Synthesis and protein kinase C inhibitory activities of acyclic balanol analogs that are highly selective for protein kinase C over protein kinase A

Article abstract of DOI:10.1021/jm960581w

A series of balanol analogs in which the perhydroazepine ring and the p- hydroxybenzamide moiety were combined into an acyclic linked unit have been prepared and evaluated for their inhibitory properties against the serine/threonine kinase PKC. Several low-micromolar to lownanomolar inhibitors of the α, β'(I), β(II), γ, δ, ε, and η PKC isozymes were prepared. In general, these acyclic balanol analogs were found to be highly selective for PKC over the serine/threonine kinase PKA. The type and number of atoms linking the benzophenone ester to the p-hydroxyphenyl group necessary for optimal PKC inhibition were investigated. The most potent compounds contained a three-carbon linker in which the carboxamide moiety of balanol had been replaced by a methylene group. The effect of placing substituents on the three-carbon chain was also investigated. The preferred compounds contained either a 2-benzene-sulfonamido (6b) or a 1-methyl (21b) substituent. The preferred compounds 6b and 21b were tested against a panel of serine/threonine kinases and found to be highly selective for PKC. The more active enantiomer of 6b, (S)-12b, was 3-10-fold more active than the R- enantiomer against the PKC isozymes. The effect of making the analogs more rigid by making the three-carbon chain part of a five-membered ring, but with retention of the methylene replacement for the carboxamide moiety, led to potent PKC inhibitors including anti-substituted pyrrolidine analog 35b and the most potent PKC inhibitor in the series, anti-substituted cyclopentane analog 29b. The anti cyclopentane analog 29b was a low-micromolar inhibitor of the PMA-induced superoxide burst in neutrophils, and its carboxylic ester was a high-nanomolar inhibitor of neutrophils. Finally esterification of 21b, (S)-12b, and 35b turned these potent PKC inhibitors into low-micromolar inhibitors of neutrophils.

Full text of DOI:10.1021/jm960581w

J . Med. Chem. 1996, 39, 5215-5227
5215
Syn th esis a n d P r otein Kin a se C In h ibitor y Activities of Acyclic Ba la n ol An a logs
Th a t Ar e High ly Selective for P r otein Kin a se C over P r otein Kin a se A
J ean M. Defauw,* Marcia M. Murphy, G. Erik J agdmann, J r., Hong Hu, J ohn W. Lampe, Sean P. Hollinshead,
Thomas J . Mitchell, Heidi M. Crane, J ulia M. Heerding, J ose´ S. Mendoza, J efferson E. Davis, J ames W. Darges,
Frederick R. Hubbard, and Steven E. Hall
Sphinx Pharmaceuticals, a Division of Eli Lilly and Company, 4615 University Drive, Durham, North Carolina 27707
Received August 7, 1996X
A series of balanol analogs in which the perhydroazepine ring and the p-hydroxybenzamide
moiety were combined into an acyclic linked unit have been prepared and evaluated for their
inhibitory properties against the serine/threonine kinase PKC. Several low-micromolar to low-
nanomolar inhibitors of the R, âI, âII, γ, δ, ꢀ, and η PKC isozymes were prepared. In general,
these acyclic balanol analogs were found to be highly selective for PKC over the serine/threonine
kinase PKA. The type and number of atoms linking the benzophenone ester to the
p-hydroxyphenyl group necessary for optimal PKC inhibition were investigated. The most
potent compounds contained a three-carbon linker in which the carboxamide moiety of balanol
had been replaced by a methylene group. The effect of placing substituents on the three-
carbon chain was also investigated. The preferred compounds contained either a 2-benzene-
sulfonamido (6b) or a 1-methyl (21b) substituent. The preferred compounds 6b and 21b were
tested against a panel of serine/threonine kinases and found to be highly selective for PKC.
The more active enantiomer of 6b, (S)-12b, was 3-10-fold more active than the R-enantiomer
against the PKC isozymes. The effect of making the analogs more rigid by making the three-
carbon chain part of a five-membered ring, but with retention of the methylene replacement
for the carboxamide moiety, led to potent PKC inhibitors including anti-substituted pyrrolidine
analog 35b and the most potent PKC inhibitor in the series, anti-substituted cyclopentane
analog 29b. The anti cyclopentane analog 29b was a low-micromolar inhibitor of the PMA-
induced superoxide burst in neutrophils, and its carboxylic ester was a high-nanomolar inhibitor
of neutrophils. Finally esterification of 21b, (S)-12b, and 35b turned these potent PKC
inhibitors into low-micromolar inhibitors of neutrophils.
In tr od u ction
Protein kinase C (PKC) is a family of phospholipid
dependent, serine/threonine specific kinases that plays
a key role in signal transduction pathways which lead
to cellular proliferation and differentiation.1 Activated
PKC phosphorylates numerous proteins which are
involved in many biological systems, and enhanced
activation of PKC has been implicated in many disease
states. Inhibitors of PKC may be useful in therapeutic
treatment of a number of diseases such as cancer,
asthma, rheumatoid arthritis, diabetic complications,
psoriasis, and central nervous system disorders.2
As part of our efforts directed toward finding thera-
peutic agents for the treatment of PKC-mediated dis-
orders, we discovered balanol, (-)-1, one of the most
potent PKC inhibitors. Balanol was isolated here as a
metabolite of the fungus Verticillium balanoides3 and
later from a species of Fusarium at Nippon Roche.4 Its
low-nanomolar potency and novel structure make bal-
anol an exciting target for total synthesis and structure-
activity relationship (SAR) studies. These reasons
coupled with its low availability from natural sources
have led to three total syntheses of balanol: from our
laboratory,5 from Nicolaou’s,6 and by a team at Rhone-
Poulenc Rorer.7
replacements were desired for both the benzophenone
ester and the perhydroazepine portions.
Balanol has been shown to be an ATP competitive
inhibitor of PKC.4,8 Balanol is not selective for PKC
over PKA, the c-AMP dependent kinase,8,9 which is not
surprising considering the high degree of homology
found among the catalytic sites of protein kinases. In
order for an inhibitor to be a useful therapeutic agent,
it will need to be selective for one kinase. Herein, we
would like to report the syntheses and activity of potent
PKC inhibitors which are selective for PKC over PKA
and in which the perhydroazepine ring and the p-
hydroxybenzamide moiety have been combined into an
acyclic linked unit.
Balanol consists of three different parts: the tetra-
substituted benzophenone, the p-hydroxybenzamide
moiety, and the perhydroazepine ring. Due to the
complexity of their synthesis, more readily accessible
Ch em istr y
In general, the acyclic analogs were made by first
preparing the perhydroazepine ring-hydroxybenzamide
replacement which was then coupled to a protected, fully
functionalized benzophenone. The syntheses of the
X Abstract published in Advance ACS Abstracts, November 15, 1996.
S0022-2623(96)00581-X CCC: $12.00 © 1996 American Chemical Society

5216 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Ta ble 1. Protected Benzophenones
Defauw et al.
Ta ble 2a
R1
R2
2a
2b
2c
2d
Cl
OH
Cl
OBn
OBn
OMe
OBn
1-imidazoyl
R1
PhSO2
R2
R3
X
Sch em e 1a
6
6b
MOM
H
MOM
CH2
CH2
PhSO2
7
7b
8
8b
MeSO2
MeSO2
PhCO
PhCO
PhSO2
PhSO2
PhSO2
PhSO2
PhSO2
PhSO2
PhSO2
Me
Me
2-C10H7SO2
2-C10H7SO2
BnOC6H4CO
HOC6H4CO
PhSO2
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
H
H
TBDMS
MOM
Cbz
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
(S)-12
(S)-12b
(S)-12c
(R)-12
(R)-12b
13
13b
15
15b
16
Cbz
Cbz
Cbz
H
CH2
CH2
CH2
CH2
CH2
CH2
OCH2
OCH2
a
Reagents: (a) p-BnOC6H4COCl, TEA, DMAP, DCM; (b) 2a ,
Et3N, DMAP, DCM; (c) H2, Pd(OH)2, EtOAc, EtOH.
16b
17
17b
26
Cbz
Bn
appropriately protected benzophenones 2 (Table 1) have
been previously reported.10
H
H
H
26b
PhSO2
The acylic analog 3, which most closely resembles
balanol, was prepared as outlined in Scheme 1. Analog
3 was synthesized from ethanolamine (4) by sequential
N-acylation with p-(benzyloxy)benzoyl chloride, O-acy-
lation with benzophenone acid chloride 2a , and hydro-
genation using Pearlman’s catalyst.
a
R4 ) H except for (S)-12c, where R4 ) CH3. Coupling method
A was used except in the preparation of 6b and 8b, for which
coupling method B was utilized. See text for descriptions of the
coupling methods.
Sch em e 2a
For the remainder of this section, the syntheses of the
perhydroazepine-hydroxybenzamide replacements (re-
ferred to as alcohols) are described first. The couplings
of the alcohols to the benzophenone portion are then
summarized in Tables 2-4.
Alcohols 6-8 (Table 2) were prepared as outlined in
eq 1 of Scheme 2. Starting with tyrosine methyl ester
hydrochloride (9), they were synthesized by first func-
tionalizing the nitrogen, followed by protecting the
phenol, and lastly reducing the carboxylic ester to an
alcohol as illustrated for alcohol 6. Treatment of
racemic tyrosine methyl ester hydrochloride (9) in a
biphasic mixture of aqueous 1 N NaOH (2.3 equiv) and
DCM with benzenesulfonyl chloride (1.1 equiv) over 2
h gave a mixture of N,O-bis(phenylsulfonyl)-DL-tyrosine
methyl ester, N-(phenylsulfonyl)-DL-tyrosine methyl
ester, and their corresponding acids. Stirring the
mixture of products in a solution of aqueous KOH in
ethanol cleaved the methyl ester and the O-sulfonyl
group to give 10 (40% over two steps). It was later found
that the yield could be improved to about 60% by using
2-3 equiv of benzenesulfonyl chloride. Treatment of
10 with chloromethyl methyl ether and N,N-diisopro-
pylethylamine in CH3CN gave trialkylated ester 11.
Reduction with LiBH4 then gave the corresponding
alcohol 6.
a
Reagents: (a) PhSO2Cl, 1 N NaOH, DCM; (b) 1 N KOH/3:1
EtOH:H2O; (c) MOMCl, iPr2NEt, CH3CN; (d) LiBH4, THF; (e) BH3,
THF, reflux; (f) CBzCl, Et3N, DCM; (g) HCHO, NaBH4.
The synthetic strategy used to prepare alcohols 12
and 13 (Table 2) became the preferred method (eq 2,
Scheme 2). These alcohols were prepared by first
functionalizing the nitrogen, then reducing the carboxy-
lic acid, and finally selectively protecting the phenol as
its Cbz derivative. In the example shown, compound
L-10 was derived from L-9 in the same manner as

PKC Inhibition by Balanol Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5217
Sch em e 3a
Sch em e 4a
a
Reagents: (a) PhSO2Cl, iPrNEt, THF; (b) TBDMSCl, ImH,
DMF; (c) LiBH4; (d) p-BnOC6H4OH, Ph3P, DEAD, THF; (e) TBAF,
THF.
Sch em e 5a
a
Reagents: (a) MOMCl, iPr2NEt, CH3CN; (b) [H], THF; (c)
NaSEt, DMF, reflux.
described above for the preparation of DL-10 from DL-9.
The reason the conditions used to hydrolyze the inter-
mediate N,O-bissulfonylated tyrosine did not lead to
racemization is most likely because the sulfonamide was
deprotonated during the hydrolysis. The carboxylic acid
L-10 was then reduced with borane which provided (S)-
14. The phenol of (S)-14 was then selectively protected
in the presence of the primary alcohol using CbzCl (1.5
equiv) and Et3N in DCM to afford (S)-12. This selective
protection of the phenol in the presence of the primary
alcohol proved useful for the synthesis of several acyclic
analogs. The Cbz group was the preferred protecting
group since all of the protecting groups could be removed
from the penultimate intermediate in a single step by
hydrogenation. Note that (R)-12 was prepared from D-9
in the exact same way as described for the preparation
of (S)-12 from L-9. The alcohols (S)-12 and (R)-12 were
prepared with at least 95% ee.11
a
Reagents: (a) 4-BnOC6H4CHO, KOH, H2O, reflux; (b) NaBH4,
THF, MeOH; (c) H2, Raney Ni; (d) CbzCl, Et3N, DCM.
Sch em e 6a
Alcohols 15-17 (Table 2) were prepared using the
following synthetic strategy: The ester was first reduced
to provide the primary alcohol, the nitrogen was then
funtionalized, and finally the phenol was protected (eq
3, Scheme 2). The synthesis of the illustrated example,
15, began with LiBH4 reduction of 9. Reductive alky-
lation of 18 then provided dimethylated amine 19. The
phenol of 19 was then selectively protected using CbzCl
which afforded 15.
The preparation of alcohols 20-22 is described in
Scheme 3. Protection of the phenol 23 as its meth-
oxymethyl ether followed by LiBH4 reduction of the
ester functionality gave alcohol 20. Similarly, phenol
24 was protected as a methoxymethyl ether, and then
the carbonyl was reduced with LiAlH4 to yield alcohol
21. Demethylation of the methyl ether 25 followed by
simultaneous esterfication and etherification using chlo-
romethyl methyl ether and subsequent LiAlH4 reduction
of the ester functionality led to alcohol 22.
The synthesis of alcohol 26 began with N-sulfonyla-
tion of serine methyl ester hydrochloride (27) (Scheme
4). The primary alcohol was then protected as its tert-
butyldimethylsilyl ether. Reduction of the carboxylic
ester gave 28. The (benzyloxy)phenyl ether was intro-
duced into 28 using Mitsonobu chemistry, and then the
silyl protecting group was removed to give 26.
Alcohols 29 and 30 were prepared as illustrated in
Scheme 5. Aldol condensation of cyclopentanone 31
with p-(benzyloxy)benzaldehyde provided the desired
aldol product 32 in 61% yield along with some cyclo-
a
Reagents: (a) 4-BnOC6H4CHO, LiN(SiMe3)2, THF, -50 to 35
°C; (b) NaBH4, THF, MeOH; (c) H2, Raney Ni; (d) 4-MeOC6H4CH2Cl,
K2CO3, DMF, 65 °C; (e) LiAlH4, THF, reflux.
pentanone self-condensation product and double-aldol
product. Reduction of the carbonyl group of 32 using
NaBH4 followed by Raney nickel hydrogenation of the
olefin provided a 1:1.3 mixture of the anti- and syn-
substituted cyclopentanones 33 and 34, which were
easily separated by column chromatography. The phe-
nol functionalities of both 33 and 34, which had lost
their protecting groups during the olefin reduction step,
were then reprotected as their Cbz derivatives 29 and
30.
The synthesis of alcohol 35 is shown in Scheme 6.
Dione 36 was obtained by N-benzylation and subsequent
enol ether hydrolysis of commercially available 4-meth-
oxy-3-pyrrolin-2-one. As in the preceding case, dione
36 was condensed with p-(benzyloxy)benzaldehyde, and
then the ketone and the olefin were sequentially reduced
to provide a 2.75:1 mixture of the anti- and syn-
substituted pyrrolidinones 37 and 38 which were easily
separated by column chromatography. The phenol of
the desired anti isomer 37 was reprotected as its
p-methoxybenzyl ether and the amide functionality was
reduced to an amine using LiAlH4, providing 35. The
last coupling precursor 39 (Table 4) was prepared in a
single step by the addition of p-(benzyloxy)phenol to
cyclopentene oxide (40).

