle. Compounds were dissolved in 0.2 mL of acetone
and either were applied once or else were applied twice weekly
for a total of four applications. Two animals were treated at
each dose of compound, and 72 h after the last application,
the animals were euthanized. Two portions of treated skin
were removed from each animal, fixed in neutral buffered
formalin, and stained with hematoxylin and eosin for histo-
logical analysis (staining of sections was performed by Ameri-
can Histolabs, Gaithersburg, MD). TPA (12-O-tetradecanoyl-
phorbol 13-acetate) and mezerein were from LC Laboratories
(Woburn, MA).
Ch em istr y. Analytical and preparative thin-layer chroma-
tography (TLC) was performed in a solvent-vapor-saturated
chamber on EM Science silica gel 60 F-254 plates. Spots were
visualized by UV. Melting points (uncorrected) were deter-
mined in open capillaries on a Thomas-Hoover apparatus.
Infrared spectra were obtained on an ATI Mattson Genesis
series spectrometer in KBr pellets. NMR spectra were recorded
on a Varian instrument (1H frequency of 300 MHz) using TMS
as an internal standard (1H and 13C) or CFCl3 as an internal
or external standard. Elemental analyses were performed by
Micro-Analysis, Inc., and the results were within 0.4% of the
theoretical value. Mass spectra were obtained on a Shimadzu
QP-5000 mass spectrometer using a direct inlet probe and an
electron beam energy of 70 eV. Determinations of purity by
HPLC were performed with a Shimadzu LC-10 AD pump and
a Waters 484 tunable absorbance detector using the following
conditions: (A) Supelco Discovery RP amide C16 250 mm ×
3.0 mm; flow rate ) 0.5 mL/min; detection at 280 nm; 0-30
min, 40-80% acetonitrile in water; 30-60 min, 80% aceto-
nitrile in water; (B) Waters µBondapak C18 300 mm × 7.8 mm;
flow rate ) 2.8 mL/min; detection at 280 nm; 0-30 min, 40-
80% acetonitrile in water; 30-60 min, 80% acetonitrile in
water; (C) Supelco Discovery RP amide C16 250 mm × 3.0 mm;
flow rate ) 0.4 mL/min; detection at 280 nm; 0-15 min,
0-100% methanol in water; 15-50 min, methanol.
(2S,5S)-(E,E)-8-(2,4-Hexa d ien oyla m in o)ben zola cta m V
(5a ). The title compound was obtained according to the general
procedure in 38% yield as yellow oil: [R]20 -253 (c 0.80,
D
1
MeOH); IR (KBr) 1646, 1539, 1506 cm-1; H NMR (CDCl3) δ
1.06 (d, 3H, J ) 6.8 Hz), 1.11 (d, 3H, J ) 6.8 Hz), 1.85 (d, 3H,
J ) 6.1 Hz), 2.38 (m, 1H), 2.60 (s, 3H), 2.63, 2.85 (ABq, 2 H,
J
) 15.6 Hz, both parts d with J ) 10.5 and 5.4 Hz,
respectively), 3.32 (d, 1H, J ) 5.6 Hz), 3.47-3.56 (m, 1H),
3.64-3.73 (m, 1H), 4.06 (br s, 1H), 4.94 (br s, 1H), 5.96 (d, 1
H, J ) 15.1 Hz), 6.03-6.25 (m, 2H), 6.37 (s, 1H), 6.96 (d, 1H,
J ) 8.8 Hz), 7.23 (dd, 1H, J ) 10.3, 14.9 Hz), 7.43 (br d, 1H,
J ) 7.3 Hz), 7.61 (dd, 1H, J ) 2.0, 8.3 Hz), 8.02 (br s, 1H); 13
C
NMR (CDCl3) δ 18.2, 18.6, 20.9, 28.9, 37.0, 38.9, 52.5, 64.2,
78.1, 119.7, 122.4, 122.6, 123.0, 129.9, 134.3, 135.3, 137.9,
141.6, 147.7, 164.7, 174.6; MS m/z 371 (M+, 13%), 340, 276,
255, 95, 44 (100%); HPLC retention time 9.5 min (96.1%
purity) using conditions A, 29.6 min (96.9% purity) using
conditions B.
