Synthesis and protein kinase C inhibitory activities of balanol analogs with replacement of the perhydroazepine moiety

Article abstract of DOI:10.1021/jm960497g

Balanol is a potent protein kinase C (PKC) inhibitor that is structurally composed of a benzophenone diacid, a 4-hydroxybenzamide, and a perhydroazepine ring. A number of balanol analogs in which the perhydroazepine moiety is replaced have been synthesized and their biological activities evaluated against both PKC and cAMP-dependent kinase (PKA). The results suggested that the activity and the isozyme/kinase selectivity of these compounds are largely related to the conformation about this nonaromatic structural element of the molecules.

Full text of DOI:10.1021/jm960497g

226
J . Med. Chem. 1997, 40, 226-235
Syn th esis a n d P r otein Kin a se C In h ibitor y Activities of Ba la n ol An a logs w ith
Rep la cem en t of th e P er h yd r oa zep in e Moiety1
Yen-Shi Lai,* J ose´ S. Mendoza, G. Erik J agdmann, J r., David S. Menaldino, Christopher K. Biggers,
J ulia M. Heerding, J oseph W. Wilson, Steven E. Hall, J ack B. J iang, William P. J anzen, and
Lawrence M. Ballas
Sphinx Pharmaceuticals, A Division of Eli Lilly & Company, 4615 University Drive, Durham, North Carolina 27707
Received J uly 8, 1996X
Balanol is a potent protein kinase C (PKC) inhibitor that is structurally composed of a
benzophenone diacid, a 4-hydroxybenzamide, and a perhydroazepine ring. A number of balanol
analogs in which the perhydroazepine moiety is replaced have been synthesized and their
biological activities evaluated against both PKC and cAMP-dependent kinase (PKA). The
results suggested that the activity and the isozyme/kinase selectivity of these compounds are
largely related to the conformation about this nonaromatic structural element of the molecules.
In tr od u ction
namely, a tetrasubstituted benzophenone diacid, a
trans-3,4-aminohydroxyperhydroazepine, and a 4-hy-
droxybenzoic acid. This convenient analysis served well
as a guideline in planning the total synthesis of balanol
and its analogs and also identified the three major
subjects of our investigations regarding the structure-
activity relationship (SAR) of these interesting mol-
ecules. This manuscipt describes the SAR of a series
of azepine replacements9 in which the other two do-
mains are those found in balanol itself. Modifications
include replacement or functionalization of the azepine
nitrogen or its equivalent, variation of ring size, and
introduction of conformational constraints.
Protein kinase C (PKC) is a family of structurally
related serine/threonine specific kinases widely distrib-
uted in tissues and cells. It is activated by diacylglycerol
(DAG) originating from receptor-mediated hydrolysis of
membrane inositol phospholipids, a process that is
responsible for initiating a cascade of cellular responses
to extracellular stimuli such as hormones, neurotrans-
mitters, and growth factors. Activated PKC catalyzes
transfer of the γ-phosphate of ATP to substrate proteins
and, by doing so, relays transmembrane signals to
biological systems involved in a number of cellular
processes including gene expression, cell proliferation,
and differentiation.2 PKC is a major receptor for tumor-
promoting phorbol ester which activates PKC in a way
similar to DAG,3 and unregulated activation of PKC has
been related to a range of disease states including
central nervous system (CNS) diseases, cardiovascular
disorders, diabetes, asthma, and HIV infections.4 Given
the importance of PKC in cellular biochemistry, inhibi-
tors which are not only specific for this family of
enzymes but also selective for only one of the isozymes
within this family are desirable as tools for probing
pertinent biological systems and, moreover, may allow
development of specific therapeutic agents for treatment
of human diseases.5
Ch em istr y
Synthesis of each balanol analog followed a uniform
theme in which the azepine replacement was condensed
with the 4-hydroxybenzoyl residue followed by coupling
of the protected benzophenone (Scheme 1). 4-(Benzyl-
oxy)benzoic acid (3) is readily available from 4-hydroxy-
benzoic acid, and the benzophenone unit is available as
5 by a multigram-scale synthesis developed in our
laboratories.10 All the hydroxyl groups in these two
fragments were strategically protected with a benzyl
group so the final deprotection step was simplified to a
single catalytic hydrogenolysis operation. This tactic
was also applied to heteroatoms in the azepine replace-
ment when applicable. Benzoic acid 3 was converted
to the corresponding acid chloride or acylimidazole and
coupled with an azepine replacement at the C.3 amino
site. Occasionally this resulted in concomitant acylation
of the vicinal hydroxyl group, and the crude products
were treated with NaOH to provide the desired alcohols.
Benzophenone acid 5 was usually converted to the
corresponding acid chloride immediately before use and
was coupled to amido alcohol 4. With these common
synthetic steps to complete the syntheses, the major
task was reduced to construction of the desired azepine
replacements 2 featuring a trans-vicinal amino alcohol
substructure. The syntheses of these required elements
are shown in Schemes 2-7.
Balanol, (-)-1, recently isolated in our laboratories
from the fungus Verticillium balanoides,6 is a new class
of PKC inhibitor that inhibits PKC at low-nanomolar
concentrations.7 Its unique structure was elucidated
based on spectroscopic data and confirmed by an asym-
metric total synthesis.8 Balanol may be disassembled
For the most part, the vicinal amino alcohol was
secured by stereospecific epoxide opening with an amine
or the equivalent. The epoxides were in turn obtained
by mCPBA epoxidation of an appropriate olefin. The
exceptions are syntheses of analogs 37-41 (Scheme 6),
into three distinctive elements on a retrosynthetic basis,
X Abstract published in Advance ACS Abstracts, December 15, 1996.
S0022-2623(96)00497-9 CCC: $14.00 © 1997 American Chemical Society

Synthesis and PKC Activity of Balanol Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 227
Sch em e 1
Ta ble 1. Kinase Inhibition by Balanol (IC50 values, µM)a
that the natural, (-)-enantiomer of balanol is at least
20-100-fold more potent than the (+)-enantiomer8b and
that the two aromatic side chains of the molecule as well
as their trans relative stereochemistry are indispensable
for optimal potency.13
compd
R
â1
â2
γ
δ
ꢀ
η
ú
PKA
(()-1 0.07 0.03 0.03 0.03 0.02 0.04 0.02
(-)-1 0.03 0.01 0.01 0.02 0.004 0.01 0.003
3.5
7.4 0.05
5.2
(+)-1 3.0 0.5 0.88 0.4 0.42 1.0 0.26 >150
a
The azepine nitrogen atom appeared to be significant
for the activity of balanol, as can be seen from Table 2
in which all four analogs with this atom replaced
showed loss of activity. The changes in activity typically
fell in the range of 50-100-fold and were isozyme-
dependent in some cases. Thus, unlike the sulfone
analog 16, which was less active than racemic balanol
against all eight isozymes, the sulfide analog 15 and,
in particular, the ether analog 14 remained relatively
active against the δ and η isozymes. Interestingly the
carbocyclic analog 17 was only 2-10-fold reduced in
potency compared to racemic balanol, and the IC50
values were maintained at a submicromolar level. This
strongly suggests that there is an offset of any signifi-
cant change in electronic properties associated with the
-NH- to -CH2- substitution. The seven-membered
ring is generally recognized as a conformationally
dynamic ring system. This would allow more confor-
mational variations among these balanol analogs and
can lead to major changes in their bioactivities via
differences in spatial projection of the aromatic side
chains. Thus the sulfur and oxygen substituents in
analogs 14-16 may not otherwise confer poor activities
to these analogs. Compounds 14-17, like balanol itself,
showed no significant selectivity between PKC and
PKA. However, unlike balanol and most of its analogs
which inhibit PKC better than PKA, the ether analog
14 appeared to be a better PKA inhibitor than PKC
inhibitor, with exceptions in the PKC-δ and -η cases.