5218 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Defauw et al.
Ta ble 3
Ta ble 4a
R1
R2
R3
H
X
X
R1
R2
20
20b
21
21b
21c
22
H
H
Me
Me
Me
H
MOM
CH2
CH2
CH2
CH2
CH2
(CH2)2
(CH2)2
29 (anti)
CH2
CH2
CH2
CH2
CH2
NBn
NH
NH
CH2
CH2
Cbz
MOM
MOM
29b (anti)
29c (anti)
30 (syn)
30b (syn)
35 (anti)
35b (anti)
35c (anti)
39 (anti)
39b (anti)
H
CH3
H
Me
Cbz
H
22b
H
H
PMB
H
CH3
a
See text for a description of the coupling method.
Bn
Tables 2-4 summarize the general procedures used
to couple the alcohols to the benzophenones. The
alcohols listed in Table 2 were coupled to the benzophe-
none portion of the molecule using one of two methods.12
Method A consisted of acylating the alcohol with ben-
zophenone acid chloride 2a or 2c in the presence of
catalytic DMAP and either Et3N or N,N-diisopropyl-
ethylamine, with DCM as the solvent. Method B
consisted of 1,1′-carbonyldiimidazole coupling of the
alcohol with benzophenone acid 2b with DBU as the
base and THF as the solvent. After coupling, any
methoxymethyl protecting groups were removed with
HCl, and any silyl protecting groups were removed
using tetrabutylammonium fluoride. The benzyl and
any benzyloxycarbonyl protecting groups were then
removed by catalytic hydrogenation to provide the
acyclic balanol analogs.
H
a
Z ) CH2 except for 39 and 39b, for which Z ) O. Coupling
method A was used except in the preparation of 35b and 35c, for
which coupling method B was utilized. See text for descriptions
of the coupling methods.
kinases, the effect of replacing the perhydroazepine ring
with simple readily accessible acyclic units was inves-
tigated.
Substitution of the perhydroazepine linker of balanol
with an ethyl linker as in 3 (Table 5) resulted in a 10-
300-fold loss in activity when compared to racemic
balanol (()-1. This is especially significant since cyclo-
pentane analog 4114 is just as potent as racemic balanol
(more potent for the δ and η isozymes). Taken together
these results suggest that inhibition of PKC is strongly
dependent upon the spatial orientation of the benzophe-
none ester and the p-hydroxybenzamide substituents.
This is supported by a study which demonstrated a
marked effect on PKC inhibitory activity as the perhy-
droazepine was contracted to six- and five-membered
rings.14 Thus it initially appeared that spatial orienta-
tion of the benzophenone ester and the p-hydroxyben-
zamide substituents needed for potent PKC inhibition
was not energetically favorable in an open chain form.
It occurred to us that a tyrosine balanol analog might
provide a readily accessible perhydroazepine replace-
ment. A tyrosine analog was appealing for several
reasons. Both enantiomers of tyrosine are commercially
available, and this amino acid has a p-hydroxybenzyl
group which might provide a suitable replacement for
the p-hydroxybenzamide side chain of balanol. It
contains a carboxylic acid which, after reduction to an
alcohol, could be used for introduction of the benzophe-
none. Finally, it possesses a nitrogen which provided
a handle for the introduction of several different groups.
N-(Phenylsulfonyl)tyrosine was chosen because it had
been observed that, although sulfonamide derivatives
of balanol such as 42 (Table 6) were less potent PKC
inhibitors, they were selective for PKC over PKA.15 In
addition, many of the sulfonamide derivatives of balanol
were selective for âI and âII among the PKC isozymes
as exemplified by 42.15 The tyrosine analog 6b (Table
7) was as potent as racemic balanol against the âII and
η isozymes while only 2-9 times less potent against the
remaining isozymes. In addition, 6b was inactive
The alcohols shown in Table 3 were coupled to the
benzophenone portion of the molecule using method A
as described for Table 2 above. The methoxymethyl
groups were then removed with aqueous HCl/THF, and
subsequently the benzyl and benzyloxycarbonyl protect-
ing groups were removed by hydrogenation using Pearl-
man’s catalyst to provide the target compounds.
The target compounds in Table 4 were prepared by
first coupling the alcohols with the benzophenone por-
tion using method A as described above followed by
removal of the benzyl and Cbz protecting groups using
catalytic hydrogenation. Target compounds 35b,c were
exceptions. Compound 35b was prepared by coupling
the lithium salt of 35 with the preformed imidazolide
2d (method C). The benzyl protecting groups were then
removed by catalytic hydrogenation followed by removal
of the p-methoxybenzyl group with TFA which provided
analog 35b. Treatment of analog 35b with thionyl
chloride in absolute MeOH provided its methyl ester 35c
in 52% yield.
Discu ssion
Although efficient syntheses5-7,13 of the appropriately
substituted perhydroazepine ring necessary to make
balanol had been devised, they were longer than desired
for large scale preparation of research materials. For
this reason, and more importantly the possibility that
modification could lead to selectivity for PKC over other

PKC Inhibition by Balanol Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5219
Ta ble 5. PKC and PKA Inhibitory Activities (IC50s) of Two-Carbon Amide-Linked Acyclic Analogs (µM)
PKC
R1
R2
R
âI
âII
γ
δ
ꢀ
η
PKA
3
H
H
11
4.5
7.5
6.6
0.41
13
0.23
8.4
(()-1
41
CH2CH2CH2NHCH2
CH2CH2CH2
0.067
0.04
0.03
0.04
0.03
0.05
0.03
0.01
0.023
0.0009
0.038
0.05
0.02
0.0006
0.06
0.03
Ta ble 6. PKC Inhibitory Activities (IC50s) of the N-Tosyl
Derivative (42) of Balanol (µM)
more resistant to enzymatic hydrolysis of the ester
linkage, was essentially as active against PKC as the
racemic tyrosine analog 6b. Importantly, it shared the
tyrosine analog’s selectivity for PKC over PKA.
Analog 17b was designed to investigate the possibility
that the benzenesulfonamide moiety present in 6b was
actually serving as a replacement for the p-hydroxy-
benzamide side chain in balanol while the p-hydrox-
yphenyl of 6b was actually serving as a replacement
for the perhydroazepine ring of balanol. The low
activity of 17b suggests that this is unlikely.
The effect that the number and type of atoms in the
linker between the benzophenone ester and the p-
hydroxyphenyl had on activity was also investigated
(Table 8). Shortening the length of the three-carbon
linker in 6b by one carbon as in 13b greatly reduced
activity (22- to >455-fold), while increasing the length
by adding an oxygen as in 26b resulted in a large loss
in activity (6-164-fold). Increasing the length of the
chain between the benzophenone ester and the p-
hydroxyphenyl by one carbon reduced potency in the
straight chain series (22b vs 20b).
PKC
R
âI
âII
γ
δ
ꢀ
η
PKA
42
6.1
0.07
0.02
3.5
4.2
15
0.10-1.5
>150
against PKA at the highest concentration tested (50 µM)
but, unlike its congener 42, did not show selectivity for
the â isozymes.
The two enantiomers of 6b were then prepared in
hopes of finding increased activity since the natural
isomer of balanol was 25-100-fold more potent against
PKC than the unnatural isomer.5d Indeed the S-
enantiomer of the tyrosine analog (S)-12b was found to
be 4-13-fold more potent than racemic 6b against the
âI, âII, δ, and η isozymes (the activities against R, γ, and
ꢀ were about the same). The more active S-enantiomer
(S)-12b was 3-10-fold more potent than the R-enanti-
omer (R)-12b, not quite as large of a difference as seen
between balanol and its enantiomer.
Given the improved kinase selectivity of 6b, further
modifications were investigated. The size of the sul-
fonamide group had varying effects on the inhibitory
activities of the individual PKC isozymes. Replacement
of phenylsulfonamide with methylsulfonamide (7b) did
not affect activity toward âI; however, activity against
the rest of the isozymes decreased 10-210-fold. Alter-
natively, increasing the size to naphthylsulfonamide
(16b) resulted in a 10-1750-fold loss in activity. Simply
replacing the benzenesulfonamide with benzenecar-
boxamide (8b) resulted in a 90-900-fold loss in activity
for the isozymes tested, whereas replacement with
dimethylamino (15b) resulted in a smaller loss in
activity (4-100-fold).
Since the acyclic analogs were selective for PKC over
PKA, the two best racemic analogs, 6b and 21b, were
tested against a panel of other kinases. Table 9
compares their inhibitory activities along with those of
synthetic (-)-balanol against four kinases. Protein
kinase A, casein kinase, and Ca2+/calmodulin (CaM)
dependent kinase are, like PKC, serine/threonine spe-
cific kinases, while src tyrosine kinase is a tyrosine
specific kinase. Neither balanol nor the acyclic analogs
6b and 21b inhibited casein kinase at the highest
concentrations tested. While balanol was a potent
inhibitor of CaM kinase (30 nM), the tyrosine analog
6b was a weak inhibitor (32 000 nM, which is 150-
1600-fold less than its activity toward the PKC isozymes),
and 21b did not inhibit at all at the highest concentra-
tion tested (50 µM). Neither 6b nor balanol inhibited
src tyrosine kinase. Balanol has also been reported to
be ineffective against the epidermal growth factor
receptor (EGFR) protein tyrosine kinase.8 Thus, like
balanol, 6b appears to be ineffective against tyrosine
protein kinases. Unlike balanol, 6b and 21b appear to
be selective for PKC over other serine/threonine kinases
in general, at least among the kinases tested.
Due to the surprisingly good activity of the simple
methyl-branched analog 21b, several less flexible ana-
logs were prepared in which part of the three-carbon
linkage was tied back in a five-membered ring (Table
10).16 It was proposed that the rigidity might provide
greater activity and that with secondary alcohol ester
linkages they might be more hydrolytically stable than
Interestingly, the simple three-carbon-linked analog
20b which lacks the benzenesulfonamide unit was fairly
potent. Compound 20b was as potent as 6b against the
âI and η isozymes while only 4-17-fold less potent
against the remaining isozymes. The simple methyl-
branched analog 21b, which would be expected to be