(2S,5S)-8-(Hexa n oyla m in o)ben zola cta m V (5b). The
title compound was obtained according to the general proce-
dure in 35% yield as colorless oil: [R]20D -166 (c 0.85, MeOH);
IR (KBr) 3290 (br), 1650, 1540, 1506 cm-1; 1H NMR (CDCl3) δ
0.92 (t, 3H, J ) 6.8 Hz), 1.02 (d, 3H, J ) 6.8 Hz), 1.10 (d, 3H,
J ) 6.8 Hz), 1.27-1.42 (m, 4H), 1.68 (m, 2H), 2.27 (t, 2H, J )
7.4 Hz), 2.38 (m, 1H), 2.64 (s, 3H), 2.72, 2.88 (ABq, 2H, J )
15.6 Hz, both parts d with J ) 9.8 and 4.8 Hz, respectively),
3.32 (d, 1H, J ) 5.9 Hz), 3.46-3.56 (m, 1H), 3.64-3.73 (m,
1H), 4.01 (br s, 1H), 4.85 (br s, 1H), 6.50 (s, 1H), 7.00 (d, 1H,
J ) 8.8 Hz), 7.31 (d, 1H, J ) 7.1 Hz), 7.48 (dd, 1H, J ) 2.2,
8.8 Hz), 7.69 (s, 1H); 13C NMR (CDCl3) δ 14.0, 18.4, 20.7, 22.5,
25.4, 28.8, 31.5, 37.1, 37.4, 38.6, 52.6, 64.3, 77.3, 119.7, 122.6,
134.2, 134.8, 147.8, 171.9, 174.4; MS m/z 375 (M+, 14%), 344,
304, 259, 147, 44 (100%); HPLC retention time 11.6 min (98.5%
purity) using conditions A, 29.6 min (98.9% purity) using
conditions C.
(2S,5S)-O-Acetyl-8-n itr oben zola cta m V (2). A mixture
of (2S,5S)-O-acetylbenzolactam V (1) (280 mg, 1.06 mmol) in
Ac2O (1.2 mL) was stirred at 0 °C. To this solution was added
dropwise over 5 min a chilled mixture of Ac2O (1.2 mL) and
70% HNO3 (268 mg, 3.11 mmol). The reaction was allowed to
proceed for 4-5 min at 0 °C and then was stopped by adding
water (50 mL) in one portion to the vigorously stirred solution.
Extraction with EtOAc (4 × 40 mL) followed by evaporation
and column chromatography on silica gel (1:1 EtOAc/hexane
as eluent) provided 340 mg (92%) of the product: mp 180-
(2S,5S)-(E,E)-8-(5-P h en yl-2,4-p en ta d ien oyla m in o)ben -
zola cta m V (5c). The title compound was obtained according
to the general procedure in 31% yield as a yellow oil: [R]20
D
-187 (c 0.56, MeOH); IR (KBr) 1653, 1540, 1507 cm-1; 1H NMR
(CDCl3) δ 1.07 (d, 3H, J ) 6.8 Hz), 1.12 (d, 3H, J ) 6.8 Hz),
2.40 (m, 1H), 2.63 (s, 3H), 2.70, 2.88 (ABq, 1H, J ) 15.8 Hz,
both parts d with J ) 10.3 and 5.2 Hz, respectively), 3.36 (d,
1H, J ) 5.6 Hz), 3.48-3.58 (m, 1H), 3.66-3.76 (m, 2H), 4.88
(br s, 1H), 6.18 (d, 1H, J ) 14.9 Hz), 6.51 (s, 1H), 6.81-6.95
(m, 2H), 7.00 (d, 1H, J ) 8.8 Hz), 7.25-7.49 (m, 7H), 7.58 (d,
1H, J ) 7.6 Hz), 7.92 (s, 1H); 13C NMR (CDCl3) δ 18.4, 20.9,
28.8, 37.1, 38.6, 52.7, 64.4, 77.5, 119.8, 122.6, 122.8, 125.0,
126.6, 127.0, 128.7, 128.8, 134.2, 134.9, 136.3, 139.3, 141.3,
147.9, 164.3, 174.6; MS m/z 433 (M+, 46%), 402, 362, 317, 276,