All compounds tested are synthetic.
in which a monooxime of a 1,2-diketone, obtained either
from commercial sources or by R-nitrosation of com-
mercial ketones, was reduced with sodium to provide
the required substructure. These reduction reactions
were usually nonstereospecific, and the trans stereo-
isomers were separated from the cis isomers by chro-
matography after N-acylation with benzoic acid 3.
Clean separation of 30R and 30â proved to be difficult
so that only one of the two epimers could be obtained
in pure form. Compound 31â was obtained as a single
diastereomer due to the stereospecificity of the chem-
istry used for its synthesis (step o, Scheme 4), and its
epimer 31R was not prepared. The stereochemical
assignments for analogs 30-33 and 37-41 were based
on 1H NMR or established information in the litera-
ture.11 However, due to a lack of unequivocal H NMR
1
information and available literature precedent, the
relative stereochemistry of 30R and 30â and the exo/
endo stereochemistry of 41 were assigned on an arbi-
trary basis. It should also be noted that analog 39 was
derived from optically pure starting material, and as
far as the C.3-C.4 region (structure 2) of the molecule
is concerned, the absolute configuration and thus the
biological activity of this analog are appropriate for
comparison with (-)-balanol.
Resu lts a n d Discu ssion
A dramatic fluctuation in potency was observed with
changes in ring size (Table 3). Reduction of ring size
by one methylene unit to analog 23 was accompanied
by a 70-100-fold reduction in activity against six of the
eight PKC isozymes. This compound, like analog 14,
is a selective inhibitor for the δ and η isozymes, by a
factor of 20-100, and a better inhibitor for PKA over
PKC for all isozymes except δ and η. Further shrinkage
in ring size produced an opposite effect, rendering the
Kinase inhibitory activities of balanol and various sets
of analogs against PKC isozymes R, â1, â2, γ, δ, ꢀ, η,
and ú, as well as cAMP-dependent kinase (PKA), are
shown in Tables 1-6. Balanol itself is one of the most
potent PKC inhibitors known to date.12 However,
except for its relative inactivity against the ú isozyme,
a property shared by all balanol analogs, balanol falls
short of isozyme selectivity as well as kinase selectivity
between PKC and PKA. Our SAR studies also showed

228 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Sch em e 2. Synthesis of Precursors to Analogs 14-17a
Lai et al.
a
Conditions: (a) Bn2NAlEt2, CH2Cl2, rt; or NaN3, NH4Cl, MeOH-H2O, 65 °C; (b) TBDMS-Cl, imidazole, DMF, rt; (c) OsO4, NMO,
acetone-H2O, rt; (d) NaIO4, THF-H2O, rt; then NaBH4, Et2O-MeOH, 5 °C; (e) MeLi, THF; then TsCl, Et3N, rt; (f) KO2, 18-crown-6,
DMSO, rt; (g) BuLi, PhCH3, reflux; (h) H2, Pd(OH)2-c, MeOH, rt; (i) 4-(benzyloxy)benzoic acid, 1,1′-carbonyldiimidazole, THF, rt; (j) Bu4NF,
THF, rt; (k) O3, CH2Cl2-MeOH, -78 °C; then NaBH4; (l) MeSO2Cl, Et3N, CH2Cl2, rt; (m) Li2S, Et3N, MeOH, reflux; (n) LiAlH4, THF, rt;
(o) 4-(benzyloxy)benzoic acid, 1,1′-carbonyldiimidazole, THF; then 1 N aq NaOH, MeOH-THF, rt; (p) 6, Et3N, DMAP, CH2Cl2, rt; (q)
CH3CO3H, CH3CO2H-CH2Cl2, rt; (r) CH3CO3H, NaOAc, Na2CO3, CH2Cl2, 5 °C-rt; (s) NaN3, NH4Cl, MeOH-H2O, reflux; (t) TBDMS-Cl,
imidazole, DMF, rt; (u) H2, 5% Pd-C, MeOH, rt; (v) 4-(benzyloxy)benzoic acid, 1,1′-carbonyldiimidazole, THF, rt; (w) Bu4NF, THF, rt.
Ta ble 2. Kinase Inhibition by Heteroatom Analogs (IC50
values, µM)
stantiating a ring size preference of 5 g 7 > 6. Both 22
and 24 were more active against the δ and η isozymes
than the other isozymes, but neither was as selective
as piperidine 23.
The two five-membered ring analogs 22 and 24 are
attractive not only for their impressive potency but also
for their ease of preparation. By using aqueous am-
monia in the epoxide-opening reactions (step d, Scheme
3), it took only one and three steps, respectively, to reach
the required intermediates (18, n ) 1, X ) -CH2- and
-NCbz-) from commercially available materials. This
compared very favorably to a seven-step synthesis of the
corresponding azepine amino alcohol.14 Since large
amounts of these intermediates became readily avail-
able, we elected to use the five-membered ring system
as an advanced lead structure on which much of our
later SAR studies were based. Related to this are
compounds 30-33 which represent analogs of 24 with
additional substituents carrying an amino or a hydroxyl
group. The amine analogs 31 and 33 can also be treated
as extensions of pyrrolidine 22 in which the endocyclic
nitrogen atom is repositioned.
compd
R
â1
â2
γ
δ
ꢀ
η
ú
PKA
(()-1 0.07 0.03 0.03 0.03 0.02 0.04 0.02
3.5
0.01 >150
3.3 3.8 2.4 1.0 0.09 5.9 0.1 121
9.6 5.2 3.6 4.3 4.0 45 0.8 >150
0.27 0.22 0.43 0.06 0.09 0.28 0.06 >150
14
15
16
17
6.7 2.5 3.3 1.0 0.09 16
0.45
7.5
38
1.4
pyrrolidine analog 22 a marginally more potent inhibi-
tor than balanol. Consistent with the observation that
the azepine nitrogen could be replaced with carbon,
analog 24 was found to be as potent as 22 in most cases
and more potent than 22 against the δ and η isozymes.
In addition, 24 is 5-8 times better in potency than its
seven-membered counterpart, analog 17, further sub-
As shown in Table 4, compound 24 remained the most
potent PKC-δ and -η inhibitor among all cyclopentane-

Synthesis and PKC Activity of Balanol Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 229
Sch em e 3. Synthesis of Precursors to Analogs 22-24a
a
Conditions: (a) mCPBA, CH2Cl2, rt; (b) NaN3, NH4Cl, MeOH-H2O, reflux; (c) PPh3, THF, rt; (d) aq NH3, 65 °C; (e) 4-(benzyloxy)benzoic
acid, oxalyl chloride, aq NaOH, CH2Cl2, rt.
Ta ble 3. Kinase Inhibition by Ring Size Analogs (IC50 values,
µM)
isozyme selectivity and were found to be as active PKA
inhibitors as PKC inhibitors.
A number of conformationally constrained analogs,
37-41, were also examined. Bicyclic analog 37 was, for
the most part, 3-7-fold less potent than racemic balanol,
and analog 38 was essentially as potent as 24, except
with PKC-δ and -η. These results may not be a fair
measure of the steric/lipophilic effect associated with the
additional methylene groups in analogs 37 and 38, since
conformational changes from balanol and 24 are ex-
pected with these bicyclic analogs. On the other hand,
compd
R
â1
â2
γ
δ
ꢀ
η
ú
PKA
a comparison between 38 and its bulkier congeners 39
and 40 does reveal an activity-reducing steric/lipophilic
effect against certain PKC isozymes. Interestingly, the
reduction in activity was 2-8 times greater with 39
than 40; the difference may actually be even greater
considering the fact that 39 is a single enantiomer with
the correct absolute configuration, while 40 is racemic.