5220 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Defauw et al.
Ta ble 7. Effects of R Groups on the PKC and PKA Inhibitory Activities (IC50s) of Three-Carbon-Linked Acyclic Balanol Analogs
(µM)a
PKC
R1
R2
R
âI
âII
γ
δ
ꢀ
η
PKA
6b
H
H
H
H
H
H
H
H
NHSO2C6H5
NHSO2C6H5
NHSO2C6H5
NHSO2CH3
NHSO2-2-naphthyl
NHCOC6H5
N(CH3)2
0.11
0.21
1.9
23
3.2
45
4.0
1.9
0.41
18
0.03
0.22
0.03
0.23
0.22
2.8
0.03
0.007
0.02
0.43
1.8
0.22
0.47
2.6
5.0
2.7
NT
4.2
0.83
0.25
2.9
0.04
0.003
0.03
0.41
2.6
0.32
0.46
2.3
0.02
0.003
0.02
0.39
35
18
1.4
<0.05
0.02
2.3
0.003
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.04
(S)-12b
(R)-12b
7b
16b
8b
15b
20b
21b
17b
>50
39
>50
30
NT
2.8
22
0.88
0.21
0.04
3.6
0.50
0.27
0.03
3.0
0.27
0.13
0.03
0.82
0.02
H
H
4.1
0.28
>50
0.02
CH3
H
NHCOC6H4-4-OH
(-)-1
0.01
0.01
0.01
a
NT ) not tested.
Ta ble 8. Effects of the Length and Type of Linker on PKC and PKA Inhibitory Activities (IC50s, µM)a
PKC
R2
Z
R
âI
11
âII
γ
δ
ꢀ
η
PKA
13b
26b
22b
6b
NHSO2C6H5
NHSO2C6H5
>50
18
2.8
4.8
1.6
1.4
0.22
0.83
1.9
>50
47
30
3.3
0.50
0.25
0.02
<0.05
>50
>50
>50
>50
>50
CH2O
CH2CH2
CH2
1.6
0.83
0.37
0.03
0.27
0.22
0.29
0.04
0.13
H
3.3
0.11
1.9
0.96
0.22
0.21
NHSO2C6H5
H
0.32
4.1
20b
CH2
a
NT ) not tested.
Ta ble 9. Comparison of the Kinase Inhibitory Properties
(IC50s) of 6b and 21b versus Balanol (µM)a
Several acyclic balanol analogs which were potent
inhibitors of the isolated enzyme and selective for PKC
over PKA were investigated in cellular assays. The
neutrophil assay, an assay which measures the inhibi-
tion of the phorbol-12-myristate-13-acetate (PMA)-
induced release of superoxide in human neutrophils, a
process thought to be mediated by PKC,18 was chosen.
Like balanol, most of the acyclic analogs initially tested
were not active in this assay at the highest concentra-
tion tested (10 µM). However, both the anti and syn
cyclopentane analogs 29b and 30b were low-micromolar
inhibitors of the superoxide burst in neutrophils (3.1 and
10 µM, respectively, Table 10), and the carboxylic ester
of the anti cyclopentane analog, 29c, was a high-
nanomolar inhibitor of neutrophils (260 nM). In addi-
tion, esterification of the tyrosine analog (S)-12b, methyl-
substituted acyclic analog 21b, and pyrrolidine analog
35b provided low-micromolar inhibitors of the neutro-
phil response, (S)-12c, 21c, and 35c, respectively (Table
11). Previous studies in our laboratories had shown
that while masking the carboxylic acid group of balanol
analogs as an ester slightly decreased PKC inhibitory
activities,19 it greatly enhanced the cellular activity.10b
PKA
casein kinase
CaM kinase
srcTyrK
(-)-1
6b
21b
0.30-0.80
>50
>50
>50
>50
>50
0.03-0.05
>10
>12.5
NT
32
>50
a
NT ) not tested.
6b. The anti cyclopentane 29b analog was 1-10-fold
more active than racemic balanol against the PKC
isozymes with the exception of ꢀ (it was 3-fold less active
than racemic balanol) and 3-55-fold more active than
6b. The syn cyclopentane 30b was 3-7-fold less active
than the anti derivative, not as large of a difference as
was anticipated since a marked preference for an anti
relationship between the benzophenone and the p-
hydroxyphenyl moieties has been observed in other
balanol analogs.17 The anti pyrrolidine analog 35b had
activity similar to racemic balanol (within (5-fold) with
the exception of ꢀ (it was 50-fold less active than racemic
balanol). The anti cyclopentane ether 39b also had
similar activity to balanol (within (3-fold) with the
exceptions of δ and ꢀ (against which it was 8- and 6-fold
less active, respectively, than racemic balanol). The anti
cyclopentane analog 29b and the anti cyclopentane
ether 39b were not active against PKA, while the
pyrrolidine analog 35b displayed modest activity (IC50
) 4.3 µM).
Con clu sion s
In conclusion we have prepared a series of balanol
analogs in which the perhydroazepine ring and the
p-hydroxybenzamide moiety have been combined into
a simple, readily accessible acyclic linked unit. A three-

PKC Inhibition by Balanol Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5221
Ta ble 10. Effects of Making the Three-Carbon Chain Part of a Five-Membered Ring on PKC, PKA, and Neutrophil Inhibitory
Activities (IC50s, µM)
PKC
X
Z
R
âI
âII
γ
δ
ꢀ
η
PKA
neutrophils
29b (anti)
30b (syn)
35b (anti)
39b (anti)
CH2
CH2
NH
CH2
CH2
CH2
O
0.030
0.160
0.260
0.220
0.004
0.020
0.010
0.020
0.003
0.020
0.030
0.020
0.040
0.130
0.040
0.250
0.005
0.030
0.005
0.020
0.120
0.500
1.8
0.003
0.010
0.020
0.010
>50
>50
4.3
3.1-4.2
10
>10
>10
CH2
0.240
>50
Ta ble 11. Effects of Esterification on PKC and Neutrophil Inhibitory Activities (IC50s, µM)
PKC
R1
R2
R
âI
âII
γ
δ
ꢀ
η
PKA
neutrophils
(S)-12c
21c
29c
35c
6b
H
H
NHSO2C6H5
H
CH2CH2CH2 (anti)
CH2NHCH2 (anti)
18
7.8
1.4
2.2
2.2
0.34
0.75
0.09
0.25
0.03
4.5
4.1
1.7
0.38
0.22
0.36
0.30
0.05
0.10
0.04
38
13
4.5
4.0
0.32
<0.05
0.17
0.04
0.50
0.02
>50
NT
>50
4.1
0.93-1.1a
1.3-2.7a
0.26a
0.46
0.10
0.11
0.22
4.3
0.11
>50
>10
a
1 h preincubation at 4 °C. NT ) not tested.
CH3CN in H2O + 0.1% TFA; B, 100% CH3CN) were employed.
The solvent was then removed by lyophilization. For inter-
mediates, column chromatography was performed using silica
gel 60 (230-400 mesh, E. Merck) at ca. 5 psig. Proton (1H)
NMRs were obtained on a 300 MHz Varian Gemini instru-
ment. Proton chemical shifts are reported in δ values (parts
per million) relative to tetramethylsilane. Mass spectra were
performed on a Finnigan MAT 211 mass spectrometer in the
FAB (positive) mode by Analytical Instrument Group, Inc.,
Raleigh, NC. Optical rotations were recorded using a Perkin-
Elmer polarimeter 241. Data are reported as follows: [R]D
(concentration in g/100 mL, solvent). Elemental analyses were
obtained on a Carlo Erba CHNS elemental analyzer 1108, and
the results are within 0.4% of the theoretical values, except
where noted. Melting points were determined in open glass
capillaries utilizing a Mel-temp apparatus and are uncorrected.
Solvents and reagents were obtained from commercial sup-
pliers and used as received. Dichloromethane is abbreviated
as DCM.
carbon linkage between the benzophenone ester and the
p-hydroxyphenyl substituent, in which the carboxamide
moiety has been replaced by a methylene, was found to
be optimal. Substitution on the three-carbon chain was
investigated, and the most active analogs found were
the 1-methyl-substituted analog 21b, the 2-benzensul-
fonamido- substituted analog 6b, and its enantiomers
(S)-12b and (R)-12b. The more potent enantiomer, (S)-
12b, had low-nanomolar activity against three of the
PKC isozymes and was 3-10-fold more active than the
R-enantiomer (R)-12b against the PKC isozymes over-
all. In general, the acyclic analogs have been shown to
be highly selective for PKC over PKA. Moreover,
compounds 21b and 6b appeared to be selective for PKC
over other serine/threonine kinases in general. Rein-
troduction of a ring for the perhydroazepine replacement
in the form of an anti-substituted pyrrolidine and an
anti-substituted cyclopentane ring, but with retention
of the methylene replacement for the carboxamide
moiety, led to the potent analog 35b and the most potent
analog 29b, respectively. Besides being equipotent to
balanol against PKC and selective for PKC over PKA,
the anti cyclopentane analog 29b was a low-micromolar
inhibitor of neutrophils, and its carboxylic ester 29c was
a high-nanomolar inhibitor of neutrophils. In addition
to having a better pharmacological profile, 29b was also
much easier to synthesize than balanol itself. Finally
esterification of 21b, (S)-12b, and 35b turned these
potent PKC inhibitors into low-micromolar inhibitors
of the superoxide burst in neutrophils.
Ben zyl 2-[[4-[[2-[[[4-(Ben zyloxy)p h en yl]ca r bon yl]a m i-
n o]eth oxy]car bon yl]-2,6-bis(ben zyloxy)ph en yl]car bon yl]-
3-(ben zyloxy)ben zoa te (5). To an ice cold solution of
ethanolamine (4) (122 mg, 2 mmol) in THF (6 mL) was added
2.0 N KOH (1 mL). Slowly, 4-(benzyloxy)benzoyl chloride (493
mg, 2 mmol) was added. After the reaction mixture stirred
for 4 h at room temperature, it was diluted with DCM and
washed with H2O and brine. The organic layer was dried (Na2-
SO4) and concentrated in vacuo. The residue was triturated
with Et2O/hexanes to give N-(2-hydroxyethyl)-4-(benzyloxy)-
benzamide (43) as a white solid (410 mg, 76%): mp 141-143
1
°C; H NMR (CD3OD) δ 7.79 (d, 2H, J ) 9 Hz), 7.36 (m, 5H),
7.04 (d, 2H, J ) 9 Hz), 5.13 (s, 2H), 3.69 (t, 2H, J ) 5.9 Hz),
3.47 (t, 2H, J ) 5.8 Hz). Anal. (C16H17NO3) C, H, N.
To a stirred solution of 4-[6-(benzyloxy)-2-(benzyloxycarbo-
nyl)benzoyl]-3,5-bis(benzyloxy)benzoic acid (2b) (339 mg, 0.50
mmol) in DCM (5 mL) was added DMF (3 drops) followed by
oxalyl chloride (0.75 mL, 2.0 M in DCM, 1.5 mmol) dropwise
at room temperature under N2. The reaction mixture was then
stirred at room temperature for 2 h and the solvent evaporated
in vacuo. The residue of benzyl 2-[[4-(chloroformyl)-3,5-bis-
Exp er im en ta l Section
All final products were purified on a Rainin HPLC using
Dynamax-60 C18 columns (unless otherwise noted). Appropri-
ate linear gradients of the solvent systems A and B (A, 5%