157, 128, 44 (100%); HPLC retention time 20.3 min (98.3%
182 °C, yellow prisms (EtOH); [R]20 -1017 (c 1.44, CHCl3);
D
IR (KBr) 1743, 1670, 1505, 1326, 1231 cm-1; 1H NMR (CDCl3)
δ 0.77 (d, 3H, J ) 6.8 Hz), 1.06 (d, 3H, J ) 6.3 Hz), 2.13 (s,
3H), 2.50 (m, 1H), 2.93 (s, 3H), 2.96 (d, 1H, J ) 17.5 Hz), 3.44
(dd, 1H, J ) 7.2, 17.5 Hz), 3.63 (d, 1H, J ) 10.3 Hz), 3.68 (m,
1H), 3.94 (dd, 1H, J ) 9.4, 11.1 Hz), 4.29 (dd, 1H, J ) 4.1,

Benzolactam-Based PKC Modulators
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3 371
purity) using conditions A, 26.0 min (96.4% purity) using
conditions B.
1H, J ) 5.4 Hz), 3.53-3.62 (m, 1H), 3.76 (br d, 1H, J ) 10.5
Hz), 3.88 (br s, 1H), 5.01 (br s, 1H), 6.31 (d, 1H, J ) 14.9 Hz),
6.35 (br s, 1H), 6.83-7.08 (m, 3H), 7.43 (dd, 1H, J ) 10.5, 14.9
Hz), 7.55 (br s, 1H), 7.65 (d, 1H, J ) 8.3 Hz), 7.76 (s, 1H), 7.83
(s, 2H), 8.04 (br s, 1H); 19F NMR (CDCl3) δ -63.5 (m); 13C NMR
(CDCl3) δ 18.1, 21.0, 29.0, 37.0, 39.2, 52.4, 64.0, 78.9, 119.8,
121.7 (sept, J ) 3.4 Hz), 122.4, 123.1 (q, J ) 273 Hz), 123.3,
126.5 (narrow m), 127.8, 130.2, 132.1 (q, J ) 33.4 Hz), 134.8,
135.2, 135.3, 138.4, 139.7, 148.1, 163.7, 174.9; MS m/z 569 (M+,
3%), 453, 279, 245, 167, 149, 44 (100%), 177; HPLC retention
time 31.3 min (98.4% purity) using conditions A, 32.1 min
(96.7% purity) using conditions C.
(2S,5S)-8-(5-P h en ylp en t a n oyla m in o)b en zola ct a m
V
(5d ). A mixture of 5c (40.0 mg, 92 µmol), Pd/C (10%) (20 mg),
and methanol (20 mL) was shaken under 3.3 atm of H2 at room
temperature for 19 h. Filtration from the catalyst, evaporation,
and thin-layer chromatography (EtOAc as eluent) provided 5d
(36.1 mg, 90%) as a colorless oil: [R]25 -129 (c 1.8, MeOH);
D
1
IR (KBr) 1652, 1541, 1507 cm-1; H NMR (CDCl3) δ 1.02 (d,
3H, J ) 6.8 Hz), 1.09 (d, 3H, J ) 6.6 Hz), 1.61-1.79 (m, 4H),
2.20-2.42 (m, 3H), 2.60 (s, 3H), 2.61-2.68 (m, 2H), 2.68, 2.85
(ABq, 2H, J ) 16.0 Hz, both parts d with J ) 10.0 and 5.0 Hz,
respectively), 3.28 (d, 1H, J ) 5.6 Hz), 3.45-3.54 (m, 1H),
3.62-3.70 (m, 1H), 4.06 (br s, 1H), 4.85 (br s, 1H), 6.46 (s, 1H),
6.93 (d, 1H, J ) 8.5 Hz), 7.13-7.21 (m, 3 H), 7.24-7.34 (m,
3H), 7.45 (dd, 1H, J ) 1.6, 8.7 Hz), 7.72 (s, 1H); 13C NMR
(CDCl3) δ 18.3, 20.8, 25.3, 28.8, 31.2, 35.7, 37.1, 37.2, 38.6,
52.6, 64.2, 77.5, 119.7, 122.5, 122.6, 125.7, 128.3, 128.4, 134.3,
134.8, 142.2, 147.8, 171.6, 174.5; MS m/z 437 (M+, 12%), 406,
366, 321, 261, 147, 91, 44 (100%); HPLC retention time 19.9
min (97.9% purity) using conditions A, 30.4 min (97.3% purity)
using conditions C.