Except in the case of the ꢀ isozyme, 40 is very close to
38 in activity, suggesting the added volume/hydropho-
bicity associated with the three extra methylene units
in 40 and, perhaps, the two additional methylene units
in 38 was not much a significant negative factor for
activity. The three methyl groups in 39 are positioned
in different regions of the molecule from the three
methylene groups of 40. The poorer activity of 39
seemed indicative of a low tolerance for steric bulk in
these regions of the molecules. Also, due to the confor-
mational rigidity and the particular shape of 39, some
of these methyl groups may be sufficiently close in space
to the benzophenone to interfere with the local confor-
mation of the aromatic side chains. This is likely to
contribute to lowering the activity of 39 relative to 38
and 40 and apparently is not as likely to occur in
compounds such as 32 and 33 because of less structural
restraint in these molecules. The ꢀ isozyme seemed to
be especially sensitive to these steric effects in that an
exceptional 120- and 30-fold drop in potency relative to
38 was observed with 39 and 40, respectively. Com-
pound 41 is inactive and may well be another example
of the ring size effect which predicts a six-membered
ring to be inactive. None of these analogs showed
isozyme selectivity, but there is an overall better
selectivity for PKC over PKA with these compounds, in
particular 39 and 40.
(()-1 0.07 0.03 0.03 0.03 0.02 0.04 0.02 3.5
22
23
24
0.022 0.017 0.033 0.013 0.005 0.01 0.004 >0.15 0.07
5.1 2.2 4.7 2.2 0.05 1.8 0.09 >150 0.35
0.04 0.04 0.05 0.01 0.001 0.05 0.001 22 0.03
based analogs. However, analogs 31â, 32R, 33R, and
33â were clearly more active than 24 in the â1 and â2
cases and marginally so in some other instances.
Analogs 30-33 differ from one another in relative
stereochemistry as well as nature of the additional
fuctional groups, and this seemed to be reflected in their
biological activities, albeit in a complex way. The
difference caused by stereochemistry is not evident with
31 and the unresolved pair 30R/30â but is manifested
in the decreasing potency of 32R > 24 > 32â, where the
gaps ranged from 5- to 15-fold in magnitude (not
including PKC-ꢀ). The pair 33R/33â behaved similarly
with PKC-γ and -δ but was quite comparable to each
other in the other cases. Comparisons of analogs 30 and
31â, as well as 32â and 33â, suggested a preference for
an amino group over a hydroxyl group. However there
is the exceptional pair 32R and 33R. It is likely that
the functional group preference is under regulation of
the relative stereochemistry of the functionalized sub-
stituents. The four amine analogs 22, 31â, 33R, and
33â inhibited PKC with small and isozyme-dependent
differences. In essence the exocyclic amines tend to be
better PKC-â1, -â2, and -γ inhibitors and were otherwise
comparable to the endocyclic 22. Chain length may
have an influence on activity, as shown by analogs 32R
and 30, but it is quite obscured whether there is a trend
or not. In summary, these additional substituents were
effective in fine tuning the biological activity. Stereo-
chemistry around the cyclopentane rings appeared to
be important for bioactivity, but it is not as clear
whether other factors such as hydrogen bonding are
operational in these cases, a similar situation to what
was found among balanol and 14-17 and between 22
and 24. These cyclopentane analogs had no significant
The pyrrolidine nitrogen of analog 22, though replace-
able with a methylene group, is valuable in providing a
site of modification to more elaborated and potentially
better analogs. Compounds 47-52 are examples in
which selected functional groups were attached to this
nitrogen atom. The N-isopropyl analog 47 was 2-10-

230 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Sch em e 4. Synthesis of Precursors to Analogs 30 and 31a
Lai et al.
a
Conditions: (a) HOCH2CH2OH, PPTS, PhH, reflux; (b) mCPBA, CH2Cl2, rt; (c) BnNH2, LiClO4, CH3CN, 60 °C; (d) H2, 5% Pd-C,
EtOAc, rt; (e) 3, CDI, THF, rt, aq NaOH workup; (f) 5, Et3N, DMAP, CH2Cl2, rt; (g) Pd(CH3CN)2Cl2, CHCl3, rt; (h) NaBH4, MeOH, 0 °C,
gave 2:1 mixture; (i) H2, Pd(OH)2-C, MeOH-THF, rt; (j) HPLC; (k) (COCl)2, DMF, CH2Cl2, rt; (l) NH4OH, rt; (m) (CF2CO2)2PhI, CH3CN,
H2O, rt; (n) BnOCOCl, aq NaOH, CH2Cl2, rt; (o) mCPBA, CH2Cl2, 0 °C-rt; (p) NaN3, NH4Cl, H2O, MeOH, 50 °C; (q) Zn, AcOH, H2O,
EtOH, rt; (r) 3, CDI, THF, rt; aq NaOH workup.
Sch em e 5. Synthesis of Precursors to Analogs 32 and 33a
a
Conditions: (a) 4-(benzyloxy)benzoyl chloride, aq KOH, THF, rt; (b) SiO2 chromatography; (c) 5, Et3N, DMAP, CH2Cl2, rt; (d) Bu4NF,
THF, rt; (e) (COCl)2, Me2SO, Et3N, 0 °C-rt, SiO2 chromatography; (f) BnNH2, NaB(OAc)3H, ClCH2CH2Cl, rt; (g) chromatography on
SiO2.
fold less potent than the parent compound 22, and
analogs 48-52 were also found to show loss of activity
compared to 22, typically by a factor of 20-50. The
N-substituents in these analogs are comparable in size,
so steric bulk is not likely responsible for the reduced
activity of 48-52 relative to 47. Rather, the way the
pyrrolidine nitrogen is blocked may have more bearing
on the reduction in activity. The partial π character of
the pyrrolidinium amido N-C bonds of 48-52 may
influence conformation about the pyrrolidine rings
differently from the isopropyl group of 47 and contribute
to their lower activity relative to 47. In addition 47
differs from 48-52 in that it has a basic nitrogen and
the isopropyl group is relatively nonpolar. The polarity
of the N-substituents in 48-52 is such that analogs with
an exposed partial negative charge and/or hydrogen
bond acceptor in that area of the molecule may not be
particularly preferred for binding. This also seemed to
be consistent with the better activities of analogs 30-
33. Finally, none of these N-substituted compounds is
significantly isozyme selective, and none of them, but
50, showed significant kinase selectivity.
Con clu sion
The perhydroazepine moiety of balanol possesses
special properties that are important to its potency
against PKC and PKA. We have carried out SAR
studies around this structural element in an attempt
to define these properties. It was pointed out that the
azepine nitrogen is replaceable as long as the replace-
ment is able to raise the two aromatic side chains in a
stereochemically correct manner. A deviated conforma-

Synthesis and PKC Activity of Balanol Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 231
Ta ble 4. Kinase Inhibition by Cyclopentane Analogs (IC50
values, µM)
saturated aqueous K2CO3 (3 × 5 mL). The organic layer was
dried (MgSO4) and evaporated to give a white solid which was
recrystallized from a hot mixture of EtOAc-THF-hexanes (10:
2:3) to give a white powder (61 mg, 79%): mp 189-193 °C; 1H
NMR (CDCl3) δ 7.74 (d, J ) 8.8 Hz, 2H), 7.01-7.48 (m, 28H),
6.94 (t, J ) 7.9 Hz, 2H), 6.82 (d, J ) 8.8 Hz, 2H), 5.47 (t, J )
8.0 Hz, 1H), 5.13 (s, 2H), 5.07 (s, 2H), 4.98 (m, 1H), 4.80 (s,
4H), 4.70 (s, 2H), 3.62 (dd, J ) 16.0, 4.5 Hz, 1H), 3.51 (dm, J
) 15.7 Hz, 1H), 3.09-3.28 (m, 2H), 2.05-2.29 (m, 4H). Anal.
(C63H55NO12S) C, H, N.
Gen er a l P r oced u r e for Hyd r ogen olysis of 6. 6 (0.1 M,
1 equiv) was dissolved in ethyl acetate-EtOH (1:1 to 1:6) and
placed in a round-bottom flask or Parr bottle. The vessel was
purged with N2, and Pearlmann’s catalyst (0.01-0.1 equiv) was
added. The mixture was either stirred under 1 atm of H2
(balloon) or charged with H2 (40-50 psi) and shaken on a Parr
apparatus for 16-24 h. The reaction mixture was diluted with
ethanol and filtered through Celite, and the filter cake was
washed and kept moist with more ethanol. The filtrate and
washes were combined and concentrated under reduced pres-
sure. The residue was purified by HPLC using a linear
gradient of increasing CH3CN in H2O containing 0.1% trifluo-
roacetic acid as the eluent, and the collected fractions were
combined, concentrated under reduced pressure to remove
most of the solvents, and lyophilized to give the final product.