5222 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Defauw et al.
(benzyloxy)phenyl]carbonyl]-3-(benzyloxy)benzoate (2a ) was
used “as is” in the following reaction without any further
purification.
(t, 1H, J ) 6.5 Hz), 2.87 (dd, 1H, J ) 9.5, 13.5 Hz), 2.54, (dd,
1H, J ) 5, 13.5 Hz).
(S)-3-(4-Hyd r oxyp h en yl)-2-[(p h en ylsu lfon yl)a m in o]-1-
p r op a n ol [(S)-14]. To a solution of L-10 (12.2 g, 42.2 mmol)
in anhydrous THF (500 mL) under an atmosphere of N2 was
added BH3‚THF complex (253 mL, 1.0 M in THF, 253 mmol),
and the reaction mixture was heated at reflux overnight. The
reaction was quenched by the dropwise addition of saturated
aqueous NH4Cl. The reaction mixture was diluted with EtOAc
and washed with 1 N HCl and brine (75 mL). The EtOAc layer
was dried (MgSO4) and concentrated. The crude product was
purified by column chromatography (hexane:EtOAc, 1:1) to
provide a white solid of (S)-14 (10.4 g, 80%): 1H NMR (DMSO-
d6) δ 9.13 (br s, 1H), 7.68-7.42 (m, 5H), 6.82 (d, 2H, J ) 8.5
Hz), 6.55 (d, 2H, J ) 8.5 Hz), 3.30-3.10 (m, 3H), 2.67 (dd, 1H,
To a stirred mixture of 43 (136 mg, 0.50 mmol) in DCM (10
mL) were added DMAP (8 mg, 65 µmol) and Et3N (0.20 mL,
1.5 mmol) followed by the slow addition of a solution of freshly
prepared 2a from above in DCM (5 mL). The reaction mixture
was stirred at room temperature for 2 h under N2, and then
the solvent was evaporated in vacuo. The crude product was
purified by column chromatography (hexane:EtOAc, 7:3 then
1:1) to afford 5 (400 mg, 86%).
2-[[2,6-Dih yd r oxy-4-[[2-[[(4-h yd r oxyp h en yl)ca r bon yl]-
am in o]eth oxy]car bon yl]ph en yl]car bon yl]-3-h ydr oxyben -
zoic Acid (3). To a solution of 5 (380 mg, 407 µmol) in EtOAc:
EtOH (3:1, 8 mL) was added Pd(OH)2 (30 mg). The reaction
mixture was then stirred at room temperature under H2 (1
atm) for 18 h. The mixture was filtered through Celite and
the solvent evaporated in vacuo. The residue was purified by
column chromatography [EtOAc (100%), MeOH:EtOAc (5:95,
10:90), and then MeOH (100%)] to give 3 (130 mg, 66%) as a
J ) 14, 6 Hz), 2.31 (dd, 1H, J ) 14, 6 Hz). Anal. (C15H17
NO4S) C, H, N.
-
Gen er a l P r oced u r e for th e Cbz P r otection of P h e-
n ols: (S)-3-[4-[(Ben zyloxyca r bon yl)oxy]p h en yl]-2-[(p h e-
n ylsu lfon yl)a m in o]-1-p r op a n ol [(S)-12] a n d (R)- 3-[4-
[ ( B e n z y l o x y c a r b o n y l ) o x y ] p h e n y l ] -2 -[ ( p h e n y l -
su lfon yl)a m in o]-1-p r op a n ol [(R)-12]. To a suspension of
(S)-14 (9.15 g, 29.8 mmol) and Et3N (6.22 mL, 44.6 mmol) in
anhydrous THF (200 mL) was added benzyl chloroformate
(4.46 mL, 31.3 mmol) at 0 °C. After stirring for 30 min, the
reaction mixture was diluted with EtOAc and washed with
water and brine. The EtOAc layer was dried (MgSO4) and
concentrated to afford (S)-12 as a white solid (11.5 g, 87%):
[R]D ) -20° (0.51, EtOAc);11 1H NMR (CDCl3) δ 7.69 (d, 2H, J
) 8 Hz), 7.58-7.38 (m, 8H), 6.98 (s, 4H), 5.28 (s, 2H), 4.78 (d,
1H, J ) 7 Hz), 3.69-3.39 (m, 3H), 2.81 (dd, 1H, J ) 7, 14 Hz),
2.69 (dd, 1H, J ) 7, 14 Hz), 2.01 (dd, 1H, J ) 6, 5.5 Hz). Anal.
(C23H23NO6S) C, H, N, S.
1
light yellow powder: mp 198-205 °C dec; H NMR (CD3OD)
δ 7.68 (d, 2H, J ) 8.6 Hz), 7.37 (d, 1H, J ) 7.6 Hz), 7.19 (t,
1H, J ) 7.8 Hz), 6.93 (s, 2H), 7.07 (d, 1H, J ) 8.1 Hz), 6.80 (d,
2H, J ) 8.7 Hz), 4.42 (t, 2H, J ) 6 Hz), 3.70 (t, 2H, J ) 6 Hz);
MS m/ z 482 (M + 1).20
N-(P h en ylsu lfon yl)-DL-tyr osin e (10). To a suspension of
9 (5.0 g, 21.6 mmol) in DCM (25 mL) was added benzenesulfo-
nyl chloride (3.0 mL, 23.7 mmol) followed by 1 N NaOH (45
mL). After 2 h, the mixture was poured into EtOAc (200 mL)
and washed with water (2 × 100 mL) followed by brine (100
mL). The organic layer was dried (MgSO4) and concentrated.
Column chromatography (EtOAc/hexanes, 30-50%) provided
the N,O-bis(phenylsulfonyl)-DL-tyrosine (44) as a white foam
(2.93 g, 40%).
Treatment of (R)-14 using the same conditions provided (R)-
12: [R]D ) 20° (0.66, EtOAc).11
Intermediate 44 was dissolved in a solution of 1 N KOH/
EtOH/water (1.4 g/20 mL/2 mL) and heated at 60 °C. After
24 h, another 1.4 g of KOH was added to the system. After
another 24 h, the solvent was evaporated and the byproduct
was recrystallized from ethanol. The side product (benzene-
sulfonic acid) was filtered off, and the filtrate was evaporated
to dryness. The residue was triturated with toluene (5×) and
then dried in a vacuum oven for 16 h. A tan solid of 10 (4.72
g, quantitative) was obtained: mp 140 °C; 1H NMR (CDCl3) δ
7.64 (d, 1H, J ) 9 Hz), 7.63 (s, 1H), 7.56-7.51 (m, 1H), 7.43
(d, 1H, J ) 8 Hz), 7.39 (s, 1H), 6.92 (d, 2H, J ) 8.5 Hz), 6.60
(d, 2H, J ) 8.5 Hz), 3.96 (dd, 1H, J ) 8, 5 Hz), 2.94 (dd, 1H,
(()-2-Am in o-3-(4-h ydr oxyph en yl)-1-pr opan ol (18). Com-
pound 9 was reduced using the same procedure described for
the preparation of 6 from 11 which provided alcohol 18 (2.0 g,
quantitative yield): 1H NMR (DMSO-d6) δ 9.20 (br s, 1H) 6.96
(d, 2H, J ) 8.3 Hz), 6.67 (d, 2H, J ) 8.3 Hz), 5.05-4.70 (m,
2H), 3.52-3.15 (m, 3H), 2.9 (d, 1H, J ) 12 Hz), 2.65-2.46 (m,
2H). Anal. (C9H13NO2‚1.6H2O) C, H, N.
(()-2-(N,N-Dim eth ylam in o)-3-(4-h ydr oxyph en yl)-1-pr o-
p a n ol (19). A solution of 18 (0.38 g, 2.26 mmol) in MeOH (2
mL) was treated with formaldehyde (0.40 mL, 37% aqueous,
15.0 mmol) and 1 drop of HOAc. Sodium borohydride (427
mg, 11.3 mmol) was added portionwise, and the reaction
mixture was allowed to stir at room temperature for 20 min.
A slurry was obtained. The reaction mixture was diluted with
DCM (2 mL) and stirred for an additional 1 h. Solvents were
removed in vacuo; the resulting residue was quenched with
water (3 mL) and then extracted with DCM (8 × 15 mL). The
combined organic layers were dried (Na2SO4) and concentrated
J ) 13.5, 5 Hz), 2.74 (dd, 1H, J ) 13.5, 8 Hz). Anal. (C15H15
-
NO5S) C, H, N, S.
N-(P h en ylsu lfon yl)-N,O-bis(m eth oxym eth ylen e)-DL-ty-
r osin e Meth oxym eth ylen e Ester (11). To a suspension of
10 (4.6 g, 14.3 mmol) and N,N-diisopropylethylamine (6.3 mL,
35.8 mmol) in CH3CN (70 mL) at 0 °C was slowly added
chloromethyl methyl ether (2.7 mL, 35.8 mmol). After 1.5 h
the solid material was filtered off and discarded. The filtrate
was concentrated, poured into a 1:1 mixture of saturated
aqueous NH4Cl, and then extracted with EtOAc (300 mL). The
organic layer was washed with brine (75 mL) and then dried
(MgSO4). After concentration, the crude oil was chromato-
graphed (EtOAc/hexanes, 25%) to give 11 as a yellow oil (1.17
g, 46%).
Gen er a l P r oced u r e for th e LiBH4 Red u ction of Ca r -
boxylic Ester s to Alcoh ols: (()-3-[4-[(Meth oxym eth yl-
en e)oxy]p h en yl]-2-[N-[(m eth oxym eth ylen e)oxy]-N-(p h e-
n ylsu lfon yl)a m in o]-1-p r op a n ol (6). To a solution of 11
(1.16 g, 2.56 mmol) in THF (9 mL) was added LiBH4 (1.5 mL,
2 M in THF, 3.00 mmol). After 18 h, the reaction was
quenched with saturated aqueous NH4Cl (9 mL) and the
mixture poured into EtOAc (60 mL). After washing with brine
(30 mL), the organic layer was dried (Na2SO4), concentrated
to a clear oil, and then dried under high vacuum to give 6 (901
mg, 92%): 1H NMR (CDCl3) δ 7.82 (d, 1H, J ) 8 Hz), 7.81 (d,
1H, J ) 8.5 Hz), 7.61-7.56 (m, 1H), 7.50 (d, 1H, J ) 8 Hz),
7.48 (d, 1H, J ) 13 Hz), 6.89 (s, 2H), 6.88 (s, 2H), 5.14 (s, 2H),
5.09 (d, 1H, J ) 11.5 Hz), 4.65 (d, 1H, J ) 11.5 Hz), 4.02-
3.90 (m, 1H), 3.64-3.50 (m, 1H), 3.48 (s, 3H), 3.46 (s, 3H), 3.13
to afford 19 as a white solid (353 mg, 80%). Anal. (C11H17
NO2‚0.35H2O) C, H, N.
-
(()-2-(N,N-Dim eth yla m in o)-3-[4-[(ben zyloxyca r bon yl)-
oxy]p h en yl]-1-p r op a n ol (15). Compound 15 was prepared
from 19 using the general procedure described for the prepara-
tion of (S)-12 from (S)-14. After stirring at room temperature
for 2 h, the reaction mixture was loaded onto a silica gel
column and eluted with 5-10% MeOH/DCM to afford 15 (340
mg, 60%).
3-[4-[(Meth oxym eth ylen e)oxy]p h en yl]-1-p r op a n ol (20).
To a solution of methyl 3-(4-hydroxyphenyl)propanoate (23)
(2.00 g, 11.0 mmol) in CH3CN (15 mL) under an atmosphere
of N2 at 0 °C was added N,N-diisopropylethylamine (2.89 mL,
11.6 mmol) followed by the dropwise addition of chloromethyl
methyl ether (1.26 mL, 11.6 mmol) over 10 min. The reaction
mixture was allowed to stir at room temperature for 96 h
during which time additional N,N-diisopropylethylamine (11.6
mL, 64.4 mmol) and chloromethyl methyl ether (5.04 mL, 64.4
mmol) were added over four portions. The reaction mixture
was diluted with EtOAc (350 mL) and washed with water (3
× 50 mL), 1 N NaOH (4 × 50 mL), water (20 mL), and brine
(50 mL). The EtOAc layer was dried (MgSO4) and concen-
trated to provide an oil of methyl 3-[4-[(methoxymethylene)-