(2S,5S)-8-[5-[3,5-Bis(t r iflu or om et h yl)p h en yl]p en t a n -
oyla m in o]ben zola cta m V (5h ). A mixture of 5g (40.0 mg,
70 µmol), Pd/C (10%) (20 mg), and methanol (20 mL) was
stirred under 1 atm of H2 at room temperature for 0.5 h.
Filtration from the catalyst, evaporation, and thin-layer chro-
matography (EtOAc as eluent) provided 5h (37.5 mg, 93%) as
a colorless oil: [R]25D -101 (c 0.26, MeOH); IR (KBr) 3410 (br),
1
1642, 1540, 1505, 1380, 1280, 1128 cm-1; H NMR (CDCl3) δ
1.01 (d, 3H, J ) 6.8 Hz), 1.10 (d, 3H, J ) 6.6 Hz), 1.69-1.82
(m, 4H), 2.28-2.45 (m, 3H), 2.64 (s, 3H), 2.71-2.84 (m, 3H),
2.88 (dd, 1H, J ) 5.3, 16.0 Hz), 3.31 (d, 1H, J ) 6.1 Hz), 3.48-
3.60 (m, 2H), 3.66-3.78 (m, 1H), 4.81 (br s, 1H), 6.48 (br s,
1H), 6.97 (d, 1H, J ) 8.8 Hz), 7.24 (br s, 1H), 7.39-7.47 (m,
2H), 7.64 (s, 2H), 7.70 (s, 1H); 19F NMR (CDCl3) δ -63.2 (m);
13C NMR (CDCl3) δ 18.5, 20.7, 25.1, 28.8, 30.7, 35.4, 36.9, 37.1,
38.4, 52.7, 64.4, 77.2, 119.7, 119.9 (sept, J ) 3.8 Hz), 122.3,
122.7, 123.4 (q, J ) 272 Hz), 128.5 (narrow m), 131.5 (q, J )
32.7 Hz), 134.2, 134.4, 144.6, 148.0, 171.0, 174.3; MS m/z 573
(M+, 4%), 542, 502, 403, 261, 227, 44 (100%); HPLC retention
time 29.3 min (99.0% purity) using conditions A, 31.7 min
(98.3% purity) using conditions C.
(2S,5S)-(E,E)-8-[5-[4-(Tr iflu or om eth yl)p h en yl]-2,4-p en -
ta d ien oyla m in o]ben zola cta m V (5e). A mixture of methyl
(E,E)-5-[(4-trifluoromethyl)phenyl]-2,4-pentadienoate45 (0.91 g,
3.55 mmol), KOH (0.60 g, 10.7 mmol), methanol (30 mL), and
water (2 mL) was stirred at room temperature for 22 h.