(()-tr a n s-3-(4-Hyd r oxyben za m id o)-4-[[4-[2-h yd r oxy-6-
(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih yd r oxyben zoyl]oxy]-
p er h yd r ooxep in e (14): yellow solid (61 mg, 94%); mp 204
compd
R
â1
â2
γ
δ
ꢀ
η
ú
PKA
30R+â 0.04 0.02 0.01 0.02 0.005 0.1 0.003 16
0.13
0.25
30â
31â
32R
32â
33R
33â
0.05 0.03 0.02 0.03 0.006 0.15 0.004 13
0.02 0.004 0.002 0.005 0.005 0.01 0.003 1.2 0.05
0.01 0.004 0.003 0.005 0.004 0.16 0.002 39
0.08 0.06 0.02 0.05 0.02 0.27 0.02 34
0.02 0.004 0.004 0.005 0.006 0.03 0.003 2.8 0.17
0.02 0.005 0.005 0.01 0.02 0.03 0.003 3.1 0.09
0.23
0.22
tion about the azepine replacement may result in
decrease in activity but may also be a source of isozyme
selectivity for PKC-δ and -η, as shown by analogs 14
and 23. In terms of ring size, five is optimal for an
azepine replacement, seven-membered rings are equally
good but are less predictable due to their conformational
flexibility, and six-membered rings are inactive against
all but the δ and η isozymes. Rational design combining
the above replaceability rule and ring size effect may
generate simple yet potent inhibitors such as 24, which
is also the most potent inhibitor against the δ and η
isozymes among all balanol analogs. Derivatization of
a readily available azepine replacement such as pyrro-
lidine and cyclopentane has been a many faceted issue
but has proven to be an effective means of bringing
about enhanced activity, such as that of 31â, 32R, 33R,
and 33â, and kinase selectivity, of 39 and 40, for
example. Ultimately these azepine-replaced analogs of
balanol provide a model via which the nature of the
interactions between balanol and PKC, as well as
structural requirements for better binding, can be
explored. A molecular modeling approach toward the
issue of conformation is currently in progress in our
laboratories.
1
°C dec; H NMR (CD3OD) δ 7.58 (d, J ) 8.6 Hz, 2H), 7.31 (d,
J ) 7.4 Hz, 1H), 7.15 (t, J ) 7.8 Hz, 1H), 6.87 (s, 2H), 6.81 (d,
J ) 8.1 Hz, 1H), 6.74 (d, J ) 8.7 Hz, 2H), 5.20 (m, 1H), 4.34
(m, 1H), 3.75-3.84 (m, 2H), 3.64-3.73 (m, 2H), 1.89-2.02 (m,
2H), 1.79-1.88 (m, 2H); IR (KBr, cm-1) 1702, 1679, 1649, 1641;
FABMS m/z ) 552 (M + 1). Anal. (C28H25NO11‚1.4H2O‚0.4CF3-
CO2H) C, H, N.
(()-tr a n s-3-(4-Hyd r oxyben za m id o)-4-[[4-[2-h yd r oxy-6-
(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih yd r oxyben zoyl]oxy]-
p er h yd r oth iep in e (15): yellow solid (25 mg, 35%); mp 196
1
°C dec; H NMR (CD3OD) δ 7.62 (d, J ) 8.6 Hz, 2H), 7.32 (d,
J ) 7.1 Hz, 1H), 7.18 (t, J ) 7.9 Hz, 1H), 6.88 (s, 2H), 6.84 (d,
J ) 8.1 Hz, 1H), 6.77 (d, J ) 8.6 Hz, 2H), 5.36 (tm, J ) 8.6
Hz, 1H), 4.60 (m, 1H), 2.89-2.96 (m, 2H), 2.69-2.79 (m, 2H),
2.11-2.22 (m, 2H), 1.93-2.08 (m, 2H); IR (KBr, cm-1) 1706,
1689, 1633; FABMS m/z ) 568 (M + 1). Anal. (C28H25
NO10S‚2H2O‚0.25CF3CO2H) C, H, N.
-
(()-1,1-Dioxo-tr a n s-3-(4-h yd r oxyb en za m id o)-4-[[4-[2-
h ydr oxy-6-(h ydr oxycar bon yl)ben zoyl]-3,5-dih ydr oxyben -
zoyl]oxy]p er h yd r oth iep in e (16): yellow solid (33 mg, 95%);
1
mp 198 °C dec; H NMR (CD3OD) δ 7.61 (d, J ) 8.7 Hz, 2H),
7.30 (d, J ) 7.7 Hz, 1H), 7.19 (t, J ) 7.8 Hz, 1H), 6.91 (s, 2H),
6.83 (d, J ) 7.7 Hz, 1H), 6.77 (d, J ) 8.7 Hz, 2H), 5.50 (tm, J
) 8.6 Hz, 1H), 4.61 (m, 1H), 3.77 (dd, J ) 15.8, 7.4 Hz, 1H),
3.49 (dm, J ) 15.3 Hz, 1H), 3.28-3.46 (m, 2H), 2.06-2.22 (m,
4H); IR (KBr, cm-1) 1718, 1686, 1635; FABMS m/z ) 599 (M).
Anal. (C28H25NO12S‚2.4H2O) C, H, N.
Exp er im en ta l Section
Gen er a l. Melting points were determined with either a
Mel-Temp II or an Electrothermal melting point apparatus
and are uncorrected. 1H NMR spectra were recorded on a
Varian Gemini 300 instrument at 300 MHz. Chemical shifts
are reported in part per million (ppm) downfield relative to
tetramethylsilane. FTIR spectra were measured with a Matt-
son Galaxy 5000 spectrometer calibrated against polystyrene
standard. Elemental analysis was performed in house with a
Carlo Erba EA1108 analyzer. Mass spectra were obtained
from Analytical Instrument Group, Raleigh, NC. Flash chro-
matography was performed on silica gel 60 purchased from
EM Science, and preparative HPLC was carried out with a
Rainin HPLC system on a C-18 reverse phase column. All
anhydrous reactions were run under an atmosphere of N2 in
oven-dried glassware.
(()-1,1-Dioxo-tr a n s-3-[4-(ben zyloxy)ben za m id o]-4-[[4-
[2-(ben zyloxy)-6-[(ben zyloxy)ca r bon yl]ben zoyl]-3,5-bis-
(ben zyloxy)ben zoyl]oxy]p er h yd r oth iep in e (12). Peroxy-
acetic acid (32 wt % in acetic acid, 37 mg, 0.155 mmol) was
added to a solution of 11 (75 mg, 0.074 mmol) in CH2Cl2 (0.7
mL). The resultant mixture was stirred at room temperature
for 1 h, diluted with CH2Cl2 (10 mL), and washed with
(()-tr a n s-1-(4-Hyd r oxyben za m id o)-2-[[4-[2-h yd r oxy-6-
(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih yd r oxyben zoyl]oxy]-
cycloh ep ta n e (17): yellow solid (83 mg, 95%); mp 186 °C dec;
1H NMR (CD3OD) δ 7.55 (d, J ) 8.7 Hz, 2H), 7.38 (d, J ) 7.4
Hz, 1H), 7.19 (t, J ) 7.9 Hz, 1H), 6.90 (d, J ) 7.8 Hz, 1H),
6.84 (s, 2H), 6.72 (d, J ) 8.7 Hz, 2H), 5.13 (m, 1H), 4.29 (m,
2H), 1.53-1.90 (m, 10H). Anal. (C29H27NO10‚H2O) C, H, N.