PKC Inhibition by Balanol Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5223
oxy]phenyl]-1-propanoate (45) (625 mg, 90%) which was used
as is in the next step: 1H NMR (CDCl3) δ 7.13 (d, 2H, J ) 9
Hz), 6.97 (d, 2H, J ) 9 Hz), 5.16 (s, 2H), 3.68 (s, 3H), 3.48 (s,
3H), 2.91 (t, 2H, J ) 8 Hz), 2.61 (t, 2H, J ) 8 Hz).
Compound 20 was obtained from 45 using a method similar
to that described for the preparation of 6 from 11. The crude
residue was purified by column chromatography (MeOH/
CHCl3, 2%) to provide 20 as an oil (1.55 g, 82%): 1H NMR
(CDCl3) δ 7.12 (d, 2H, J ) 9 Hz), 6.97 (d, 2H, J ) 9 Hz), 5.16
(s, 2H), 3.68 (dt, 2H, J ) 6, 5 Hz), 3.48 (s, 3H), 2.67 (t, 2H, J
) 7 Hz), 1.88 (m, 2H), 1.28 (t, 1H, J ) 6 Hz). Anal. (C11H16O3)
C, H.
(()-4-[4-[(Met h oxym et h ylen e)oxy]p h en yl]-2-b u t a n ol
(21). Using conditions similar to those described for the
preparation 45 from 23, 24 was converted into 4-[4-[(meth-
oxymethylene)oxy]phenyl]-2-butanone (46) (2.67 g, 70%), a
golden yellow oil: 1H NMR (CDCl3) δ 7.11 (d, 2H, J ) 9 Hz),
6.96 (d, 2H, J ) 9 Hz), 5.15 (s, 2H), 3.48 (s, 3H), 2.89-2.82
(m, 2H), 2.78-2.71 (m, 2H), 2.14 (s, 3H). Anal. (C12H16O3) C,
H.
20 h the mixture was diluted with ether (200 mL), washed
with 2 N HCl and brine, dried (MgSO4), and evaporated to a
crude residue. Recrystallization from EtOAc/hexanes afforded
4.05 g (63%) of N-(phenylsulfonyl)-DL-serine methyl ester (49)
as a white solid: mp 93-96 °C. Anal. (C10H13NO5S) C, H, N,
S.
To a solution of 49 (1.04 g, 4.01 mmol) in DMF (4 mL) were
added imidazole (0.408 g, 5.99 mmol) and tert-butyldimethyl-
silyl chloride (0.603 g, 4.00 mmol), and the mixture was stirred
at room temperature under a N2 atmosphere. After 16 h the
mixture was diluted with EtOAc (50 mL), washed with water
and brine, dried (MgSO4), and evaporated to give 1.47 g (99%)
of O-(tert-butyldimethylsilyl)-N-(phenylsulfonyl)-DL-serine meth-
yl ester (50) as a white solid: mp 57-59 °C. Anal. (C16H27
NO5SSi) C, H, N, S.
-
Treatment of 50 with LiBH4, using conditions similar to the
general procedure described for the reduction of 11 to 6,
provided 0.70 g (100%) of 28 as a colorless oil, which was
carried on without further purification.
(()-N-[2-[4-(Ben zyloxy)p h en oxy]-1-(h yd r oxym et h yl)-
eth yl]ben zen esu lfon a m id e (26). To a solution of 28 (0.173
g, 0.501 mmol) in THF (2 mL) at 0 °C were added 4-(benzy-
loxy)phenol (0.100 g, 0.499 mmol), Ph3P (0.170 g, 0.648 mmol),
and diethyl azodicarboxylate (102 µL, 0.648 mmol), and the
mixture was stirred at room temperature under a N2 atmo-
sphere for 4 h, after which it was heated to reflux. After 16 h
the mixture was cooled to room temperature and treated with
KOtBu (0.5 mL, 1.0 M in THF, 0.5 mmol). The deep red
mixture was stirred for an additional 24 h at room tempera-
ture, after which it was diluted with EtOAc (20 mL), washed
with 2 N HCl and brine, dried (MgSO4), and evaporated to
give a residue. The crude product was chromatographed
(hexane:EtOAc, 4:1) to give 78.6 mg of partially purified (()-
N-[2-[4-(benzyloxy)phenoxy]-1-[[(tert-butyldimethylsilyl)oxy]-
methyl]ethyl]benzenesulfonamide (51), which was carried on
as is.
The above 78.6 mg of partially purified 51 was dissolved in
THF (1 mL), and the solution was treated with tetrabutylam-
monium fluoride (0.224 mL, 1.0 M in THF, 0.224 mmol). The
mixture was stirred for 3 h at room temperature, after which
it was diluted with 20 mL of EtOAc, washed with water and
brine, dried (MgSO4), and evaporated to a residue. Column
chromatography (DCM:EtOAc, 4:1) afforded 17.8 mg (8.6%
overall) of 26 as a glass, which was used without further
characterization.
(()-a n ti-2-(4-Hyd r oxyben zyl)cyclop en ta n ol (33) a n d
(()-syn -2-(4-Hydr oxyben zyl)cyclopen tan ol (34). To 4-(ben-
zyloxy)benzaldehyde (12.4 g, 58.2 mmol) was added a solution
of KOH (1.45 g, 25.9 mmol) in H2O (25.5 mL), and the
suspension was heated to 65 °C. Cyclopentanone (31) (5.72
mL, 64.7 mmol) was added dropwise over 0.25 h, and the
reaction mixture was brought to reflux. After refluxing for 5
h the reaction mixture was allowed to stir overnight at room
temperature. After acidifying with 1 N HCl (150 mL) and
diluting with EtOAc (700 mL), a suspension was obtained. The
suspension was filtered, and the biphasic filtrate was dis-
carded. The solid was triturated with CHCl3 (500 mL),
filtered, and discarded. The filtrate was washed with brine
(100 mL), dried (MgSO4), and concentrated to provide aldol
adduct 32 (9.38 g, 61%), a yellow solid, which was used as is
in the next step: 1H NMR (CDCl3) δ 7.60-7.32 (m, 8H), 7.02
(d, 2H, J ) 9 Hz), 5.12 (s, 2H), 2.99-2.92 (m, 2H), 2.41 (t, 2H,
J ) 6 Hz), 2.08-1.98 (m, 2H).
To a suspension of the aldol adduct 32 (9.37 g, 33.6 mmol)
in anhydrous 1:1 THF:MeOH (240 mL) under an atmosphere
of N2 at 0 °C was added NaBH4 (1.76 g, 46.5 mmol), and the
reaction mixture was allowed to stir while warming to room
temperature over 4 h. The reaction mixture was filtered, and
the solid was washed with MeOH. The volatiles were removed
from the filtrate, and the resulting residue was purified by
column chromatography (hexane:EtOAc, 5:1-2:1) to provide
an off-white solid of the allylic alcohol (8.21 g, 87%): mp 89-
91 °C; 1H NMR (CDCl3) δ 7.49-7.30 (m, 7H), 6.97 (d, 2H, J )
9 Hz), 6.52 (s, 1H), 5.68 (s, 2H), 4.60 (br s, 1H), 2.80-2.48 (m,
2H), 2.07-1.60 (m, 4H). Anal. (C19H20O2) C, H.
To a solution of 46 (1.25 g, 6.00 mmol) in anhydrous THF
(75 mL) under an atmosphere of N2 at 0 °C was added LiAlH4
(6.60 mL, 1 M in THF, 6.60 mmol) dropwise over 10 min. The
reaction mixture was allowed to stir while warming to room
temperature over 2 h. The reaction was quenched by the
sequential dropwise addition of water (250 µL), 15% NaOH
(250 µL), and water (875 µL). The reaction mixture was
diluted with EtOAc (200 mL), dried (MgSO4), and concentrated
to provide a colorless oil of 21 (1.26 g, 100%): 1H NMR (CDCl3)
δ 7.13 (d, 2H, J ) 9 Hz), 6.97 (d, 2H, J ) 9 Hz), 5.16 (s, 2H),
3.88-3.78 (m, 1H), 3.48 (s, 3H), 2.78-2.57 (m, 2H), 1.79-1.70
(m, 2H), 1.23 (d, 3H, J ) 6 Hz). Anal. (C12H18O3) C, H.
4-[4-[(Meth oxym eth ylen e)oxy]p h en yl]bu ta n ol (22). To
a suspension of NaH (1.65 g, 60% in mineral oil, 41.2 mmol)
in anhydrous DMF (30 mL) under an atmosphere of N2, at 0
°C, was added ethanethiol (3.05 mL, 41.2 mmol) dropwise over
20 min. The reaction mixture was allowed to warm to room
temperature over 1 h. After recooling to 0 °C a solution of
4-(4-methoxyphenyl)butyric acid (25) (2.00 g, 10.3 mmol) in
anhydrous DMF (15 mL) was added dropwise over 15 min.
The reaction mixture was heated at reflux for 7 h. After
cooling to room temperature the reaction was quenched with
2 N HCl (100 mL) and the mixture was diluted with EtOAc
(350 mL). The layers were separated, and the EtOAc layer
was washed with 2 N HCl (100 mL), water (5 × 75 mL), and
1 N NaOH (3 × 75 mL). The combined 1 N NaOH layers were
acidified with 1 N HCl and then extracted with EtOAc (2 ×
125 mL). The latter two EtOAc layers were combined and
washed with water (2 × 50 mL) and brine (50 mL). The EtOAc
layer was dried (MgSO4) and concentrated to provide a white
solid of 4-(4-hydroxyphenyl)butanoic acid (47) (1.69 g, 91%):
1
mp 106-108 °C; H NMR (DMSO-d6) δ 12.02 (br s, 1H), 9.18
(br s, 1H), 6.96 (d, 2H, J ) 8.5 Hz), 6.66 (d, 2H, J ) 8.5 Hz),
2.45 (t, 2H, J ) 7.5 Hz), 2.17 (t, 2H, J ) 7.5 Hz), 1.72 (tt, 2H,
J ) 7.5, 7.5 Hz). Anal. (C10H12O3) C, H.
Using conditions similar to those described for the prepara-
tion of 45 from 23, 47 was converted into a golden yellow oil
ofmethoxymethyleneoxy 4-[4-[(methoxymethylene)oxy]phenyl]-
butanoate (48) (1.67 g, 69%) which was used as is in the next
step: 1H NMR (CDCl3) δ 7.10 (d, 2H, J ) 8.5 Hz), 6.97 (d, 2H,
J ) 8.5 Hz), 5.23 (s, 2H), 5.16 (s, 2H), 3.48 (s, 3H), 3.47 (s,
3H), 2.62 (t, 2H, J ) 7.5 Hz), 2.37 (t, 2H, J ) 7.5 Hz), 1.95 (tt,
2H, J ) 7.5, 7.5 Hz).
Lithium aluminum hydride reduction of 48 using a proce-
dure similar to the one described for the preparation of 21 from
46 afforded a colorless oil of 22 (540 mg, 96%): 1H NMR
(CDCl3) δ 7.10 (d, 2H, J ) 8.5 Hz), 6.96 (d, 2H, J ) 8.5 Hz),
5.16 (s, 2H), 3.65 (t, 2H, J ) 6 Hz), 3.48 (s, 3H), 2.60 (t, 2H, J
) 7 Hz), 1.56-1.74 (m, 4H).
(()-N-[2-[(ter t-Bu tylsilyl)oxy]-1-(h ydr oxym eth yl)eth yl]-
ben zen esu lfon a m id e (28). To a suspension of 27 (3.89 g,
25.0 mmol) in THF (100 mL) at 0 °C was added benzenesulfo-
nyl chloride (3.5 mL, 27 mmol). To this mixture was added
dropwise over 10 min a solution of N,N-diisopropylethylamine
(9.6 mL, 55 mmol) in THF (10 mL), and the mixture was
stirred for 1.5 h at 0 °C and then at room temperature. After

5224 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Defauw et al.
To a solution of the allylic alcohol (4.00 g, 4.30 mmol) in
absolute EtOH (75 mL) under an atmosphere of N2 was added
Raney nickel (1/3 tsp). The reaction mixture was placed under
H2 (55 psi) overnight. The reaction mixture was filtered
through a pad of Celite and concentrated. The residue was
purified by column chromatography (hexane:EtOAc, 4:1-2.5:
1) which provided a white solid of the anti alcohol 33 (1.10 g,
vacuo. The residue was taken up in 1:1 MeOH:EtOH (40 mL),
placed in a Parr bottle, and treated with Raney nickel (1/2 tsp).
The bottle was charged with H2 on a Parr apparatus, shaken
at 45 psi for 1.5 h, and then carefully evacuated of H2. The
solution was filtered through Celite, and the filter cake was
rinsed with methanol (but not allowed to dry). The filtrate
was concentrated in vacuo, and the residue was chromato-
graphed on silica gel (2-propanol/DCM, 5%, 10%, and then
15%) to afford the anti isomer 37 (0.578 g, 69%) as a colorless
glass followed by a minor amount (0.21 g, 25%) of the syn
isomer 38.
(()-a n ti-3-Hydr oxy-4-[4-[(4-m eth oxyben zyl)oxy]ben zyl]-
1-ben zylp yr r olid in e (35). A solution of 37 (0.297 mg, 1.0
mmol) in anhydrous DMF (2.5 mL) under N2 was treated with
4-methoxybenzyl chloride (0.19 g, 1.2 mmol) and K2CO3 (0.166
g, 1.2 mmol), and the mixture was heated to 65 ( 5 °C for 2.5
h. Additional 4-methoxybenzyl chloride (30 mg, 0.2 mmol) was
added, and heating was continued for 4.5 h longer. The
mixture was cooled and added to water (20 mL), whereupon a
precipitate formed. This was collected by filtration, washed
with water, air-dried, dissolved in DCM, dried (Na2SO4), and
concentrated in vacuo. The residual solid was chromato-
graphed on silica gel (2-propanol/DCM, 3%) to afford (()-anti-
4-hydroxy-3-[4-[(4-methoxybenzyl)oxy]benzyl]-1-benzylpyrrolidin-
2-one (54) (0.34 g, 81%) as a white solid.
A cooled (-5 °C) solution of 54 (0.313 g, 0.75 mmol) in
anhydrous THF (4 mL) under N2 was treated dropwise with
1.0 N LiAlH4/THF (1.9 mL, 1.9 mmol) at a rate to keep the
pot temperature below 10 °C, then allowed to warm to room
temperature, and stirred overnight. The mixture was refluxed
for 3 h, cooled (5 °C), and treated dropwise sequentially with
water (0.08 mL), 15% NaOH (0.08 mL), and water (0.24 mL).
The suspension was stirred for 30 min and filtered, and the
filter cake was washed with THF. The filtrate was concen-
trated in vacuo, and the residue was chromatographed on silica
gel (MeOH/DCM, 10%) to afford 35 (0.246 g, 81%) as an off-
white solid.
(()-a n ti-1-[4-(Ben zyloxy)ph en oxy]-2-h ydr oxycyclopen -
ta n e (39). A mixture of 4-(benzyloxy)phenol (0.402 g, 2.01
mmol), cyclopentene oxide (40) (43.6 mL, 5.00 mmol), and DBU
(74.8 mL, 5.00 mmol) was heated to nearly 100 °C under an
atmosphere of N2. After 3 h additional cyclopentene oxide
(43.6 mL, 5.00 mmol) was added, and heating was continued
for 40 h. The crude reaction mixture was chromatographed
twice on silica gel (DCM/hexane/EtOAc, 90/8/2, and then DCM/
EtOAc, 97/3) to give 39 (119 mg, 42%) as a white solid: mp
76-79 °C. Anal. (C18H20O3) C, H.
Meth od A. (S)-Ben zyl 2-[[4-[[[3-[4-[(Ben zyloxyca r bo-
n yl)oxy]p h en yl]-2-[(p h en ylsu lfon yl)a m in o]p r op yl]oxy]-
ca r bon yl]-2,6-bis(ben zyloxy)p h en yl]ca r bon yl]-3-(ben zy-
loxy)b en zoa t e (55) a n d (R)-Ben zyl 2-[[4-[[[3-[4-[(Ben -
zyloxyca r bon yl)oxy]p h en yl]-2-[(p h en ylsu lfon yl)a m in o]-
pr opyl]oxy]car bon yl]-2,6-bis(ben zyloxy)ph en yl]car bon yl]-
3-(ben zyloxy)ben zoa te (56). To a solution of (S)-12 (197 mg,
0.334 mmol), Et3N (140 µL, 1.00 mmol), and DMAP (5.3 mg,
43.5 µmol) in DCM (5 mL) under an atmosphere of N2 was
added a solution of freshly prepared 2a (201 mg, 0.290 mmol)
in DCM (7 mL) at 0 °C. The ice bath was removed, and the
reaction mixture was allowed to stir overnight. The reaction
mixture was diluted with EtOAc (75 mL) and washed with
0.5 N HCl (2 × 20 mL) and brine (25 mL). The EtOAc layer
was dried (MgSO4) and concentrated. The crude residue was
purified by column chromatography (DCM-2% acetone/DCM)
to provide 55 (259 mg, 81%): mp 61-64 °C; [R]D ) -1.40°
(0.43, CHCl3);11 1H NMR (CDCl3) δ 7.67 (d, 2H, J ) 8 Hz),
7.50-7.02 (m, 34H), 6.96 (dd, 1H, J ) 8, 2 Hz), 6.90 (d, 2H, J
) 7 Hz), 5.28 (s, 2H), 5.16 (s, 2H), 4.81 (s, 4H), 4.75 (s, 2H),
4.32-4.10 (m, 2H), 3.89-3.78 (m, 1H), 2.81 (d, 2H, J ) 7 Hz).
Anal. (C66H55NO13S) C, H, N, S.
1
40%): mp 104-106 °C; H NMR (CDCl3) δ 7.10 (d, 2H, J ) 9
Hz), 6.76 (d, 2H, J ) 9 Hz), 4.10 (ddd, 1H, J ) 6, 4, 1 Hz),
2.78 (dd, 1H, J ) 8, 15 Hz), 2.63 (dd, 1H, J ) 7.5, 15 Hz),
2.05-1.42 (m, 7H). Anal. (C12H16O2) C, H.
Further elution provided a white solid of the syn alcohol 34
1
(1.46 g, 53%): mp 79-81 °C; H NMR (CDCl3) δ 7.05 (d, 2H,
J ) 8.5 Hz), 6.72 (d, 2H, J ) 8.5 Hz), 3.93 (ddd, 1H, J ) 9, 6,
4 Hz), 2.67-2.49 (m, 2H), 2.08-1.51 (m, 7H). Anal. (C12H16O2)
C, H.
(()-a n t i-2-[4-[(Be n zyloxyca r b on yl)oxy]b e n zyl]cyc-
lop en ta n ol (29). Treatment of 33 under the same conditions
described for the preparation of (S)-12 from (S)-14 provided a
colorless oil of 29 (1.15 g, 99%): 1H NMR (CDCl3) δ 7.49-7.35
(m, 5H), 7.19 (d, 2H, J ) 9 Hz), 7.09 (d, 2H, J ) 9 Hz), 5.28 (s,
2H), 4.14-4.05 (m, 1H), 2.86 (dd, 1H, J ) 8, 15 Hz), 2.68 (dd,
1H, J ) 8, 15 Hz), 2.05-1.43 (m, 7H). Anal. (C20H22O4) C, H.
(()-syn -2-[4-[(Be n zyloxyc a r b on yl)oxy]b e n zyl]c yc -
lop en ta n ol (30). Treatment of 34 under the same conditions
described for the preparation of (S)-12 from (S)-14 provided a
colorless oil of 30 (1.28 g, 100%): 1H NMR (CDCl3) δ 7.49-
7.35 (m, 5H), 7.20 (d, 2H, J ) 9 Hz), 7.10 (d, 2H, J ) 9 Hz),
5.27 (s, 2H), 3.90 (ddd, 1H, J ) 6, 6, 6 Hz), 2.80 (dd, 1H, J )
6.5, 15 Hz), 2.50 (dd, 1H, J ) 9, 15 Hz), 2.10-1.52 (m, 7H).
Anal. (C20H22O4) C, H.
1-(P h en ylm eth yl)p yr r olid in e-2,4-d ion e (36). A solution
of solid KOtBu (10.0 g, 89 mmol) in anhydrous THF (165 mL)
under N2 was cooled on an ice bath and treated in portions
with 4-methoxy-3-pyrrolin-2-one (9.04 g, 80 mmol) so that the
pot temperature remained below 10 °C. After the addition,
the mixture was stirred at 5 °C for 15 min and then treated
dropwise with a solution of benzyl bromide (10.7 mL, 15.4 g,
90 mmol) in anhydrous THF (15 mL). The mixture was heated
to 50 °C, stirred for 2 h, then cooled (10 °C), treated with
saturated aqueous NaHCO3 (35 mL), and filtered through
Celite. The filtrate was separated, and the lower aqueous
layer was discarded. The organic solution was dried (MgSO4)
and concentrated in vacuo. Column chromatography (DCM:
acetone, 2:1) provided 1-benzyl-4-methoxy-3-pyrrolin-2-one (52)
(11.1 g, 68%) as a tan solid.
A solution of 52 (4.06 g, 20 mmol) in DCM (40 mL) was
treated with 50% aqueous H2SO4 (16 g), and the biphasic
mixture was stirred for 15 h. Additional DCM (60 mL) was
added, and the organic layer was separated. The acidic
aqueous layer was extracted with DCM (30 mL), and the
combined organic solution was washed with water (2 × 30 mL,
each back-washed with DCM), dried (Na2SO4), and concen-
trated in vacuo to afford 36 (3.67 g, 97%) as a viscous oil
containing 8% unhydrolyzed enol ether.
(()-a n ti-4-Hyd r oxy-3-(4-h yd r oxyben zyl)-1-ben zylp yr -
r olid in -2-on e (37). A cooled (-60 °C) solution of 36 (0.378
g, 2.0 mmol) and 4-(benzyloxy)benzaldehyde (0.467 g, 2.2
mmol) in anhydrous THF (4 mL) under N2 was treated
dropwise with lithium bis(trimethylsilyl)amide (2.0 mL, 1 M
in THF, 2.0 mmol) so as to keep the pot temperature below
-50 °C; then the mixture was allowed to warm to 35 °C over
30 min and was stirred at room temperature for 3 h. The
solution was plunged into a stirred mixture of saturated
aqueous NaHCO3 (10 mL) and DCM (15 mL) and then stirred
for 5 min, during which time the organic layer became bright
yellow and was separated. The aqueous layer was extracted
with DCM (15 mL), and the combined organic layers were
dried (Na2SO4) and concentrated in vacuo. The residue was
chromatographed (acetone/DCM, 1-2%) to afford 3-[4-(ben-
zyloxy)benzyl]-1-benzylpyrrolidine-2,4-dione (53) (0.35 g, 46%)
as a bright yellow solid: mp 135-137 °C.
Compound 56 was obtained from (R)-12 using the same
procedure: [R]D ) 1.10° (0.42, CHCl3).11
A cooled (5 °C) suspension of 53 (1.07 g, 2.8 mmol) in 2:1
DCM:methanol (15 mL) was treated with NaBH4 (granular,
0.14 g, 3.7 mmol), and the mixture was warmed to room
temperature, stirred for 15 min, and then concentrated in
Gen er a l P r oced u r e for th e P a lla d iu m (II) Hyd r oxid e
on Ca r bon -Ca ta lyzed Deben zyla tion : (S)-2-[[2,6-Dih y-
d r oxy-4-[[[3-(4-h yd r oxyp h en yl)-2-[(p h en ylsu lfon yl)a m i-
n o]p r op yl]oxy]ca r b on yl]p h en yl]ca r b on yl]-3-h yd r oxy-