Solvents were evaporated, and the residue was dissolved in a
minimum amount of water. The resulting solution was acidi-
fied with 3 M HCl. The precipitate that formed immediately
was separated by filtration and dried under vacuum. This
product (0.74 g, 3.06 mmol) and SOCl2 (6.6 mL) were heated
at 70 °C for 3 h. Evaporation of the excess of SOCl2 provided
the crude acid chloride, which was used in the synthesis of
compound 5e (see general procedure above) without purifica-
(2S,5S)-(E,E,E)-8-[5-[4-(3,3,4,4,5,5,6,6,7,7,8,8,8-Tr id eca -
flu or ooct-1-en yl)p h en yl]-2,4-p en ta d ien oyla m in o]ben zo-
la cta m V (5i). The title compound was obtained according to
tion. Compound 5e: yield 42%; yellow oil; [R]20 -165 (c 0.52,
D
MeOH); IR (KBr) 1653, 1541, 1507, 1324 cm-1
;
1H NMR
the general procedure in 41% yield as a yellow oil: [R]20 -87
D
(c 0.185, MeOH); 1H NMR (CDCl3) δ 1.09 (d, 3H, J ) 6.8 Hz),
1.13 (d, 3H, J ) 6.8 Hz), 2.41 (m, 1H), 2.63 (s, 3H), 2.67-2.77
(m, 1H), 2.90 (dd, 1H, J ) 5.3, 16.0 Hz), 3.36 (d, 1H, J ) 5.6
Hz), 3.50-3.61 (m, 1H), 3.68-3.79 (m, 2H), 4.95 (br s, 1H),
6.20 (dt, 1H, J ) 11.8 Hz (t), 16.1 Hz (d)), 6.22 (d, 1H, J )
14.6 Hz), 6.39 (br s, 1H), 6.83, 6.95 (ABq, 2H, J ) 15.6 Hz, B
part d with J ) 10.7 Hz), 6.95 (dd, 1H, J ) 10.6, 15.5 Hz),
7.00 (d, 1H, J ) 8.8 Hz), 7.14 (dt, 1H, J ) 16.1 Hz (d), 2.4 Hz
(CDCl3) δ 1.11 (d, 3H, J ) 7.1 Hz), 1.14 (d, 3H, J ) 6.8 Hz),
2.41 (m, 1H), 2.62 (s, 3H), 2.67, 2.90 (ABq, 2H, J ) 15.8 Hz,
both parts d with J ) 5.7 and 10.2 Hz, respectively), 3.35 (d,
1H, J ) 5.4 Hz), 3.52-3.61 (m, 1H), 3.69-3.78 (m, 1H), 3.93
(br s, 1H), 5.00 (br s, 1H), 6.26 (d, 1H, J ) 14.9 Hz), 6.33 (s,
1H), 6.80-7.02 (m, 3H), 7.38-7.68 (m, 7 H), 8.05 (br s, 1H);
13C NMR (CDCl3) 18.1, 21.0, 28.9, 37.0, 39.1, 52.5, 64.1, 78.6,
119.8, 122.4, 123.2, 124.0 (q, J ) 272 Hz), 125.6 (q, J ) 3.5
Hz), 126.7, 127.0, 128.9, 130.1 (q, J ) 32.7 Hz), 134.7, 137.2,
139.7, 140.4, 147.9, 164.1, 174.8; MS m/z 501 (M+, 94%), 470,
430, 415, 385, 276, 225 (100%), 177; HPLC retention time 26.0
min (98.9% purity) using conditions A, 20.2 min (97.3% purity)
using conditions B.
(t)), 7.38-7.54 (m, 6H), 7.58-7.66 (m, 1H), 7.93 (br s, 1H); 19
F
NMR (CDCl3) δ -126.7 (m, 2F), -123.6 (m, 2F), -123.4 (br s,
2F), -122.1 (br s, 2F), -111.6 (m, 2F), -81.3 (m, 3F); 13C NMR
(CDCl3) δ 18.3, 21.0, 28.9, 37.0, 38.9, 52.6, 64.3, 78.1, 114.4 (t,
J ) 22.9 Hz), 119.9, 122.5, 123.0, 126.1, 127.5, 128.0, 128.1,
133.6, 134.4, 135.1, 137.9, 138.2, 139.0 (t, J ) 9.1 Hz), 140.8,
147.9, 164.1, 174.7; HPLC retention time 43.8 min (96.8%
purity) using conditions A, 33.3 min (99.0% purity) using
conditions C.