(()-tr a n s-3-(4-Hyd r oxyben za m id o)-4-[[4-[2-h yd r oxy-6-
(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih yd r oxyben zoyl]oxy]-
p yr r olid in e (22): yellow solid (88 mg, 72%); mp 197-199 °C;
1H NMR (CD3OD) δ 7.73 (d, J ) 8.7 Hz, 2H), 7.48 (d, J ) 7.7
Hz, 1H), 7.24 (t, J ) 8.0 Hz, 1H), 7.02 (d, J ) 8.2 Hz, 1H),
6.94 (s, 2H), 6.79 (d, J ) 8.7 Hz, 2H), 5.58 (m, 1H), 4.66 (m,
1H), 4.93 (dd, J ) 13.4, 5.3 Hz, 1H), 3.82 (dd, J ) 13.0, 7.0
Hz, 1H), 3.60 (apparent dd, J ) 12.6, 4.4 Hz, 2H); IR (KBr,
cm-1) 1722, 1665, 1633. Anal. (C26H22N2O10‚CF3CO2H) C, H,
N.
(()-tr a n s-3-(4-Hyd r oxyben za m id o)-4-[[4-[2-h yd r oxy-6-
(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih yd r oxyben zoyl]oxy]-
p ip er id in e (23): yellow solid (60 mg, 50%); mp 216-220 °C
dec; 1H NMR (CD3OD) δ 7.43 (d, J ) 8.7 Hz, 2H), 7.29 (d, J )

232 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Lai et al.
Ta ble 5. Kinase Inhibition by Conformationally Constrained Analogs (IC50 values, µM)
compd
R
â1
â2
γ
δ
ꢀ
η
ú
PKA
37
38
39
40
41
0.41
0.06
0.41
0.1
0.026
0.05
0.31
0.15
4.4
0.069
0.03
0.08
0.03
0.14
0.04
0.21
0.05
4.6
0.11
0.02
0.34
0.05
5.0
0.17
0.03
3.6
0.97
27
0.026
0.02
0.31
0.05
2.8
22.5
37
>50
37
1.7
1.9
26
32
>50
34
14
>50
Sch em e 6. Synthesis of Precursors to Analogs 37-41a
7.03 (d, J ) 8.0 Hz, 1H), 6.94 (s, 2H), 6.82 (d, J ) 8.7 Hz, 2H),
5.32 (dt, J ) 12.1, 6.0 Hz, 1H), 4.87 (m, 1H), 4.41 (m, 1H),
2.66 (td, J ) 14.5, 6.3 Hz, 1H), 2.20 (tm, J ) 8.0 Hz, 1H), 1.97
(ddd, J ) 14.5, 8.9, 4.8 Hz, 1H), 1.79 (dm, J ) 14.6 Hz, 1H);
IR (KBr, cm-1) 1703, 1634, 1606; FABMS m/z ) 538 (M + 1).
Anal. (C27H23NO11‚0.8H2O‚0.1CF3CO2H) C, H, N.
30r + 30â: yellow solid; mp 184-189 °C; FABMS m/z )
538 (M + 1). Anal. (C27H23NO11‚1.0H2O‚0.2CF3CO2H) C, H,
N. 1H NMR (CD3OD) for 30R from spectrum of the mixture:
δ 5.45 (m, 1H), 4.53 (m, 1H), 2.55 (m, 1H), 2.26 (m, 1H), 2.08
(m, 1H), 1.75 (m, 1H); the aromatic region was indistinguish-
able from that of 30â.
(()-(1â,3r,4â)-1-Am in o-3-(4-h yd r oxyben za m id o)-4-[[4-
[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih yd r oxy-
ben zoyl]oxy]cyclop en ta n e (31â): yellow solid (151 mg,
72%); mp 197 °C dec; 1H NMR (CD3OD) δ 7.90 (d, J ) 8.9 Hz,
2H), 7.52 (d, J ) 7.6 Hz, 1H), 7.30 (t, J ) 8.0 Hz, 1H), 7.05 (d,
J ) 6.3 Hz, 1H), 6.99 (d, J ) 8.7 Hz, 2H), 6.94 (s, 2H), 5.67
(m, 1H), 5.59 (d, J ) 6.5 Hz, 1H), 4.84 (m, 1H), 4.21 (m, 1H),
3.07 (dm, J ) 16.8 Hz, 1H), 2.56 (tm, J ) 13.3 Hz, 1H), 2.46
(tm, J ) 13.4 Hz, 1H); IR (KBr, cm-1) 1776, 1680, 1606;
FABMS m/z ) 537 (M + 1). Anal. (C27H24N2O10‚1.2H2O‚1.5CF3-
CO2H) C, H, N.
(()-(1r,3r,4â)-1-(Hyd r oxym eth yl)-3-(4-h yd r oxyben za -
m id o)-4-[[4-[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-3,5-
d ih yd r oxyben zoyl]oxy]cyclop en ta n e (32r): yellow solid
(160 mg, 72%); mp 180-184 °C dec; 1H NMR (CD3OD) δ 7.47
(d, J ) 8.8 Hz, 2H), 7.28 (d, J ) 6.6 Hz, 1H), 7.06 (t, J ) 7.9
Hz, 1H), 6.80 (d, J ) 8.7 Hz, 1H), 6.69 (s, 2H), 6.59 (d, J ) 8.8
Hz, 2H), 5.06 (m, 1H), 4.32 (m, 1H), 3.37 (d, J ) 5.1 Hz, 2H),
2.30-2.11 (m, 2H), 1.91-1.67 (m, 2H), 1.36-1.25 (m, 1H); IR
(KBr, cm-1) 3373, 1704, 1633, 1605, 1507, 1425, 1369, 1244,
a
Conditions: (a) TMSCHN2, THF-MeOH, 5 °C-rt; (b) LiN(T-
MS)2, n-butylnitrile, various solvents, 60 °C-rt; (c) Na, EtOH,
heat; 4-(benzyloxy)benzoyl chloride, 1 N NaOH, toluene, rt.
1199, 762; FABMS m/z ) 552 (M + 1). Anal. (C28H25
NO11‚1.0H2O‚0.3CF3CO2H) C, H, N.
-
7.6 Hz, 1H), 7.06 (t, J ) 8.0 Hz, 1H), 6.82 (d, J ) 8.4 Hz, 1H),
6.70 (s, 2H), 6.58 (d, J ) 8.6 Hz, 2H), 5.11 (m, 1H), 4.28 (m,
1H), 3.54 (ddm, J ) 12.2, 4.1 Hz, 1H), 3.30 (m, 1H), 2.99-
3.11 (m, 2H), 2.08 (ddm, J ) 13.7, 3.7 Hz, 1H), 1.87 (m, 1H);
IR (KBr, cm-1) 1720, 1677, 1636, 1607, 1510, 1428, 1376, 1234;
FABMS m/z 537 (M + 1). Anal. (C27H24N2O10‚2.9 H2O‚0.9 CF3-
CO2H) C, H, N.
(()-(1â,3r,4â)-1-(Hyd r oxym eth yl)-3-(4-h yd r oxyben za -
m id o)-4-[[4-[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-3,5-
d ih yd r oxyben zoyl]oxy]cyclop en ta n e (32â): yellow solid
1
(85 mg, 79%); mp 159-168 °C dec; H NMR (CD3OD) δ 7.47
(d, J ) 8.7 Hz, 2H), 7.28 (d, J ) 7.7 Hz, 1H), 7.05 (t, J ) 8.0
Hz, 1H), 6.80 (d, J ) 7.4 Hz, 1H), 6.68 (s, 2H), 6.58 (d, J ) 8.7
Hz, 2H), 5.11 (m, 1H), 4.34 (m, 1H), 3.33 (d, J ) 6.0 Hz, 2H),
(()-tr a n s-1-(4-Hyd r oxyben za m id o)-2-[[4-[2-h yd r oxy-6-
(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih yd r oxyben zoy]loxy]-
cyclop en ta n e (24): yellow solid (168 mg, 85%); mp 198 °C
dec; 1H NMR (CD3OD) δ 7.48 (d, J ) 8.7 Hz, 2H), 7.28 (d, J )
7.7 Hz, 1H), 7.06 (t, J ) 7.9 Hz, 1H), 6.81 (d, J ) 7.1 Hz, 1H),
6.69 (s, 2H), 6.60 (d, J ) 8.8 Hz, 2H), 5.07 (m, 1H), 4.29 (m,
2.11 (m, 2H), 1.80-1.59 (m, 2H), 1.38 (m, 1H); IR (KBr, cm-1
)
3398, 1704, 1632, 1606, 1507, 1426, 1369, 1244, 1200, 763;
FABMS m/z ) 552 (M + 1). Anal. (C28H25NO11‚1.0H2O‚0.3CF3-
CO2H) C, H, N.