PKC Inhibition by Balanol Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5225
ben zoic Acid [(S)-12b] a n d (R)-2-[[2,6-Dih yd r oxy-4-[[[3-
( 4 -h y d r o x y p h e n y l ) -2 -[ ( p h e n y l s u l f o n y l ) a m i n o ] -
p r op yl]oxy]ca r b on yl]p h en yl]ca r b on yl]-3-h yd r oxyb en -
zoic Acid [(R)-12b]. To a solution of 55 (252 mg, 0.229 mmol)
and TFA (53 µL) in 1:1 EtOAc:EtOH (20 mL) under an
atmosphere of N2 was added Pearlman’s catalyst (63 mg, 20%
by wt on C, 25% by wt), and the reaction mixture was placed
under 1 atmosphere of H2. The reaction mixture was allowed
to stir overnight. Purification by HPLC provided (S)-12b (120
mg, 83%): mp 148-150 °C; [R]D ) 1.02° (0.59, EtOH);11 1H
NMR (CD3OD) δ 7.66 (d, 2H, J ) 8 Hz), 7.52 (d, 1H, J ) 8
Hz), 7.44 (dd, 1H, J ) 8, 8 Hz), 7.37-7.25 (m, 3H), 7.04 (d,
1H, J ) 8 Hz), 6.93 (d, 2H, J ) 9 Hz), 6.78 (s, 2H), 6.62 (d,
2H, J ) 8 Hz), 4.20 (dd, 1H, J ) 4, 11 Hz), 4.07 (d, 1H, J ) 7,
11 Hz), 3.77-3.68 (m, 1H), 2.83-2.66 (m, 2H); HRMS m/ z
608.1247 [calcd for C30H25NO11S (M + 1) 608.1227]. Anal.
(C30H25NO11S‚1.5H2O) C, H, N, S.
7.9, 8.0 Hz), 6.91 (d, 2H, J ) 8.4 Hz), 6.82 (d, 1H, J ) 7.2 Hz),
6.77 (s, 2H), 6.54 (d, 2H, J ) 8.4 Hz), 4.11 (dd, 1H, J ) 11.2,
4.8 Hz), 3.99 (dd, 1H, J ) 11.3, 6.1 Hz), 3.70-3.60 (m, 1H),
2.70 (dd, 1H, J ) 13.9, 5.6 Hz), 2.48 (dd, 1H, J ) 13.8, 9.0
Hz), 2.26 (s, 3H); HRMS m/ z 546.1060 [calcd for C25H24NO11
S
(M + 1) 546.1070]. Anal. (C25H23NO11S‚0.4TFA‚0.5H2O) C,
H, N, S.
(()-2-[[2,6-Dih yd r oxy-4-[[[3-(4-h yd r oxyp h en yl)-2-[(p h e-
n y lc a r b o n y l)a m i n o ]p r o p y l]o x y ]c a r b o n y l]p h e n y l]-
ca r bon yl]-3-h yd r oxyben zoic a cid (8b): mp 140-148 °C dec;
1H NMR (CD3OD) δ 8.44 (d, NH), 7.68 (d, 2H), 7.45 (m, 4H),
7.26 (t, 1H), 7.12 (d, 2H), 7.06 (d, 1H), 6.92 (s, 2H), 6.71 (d,
2H), 4.61 (m, 1H), 4.44 (dd, 1H), 4.30 (dd, 1H), 2.91 (m, 2H);
HRMS m/ z 572.1551 [calcd for C31H26NO10 (M + 1) 572.1557].
Anal. (C31H25NO10‚0.3TFA‚H2O‚0.1CH3CN) C, H, N.
(()-2-[[2,6-Dih yd r oxy-4-[[2-(4-h yd r oxyp h en yl)-2-[(p h e-
n ylsu lfon yl)a m in o]eth oxy]ca r bon yl]p h en yl]ca r bon yl]-3-
1
(R)-12b was obtained from 56 using the same procedure (94
mg, 74%): mp 148-150 °C; [R]D ) -2.86° (0.70, EtOH);11
HRMS m/ z 608.1253 [calcd for C30H25NO11S (M + 1) 608.1227].
Anal. (C30H25NO11S‚2H2O) C, H, N, S.
h yd r oxyben zoic a cid (13b): mp 144-146 °C dec; H NMR
(CD3OD) δ 7.66 (d, 2H, J ) 7 Hz), 7.52 (d, 1H, J ) 8 Hz), 7.42-
7.24 (m, 4H), 7.12-7.03 (m, 3H), 6.72 (s, 2H), 6.67 (d, 2H, J )
9 Hz), 4.68-4.61 (m, 1H), 4.37-4.21 (m, 2H); MS m/ z 594 (M
+ H). Anal. (C29H23NO11S‚0.75H2O) C, H, N, S.
(S)-Meth yl 2-[[2,6-Dih yd r oxy-4-[[[3-(4-h yd r oxyp h en yl)-
2-[(p h en ylsu lfon yl)a m in o]p r op yl]oxy]ca r bon yl]p h en yl]-
ca r bon yl]-3-h yd r oxyben zoa te [(S)-12c]: mp 123-125 °C;
[R]D ) -3.95° (0.38, EtOH);11 1H NMR (CD3OD) δ 7.68 (d, 2H,
J ) 8 Hz), 7.51 (d, 1H, J ) 7.5 Hz), 7.45 (d, 1H, J ) 7.5 Hz),
7.38-7.28 (m, 3H), 7.07 (d, 1H, J ) 8 Hz), 6.94 (d, 2H, J ) 8.5
Hz), 6.82 (s, 2H), 6.63 (d, 2H, J ) 8.5 Hz), 4.22 (dd, 1H, J )
11, 4.5 Hz), 4.10 (dd, 1H, J ) 11, 6.5 Hz), 3.81-3.69 (m, 4H),
(()-2-[[2,6-Dih yd r oxy-4-[[[2-(N,N-d im eth yla m in o)-3-(4-
h yd r oxyp h en yl)p r op yl]oxy]ca r bon yl]p h en yl]ca r bon yl]-
3-h yd r oxyben zoic a cid , tr iflu or oa cetic a cid sa lt (15b):
mp 124-127 °C dec; 1H NMR (CD3OD) δ 7.49 (d, 1H, J ) 8.7
Hz), 7.28 (t, 1H, J ) 8.7 Hz), 7.12 (d, 2H, J ) 8.6 Hz), 7.03 (d,
1H, J ) 8.7 Hz), 6.97 (s, 2H), 6.76 (d, 2H, J ) 8.6,), 4.52 (dd,
1H, J ) 13.7, 2.4 Hz), 4.39 (dd, 1H, J ) 13.7, 5.8 Hz), 3.94 (m,
1H), 3.21 (m, 1H), 2.98 (m, 1H), 3.03 (s, 6H); MS m/ z 496 (M
+ 1). Anal. (C26H25NO9‚1.6TFA‚1.0H2O) C, H, N.
2.84-2.66 (m, 2H); MS m/ z 622 (M + 1). Anal. (C31H27
NO11S‚0.2TFA‚0.8H2O) C, H, N, S.
-
Meth od B. (()-Ben zyl 2-[[2,6-Bis(ben zyloxy)-4-[[[2-[N-
(m e t h oxym e t h yle n e )-N -(p h e n ylsu lfon yl)a m in o]-3-[4-
[(m eth oxym eth ylen e)oxy]p h en yl]p r op yl]oxy]ca r bon yl]-
p h en yl]ca r bon yl]-3-(ben zyloxy)ben zoa te (57). A solution
of 2b (370 mg, 0.545 mmol) in THF (6 mL) was treated with
1,1′-carbonyldiimidazole (97 mg, 0.600 mmol) and stirred for
6 h at room temperature. A mixture of 6 (313 mg, 0.818 mmol)
and DBU (90 µL, 0.600 mmol) in THF (6 mL) was then added
to the reaction mixture. After 72 h, the mixture was poured
into EtOAc (50 mL) and washed with water (50 mL) followed
by brine (25 mL). The organic layer was dried (MgSO4) and
concentrated. The crude material was purified by chroma-
tography (EtOAc/hexanes, 25-40%) and then purified further
on a chromatotron (acetone/DCM, 1%) to afford 295 mg of 57
as a clear glass (51%): 1H NMR (CDCl3) δ 7.72 (d, 2H, J ) 7
Hz), 7.43 (d, 1H, J ) 7.5 Hz), 7.36 (d, 2H, J ) 8 Hz), 7.28-
7.18 (m, 16H), 7.15 (d, 1H, J ) 6 Hz), 7.12-7.08 (m, 6H), 6.96
(d, 1H, J ) 2 Hz), 6.92 (d, 4H, J ) 3.5 Hz), 6.88 (d, 1H, J )
1.5 Hz), 5.15 (s, 2H), 5.14 (s, 2H), 4.90 (s, 2H), 4.83 (s, 4H),
4.75 (s, 2H), 4.43-4.24 (m, 3H), 3.48 (s, 3H), 3.35 (s, 3H), 2.88-
2.70 (m, 2H). Anal. (C62H57NO13S) C, H, N, S.
(()-2-[[2,6-Dih ydr oxy-4-[[[3-(4-h ydr oxyph en yl)-2-[(n aph -
t h y ls u lfo n y l)a m in o ]p r o p y l]o x y ]c a r b o n y l]p h e n y l]-
ca r bon yl]-3-h yd r oxyben zoic a cid (16b): MS m/ z 658 (M
+ 1).
(()-2-[[2,6-Dih yd r oxy-4-[[[3-(4-h yd r oxyp h en yl)-2-[[(4-
h yd r oxyp h en yl)ca r bon yl]a m in o]p r op yl]oxy]ca r bon yl]-
p h en yl]ca r bon yl]-3-h yd r oxyben zoic a cid (17b): mp 133-
137 °C dec; 1H NMR (CD3OD) δ 7.60 (d, 2H, J ) 8.7 Hz), 7.48
(d, 1H, J ) 7.9 Hz), 7.26 (t, 1H, J ) 7.7 Hz), 7.09 (d, 2H, J )
8.4 Hz), 7.00 (d, 1H, J ) 7.9 Hz), 6.91 (s, 2H), 6.78 (d, 2H, J
) 8.7 Hz), 6.69 (d, 2H, J ) 8.4 Hz), 4.58 (m, 1H), 4.39 (dd, 1H,
J ) 13.9, 4.4 Hz), 4.28 (dd, 1H, J ) 13.9, 7.3 Hz), 3.00-2.80
(m, 2H); MS m/ z 588 (M
0.8TFA‚1.0CH3CN) C, H, N.
+ 1). Anal. (C31H25NO11‚
2-[[2,6-Dih ydr oxy-4-[[[3-(4-h ydr oxyph en yl)pr opyl]oxy]-
ca r bon yl]p h en yl]ca r bon yl]-3-h yd r oxyben zoic a cid (20b):
1
mp 124-127 °C; H NMR (CD3OD) δ 7.51 (d, 1H, J ) 8 Hz),
7.28 (dd, 1H, J ) 8, 8 Hz), 7.07-7.01 (m, 3H), 6.93 (s, 2H),
6.71 (d, 2H, J ) 8 Hz), 4.26 (t, 2H, J ) 7 Hz), 2.68 (t, 2H, J )
7 Hz), 2.02 (tt, 2H, J ) 7, 7 Hz); MS m/ z 453 (M + H). Anal.
(C24H20O9‚0.5H2O) C, H.
(()-Ben zyl 2-[[2,6-Bis(ben zyloxy)-4-[[[3-(4-h yd r oxyp h e-
n yl)-2-[(p h e n ylsu lfon yl)a m in o]p r op yl]oxy]ca r b on yl]-
p h en yl]ca r bon yl]-3-(ben zyloxy)ben zoa te (58). Compound
57 (280 mg, 0.265 mmol) was dissolved in ∼0.5 N HCl/dioxane
(20 mL), and 2 drops of concentrated HCl was added. After
24 h, the mixture was concentrated to an oil and dried under
high vacuum to give 255 mg (100%) of 58.
(()-2-[[2,6-Dih yd r oxy-4-[[[3-(4-h yd r oxyp h en yl)-1-m eth -
ylp r op yl]oxy]ca r bon yl]p h en yl]ca r bon yl]-3-h yd r oxyben -
zoic a cid (21b): mp 130-133 °C; 1H NMR (CD3OD) δ 7.51
(d, 1H, J ) 8 Hz), 7.28 (dd, 1H, J ) 8, 8 Hz), 7.03 (d, 1H, J )
8 Hz), 6.99 (d, 2H, J ) 8.5 Hz), 6.91 (s, 2H), 6.68 (d, 2H, J )
8.5 Hz), 5.10-5.00 (m, 1H), 2.71-2.51 (m, 2H), 2.08-1.81 (m,
2H), 1.33 (d, 3H, J ) 6 Hz); MS m/ z 467 (M + H). Anal.
(C25H22O9‚0.5H2O) C, H.
(()-Meth yl 2-[[2,6-d ih yd r oxy-4-[[[3-(4-h yd r oxyp h en yl)-
1-m eth ylpr opyl]oxy]car bon yl]ph en yl]car bon yl]-3-h ydr ox-
yben zoa te (21c): mp 92-95 °C dec; 1H NMR (CD3OD) δ 7.48
(dd, 1H, J ) 8, 1 Hz), 7.29 (dd, 1H, J ) 8, 8 Hz), 7.05 (dd, 1H,
J ) 8, 1 Hz), 6.99 (d, 2H, J ) 8.5 Hz), 6.92 (s, 2H), 6.68 (d,
2H, J ) 8.5 Hz), 5.10-4.98 (m, 1H), 3.72 (s, 3H), 2.71-2.53
(m, 2H), 2.08-1.81 (m, 2H), 1.34 (d, 3H, J ) 6 Hz); MS m/ z
481 (M + H). Anal. (C26H24O9‚0.5H2O) C, H.
(()-2-[[2,6-Dih yd r oxy-4-[[[3-(4-h yd r oxyp h en yl)-2-[(p h e-
n y ls u lfo n y l)a m i n o ]p r o p y l]o x y ]c a r b o n y l]p h e n y l]-
ca r bon yl]-3-h yd r oxyben zoic Acid (6b). To a suspension
of 58 (250 mg, 0.258 mmol) in 15:1 MeOH:EtOAc (32 mL) was
added Pearlman’s catalyst (50 mg, 20% by wt). The suspension
was stirred under a H2 atmosphere at ambient conditions for
20 h. The catalyst was filtered through Celite, and the filtrate
was concentrated. The crude material was purified by reverse
phase HPLC (0-100% B over 1 h; A ) 5% CH3CN in water
with 0.1% TFA, B ) CH3CN) to give 60 mg of 6b as a light
1
yellow powder (38%): mp 143-145 °C; H NMR see (S)-12b;
2-[[2,6-Dih yd r oxy-4-[[[4-(4-h yd r oxyp h en yl)bu tyl]oxy]-
ca r bon yl]p h en yl]ca r bon yl]-3-h yd r oxyben zoic a cid (22b):
MS m/ z 608 (M + 1). Anal. (C30H25NO11S‚0.2TFA‚0.8H2O)
C, H, N, S.
1
mp 124-127 °C; H NMR (CD3OD) δ 7.50 (d, 1H, J ) 8 Hz),
(()-2-[[2,6-Dih yd r oxy-4-[[[3-(4-h yd r oxyp h en yl)-2-[(m e-
t h y ls u lfo n y l)a m in o ]p r o p y l]o x y ]c a r b o n y l]p h e n y l]-
ca r bon yl]-3-h yd r oxyben zoic a cid (7b): mp 92-100 °C dec;
1H NMR (CD3OD) δ 7.29 (d, 1H, J ) 8.7 Hz), 7.07 (dd, 1H J )
7.27 (dd, 1H, J ) 8 Hz), 7.03 (d, 1H, J ) 8 Hz), 7.01 (d, 2H, J
) 8.5 Hz), 6.89 (s, 2H), 6.69 (d, 2H, J ) 8.5 Hz), 4.29 (t, 2H, J
) 6 Hz), 2.59 (t, 2H, J ) 7 Hz), 1.81-1.66 (m, 4H); MS m/ z
467 (M + H). Anal. (C25H22O9‚0.5H2O) C, H.