(2S,5S)-8-[5-[4-(Tr iflu or om e t h yl)p h e n yl]p e n t a n oyl-
a m in o]ben zola cta m V (5f): [R]25 -127 (c 0.70, MeOH); IR
D
(KBr) 3290 (br), 1649, 1504, 1326, 1121, 1068 cm-1; H NMR
1
(CDCl3) δ 1.02 (d, 3H, J ) 6.8 Hz), 1.10 (d, 3H, J ) 6.8 Hz),
1.63-1.82 (m, 4H), 2.21-2.43 (m, 3H), 2.60 (s, 3H), 2.65-2.78
(m, 3H), 2.87 (dd, 1H, J ) 5.0, 15.7 Hz), 3.28 (d, 1H, J ) 5.6
Hz), 3.47-3.59 (m, 1H), 3.65-3.75 (m, 1H), 3.90 (br s, 1H),
4.88 (br s, 1H), 6.38 (br s, 1H), 6.94 (d, 1H, J ) 8.5 Hz), 7.29
(d, 2H, J ) 8.1 Hz), 7.36 (br s, 1H), 7.47 (d, 1H, J ) 8.8 Hz),
7.52 (d, 2H, J ) 8.3 Hz), 7.59 (br s, 1H); 13C NMR (CDCl3) δ
18.2, 20.8, 25.2, 28.8, 30.8, 35.5, 37.0, 38.9, 52.5, 64.1, 78.1,
119.6, 122.5, 122.7, 124.3 (q, J ) 272 Hz), 125.2 (q, J ) 3.5
Hz), 128.1 (q, J ) 32.2 Hz), 128.7, 134.5, 135.0, 146.3, 147.8,
171.4, 174.5; MS m/z 505 (M+, 32%), 474, 462, 434, 419, 389,
261, 159, 44 (100%); HPLC retention time 24.9 min (99.7%
purity) using conditions A, 19.0 min (99.5% purity) using
conditions B.
(2S,5S)-8-[5-[4-(3,3,4,4,5,5,6,6,7,7,8,8,8-Tr id eca flu or o-
octyl)p h en yl]p en ta n oyla m in o]ben zola cta m V (5j). A mix-
ture of 5i (40.0 mg, 51 µmol), Pd/C (10%) (20 mg), and
methanol (20 mL) was stirred under 1 atm of H2 at room
temperature for 0.5 h. Filtration from the catalyst, evapora-
tion, and thin-layer chromatography (EtOAc as eluent) pro-
vided 5j (38.6 mg, 96%) as a colorless oil: [R]20 -88 (c 1.24,
D
1
MeOH); H NMR (CDCl3) δ 1.02 (d, 3H, J ) 6.8 Hz), 1.10 (d,
3H, J ) 6.8 Hz), 1.60-1.80 (m, 4H), 2.23-2.44 (m, 5H), 2.62
(s, 3 H), 2.62-2.66 (m, 1 H), 2.73 (dd, 1 H, J ) 9.9, 16.0 Hz),
2.81-2.92 (m, 4H), 3.30 (d, 1H, J ) 6.1 Hz), 3.47-3.58 (m,
1H), 3.65-3.82 (m, 2H), 4.84 (br s, 1H), 6.45 (br s, 1H), 6.95
(d, 1H, J ) 8.5 Hz), 7.07-7.17 (m, 4H), 7.28-7.36 (m, 1H),
7.44 (br d, 1H, J ) 8.8 Hz), 7.54 (br s, 1H); 19F NMR (CDCl3)
δ -126.7 (m, 2F), 124.1 (br s, 2F), -123.4 (br s, 2F), -122.4
(br s, 2F), -115.2 (m, 2F), -81.3 (m, 3F); 13C NMR (CDCl3,
weak multiplets of fluorinated C omitted) δ 18.2, 20.8, 25.3,
25.9 (t, J ) 4.0 Hz), 28.9, 31.2, 33.0 (t, J ) 22.2 Hz), 35.3,
37.0, 37.2, 38.9, 52.5, 64.1, 78.2, 119.7, 122.4, 122.7, 128.2,
128.8, 134.5, 135.1, 136.4, 140.7, 147.7, 171.6, 174.6; HPLC
(2S,5S)-(E,E)-8-[5-[3,5-Bis(tr iflu or om eth yl)p h en yl]-2,4-
p en ta d ien oyla m in o]ben zola cta m V (5g). The title com-
pound was obtained according to the general procedure in 42%
yield as a yellow oil: [R]20 -149 (c 0.25, MeOH); 1H NMR
D
(CDCl3) δ 1.11 (d, 3H, J ) 6.8 Hz), 1.14 (d, 3H, J ) 6.8 Hz),
2.43 (m, 1H), 2.63 (s, 3H), 2.70, 2.91 (ABq, 2H, J ) 15.8 Hz,
both parts d with J ) 10.3 and 5.5 Hz, respectively), 3.36 (d,

372 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 3