(()-(1r,3r,4â)-1-(Am in om eth yl)-3-(4-h yd r oxyben za m i-
d o)-4-[[4-[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-3,5-d i-
h yd r oxyben zoyl]oxy]cyclop en ta n e (33r): yellow solid (60
mg, 50%); mp 164-170 °C dec; 1H NMR (CD3OD) δ 7.48 (d, J
) 8.8 Hz, 2H), 7.28 (d, J ) 7.7 Hz, 1H), 7.06 (t, J ) 8.0 Hz,
1H), 6.81 (d, J ) 8.2 Hz, 1H), 6.70 (s, 2H), 6.60 (d, J ) 8.7 Hz,
2H), 5.16 (m, 1H), 4.34 (m, 1H), 2.82 (d, J ) 6.6 Hz, 2H), 2.28
(m, 2H), 1.87 (m, 2H), 1.29 (m, 1H); IR (KBr, cm-1) 3391, 3304,
3187, 2978, 1682, 1633, 1606, 1507, 1427, 1371, 1244, 1197,
1H), 1.99-2.01 (m, 2H), 1.48-1.70 (m, 4H); IR (KBr, cm-1
)
1703, 1633, 1606, 1507, 1425, 1373, 1245, 1200; FABMS m/z
522 (M + 1). Anal. (C27H23NO10‚2.0H2O‚0.5C2H6O) C, H, N.
(()-(1â,3r,4â)-1-H yd r oxy-3-(4-h yd r oxyb en za m id o)-4-
[[4-[2-h ydr oxy-6-(h ydr oxycar bon yl)ben zoyl]-3,5-dih ydr ox-
yben zoyl]oxy]cyclop en ta n e (30â a n d 30r + 30â). 30â:
yellow solid; mp 189-192 °C; 1H NMR (CD3OD) δ 7.70 (d, J )
8.7 Hz, 2H), 7.51 (d, J ) 7.7 Hz, 1H), 7.29 (t, J ) 7.5 Hz, 1H),

Synthesis and PKC Activity of Balanol Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 233
Sch em e 7. Synthesis of Precursors to Analogs 47-52a
a
Conditions: (a) (tBoc)2O, DMAP, CH2Cl2; (b) mCPBA, CH2Cl2; (c) NaN3, NH4Cl, MeOH-H2O; (d) H2, Pd-C, EtOAc; (e) 3, CDI, THF;
then NaOH, H2O-MeOH; (f) CF3CO2H, CH2Cl2; (g) acetone, NaBH4, AcOH; (h) Ac2O; (i) (CF3CO)2O; (j) CH3NCO, NEt3, MeOH; (k)
CH3OCOCl, pyridine; (l) CH3SO2Cl, pyridine, CH2Cl2; (m) mCPBA, CH2Cl2; (n) TMSN3, Ti(OiPr)4, CH2Cl2; 1 N HCl; (o) H2, 10% Pd-C,
MeOH; (p) 3, (COCl)2, aq NaOH, CH2Cl2.
Ta ble 6. Kinase Inhibition by N-Substituted Pyrrolidine
Analogs (IC50 values, µM)
(()-tr a n s-1-Aza -3-(4-h yd r oxyb en za m id o)-4-[[4-[2-h y-
d r o x y -6-(h y d r o x y c a r b o n y l)b e n zo y l]-3,5-d ih y d r o x y -
ben zoyl]oxy]b icyclo[3.2.2]n on a n e (37): yellow solid (7.6
1
mg, 22%); mp 260-265 °C dec; H NMR (DMSO-d6) δ 11.66
(s, 2H), 10.06 (s, 1H), 9.85 (s, 1H), 8.33 (d, J ) 8 Hz, 1H), 7.62
(d, J ) 9 Hz, 2H), 7.36 (d, J ) 8 Hz, 1H), 7.28 (t, J ) 8 Hz,
1H), 7.05 (d, J ) 8 Hz, 1H), 6.80 (m, 4H), 5.28 (d, J ) 8 Hz,
1H), 4.70 (m, 1H), 3.15-3.70 (m, 6H), 2.70 (m, 1H), 1.80-2.40
(m, 4H); FABMS m/z ) 577 (M + 1).
(()-tr a n s-2-[[4-[2-H yd r oxy-6-(h yd r oxyca r b on yl)b en -
zoyl]-3,5-dih ydr oxyben zoyl]oxy]-3-(4-h ydr oxyben zam ido)-
bicyclo[2.2.1]h ep ta n e (38): yellow solid (110 mg, 68%); mp
185-193 °C dec; 1H NMR (CD3OD) δ 7.54 (d, J ) 8.7 Hz, 2H),
7.29 (d, J ) 7.6 Hz, 1H), 7.06 (t, J ) 8.1 Hz, 1H), 6.82 (d, J )
8.2 Hz, 1H), 6.70 (s, 2H), 6.61 (d, J ) 8.7 Hz, 2H), 4.59 (s,
1H), 3.98 (br s, 1H), 2.42 (br s, 1H), 2.19 (d, J ) 4.4 Hz, 1H),
compd
R
â1
â2
γ
δ
ꢀ
η
ú
PKA
47
48
49
50
51
52
0.24 0.06 0.07 0.09 0.04 0.05
1.2 0.71 0.82 0.39 0.06 3.7
0.43 0.36 0.26 0.26 0.1
0.54 0.81 2.7 0.54 0.1
2.2 0.68 1.8 0.41 0.04 3.6
0.91 0.33 0.35 0.4 0.035 1.3
0.004
0.03 >150
37 <0.33
1.65 (d, J ) 9.9 Hz, 1H), 1.51-1.22 (m, 5H); IR (KBr, cm-1
)
3.6
2.6
3369, 2963, 1703, 1635, 1607, 1505, 1425, 1365, 1238, 1176,
762; FABMS m/z ) 548 (M + 1). Anal. (C29H25NO10) C, H,
N.
0.25 <0.01
4.8
56
0.09 >150 13
0.13
0.062 >150
>50
3.0
4.5
(+)-tr a n s-2-[[4-[2-H yd r oxy-6-(h yd r oxyca r b on yl)b en -
zoyl]-3,5-dih ydr oxyben zoyl]oxy]-3-(4-h ydr oxyben zam ido)-
1,7,7-tr im eth ylbicyclo[2.2.1]h ep ta n e (39): yellow solid (26
mg, 46%); [R]D +101.84° (c 0.38, MeOH, room temperature);
1141, 764; FABMS m/z
(C28H26N2O10‚1.8CF3CO2H) C, H, N.
)
551 (M
+
1). Anal.