5226 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26
Defauw et al.
(()-2-[[2,6-Dih ydr oxy-4-[[[3-(4-h ydr oxyph en oxy)-2-[(ph e-
n y ls u lfo n y l)a m i n o ]p r o p y l]o x y ]c a r b o n y l]p h e n y l]-
ca r bon yl]-3-h yd r oxyben zoic a cid (26b): mp 114-135 °C;
1H NMR (CD3OD) δ 7.80 (d, 2H, J ) 7 Hz), 7.52 (dd, 1H, J )
1, 9 Hz), 7.48-7.34 (m, 3H), 7.29 (t, 1H, J ) 10 Hz), 7.04 (dd,
1H, J ) 1, 8 Hz), 6.71 (s, 2H), 6.70-6.65 (m, 4H), 4.43 (dd,
1H, J ) 4, 12 Hz), 4.32 (dd, 1H, J ) 7, 12 Hz), 4.04-3.98 (m,
7.09 (d, 1H, J ) 8 Hz), 6.98 (d, 2H, J ) 8 Hz), 6.97 (s, 2H),
6.66 (d, 2H, J ) 8 Hz), 5.27 (br s, 1H), 3.30-3.60 (m, 3H),
3.10 (m, 1H), 2.70 (m, 3H); MS m/ z 494 (M + H). Anal.
(C26H23NO9‚1.3TFA‚H2O) C, H, N.
(()-a n ti-3-[[3,5-Dih yd r oxy-4-[2-h yd r oxy-6-(m et h oxy-
ca r bon yl)ben zoyl]-ben zoyl]oxy]-4-(4-h yd r oxyben zyl)p yr -
r olid in e, Tr iflu or oa cetic Acid Sa lt (35c). A cooled (-20
°C) solution of 35b (63 mg, 0.095 mmol) in absolute MeOH
(1.5 mL) was treated dropwise with thionyl chloride (0.15 mL,
2.0 mmol), placed under a drying tube, then warmed to 50 °C
over 30 min, and stirred at 50 °C for 2 h. The solution was
concentrated in vacuo, and the residue was dissolved in DMF
(0.5 mL) and purified by C18 HPLC (0-100% B over 1 h; A )
5% CH3CN/H2O + 0.5% TFA, B ) CH3CN) to afford 35c (0.034
g, 52%) as a voluminous orange solid: mp 144-147 °C; Rf (75:
1H), 3.95-3.89 (m, 2H); MS m/ z 623 (M + H). Anal. (C30H25
NO12S‚0.1TFA‚1.2H2O) C, H, N, S.
-
(()-a n ti-2-[[2,6-Dih yd r oxy-4-[[[2-(4-h yd r oxyben zyl)cy-
clopen tyl]oxy]car bon yl]ph en yl]car bon yl]-3-h ydr oxyben -
zoic a cid (29b): mp 141-144 °C; 1H NMR (CD3OD) δ 7.51
(d, 1H, J ) 8 Hz), 7.28 (dd, 1H, J ) 8, 8 Hz), 7.04 (d, 1H, J )
8 Hz), 6.95 (s, 2H), 6.94 (d, 2H, J ) 8.5 Hz), 6.64 (d, 2H, J )
8.5 Hz), 5.15 (dd, 1H, J ) 4, 4 Hz), 2.76 (dd, 1H, J ) 8, 15
Hz), 2.62 (dd, 1H, J ) 8, 15 Hz), 2.30-2.22 (m, 1H), 2.06-
1.56 (m, 6H); MS m/ z 493 (M + H). Anal. (C27H24O9‚1.25H2O)
C, H.
(()-a n ti-Met h yl 2-[[2,6-d ih yd r oxy-4-[[[2-(4-h yd r oxy-
b en zyl)cyclop en t yl]oxy]ca r b on yl]p h en yl]ca r b on yl]-3-
h yd r oxyben zoa te (29c): mp 111-114 °C; 1H NMR (CD3OD)
δ 7.48 (dd, 1H, J ) 8, 1 Hz), 7.29 (dd, 1H, J ) 8, 8 Hz), 7.05
(dd, 1H, J ) 8, 1 Hz), 6.96 (s, 2H), 6.95 (d, 2H, J ) 8.5 Hz),
6.65 (d, 2H, J ) 8.5 Hz), 5.15 (dd, 1H, J ) 4, 4 Hz), 3.72 (s,
3H), 2.77 (dd, 1H, J ) 8, 14 Hz), 2.63 (dd, 1H, J ) 8, 14 Hz),
2.31-2.28 (m, 1H), 2.06-1.57 (m, 6H); MS m/z 507 (M + H).
Anal. (C28H26O9‚1.25H2O) C, H.
1
24:1 DCM:MeOH:NH3) 0.50; H NMR (DMSO-d6) δ 11.82 (s,
2H), 10.14 (s, 1H), 9.34 (s, 1H), 9.18 (br s, 1H), 9.00 (br s, 1H),
7.44 (d, 1H, J ) 8 Hz), 7.35 (t, 1H, J ) 8 Hz), 7.17 (d, 1H, J )
8 Hz), 7.05 (s, 2H), 6.99 (d, 2H, J ) 8 Hz), 6.71 (d, 2H, J ) 8
Hz), 5.30 (br s, 1H), 3.69 (s, 3H), 3.30-3.60 (m, 3H), 3.05-
3.20 (m, 1H), 2.70 (m, 3H); MS m/ z 508 (M + H). Anal.
(C27H25NO9‚1.4TFA‚H2O) C, H, N.
(()-a n ti-2-[[2,6-Dih yd r oxy-4-[[[2-[(4-h yd r oxyp h en yl)-
oxy]cyclop en t yl]oxyc]a r b on yl]p h en yl]ca r b on yl]-3-h y-
d r oxyben zoic a cid (39b): mp 114-138 °C; MS m/ z 494 (M
+ H). Anal. (C26H22O10‚0.75 H2O‚CH3CN) C, H, N.
P r otein Kin a se C Exp r ession a n d P u r ifica tion . The
R, âI, âII, γ, δ, ꢀ, and η recombinant human PKC enzymes were
produced using a baculovirus expression system in SF9 cells.20
The Ca2+ independent isozymes (δ, ꢀ, and η) were purified as
described in the literature by Bronson et al.21 The Ca2+
dependent isozymes (R, âI, âII, and γ) were purified using a
modification of a method described by Kochs et al.22 After the
Ca2+ dependent isozyme was released by EGTA treatment, it
was purified on a Poros Q (Perspective Biosystems) anion
exchange column using 0-500 mM NaCl. Each fraction was
assayed for PKC activity, and the peak activity for each
recombinant PKC was pooled and used in these studies.
Purities range from 50% to 90% depending on isozyme subtype.
P r otein Kin a se C Assa y. PKC was assayed by quantitat-
ing the incorporation of 32P from [γ-32P]ATP into histone type
IIIS. The reaction mixture (250 µL) contained 30 mg of
phosphatidylserine (Avanti), 20 mM Hepes buffer (pH 7.5;
Sigma), 10 mM MgCl2, 47.5 µM EGTA, 100 mM CaCl2, 200
µg/mL histone (Sigma), 10 µL of DMSO or 10 µL of a solution
of inhibitor in DMSO, 30 µM [γ-32P]ATP (DuPont), the enzyme,
and diacylgylcerol. The amount of diacylglycerol necessary for
50% maximal activation of the enzyme was used. This amount
was isozyme dependent. The assay was incubated for 10 min
at 30 °C and terminated with 500 µL of 25% trichloroacetic
acid and 100 µL of bovine serum albumin (1 mg/mL; Sigma).
The reaction mixtures were filtered onto glass fiber filters and
quantified by counting in a â scintillation counter. The
reported IC50s are single determinations. Most IC50s were
determined by testing the compounds at 0.05, 0.5, 5, and 50
µM and then at 0.5, 5, 50, and 500 nM, respectively. The more
pertinent of the two values was reported. Assay controls
included a maximal lipid-activated PKC assay and a no-lipid
PKC assay. The no-lipid activity was subtracted from the
maximal lipid dependent activity to account for background
nonspecific kinase activities. The PKC inhibitor sphingosine,
which inhibits all the PKC isozymes, was included as a control
inhibitor for all of the PKC assays.23
(()-syn -2-[[2,6-Dih yd r oxy-4-[[[2-(4-h yd r oxyben zyl)cy-
clopen tyl]oxy]car bon yl]ph en yl]car bon yl]-3-h ydr oxyben -
zoic a cid (30b): mp 129-132 °C; 1H NMR (CD3OD) δ 7.50
(d, 1H, J ) 8 Hz), 7.27 (dd, 1H, J ) 8, 8 Hz), 7.02 (d, 1H, J )
8 Hz), 7.00 (d, 2H, J ) 8.5 Hz), 6.83 (s, 2H), 6.67 (d, 2H, J )
8.5 Hz), 5.05-4.95 (m, 1H), 2.75 (dd, 1H, J ) 6, 14 Hz), 2.49
(dd, 1H, J ) 9, 14 Hz), 2.41-2.38 (m, 1H), 2.18-2.02 (m, 1H),
1.93-1.68 (m, 4H), 1.45-1.34 (m, 1H); MS m/ z 493 (M + H)+;
analytical HPLC eluted in 24.3 min (area ) 100%) on C18
using 0-100% B over 45 min with A ) 5% CH3CN + 0.1%
TFA in water and B ) CH3CN. Anal. (C27H24O9‚1H2O‚0.1DMF)
C, H, N.
Meth od C. (()-a n ti-3-[[3,5-Bis(ben zyloxy)-4-[6-(ben -
zyloxyca r b on yl)-2-(b en zyloxy)b en zoyl]b en zoyl]oxy]-4-
[4-[(4-m et h oxyb en zyl)oxy]b en zyl]-1-b en zylp yr r olid in e
(59). A cooled (-20 °C) solution of 35 (0.34 g, 0.84 mmol) in
anhydrous THF (5 mL) under N2 was treated with nBuLi/
hexane (0.35 mL, 2.45 N, 0.86 mmol); then the mixture was
stirred at room temperature for 10 min, concentrated in vacuo,
and placed under high vacuum. Meanwhile, a solution of 2b
(0.62 g, 0.90 mmol) in anhydrous THF (3 mL) under N2 was
treated with 1,1′-carbonyldiimidazole (0.17 g, 1.05 mmol) and
then stirred for 2 h at room temperature after gentle warming
with a heat gun. This acylimidazole solution was transferred
into the flask containing the above alcoholate under N2, and
the mixture was stirred at room temperature for 16 h and at
55 °C for 1 h. The solution was concentrated in vacuo, and
the residue was chromatographed (EtOAc:hexane, 1:1) to
afford 59 (0.66 g, 74%) as a white foam.
(()-a n ti-3-[[4-(6-Ca r b oxy-2-h yd r oxyb en zoyl)-3,5-h y-
d r oxyben zoyl]oxy]-4-(4-h yd r oxyben zyl)p yr r olid in e, Tr i-
flu or oa cetic Acid Sa lt (35b). A solution of 59 (0.70 g, 0.658
mmol) in 3:1 EtOH:EtOAc (40 mL) in a 500 mL Parr bottle
was purged with N2. Trifluoroacetic acid (0.15 mL) was added
followed by 20% Pd(OH)2/C (0.2 g), and the vessel was
promptly charged without delay with H2 (45 psi) on a Parr
apparatus and shaken for 18 h. The bottle was carefully
evacuated of H2, the solution was filtered through Celite, and
then the filter cake was washed with EtOH but n ot allowed
to dry. The filtrate was concentrated in vacuo to a yellow
foam, dissolved in TFA (12 mL), and then heated to 55 °C for
2 h under N2 in order to remove the 4-methoxybenzyl protect-
ing group. The solution was concentrated in vacuo and the
residue purified by C18 HPLC (0-100% B over 1 h; A ) 5%
CH3CN/H2O + 0.5% TFA, B ) CH3CN) to afford 35b (0.235 g,
54%) as a voluminous yellow solid: mp 186-191 °C; Rf (6:1:1
cAMP Dep en d en t P r otein Kin a se Assa y. The assay
components were in a total volume of 250 µL: 20 mM Hepes
buffer (pH 7.5; Sigma), 200 µg/mL histone type HL (Worthing-
ton), 10 mM MgCl2 (Sigma), 10 µL of DMSO or 10 µL of a
solution of inhibitor in DMSO, and 30 µM [γ-32P]ATP (DuPont).
The reaction was initiated by the addition of bovine heart
cAMP dependent kinase catalytic subunit (Sigma), incubated
at 30 °C for 10 min, and stopped by adding 0.5 mL of ice cold
trichloroacetic 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
cell harvester and quantified by counting in a â scintillation
counter.
1
nBuOH:AcOH:H2O) 0.50; H NMR (DMSO-d6) δ 12.95 (br s,
1H), 11.79 (s, 2H), 9.96 (s, 1H), 9.28 (s, 1H), 9.15 (br s, 1H),
9.00 (br s, 1H), 7.40 (d, 1H, J ) 8 Hz), 7.30 (t, 1H, J ) 8 Hz),