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retention time 44.1 min (96.6% purity) using conditions A, 33.6
min (98.9% purity) using conditions C.
Dock in g a n d MD Sim u la tion s. The 3D model of PKCR61
was obtained as described earlier.62 The docking was per-
formed using the FlexX module in Sybyl6.8.63,64 The molecules
were docked to the binding site of PKCR C1b domain formed
by loop A comprising Thr 108, Tyr 109, Ser 110, Ser 111, Pro
112, Thr 113, and Phe 114 and by loop B comprising amino
acids Leu 121, Leu 122, Tyr 123, Gly 124, Leu 125, Ile 126,
His 127, and Gln 128. To simplify the analysis, the long alkyl
tail in BL and ligand 5 was truncated, and the resulting two
molecules were used for further studies. The 30 best docking
solutions based on the FlexX docking functions were saved by
FlexX. To produce a more robust evaluation of ligand-receptor
interactions, a consensus of all five scoring functions (FlexX,65
PMF,66 GOLD,67,68 Chemscore,69 and DOCK70) available in
CScore was generated using the CScore module in Sybyl6.8.64
Among the 30 best solutions, only those that have a consensus
of three or more functions and are ranked highest by a
combination of GOLD, DOCK, and FlexX scores were taken
into further consideration. The poses of BL and ligand 5 with
the highest PMF score are shown in Figure 3 and were used
for further MD simulations.
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The molecules of BL and ligand 5 in the PKCR C1b domain
obtained during the docking studies were minimized using the
Tripos force field in Sybyl6.864 and the Powell method for 5000
steps or until the gradient is lower than 0.01 kcal mol-1 Å-1
.
All simulations were performed with the molecular dynamics
module (Sybyl6.8) in a vacuum with a constant dielectric
constant ꢀ ) 1 using the Tripos force field. The ligands were
allowed to be fully flexible, while in PKCR, only residues within
an 8 Å radius of the ligand were made fully flexible keeping
the other residues fixed. The following parameters were used
for MD: 0.5 fs time step, snapshot each 50 fs, Bolzmann initial
velocities, constraint of all bonds to hydrogen atoms using the
SHAKE algorithm.71 The heating phase in the NTP ensemble
of 50 ns was followed by a 200 ns equilibration phase and a
100 ns production phase. All molecular modeling studies were
performed on a SGI Octane with two 300 MHz MIPS R12000
processors. The results of molecular modeling studies are
summarized in Table 3.
Ack n ow led gm en t. We are indebted to the National
Institutes of Health (Grant CA79601) for support of this
work. We thank Dr. Werner Tu¨ckmantel for proofread-
ing the manuscript.
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