1
mp 192-196 °C dec; H NMR (CD3OD) δ 7.53 (d, J ) 8.7 Hz,
(()-(1â,3r,4â)-1-(Am in om eth yl)-3-(4-h yd r oxyben za m i-
d o)-4-[[4-[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-3,5-d i-
h yd r oxyben zoyl]oxy]cyclop en ta n e (33â): yellow solid (17
mg, 24%); mp 165-169 °C dec; 1H NMR (CD3OD) δ 7.48 (d, J
) 8.7 Hz, 2H), 7.28 (d, J ) 7.8 Hz, 1H), 7.06 (t, J ) 7.9 Hz,
1H), 6.81 (d, J ) 8.2 Hz, 1H), 6.69 (s, 2H), 6.60 (d, J ) 8.7 Hz,
2H), 5.17 (m, 1H), 4.37 (m, 1H), 2.82 (d, J ) 7.0 Hz, 2H), 2.37
(m, 2H), 1.83 (m, 2H), 1.41 (m, 1H); IR (KBr, cm-1) 3443, 3243,
1680, 1633, 1607, 1508, 1427, 1243, 1199, 1141, 919, 764;
FABMS m/z ) 551 (M + 1). Anal. (C28H26N2O10‚2.0H2O‚1.5CF3-
CO2H) C, H, N.
2H), 7.28 (d, J ) 8.2 Hz, 1H), 7.06 (t, J ) 8.1 Hz, 1H), 6.81 (d,
J ) 8.1 Hz, 1H), 6.70 (s, 2H), 6.61 (d, J ) 8.7 Hz, 2H), 4.79 (d,
J ) 3.5 Hz, 1H), 4.41 (m, 1H), 1.89 (br s, 1H), 1.56-1.21 (m,
4H), 1.07 (s, 3H), 0.76 (s, 3H), 0.71 (s, 3H); IR (KBr, cm-1
)
3387, 3308, 2961, 1703, 1634, 1607, 1505, 1425, 1368, 1238,
1176, 764; FABMS m/z ) 590 (M + 1). Anal. (C32H31
NO10‚0.5CF3CO2H) C, H, N.
-
(()-8-exo-[[4-[2-Hyd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-
3,5-dih ydr oxyben zoyl]oxy]-9-en do-(4-h ydr oxyben zam ido)-
tr icyclo[5.2.1.02,6]d eca n e (40): yellow solid (166 mg, 82%);

234 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Lai et al.
(6-line mult, 2H), 3.2-3.4 (m, 2H), 2.53 (s, 3H); IR (KBr, cm-1
mp 195-200 °C dec; 1H NMR (DMSO-d6) δ 11.66 (s, 2H), 9.97
(br s, 1H), 9.88 (s, 1H), 8.35 (d, J ) 7 Hz, 1H), 7.75 (d, J ) 9
Hz, 2H), 7.38 (d, J ) 8 Hz, 1H), 7.28 (t, J ) 8 Hz, 1H), 7.06 (d,
J ) 8 Hz, 1H), 6.83 (s, 2H), 6.79 (d, J ) 9 Hz, 2H), 4.83 (d, J
) 2.5 Hz, 1H), 4.11 (m, 1H), 2.25 (d, J ) 4 Hz, 1H), 2.10-2.20
(m, 1H), 2.07 (br s, 2H), 1.60-2.00 (m, 3H), 1.48 (br s, 2H),
1.10-1.30 (m, 1H), 0.90-1.10 (m, 2H); IR (KBr, cm-1) 1703,
)
3385, 1714, 1605, 1236, 763 cm-1; HRMS m/z calcd for
C28H25N3O11 580.1567, found 580.1481. Anal. (C28H25N3O11
0.8H2O‚0.8CF3CO2H) C, H, N.
‚
(()-1-(Meth ylsu lfon yl)-tr a n s-3-(4-h yd r oxyben za m id o)-
4-[[4-[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih y-
d r oxyben zoyl]oxy]p yr r olid in e (52): yellow solid (137 mg,
1
1635, 1608; FABMS m/z ) 588 (M + 1). Anal. (C32H29
NO10‚1.0H2O‚0.25CF3CO2H) C, H, N.
-
74%); mp 179-187 °C dec; H NMR (CD3OD) δ 7.53 (d, J )
8.7 Hz, 2H), 7.29 (d, J ) 7.6 Hz, 1H), 7.07 (dd, J ) 7.9, 8.1 Hz,
1H), 6.82 (d, J ) 8.2 Hz, 1H), 6.73 (s, 2H), 6.62 (d, J ) 8.7 Hz,
2H), 5.29 (dt, J ) 5.5, 3.1 Hz, 1H), 4.48 (dt, J ) 6.2, 4.2 Hz,
1H), 3.73 (dd, J ) 12.0, 5.5 Hz, 1H), 3.67 (dd, J ) 10.7, 6.9
Hz, 1H), 3.35 (dd, J ) 12.0, 2.8 Hz, 1H), 3.28 (dd, J ) 10.6,
4.4 Hz, 1H), 2.75 (s, 3H); FABMS m/z ) 601 (M + 1); IR (KBr,
(()-tr a n s-3-[[4-[2-H yd r oxy-6-(h yd r oxyca r b on yl)b en -
zoyl]-3,5-dih ydr oxyben zoyl]oxy]-2-(4-h ydr oxyben zam ido)-
8-m eth yl-8-a za bicyclo[3.2.1]octa n e (41): yellow solid (120
mg, 82%); mp 250 °C dec; 1H NMR (DMSO-d6) δ 11.50 -12.20
(br s, 2H), 9.50-10.20 (br s, 2H), 8.11 (d, J ) 8 Hz, 1H), 7.58
(d, J ) 9 Hz, 2H), 7.20-7.35 (m, 2H), 6.98 (d, J ) 8 Hz, 1H),
6.86 (s, 2H), 6.75 (d, J ) 9 Hz, 2H), 5.34 (m, 1H), 4.67 (m,
1H), 3.60 (m, 2H), 2.59 (s, 3H), 1.90-2.80 (m, 6H); IR (KBr,
cm-1) 1712, 1637, 1608; FABMS m/z ) 577 (M + 1). Anal.
(C30H28N2O10‚1.3H2O‚0.5CF3CO2H) C, H, N.
(()-1-Isop r op yl-tr a n s-3-(4-h yd r oxyben za m id o)-4-[[4-[2-
h ydr oxy-6-(h ydr oxycar bon yl)ben zoyl]-3,5-dih ydr oxyben -
zoyl]oxy]p yr r olid in e (47): yellow solid (32 mg, 59%); mp
194-198 °C dec; 1H NMR (CD3OD) δ 7.78 (d, J ) 8.7 Hz, 2H),
7.52 (d, J ) 7.7 Hz, 1H), 7.30 (t, J ) 8.0 Hz, 1H), 7.05 (d, J )
8.2 Hz, 1H), 7.00 (s, 2H), 6.86 (d, J ) 8.7 Hz, 2H), 5.69 (m,
1H), 4.68 (m, 1H), 3.93-4.04 (m, 2H), 3.55-3.92 (m, 2H), 3.60
(quint, J ) 6.5 Hz, 1H), 1.45 (d, J ) 6.5 Hz, 3H), 1.44 (d, J )
6.4 Hz, 3H); IR (KBr, cm-1) 1705, 1676, 1636, 1607; FABMS
m/z ) 565 (M + 1). Anal. (C29H28N2O10‚1.0H2O‚1.0CF3CO2H)
C, H, N.
(()-1-Acetyl-tr a n s-3-(4-h yd r oxyben za m id o)-4-[[4-[2-h y-
d r oxy-6-(h yd r oxyca r b on yl)b en zoyl]-3,5-d ih yd r oxyb en -
zoyl]oxy]p yr r olid in e (48): yellow solid (43 mg, 59%); mp
196-200 °C dec; 1H NMR (CD3OD) δ 7.71 (d, J ) 8.6 Hz, 2H),
7.48 (d, J ) 7.7 Hz, 1H), 7.26 (t, J ) 7.9 Hz, 1H), 7.02 (d, J )
7.3 Hz, 1H), 6.91 (s, 2H), 6.80 (d, J ) 8.6 Hz, 2H), 5.46 (dm, J
) 10.4 Hz, 1H), 4.70 (tm, J ) 10.4 Hz, 1H), 4.07 (dt, J ) 11.1,
5.3 Hz, 1H), 3.93 (dt, J ) 12.4, 5.5 Hz, 1H), 3.60-3.72 (m,
2H), 2.05 and 2.08 (both s, 3H, rotamers); IR (KBr, cm-1) 1716,
cm-1
(C27H24N2O12S‚2.5H2O) C, H, N.