PKC Inhibition by Balanol Analogs
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 26 5227
(8) Koide, K.; Bunnage, M. E.; Paloma, L. G.; Kanter, J . R.; Taylor,
S. S.; Brunton, L. L.; Nicolaou, K. C. Molecular Design and
Biological Activity of Potent and Selective Protein Kinase
Inhibitors Related to Balanol. Chem. Biol. 1995, 2, 601-608.
(9) Casnellie, J . E. Protein Kinase Inhibitors: Probes for the
Functions of Protein Phosphorylation. Adv. Pharmacol. 1991,
22, 167-205.
(10) (a) Hollinshead, S. P.; Nichols, J . B.; Wilson, J . W. Two Practical
Syntheses of Sterically Congested Benzophenones. J . Org. Chem.
1994, 59, 6703-6709. (b) Crane, H. M.; Menaldino, D. S.;
J agdmann, J r., G. E.; Darges, J . W.; Buben, J . A. Increasing
the Cellular PKC Activity of Balanol: A Study of Ester Analogs.
Bioorg. Med. Chem. Lett. 1995, 5, 2133-2138.
Ca sein P r otein Kin a se II Assa y. 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), 10 mM
MgCl2 (Sigma), 10 µL of DMSO or 10 µL of a solution of
inhibitor in DMSO, and 30 µM [γ-32P]ATP (DuPont). The
reaction was initiated by addition of casein protein kinase II
(isolated from rat brain homogenate), incubated at room
temperature for 10 min, and 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). The precipitate
was collected by vacuum filtration on glass fiber filters and
quantified by counting in a â scintillation counter.
Ca 2+ Ca lm od u lin Dep en d en t P r otein Kin a se Assa y.
Assay conditions were as follows: 50 mM Tris-HCl (pH 7.5),
5 mM â-mercaptoethanol, 400 µM EGTA, 700 µM calcium
chloride (Sigma), 10 mM MgCl2 (Sigma), 200 µg/mL histone
type HA (Worthington), 10 µL of DMSO or 10 µL of a solution
of inhibitor in DMSO, and 30 µM [γ-32P]ATP (DuPont). The
reaction was initiated by addition of Ca2+ calmodulin depend-
ent protein kinase (isolated from rat brain homogenate),
incubated at room temperature for 10 min, and stopped by
adding 0.5 mL of ice cold trichloroacetic acid (Amresco)
followed by 100 µL of 1 mg/mL bovine serum albumin (Sigma).
The precipitate was collected by vacuum filtration on GFC
filters and quantified by counting in a â scintillation counter.
sr c P r otein Tyr osin e Kin a se Assa y. The assay was
carried out according to a protocol obtained from Oncogene
Science, Inc. except that Triton X-100 was used in place of
Brij 35. Protein tyrosine kinase (p60c-src, #PK03) and Raytide
kinase substrate (#PK02) were obtained from Oncogene Sci-
ence.
Neu tr op h il Assa y. Phorbol-12-myristate-13-acetate (PMA;
15 ng/mL) was added to lucigenin in reaction HBSS containing
a neutrophil suspension (2 × 106 cells/mL). Cuvettes were
loaded into a luminometer, and chemiluminescence at 550 nm
was measured for 15 cycles at 37 °C. Determinations made
in the presence of test compounds were compared to maximum
response values. IC50s were determined using a four-point
curve of 10-fold dilutions.
(11) (S)-12 and (R)-12 were determined to be at least 95% ee by chiral
HPLC using a Chiralcel OA column with 50% hexane/2-propanol
as the solvent system. Enantiomers (S)-12 and (R)-12 eluted at
14.85 and l9.02 min, respectively. The enantiomeric purity could
have been greater than 95%; however, base-line resolution
between the two peaks was not quite achieved. Chiral purity
was determined at this point by chiral HPLC because the optical
rotations of the final products (S)-12b, (R)-12b, and (S)-12c were
small and solvent dependent.
(12) Note that in the preparation of 16b it was possible to couple
the benzophenone acid chloride 2b selectively with the primary
alcohol of 16 in the presence of the unprotected phenol.
(13) (a) Naito, T.; Mayumi, T. M.; Tajiri, K.; Ninomiya, I.; Kiguchi,
T. A Novel and Chiral Synthesis of Both Enantiomers of trans-
3-Amino-4-hydroxyhexahydroazepine, a Key Intermediate for
the Synthesis of Balanol. Chem. Pharm. Bull. 1996, 44, 624-
626. (b) Mu¨ller, A.; Takyar, D. K.; Witt, S.; Ko¨nig, W. A.
Synthesis of (3R,4R)-3-Amino-4-hydroxyhexahydroazepine, the
Chiral Constituent of the Antibiotic Ophiocordin. Liebigs Ann.
Chem. l993, 651-655.
(14) Lai, Y. S.; Menaldino, D. S.; Nichols, J . B.; J agdmann, G. E.,
J r.; Mylott, Gillespie, J .; Hall, S. H. Ring Size Effect in the PKC
Inhibitory Activities of Perhydroazepine Analogs of Balanol.
Bioorg. Med. Chem. Lett. 1995, 5, 2151-2154.
(15) Unpublished results.
(16) J agdmann, G. E., J r.; Defauw, J . M.; Lampe, J . W.; Darges, K.
K. Potent and Selective PKC Inhibitory 5-Membered Ring
Analogs of Balanol With Replacement of the Carboxamide
Moiety. Bioorg. Med. Chem. Lett. 1996, 6, 1759-1764.
(17) (a) Mendoza, J . S.; J agdmann, G. E., J r.; Gosnell, P. A. Synthesis
and Biological Evaluation of Conformationally Constrained
Bicyclic and Tricyclic Balanol Analogues as Inhibitors of Protein
Kinase C. Bioorg. Med. Chem. Lett. 1995, 5, 2211-2216. (b) Hu,
H.; Hollinshead, S. P.; Hall, S. E.; Kalter, K.; Ballas, L. M.
Synthesis and Protein Kinase C Inhibitory Activities of Indane
Analogs of Balanol. Bioorg. Med. Chem. Lett. 1996, 6, 973-978.
(18) Twomey, B.; Muid, R. E.; Nixon, J . S.; Sedgwick, A. D.;
Wilkinson, S. E.; Dale, M. M. The effect of New Potent Selective
Inhibitors of Protein Kinase C on the Neutrophil Respiratory
Burst. Biochem. Biophys. Res. Commun. 1990, 171, 1087-1092.
(19) Defauw, J . M.; Lampe, J . W.; Hu, H.; Lynch, M. P.; Heerding,
J . M.; Biggers, C. K.; Menaldino, D. S.; Murphy, M. M.;
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G.; Lai, Y.-S.; J anzen, W. P.; Hall, S. E. Synthesis and Protein
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(20) Since it was necessary to use 100% MeOH to elute 3 off the
column, it was contaminated with silica; thus, it was not possible
to obtain a satisfactory elemental analysis. Because of this, all
subsequent analogs were purified by reverse phase HPLC.
(21) Basta, P.; Strickland, M. B.; Holmes, W.; Loomis, C. R.; Ballas,
L. M.; Burns, D. J . Sequence and Expression of Human Protein
Kinase C-ꢀ. Biochem. Biophys. Acta 1992, 1132, 154-160.
(22) Bronson, D. D.; Daniels, D. M.; Dixon, J . T.; Redick, C. C.;
Haaland, P. D. Virtual Kinetics: Using Statistical Experimental
Design for Rapid Analysis of Enzyme Inhibitor Mechanisms.
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