)
3394,
1708,
1607,
1235,
762.
Anal.
P r otein Kin a se C Exp r ession a n d P u r ifica tion . The
R, â1, â2, γ, δ, ꢀ, η, and ú recombinant human PKC enzymes
were produced using a baculovirus expression system in SF9
cells.15 The Ca2+-independent isozymes (δ, ꢀ, η, and ú) were
purified as described in the literature by Bronson et al.16 The
Ca2+-dependent isozymes (R, â1, â2, and γ) were purified using
a modification of a method described by Kochs et al.17 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 a n d cAMP -Dep en d en t Kin a se As-
sa ys. PKC was assayed by quantitating the incorporation of
32P from [γ-32P]ATP into histone type IIIS. The reaction
mixture (250 µL) contained 30 µg of phosphatidylserine
(Avanti), 20 mM Hepes buffer (pH 7.5; Sigma), 10 mM MgCl2,
47.5 µM EGTA, 100 µM CaCl2, 200 µg/mL histone (Sigma), 10
µL of DMSO or compound in DMSO, 30 µM [32P]ATP (DuPont),
the enzyme, and diacylglycerol. The amount of diacylglycerol
necessary for 50% maximal activation of the enzyme was used.
The assay was performed 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 concentration of compounds tested
to estimate IC50 values ranged from 0.1 nM to 150 µM and
depended on the enzyme employed in the assay and the
compound. The assays always started at the highest concen-
trations, and compounds were retested at lower concentrations
until IC50 values could be determined. Most of the IC50 values
were results of single-point determinations at four concentra-
tions. 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 the PKC assays.18
The cAMP-dependent protein kinase assay was performed as
previously described.19
1633, 1606; FABMS m/z ) 565 (M + 1). Anal. (C28H24N2O11
1.0H2O) C, H, N.
‚
(()-1-(Tr iflu or oa cetyl)-tr a n s-3-(4-h yd r oxyben za m id o)-
4-[[4-[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-3,5-d ih y-
d r oxyben zoyl]oxy]p yr r olid in e (49): yellow solid (51 mg,
1
59%); mp 167-172 °C dec; H NMR (CD3OD) δ 7.74 (d, J )
8.1 Hz, 2H), 7.49 (d, J ) 7.7 Hz, 1H), 7.27 (t, J ) 7.9 Hz, 1H),
7.02 (d, J ) 8.2 Hz, 1H), 6.93 (s, 2H), 6.83 (d, J ) 8.4 Hz, 2H),
5.52 and 5.55 (both m, 1H, rotamers), 4.74 and 4.80 (both m,
1H, rotamers), 4.21 (m, 1H), 4.11 (m, 1H), 3.90 and 3.94 (both
br s, 1H, rotamers), 3.77 and 3.82 (both br s, 1H, rotamers);
IR (KBr, cm-1) 1688, 1635, 1607; FABMS m/z ) 619 (M + 1).
Anal. (C28H21N2O11F3‚1.25H2O) C, H, N.
(()-1-(Meth oxyca r bon yl)-tr a n s-3-(4-h yd r oxyben za m i-
d o)-4-[[4-[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-3,5-d i-
h yd r oxyben zoyl]oxy]p yr r olid in e (50). Anhydrous pyridine
(0.25 mL) was added to a stirred mixture of 22 (0.051 g, 0.080
mmol) and methyl chloroformate (12 µL, 0.160 mmoL) at 0 °C
under N2. The resulting mixture was stirred at 0 °C for 2 h,
allowed to warm to room temperature, and stirred for 16 h.
The solution was then concentrated in vacuo. The residue was
chromatographed on a 41 × 300 mm C-18 column (solvent A,
95:5 water/acetonitrile + 0.1% TFA; solvent B, 100% acetoni-
trile; gradient, 0-100% B over 60 min; flow, 25 mL/min)
affording the title compound (4.2 mg, 9%) as a yellow gum; 1H
NMR (CD3OD) δ 7.52 (dd, J ) 7.7, 2.1 Hz, 2H), 7.29 (d, J )
7.7 Hz, 1H), 7.07 (t, J ) 7.8 Hz, 1H), 6.82 (d, J ) 8.0 Hz, 1H),
6.70 (s, 2H), 6.61 (dd, J ) 6.9, 2.0 Hz, 2H), 5.21-5.23 (m, 1H),
4.45-4.47 (m, 1H), 3.67-3.75 (m, 2H), 3.51 (s, 3H), 3.36 (dd,
J ) 12.4, 2.4 Hz, 2H); FABMS m/z ) 581 (M + 1).
Ack n ow led gm en t . The authors thank Thomas
Mitchell for his assistance in the physical characteriza-
tion of the compounds used in this study.
Su p p or tin g In for m a tion Ava ila ble: Listing of the ex-
perimental details for the preparation of all intermediates (19
pages). Ordering information is given on any current mast-
head page.
Refer en ces
(1) Preliminary communications published in (a) Lai, Y.-S.; Stamper,
M. Heteroatom effect in the PKC inhibitory activities of perhy-
droazepine analogs of balanol. Bioorg. Med. Chem. Lett. 1995,
5, 2147-2150. (b) Lai, Y.-S.; Menaldino, D. S.; Nichols, J . B.;
J agdmann, G. E., J r.; Myllot, F.; Gillespie, J .; Hall, S. E. Ring
size effect in the PKC inhibitory activities of perhydroazepine
analogs of balanol. Bioorg. Med. Chem. Lett. 1995, 5, 2151-2154.
(c) Lai, Y.-S.; Mendoza, J . S.; Hubbard, F.; Kalter, K. Synthesis
and PKC inhibitory activities of balanol analogs with a cyclo-
(()-1-[(Meth yla m in o)ca r bon yl]-tr a n s-3-(4-h yd r oxyben -
za m id o)-4-[[4-[2-h yd r oxy-6-(h yd r oxyca r bon yl)ben zoyl]-
3,5-d ih yd r oxyben zoyl]oxy]p yr r olid in e (51): yellow pow-
1
der (68.7 mg, 77%); mp 178-198 °C dec; H NMR (CD3OD) δ
7.53 (d, J ) 8.7 Hz, 2H), 7.30 (d, J ) 7.7 Hz, 1H), 7.06 (dd, J
) 8.1, 7.9 Hz, 1H), 6.80 (d, J ) 8.3 Hz, 1H), 6.70 (s, 2H), 6.62
(d, J ) 8.7 Hz, 2H), 5.2-5.3 (m, 1H), 4.4-4.5 (m, 1H), 3.6-3.8

Synthesis and PKC Activity of Balanol Analogs
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diabetic complications. Decreased cytotoxicity due to potent
inhibition of only one of the PKC isozymes may be of significant
benefit in some diseases. As PKC’s relevance in specific diseases
evolves, the importance of developing specific inhibitors will yield
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ever, we have no evidence that these compounds are acting like
calphostin. Although the assays were not performed in the dark,
light was minimal. Calphostin tested under these conditions
values 10-20-fold less than what was reported
resulted in IC50
in the literature. Kinetics were performed on the parent balanol
compound, and this compound appeared to be competitive with
ATP. Also, for a virtual kinetic study resulting in a similar
conclusion, see ref 16.
(8) (a) Lampe, J . W.; Hughes, P. F.; Biggers, C. K.; Smith, S. H.;
Hu, H. Total synthesis of (-)-balanol. J . Org. Chem. 1994, 59,
5147-5148. (b) Lampe, J . W.; Hughes, P. F.; Biggers, C. K.;
Smith, S. H.; Hu, H. Total synthesis of (-)- and (+)-balanol. J .
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(11) See ref 1 and discussion therein.
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(13) Unpublished results.
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whether they are inhibitors of ATP binding, and the possibility
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was very slow. Conclusive experiments could not be performed
due to the instability of the enzyme under these assay conditions.
The light sensitivity of these compounds was not tested. How-
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